102

Mycorrhiza application in sustainable agriculture and natural systems

Working groups 2 and 4 meeting

17-19 September

Aristotle University of Thessaloniki, School of Forestry and Natural Environment,

Forest Soil Lab. Thessaloniki, Greece

Organising committee: Jacqueline Baar, Victoria Estaun, Ibrachim Ortas, Michail Orfanoudakis, Dimitrios

Alifragis

Hotel Philippion

2

environmental factors on the development of these symbiontic fungi. If the set-up of such

studies is systematic, statistical analysis methods can be applied relating biological

components with abiotic factors providing data that are useful for the production and

application of AM fungi. In such an approach, environmental factors are of major importance.

For mycorrhizal studies, there are two major groups of environmental factors that can be

distinguished clearly affecting the development of the fungi. One major environmental factor

in mycorrhizal symbiosis comprises the host plant, and the other major factor is the soil

environment (see fig 1).

Systematic set-up

Fig. 1. Systematic studies on the relations between the environmental factors ―host plant‖and

―soil‖ and AM fungi provide more insight in the development of AM fungi enabling

optimization of production and application of AM fungi.

The environmental factor “host plant”

An essential environmental factor in mycorrhizal symbiosis is the host plant. In fact, the

symbiosis is as much dependant on the host plant as on the fungus. Approaching mycorrhizal

symbiosis from the plant, the plant influences considerably the effectiveness of the symbiosis

by its susceptibility. In various experiments, it has been shown that plant species vary greatly

in their responsiveness to mycorrhizal fungi (Van der Heijden et al. 1998). Also, crops can

vary in their mycorrhizal responsiveness as has been reported for a variety of agricultural

crops (Ryan & Graham 2002). For agricultural purposes, breeding programs have resulted in

varieties or cultivars with a range of genetic differences. Variation in mycorrhizal dependency

of Citrus rootstocks to AM fungi has been reported. However, some of the breeding programs

have resulted in poor responses of AM fungi to cultivars of major main crops of economic

value. Examples are corn (Zea mays), oat (Avena sativa), barley (Hordeum vulgare) and

wheat (Triticum aestivum) (Hetrick et al. 1993; Kaeppler et al. 2000; Ryan & Graham 2002).

The breeding of agricultural crops has been carried out for raising resistance to fungal and

bacterial pathogens. Unfortunately, successful breeding against pathogens was accompanied

with suppression of AM fungal colonization and responsiveness (Ryan & Graham 2002). As a

consequence, incompatible reactions between host plants and certain AM fungi have been

observed. Hetrick et al. (1993) noted that breeding of wheat cultivars has resulted in high

dependency on fertilizers and non-responsiveness to AM fungal colonization.

As in many biological processes, numerous genes could be involved in the symbiosis between

host plants and mycorrhizal fungi. Experiments with different mycorrhiza-defective plant

mutants indicate that root colonization of AM fungi is controlled by a large number of genes

(Gollotte et al. 2002). Thus far, most researchers have focused on the identification of plant

genes controlling essential steps in the symbiosis between host plant and AM fungus

(Balestrina & Lanfranco 2006). A number of the mycorrhiza-regulated genes have been

identified by this approach, but there are still plants genes involved in the mycorrhization

process unknown (Gollotte et al. 2002).

An additional applied approach could be setting up breeding programs aiming for the

development of crop varieties that are susceptible for AM fungi. Evidence is growing that

wild accessions or old crop cultivars show more susceptibility to AM fungi than modern

cultivars of these species, indicating that mycorrhizal responsiveness may have been bred out

Abundance and diversity

of arbuscular

mycorrhizal fungi

Environmental factors

host plant

soil

3

of some crops (Kik, oral. comm.). These studies demonstrate that genetic traits determining

mycorrhizal responsiveness exist in the plants as well as in the fungi involved in this

symbiosis. This indicates the clear needs of combining the knowledge of plant geneticists and

breeders with scientists working on AM fungi in order to develop an understanding of the

plant genetic basis for mycorrhizal responsiveness.

Cooperation between plant geneticists, plant breeders, and mycorrhizal researchers is one of

the aims of the European network COST Action 870 enabling the set-up of programs for

searching and developing crop varieties susceptible for AM fungi. Determining differences of

various crop varieties in their response to AM fungi fits well into such programs. These can

form the basis for developing more optimal combinations of mycorrhizal responsiveness of

crop varieties to the most beneficial AM fungi. Further studies are needed to determine the

genetic traits in the diverse crops that are of economic value worldwide. Thus far, several

studies on the responses of different crops varieties to AM fungi have been carried out, like in

The Netherlands (Baar & Ozinga, 2007). Also, some studies for plant breeding have started to

study the basis for developing crop varieties susceptible for AM fungi in Europe. Still, more

studies on different crops are needed.

The environmental factor “soil”

Soil is the other important determining environmental factor for the development of

mycorrhizal fungi. This environmental factor comprises different soil factors described either

by the abiotic chemical and physical components, or by the biotic soil components. The

abiotic factors include chemical and physical composition as well as moisture content of the

soil. The vast majority of soils in the world contain AM fungi, but the diversity and

abundance can vary. There are some studies showing that soil mineral content and structure

can affect AM fungal communities (Johnson et al., 1992; Oehl et al. 2005). Johnson et al.

(1992) showed that the occurrence of six AM fungal species was influenced by soil type. The

study by Oehl (2005) revealed that the AM fungal communities changed with soil depth and

that different AM fungal species were observed in different soil layers.

Generally, the development of AM fungi and their effects on plant growth are greater in soils

with relatively low nutrient content, particularly with low nitrogen and phosphorus levels,

than in soils with relatively high nutrient content. As shown in various studies, high levels of

nitrogen and phosphorus, often caused by intensive fertilization with chemical fertilizers and

live-stock manure, reduces development of root colonization of AM fungi, but the magnitude

of the effect is strongly affected by the fungi studied and by other environmental conditions

(Baar & Ozinga, 2007). This is illustrated by a study by Egerton-Warburton & Allen (2000)

showing that enhanced soil nitrogen concentrations changed the composition of the AM

fungal communities in coastal vegetation communities in southern California and that the

abundance of AM fungal spores was reduced by nitrogen enrichment. In a study in The

Netherlands, it was found that grasslands with Lolium perenne L. intensively fertilized with

live-stock manure with high levels of nitrogen and phosphate for several decades, contained

less than 1% of AM fungi and were colonized with oomycetous fungi (Baar & Ozinga, 2007).

A more recent study in 2007 showed that colonization by AM fungi in a L. perenne grassland

was reduced by intensive fertilization with live-stock manure for over twenty years (see Table

1.).

% AC % VC

Non-fertilized grassland 20.7 19.9

Fertilized grassland 12.6 12.3

4

Table 1. Reduced percentage of colonization of AM fungi in grass roots expressed as % AC

for the amount of arbuscules and % VC for the amount of vesicles by intensive fertilization

with live-stock manure. Samples were obtained in 2007 and colonization levels were

determined microscopically after staining according to McGonigle (1990).

The studies carried out thus far have provided us with knowledge on the development of

mycorrhizal fungi in relation to the chemical and physical composition of the soil. However,

the number of studies relating AM fungal development with chemical and physical soil

composition in a systematic way is limited (Estaún et al. 2002).

Fig. 2. Sampling soil for a study to relate the abundance and diversity of AM fungi to the

chemical soil conditions in The Netherlands.

Setting up more systematic studies for unravelling the effects of soil variables on the AM

fungal development is one of the aims of the European network COST Action 870. If the set-

up of the studies is systematic, statistical analysis methods can be applied to relate the

abundance and diversity of AM fungi with the chemical and physical soil composition. Such

systematic studies provide data that are useful for the production and application of AM fungi.

Within the European network COST Action 870, new studies are set up for relating the

chemical soil composition to the development of AM fungi.

References Baar J, Ozinga WA, 2007. Mycorrhizal fungi key factor for sustainable agriculture and nature

(In Dutch). KNNV-uitgeverij, Zeist, The Netherlands.

Balestrini R, Lanfranco L, 2007. Fungal and plant gene expression in arbuscular mycorrhizal

symbiosis. Mycorrhiza 17:153-153.

Egerton-Warburton, LM, Allen EB, 2000. Shifts in arbuscular mycorrhizal communities

along an anthropogenic nitrogen deposition gradient. Ecological Applications 10: 484-496.

Estaún V, Camprubi A, Joner EJ, 2002. Selecting arbuscular mycorrhizal fungi for field

application. In: Gianinazzi S, Schüepp H, Barea JM, Haselwandter K (eds) Mycorrhizal

Technology in Agriculture. Birkhäuser Verlag, Basel, pp. 249-259.

Gollotte A, Brechenmacher L, Weidmann S, Franken P, Gianinazzi-Pearson V, 2002. Plant

genes involved in arbuscular mycorrhiza formation and functioning. In: Gianinazzi S,

Schüepp H, Barea JM, Haselwandter K (eds) Mycorrhizal Technology in Agriculture.

Birkhäuser Verlag, Basel, pp 87-102.

Hetrick BAD, Wilson GWT, Cox TS, 1993. Mycorrhizal dependence of modern wheat

cultivars and ancestors: a synthesis. Canadian Journal of Botany 71: 512-518.

Johnson NC, Tilman D, Wedin D, 1992. Plant and soil controls on mycorrhizal fungal

communities. Ecology 73: 2034-2042.

5

Kaeppler SM, Parke JL, Mueller SM, Senior L, Struber C, Tracy WF, 2000. Variation among

maize inbred lines and detection of quantitative trait loci for growth at low phosphorus and

responsiveness to arbuscular mycorrhizal fungi. Crop Science 40:358-364

McGonigle TP, Miller MH, Evans DG, Fairchild DL, Swan JA, 1990. A new method which

gives an objective measure of colonization of roots by vesicular-arbuscular mycorrhizal fungi.

New Phytologist 115: 495-501.

Oehl F, Sieverding E,

Ineichen K,

Ris E-A,

Boller T,

Wiemken A, 2005. Community structure

of arbuscular mycorrhizal fungi at different soil depths in extensively and intensively

managed agroecosystems. New Phytologist 2005: 273-283.

Ryan MH, Graham JH, 2002. Is there a role for arbuscular mycorrhizal fungi in production

agriculture? Plant and Soil 244: 263-271

Van der Heijden MGA, Boller T, Wiemken A, Sanders IS, 1998. Different arbuscular

mycorrhizal fungal species are potential determinants of plant community structure. Ecology

79: 2082-2091.

6

Introduction about mycorrhizal work in Greece and future possibilities.

Orfanoudakis M.

Forest Soil Lab, School of Forestry and Natural Environment, Aristotle University of

Thessaloniki, Greece PO BOX 271

Introduction

The soil is probably the most important natural resource. The soil supports the plant growth

by providing the necessary nutrient and water. However the terrestrial ecosystems are losing

significant amounts of this unique resource. Human activities such as roads building

structures and others lead to loss of surface material, destruction of the organic matter with

consequences to several other natural resources and to the atmospheric gases.

From the other hand Modern agriculture made possible for large land sites to be in crop

production system covering the needs in feeding of an expanding world population. However

the modern systems become more depended upon fertilisers, agrochemicals and machinery

covering the limited availability in nitrogen phosphate and other plant nutrients (Atkinson et

al 1996. With such practises significant increase to crop production achieved even at place

were the natural ability of the soil to crop production was limited. The desire for

maximisation became a necessity. The desire for more quantity easily directs to bad

fertilisation management systems. With this significant effect to the agricultural economy,

due to the increase spends on chemicals and to the natural resources due to the loss of the

―extra‖ fertilisers occurred. In addition the bad soil management often led to degradation and

to the loss of valuable surface soil material. Such mechanisms could increase to loss of

phosphate from the soils. Gradually the soil as natural resource becomes less fertile, unable to

sustain the human desire for development. From the times of the antiquities people in Greece

were worried by the loss of soil mass and the loss of productivity. (Plato Kritias 111b From

Alifragis 2008).These soil changes are common to the Mediterranean regions. The loss of the

surface soil leads to a loss of significant AMF number of species. Thus are possible due to the

loos of material, increase to the soil temperatures, and the lack of plant species diversity.

Farming applications

Sustainable agriculture is not just a modern idea about farming but rather a necessity in order

to maintain the productivity of the poor framing lands. At a global economy environment to

maintain of the maximisation at poor sites is like a dream of a summer night. Managing the

soil in favour of AMF diversity is the key to sustainable farming systems.

The soil history of the Mediterranean regions however, suggests that the natural occurring

AMF population is reduced, significantly, due to the loss of surface soil material or from the

extensive use of fertilisers and other agrochemicals. In particular at the second case the

remaining AMF population will be from those species fitting better to the environment but

with those fitting better to the agrochemicals applied.

Selective AMF species application could be an important solution to the problem. The

selective species could be imported to the farming system either as selective inoculum or as

pre inoculated plants. In both forms could improve the survivability of the host and enchase

growth particularly in sites of medium fertility (Orfanoudakis et al unpublished).

Natural system applications.

Mycorrhizal applications are applicable to the natural systems. As it was prolonged before the

degradation is a significant problem of the Mediterranean regions The loss of surface soil

layers and the exposure of the raw rock material creates harsh soil condition for the green

plants to grow. Heavy metals high soil surface temperatures lack of nutrient availability and

the absence of enough water are among the problems should be compensate by vegetation.

7

The role of AMF upon such environments is well documented. However there are not enough

applications in the field.

The management of the natural occurring AMF population could give an advantage.

Presumably the autochthonous species are important to the vegetation establishment.

Additionally AMF could be applied as selective inoculum and in pre-inoculated plant

The effects of the mycorrhizosphere created increasing the availability of phosphate and other

nutrients (Fig 1)

Figure 1: Effects extractable P after inoculation with different arbuscular mycorrhizal fungi at the three years of the experiment. Glomus intraradices (empty), Gigaspora margarita (lined), Acaulospora longula (squered), mix of BEG isolates (sphere), indigenous AMF (filled).Bars are standard error. Data points marked with an asterisk are significantly different from each other (P < 0.05).

Such differences could lead the formation of the plant community. In particular as it is

described in Fig 1 the variations of the mycorrhizosphere ability the increase the extractable P

from the inorganic soil material could give an advantage to the plants with the highest

biological compatibility. Such examples were demonstrated in the past (Van der Heijden et al

1998). Managing this we could drive the natural population to desirable plant diversity.

Introduction of exotic AMF species at new sites will eventually turn in favour of the

autochthonous fungi. (Qing Yao et al 2007) The lack of enough data upon such mechanism is

not well documented and lots of future work is needed on how soon the indigenous species

will dominate again or for how long the superiority of ―selective‖ AMF will occur. Also when

applications of selective AMF inoculum occur we need to investigate how interacts with the

local plant species. Particularly at natural systems, that could means the AMF application

could be in favour of not desirable plant species.

References

Atkinson, D. and Watson C.A 1996. The environmental impact of intensive systems of animal

production in the low lands. Animal Science 65: 353-361

Alifragis D. 2008 The soil : Genesis-Properties- Clasification. Book I Thessloniki 2008

0

20

40

60

80

100

120

140

1 2 3

P m

g/1

00

g a

sper

cen

tag

e o

f th

e co

ntr

ols

*

*

*

** *

*

8

Qing Yao, Hong-Hui Zhu, You-Li Ho and Liang-Qiu Li 2008 Differential influence of native

and introduced arbuscular mycorrhizal fungi on growth of dominant and subordinate plants.

Plant Ecology 196, 261-268

9

What determines a quality mycorrhizal inoculant?

Peter Moutoglis

BioΣyneterra Solutions Inc., Lanaudiere Industrial and Experimental Carrefour 801 route

344, P.O. Box 3158, L’Assomption, Quebec, J5W 4M9, CANADA

What determines a quality inoculant? Several presentations have addressed this question over

the past years (Adholeya, 2006; Baar et al., 2008; Blal & Parat, 2003; Gagné & Moutoglis,

2006; Gollotte et al., 2008; Gianinazzi & Vosátka, 2004; Moutoglis et al, 2003; Quinn &

Blal, 2003) and common ideas that are consistent in all are the following: 1) pathogen-free,

non-contaminated inoculant ; 2) inoculant that contains mycorrhizal propagules in the form of

spores, colonized roots, hyphae or combinations thereof; 3) methodology(ies) by which the

active ingredient (propagules) can be quantified; 4) clearly defined application rates based on

statistically significant responses to tested claims; 5) product that has a validated and defined

shelf life and storage conditions throughout the supply chain.

Most of these criteria are consistent with the Canadian regulatory system which ensures that

products are safe for humans, plants and the environment, efficacious for their intended

purpose(s) and properly labeled. Efficacy is defined as the ability to fulfill any label claims,

supported by scientifically valid efficacy data, and to produce a desired or intended result

based on the labeled guarantees and directions for use. This definition includes the ability to

clearly demonstrate a benefit to the end user from the application of the product. In addition to

specific claims and guarantee(s), each usage pattern or direction for application on the product

label should be supported by scientifically valid efficacy data. Anecdotal or testimonial

evidence is not to be considered as a scientifically valid form of efficacy data (Government of

Canada, Canadian Food Inspection Agency, 2008) but as a highly valuable marketing tool.

An example will be presented from field studies of different species of saplings inoculated

with ectomycorrhizal inoculants from the selection, production, application, validation and

registration process. Small scale nutrient uptake, biomass and stress resistance variables were

tested and measured for screening. In addition, the collar diameter and height of the saplings

and, in some cases, the dry mass were measured both in small and large scale field

experiments. The studies and analyses show that the mycorrhizal inoculant conformed to the

required ―quality‖ standards and resulted in statistically significant responses for the treated

saplings. The efficacy data was submitted and the mycorrhizal product was registered for sale

in Canada. The ultimate goal was to translate this data into estimates for the potential

reductions in greenhouse gases that may result from the mycorrhizal technology which could

be registered as Emission Reduction Credits (ERC) and sold to potential clients under a

defined carbon trading system. ERC determination was achieved in a three step process.

Firstly, the acceleration in growth that results from mycorrhizal inoculation was estimated.

Then, the effects the acceleration has on stands were modeled. Finally, the effects from stands

to forests were extrapolated.

Mycorrhizal technology for inoculating seedlings has been shown, from controlled

experiments, to accelerate growth of trees by between 0 and 28%. On average, the

acceleration is 10% for Jack Pine, 8% for Black Spruce and 16% for White Spruce (Table 1).

This results in substantial increases in sequestration over a rotation. In the simplest situation,

at the inoculated stand, we have shown through modeling that inoculation reduces net

greenhouse gas emissions by 19.3, 21.2 and 27.4 t CO2e / ha for Jack Pine, Black Spruce and

10

White Spruce respectively (Table 2). Extrapolating to the forest, the inoculated stands

produce more fiber and decreases harvesting elsewhere in the forest. Modeling suggests that

net greenhouse gas emissions would be reduced by 23.5, 24.7, and 31.7 t CO2e / ha for Jack

Pine, Black Spruce and White Spruce respectively (Table 3).

Mycorrhizal manufacturers, blenders, distributors, retailers and end users have increased over

the last five to ten years. The industry is growing at a significant rate in several countries

around the world, the majority of which are not regulated, nor do they enforce any quality

standards for these types of products. The above case study is but one of many examples that

there are good quality inoculants that do what they claim and how they can be applied in

innovative ways. Industry can be proactive and along with collaborative efforts with

researchers and regulators, a voluntary self-regulating quality system can be conceived and

put into effect. This would give rise to increased credibility to the technology and the various

products and companies that would voluntarily take part in such an endeavor which would

lead to increased acceptance, less skepticism, further research funding, job creation and

greater revenues. Such a project could be mediated by an objective, collective, international

organization like the International Mycorrhizal Society (IMS) with support from the

Mycorrhizal Commercial Relations Committee (MCRC) and COST 870, WG2, Quality

control of AM fungal inoculum.

Acknowledgement

This presentation would not have been possible if not for the close collaborative partnership

shared with Mark Kean, Mikro-Tek Inc., Woodrising Consulting Inc. and Canada‘s climate

change Technology Early Action Measures (TEAM).

References Adholeya, A. 2006. In vitro mass production technology for arbuscular mycorrhizal fungi:

Scientific and industrial aspects. 5th

International Conference on Mycorrhiza. Mycorrhiza for

Science and Society. Granada, Spain, July 23-27, 2006. p. 227

Baar, J., Steffen, F., Huig Bergsma, H. & Carpay, B. 2008. Novel approaches to enhance

application of arbuscular mycorrhizal fungi for the development of sustainable agricultural

11

and landscape systems: experiences from The Netherlands. Proceedings of COST 870

meeting: From production to application of arbuscular mycorrhizal fungi in agricultural

systems: a multidisciplinary approach. Denmark, May 27-30, 2008, p. 27.

Blal, B. & Parat J. 2003. Production, application and regulation of commercial AMF

inoculants in agriculture. The Fourth International Conference on Mycorrhizae (ICOM4).

Montreal, August 10-15, 2003, p. 335.

Gagné, S. & Moutoglis, P. 2006 Challenges for development of mycorrhizal inoculants

adapted for specific markets. 5th

International Conference on Mycorrhiza. Mycorrhiza for

Science and Society. Granada, Spain, July 23-27, 2006. p. 228

Gianinazzi S. & Vosatka M. 2004. Inoculum of arbuscular mycorrhizal fungi for production

systems: science meets business. Can J Bot 82: 1264-1271.

Gollotte, A., Mercy L., Secco, B., Laurent, J., Prost, M., Gianinazzi S. & Lemoine, M-C.

2008. Raspberry biotisation for quality plant production. Proceedings of COST 870 meeting:

From production to application of arbuscular mycorrhizal fungi in agricultural systems: a

multidisciplinary approach. Denmark, May 27-30, 2008, p.11.

Government of Canada, Canadian Food Inspection Agency, 2008. Trade Memoranda

T-4-108: Efficacy data requirements for fertilizers and supplements regulated under the

Fertilizers Act.

Moutoglis, P., Béland, M., Gagné, S. 2003. Challenges in commercializing AM inocula in

the retail market. The Fourth International Conference on Mycorrhizae (ICOM4). Montreal,

Canada. August 10-15, 2003, p. 601.

Quinn, J. & Blal, B. 2003. Commercial challenges of ectomycorrhizal fungi. The Fourth

International Conference on Mycorrhizae (ICOM4). Montreal, August 10-15, 2003, p. 735.

12

Mycorrhizal inoculation of grapevines in replant soils: improved field

application and plant performance.

Nogales A., Camprubí A., Estaún V., Calvet C.

IRTA, Recerca i Tecnologia Agroalimentàries, Ctra. de Cabrils Km 2, E-08348 Cabrils,

Barcelona, Spain.

Introduction Soilborne plant pathogens and abiotic stress factors, such as bad drainage, toxic metabolites or

extreme pH are causal agents that contribute to the severity of the vineyard‘s replant disease.

Several species of fungi are associated with the syndrome and among them, the root rot

fungus Armillaria mellea (Vahl ex Fr.) Kummer is considered the principal cause of soil

fatigue in spanish vineyards.

Considering the fact that there are no commercial rootstocks conferring protection in replant

situations, few control measures are available. Soil fumigation is banned due to high cost and

environmental concerns and long term fallow is strongly recommended before planting, but

growers are not willing to wait in intensive production areas.

The mycorrhizal inoculation of grapevines under controlled conditions has been achieved by

many authors and the beneficial effects on plant growth promotion proved (Linderman and

Davis, 2001; Aguín et al, 2004), thus, the use of arbuscular mycorrhizal fungi (AMF) to

obtain plants with increased capacity to withstand replant stress has been proposed as a

biotechnological alternative.

Two consecutive applied research projects, starting in 2000, have been conducted at IRTA,

Barcelona, involving growers of several wine production areas in Northeastern Spain. The

final purpose was to evaluate mycorrhizal inoculation in replant vineyards by using several

inoculation methods, by comparing different AMF isolates, by testing the agronomic response

of commercial vine rootstocks of different genetic origin, and by establishing the field

performance of mycorrhizal grapevines in replant vineyards with identified replant

contributing factors or pathological causal agents. Some of the results obtained are

summarized in this presentation, focused on rootstock screening and field growth

performance of inoculated mycorrhizal vines.

Mycorrhizal inoculation of grapevine rootstocks suitable for mediterranean soils:

evaluation of their growth response Five commercial rootstocks tolerant to high lime soil contents and commonly used in

mediterranean production areas were inoculated with three Glomus intraradices isolates, two

of them obtained from vineyard soils and the registered BEG 72 isolated from similar edaphic

and climatic conditions. Hardwood cuttings from Richter 110 (Vitis berlandieri Planch. x

Vitis rupestris Scheele), SO4 (V. berlandieri x Vitis riparia Mich.), 41B (V. berlandieri x

Vitis vinifera L.), 140 Ruggeri (V. berlandieri x V. rupestris) , and 1103 Paulsen ( V.

berlandieri x V. rupestris), were rooted in perlite beds (Figure 1) and 15 plants per treatment

were either individually inoculated with the mycorrhizal fungi or fertilized with P (0,035 g

KH2PO4/Kg substrate) once transplanted to 2 L volume containers filled with a pasteurized

substrate mixture (sandy soil, quartz sand and sphagnum peat; 3:2:1, v/v).

After six months growth under greenhouse and shadowhouse conditions, plants were

harvested and growth parameters measured, and the mycorrhizal colonization achieved was

estimated in their root systems.

13

Figure 1 Figure 2

Results obtained for shoot dry weight (Figure 3) after the rootstocks screening defined the

high mycorrhizal aptitude of the most commonly used vine rootstocks in commercial

mediterranean vineyards and the effectivity of the mycorrhizal fungi used as inoculum source.

Figure 3

Field experiments

Nursery Inoculation of Merlot plants with Glomus intraradices BEG 72 and post transplant

growth response in a high lime content replant soil (Calvet et al., 2007)

Plants from the cultivar Merlot grafted on the rootstock SO4 were grown in forest pots filled

with a sphagnum peat-perlite mixture (1:1,v/v) and inoculated with G. intraradices BEG 72

(Figure 4). Two months later, when 15 plants per treatment were transplanted at random to the

field, the mycorrhizal inoculation had caused a significant growth depression in plant shoots,

but only five months after the plants establishment in the high lime content replant soil,

mycorrhizal plants (Figure 5) outgrew the noninoculated control plants (Figure 6) and their

biomass was significantly higher, despite the container‘s volume used in the nursery.

14

Figure 4

One year later, the difference in shoot biomass was still significant between treatments, and

moreover, the foliar relative chlorophyll content recorded demonstrated the presence of a

higher pigment concentration in plants previously inoculated with G. intraradices.

Figure 5 Figure 6

Field inoculation of grapevines in a replanted vineyard soil infested by A. mellea

(Camprubí et al., 2008)

Cabernet Sauvignon plants grafted on Richter 110 were planted in a high pH replant soil

heavily infested by the root-rot fungus A. mellea and with an estimated number of

mycorrhizal propagules of 114 in 100 ml. Seventy-five grapevines per treatment were

established in the field empty loci left by dead plants previously removed. Four treatments

were considered: non inoculated plants, and inoculation with one of the isolates tested in

rootstock evaluation (Figure 3), G. intraradices BEG 72 and two native G. intraradices

refered as isolate 1 and isolate 2. One hundred grams of fungal inocula developed on

―Terragreen®‖ were placed under the plants, but the traditional planting method involving

water flooding around the plants was modified in order to avoid the inocula dispersion and

plants were only watered after planting.

After 8 months growth, vines were pruned and their shoot biomass recorded. Despite the

presence of mycorrhizal propagules in the field soil, G. intraradices BEG 72 significantly

increased the growth of plants (Figure 7), while the other two introduced AM fungi did not.

The results demonstrated that in the field not all the AMF are equally efficient at increasing

plant growth, even if they belong to the same species, and despite their identical performance

when they colonized the same rootstock, Richter 110, under controlled conditions (Figure 3).

15

Figure 7

0

1

2

3

4

5

6

Control Isolate 1 Isolate 2 BEG 72

Dry

weig

ht

(g)

a a

ab

b

Development of new inoculum formulations

The implementation of mycorrhizae into the vineyard agronomical practices pointed out the

need to adapt the inoculation method to the traditional mechanized planting system.

Experimental research has been undertaken to obtain solid formulated products based on the

use of biodegradable organic polymers including mycorrhizal propagules which can be easily

delivered in the water hole when planting grapevines (Figure 8).

Figure 8

Acknowledgements

Financial support from INIA (―Instituto Nacional de Investigación y Tecnología Agraria y

Alimentaria‖) grant RTA-04-027-C2 and ―Miguel Torres S.A.‖ is acknowledged.

References

Aguín O, Mansilla P, Vilariño A, Sainz MJ, 2004. Effects of mycorrhizal inoculation on root

morphology and nursery production of three grapevine rootstocks. Am J EnolVitic 55:108-

111

Calvet C, Camprubí A, Estaún V, Luque J, De Herralde F, Biel C, Savé R, Garcia-Figueres

F, 2007. Aplicación de la simbiosis micorriza arbuscular al cultivo de la vid. Viticultura

Enologia Profesional 110: 1-7

Camprubí A, Estaún V, Nogales A, Garcia-Figueres F, Pitet M, Calvet C, 2008. Response of

the grapevine rootstock Richter 110 to inoculation with native and selected arbuscular

mycorrhizal fungi and growth performance in a replant vineyard. Mycorrhiza 2008,18: 211-

216

Linderman RG, Davis EA, 2001.Comparative response of selected grapevine rootstocks and

cultivars to inoculation with different mycorrhizal fungi. Am J EnolVitic 52:8-11.

16

Use of mycorrhizal inoculum in heavy metal rich industrial wastes

Turnau K.1, Wojtczak G.

1, Ostachowicz B.

2, Ryszka P.

1

1

Institute of Environmental Sciences, Jagiellonian University, ul. Gronostajowa 7, 30-387

Kraków, Poland, 2

AGH University of Science and Technology, Faculty of Physics and Applied Computer

Science, Department of Nuclear Methods. Mickiewicza 30 30-059 Krakow, Poland

Abstract

Results of the experiments concerning the use of mycorrhizal inoculum in phytoremediation

of heavy metal rich industrial wastes are reported. They include simple AMF inoculation and

seeding the area with commercial grass cultivars and introduction of pre-adapted, mycorrhizal

seedlings of plants originating from xerothermic grasslands. Heavy metal uptake studies

(TXRF) were used to select plant species that exclude most potentially toxic metals from the

shoots.

Introduction

Post-flotation wastes rich in heavy metals are an example of harsh substratum difficult for any

biological reclamation (Turnau et al. 2006, Strzyszcz 2003). The slopes of the heap are not

only toxic for plants, but also slide down and are extremely difficult to stabilize. The dust

originating from the waste heap area often contains high levels of Zn, Pb and Cd which poses

serious health hazards for plants and animals. Phytostabilization of such areas is of utmost

importance. Typical remediation practices consist of covering the waste with a layer of soil,

transported mostly from another area, to prevent erosion. The next step consists of the

introduction of trees, and grasses such as Lolium perenne. Although most of these plants are

known to form mycorrhiza or other mutualistic associations, typically no inoculation is being

carried out. After a few years, especially if the area is not being additionally watered, the soil

cover is destroyed and the vegetation is lost, leaving bare spaces of industrial waste. Research

on the plants mycorrhizal status occurring on the Zn-Pb waste in Chrzanów has been carried

out for the last 15 years (reviewed by Turnau et al. 2006). Several experiments were

performed including the introduction of mycorrhizal inoculum followed by seeding various

grasses, and also the introduction of mycorrhizal plants originating from xerothermic

grasslands. These plants together with the inoculum were introduced directly to the waste

without using a soil cover, which is a very expensive practice and demands continuous care

and the use of large volumes of water. The main aim was to select proper cultivars or plant

species that are able to survive under these harsh conditions (see also Turnau et al 2008) and

will avoid the accumulation of toxic elements in shoots. As the nonmycorrhizal plants were

mostly not able to survive under such conditions (field and laboratory data) the experiments

were carried out focusing on mycorrhiza-assisted plants.

Methods

Experimental plots were established in autumn 2003 and 2004 in Chrzanów (wastes of the ZG

Trzebionka Mining Company). They were devoid of vegetation due to either accidental spills

of the sedimental pulp or to mechanical destruction of the surface layer. The tailing material is

characterized by 75% carbonate, high concentrations of Ca2+

, SO42-

and low Na+, K

+, N, P,

Mg2+

, Cl- and HCO3

-. The substratum of the wastes is alkaline (pH 7.4) (Orlowska et al.

2005). Two experiments were carried out. The first one, started in 2003, was carried out on 14

plots each of 20 m2. A part of these plots was additionally treated with AgroHydroGel

(Agroidea Polska). The first seven plots were inoculated each with 15 l of inoculum

17

SYMBIVIT (obtained from SYMbio-M, Czech Republic) containing minimum 20 spores per

ml of five AM fungal strains (Glomus mosseae CM1, and K1, G. intraradices PH5, G.

claroideum BEG 96 and G. etunicatum BEG 136), originating from polluted areas. After

inoculation, the plots were covered with an about 5 cm deep layer of substratum and each plot

was seeded with one of the following grass species: Lolium perenne L. cv. Inca and Solen,

Festuca rubra L. cv. Leo and cv Nimba, Poa pratensis L. var. Alicja, Festuca ovina L. cv.

Spartan. The second experiment was started in 2004 and concerned plants originating from

dry calcareous grassland located in Kalina-Lisiniec (Wyżyna Miechowska, Southern Poland),

members of the following species: Achillea millefolium L., Agropyron intermedium (Host) P.

Beauv., Agrostis capillaris L., Anthyllis vulneraria L., Astragalus cicer L., Brachypodium

pinnatum (L.) P. Beauv., Bromus inermis Leyss., Cirsium pannonicum (L.) Link, Convallaria

majalis L., Dianthus carthusianorum L., Fragaria vesca L., Fragaria viridis Duchesne Inula

ensifolia L., Libanotis montana Crantz, Onobrychis viciifolia Scop., Ononis arvensis L.,

Plantago media L., Verbascum thapsus L. and Veronica spicata L. Two months-old plants

that were introduced on the wastes were pre-adapted by cultivation in soil mixed 4:1 v/v with

the industrial waste substratum and were planted in rows 15 cm apart. The plants were

growing there for almost three years. In October 2007 the shoots (N=5) were collected. Roots

were left to allow further growth of the plants. The analysis of metal content in plant material

was done using Total Reflection X-ray fluorescence (TXRF) (Hołyńska et al. 1998). For the

acid digestion the plant samples were incubated at 185 C for 8 hours. 2 μl of sample were

applied onto the clean quartz reflector and measured with a TXRF spectrometer equipped

with a Mo X-ray tube. Quantitative analysis was performed using an internal standard (Se).

Plate 1. Fig. 1-4. Experimental plots on Zn-Pb Trzebionka waste (Southern Poland).

Fig. 1. Grasses growing after introduction of inoculum just before the beginning of dry

period.

Fig. 2. Verbascum thapsus (in the front row - arrowhead) and Brachypodium pinnatum

introduced into experimental plots.

Fig. 3. Hieracium pilosella developing within the grass tufts (Festuca ovina).

Fig. 4. Verbascum thapsus growing from seeds produced by introduced plants and self-

sown within the tufts of Brachypodium pinnatum.

Results

The introduction of seeds of seven grass cultivars into the metal-rich experimental plots,

where the inoculum was applied, was totally unsuccesful. Although the seeds germinated (fig.

1), the seedlings died during the first longer dry period when the plants were ca. 1 month old.

18

None of the plant cultivars introduced survived the summer period. Slightly more tolerant to

waste conditions were plants on plots treated with AgroHydroGel, but they also did not

survive till the autumn. Only a few plants appeared in the plots inoculated with AMF but they

were probably from seeds that got to the waste from the surroundings or originated from

seeds mixed accidentally into the seed batch used. All these plants were strongly mycorrhizal,

although they were not expanding much. In the places where they appeared in the following

seasons, some other accompanying plants established, such as Hieracium pilosella and

Hieracium aurantiacum. Especially successful was H. pilosella that usually formed new

seedlings on the top of the partly dried tufts and the ramets were formed outside (fig. 3). Such

ramets sometimes disappeared while it was hot and dry, but after rain they were usually

rebuilt from the remaining parts.

Also the introduction of xerothermic plants as seeds was mostly unsuccessful, even when

mature specimens of grasses transferred from industrial wastes were introduced into the plots

to maintain a source of inoculum and to increase the moisture of the place (―nurse plants‖, fig.

3, 4). Although germination was observed, the seedlings did not survive the large changes of

temperature and moisture during late spring and summer. Tufts of grasses transferred from

older parts of the wastes were left on the plots, and if they did not perform well themselves,

they allowed for the appearance of seedlings of other plant species in the place of the dying

tufts. This was observed again in the case of H. pilosella that developed flowers and seeds on

the top or close to tufts of Festuca ovina. Both plant species were found to be strongly

mycorrhizal and the parameters of the plant performance were similar to populations

developing on non-polluted soils.

Plants introduced into industrial wastes in the form of seedlings that were pre-adapted by

including a fraction of the waste substratum within the soil mix survived better than those

cultivated only on non-polluted soil (fig. 2). This was the reason that the pre-adaptation was

selected. Most of the introduced plants survived till the end of the experiment. Except for

Verbascum thapsus, all other plants propagated vegetatively. They were producing flowers

and seeds but new seedlings were not observed during the study period. V. thapsus, a bi-annul

plant, was the only one among the introduced species, that spread due to seeds produced by

plants introduced into the wastes and formed new seedlings (fig. 4). In laboratory conditions,

over 80% of seeds of this plant produced on the waste germinated on wet filter paper within a

few days.

Among plants introduced into the plots several were efficient accumulators of Pb and Zn. The

highest content of Pb was found in the case of V. thapsus (over 1100 mg kg-1

), Veronica

spicata (400 mg kg-1

), Cirsium pannonicum (360 mg kg-1

) and Plantago media (300 mg kg-1

).

The highest Zn content was found in V. thapsus (5400 mg kg-1

), V. spicata (1450 mg kg-1

),

Inula ensifolia (960 mg kg-1

), C. pannonicum (1700 mg kg-1

), P. media (1450 mg kg-1

),

Dianthus carthusianorum (870 mg kg-1

), Fragaria viridis (936 mg kg-1

). All studied plants

contained Zn mostly much above 200 mg kg-1

, Pb above 30 mg kg-1

and As (up to 50 mg kg-

1). 60% of plant species accumulated also above 70 mg kg

-1 of Mn. A relatively low content of

potentially toxic metals was found in the case of grasses such as Melica transsilvanica,

Bromus innermis, Agropyron intermedium and Anthyllis vulneraria (a member of Fabaceae).

Discussion

The results presented above showed that simple introduction of mycorrhizal inoculum was not

enough in the case of metal-rich industrial wastes. Also the addition of hydrogel was not very

helpful. As shown previously, selected plants originating from xerothermic grasslands (Turnau

et al. 2008) can be used for phytostabilization. Certainly these plants are able to tolerate

drought and high temperature. All the plants used in the experiment at Trzebionka waste

belong to a group of pseudometalophytes that usually should show the metal exclusion

19

strategy, comprising the avoidance of metal uptake and restriction of metal transport to the

shoots. Plants used in phytostabilization should have low contents of toxic elements in shoots,

to avoid contamination of the food chain. According to the presently reported data, all plant

species had as high contents of Pb, Zn and As in shoots that are usually not considered as

suitable for animal food. Such concentrations are, however, common in industrial areas. Still,

we should try to select species that contain as little metals as possible. Among the studied

species the most useful would be Melica transsilvanica, Bromus innermis, Agropyron

intermedium and Anthyllis vulneraria. In those plants also, no differences in photosynthesis

were shown while comparing plants from the waste with those growing on xerothermic

grasslands. On the contrary, very high accumulation of potentially toxic metals was found in

the case of Verbascum thapsus. The possibility to use this species in phytoextraction will be

checked in future. Most plants originating from dry calcareous grasslands were successfully

performing on the waste only if they were introduced as seedlings, what implies higher costs

of introduction. However, these costs are still lower than in case of covering the waste with

additional soil layer transported from other areas and constant watering the site. Plants from

xerothermic grasslands are tolerant enough to heavy metals to survive in vegetative form and

even to produce seeds. Only one species of those (V. thapsus) was able to multiply using its

seeds and even more, these seeds were highly vital. For comparison, plants that are adapted to

growth on wastes such as Silene vulgaris produce seeds that are able to germinate only in

45% while seeds produced in Botanical Garden germinate in 89% (Wierzbicka and Panufnik

1998). Studying the mechanisms of tolerance of V. thapsus should be the next aim of our

research.

Acknowledgements: We greatly acknowledge Dr. Anna Jurkiewicz (Aarhus University, DK)

for the linguistic comments on this manuscript.This research based on the experiment

established within the framework of the Polish Ministry of Scientific Research and

Information Technology 2P04G 003 27 and was carried out further under the project 197/N-

COST/2008/0.

References

Hołynska B, Ostachowicz B, Ostachowicz J, Samek L, Wachniew P, Obidowicz A,

Wobrauschek P, Streli C, Halmetschlager G, 1998. Characterisation of 210Pb dated peat core

by various X-ray fluorescence techniques. Sci. Total Environ. 218, 239–248.

Orlowska E, Jurkiewicz A, Anielska T, Godzik B, Turnau K, 2005. Influence of different

arbuscular mycorrhizal fungal (AMF) strains on heavy metal uptake by Plantago lanceolata

L. Pol. Bot. Stud. 19:65–72.

Strzyszcz Z, 2003. Some problems of the reclamation of waste heaps of zinc and lead ore

exploitation in southern Poland. Z. Geol. Wissenschaft. 31: 167-173.

Turnau K, Orlowska E, Ryszka P, Zubek S, Anielska T, Gawronski S, Jurkiewicz A, 2006.

Role of mycorrhizal fungi in phytoremediation and toxicity monitoring of heavy metal rich

industrial wastes in Southern Poland. In Soil and Water Pollution Monitoring, Protection and

Remediation. Eds. I Twardowska H E Allen and M M Häggblom. pp 533–551. Springer,

Dordrecht.

Turnau K, Anielska T, Ryszka P, Gawronski S, Ostachowicz B, Jurkiewicz A, 2008.

Establishment of arbuscular mycorrhizal plants originating from xerothermic grasslands on

heavy metal rich industrial wastes – new solution for waste revegetation. Plant Soil 305: 267-

280.

Wierzbicka M, Panufnik D, 1998. The adaptation of Silene vulgaris to growth on a calamine

waste heap (S. Poland). Environ. Pollution 101: 415-426.

20

New outlooks in mycorrhiza applications

Silvio Gianinazzi1, Odile Huchette

2, Vivienne Gianinazzi-Pearson

1

1UMR INRA 1088/CNRS 5184/U.Bourgogne, Plante-Microbe-Environnement, INRA-CMSE,

BP 86510, 21065 Dijon Cédex, France 2Dijon Céréales/COOPD’OR R&D, INRA, BP 86510, 21065 Dijon Cedex, France

E-mail: silvio.gianinazzi@dijon.inra.fr

Until now the application of mycorrhiza in plant production systems has focussed principally

on compensating for nutrient deficiency or improving growth and productivity in presence of

reduced chemical inputs (Gianinazzi & Vosatka 2004). However, tissues of arbuscular

mycorrhizal (AM) plants often have higher mineral contents and several works in the past

have reported enrichment in secondary flavonoid metabolites (Morandi et al. 1984, Harrison

& Dixon 1994), the antioxidant activity of which is well-known. The protective effect of

phytochemicals or antioxidant activities is frequently associated to health benefits of fruit or

vegetables having pharmaceutical properties. With the growing interest in such aliments,

researchers and producers have recently begun to address the question whether mycorrhizal

plants are of better nutritional and/or health quality. For example, Khaosaad et al. (2006) and

Copetta et al. (2006) reported that AM fungi can increase amounts of essential oils in basil

and oregano, independent of an improved P status of plants, and we have shown increased

carotene contents in sweet potato following the introduction of AM fungal inoculants under

field conditions (Farmer et al. 2007).

Onions are a rich source of flavonoids and sulfur-containing compounds, both of which are

considered to be potentially health-promoting through their activity, for example, as

antioxidants, antimicrobiotics or potential anticancer agents (Corzo-Martinez et al. 2007). In

this context, Perner et al. (2008) have provided evidence that major quercetin flavonoids

accumulate to a greater extent in bulbs of mycorrhizal as compared to non mycorrhizal

onions. In order to further evaluate the effect of mycorrhiza on the production of beneficial

compounds in onions, we have focussed studies on several health and flavour-related

organosulfur compounds. For this purpose, greenhouse grown onions (cv Kador) were

inoculated with either G. intraradices BEG141 or G. mosseae BEG12 in bedflats; plant

growth, mycorrhizal colonisation and concentration of 2-propenyl cysteine sulfoxide

(isoalliine), γ-glutamyl-S-(trans-1-propenyl)-L-cysteine (isoalliine precursor) and methyl

cysteine sulfoxide were quantified.

Inoculation with either AM fungus significantly enhanced the accumulation of isoalliine in

onion bulbs and G. mosseae had a positive effect on methyl cysteine sulfoxide concentrations

(Table 1). However, neither AM fungus affected the production of γ-glutamyl-S-(trans-1-

propenyl)-L-cysteine. These effects were observed independent of plant growth which was

not significantly increased in spite of high mycorrhizal colonisation of root systems.

The positive effect of both AM fungi on the accumulation of isoalliine was confirmed over

two years of experimentation (Figure 1). Since N supply can influence the quality and/or

flavour of onions, we also evaluated mycorrhizal effects on isoalliine accumulation in the

21

presence of reduced N fertilisation (50%). Under these conditions, both G. intraradices and

G. mosseae continued to increase isoalliine concentrations in onion bulbs (+35 to 48%).

Treatment MeCSO GLUPeCS PeCSO % M

Ni 1.68a 1.31a 7.56a 0a

Gi 1.84a 1.06a 9.81b 85.9b

Gm 3.47b 1.29a 14.16c 68.2b

Table 1 : Effects of mycorrhizal inoculation (Gi, G. intraradices ; Gm, G. mosseae ; Ni, non

inoculated) on the concentration (nmol/mg) of sulfur compounds in onion bulbs cv Kador :

MeCSO, methyl cysteine sulfoxide ; PeCSO, 2-propenyl cysteine sulfoxide (isoalliine) ;

GLUPeCs, γ-glutamyl-S-(trans-1-propenyl)-L-cysteine (isoalliine precursor). Experiment

2006, normal N fertilisation (mg/l) : Ca(NO3)2 4H2O, 649 ; KNO3, 465 ; (NH4) 2H2PO4, 59.

Different letters in columns indicate significantly different values (P=0.05).

0

5

10

15

20

K_Ni K_Gi K_Gm

Figure 1: Effects of mycorrhizal inoculation (Gi, G. intraradices ; Gm, G. mosseae ; Ni, non

inoculated) on the concentration (nmol/mg) of 2-propenyl cysteine sulfoxide (isoalliine) in

onion bulbs cv Kador in two independent experiments (black columns, 2005 ; white columns,

2006). Normal N fertilisation (see Table 1).

These observations reinforce the evidence for a role of AM in enhancing food quality and

provide a new outlook for the exploitation of this symbiosis to define systems for the

production of crops with high nutritional and/or health value. The fact that this beneficial

effect on food quality is expressed also at a low level of fertilizers is particularly relevant for

the Mediterranean countries where agroecosystems are often fragile.

References

Copetta A, Lingua G, Berta G (2006) Effects of three AM fungi on growth, distribution of

glandula hairs, and essential oil production in Ocimum basilicum L. var Genovese.

Mycorrhiza 16: 485-494.

Corzo-Martinez M, Corzo N, Villamiel M (2007) Biological properties of onions and garlic.

Trends food Sci Technol 18: 609-625.

22

Farmer MJ, Li X, Feng, G, Zhao B, Chatagnier O, Gianinazzi S, Gianinazzi-Pearson V, van

Tuinen D (2007) Molecular monitoring of field-inoculated AMF to evaluate persistence in

sweet potato crops in China. Appl Soil Ecol 35: 599-609.

Gianinazzi S, Vosatka M (2004) Inoculum of arbuscular mycorrhizal fungi for production

systems: science meets business. Can J Bot 82: 1264-1271.

Harrison MJ, Dixon RA (1994) Spatial patterns of expression and flavonoid/isoflavonoid

pathway genes during interactions between roots of Medicago truncatula and the mycorrhizal

fungus Glomus versiforme. Plant J 6, 9-20.

Khaosaad T, Vierhilig H, Nell M, Zitterl-Eglseer K, Novak J (2006) Arbuscular mycorrhiza

alter the concentration of essential oils in oregano (Origanum sp., Lamiaceae). Mycorrhiza

16: 443-446.

Morandi D, Bailey JA, Gianinazzi-Pearson V (1984) Isoflavonoid accumulation in soytbean

roots infected with vesicular-arbuscular mycorrhizal fungi. Physiol Plant Pathol 24: 357-364.

Perner H, Rohn S, Driemel G, Batt N, Schwarz D, Kroh LW, George E (2008) Effect of

nitrogen species supply and mycorrhizal colonization on organosulfur and phenolic

compounds in onion. J Agric Food 56: 3538-3545.

23

Production of AMF’s and growing needs in plant production Adholeya Alok, Reena Singh & Shanuja Beri

Abstract India, which spreads over 329 million ha (hectares), has about 114 million ha of land presently

under cultivation. Meeting the ever-increasing demand for food production in this second most

populous country of the world is a major challenge. The land under cultivation has almost reached

its saturation point with respect to productivity. This is due to the practice of intensive agriculture,

which includes excessive application of fertilizers and pesticides, and introduction of modern,

high yielding varieties, which generally are also highly demanding. Therefore, to further increase

crop productivity, either land productivity needs to be increased or additional lands, presently

under fallow or wasteland categories, need to be brought under cultivation. This can be achieved

by adopting suitable technologies. Mycorrhiza technology is one such successful technology,

capable of wasteland reclamation and beneficial in agriculture owing to its contribution to the

plant with regard to nutritional mobilization properties.

The Centre for Mycorrhizal Research, TERI, has developed the monoaxenic technology for mass

production of AM (arbuscular mycorrhizal) inoculum. This technology exploits host roots

genetically modified by using the bacterium Agrobacterium rhizogenes carrying Ri T-DNA

plasmid to mass-produce viable, healthy, genetically pure, and high-quality fungal propagules in

vitro in a sterile environment. (Figure 1).

Mass production of AM fungi has been achieved with several species of genes glomous,

gigaspore and_scetulospora, but G. intraradices remains the most promising, with increased spore

production obtained since the early investigations on monoxenic cultivation until today. In 1992,

Chabot et al. established cultures from surface sterilized spores as starter material and produced

750 spores in 30 ml medium after a period of 4 months of growth in a mono-compartmental Petri

plate system. Using sheared roots as starter inoculum, Diop et al. (1994) obtained approximately

890 spores after 3 months of incubation. An advanced mode of airlift bioreactor-based production

was adopted by Jolicoeur et al. (1999). These authors recovered 12,400 spores per litre of

medium. St Arnaud et al. (1996) obtained 15,000 spores in a bicompartmental Petri plate in 3–4

months. This bi-compartmental system was improved by Douds (2002) by replacing the

mediumin the distal compartment by fresh medium at regular intervals. With this procedure, this

author obtained 65,000 spores in the distal side of the bi-compartment in a period of 7

months.With the technology developed at the Centre for Mycorrhizal Research, The Energy and

Resources Institute (TERI), New-Delhi, India, the recovery of infective propagules approximated

250,000–300,000 spores in 3 months in 100 ml of medium (Adholeya et al. 2006). The TERI

technology here adopts optimization at different levels, identifying the rate-limiting factors

leading to the bulk production for commercial utilization. The AM fungi in genus Glomus provide

the possibility of using colonized roots as inoculum material. This was also optimized in parallel

to achieve higher root colonization, up to 70–80% (Tiwari and Adholeya 2003). The

subcultivation of the

24

Figure 1: TERI‘s Mycorrhiza Technology

Figure 2: Testing of mycorrhizal fertilizer with wheat

Figure 3: Testing of mycorrhizal fertilizer with rice root organ and its harvest have been attained at 4 and 12 weeks respectively. Such improvement

allows higher spore and propagule recovery when compared with the unit volume of media in

earlier published research. This also facilitates the efficient utilization of space and energy in the

production system, i.e. solid-state fermentation.

Many process controls were developed in order to reduce the levels of contamination (generally

from 10–15% to 3–5%, common under tropical conditions).

25

The technology is economical and does not require any heavy hardware and infrastructureand

been transferred to five Industries. The technology has received many awards including the

‗Biotech Product and Process Development and Commercialization Award‘ by DBT (Department

of Biotechnology, Ministry of Science and Technology, Government of India)) in 2004 and has

led to the development of first-ever mycorrhizal product from in vitro based technology. The

product being produced on low cost with comparatively better efficiency, found major market in

North America and Europe.

The technology is an innovative invention offering a partial substitute to chemical fertilizers. This

provides an edge to plants to thrive better and offer enhanced yield and establishment in nutrient

poor conditions. This fungal microbe, which forms a symbiotic, non-pathogenic, permanent

association between the roots of land plants, is an appropriate partial substitute to mineral

fertilizers and promotes yield significantly. This is extremely beneficial to almost all cultivated

plants as it has a broad host range in contrast to other products available (not equivalently

comparable). It is easy in application similar to chemical fertilizers. Its cost of production is

highly competitive to other products and offers economic, environmental and wide spread use,

advantage to farmers/growers and commercial production units.

•

Mycorrhiza is a broad-spectrum non-specific organism. A single species is known to colonize

85% of land plants.

It has a broad ecological adaptability and is known to occur in deserts as well as arctic, temperate,

tropical and other inhospitable habitats.

It facilitates better uptake of nutrients like phosphorus and immobile trace elements like zinc,

molybdenum, etc, leading to better nutrition for plants.

It offers tolerance against a range of soil stresses like heavy metal toxicity, salinity, drought, and

high soil temperatures. This enhances the chances of plant survival immensely.

It offers higher resistance to various soil and root-borne pathogens, thus becoming a potential

disease control agent.

It helps in soil conservation and soil structure stabilization, thus restoring land productivity.

Mycorrhiza based product do not require to be kept at low temperature, hence provide major

handling and application even with highest efficiency.

Mycorrhizal fungi can utilize phosphorus from extremely low concentrations, even from

unavailable sources, and provide an alternative to offset the high cost of phosphate fertilizer input.

To meet the requirement of 170.26 million ha of total land present in India (land under

cultivation) and future land (land that can be brought under cultivation) for cultivation in a

sustainable way, the mycorrhizal requirement is 2128.26 million tonnes annually. Few Industries

in India have now initiated the production of mycorrhiza. The constructed area requirement is

5000 square feet to produce 200 tonne/annum and 12,000 square feet to produce 1000

tonne/annum

Extensive field trials were conducted with successful results using mycorrhiza on different crops

in different agro-climatic regions of India (Figures 2-5). The trials conducted under different

programmes supported by the Ministry of Environment and Forests, the Ministry of Science and

Technology in different regions across the country also met with success. These results proved the

commercial viability of the technology. However, the demand for mycorrhiza requirement is too

high to be met by just a few industries.

26

Figure 4: Testing of mycorrhizal fertilizer with potato

Quality control and regulation of mycorrhizal biofertilizer In India and comparable countries, most commercial organic fertilizers are not covered by the

type of national or international standards which govern the quality of chemical fertilizers. Thus,

specific protocols for quality control of AM fungal inocula need to be developed and standardized

for application. This is essential not only as a guarantee for producers and users but also for the

protection of ecosystems. Moreover, this would also help in quality management and assessment

of inoculum potential with every batch of inocula produced.

Quality control of commercial AM fungal inoculum is extremely important for developing faith

among the user community, along with its effectively demonstrated potentials. Unless this is

achieved, the potentials will remain unexplored among the other biofertilizers. It is important to

evaluate the produced inoculum from commercial units with certain reference values to ensure the

strict adherence to the protocols and methodologies recommended by recognized and independent

laboratories. This is most vital, as several handling errors occur at the industrial level during

technology adoption and

Figure 5: Testing of mycorrhizal fertilizer with Sugarcane

27

implementation, causing subsequent problems in product quality, which may lead to the

dissatisfaction of both the end users and producers.

For the mass production of AM fungi, critical benchmarks at all stages of inoculum development,

covering all possible parameters desirable for ensured production, are identified. These include

viability checks at processing stages until the formulation stage, ranging from the colonization of

host roots, weight of dried inoculum at harvest, propagule estimations, infectivity potential of

crude and formulated diluted inoculum, formulation conditions like temperature and suitable

storage conditions. Such benchmarks also help institutionalized process efficiency at the

production level. Once the commercial launch of the formulation is achieved, both the developer

of the technology and the distributing industries share equal responsibilities for the authenticity

and performance of commercialized products, and must continue to work together to evaluate

responses obtained in the field by the end users. This would ensure confidence building and

continuous use of these products over the years. It is important to regularly validate product

performance, customer satisfaction and willingness for future use, to monitor the effectiveness of

the inoculum. TERI together with Government of India recently initiated the process of standards

for mycorrhiza bond product and evaluation methodologies

Acknowledgements The author wish to thank financial contribution for the project from SDC,

Government of Switzerland & the DBT, Government of India under the Indo-Swiss Collaboration

in Biotechnology

References: Diop TA, Plenchette C, Strullu DG (1994) Dual axenic culture of sheared root inocula of

vesicular arbuscular mycorrhizal fungi associated with tomato roots. Mycorrhiza 5:17–22

Douds DD Jr (2002) Increased spore production by Glomus intraradices in the split-plate

monoxenic culture systemby repeated harvest, gel replacement, and re-supply of glucose to the

mycorrhiza. Mycorrhiza 12:163–167

Jolicoeur M, Williams RD, Chavarie C, Jortin JA, Archambault J (1999) Production of Glomus

intraradices propagules, an arbuscular mycorrhizal fungus, in a airlift bioreactor. Biotechnol

Bioeng 63:224–232

Tiwari P, Adholeya A (2003) Host dependent differential spread of Glomus intraradices on

various Ri T-DNA transformed roots in vitro.Mycol Prog 2:171–177

28

Mycorrhizal application in Greece -the issues and future plans

Reiko SHIBATA (VIORYL SA, Greece)

Abstract

The sensitivity of consumers about matters pertaining to food safety as well as environmental

protection has increased, and resulted in the development of organic farming system in the

last few years. Utilization of mycorrhizal symbioses should be considered in the system, due

to reduction of application of chemical fertilizers, pesticides and irrigation.

Very few scientific papers and researches are available in Greece, and it has been always

difficult to find answers to critical questions concerning mycorrhizal application in the fields.

However, in terms of the climate, under long dry summer and very poor winter rainfall,

mycorrhizal symbioses could play an important role in the agricultural system.

Since, we have few reported data, it is important initially to establish an experiment focusing

on the presence or absence of mycorrhizal symbioses naturally occurring in the fields.

Collecting these data will guide us to the next step which is investigating the possible benefits

of applying commercial mycorrhizal inoculum. The final step will be to evaluate the

outcomes of mycorrhizal symbioses in the field and create a database for future research and

the development.

VIORYL was established in 1946, and since the early years we have been focusing on organic

farming system, developing organic insects trapping system with synthesized pheromone, bird

repellents, fertilizers etc. In the last years we have launched mycorrhizal research in Greece

and we are conducting experiments applying commercial mycorrhizal inoculum to fields and

evaluating the results. Even though it is very early to present any data, we are expecting

mycorrhizal inoculum to be a productive and reliable way achieving reduced application of

chemical fertilizers, pesticides and irrigation.

In this meeting, we shall present issues we are facing, future plans and challenges that lie

ahead of us.

29

Quality Control through Record Keeping and Vouchers and a tale of

confusion with Glomus intraradices.

Christopher Walker,

Royal Botanic Garden Edinburgh, 20A Inverleith Row, Edinburgh EH3 5LR, UK.

Different AMF have different effects on different host plants. Some have unbalanced

associations, taking more from the host than they return (e.g. Modjo & Hendrix 1986; Lerat et

al. 2003), sometimes depending on available substrate P concentration (Peng et al. 1993).

Whole ecosystems may be affected by the identity of available AMF (Grime et al. 1987) and

conversely, AMF diversity may be influenced by their ability to interact with particular hosts

(Eom et al. 2000; Scheublin et al. 2007).

There are edaphic and anthropogenic effects that influence AMF in different ways. As

examples Glomus mosseae, may be favoured by agricultural disturbance and there are

complex effects of agricultural practice on species retrieved from examination of spores

produced from a variety of agricultural soils (Oehl et al. 2003) and laboratory studies have

shown significant effects of different disturbance events (P or OM addition) (Boddington &

Dodd 2000). In the field, AMF species recovered from soil were significantly affected by

experimental changes to soil pH (Wang et al. 1985).

Other AMF may require particular predispositions to germinate efficiently (Lous & Lim

1988). Acaulospora laevis from Australia requires a period of drying before the spores will

germinate (Tommerup 1983). However, A. laevis from the Pacific Northwest, its type

location, may never experience such an extended period of drought. The species is found in

the UK and in many other lcoations in which extended dry periods are infrequent or unlikely.

So perhaps species alone is not an adequate definer of at least some physiological aspects of

the fungus. Similarly, if all the organisms described in the work of Croll et al. (2008) really

are G. intraradices, species is an inadequate taxonomic level for defining, and thus predicting,

the interaction with plants, as some individuals appear to associate predominantly with

particular host species.

There is evidence that competitive effects in pot culture (and therefore presumably in mass

inoculum cultivation) can result in changes in AMF populations, possibly resulting in the

disappearance of some species in favour of others over time. If production systems are based

on open pots or beds, or even field beds, there is a high probability of contamination by other

AMF or even by pathogens of plant or fungus. These factors make it important for quality

control and quality assurance that the organisms present in inoculum are identified and

characterised, and their interactions are understood. Good record keeping with adequate

vouchers can be used to demonstrate that each batch of inoculum is free from such organisms.

Such quality control can contribute greatly to assuring high quality inoculum and to

prevention of transmission of deleterious organisms.

At the species level, this can usually, but not always, be achieved through morphological

recognition of the spores. However the existence of cryptic speciation implies that it may be

necessary to use molecular tools to identify the species and individuals concerned. Depending

on the complexity of the species-strain mixture, more or less effort is required. For a single

species culture, a well defined species such as Scutellospora reticulata presents much less of a

problem than, shall we say, a member of the complex and inadequately defined species

groups such as those containing G. intraradices, G. mosseae or G. etunicatum. Mixed cultures

of well characterised organisms with widely different morphologies are easier to verify than

mixed cultures of very similar organisms. This is exemplified by one of the so-called G.

intraradices cultures, where a supposed ‗isolate‘ is represented by different morphological

species depending on the secondary source of the culture. Fungi identified as G. mosseae have

30

been shown to be widely different in their behaviour and plant growth interactions

(Giovannetti et al. 2003; Lerat et al. 2003), but whether this is erroneous identification or

genuine intraspecific difference remains moot.

The example of G. intraradices above merits more detailed explanation. An organism from

Pont Rouge, Canada with this name has been extensively reported on under two different

reference numbers, DAOM 197198 (Chabot et al. 1992) and DAOM 181602 (Corradi &

Sanders 2006, Alcan et al. 2006). Both numbers were listed on 19 Dec 2006 as representing

the same organism in the website of GINCO-CAN, although this site is no longer active

[http://res2.agr.ca/ecorc/ginco-can/details_e.asp?SpeciesID=36]. Examination of cultures (Fig

1) from different sources indicate that two different organisms are present under the same

identifier. This is confounded by a misunderstanding of the meaning of DAOM numbers that

exemplifies how a flawed documentation process can result in breakdown of quality control

of all. DAOM numbers are herbarium voucher numbers. Such identities are given to samples

of an organism when they are lodged in a herbarium, and represent only a snapshot of what

was present in a culture at that particular time, and at that time only. They should never be

used as culture identifiers. A voucher number is a historical record of what was present at a

particular sampling, but cannot be used as evidence for what will be found the future. It can

be used as a quality control check to verify (or otherwise) if a later subculture still has the

same morphological characteristics. It may be that records are available to trace the life

history of the numerous cultures of the Pont Rouge fungus (or fungi) used under these

numbers, but if so, they do not seem to be in the public domain.

Fig 1. Morphologically different cultures, both named Glomus intraradices, and both with the

same voucher number in their ancestry. Upper from Canada via Australia. Lower, from

Canada via Europe. Left, typical spores in PVLG. Right, typical spores in PVLG with

Melzer's reagent.

31

Record Keeping

A database or other record keeping system can easily be developed to solve this problem, and

to provide quality assurance in respect of cultures throughout their subculturing histories. For

example, the records maintained manually at the University of Western Australia for many

decades provide a full and detailed account of the life histories of WUM cultures maintained

at that institution, though not those of the same organisms after distribution elsewhere. A

database suitable for this purpose is described here. It is based on data of origin,

establishment, isolation, sampling history and culturing history.

Origin

For any AMF culture, an original sample must have been collected by some individual from

some geographic location. The database therefore starts with this information. Geographic

origin is further subdivided so that separate samples from the same location can be identified.

There is confusion with the Pont Ruge cultures in this respect. One source indicates that it

was collected originally by S. Parent (personal communication with S. Chabot in July 1987

from DAOM records), but the GINCO-CAN website indicated it was collected by two people

C. Plenchette and V. Furlan. There is agreement that it was collected from Pont Rouge,

Québec. There is agreement that it was from a Fraxinus americana woodland.

Vouchers

The database provides unique voucher numbers to specimens taken from the original samples,

thus allowing provision of a record, with voucher specimens, of the different species or spore

types present in the soil or substrate at the time of sampling. The results of any trap cultures

can then be compared, along with appropriate uninoculated controls to ensure contaminant

organisms have not been inadvertently established in culture. The voucher specimens

numbers are linked to the origin numbers by way of the sample numbers. Vouchers are very

important for quality control of cultures.

Culture attempts

Establishment of cultures is often first carried out by trapping in open pots with a host plant

(Walker 1999), although it may be with single spores from the soil in some type of sealed

system. Whichever method is chosen, the database provides unique Attempt numbers which

are related in the system through sample numbers to origin numbers (and then to collector).

Any vouchers that were made from the sample are recorded and linked with the attempt

number for comparison at a later date. These Attempt numbers therefore are clearly distinct

from, but linked to the vouchers. For the Pont Rouge cultures, I could obtain no record of

original culture establishment, but it seems likely it was a soil trap. Doubtless, records do

exist, but they do not appear to be in the public domain. Soil traps, of course, cannot be relied

upon to provide pure cultures of a single organism. The original type culture of the species G.

intraradices was trapped from a Florida citrus plantation by using root fragments and was not

purified before the species was described.

Subculture attempts

Should the original culture attempts be successful, then further subcultures, including

purification and isolation where appropriate, can be recorded in the database. Each subculture

attempt receives a number, unique and linked through the database to its parent culture, and

hence to all other related data in the system. The database also provides the subculture‘s

unique parental number. It is interesting to note that the number DAOM 197198 was given in

1992, although the original culture appears to have been established in 1987 from DAOM

records. I have not been able to establish the date when DAOM 181602 was issued. The

32

provision of linked numbers for subcultures in the database allows the ancestry of each

subculture to be established, showing its relationship with other cultures, both in the past and

for the future. Voucher specimens can be taken from attempts at any time and numbered,

automatically linking them with their historical records.

Purification Establishment of a trap or other multi-propagaule culture is a first step only. The culture must

then be purified. Often this results in what is known euphemistically as a ‗single species

culture‘. By the nature of AMF, such a culture from root fragments or multiple spores cannot

be reliably considered to be an isolate because it is likely that more than one organism is

involved. Indeed, cultures of species from one genus can appear from single spore isolate

attempts from a spore of a different one as smaller spores may be occupying the space

provided in the lumen of larger, dead specimens. It is difficult to select spores of the same

species under a dissecting microscope. Consequently, although it is possible that only a single

organism has been established in culture from a multi-spore attempt, it is impossible to be

absolutely certain. I have not been able to find out how or when any purification attempt was

made for the Pont Rouge culture .

Isolation

Isolation is the separation of a fungus by single propagule into a culture that, for an asexual

organism, represents an individual organism. This is normally achieved by establishing single

spore cultures which of course must be maintained in contamination-free conditions (both

actual an theoretical) so its identity and purity can be assured. There seems to be no evidence

that the Pont Rouge culture is an isolate, though it may be. It is certainly frequently referred to

as such (e.g., Croll et al. 2008). The database provides a field to enter details of whether a

culture is, for example, a trap, a multi-spore attempt, or an isolation attempt. Once the culture

is established as an isolate, it can be provided with a further unique number within the

database that is linked to the appropriate Attempt number, and thus to its history.

Conclusion

Confusion such as that described above for the so-called G. intraradices from Pont Rouge can

be avoided if full records are maintained and appropriate vouchers kept. A purpose-built

database can be used for this, but a paper-based system or a simple system using a spreadsheet

would also be adequate. Vouchers ideally should be placed in a public domain herbarium, but

clearly if commercially sensitive cultures are used, they may be kept in a private collection

used only by the company concerned. The following protocol is offered as a guide for record

keeping and specimen preservation.

1. Origin registration Record the collector name and the geographical location of the

original sample.

2. Sample identification. Detail the specific sample including, date of collection, and

ecological and edaphic characteristics such as nearby AMF plants, soil pH, available P and

organic matter.

3. Purification and isolation. Identify the original cultures, each one uniquely labelled.

Record the details of substrate type and treatment, container size, host plant, etc. Ideally, the

culture attempts should be maintained in a sealed system (e.g. Walker & Vestberg 1994).

Suitable control pots need to be established to check for contamination from such problems as

incomplete substrate disinfestations, carry over of contaminant spores in water or movement

of propagules by insects or animals.

33

4. Subculturing. When the original attempt at pot culturing is successfully producing spores,

use these in a process of establishing isolates. This can be directly from single spores in the

first instance, or can be through one or more multi-spore cultures. Only when a single spore

culture is successful should it be called an isolate. These will all be subcultures of the original

isolation attempt, and a numbering system can be devised to reflect this.

5. Voucher specimens. Ideally, the original location would be sampled to establish which

species are present. Samples can be taken and spores extracted, mounted on microscope

slides, numbered serially, photographed, and preserved as evidence. In addition, it is useful to

preserve the remains of the original sample by drying or deep-freezing in case future

investigations are needed. Not all species present will be sporulating at any given time, so, for

example, a soil trap may produce species that have not been seen earlier. Similarly, root

fragments used for establishing cultures may yield species that were not sporulating at the

time of original sampling. Once AM are established in the original traps, vouchers can be

made to verify what has been found. These vouchers can include microscope slides and

photographic images, but also dried or frozen subsamples for later further investigation. The

original traps can be maintained until successful isolates are obtained, and then might be

discarded, although ideally they would be dried or frozen in case of future needs.

With good vouchers, isolation attempts can be compared to ensure that the cultures obtained

correspond to the expected outcome. Differences might be because of external contamination

(which will normally show up in the controls) or perhaps because apparently healthy spores

were in fact dead and contained internal spores (Koske 1984) that were successful in

establishing a symbiosis.

Keeping these detailed records may seem tedious, but there are large gains in quality control

and quality assurance to be obtained. Systems and record maintenance are at the root of such

quality factors.

References Alkan N, Gadkar V,Yarden O,Yoram Kapulnik Y 2006 Analysis of Quantitative Interactions

between Two Species of Arbuscular Mycorrhizal Fungi, Glomus mosseae and G. intraradices,

by Real-Time PCR. Applied and Environmental Microbiology 72: 4192–4199.

Boddington CL, Dodd JC 2000 The effect of agricultural practices on the development of

indigenous arbuscular mycorrhizal fungi. II. Studies in experimental microcosms. Plant and

Soil 218: 145-157.

Chabot S, Bécard G, Piché Y 1992 Life cycle of Glomus intraradix [sic] in root organ culture.

Mycologia 84: 315-321.

Croll D, Wille L, Gamper HA, Mathimaran N, Lammers PJ, Corradi N,

Eom A-H, Hartnett, DCWilson GWT 2000. Host Plant Species Effects on Arbuscular

Mycorrhizal Fungal Communities in Tallgrass Prairie. Oecologia 122: 435-444.

Giovannetti M, Sbrana C, Strani P, Agnolucci M, Rinaudo V, Avio L 2003 Genetic Diversity

of Isolates of Glomus mosseae from Different Geographic Areas Detected by Vegetative

Compatibility Testing and Biochemical and Molecular Analysis. Applied and Environmental

Microbiology 69: 616-624.

Grime JP, Mackey JML, Hillier SH, Read DJ 1987 Floristic diversity in a model system using

experimental microcosms. Nature 328:420-422.

Koske RE 1984 Spores of VAM fungi inside spores of VAM fungi. Mycologia 76: 853-862.

Lerat S, Lapointe L, Piché Y, Vierheilig H 2003 Variable carbon-sink strength of different

Glomus mosseae strains colonizing barley roots Canadian Journal of Botany 81: 886–889.

Modjo HS, Hendrix JW 1986 The mycorrhizal fungus Glomus macrocarpum as a cause of

tobacco stunt disease. Phytopathology 76: 688-691.

34

Peng S, Eissenstat DM, Graham JH, Williams K, Hodge NC 1993. Growth depression in

mycorrhizal citrus at High-Phosphorus supply. Plant Physiology 101: 1063-1071.

SandersIR 2008 Genetic diversity and host plant preferences revealed by simple sequence

repeat and mitochondrial markers in a population of the arbuscular mycorrhizal fungus

Glomus intraradices. New Phytologist 178: 672–687.

Scheublin TR, van Logtestijn RSP, van der Heijden MGA 2007 Presence and identity of

arbuscular mycorrhizal fungi influence competitive interactions between plant species.

Journal of Ecology 95: 631–638

Walker C, Vestberg M 1994 A simple and inexpensive method for producing and maintaining

closed pot cultures of arbuscular mycorrhizal fungi. Agricultural Science in Finland 3, 233-

240.

Wang GM, Stribley DT, Tinker PB, Walker C 1985 Soil pH and vesicular-arbuscular

mycorrhiza. pp. 219-224 IN British Ecological Society Special Symposium - Ecological

Interactions in the soil environment: Plants, Microbes and Animals. Ed. by A. H. Fitter.

Blackwell, Oxford.

35

AM fungi in planta : 20 years of progress in quality of cultures in vitro

Strullu D.G. (1), Barbas V. (2) and Strullu-Derrien C. (1)

(1) Laboratoire Mycorhizes, Université d’Angers,2Bd Lavoisier, 49045 Angers Cedex,

France

(2) Department of genetics, University of Thessaloniki, Greece

Abstract.

The relationship between fungi and plants is known to be ancient; paramycorrhizas occur in

the plants from the Rhynie Chert (400 million years). Eumycorrhizas have been recorded in

300 million-year-old petrified Cordaites which showed endophytic fungi in their rootlets. In

modern eumycorrhizas the intraradical form comprises hyphae, arbuscules and sometimes

vesicles. In 1986, Strullu and Romand achieved the first subcultivation of AM fungi, isolated

from mycorrhizal roots. That result and other steps - including Agrobacterium rhizogenes

transformed roots, somatic embryos, rhizogenous calli, somatic hybrids - allowed the

improvement of the indefinite culture of those micro-organisms and the production of high

quality inocula. These technologies require very strict controlled measures: viability, identity,

purity and stability. Specific protocols for quality control of AM fungal inocula are now

developed for applications. The use of in vitro cultures for inocula production appears now

totally acceptable in respect to environmental and legal aspects. AM fungi inocula are now

used worldwide to the satisfaction of an increasing number of plant growers and farmers.

The relationship between fungi and plants is known to be ancient; paramycorrhizas occur in

the plants from the Rhynie Chert (Scotland, 400 million years). The fungus Glomites

rhyniensis is know from Aglaophyton sporophyte and Lyonophyton gametoppyte.

Eumycorrhizas have been recorded in 300 million-year-old petrified Cordaites from

Grand‘Croix (France) which show endophytic fungi in their rootlets. The fossil association

shows intracellular hyphae within the cells and typical arbuscules with thick trunks and

narrow branches (Strullu-Derrien and Strullu, 2007). In modern eumycorrhizas the

intraradical form comprises intercellular hyphae, coils, arbuscules and sometimes vesicles.

Bryophytes, lycopods, ferns gymnosperms and angiosperms develop arbuscular mycorrhizas.

It is now recognised that the AM fungi should de placed in the Glomeromycota.

Mosse and Hepper (1975) reported the first co-culture between a contaminant-free inoculum

and a root organ. Strullu and Romand (1986) achieved the first subcultivation of Glomus

fasciculatus, isolated from mycorrhizal roots of Fragaria and the same authors subcultivated

Glomus intraradices regrowth from isolated vesicles. So the first continuous cultures of high-

number vesicle-forming Glomus species was performed. After Declerck, Seguin and Dalpé

(2005) and only referring to published papers, 15 species have been cultivated with success

under monoxenic conditions with production of mature spores and only 9 have been

subcultivated (Figure 1).

That result and other steps - including Agrobacterium rhizogenes transformed roots, somatic

embryos, rhizogenous calli, somatic hybrids - allowed the improvement of the indefinite

culture of those micro-organisms and the production of high quality inocula. These

technologies require very strict, controlled measures to guarantee the intrinsic properties of

identified strains over generations (see Declerck, Seguin and Dalpé, 2005). The viability can

be confirmed through spore or vesicle germination tests. Three media could be used for these

tests : SR87 medium ( Strullu and Romand, 1987) for isolated vesicles, SRM medium

(Declerck et al, 1998 modified from Strullu and Romand, 1986) for spores and M medium

36

(Bécard and Fortin, 1988). Host are transformed or non-transformed excised roots (Daucus

carota, Lycoperdicon esculentum, Allium cepa, Trifolium pratense, Medicago truncatula).

Figure 1. Monoxenic cultures of AM fungi with production of mature spores

and possibilities of subcultivation (after Declerck, Seguin and Dalpé, 2005)

The identity of the fungal strains is mainly based on the spore morphology but the continuous

cultures increase the interest of taxonomy by opening the possibility of using new material

and tools as sporulation, mycelium morphology, biochemistry, molecular biology. The purity

of AM fungi strains can be confirmed by starting the continuous cultures from single vesicle

(Strullu and Romand, 1987; Declerck et al, 1998) or single spore ( Declerck et al, 1998). The

stability of AM fungi in monoxenic cultures needs to be ascertained by long-term

preservation. The situation is quite different for Acaulosopra, Gigaspora or Glomus since

only the later could be cultivated over several generations. Generally there is no important

variability of infectivity but some decreases of sporulation and colonisation rates can been

detected. Different methods for long-term preservation are currently used..

The monoxenic culture of AM fungi with transformed roots is an excellent method for

providing spores; so mass production of these spores of high quality and lower production

cost has been developed. Commercial inoculum production of sterile AM fungal spores has

been increasing during the last years. However, the use of spores produced from

Agrobacterium rhizogenes transformed roots for the inoculation in field is sometimes

restricted. As ecologists seek answers to pratical problems related to transgenic

biotechnology, approaches to increase spore production using chemical methods and new

methods of dual culture have been suggested. A large-scale inoculum production using

rhizogenous calli is show in figure 3.

37

Figure 3. Schematic model of monoxenic culture of AM fungi

using rhizogenous calli

This method can be divided into four stages: (1) starting AM fungal material: single spores,

single vesicles, mycorrhizal roots (2) plant culture tissues as somatic embryos, somatic

hybrids, (3) inoculation, (4) growth of the rhizogenous calli and spore production (figure 4).

The potential improvement of production performance is tested for each stage of this method

(Strullu and Barbas, 2004).

Figure 4. Spore production using rhizogenous calli

The methodologies for in vitro cultures on root organs suffer from other limitations as the

absence of photosynthetis tissues which unable the photosynthetate transfer from the plant to

the AM fungus. The techniques of in vitro culture enable us to produce somatic embryos for

many plant species and when these embryos are encased in a biodegradable nutritive capsule,

artificial seeds are produced. Artificial seeds of alfalfa (Medicago sativa) were inoculated

38

with Glomus and the AM fungus regenerated from root fragments formed typical mycorrhizas

with arbuscules, vesicles and spores (Strullu et al, 1989). An other autotrophic culture system

in which an autotrophic plant (Solanum tuberosum) was associated to an AM fungus was

developed (Voets et al, 2005).

For the inoculum production, the low productivity of aseptic culture which raises the cost has

been an important obstacle to putting inoculation in large-scale industrial practice. In the field

of AM fungi inoculum it is necessary to check that the microorganism is present in plant, that

it develops typical symbiotic structure and at least causes the plant response. Specific

protocols for quality control of AM fungal inocula are now developed for horticultural and

agricultural applications. Colorimetric tests, especially those using non toxic products, allow

the control of the symbiotic stages. So far, few studies have examined the identity of the

intraradical form in planta. An interesting objective will be to use and evaluate different

molecular methods to identify the intraradical form of the AM fungi after inoculation .

The associations are valuable for the plant particularly in stress conditions. The external

hyphae have the ability to take up phosphate and to transport it in planta structures were it is

released to the plant cells. The AM associations also increase the water uptake, this is

associated with a lower transpiral flux and a better ability to extract water from substrates.

The use of in vitro cultures for inocula production appears currently acceptable in respect to

environmental and legal aspects. AM fungi inocula are now used worldwide to the

satisfaction of an increasing number of plant growers and farmers.

References

Bécard G.and Fortin J.A. (1988).Early events of vesicular mycorrhizas formation in Ri-T-

DNA transformed roots. New Phytol., 108, 211-218.

Declerck S., Seguin S. and Dalpé Y. (2005).The monoxenic culture of arbuscular mycorrhizal

fungi. In : In Vitro Culture of Mycorrhizas. Editors S. Declerck, D.G. Strullu and J.A. Fortin.

Springer. Soil Biology. 341-375.

Declerck S., Strullu D.G.and Plenchette C. (1998). Monoxenic culture of the intraradical

forms of Glomus sp. isolated from tropical ecosystem : a proposed methodology for

germplasm collection. Mycologia, 90 (4), 579-585.

Mosse B. and Hepper C. (1975). Vesicular arbuscular mycorrhizal infections in root organs

cultures. Physiol. Plant Pathol., 5, 215-223.

Strullu D.G.and Romand C. (1986). Méthode d‘obtention d‘endomycorhizes à vésicules et

arbuscules en conditions axéniques. C.R. Acad. Sci., 303, 245-250.

Strullu D.G. and Romand C.(1987). Culture axénique de vésicules isolées à partir

d‘endomycorhizes et ré-association in vitro à des racines de tomate. C.R. Acad. Sci., 305, 15-

19.

Strullu D.G. and Barbas V. (2004). Method for in vitro production of mycorrhizal fungi,

mycocallus and mycorrhized biological support obtained thus. PTC Application,

FR2004/001570.

Strullu D.G., Romand C., Callac P., Téoulé E. and Demarly Y. (1989). Mycorrhizal synthesis

between Glomus spp. and artificial seeds of alfalfa. New Phytol. 113, 545-548.

Strullu-Derrien C. and Strullu D.G. (2007). Mycorrhization of fossil and living plants. C.R.

Palevol., 6-7, 483-494.

Voets L, Dupré du Boulois H., Renard L., Strullu D.G and Declerck S. (2005). Development

of an autotrophic culture system for the in vitro mycorhization of potato plantlets. FEMS

Microbiology Letters, 248 (1), 111-118.

39

How much diversity do we need in arbuscular mycorrhizal fungi (AMF)

inoculum?

Gosling P., Jones J., Bending G.D.

Warwick HRI, University of Warwick, Wellesbourne, CV35 9EF, UK.

Abstract

Evidence for functional diversity in the arbuscular mycorrhizal fungi (AMF) is now

established at all taxonomic levels, from family right down to different strains within a single

species. Specific species and strains of AMF have been shown to be more or less effective at

relieving plant stress from soil nutrient limitation, soil pests and disease, salinity and heavy

metals. As a result, AMF diversity is important in determining the benefits gained from AMF

in natural ecosystems. However, the role of AMF diversity in delivering benefits to crop

plants has received less attention. We examined the effect of between 1 and 7 species of AMF

on the growth of onion and clover in the glasshouse on soil from an organically managed

horticultural system. Growth of onion was increased by AMF, but there was no improvement

in growth by applying more than two species. Three Glomus species significantly increased

growth, but neither Paraglomus occultum, Scutellospora fulgida or Acaulospora spinosa

significantly increased growth. The degree of AMF colonisation was not affected by species

of AMF present. Growth of clover was not influenced by application of AMF.

Introduction

The traditional view of arbuscular mycorrhizal fungi as generalists with wide geographical

and host ranges and little functional speciality has been abandoned in the last two decades as

more and more evidence of functional speciality and host specificity has emerged. The advent

of molecular techniques in particular, has enabled direct investigation of which AMF species

are colonising which plants, revealing complex relationships between the AMF community in

the soil and that inhabiting plant roots. Increasing evidence of functional diversity and host

specificity has led to the realisation that AMF diversity is likely to be important in structuring

plant communities and experimental evidence supports this view (Klironomos 2003). While

evidence of the importance of AMF diversity in plant communities was accumulating there

was also an increasing realisation that agroecosystems are depleted in AMF, both in terms of

colonisation potential and species diversity (Helgasson et al 1998). There is some limited

evidence that this may lead to reduced productivity (Johnson et al 1992), though this is far

from conclusive. Nevertheless, a paradigm has developed suggesting that productivity and

sustainability of agroecosystems can be improved by enhancing AMF diversity, either

through managing the system to enhance the native AMF community or through the

introduction of new species/strains of AMF through the use of inoculum. However, there is

little evidence to suggest what level of AMF diversity is required to maximise benefits or

which species of AMF may be most effective. This is clearly a problem when assessing the

consequences of reduced AMF diversity or deciding upon the most effective mix of AMF to

apply as an inoculum. We sought to assess the degree of AMF diversity required in to gain

maximum benefit to two crop plants grown in a low nutrient soil.

Materials and methods

We selected a sandy loam soil that had been under organic management in a horticultural

rotation for 18 years. During this time it had received no animal manures or other

supplementary fertilizers. As a consequence total (459 mg kg-1

) and extractable (8.1 mg kg-1

,

Olsen extraction) soil P concentrations were low, other soil characteristics were; extractable K

63 mg kg-1

, pH 6.10 (H2O), total organic carbon 2.01% and total N 0.21%. Soil was passed

40

thorough 3 mm sieve and irradiated with 10 k Gy radiation. Irradiated soil was moistened to

60% WHC and placed into 11cm plastic pots (753g dry wt). All pots received 10 ml of soil

inoculum made from non-irradiated soil/water slurry filtered through a 38μm sieve. Pots were

then inoculated with AMF. Inoculum consisted of root fragments, hyphae and spores in 50/50

silver sand/Terragreen mix in which Plantago lanceolata had been grown with a single AMF

species. Between zero and 7 species of AMF were inoculated into each pot with total weight

of inoculum being the same for all pots (20g), divided equally between the different AMF

species added. Species used are shown in Table 1. The species selection was intended to

reflect the type of mixture usually found in commercial inoculum, that is, mostly Glomus

species with a single species from two or three other genera. Control pots received 20 g of

twice autoclaved AMF inoculum (a mixture of all 7 AMF species), plus 5ml of a 38μm filtrate

of this mixed inoculum. The design was fully factorial with all possible combinations of

species. The resources required to replicate such an experiment would be great and thus there

was no replication except for control treatments and the treatment receiving all 7 species,

which were replicated three times.

Two plant species were used, white clover (Trifolium repens L.) and onion (Allium cepa L.)

White clover is an important pasture species, particularly in northern Europe and its use is

increasing in response to increasing fertiliser prices. Onion is a globally important vegetable

crop. Within the EU production is concentrated in Mediterranean countries with Spain alone

accounting for approximately one quarter of EU and 2% of world production. Both crops are

strongly mycorrhizal and can be expected to respond positively to inoculation with AMF.

Three pre-germinated seeds were added to each pot, which was reduced to two plants once

fully established (after around one week). Pots were placed in a glasshouse and watered three

times weekly to maintain soil moisture content. Plants were harvested after 15 weeks for

onion and 21 weeks for clover. Roots and shoots were separated. A sub sample of roots was

retained for determining colonisation using the grid line intersect method, the remaining roots

and shoots were dried at 90oC and weighed.

Table 1. AMF species used

Species Culture

Glomus manihotis FL879

Glomus intraradices BEG144

Glomus caledonium BEG20

Glomus mosseae BEG12

Acaulospora spinosa NC501

Paraglomus occultum WV224

Scutellospora fulgida VA103B

Results

Growth of both onions and clover was satisfactory. By the end of the experiment bulb

formation had begun in onion, though it was not very advanced, while clover had begun to

flower, with around one quarter of plants flowering to some extent.

Figure 1 shows onion shoot dry weight. There was no significant difference in shoot weight

between zero and 1 species of AMF or between 1 and 2 species, but 2 through to 7 species all

produced significantly more dry weight than zero and 3 through to 7 species produced

significantly more than 1. When the effect of individual AMF species was considered alone

Glomus mosseae, Glomus intraradices and Glomus caledonium produced a significant

41

increase in shoot weight. Glomus manihotis was the next most significant (P 0.086), none of

the other species approached a significant level.

0 1 2 3 4 5 6 70

0.5

1

1.5

2

2.5

3

3.5

Number of species

Sh

oo

t d

ry w

t. (

g)

Figure 1. Mean shoot dry weight of onion for all species combinations. Error bars are ± 1 standard error.

There were few specific species mixtures producing a significant increase in shoot weight. Of

those that did they were equally likely to contain the Paraglomus, Acaulospora or

Scutellospora species or just a combination of Glomus species. Root dry weight (data not

shown) was not influenced by AMF (P 0.066). Total plant dry weight followed an almost

identical pattern to shoot dry weight although there was a subtle difference in that total plant

dry weight was significantly increased by Glomus mosseae and Glomus intraradices and

Glomus manihotis, but not Glomus caledonium (P 0.081). The largest increase was with

Glomus mosseae and the smallest (significant) increase was with Glomus manihotis.

Examination of colonisation revealed no significant effect of AMF species or number on root

length colonised.

Figure 2 shows shoot dry weight of clover. The number of AMF species had no significant

affect on clover shoot dry weight (P 0.427) the effect on root dry weight was similarly non

significant (P 0.365). As far as individual species is concerned only G. mosseae significantly

influenced growth (both shoot and root) and this was a reduction in growth. No combination

of species produced a significant increase or decrease in growth.

0 1 2 3 4 5 6 70

1

2

3

4

5

6

7

Number of species

Sh

oo

t d

ry w

t. (

g)

Figure 2. Mean shoot dry weight of clover for all species combinations. Error bars are ± 1 standard error.

42

Conclusions

The evidence in the literature for functional diversity in AMF and host/AMF specificity is

strong (Klironomos 2003, Munkvold et al 2004, Maherali and Klironomos 2007). Thus some

authors have suggested that high AMF diversity is required in agroecosystems to maximise

the benefits that AMF can bring. However, most intensive agroecosystems operate under low

plant stress conditions and it is not clear if a diverse AMF community provides a significant

benefit. Our results suggest that little benefit may be gained from high AMF diversity under

conditions of low stress and that AMF may be parasitic on some plant species under these

conditions. Furthermore, our results suggest that Glomus species are likely to have the largest

influence of host plant growth, whether positive or negative. Although in soils with different

stress factors, such as high disease pressure, this may not be the case.

References Helgason T, Daniell TJ, Husband R, Fitter AH, Young JPW, 1998. Ploughing up the wood-

wide web? Nature 355: 431-431.

Johnson NC, Copeland PJ, Crookston RK, Pfleger FL, 1992. Mycorrhizae - possible

explanation for yield decline with continuous corn and soybean. Agronomy Journal 84:387-

390.

Klironomos JN, 2003. Variation in plant response to native and exotic arbuscular mycorrhizal

fungi. Ecology 84: 2292-2301.

Maherali H, Klironomos, JN, 2007. Influence of Phylogeny on fungal community assembly

and ecosystem functioning. Science 316: 1746-1748.

Munkvold L, Kjoller R, Vestberg M, Rosendahl S, Jakobsen I, 2004. High functional

diversity within species of arbuscular mycorrhizal fungi. New Phytologist 164: 357-364.

43

Monitoring the structure and dynamics of arbuscular mycorrhizal fungus

communities using Terminal Restriction Fragment Length Polymorphism

of 18S rRNA genes

Gary D. Bending1, Paul Gosling

1 and Giles King Salter

2

1Warwick HRI, University of Warwick, Wellesbourne Warwick CV35 9EF, UK

2School of Biology and Environmental Science, University College Dublin, Dublin 4Ireland

Abstract

Commercial AMF inocula are typically introduced into soil environments containing existing

native species of AMF. Methods are needed to measure the extent to which inoculant AMF

are able to successfully compete with native strains to colonise the roots of plants. Since

identification of fungal species colonising plant roots is not possible using staining and

morphological analysis, molecular approaches based on analysis of fungal nucleic acids offers

the best way forward. PCR primers are available for amplification of specific strains, species,

genera or whole communities of AMF. Since AMF inocula contain diverse strains belonging

to several genera, community based profiling approaches could offer a convenient means to

investigate the success of inoculant fungi.

Although a number of methods are available to profile the composition of complex microbial

communities, Terminal Restriction Fragment Length Polymorphism (TRFLP) of 18S rRNA

genes offers a cheap, high throughput approach which can provide identification and semi-

quantitative estimation of AMF community members. In the technique, DNA is amplified

using fluorescently labelled primers. Amplicons are digested using restriction enzymes and

the size and quantity of Terminal Restriction Fragments (TRF) determined on a sequencer

using capillary electrophoresis. TRF can be related to specific organisms by reference to TRF

produced from sequenced clones derived from the sample or strains described in DNA

databases. In the current paper we describe application of the 18S rRNA-TRFLP technique to

investigate the structure and dynamics of AMF communities in agricultural systems.

The influence of soil phosphorus (P) status on AMF associated with maize and soybean was

studied in a field experiment at Wellesbourne. A P gradient was established and maintained

by application of phosphate fertiliser to an arable field to provide UK soil phosphorus indices

between 1 and 8 (between 10 and 200 mg available P kg-1

). Five years following

establishment of the gradient, crops of maize and soybean were grown. Roots of each crop

were taken at pre-flowering, flowering and just prior to harvest. DNA was extracted from

roots and TRFLP performed using the primers AML1 and AML2, which amplify AMF with

the exception of the Archaeosporales.

It was shown that the total number of TRF was affected by plant species, with more TRF, and

hence higher diversity, in soybean relative to maize. Number of TRF was unaffected by

sampling time. Elevated soil P status reduced number of TRF in soybean by up to 80 %, but

had no effect on number of TRF associated with maize roots. Multi Dimensional Scaling

(MDS) showed that the structure of communities was significantly affected by crop type but

not sampling time. The structure of AMF communities at the highest P level was significantly

different to that at lower P levels for both crops.

Cloning demonstrated that the dominant members of the community were Glomus

caledonium, G. etunicatum, G. sinuosum and G. mosseae. Restriction analysis of the cloned

sequences revealed that the G. caledonium community consisted of 4 clone types, each of

44

which gave different TRF, while the G. etunicatum community consisted of 2 clone types,

each of which gave distinct TRF. Relating the individual clone types to TRFLP profiles

enabled the response of the different species, and for G. caledonium and G. etunicatum, the

different clone types, to crop type and soil P status to be determined.

The applicability of 18S rRNA TRFLP to differentiate between AMF found in inocula and

native communities will be discussed.

45

Characterization of Mediterranean AM: initiation of a novel functional

marker approach

Vicente C.2

and Arnholdt-Schmitt B.1*

Corresponding author: * email: eu_chair@uevora.pt

1EU Marie Curie Chair, ICAM, University of Évora, Portugal

2Instituto Nacional de Recursos Biológicos L-INIA Elvas (Ex-Estação de Melhoramento de

Plantas) Estrada Gil Vaz Apartado 6. 7350-951 Elvas, Portugal

Introduction

The Mediterranean climate, predominant in the Mediterranean basin, is characterized by cold

winters and warm summers with an enormous irregularity in the distribution of rainfall in

space and time. In accordance with summer drought, this system can be divided into three

regions: an arid region to the south; semi-arid regions in the eastern littorals, the western

Spanish coast and large islands; and a humid region to the north (Makhzoumi, 1999). The

complex interactions between rainfall and temperature, together with geology, topography,

soil and vegetation cover, account for the Mediterranean ecosystems fragility and

susceptibility to degradation (Makhzoumi, 1999). The intensification of desertification

conditions, rather by natural or anthropogenic processes, imposes the search for new

alternatives for soil rehabilitation and recuperation of vegetation diversity. The application of

arbuscular fungi (AMF) in agricultural systems comprehends mainly in the recuperation of

poor and degraded soils and in the establishment of new practices for the recovering of plant

populations. Besides the well-known advantage of phosphorus uptake, AM fungi can also

benefit plants by: increased shoot and root biomass; increased uptake of other nutrients

(nitrogen and cooper), limited uptake of toxic heavy metals, increased resistance to

pathogens; improved defence against or altered interactions with herbivores; and one of the

most ambitious and controversial profit, improved plant-water relations (Newsham et al.,

1995).

Applications of AMF in Mediterranean ecosystems

AMF in the recovering of desertified Mediterranean Ecosystems

Several studies have been developed in attempt to decrease the pressure of desert-like

conditions along the Mediterranean systems, emphasizing the importance of mycorrhizal

symbioses in the rehabilitation/restoration of degraded ecosystems. Herrera et al. (1993),

Requena et al. (1996) as well as Azcón-Aguillar et al. (2003) highlighted the importance of

natural mycorrhizal potential associated with woody species from the natural succession in

semiarid ecosystems. In addition, Alguacil et al. (2005) proved that the establishment of

mycorrhizal shrub species favours the reactivation of soil microbial activity, which is linked

to an increase of aggregates stability and an acceleration of nutrient cycles. Concerning AMF

diversity in the Mediterranean regions, Ferrol et al. (2004) analysed one of the most

representative shrub species, Pistacea lenticus, which is a also target plant for re-vegetation

programmes. The author found that diversity was confined especially to Glomus (G.

constrictum, G. viscosum, G. claroideum, G. mosseae) and Paraglomus occultum, typical

species from Mediterranean systems except P. occultum.

46

AMF in important agronomic cultures of Mediterranean ecosystems

Here we focus on two important Mediterranean species: the emblematic Olea europea L and

Cicer arietinum L. AM inoculation is known to increase survival rate and development of

micro-propagated plantlets (Azcón-Aguilar and Barea, 1997). AM application is thus

important for the establishment of young olive trees. Santos-Antunes (2002) reported that the

inoculation of olive plantlets just before field sowing, can produce higher rates of

development in the field and also better chances to survive to an eventual drought period or

attack by pathogenic agent. Assessing the effectiveness of native fungal isolates as inoculants

of olive trees, Calvente and co-workers (2004) found that AM fungi diversity in the

rhizosphere was considerably low under the monoculture practice and that the degree of

responsiveness to AM inoculation not only varied depending on olive genotype but also on

the AM fungi inoculated.

The pulse crop Cicer arietinum L. (chickpea), as the third most important food legume

globally, plays an important role in the maintenance of soil fertility and for human/animal

consumption with optimal nutritional compositions (high protein content with a good

percentage of true protein digestibility) (Saxena, 1990). Chickpea productivity can be affected

by different biotic (such as pathogenic agents) and abiotic factors (water and nutrient

deficiencies), in which AM fungi inoculation may help to overcome. Rao et al. (2006)

reported that a dual inoculation of Rhizobium sp. and Glomus fasciculatium has increased the

nodulation, nitrogen and phosphorus concentration in plants and yield of chickpea. However,

care must be taken in view of the results of Weber et al. (1993). These authors observed a

negative effect of AM inoculation related to the reproductive growth. Mycorrhiza plants had

improved P uptake and vegetative growth, but failed to achieve high productivity probably

because of the terminal drought effects under Mediterranean climate conditions. Jalali and

Thareja (1981) observed the suppression of Fusarium wilt incidence in chickpea and

concluded that mycorrhizal roots were nutritionally healthier than non-mycorrhizal roots.

Jalali (1992) also reported that the presence of AM fungi in chickpea exerted a significant

impact on the spectrum of root exudation as well as plant mineral nutrition. Gahoonia et al.

(2006) underlined the need to study root traits as support for breeding drought-tolerant and

nutrient efficient high yielding varieties. These authors suggested that the combination of root

traits found in chickpea (high acid exudation, greater root-hair density and length) could

enhance the crop adaptation to marginal and dry areas.

Presentation of a novel functional approach for AM characterization

For the application of arbuscular mycorrhizal fungi in agricultural systems the following

parameters will be critical:

1. survival of the inoculum in the natural habitat

2. competitiveness with other soil organisms in the rhizosphere

3. maintenance of the genetic integrity

4. efficiency of the AMF in terms of

a. inducibility of branching

b. symbiotic effectiveness to improve availability of nutrients and water and

provide pathogen tolerance

All these parameters can potentially be influenced by the genetics of the AM fungi. Genetic

techniques are available to characterize the genomic background of AM fungi (Franken and

Requena, 2001; Ferrol et al., 2004; Hause and Fester, 2005, Turrini et al. 2008). Typically,

AMF isolates are characterized by methodologies using SSR, SSU or ITS identification.

However, single spores can carry several genetic variants (Helgason and Fitter, 2005). Large

47

spores contain typically hundreds of nuclei and high degrees of polymorphism make this task

a difficult one (Young, 2008). Van der Heijden and Scheublin (2007) point to the importance

of considering the functional diversity of AMF. The composition of AMF can have important

impact on plant performance, plant community structure and ecosystem functioning. The

authors stress a missing link between knowledge on the functional significance of AMF and

ecosystem functioning. Recently, it started that selected genes, such as phosphate transporters,

have been characterized in AM fungi (e.g. Isayenkov et al. 2004). This approach aims a

specific characteristic related to the symbiotic efficiency. We will propose working on the

alternative oxidase (AOX) as a marker to characterized biodiversity/gene diversity and at the

same time the functional integrity of AMF communities. Tamasloukht et al. (2003) speculated

on the role of alternative respiration in the pre-symbiotic phase to enable survival of AM

fungi spores and AOX is the key enzyme in this pathway. This candidate gene could serve to

monitor most of the critical parameters for application. AOX belongs to an ancient gene

family that is distributed in all kingdoms of life (McDonald, 2008). During evolution, fungi

AOX has developed separately from plants. Characterization in mycorrhiza plants will be

possible. The authors of this paper are working on AOX characterization in diverse plant

species and want to initiate research on the hypothesis that regulation of AOX from plants and

AM fungi is connected during the pre-symbiotic phase and plays a crucial role in mycorrhiza

colonisation (Arnholdt-Schmitt, 2008).

References

Arnholdt-Schmitt B, 2008. A novel gene –candidate of socio-economic interest? Proceedings

of COST 870 meeting:From production to application of arbuscular mycorrhizal fungi in

agricultural systems: a multidisciplinary approach, p. 47.

Azcón-Aguilar C, Palenzuela P, Roldán A, Bautista S, Vallejo R, and Barea JM, 2003.

Analysis of the mycorrhizal potential in the rhizosphere of representative plant species from

desertification-threatened Mediterranean shrublands. Applied Soil Ecology 22:29-37

Calvente R, Cano C, Ferrol N, Azcón-Aguilar, and Barea JM, 2004. Analysing natural

diversity of arbuscular mycorrhizal fungi in olive tree (Olea europea L.) plantations and

assessment of effectiveness of native fungal isolates as inoculants for commercial cultivars of

olive plantlets. Applied Soil Ecology 26:11-19.

Ferrol N, Calvente R, Cano C, Barea JM, Azcón-Aguilar C, 2004. Analysing arbuscular

mycorrhizal fungus Diversity in shrub-associated resource islands from a desertification-

threatened semiarid Mediterranean ecosistema. Applied Soil Ecology 25:123-133.

Ferrol N, Azcón-Aguilar C, Bargo B, Franken P, Gollotte A, González-Guerrero M, Harrier

LA, Lanfranco L, van Tuinen D, and Gianinazzi-Pearson V, 2004. Genomics of arbuscular

mycorrhizal fungi. Applied mycology and biotechnology 4:379-403.

Franken P, and Requena N, 2001. Analysis of gene expressionin arbuscular mycorrhizas: new

approacheas and challenges. New Phytologist 150:517-523.

Gahoonia TS, Rawshan A, Malhotra RS, Jahoor A, Matiur Rahman M, 2006. Root

morphological and physiological traits and nutrient uptake of chickpea genotypes. Journal of

Plant Nutrition (accepted).

Hause B, and Fester T, 2005. Molecular and cel biology of arbuscular mycorrhizal symbiosis.

Planta 221: 184-196.

Herrera MA, Salamanca CP, and Barea JM, 1993. Inoculation of Woody legumes with

selected arbuscular mycorrhizal fungi and rhizobia to recover dersertified Mediterranean

ecosystems. Applied and Environmental Microbiology 59:129-133.

Helgason T, and Fitter A, 2005. The ecology and evolution of the arbuscular mycorrhizal

fungi. Mycology 19:96-101.

48

Isayenkov S, Fester T, and Hause B 2004. Rapid determination of fungal colonization and

arbuscule formation in roots of Medicago truncatula using real-time (RT) PCR. J. Plant

Physiol. 161:1379 -1383.

Jalali BL, and Thareja ML, 1981, Suppression of Fusarium wilt of chickpea in vesicular-

arbuscular mycorrhizal inoculated soils. International Chickpea Newsletter 4:21-22.

Jalali BL, and Chand H, 1992. Chickpea wilt. In: Plant Diseases of International Importance.

Vol. 1. Diseases of Cereals and Pulses (U.S. Singh, A.N. Mukhopadhayay, J. Kumar, H.S.

Chaube, ed.), Prentice Hall, Englewood Cliffs, NJ, USA, 429–444.

McDonald A E, 2008. Alternative oxidase: an inter-kingdom perspective on the function and

regulation of this broadly distributed ‗cyanide-resistant‘ terminal oxidase.Functional Plant

Biology 35:535-552.

Makhzoumi J, 1999. The Mediterranean context. In: Makhzoumi, J. and Pungetti, G (edts).

Ecological Landscape Design and Planning: The Mediterranean context. GBR: Spon Press.

pp. 22-23.

Newsham KK, Fitter AH, and Watkinson AR, 1995. Multi-functionality and biodiversity in

arbuscular mycorrhizas, Tree 10:407-410.

Requena N, Jeffries P. and Barea AM, 1996. Assessment of natural mycorrhizal potential in a

desertified semiarid ecosystem. Applied and Envirornmental Microbiology 62: 842-847

Santos-Antunes AF, 2002, As Micorrizas e o crescimento de plantas: o caso da oliveira.

Melhoramento 38:223-230

Van der Heijden MGA, and Scheublin TR, 2007. Functional traits in mycorrhizal ecology:

their use for predicting the impact of arbuscular mycorrhizal fungal communities on plant

growth and ecosystem functioning. New phytologist 174:244-250.

Weber E, Saxena MC, George E, and Marschner H, 1993. Effect of vesicular-arbuscular

mycorrhizaon vegetative growth and harvest index of chickpea grown in northern Syria. Field

Crops Research 32:115-128.

Young JPW, 2008. The genetic diversity of intraterrestrial aliens. New Phytologist 178: 465

Tamasloukht MB, Séjalon-Delmas N, Kluever A, Jauneau A, Roux C, Bécard G, and Franken

P, 2003. Root factors induce mitochondrial-related gene expression and fungal respiration

during the development switch from asymbiosis to presymbiosis in the arbuscular

mycorrhizal fungus Gigaspora rosea. Plant Physiology 131:1468-1478.

Turrini A, Avio L, Bedini S, and Giovannetti M, 2008. In situ collection of endangered

arbuscular mychorrhizal fungi in a Mediterranean UNESCO Biosphere Reserve. Biodiversity

and Conservation 17: 643

49

Morphological and molecular characterization of some arbuscular

mycorrhizal fungi, potential candidates to use in the protection of dune

plants of the Mediterranean Sea

Błaszkowski J., Gábor M. Kovács G. M.

Department of Plant Protection, University of Agriculture

Slowackiego 17, PL-71434 Szczecin, Poland

Department of Plant Anatomy, Institute of Biology,

Eötvös Loránd University, Pázmány Péter sétány 1/C, 1117 Budapest, Hungary

Arbuscular mycorrhizal fungi of the phylum Glomeromycota are considered to belong to the

most common soil fungi in the world and associate with at least 80% of vascular land plants

(Smith and Read 1997).

Sites harbouring exceptionally abundant and diverse populations of arbuscular fungi are

maritime sand dunes, mainly because of their low nutrient content and organic components

(Dalpé 1989, Koske 1987, 1988, Nicolson and Johnston 1979, Tadych and Błaszkowski

2000).

Of many plants of dune sites, Ammophila arenaria (L.) Link is the most important sand-fixing

species in maritime dunes of Europe (Rodríguez-Echeverría and Freitas 2006), and its roots

commonly host arbuscular mycorrhizae and diverse populations of spores of the

Glomeromycota (Kowalchuk et al 2002, Nicolson and Johnston 1979, Tadych and

Błaszkowski 2000). Other dune plants, including many protected and threatened species,

generally also coexist with diverse spore populations of these fungi.

The association of arbuscular mycorrhizal fungi with maritime dune plants may be of

considerable ecological significance for their establishment and growth, because these fungi

enhance plant nutrient uptake, increase plant tolerance to drought and salt stress, and protect

them against soil pathogens and nematodes (Koske et al. 2004).

At least 32 newly described species of arbuscular mycorrhizal fungi have originally been

associated with roots of dune plants and many others have occurred in maritime dunes

(Błaszkowski 2003; Sridhar and Beena 2001).

Of the almost 70% of the described species of the Glomeromycota found by the first author in

dune sites of the Mediterranean Sea located in, e. g., Portugal, Spain, France, Italy, Greece,

Turkey, Israel, Cyprus and northern Africa, the fungi most frequently occurring in the field

conditions and easily and abundantly sporulating in both pot trap and one-species-cultures

were Archaeospora trappei, A. delicata, A. lacunosa, A. paulinae, A. scrobiculata, A. spinosa,

Glomus aggregatum, G. aurantium, G. drummondii, G. constrictum, G. corymbiforme, G.

eburneum, G. fasciculatum, G. irregulare, G. intraradices, G. lamellosum, G. pustulatum, G.

trimurales, G. versiforme, G. walkeri, G. xanthium, Scutellospora calospora, S. fulgida, S.

pellucida, and S. persica. Their wide distribution and easy cultivation in greenhouse

conditions indicate them to be the best candidates to use in production of inoculum assigned

for the preservation of protected and threatened dune plant species.

During the presentation, the most important diagnostic morphological and molecular

properties of some of the species listed above will be shown. The species will be also

compared with those with which they may easily be confused. Additionally, the steps of

formulation of an inoculum and its incorporation in the field conditions will be proposed and

discussed.

50

References

Błaszkowski J, 2003. Arbuscular mycorrhizal fungi (Glomeromycota), Endogone, and

Complexipes species deposited in the Department of Plant Pathology, University of

Agriculture in Szczecin, Poland.

http://www.agro.ar.szczecin.pl/~jblaszkowski/.

Koske RE, Gemma JN, Corkidi L, Sigüenza C, Rinkón E, 2004. Arbuscular mycorrhizas in

coastal dunes. In: M. I. Martínez, N. P. Psuty (eds.). Coastal dunes, ecology and conservation.

Ecol. Studies 171: 173-187.

Kowalchuk GA, De Souza FA, Van Veen JA, 2002. Community analysis of arbuscular

mycorrhizal fungi associated with Ammophila arenaria in Duch coastal sand dunes.

Molecular Ecology 11: 571-581.

Nicolson TH, Johnston C, 1979. Mycorrhiza in Gramineae. III. Glomus fasciculatum as the

endophyte of pioneer grasses in maritime sand dunes. Trans. Br. Mycol. Soc. 72: 261-268.

Rodríguez-Echeverría S, Freitas H, 2006. Diversity of AMF associated with Ammophila

arenaria spp. arundinacea in Portuguese sand dunes. Mycorrhiza 16: 543-552.

Sridhar KR, Beena KR, 2001. Arbuscular mycorrhizal research in coastal sand dunes: a

review. Proc. Nat. Acad. Sci. India. 71: 179-205.

Tadych M, Błaszkowski J, 2000. Arbuscular fungi and mycorrhizae (Glomales) of the

Slowiński National Park, Poland. Mycotaxon 74: 463-483.

51

The real-time quantitative PCR assay: from its detection of fungi

pathogenic on trees to its use detecting AMF

Luchi N., Capretti P.

Department of Agricultural Biotechnology, Plant Pathology Section, University of Florence.

Piazzale delle Cascine 28, I-50144 Florence, Italy

Abstract

Molecular techniques have been particularly useful to detect specific micro-organisms in

different substrates (i.e. wood, soil, water) without the need to isolate a pure culture.

Molecular techniques are more reliable than the traditional methods, which have several

limitations in that they are difficult to standardise, usually do not give quantitative results

(O‘Connel et al., 1998; Loric et al., 1995) and tend to be cumbersome and time–consuming,

especially if hundreds of samples have to be analysed (Simon et al., 1992).

In the case of fungal communities that colonise plants, some species may be difficult to

isolate, while other species are masked by endophytic micro-organisms that overgrow them

on agar media (Catal et al., 2001).

Over the last few years the use of PCR techniques has greatly increased and expanded,

including new PCR approaches such as real time quantitative PCR (rt-qPCR), which has

proved to be a useful tool to quantify amplification products. The main feature of rt-qPCR is

that the PCR products are detected at each cycle, in real-time, allowing them to be quantified

during the exponential phase of the run. This is unlike classical PCR, where quantification is

carried out only at the end-point (or plateau phase) of the run (Heid et al., 1996), so that their

accuracy is lower.

The sensitivity of rt-qPCR is mainly due to a different ‗chemistry,‘ which uses fluorogenic

molecules that release fluorescence during PCR synthesis. Some fluorogenic molecules are

single molecules (such as for example SYBR green) that intercalate in a double–stranded

DNA, but some are also labelled oligonucleotides that bind with a specific region of the DNA

target (Bustin, 2000).

Rt-qPCR has been applied in plant pathology to detect fungal pathogens and for other

purposes. In this field rt-qPCR has furthered our understanding of plant–pathogen interactions

(Schena et al., 2004).

An interesting feature that has been found is that some fungi, including pathogenic fungi,

have a ‗latent phase‘ in standing trees, when they live in those trees but do not cause any

symptoms. When these trees become debilitated for some reason, however (mostly by

drought) the fungus starts to grow, and to colonize and damage the host-tissue. Diagnostic

methods based on DNA may be able to detect infections with such fungi at an early stage.

During the last few years, real-time PCR has therefore been developed to detect fungi in

apparently healthy plant tissue, in three different pathosystems: Diplodia pinea on Pinus

nigra (Maresi et al., 2007), Biscogniauxia mediterranea on Quercus cerris, Q. ilex (Luchi et

al., 2005) and Biscogniauxia nummularia on Fagus sylvatica (Luchi et al., 2006).

The ABI Prism 7700 Sequence Detection System with the TaqManTM

assay has proved to be

a rapid and sensitive means to quantify amplification products in real-time PCR. This system

uses a dual-labelled fluorogenic probe that anneals between the two primers. The probe

contains two fluorescent dyes covalently linked at its ends: at the 5‘ end there is a reporter

[FAM (6-carboxy-fluorescein)] and at the 3‘ end there is a quencher [TAMRA (6-carboxy-

tetramethyl-rhodamine)]. Reporter and quencher are excited by laser during PCR. After a

52

denaturation step, the fluorogenic probe hybridises to the template DNA within the region

defined by the forward and reverse primers. During the polymerisation steps, the 5‘-3‘

exonuclease activity of the Taq-polimerase cleaves the probe (Holland et al., 1991). The

fluorescent signal from the free reporters, detected during PCR thermocycling, allows

quantification of the template copies.

Computerised systems such as the ABI Prism 7700 Sequence Detection System make it

possible to analyse a great number of samples at a time (up to 96 per run) and to reduce

processing times quite substantially (to about 2 hours). Higher initial costs are offset by the

greater number of samples processed.

Research on forest tree pathogens showed that with this system lower amounts of fungal DNA

(less than 10 pg) could still be detected in healthy shoots. The technique was sensitive enough

to detect fungal DNA from an ascospore suspension (Luchi et al., 2005). The data showed

that the rt-qPCR detected low quantities of amplified DNA. The studies confirmed the

reliability of this technique for tree diseases and suggested that it could also be used on

samples collected from natural populations. Sensitive molecular techniques such as rt-qPCR,

make it possible to identify a specific organism in a DNA mixture consisting of plant tissue

and other micro-organisms that usually live into the host. It is also possible to monitor

harmful fungal parasites, particularly those that have a long latency phases without showing

any symptoms, such as fungal endophytes.

In forest pathology, when monitoring fungal diseases, this molecular approach gives useful

information before the diseases are clearly evident in the landscape, and it can also be utilised

in probing the relationship between disease and environmental parameters, which may

promote fungal spread.

So far there have been only a few studies dealing with the application of rt-qPCR to

arbuscular mycorrizal fungi (AMF).The rt-qPCR technique can be used to test: a) AMF

inoculum; b) AMF in the soil; c) the interaction between AMF and the host.

a) AMF inoculum. It is important to develop a standardised control system to analyse the

inoculum. Conventional methods, such as MPN, only have a limited capacity to estimate the

number of inoculum spores (Baar, 2007). The rt-qPCR assay could determine the quality and

the quantity of the fungal inoculum. The assay has already been used reliably to determine the

spore dilution of B. mediterranea (Luchi et al., 2005) and G. mossae (Bohm et al., 1999). The

rt-qPCR assay also requires only minute quantities of starting material, reducing loss of

inoculum.

b) AMF in the soil. The amount of AMF in the soil may be affected by cultural practices and

by soil management, with negative consequences on the abundance and diversity of the AMF.

The rt-qPCR technique can be useful to monitor changes in the AMF community (Cavagnaro

et al., 2007).

c) AMF in planta. The most common tool conventionally used to detect AMF in host tissue is

light microscopy. However, this method does not distinguish between fungal species or

quantify their presence at species level. As for fungal pathogens, rt-qPCR is particularly

useful to monitor the colonisation process of fungi in plants. A recent study has shown an

interesting application of rt-qPCR to detect different AMF in host plants (Alkan et al., 2006).

This method could be utilised to study gene-expression in the plant–microbe interaction. Liu

et al. (2007) has shown that it might be possible to apply rt-qPCR to study gene expression as

result of AM symbiosis.

The understanding of gene expression could be improved using a sophisticated technique

named Laser Microdissection Pressure Catapulting (LMPC), which dissects out single cell

types from microscopic tissue sections (Day et al., 2005). LMPC uses an inverted microscope

and a nitrogen laser in order to microdissect out selected cells that are subsequently levitated

53

(catapulted) into a collection cap using the same laser source. The cells so collected are then

used to extract RNA and DNA, and for downstream applications such as expression studies or

gene copy number evaluation using real-time PCR (Pinzani et al., 2006). We optimised the

LMPC technique and combined it with real-time RT-PCR in order to detect -tubulin genes

in the bark tissue of Norway spruce (Picea abies). This method is an important tool to study

gene modulation at the level of specific cells, and can be used to exploit the interaction

between plant cells with micro-organisms such as AMF.

The sensitivity and specificity of rt-qPCR represents one of the most notable aspects of this

innovative technique, which has proved to be highly efficient because of the great number of

samples that can be analysed in a single run. For the applications mentioned above, real-time

PCR is a promising technique to study the biology of AMF. This new technique may also

have implications for AM fungi, and may facilitate monitoring these micro-organisms in a

wide range of substrates

References Alkan N, Gadkar V, Yarden O, Kapulnik Y, 2006. Analysis of quantitative interactions

between two species of arbuscular mycorrhizal fungi, Glomus mosseae and G. intraradices,

by real-time PCR. Applied and Environmental Microbiology, 4192–4199.

Baar J, 2007. Innovative thoughts for the development of a quality control system. In:

Proceedings of the COST Action 870. ―Contribution of mycorrhizal fungi to agro-ecosystems:

how to bridge the gap from small-scale production to commercial utilization‖. 24-25 May

2007. Budapest, Hungary

Bohm J, Hahn A, Schubert R, Bahnweg G, Adler N, Nechwatal J, Oehlmann R, Osswald W,

1999. Real-time quantitative PCR: DNA determination in isolated spores of the mycorrhizal

fungus Glomus mosseae and monitoring of Phytophthora infestans and Phytophthora

citricola in their respective host plants. Journal of Phytopathology 147, 409-16.

Bustin SA, 2000. Absolute quantification of mRNA using real-time reverse transcription

polymerase chain reaction assays. Journal of Molecular Endocrinology 25:169-193.

Catal M, Adams GC, Chastagner GA, 2001. Detection, identification and quantification of

latent needlecast pathogens and endophytes in symptomless conifer foliage by PCR and Dot-

blot assays. In: Forest Research Institute Res. Papers. Proceedings of the IUFRO Working

Party 7.02.02 Shoot and foliage Diseases, 2001. Hyytiälä, Finland, 164-168.

Cavagnaro TR, Jackson LE, Scow KM, Hristova KR, 2007. Effects of arbuscular mycorrhizas

on ammonia oxidizing bacteria in an organic farm soil. Microbial Ecology 54:618-26.

Day RC, Grossniklaus U, Macknight RC (2005) Be more specific! Laser microdissection of

plant cells. Trends Plant Sci 10:397-406.

Heid CA, Stevens J, Livak KJ, Williams PM, 1996.Real time quantitative PCR. Genome

Research 6: 986-994.

Holland PM, Abramson RD, Watson R, Gelfand DH, 1991. Detection of specific polymerase

chain Reaction product by utilizing the 5' to 3' exonuclease activity of Thermus acquaticus.

DNA polymerase. Proceedings of the National Academy of Sciences of the United States of

America. 88: 7276-7280.

Liu J, Maldonado-Mendoza I, Lopez-Meyer M, Cheung F, Town CD, Harrison MJ, 2007.

Arbuscular mycorrhizal symbiosis is accompanied by local and systemic alterations in gene

expression and an increase in disease resistance in the shoots. Plant Journal 50:529-44.

Loric S, Dumas F, Eschwege P, Blanchet P, Benoit G, Jardin A, Lacour B, 1995. Enhanced

detection of hematogenous circulating prostatic cells in patients with prostate adenocarcinoma

by using nested reverse transcription polymerase chain reaction assay based on prostate-

specific membrane antigen. Clinical Chemistry 41: 1698-1704.

54

Luchi N, Capretti P, Pinzani P, Orlando C, Pazzagli M, 2005. Real – time PCR detection of

Biscogniauxia mediterranea in symptomless oak tissue. Letters in Applied Microbiology 41:

61-68.

Luchi N, Capretti P, Vettraino AM, Vannini A, Pinzani P, Pazzagli M, 2006. Early detection

of Biscogniauxia nummularia in symptomless European beech (Fagus sylvatica L.) by

TaqMan™ real-time PCR. Letters in Applied Microbiology 43: 33-38.

Maresi G, Luchi N, Pinzani P, Pazzagli M, Capretti P, 2007. Detection of Diplodia pinea in

asymptomatic pine shoots and its relation to the Normalized Insolation index. Forest

Pathology 37: 272-280.

O'Connell CD, Juhasz A, Kuo C, Reeder DJ, Hoon DSB, 1998. Detection of tyrosinase

mRNA in melanoma by reverse transcription-PCR and electrochemiluminescence. Clinical

Chemistry 44: 1161-1169.

Pinzani P, Orlando C, Pazzagli M 2006a. Laser-assisted microdissection for real-time PCR

sample preparation. Molecular Aspects of Medicine. 27: 140-159.

Schena L, Nigro F, Ippolito A, Gallitelli D, 2004. Real-time quantitative PCR: a new

technology to detect and study phytopathogenic and antagonistic fungi. European Journal of

Plant Pathology 110: 893–908.

Simon L, Levesque L, Lalonde RCM, 1992. Rapid quantitation by PCR of endomycorrhizal

fungi colonizing roots. PCR Methods and Applications 2: 76-80.

55

Efficacy and persistance of introduced AM fungi in rehabilitation

processes.

Estaún V, Busquets M, Calvet C. Camprubí A.

IRTA Investigació i tecnologia agroalimentàries. Protecció Vegetal. Cabrils. Cra de Cabrils

Km 2 , 08348 Cabrils (Barcelona) Spain

Introduction

In semiarid climates the establishment of a plant cover is the most important step in the

restoration of degraded areas, such as quarries or urban wastelands, to avoid further

degradation and desertification.. Under Mediterranean conditions the restoration becomes

difficult because of the many constraints associated with the climate (dry periods followed by

torrential rains) and the soil (shallow, stony soils with low organic matter and high pH). In

most instances the plants used in these projects are grown under supraoptimal conditions and

do not present any mycorrhizal symbiosis. The success level of these revegetations is low

(Sort & Alcañiz, 1996). The use of organic fertilizers such as sewage sludge has increased the

success of the plants survival (Sort & Alcañiz, 1996), however it has as a drawback

considering the development of many volunteer plants that are nonmycorrhizal and extremely

aggressive and can delay the targeted species growth and development, besides added

problems of soil and water contamination. These problems have prevented the spread of this

practice in sensitive areas. Mycorrhiza are reported to reduce the detrimental effects on plant

growth of soilassociated stresses such as lack of nutrients, high pH and climate associated

stresses such as drought and high temperatures (Requena et al, 2001; Caravaca et al, 2003;

Alguacil et al, 2005), however the effect of the symbiosis under field conditions is sparsely

documented and with diverse results (Clemente et al, 2005). The establishment of a plant

cover can be done transplanting shrubs and /or trees or casting seed in the areas to be

revegetated. Both strategies are routinely used, depending on the accessibility of the area to be

reclaimed and on economic issues. Some rehabilitation projects contemplate the use of a

nurse crop of herbaceous species to stop the erosion process and improve the soil for future

rehabilitation activities (Skousen and Zipper, 1996). In this paper we present the results of

pretransplant inoculation with AMF on the revegetation of two quarries and one urban

wasteland using native shrubs and the establishment of an herbaceous crop directly sown with

a hydroseeder as a first rehabilitation step. The urban small open space was included in the

experiment due to the growing importance of these areas in reclamation projects.

Materials and methods

Quarry revegetation The first quarry studied is located in MontRal (Tarragona, Spain; 41º

16‘N 1º07‘E). The quarry has been mined for several decades for the extraction of stone

blocks for ornamental use. The experimental area covered a terrace of 10.000m2

formed by

stocked limestone gravel and other debris. Topsoil originally retrieved from the area to allow

the excavation and stored in piles was spread over the terrace to provide a substrate for

revegetation. (Photo1)

56

Photo 1: Revegetation of the quarry with added topsoil

The second quarry studied is located in Castellar del Vallés (Barcelona Spain, 41º 36‘ N

2º03‘E). The quarry has been mined extensively to obtain ground stone for building and

production of cement. The experimental area was a dumping area with unusable ground rock

debris with no added topsoil. (Photo2)

Photo 2: Revegetation of the quarry with no added topsoil

The plants used for the experiment were: Lavandula angustifolia Mill. and Santolina

chamecyparissus L. for both quarries with Juniperus phoeniceae L. and Thymus vulgaris L.,

for the first quarry and Anthyllis citisoides and Rosmarinus officinalis L. for the second

quarry The AM inoculum used was a mixture of roots and rhizosphere substrate of leek plants

inoculated with Glomus intraradices Schenk &Smith BEG72 and grown in Terragreen. The

growth parameters evolution was estimated as plant volume for L. angustifolia, T. vulgaris, S.

chamaecyparissus, R. officinalis and A. cytisoides and as plant height for J. phoenicia. To

assess mycorrhizal colonisation composite samples were taken from rhizosphere soil with a

soil core borer in three points chosen at random for each of the repeated treatments of 6

plants, samples were observed under a binocular microscope to evaluate mycorrhizal

colonization (Koske and Gema,1989; Giovanetti and Mosse, 1980) To assess the diversity and

persistence of the introduced AM fungus in the area after 23 months growth, eight 1cm root

pieces of each of the root composite samples (inoculated and noninoculated) were used for

DNA extraction using a chelex extraction method (van Tuinen et al. 1998, Kjøller &

Rosendahl 2000). Primary PCR was performed with the eukaryote specific primers LSU0061

(LR1) and LSU0599 (NDL22) followed by nested PCR with the primer combinations

LSURK4f and LSURK7r (van Tuinen et al. 1998, Kjøller & Rosendahl 2000) and FLR3 and

FLR4 (Gollotte et al. 2004). PCR were performed as described by Kjøller & Rosendahl

(2000). All positive PCR were

sequenced using RK4f or FLR3 as sequencing primer. Parsimony analyses were conducted in

PAUP.

Urban open space revegetation The small urban space is located in Badalona (Barcelona

Spain, 41º26‘N 2º13‘E), it is a small layout, in a slope, between blocks of apartments,

underutilised and unattractive. The soil was heavily degraded and contained building debris

from nearby construction sites. The plants used for this experiment were Rosmarinus

officinalis L. inoculated with AMF or not in the nursery.

57

Hydroseeding experiment The hydroseeding system was used to improve soil coverage and

erosion control in the quarry where no topsoil was added and in a small waste land in an

urban location. The plants used were the legumes Medicago lupulina and Lotus corniculatus

and the grasses Lolium perenne, Festuca ovina and Brachypodium. phoenicoides. The fungus,

G. intraradices, was also chosen according to the results of a previous experiment (Estaún et

al, 2006). The results of the hydroseeding procedure were evaluated in the field and in

greenhouse conditions. Three months after the hydroseeding procedure, the number of grasses

and legumes in five squares of 20cm x 20cm in each of the replicated treatments in the field

locations was recorded. Plants under greenhouse conditions were harvested and the shoot

weight of legumes and grasses was recorded separately, the root weight/cm2

and the root

colonisation were determined.

Results and Discussion

Quarry revegetation All plants established the mycorrhizal symbiosis at the nursery. At

transplant inoculated and noninoculated plants were similar in size. After 8 months growth in

the quarry with added top soil all control plants sampled presented the symbiosis and there

were no differences in plant growth (Photo 3) Molecular probes show that the most

widespread fungi were G. intraradices, and Glomus microaggregatum although other fungi

were present in the roots of the control plants(Figure1a).

Figure 1a: Diversity of AM fungi detected in mycorrhizal roots of noninoculated and

inoculated shrubs 23 months after transplant in the quarry with added topsoil

In the quarry where no top soil was added all inoculated plants grew better, although 60% of

the non inoculated plants sampled presented the symbiosis. Glomus intraradices was the only

fungus detected in the roots of the inoculated plants whilst in the roots of the control plants

other fungi were found (Figure 1b).

Figure 1b: Diversity of AM fungi detected in mycorrhizal roots of noninoculated and

inoculated shrubs 23 months after transplant in the quarry with no added topsoil

58

Our results show that adding stored top soil is a good system to enhance mycorrhizal

colonisation in quarry restoration; otherwise it is important to inoculate the plants used with

an effective fungus. Molecular probes show that in the Mediterranean ecosystems studied,

with eroded soils and high pH, the AMF species diversity is very low and G. intraradices is

present in a high percentage of the samples, indicating the resilience and adaptation to these

conditions of this species.

Urban open space revegetation As many of the areas that need rehabilitation often are in

pronounced slopes, the growth and development of Rosmarinus officinalis plants inoculated

and noninoculated with

G. intraradices was evaluated in different gradient slopes. The inoculation at the nursery level

was found to increase plant growth and coverage in the two gradients of slope considered

(Figure 2 ).

Figure 2: Height of R. officinalis plants grown in 20º and 40º slopes inoculated with G.

intraradices (M) or noninoculated (C) 12 months after trasplant.

For the rehabilitation of the urban layout, R. officinalis was considered to be the most

adequate plant due to its soil and climate stress resistance and its aromatic and ornamental

properties. Nursery inoculated plants survived transplant and the summer drought stress

(Figures 3a and 3b) (Photo 4). The inoculation of plants under these circumstances is cost

effective due to the high costs of labour if plants need to be replaced.

Figure 3: Survival (a) and growth (b) of R. officinalis plants grown in a urban layout

inoculated with

G. intraradices (M) or noninoculated (C) 8 months after trasplant.

59

Photo 4: Revegetation of a urban layout with R. officinalis 8 months after plant establishment

(A. Plants inoculated with G. intraradices, B: plants noninoculated)

Hydroseeding experiment The addition of inocula increased the total weight of plants

recovered both in the greenhouse and in the field experiments (Figure 6) (Photo5). The same

results apply for the analysis of the legumes dry weight, where the weight of legumes in the

mycorrhizal treatments was increased by tenfold respect to the nonmycorrhizal treatments.

Considering the dry weight of grasses there is no significant effect of the inoculation

with G. intraradices although there is a significant effect of the inoculation in the

legumes/grasses ratio, which is higher in all mycorrhizal treatments.

Figure 4: Total number of plants and numbers of legumes and grasses in two locations (A and

B) three months after the hydroseed application. Data are means of the plants observed in five

20cm x 20 cm squares in each of the three replicates per treatments per location ± 1.98SE of

total number of plants

60

Photo 5: Hydroseeding of a urban layout 3 months after application (A: noninoculated, B:

inoculated with G. intraradices)

Conclusions

Transplanting native plants inoculated with AMF is a good system to establish shrubs and

trees in eroded semiarid lands (Requena et al,2001; Caravaca et al, 2003; Alguacil et al, 2005)

but it is impracticable when considering large areas (Greipsson and ElMayas 1999). The

integration of the AMF inoculation with the hydroseeding technique might permit the use of

mixtures of native grasses and legumes, and can facilitate the establishment of other

mycorrhizal plants (Enkhuya et al, 2005) in large or inaccessible areas where transplanting

cannot be considered as an option for rehabilitation.

References

Alguacil MM, Caravaca F, Roldán A (2005). Changes in the rhizosphere microbial activity

mediated by

native or allochtonous AM fungi in the reafforestation of a Mediterranean degraded site .

Biology and

Fertility of Soils 41, 5968

Caravaca F, Barea JM, Palenzuela J, Figueroa D, Alguacil MM, Roldán A (2003).

Establishment of shrub species in a degraded semiarid site after inoculation with native or

allochtonous arbuscular mycorrhizal fungi. Applied Soil Ecology 22, 103111

Clemente AS, Werner C, Máguas C, Cabral MS, MartinsLouçao MA, Correia O (2004).

Restoration of a limestone quarry: effect of soil amendments in the establishment of native

Mediterranean sclerophyllous shrubs. Restoration Ecology 12, 2028

Estaún V, Vicente S, Calvet C, Camprubí A , Busquets M (2007). Integration of arbuscular

mycorrhizal inoculation in hydroseeding technology. Effects on plant growth and interspecies

competition. Land Degradation and Development 18, 621630

Gollote A, van Tuinen D, Atkinson D (2004). Diversity of arbuscular mycorrhizal fungi

colonising roots of grass species Agrostis capillaries and Lolium perenne in a field

experiment. Mycorrhiza 14:111117

Giovannetti M, Mosse B (1980). An evaluation of techniques for measuring vesicular-

arbuscular mycorrhizal infection in roots. New Phytologist 87: 489500

Kjoller R, Rosendahl S (2000). Detection of arbuscular mycorrhizal fungi (Glomales) in roots

by nested PCR (polymerase chain reaction) and SSCP (single stranded conformation

polymorphism) Plant and Soil 226,189196

Koske RE, Gemma JN (1989). A modified procedure for staining roots to detect VA

mycorrhizas. Mycological Research 92, 486505

61

Requena N, PérezSolis E, AzcónAguilar C, Jeffries P, Barea JM (2001). Management of

indigenous plantmicrobe symbiosis aids restoration of desertified ecosystems. Applied and

Environmental Microbiology 67, 495498

Rosendahl S, Stukenbrock E (2004) Community structure of arbuscular mycorrhizal fungi in

undisturbed vegetation revealed by analysis of LSU rDNA sequences. Molecular Ecology 6,

821829

Sort X, Alcañiz JM (1996). Contribution of sewage sludge to erosion control in the

rehabilitation of quarries. Land Degradation and Development 7, 6976

Enkhtuya B, Poschl M, Vosatka M. 2005. Native grass facilitates mycorrhizal colonisation

and P uptake of tree seedlings in two anthropogenic substrates. Water Air and Soil Pollution

166: 217236.

Greipsson S, ElMayas H. 1999. Large scale reclamation of barren lands in Iceland by aerial

seeding . Land Degradation and Development 10:185193.

Skousen J, Zipper CE. 1996. Revegetation species and practices. Reclamation Guidelines for

Surface Mined Lands in Southwest Virginia. Powell River Project. Viginia Cooperative

Extension Publication 460

122.

62

Effects of inoculation with two AM fungi on two poplar clones grown at

high zinc concentration.

Guido Linguaa

, Valeria Todeschinia

, Cinzia Franchinb

, Stefano Castiglionec

, Stefania Biondib

,

Patrizia Torrigianib

, Giovanni D’Agostinod

, Graziella Bertaa

a

Dipartimento di Scienze dell’Ambiente e della Vita, Universita` del Piemonte Orientale

“Amedeo Avogadro”,Via Bellini 25/G, I-15100 Alessandria, Italy -

guido.lingua@mfn.unipmn.it b

Dipartimento di Biologia evoluzionistica sperimentale, Universita` di Bologna, Via Irnerio

42, I-40126 Bologna, Italy c

Dipartimento di Chimica, Università di Salerno, Stecca 7, Via Ponte don Melillo, I-84084

Fisciano (SA), Italy. d

Istituto di Virologia Vegetale del CNR, Strada delle Cacce 73, I-10100 Torino, Italy.

Abstract

Since 2003, a wide study has been conducted on the use of poplar, associated or not to AM

fungi, for phytoremediation purposes. Part of the research was conducted in field, working a

site polluted with copper and zinc (over 1000 ppm in the first layers of soil – Castiglione et al.

submitted manuscript), made available by KME Italy, part in glasshouse (Todeschini et al.

2007; Todeschini et al., submitted manuscript; Lingua et al., 2008), under more controlled

conditions, and part in vitro, under laboratory conditions (Castiglione et al., 2007; Franchin et

al., 2007).

The glasshouse experiments were carried out on non-sterile soil (in order to simulate field

conditions), supplemented or not with zinc chloride (300 mg/kg of soil), using two registered,

commercial clones of poplar: Populus alba Villafranca (VF) and Populus nigra Jean Pourtet

(JP), inoculated or not with an arbuscular mycorrhizal fungus, either Glomus mosseae or G.

intraradices (Lingua et al. 2008).

After about six months of growth, plants were harvested and analysed for their morphological

parameters, zinc concentration in their organs (root – stem – leaves), leaf polyamine

concentration (putrescine, spermine and spermidine) and for leaf morphology and

ultrastructure.

The applied zinc concentration resulted to be toxic for plants, as shown by their

morphological parameters: growth was inhibited in comparison to control (no metal) plants in

both poplar clones (Fig. 1). Spontaneously occurring mycorrhizal colonization was prevented

(VF) or strongly inhibited (JP) by the metal, but pre-inoculation resulted in colonization levels

comparable with those of plants grown without metal supplementation. It is worth nothing,

however, that arbuscule abundance was severely reduced by zinc pollution (Fig. 2).

Zinc was differentially accumulated in the two poplar clones. In general, VF accumulated

larger amounts of metal than JP. In both clones, zinc concentration was highest in the leaves,

followed by roots and shoots. Differences between the two clones were mostly evident in the

leaves, where VF accumulated over 2000 mg of zinc per kg of dry weight, while JP reached

about 1500 mg/kg. Mycorrhizal colonization slightly (but significantly) reduced zinc

concentration in the leaves of VF plants and in the shoots of JP poplars.

The two poplar clones differentially responded to zinc in pre-inoculated plants. JP plants did

not show any effect of the fungus, while VF poplars, when inoculated with G. mosseae but

63

not with G. intraradices, resulted to be similar, as far as size and morphology are concerned,

to control (no metal) plants, suggesting a protective effect of the symbiosis (Fig. 1).

These results are associated to variations in the profile of polyamine expression. In VF leaves,

where the highest amount of zinc was accumulated, the concentration of free putrescine was

greatly increased by the metal treatment, in comparison with the controls, while the

concentration of conjugated putrescine was drastically decreased. Pre-inoculated plants, in the

presence of zinc, did not show such a response and their free and conjugated putrescine

concentrations were not different from those of the controls, in spite of a leaf zinc

concentration of about 1900 mg/kg (Fig. 3).

Fig. 1 – Shoot dry weight of P. alba Villafranca (left) and of P. nigra Jean Pourtet (right).

NoMet: plants not supplemented with zinc; Zn: plants supplemented with zinc; ZnGm: plants

treated with zinc and pre-inoculated with G. mosseae; ZnGi: plants treated with zinc and pre-

inoculated with G. intraradices. Bars represent standard errors. Different letters indicate

significant differences ( p< 0.05). Other morphological parameters showed similar trends.

From Lingua et al., 2008.

Fig. 2. Mycorrhizal colonization (M%, white columns) and arbuscule abundance in the

colonized area (a%, black columns) in the root system of P. alba Villafranca (a) or of P. nigra

Jean Pourtet (b). NoMet: plants not supplemented with metal; Zn: plants supplemented with

zinc; ZnGm: plants treated with zinc and pre-inoculated with G. mosseae; ZnGi: plants treated

with zinc and pre-inoculated with G. intraradices. Bars represent standard errors. Different

letters indicate significant differences ( p < 0.05). From Lingua et al., 2008.

64

Fig. 3 - Mean concentration and standard error (bars) of free (white columns) and soluble

conjugated (black columns) putrescine in leaves of P. alba Villafranca (left) and of P. nigra

Jean Pourtet (right), treated or not with zinc, and pre-inoculated or not with G. mosseae or G.

intraradices. NoMet: plants not supplemented with zinc; Zn: plants supplemented with zinc;

ZnGm: plants treated with zinc and pre-inoculated with G. mosseae; ZnGi: plants treated with

zinc and pre-inoculated with G. intraradices. Bars represent standard errors. Different letters

indicate significant differences ( p < 0.05). Spermidine and spermine did not show similar

variations. From Lingua et al., 2008.

The observed effects, cannot be described as nutritional effects, as the phosphate

concentration in the soil and in the plants was rather high and also because of the reduced

amounts of arbuscules. Therefore, it is likely that the arbuscular mycorrhizal fungus G.

mosseae could promote some response in the plant, increasing its tolerance to zinc.

Microscopic analyses, carried out on VF leaves, with light transmission, TEM and SEM

instruments, provided additional information on the effects of zinc on the leaves and on the

modification induced by AM colonization. The thickness of the leaf lamina (and of each leaf

tissue: palisade parenchyma and spongy tissue) was increased in the presence of zinc, but this

effect was reduced in zinc treated, G. mosseae-inoculated plants, and completely reverted in

zinc treated and G. intraradices-inoculated plants. Sections of the palisade parenchyma,

parallel to the leaf surface, showed much larger intracellular spaces and detachment of the cell

walls, following zinc treatments (Fig. 4). These effects were not observed in G. mosseae

inoculated plants, but in all cases zinc accumulation mainly occurred in the cell walls, as

shown by a histo-chemical staining (Fig. 5).

Considering the ultrastructural level, some modifications were induced by the treatment with

zinc. In the first place, a large number of crystals could be observed in the parenchyma cells

close to the vascular bundles (Fig. 6). Such crystals were recognized to be calcium oxalates

combining raman spectroscopy and SEM EDS analyses. Second, primary starch in the

chloroplast was very abundant in the controls, but not in the metal-treated plants. Both these

effects were recovered in zinc treated plants, pre-inoculated with G. mosseae (Fig. 7).

The increased presence of calcium oxalate crystals might be connected with the larger

intracellular space and with the cell wall localization of zinc. Indeed, zinc is a stronger ligand

than calcium and could displace it from the cell wall, where it is usually present. Cells strictly

65

control calcium concentration; increased levels of calcium could be made physiologically and

osmotically inactive producing oxalate crystals.

The present data suggest an important role for AM fungi in increasing the plant tolerance to

metals, with relevant consequences for phytoremediation applications. However, they also

stress the importance of the right plant-fungus combination: VF poplars, more sensitive to

zinc pollution, most likely because of a larger metal accumulation, highly benefited of the

inoculation with G. mosseae, but not with G. intraradices. On the other hand, JP plants did

not show relevant effects of the mycorrhizal symbiosis.

In addition, it is noteworthy that several effects of AM colonization could be detected in the

leaves, a district of the plant that is not directly interested by the presence of the fungus. Such

effects concerned anatomy, cell wall organization, photosynthesis and sugar metabolism.

Finally, a model is proposed for the disruption of tissue organization observed in the palisade

parenchyma, where the presence of zinc, associated to the displacement of calcium (and hence

to the formation of calcium oxalate crystals) and to the variation in the concentration of free

and conjugated putrescine, could be considered responsible of the observed effects.

Fig. 4 – Section of VF poplar leaves, parallel to the surface, showing the palisade parenchyma

of control (left) and zinc treated plants. (right) Note the distance between the cell walls on the

right.

Fig. 5 (left) – Cross section of a leaf of VF poplar, zinc treated, stained with dithizone, in

order to show the localization of zinc.

66

Fig. 7 – TEM images of leaves of VF poplars, showing details of chloroplasts. From left to

right: control , zinc-trated, zinc + G. mosseae, and zinc + G. intraradices.

References Castiglione S, Franchin C, Fossati T, Lingua G, Torrigiani P, Biondi S, 2007. High zinc

concentrations reduce rooting capacity and alter metallothionein gene expression in white

poplar (Populus alba cv. Villafranca). Chemosphere 67: 1117-1126

Castiglione S, Todeschini V, Franchin C, Torrigiani P, Gastaldi D, Cicatelli A, Rinaudo C,

Berta G, Biondi S, Lingua G, submitted manuscript. Screening of a large poplar clone

collection for phytoremediation of heavy metal-contaminated soil: a field trial.

Franchin C, Fossati T, Pasquini E, Lingua G, Castiglione S, Torrigiani P, Biondi S, 2007.

High concentrations of Zn and Cu induce differential polyamine responses in

micropropagated poplar (Populus alba L. cv. Villafranca). Physiologia Plantarum 130: 77-90

Lingua G, Franchin C, Todeschini V, Castiglione S, Biondi S, Burlando B, Parravicini V,

Torrigiani P, Berta G, 2008. Arbuscular mycorrhizal fungi differentially affect the response to

high zinc concentrations of two registered poplar clones, Villafranca (Populus alba L.) and

Jean Pourtet (Populus nigra L.). Environmental Pollution 153: 137-147

Todeschini V, Franchin C, Castiglione S, Burlando B, Biondi S, Torrigiani P, Berta G, Lingua

G, 2007. Responses of two registered poplar clones to copper, after inoculation, or not, with

arbuscular mycorrhizal fungi. Caryologia 60: 146-155

Todeschini V, D‘Agostino G, Boccaleri E, Roccotiello E, Bonelli G, Berta G, Lingua G,

submitted manuscript. Leaf modifications induced by zinc accumulation in leaves of poplar

inoculated or not with arbuscular mycorrhizal fungi.

67

Challenges of AM applications in phytoremediation of post-industrial

wastes – case of Molinia caerulea

Ryszka P., Turnau K.

Institute of Environmental Sciences, Jagiellonian University, Gronostajowa 7, 30-387

Krakow, Poland

Abstract

Molinia caerulea was found in previous studies to be very useful in phytostabilisation of zinc

wastes. However, many factors might affect the success of this application. Wide range of

interactions between AMF-inoculated plants and other organisms should be considered as

introduced plants are hot spots of microbial diversity and activity. Here we present

preliminary results of investigations on M. caerulea vitality and interactions with Claviceps

purpurea, a fungus associated with graminoid plants.

Introduction Mining and the activity of different industrial branches result in the deposition of large

amounts of wastes. Remediation and restoration of areas occupied by these materials are

usually a difficult task, due to extreme chemical and physical properties. Phytostabilisation is

often used to minimize mobility of potentially toxic wastes and threats to environment and

human health. However, creation of a stable plant cover is difficult. Selection of proper plants

and substratum amendments is considered as a crucial step. Growth and survival of

introduced plants can be improved by their symbionts. Mycorrhizal fungi were shown to be

very important organisms as they supply plants with phosphorus, nitrogen and other elements,

protect them against pathogens and reduce environmental stresses such as drought or heavy

metal toxicity (Turnau et al. 2006).

Our studies (Ryszka and Turnau 2007) shown that grasses occurring spontaneously on zinc

wastes should be considered for phytostabilisation strategies . Among them, Molinia caerulea

is a very promising plant. This plant showed high survival rates when planted without any

amendments into bare zinc wastes and ability to stabilize the substratum. All investigated

plants showed extensive colonisation by AMF fungi (Ryszka and Turnau 2007). However,

application of this species on larger scale needs to be optimised and require further

investigation. Multiplication of plant material from seeds, although difficult, will not exploit

local populations of M. caerulea on zinc wastes. Inoculation with native fungal strains

ensures best performance under harsh conditions and will not increase heavy metal content

within plant tissues. Beside AMF, diazotrophs – free-living nitrogen fixing bacteria

(Reinhold-Hurek and Hurek 1998) were reported from M. caerulea root system (Hamelin et

al. 2002), these microbes can increase amount of nitrogen available to plants. As introduced

plantlets can be considered as hot spots of microbial diversity and activity on bare zinc

wastes, their interactions with wide range of microorganisms might influence the

phytostabilisation effectiveness. The aim of the present research was to characterize the

interaction between M. caerulea, AMF and a parasitic fungus Claviceps purpurea.

Material and Methods Field investigations were carried out on Trzebionka Mining Company zinc wastes (N

49°09'30", E19°25'10"). Detailed information on this site was given by Ryszka and Turnau

(2007). Molinia caerulea plants introduced during previous studies were investigated for the

presence of Claviceps purpurea ergots within inflorescences. Five classes of ergot frequency

68

were considered: 0 – no ergots; I – single ergot in the stalk; II – 2 or 3 ergots per stalk; III – 4

or 5 ergots; IV – more than 5 ergots per stalk. Intensity of leaf colour was also recorded.

Mycorrhizal colonization was estimated according to Trouvelot et al (1986)

(http://www2.dijon.inra.fr/mychintec/Mycocalc-prg/download.html). For this, roots were

collected, carefully washed and cleared in 10% KOH for 24 h at room temperature.

Subsequently, after careful washing in tap water, the roots were acidified for 1 h in 5% lactic

acid and stained for 24 h at room temperature in 0.05% aniline blue in lactic acid, in order to

visualize the fungal structures inside the roots. Material obtained this way was cut into 1 cm

pieces and mounted on slides in lactoglycerol. The following parameters were assessed in

collected root samples: frequency of mycorrhiza (F%), mycorrhizal intensity relative (M%)

and absolute (m%), arbuscule richness relative (A%) and absolute (a%).

Chlorophyll a fluorescence transients of intact leaves of M. caerulea were measured with a

Plant Efficiency Analyser (PEA) fluorimeter (Hansatech Instruments, GB). The transients,

induced by a red light of 600 W×m-2

, were recorded for 1 s, starting 50 µs after the onset of

illumination. The data was acquired every 10 µs for the first 2 ms and every 1 ms thereafter as

described by Strasser et al. (1995). Each transient was analysed according to the OJIP-test

(Strasser et al. 1995; Strasser et al. 2000). Performance index (PI) was calculated for the

characterization of PSII vitality and performance.

Results

Number of Claviceps purpurea ergots recorded in stalks of Molinia caerulea collected from

Trzebionka Mining Company zinc wastes varied from 0 to 10. Also, colour intensity of leaves

showed variation from pale green to dark green. The most intense leaf colour was noticed in

plants with higher (III–IV) number of ergots, whereas specimens with low number of ergots

were characterised by pale leaves (Table 1). Similar trend was observed in case of

photosystem II vitality. Plants with low number of ergots (0–I) showed lower performance

index than those with more infected stalks (Table 1), however no statistically significant

differences were found. A slight decrease in performance index was noticed in specimens

with medium number of ergots.

Table 1. Mycorrhizal colonization parameters (F%, M%, m%, A%, a% – see methods for

explanation) and performance index (PI) of Molinia caerulea non-infected or infected by

Claviceps purpurea and growing on zinc industrial wastes.

Frequency of

ergots per

stalk

(classes)

F% M% m% A% a% PI Colour of leaves

0 100.00 42.67

42.6

7 38.26

89.6

6 3.00 pale

I 96.39 37.85

36.1

0 28.90

69.8

4 2.51 yellow-green

II 77.16 15.60

22.4

7 11.56

76.9

8 3.50 light green

III 87.21 31.52

36.5

0 27.22

86.0

3 2.56 green

IV 90.09 34.04

35.1

0 26.39

76.0

1 4.87 dark green

69

Similar observations were done in case of mycorrhizal colonization parameters. Presence of

arbuscular mycorrhiza was noticed in all investigated samples of M. caerulea. Most values

showed decrease in plants with medium (II) number of ergots per stalk, however non-infected

specimens showed slightly higher values of all parameters than infected ones (Table 1).

Among plants with different ergot frequency no statistically significant differences were

found between parameters describing mycorrhizal colonization. However, plants with highest

performance index showed slightly lower values of mycorrhizal colonization (Table 1).

Discussion Analysis of chlorophyll a fluorescence is a useful method proven to show plant overall

performance and vitality (Strasser et al. 2000). Higher number of ergots in investigated

specimens of Molinia caerulea increased performance index and intensity of leaf colour, thus

suggesting a positive effect of presence of Claviceps purpurea within stalks. This fungus,

however, is usually regarded as a parasite, decreasing plant fitness by reduction of the number

of seeds. Seeds, however, are not so important in this particular case as this species multiplies

mostly by vegetative means while seeds need much more favourable conditions than that at

the industrial wastes. The influence of various endophytes of shoots on plant development is

broadly known. Up to our knowledge C. purpurea was never shown to stimulate plant

photosynthesis, especially at the end of vegetation period. The impact is especially strong

while the number of ergots was very high (that high frequency is mostly not observed under

the non-polluted condition in M. caerulea). On the contrary to such endophytes as

Neotyphodium/Epichloë, the mycelium of C. purpurea appears in the shoots only at the of

flower production and becomes the sink for energy while the formation of seeds starts.

Similar situation is in case of the leaf parasites that stimulate photosynthesis of the host in the

form of so called ―green islands‖. Under harsh environmental conditions such as drought, low

nutrient content, erosion and presence of heavy metals in soil any factor increasing plant

vitality should be regarded as positive for phytostabilization strategy. Frequency of C.

purpurea was not correlated with parameters of mycorrhizal colonization. Although, plants

with medium number of ergots showed lower values of these parameters than those with

either no or high number of ergots. Investigations of multiple plant-fungal interactions should

be carried out during optimisation of reclamation strategies for areas occupied by post-

industrial wastes.

Plants with highest performance index showed slightly lower values of mycorrhizal

colonization (Table 1). This may suggest lowering plant overall performance by native fungal

strains present in rhizosphere of investigated Molinia caerulea specimens. Evaluation of

native strains selected for further large-scale application of M. caerulea on zinc wastes can be

assisted by the use of chlorophyll a fluorescence.

References Hamelin J, Fromin N, Tarnawski S, Teyssier-Cuvelle S, Aragno M, 2002. nifH gene diversity

in the bacterial community associated with the rhizosphere of Molinia coerulea, an

oligonitrophilic perennial grass. Environ. Microbiol. 4: 477–481.

Reinhold-Hurek B, Hurek T, 1998. Life in grasses: diazotrophic endophytes. Trends

Microbiol. 6: 139–144.

Ryszka P, Turnau K, 2007. Arbuscular mycorrhiza of introduced and native grasses

colonizing zinc wastes: implications for restoration practices. Plant Soil 298: 219–229.

Strasser RJ, Srivastava A, Govindjee, 1995. Polyphasic chlorophyll a fluorescence transient in

plants and cyanobacteria. Photochem. Photobiol. 61: 32–42.

70

Strasser RJ, Srivastava A, Tsimilli-Michael M, 2000. The fluorescence transient as a tool to

characterise and screen photosynthetic samples. In: Yunus M, Pathre U, Mohanty P (eds),

Probing photosynthesis: mechanisms, regulation and adaptation. Taylor & Francis, London,

pp. 445–483.

Turnau K, Orlowska E, Ryszka P, Zubek S, Anielska T, Gawronski S, Jurkiewicz A, 2006.

Role of mycorrhizal fungi in phytoremediation and toxicity monitoring of heavy metal rich

industrial wastes in Southern Poland. In: Twardowska I, Allen HE, Häggblom MM (eds), Soil

and Water Pollution Monitoring, Protection and Remediation. Springer, Dordrecht, pp. 533–

551.

71

Extreme abiotic environmental factors are determining arbuscular

mycorrhizal fungal community structure at natural CO2 springs

Maček I. 1*

, Dumbrell A.J. 2, Helgason T.

2, Nelson M.

2, Fitter A.H.

2, Vodnik D.

1

1

University of Ljubljana, Biotechnical Faculty, Department of Agronomy, Jamnikarjeva 101,

SI-1001 Ljubljana, Slovenia. *

Corresponding author: irena.macek@bf.uni-lj.si. 2 Department of Biology, PO Box 373, University of York, York. YO10 5YW. UK

Abstract Hypoxia (reduced O2 availability) is a common soil characteristic, usually present in

waterlogged or flooded soil. Natural CO2 springs (mofettes) form specific environments,

where long-term geological gas emissions are present, resulting in a range of different CO2

concentrations in the atmosphere and soil [1]. The Stavešinci mofette (NE Slovenia) has been

used before as a research site where several studies on mofette gas characteristics [1, 8], plant

ecophysiology [2, 3, 4] and soil microbial community [5] have been carried out in the last

decade. However, sites of postvolcanic activities have rarely been used for studies on

arbuscular mycorrhiza [6, 7]. Extremely high soil CO2 concentration can result in soil

hypoxia, where severe and relatively constant selection pressure over a small spatial scale is

present, altering O2 availability. In order to examine locations with extreme geogenic CO2

enrichment and corresponding O2 concentrations, an area within the Stavešinci mofette has

been chosen with a final number of 55 measuring sites. Strong negative linear relationship

between soil CO2 and soil O2 concentrations was found (correlation coefficient -0.96, p =

0.0000), with maximum CO2 concentration within the selected area measured 78.2 % and

corresponding O2 concentration 6.5 % [8]. In addition to soil CO2 concentrations soil CO2

flux was measured and relatively high correlation between soil CO2 concentrations and fluxes

was found, with correlation coefficients between 0.76 and 0.88 (p = 0.0001) [8].

In order to examine arbuscular mycorrhizal (AM) root colonization, we sampled individual

plants of seven plant species (Dactylis glomerata, Poa pratensis, Setaria pumila, Echinochloa

crus-galli, Juncus effusus, Plantago lanceolata and Solidago gigantea) in approximately

100 x 100 m grassland area within the Stavešinci site, with sampling locations exposed to

control (flux below 16.8 ± 4.6 μmol CO2 m-2

s-1

) and high (flux above 151.9 ± 22.9 CO2 μmol

m-2

s-1

) soil CO2 environment during growth, and thus normoxic and potentially hypoxic

conditions in soil. Four of the sampled species (D. glomerata, P. pratensis, S. pumila and P.

lanceolata) were, in addition to the control sites, mycorrhizal also when exposed to extremely

high soil CO2 concentrations, E. crus-galli was not colonized at high CO2 exposure and two

species (J. effusus and S. gigantea) were not colonized, irrespective of the sampling site.

In addition to AM root colonization, molecular characterization of the AM community has

been performed. Data on AM fungal community structure from the four colonized species

showed a big shift in AM fungal community composition, with high ß-diversity (sequence

type turn-over) between sites. This resulted in the majority of clones screened from high CO2

(or low O2) locations exclusive to high CO2 soils. These data suggest that in the hypoxic

environment, specialists dominate the AM-fungal communities and that abiotic environmental

factors are as important as plant host in determining AM community structure and diversity.

References

Vodnik D, Kastelec D, Pfanz H, Maček I, Turk B, 2006. Small-scale spatial variation in soil

CO2 concentration in a natural carbon dioxide spring and some related plant responses.

Geoderma, 133: 309-319.

72

Vodnik D, Pfanz H, Maček I, Kastelec D, Lojen S, Batič F, 2002. Photosynthesis of cockspur

[Echinochloa crus-galli (L.) Beauv.] at sites of naturally elevated CO2 concentration.

Photosyntetica 40 (4): 575-579.

Pfanz H, Vodnik D, Wittmann C, Aschan G, Raschi A, 2004. Plants and geothermal CO2

exhalations - survival in and adaptation to a high CO2 environment. Progress in Botany 65:

499-538.

Maček I, Pfanz H, Francetič V, Batič F, Vodnik D, 2005. Root respiration response to high

CO2 concentrations in plants from natural CO2 springs. Environmental and Experimental

Botany 54: 90-99.

Videmšek U, Hagn A, Suhadolc M, Radl V, Knicker H, Schloter M, Vodnik D. Abundance

and diversity of CO2 fixing bacteria in grassland soils close to natural carbon dioxide springs.

Mycrobial Ecology, accepted.

Rillig MC, Hernandez GY, Newton PCD, 2000. Arbuscular mycorrhizae respond to elevated

atmospheric CO2 after long-term exposure: evidence from a CO2 spring in New Zealand

supports the resource balance model. Ecology Letters 3 (6): 475-478.

Appoloni S, Lekberg Y, Tercek MT, Zabinski CA, Redecker D, 2008. Molecular community

analysis of arbuscular mycorrhizal fungi in roots of geothermal soils in Yellowstone National

Park (USA). Microbial Ecology, DOI 10.1007/s00248-008-9384-9.

Vodnik D, Videmšek U, Pintar M, Maček I, Pfanz H. The characteristics of soil CO2 fluxes at

a site with natural CO2 enrichment, Geoderma, submitted.

73

Biological management of foliar diseases in pot roses

Sabine Ravnskov and John Larsen,

University of Aarhus, Institute of Integrated Pest Management, Research Centre Flakkebjerg,

DK-4200 Slagelse, Denmark. Sabine.Ravnskov@agrsci.dk; John.Larsen@agrsci.dk

Abstract.

In an overall plant protection strategy aiming at a reduced input of agrochemicals in plant

production, it is important to study possible biological alternatives for replacement. In the

production of pot roses, the foliar plant pathogens Botrytis cinerea (grey mould) and

Sphaerotheca pannonsa (powdery mildew) are causing serious damage, and significant

amounts of fungicides are used to control them. In a series of experiments the antagonistic

effect of different microorganisms and of a reduced input of phosphorus (P) on the

development of these diseases in pot roses was studied.

The results showed that the arbuscular mycorrhizal (AM) fungus Glomus intradices did not

influence on the disease incidence of powdery mildew under either high or low P conditions.

However, irrespective of the P level input, but added to the roots in combination with the

fungi Pseudozyma flocculosa or Ampelomyces quisqualis added to the leaves the AM fungus

significantly reduced the disease incidence of powdery mildew on pot roses. Similarly, the

inoculation of roots with the AM fungus G. mosseae did not influence incidence of grey

mould in pot roses, but inoculated in combination with the fungus Ulocladium atrum on

leaves the disease incidence was significantly lowered. However, in this case only under low

P conditions. In this experiment, it was also shown that the AM fungus could influence the

foliar microbial community by decreasing the biomass of Gram negative bacteria, which was

in contrast to the effect of high P concentrations in the nutrient solution, which markedly

stimulated this group of foliar bacteria.

In conclusion, the results showed that AM fungi could interact with microbiological control

agents inoculated on the leaves in relation to disease control of foliar pathogens in pot roses.

Furthermore, it was shown that AM fungi could influence the foliar microbial community of

pot roses but that the effect depended on the P level in the soil..

74

Application of Mycorrhiza to Ornamental Horticulture Crops- present and

future prospects

Hinanit Koltai, Doron Meir, Nathalie Resnick and Eitan Shlomo

Dept. of Ornamental Horticulture, ARO, the Volcani Center, Bet Dagan, 50250 Israel* Email

address: hkoltai@volcani.agri.gov.il

Smadar Wininger and Bruria Ben-Dor

Dept. of Agronomy and Natural Resources, ARO, the Volcani Center, Bet Dagan, 50250

Israel

Abstract

The application of Arbuscular Mycorrhiza Fungi (AMF) inoculum to the soil, under sub-

optimal growth conditions, has been proven to be effective in enhancement of plant growth,

and in increasing plant resistance against abiotic and biotic stress conditions. These features

of AMF were demonstrated in Israel especially for commercially grown pepper and chive.

However, for economical and practical reasons, AMF inoculum application is awaiting to be

demonstrated for many crops. Under conditions of intensive agriculture, which takes place in

Israel in semi-arid regions, under greenhouse conditions, soil disinfection methods are applied

routinely. As a result, a reduction in the native AMF population is observed. Therefore, it has

become important to introduce convenient and inexpensive method(s) for applying AMF

inoculum, which will enable the direct application of AMF inoculum in the greenhouse,

before or at the time of planting. Ornamental crops are high-cash crops, and are being grown

as part of an intensive agriculture in Israel especially in semi-arid regions, under greenhouse

conditions. Here, with the aim of expanding AMF usage for a variety of ornamental crops, we

have examined avenues for AMF application. This was conducted by examining the effect of

several inoculums types and methods of application on growth and yield of a variety of

ornamental crops. Our studies demonstrated that AMF may enhance crop growth and yield,

and prevent leaf senescence, for some of the examined species. Hence, AMF is suggested to

be useful for application under greenhouse conditions in semi arid regions, for a variety of

ornamental crops. However, some considerations should be taken once further expansion of

the application of AMF is sought for ornamental horticulture; these will be indicated in the

present talk.

75

Application of AM fungi in red fruit production

Armelle Gollotte

1, Louis Mercy

1, Benjamin Secco

1, Julie Laurent

1, Michel Prost

2, Silvio

Gianinazzi3, and Marie-Claude Lemoine

1

1. CRITT AgroEnvironnement, 17 rue Sully, 21065 Dijon Cedex, France,

gollotte@dijon.inra.fr

2. LARA Spiral, 3 rue des Mardors, 21560 Couternon, France

3. UMR Plante-Microbe-Environnement, INRA/CMSE, 17 rue Sully, 21065 Dijon Cedex,

France

Abstract.

Red fruits are becoming more and more popular due to the public awareness of their high

antioxidant molecule (AOM) content. AOM decrease risks of human diseases such as cancer,

cardiovascular and neurodegenerative diseases as well as diabetes. Producers therefore need

to increase the production of red fruits of high quality. This requires growing more plants and

optimising cultural practices for increased AOM content. In particular, the use of mineral

fertilisers and pesticides should be reduced as they can have negative effects on AOM

production whilst fungicides are not always efficient in preventing plant diseases.

Biotisation with AM fungi is one way to improve plant nutrition and to increase plant

tolerance to biotic and abiotic stresses. It is also thought to favour AOM synthesis. Our aim is

therefore to optimise biotisation of raspberry, blackcurrant and strawberry in order to gain a

long-term positive effect on plant quality in terms of growth, health and AOM content.

Inoculation with commercial inocula of AM fungi was tested at different steps of plant

culture: acclimatisation, post acclimatisation or plantation in the field. The best results were

obtained when plants were inoculated during the acclimatisation phase. Root colonisation by

AM fungi and effect on plant growth were monitored at different steps of plant culture in

order to select inocula of good quality and compatible growing media and organic fertilisers.

The plant sanitary status was assessed by PCR by checking the absence of phytopathogenic

fungi and the presence of AM fungi.

Finally, effect of varieties, biotisation and organic fertilisers on AOM production in leaves

and fruits is being characterised in order to identify agricultural practices which will most

favour fruit quality.

76

Reforestation in Cyprus – in vivo and in situ Assessment of Mycorrhization

Impact on Plants’ Vitality with the JIP-test

Tsimilli-Michael M.2, Jarraud N.

2, Strasser R.J.

1

1Bioenergetics Laboratory, University of Geneva, CH-1254 Jussy-Geneva, Switzerland;

Merope.Tsimilli-Michael@bioveg.unige.ch and Reto.Strasser@bioveg.unige.ch 2UNDP Action for Cooperation and Trust, P.O.Box 21642, Nicosia 1590, Cyprus;

Nicolas.jarraud@undp.org

Abstract

We present an in vivo and in situ analysis of plants‘ vitality, applying the JIP-test on needles

from pine trees planted, in the frame of a 3-years reforestation project in Cyprus, in

degraded/polluted soil with and without inoculation with ecto-mycorrhiza. By this test, which

translates fluorescence transients to structural and functional parameters of the photosynthetic

machinery, the impact of mycorrhization was revealed, evaluated and compared among cases.

Introduction

Mycorrhizae (ecto- and endo-) are mutualistic microsymbionts of about 90% of higher plants

in natural, semi-natural and agricultural plant communities, with a well-documented

beneficial role, especially at suboptimal for the plant soil conditions or during stress periods.

However, the success of any inoculation in practise has to be tested, since the effectiveness of

symbiosis depends on complex interactions between plant, symbionts and environmental

conditions.

Two big projects applying mycorrhization are running in Cyprus (2005-2008), under the

auspices of the United Nations Development Programme‘s Initiative, Action for Cooperation

and Trust in Cyprus (UNDP-ACT): reforestation (Innovative Biological Approaches for the

Reforestation of Environmentally Stressed Sites – IBARESS II) and organic vegetable

farming. The reforestation project was implemented with 1608 inoculated plants in total at

three sites (two watering regimes) in the Greek-Cypriot Community (south) and with 6755

inoculated and 7369 non-inoculated plants in total at four sites (no watering) in the Turkish-

Cypriot Community (north).

Mycorrhizal activity has multiple effects on the physiology and vitality of the host plant. For

the detection and evaluation of mycorrhization impact we screened, in vivo and in situ,

photosynthetic performance of hundreds of samples from several cases. We applied the JIP-

test (Tsimilli-Michael et al. 2000, Strasser et al. 2004), which quantifies plants‘ vitality

(activity – adaptability – stability) by analysing chlorophyll (Chl) a fluorescence transients

(OJIP) emitted by photosynthetic tissues and translating them to biophysical parameters and

behaviour patterns.

Materials and Methods

The presented study was done on Pinus halepensis and Pinus brutia trees, planted in two

gypsum quarries in north Cyprus: Chatos – Serdarli (G1) and Ayios Iakovos – Altinova (G2),

non-inoculated or inoculated with commercial ecto-myccorrhizae (AEGIS Ecto Gel - SYTEN

Company; Rhizopogon sp., Pisolithus sp. and Schleroderma sp. mixture) by dipping root balls

in the gel. Mycorrhization was tested and verified by visual examination of the roots.

Fluorescence transients of bushels of 10 needles, dark-adapted for 4-5 h, were measured with

a HandyPEA Analyser (Hansatech Instruments, Kings Lynn Norfolk GB) (as in Strasser et al.

77

2004). The JIP-test was applied on the representative for each case transient (average of about

100 replicates).

Results and Discussion

Chlorophyll (Chl) a fluorescence transients were processed as in Fig. 1. Inoculated and non-

inoculated plants showed no difference in F0, hence Ft/F0 is plotted in Fig. 1A, but they differ

in respect to FP (=FM). More differences are revealed by normalisations/subtractions (Fig. 1

B-D): For both species (and all other studied cases) the appearance of a negative K-band (Fig.

1B) and a negative L-band (Fig. 1C) show respectively that, compared to the non-inoculated,

the inoculated plants have a higher activity at the oxygen evolving side and a higher extent of

energetic connectivity among photosystem (PS) II units; it is worth pointing out that

connectivity increases the utilization of excitation energy and is also a factor of stability of the

photosynthetic system (Strasser et al. 2004, Tsimilli-Michael and Strasser 2008). The benefit

is shown to be much bigger in P. brutia than in P. halepensis. Figure 1D processes the I-P

phase. The insert shows that, compared to the non-inoculated, in the inoculated plants the PSI

end electron acceptor pool is bigger, while the main plot, which depicts the kinetics of

reducing the differing pools, demonstrates that inoculation results in higher rate constants; the

benefit is much bigger in P. brutia, which appears, compared to P. halepensis, to ―suffer‖

more when non-inoculated.

Figure 1. (A) The Chl a fluorescence kinetics of needles from inoculated and non-inoculated pines (control),

grown in gypsum quarry G1 and measured in July 2008, are presented as Ft/F0 on logarithmic time scale; the

characteristic steps O, J, I and P (P: maximal; all RCs closed) are marked, as well as the complementary area

(Area; indicatively for one case). The transients are further presented as kinetics of different expressions of

relative variable fluorescence, derived by the following normalisations: (B) between O and J: W t = (Ft F0)/(FJ

F0); (C) between O and 300s (K): (WK)t = (Ft F0)/(F300s F0); (D) between I and P (FP = FM): (WI-P)t =(Ft

FI)/(FM FI) and, in the insert, between O and I: (WI)t = (Ft F0)/(FI F0). In the plots (B) and (C), the

difference kinetics (inoculated minus respective control) are also depicted (right vertical axis), revealing clearly

the K-band and L-band respectively

The behaviour patterns of the photosynthetic machinery depicted in Fig. 2 permit evaluation

of the myccorhization impact on pine trees and comparison among cases. Each pattern is built

by 14 structural and functional parameters, derived from the OJIP fluorescence transients by

the JIP-test equations (Tsimilli-Michael and Strasser 2008), summarised in Fig. 3 along with

the parameters‘ definitions. All plots reveal that inoculation increases all photosynthetic

0.0

1.0

2.0

3.0

4.0

0.01 0.1 1 10 100 1000Time (ms)

F t F

0

(A) P-step (FP =FM)

I-step

J-step

O-step

0.0

0.2

0.4

0.6

0.8

1.0

0 0.5 1 1.5 2

-0.16

-0.12

-0.08

-0.04

0.00

Wt

= (F

t F

0)/(F

J

F 0)

Wt =

[(

F t

F0)

/(FJ

F 0)]

(B)

Time (ms)

K-band

W t

0.0

0.2

0.4

0.6

0.8

1.0

0 0.1 0.2 0.3

-0.12

-0.08

-0.04

0.00

Time (ms)

(WK) t

= (F

t F

0)/(F

300

s F

0)

(W

K) t[

(Ft

F0)

/(F30

0s

F0)

] (C) L-band

WK) t 1.0

1.1

1.2

30 80 130 180 230 280

0.0

0.2

0.4

0.6

0.8

1.0

30 80 130 180 230 280

Time (ms)

(WI-

P)t =

(Ft

FI)/

(FM

F

I)

(WI) t

= (F

t F

0)/(F

I F

0)

(D)

Time (ms)

Area

P.brutia P. halepensis control Myc

78

efficiencies. The most sensitive parameter, hence the most suitable as a probe, is PItotal

(performance index), which combines (Fig. 3) all conservation efficiencies and RC/ABS (RC:

PSII reaction centre). Note also that an increase of RC/ABS (decrease of Chl a molecules per

RC), as in plots C-D, is a photoprotective regulation. We also observe that: long needles have

higher performance than short (plot A); mycorrhization has the same fractional impact on

short and long needles (plot B); the benefit in P. brutia is much bigger than in P. halepensis

(as also shown by Fig. 1) and very similar at the two sites for P. halepensis (plot C); the

benefit in P. halepensis is bigger in July 2008 than in May 2007 (plot D), most probably

because there was no rainfall in May-July 2008.

Figure 2. Behaviour patterns of the photosynthetic machinery evaluating the impact of myccorhization on pine

trees and comparing it among several cases. The results refer to plants from two species, P. halepensis (ha) and

P. brutia (br), grown on degraded soil (gypsum quarries G1 and G2), with (M) or without inoculation (C;

respective control); the measurements were conducted in May 2007 and July 2008, as indicated. Plots (A) and

(B) compare also small (S) and long (L) needles; in plot (A), CS is taken as the control (C). Each pattern consists

of 14 structural and functional parameters, derived by the JIP-test from the OJIP transients (as shown in Fig. 3).

Each parameter was normalised on that of the control; hence, for each case, the deviation of the behaviour

pattern of the inoculated from that of the non-inoculated (regular polygon – control) demonstrates the fractional

impact of mycorrhization.

0.1

1

10

RE0/RC

ET0/RC

TR0/ABS

ET0/ABS

TR0/RC

EC/RC PIABS PItotal

RC/ABS

RE0/ABS

ET0/TR0

RE0/TR0 ABS/RC RE0/ET0

RE0/RC

ET0/RC

TR0/ABS

ET0/ABS

TR0/RC

EC/RC PIABS PItotal

RC/ABS

RE0/ABS

ET0/TR0

RE0/TR0 ABS/RC RE0/ET0

0

1

2

3

C (CS) MS CL ML C (respective control) ML MS

P. halepensis

Site G1

May 2007

(A)

(B)

0

1

2

3

RE0/RC

ET0/RC

TR0/ABS

ET0/ABS

TR0/RC

EC/RC PIABS PItotal

RC/ABS

RE0/ABS

ET0/TR0

RE0/TR0 ABS/RC RE0/ET0

RE0/RC

ET0/RC

TR0/ABS

ET0/ABS

TR0/RC

EC/RC PIABS PItotal

RC/ABS

RE0/ABS

ET0/TR0

RE0/TR0 ABS/RC RE0/ET0

0.1

1

10

Comparison of pine species,

year-time and polluted sites.

Long needles only.

C (respective control)

M-G1-ha

M-G2-ha

M-G1-br

C (respective control) M-May 2007 M-July 2008

2007

(C)

(D)

P. halepensis

Site G1 July 2008

79

Figure 3. A schematic summary of the JIP-test: Energy fluxes (wide arrows) and their bifurcations to fluxes for

energy conservation (grey) and dissipation (white) are demonstrated; the fluxes refer to PSII absorption (ABS),

trapping (TR), i.e. reduction of Pheo and QA, electron transport (ET) from QA to the intersystem pool and

reduction of PSI end acceptors (RE). The efficiencies/yields, defined as ratios of fluxes, are shown (line arrows)

and linked, when referring to the onset of illumination (all RCs open; subscript ―0‖), with signals from the OJIP

fluorescence transient (see Fig. 1). The derivation of the performance indexes (PIABS and PItotal), the specific

fluxes (fluxes per RC), the relative variable fluorescence Vt, and the total electron carriers per reaction centre

(EC/RC; equal to the normalized area, Sm) are also presented. [*Paillotin G (1976) J Theor Biol 58:237-252].

The results from the vitality analysis are in accordance with macroscopic findings. Compared

to non-inoculated, mortality in inoculated plants was 2-3 times smaller, the long/short needles

ratio much bigger (hence, photosynthetic performance on plant basis is even higher than what

Fig. 2 depicts) and the needles are more green and fresh; however, plants‘ height was not

promoted.

Acknowledgements: The Cyprus projects are under the auspices of the United Nations Development

Programme‘s Initiative, Action for Cooperation and Trust in Cyprus (UNDP-ACT), supported by the American

People through a grant from USAID. RJS (the scientific coordinator) acknowledges a grant from UNDP-ACT;

M.T-M and R.J.S acknowledge support by the Swiss National Science Foundation, Project Nr: 200021-116765.

Acknowledgments are also due to the Foundation for the Rehabilitation of Quarries (TASOVA) and the Cyprus

Association of Professional Foresters, as well as all the forestry experts in Cyprus who worked for the realisation

of the projects.

References

Tsimilli-Michael M, Eggenberg P, Biro B, Köves-Pechy K, Vörös I, Strasser RJ, 2000.

Synergistic and antagonistic effects of arbuscular mycorrhizal fungi and Azospirillum and

Rhizobium nitrogen-fixers on the photosynthetic activity of alfalfa, probed by the chlorophyll

a polyphasic fluorescence transient O-J-I-P. Appl Soil Ecol 15: 169-182.

Strasser RJ, Tsimilli-Michael M, Srivastava A, 2004. Analysis of the chlorophyll a

fluorescence transient. In: Papageorgiou GC, Govindjee, eds. Chlorophyll a fluorescence: a

signature of photosynthesis. Advances in Photosynthesis and Respiration Series (Govindjee –

Series Editor) vol 19. Kluwer Academic Publishers, Rotterdam, 321-362.

Tsimilli-Michael M, Strasser RJ, 2008. In Vivo assessment of plants‘ Vitality: applications in

detecting and evaluating the impact of Mycorrhization on host plants. In: Varma A, ed.

Mycorrhiza, 3rd edition, Springer, pp 679-703.

Ro Po . Eo . Ro ABS

RE0

M

I

F

F1 Pi Po )V1( I

Pt M

t

F

F

ABS

TR t M

t

F

F1 Pt Po )V1( t

quantum yield of primary photochemistry at time t,

for any state (Paillotin 1976*)

E

R R

P

E

R

Po Po ABS

TR0 M

0

F

F1 Po Po )V1( 0

Eo Po . Eo ABS

ET0 M

J

F

F1 Pj Po )V1( J

0

0

ET

RE

)FF(

)FF(

JM

IM

)V1(

)V1(

J

I

0

0

TR

ET

)FF(

)FF(

0M

JM

)V1( J

Trapping (primary

photochemistry): reducing

Pheo and QA

Electron Transport: reducing

electron acceptors of the

intersystem pool

Reduction of End acceptors

(at PSI electron

acceptor side)

ET TR-ET

RE ET-RE

TR ABS -TR

QB PQ Cyt PC

Fd NADP

Chl*

Pheo QA

ABS

The total electron carriers

(EC) per RC ratio is defined as:

0M

m FF

AreaS

RC

EC

The specific fluxes, i.e. fluxes per reaction centre (RC), are derived from the

quantum yields (fluxes per ABS) using ABS/RC, experimentally determined as:

0J

s50s300

J

00

0

0

FF

)FF(4

V

t/)V(

RC

TR

ABS/TR

RC/TR

RC

ABS

where

The relative variable

fluorescence Vt is defined as:

0M

0tt FF

FF V

Ro

RoABStotal 1

IP PI

REET

REIP

00

0ABS

ETTR

ET

TRABS

TR

ABS

RC

11

ABS

RC PI

00

0

0

0

Eo

Eo

Po

PoABS

80

Effect of mycorrhiza on the photosynthetic performance of Medicago sativa

L. cultivated on control and heavy metal rich substratum, studied in vivo

with the JIP-test

Tsimilli-Michael M.

1, Turnau K.

2, Ostachowicz B.

3, Strasser R.J.

1

1Bioenergetics Laboratory, University of Geneva, CH-1254 Jussy-Geneva, Switzerland,

Merope.Tsimilli-Michael@bioveg.unige.ch and Reto.Strasser@bioveg.unige.ch 2Institute of Environmental Sciences of the Jagiellonian University, ul Gronostajowa 7, 30-

387 Kraków, Poland, katarzyna.turnau@uj.edu.pl

3AGH University of Science and Technology, Faculty of Physics and Applied Computer

Science, Department of Nuclear Methods. Mickiewicza 30 30-059 Krakow, Poland

Abstract

Medicago sativa var. Maya was cultivated under laboratory conditions on zinc waste and non-

polluted substrata. Plants were either non-inoculated or inoculated with arbuscular

mycorrhizal fungi originating from industrial wastes or from control area. We applied the JIP-

test, an analysis of chlorophyll a fluorescence transients, to evaluate in vivo the photosynthetic

performance in intact leaves of M. sativa, grown in both substrata with and without

inoculation with G. mosseae or G. clarum. Their presence caused the increase of the heavy

metal uptake into the shoots and roots. Still the plants survived losing not more than 45% of

their overall potential (PItotal) for photosynthesis. This loss explains the inability of M. sativa

to survive on industrial wastes where the conditions are more extreme. Further work will be

done to select more appropriate symbionts using the JIP-test as a sensitive monitoring tool.

Introduction

Phytostabilization of industrial wastes rich in heavy metals involves mostly the use of grasses.

The substratum, however, is very poor in nitrogen and the use of plants that form symbiosis,

not only with mycorrhiza but also with nitrogen fixing bacteria, would be of great importance.

Experiments confirmed the potential role of arbuscular mycorrhizal fungi (AMF,

Glomeromycota) in the plant establishment on Zn-Pb rich wastes in southern Poland (Turnau

et al. 2008). The aim of the present work was to check if Medicago sativa, a plant forming

rhizobial nodules, can be supported by AMF introduction when grown on Zn-Pb rich wastes.

We compared the photosynthetic activity of mycorrhized and non-mycorrhized plants grown

in polluted and non-polluted substrata, by means of the JIP-test. This test, which analyses the

OJIP chlorophyll (Chl) a fluorescence transients, is a powerful tool for stress detection and

evaluation (Strasser et al 2004), suitable also for screening the beneficial role of symbiosis

(Tsimilli-Michael et al 2000, Strasser et al 2007, Tsimilli-Michael and Strasser 2008).

Materials and Methods

Medicago sativa var. Maya was cultivated for 5 month under laboratory conditions on zinc

waste and control (mixture of sand, garden soil and clay) substrata, both of pH ca. 7 measured

in H2O. The plants were either non-inoculated or inoculated with Glomus mosseae BEG 12

originating from non-polluted places or G. clarum UNIJAG.Pl.13 that was isolated from zinc

waste in Trzebionka. The efficiency of inoculation was checked using standard techniques.

Soil from the zinc waste contained high concentrations of heavy metals: 468 μg/g of total

cadmium, 7068 μg/g of total lead, and 53303 μg/g of total zinc (Turnau et al. 2008).

81

The analysis of metal content in plant material was done using Total Reflection X-ray

fluorescence (TXRF) (Hołyńska et al. 1998).

Chlorophyll (Chl) a fluorescence measurements were conducted with a HandyPEA Analyser

(Hansatech Instruments, Kings Lynn Norfolk GB) (for details see Strasser et al. 2004) on

dark-adapted leaves of M. sativa plants cultivated under the six conditions. For each case, the

average OJIP fluorescence transient (10-15 replicates), as representative of the case, was

translated according to the JIP-test to biophysical parameters of the photosynthetic machinery.

The JIP-test parameters used here refer to the condition of the sample at the onset of

fluorescence induction (all photosystem (PS) II reaction centers open; subscript ―0‖) and are

defined in terms of the following energy fluxes: absorption, ABS; trapping (reduction of QA,

the primary electron quinone acceptor of PSII), TR0; electron transport from QA to the

plastoquinone pool (PQ), ET0; reduction of PSI end electron acceptors, RE0. The parameters

were: (A) Specific energy fluxes, i.e. energy fluxes per PSII active reaction centre, RC. (B)

Quantum yields of primary photochemistry, TR0/ABS Po; of electron transport, ET0/ABS

Eo; of reduction of PSI end electron acceptors, RE0/ABS Ro. (C) Efficiencies/probabilities

with which a trapped exciton can move an electron from QA to PQ, ET0/TR0 Eo; of

electron transport from QA to the PSI end electron acceptors, RE0/TR0 Ro; of electron

transport from the reduced PQ to the PSI end electron acceptors, RE0/ET0 Ro. (D) Amount

of active reaction centers per absorption (arbitrary units), RC/ABS – proportional to the

probability that a Chl a molecule is functioning as RC. (E) The performance indexes PIABS

and PItotal, which are products of terms expressing ―potentials‖ for photosynthetic

performance (partial performances) at the sequential energy bifurcations from exciton to PQ

reduction and to the reduction of PSI end acceptors respectively, PIABS =

[RC/ABS].[TR0/(ABS-TR0][ET0/(TR0-ET0)] and PItotal = PIABS.[RE0/(ET0-RE0)]. The reader

can consult Tsimilli-Michael et al (these Proceedings) for the formulae and Strasser et al

(2004) for their analytical derivation.

Results and Discussion In order to compare the impact of pollution and mycorrhization on the photosynthetic

Figure 1. Efficiencies/probabilities of energy conservation in the photosynthetic machinery of plants grown

under different conditions, presented by the fractional difference of each parameter from the corresponding

value in non-mycorrhized plants grown in non-polluted soil (Non-pol/NM; control); the performance indexes

are also depicted. The conditions were: non-polluted soil – plants mycorrhized with

G. mosseae (Non-pol/Gm) or G. clarum (Non-pol/Gc); polluted soil – non-mycorrhized plants (Pol/NM)

or mycorrhized with G. mosseae (Pol/Gm) or G. clarum (Pol/Gc).

-50%

-40%

-30%

-20%

-10%

0%

Non-pol/Gc Non-pol/Gm Pol/NM Pol/Gm Pol/Gc

F

ract

ion

al

dif

feren

ce

from

Non

-pol/

NM

PItotal

PIABS

RE0/ABS

RE0/TR0

RE0/ET0

ET0/ABS

ET0/TR0

TR0/ABS

RC/ABS

82

performance of M. sativa, we present in Figs. 1 and 2 the calculated JIP-test parameters in

plants grown under the different conditions studied, relatively to non-inoculated plants grown

in non-polluted soil (Non-pol/NM; control). For each parameter, the fractional difference of

its value from the control value is depicted. Figure 1 presents the efficiencies/probabilities and

the performance indexes, while Fig. 2 the specific energy fluxes. Among all parameters, the

performance index, which combines all energy conservation efficiencies and RC/ABS, is

shown to be the most sensitive (Fig. 1), hence the most suitable as a probe. On the other hand,

TR0/ABS (= FV/FM) is almost insensitive, exhibiting a rather homeostatic behavior.

We observe that, in non-polluted soil, mycorrhization has a negative impact on all

efficiencies/probabilities (Fig. 1). Findings that mycorrhization can cause such stress effects

have been also reported and commented earlier (Tsimilli-Michael and Strasser 2002). We also

see that the impacts of G. mosseae and G. clarum are very similar and, concerning PItotal,

almost identical. The parameters related with the reduction of PSI end electron acceptors (RE-

parameters) were much more affected than the non-RE-related parameters, which are rather

insensitive. Figure 2 shows minor increases of ABS/RC; therefore, the depicted impacts on

the specific fluxes (flux/RC) follow the impacts on the respective quantum yields (flux/ABS).

The impact of pollution in non-mycorrhized plants (Pol/NM) is more extended than that of

mycorrhization in non-polluted soil (Non-pol/Gm or Non-pol/Gc), especially on non-RE-

related parameters (Fig. 1); the impact on the absorption specific flux (ABS/RC) is also much

bigger (Fig. 2). In general, ABS/RC increase may mean either inactivation of a fraction of

RCs, in which case TR0/RC is not affected, or an antenna size increase (in respect to Chl a

molecules per RC), in which case TR0/RC also increases. Therefore, the case of Pol/NM,

where the impact on TR0/RC is close to that on ABS/RC (Fig. 2), points towards the second

explanation.

In polluted soil, mycorrhization with G. mosseae (Pol/Gm) plays a beneficial role on all

efficiencies/probabilities, which, however, is not enough to compensate the pollution effect;

on the other hand, mycorrhization with G. clarum (Pol/Gc) results in a further decrease of all

RE-related parameters, bringing however a restricted improvement of the non-RE-related

(Fig. 1). Mycorrhization keeps ABS/RC higher than in the control, but lower than in the non-

mycorrhized plants (Fig. 2); the same trend is followed by TR0/RC, indicating, as above

-25%

-20%

-15%

-10%

-5%

0%

5%

10%

15%

Non-pol/Gc Non-pol/Gm Pol/NM Pol/Gm Pol/Gc

ABS/RC

TR0/RC

ET0/RC

RE0/RC

Fracti

on

al

dif

fere

nce

from

Non

-pol/

NM

Figure 2. The fractional differences of the specific energy fluxes, for the cases

presented in Fig. 1. See legend of Fig. 1 for details.

83

explained, an effect on the antenna size. Each of the other two specific fluxes shows the

combined impact on the respective quantum yield and on ABS/RC. In conclusion, our

results revealed not only the overall impact of pollution and/or mycorrhization on the

photosynthetic performance of the plants under investigation, but also the differential impacts

on parameters quantifying sequential parts of the photosynthetic process.

Though the efficiency of G. mosseae in alleviating pollution stress appears to be limited, and

G. clarum appears to have even an additional negative impact, it should be taken in

consideration that, in polluted soil, mycorrhized plants were found to accumulate, in general,

bigger amounts of heavy metals, especially in the shoots; e.g., in the Pol/Gc case, the ratios

of Pb, As and Cr concentrations to those in the Pol/NM case were 2.29, 2.26 and 1.34

respectively, while for the Pol/Gm case they were 1.12, 1.13 and 0.70 respectively).

Therefore, the beneficial role of mycorrhization is bigger that what recognized in Figs. 1 and

2. Moreover, our results show that, in polluted soil, the mycorrhized plants, though

accumulating high metal quantities, can still survive, losing not more than 35% (Pol/Gm) or

45% (Gc) of their overall photosynthetic potential (PItotal). On the other hand, the low

potential explains the inability of M. sativa to survive, even when mycorrhized, on industrial

wastes where the conditions are more extreme. Further work will be done to select, for

different plant cultivars, more appropriate symbionts, AMF and nitrogen fixing bacteria,

suitable to eliminate pollution effects; as shown by the present study, the JIP-test can serve as

a sensitive monitoring tool.

Acknowledgements: MT-M and RJS acknowledge support by the Swiss National Science

Foundation, Project Nr: 200021-116765.

References Hołynska B, Ostachowicz B, Ostachowicz J, Samek L, Wachniew P, Obidowicz A,

Wobrauschek P, Streli C, Halmetschlager G, 1998. Characterisation of 210Pb dated peat core

by various X-ray fluorescence techniques. Sci Total Environ 218: 239-248.

Strasser RJ, Tsimilli-Michael M, Srivastava A, 2004. Analysis of the chlorophyll a

fluorescence transient. In: Papageorgiou GC, Govindjee, eds. Chlorophyll a Fluorescence: a

Signature of Photosynthesis. Advances in Photosynthesis and Respiration Series (Govindjee –

Series Editor) vol 19. Kluwer Academic Publishers, Rotterdam, pp 321-362.

Tsimilli-Michael M, Strasser RJ, 2002. Mycorrhization as a stress adaptation procedure. In:

Gianinazzi S, Haselwandter K, Schüepp H, Barea JM, eds. Mycorrhiza Technology in

Agriculture: from Genes to Bioproducts. Birkhauser/Verlag, Basel/Boston/Berlin, pp 199-209

Tsimilli-Michael M, Strasser RJ, 2008. In Vivo assessment of plants‘ Vitality: applications in

detecting and evaluating the impact of Mycorrhization on host plants. In: Varma A, ed.

Mycorrhiza, 3rd edition, Springer, pp 679-703.

Tsimilli-Michael M, Eggenberg P, Biro B, Köves-Pechy K, Vörös I, Strasser RJ, 2000.

Synergistic and antagonistic effects of arbuscular mycorrhizal fungi and Azospirillum and

Rhizobium nitrogen-fixers on the photosynthetic activity of alfalfa, probed by the chlorophyll

a polyphasic fluorescence transient O-J-I-P. Appl Soil Ecol 15: 169-182.

Turnau K, Anielska T, Ryszka P, Gawronski S, Ostachowicz B, Jurkiewicz A, 2008.

Establishment of arbuscular mycorrhizal plants originating from xerothermic grasslands on

heavy metal rich industrial wastes – new solution for waste revegetation. Plant Soil 305: 267-

280.

84

Arbuscular mycorrhizal fungi and soil aggregation

Matthias C. RILLIG and Daniel L. MUMMEY

Institut für Biologie, Freie Universität Berlin, Altensteinstr. 6, D- 14195 Berlin, Germany

Division of Biological Sciences, University of Montana, Missoula MT 59812, USA

Arbuscular

Mycorrhizal fungi exert pervasive influences on terrestrial ecosystem processes; one of these

is their effect on soil structure. Soil structure describes the spatial arrangement of aggregates

and pores in the soil matrix, often by measuring stability after application of disintegrating

forces. Soil aggregation is a key parameter that influences, in turn, a number of other

ecosystem processes (like carbon storage) and soil biota distribution. Soil structure is thus an

important consideration in restoration of degraded ecosystems in the context of avoiding

erosion. Despite this important role of soil aggregation and abundant research in mycorrhizal

ecology, there are still large gaps in our understanding of mycorrhizal contribution to soil

structure.

Fundamentally, arbuscular mycorrhizal fungi (AMF) could influence soil aggregation at

different scales: (a) at the level of the plant community, for example through influencing the

composition of plant communities and its properties; (b) at the level of the individual host

plant, by influencing, for example, rhizodeposition or root growth properties; and (c) at the

level of the fungal mycelium (direct effects). At this scale, recent research focus has been on

biochemical mechanisms (e.g. glomalin-related soil protein). Based on correlative data,

glomalin-related soil protein is hypothesized to be important in stabilization of soil

macroaggregates. Recently, glomalin has been tentatively identified as a heat shock protein

homolog whose cellular location includes the hyphal wall; yet other fungal proteins and

substances may be important as well. Other, concurrently acting mechanisms include fungal

interaction with other soil biota (e.g. bacterial communities or hyphal grazers), as well as

physical mechanisms (hyphal enmeshment, alignment of particles).

By learning more about mechanisms, including a dynamic view of aggregate formation, we

hope to be able to provide the tools to apply AMF in a way that could specifically favor soil

aggregation in degraded ecosystems.

85

Potential of AM in the production of biofuel in the Mediterranean area

Vierheilig H., Sampedro I., Castellanos V.

Departamento de Microbiología del Suelo, Estación Experimental de Zaidín, CSIC, E-18008

Granada, Spain

Abstract

In recent years around the world the search for crops for the production of biofuel has been of

high priority, however, lately land use for biofuel production is seen more and more critical as

it can compete with food production.

Jatropha curcas, a member of the euphorbia family, originates from Central America. The

plant produces an oil toxic for human consumption which has been used for a long time as a

source of lamp oil and soap and most recently is promoted for the use of biofuel. The first

harvest is obtained after one year. Harvest is increasing during the first 4 years and thereafter

becomes stable for 40 - 50 years. When 4 years old between 1500 kg to 2000 kg of oil can be

obtained from Jatropha per hectar on semi-arid land. When irrigated the harvest can be

duplicated to 3000 kg to 4000 kg.

Jatropha seems to avoid a competition for land use with food plants, as it can be cultivated on

eroded, degraded farmland of low fertility or on non-arable wasteland where no other crops

grow (Fairless 2007). Jatropha plants have a deep root system which stops ground erosion and

increases water storage and being drought tolerant, it can be planted even in desert areas

(Fairless 2007; Zhang et al. 2007).

The current distribution of Jatropha shows that introduction has been most successful in the

drier regions with annual rainfall of 300-1000 mm, although they do not bother abundant rains of

up to 1800 mm per year. This high drought-resistance makes it a crop for arid and semi-arid

climates.

It occurs mainly at lower altitudes (0-500 m) in areas with an average annual temperature well

above 20°C but can grow at higher altitudes and tolerates slight frost.

Jatropha curcas grows almost anywhere, even on gravelly, sandy and saline soils. It can

thrive on the poorest stony soil. It can grow even in the crevices of rocks. The leaves shed

during the winter months form mulch around the base of the plant. The organic matter from

shed leaves enhance earth-worm activity in the soil around the root-zone of the plants, which

improves the fertility of the soil.

So far Jatropha is cultivated on a large scale in Africa, Asia and Central and South America,

however, to our knowledge no scientifically based data are available yet on the potential of

Jatropha cultivation in the Mediterranean area. In large parts of the Mediterranean, with low

rainfalls and soils with a poor fertility, climatic conditions seem favourable for the cultivation

of Jatropha.

Arbuscular mycorrhiza (AM), formed by symbiotic fungi in plant roots, are know to promote

the growth of a large variety of plants especially under extreme conditions such as drought.

The presence of AM has been suggested as a prerequisite for growth of Jatropha under harsh

climatic conditions (Sharma, 2007). To our knowledge there is only one report stating the

presence of AM fungi in roots of Jatropha curcas (Ragupathy et al. 1993). We are interested

to understand the importance of the Jatropha-AM fungus association and presently study three

different aspects of this interaction, which are described shortly below:

86

AM in Jatropha and effects on plant

No data are available yet which AM fungal structures are formed in roots of Jatropha plants

and how AM root colonization affects the growth and productivity of Jatropha plants.

Presently we perform first studies to obtain more detailed informations on the AM structures

in roots of Jatropha and on the effect of AM inoculation on Jatropha plant performance.

Phytoremediation, Jatropha and AM The suitability of Jatropha in phytoremediation has been shown with heavy metals, as

Jatropha has been reported to grow on heavy metal contaminated soils and to phytoextract

heavy metals (Juwarkar et al 2008; Kumar et al. 2008). We study the possibility to use

Jatropha in combination with AM for the phytoremediation of soils contaminated with

organic pollutants.

Usually plants grown on contaminated sites are without any economic value as they can not

be used for human consumption or to feed animal stock.

As the oil obtained from Jatropha seeds is not used for human consumption but for biofuel,

sites which are decontaminated with these plants at the same time could be used for the

production of biofuel as a by-product.

Figure 1. Jatropha is cultivated on contaminated site. This could result on one hand in a

decontamination of the site and at the same time in the production of biofuel.

Contaminated

soil which can

not be used for

crop production

Cultivation of Jatropha

(in combination with AMF)

Decontamination of soil

Production of

biofuel

Decontamination of soils and biofuel

production

1

An environmental approach as key factor for enhancing application of

arbuscular mycorrhizal fungal in agriculture systems

Jacqueline Baar,

ARCADIS Nederland BV, Rural Developments, P.O. Box 264, 6800 AG Arnhem, The

Netherlands

Abstract

Currently, growing crops in sustainable agricultural systems with reduced chemical inputs has

increasing interest. The application of high loads of chemical fertilizers and live stock manure

has become more and more under pressure. Evidence is growing that addition of high nutrient

levels in agricultural systems have destructive effects on the diversity above- and

belowground. The biotic below-ground soil communities including earth worms, nematodes

and fungi are reduced by intensive fertilization, particularly with fluid live-stock manure.

Such intensive fertilization regimes do not meet the requirements for sustainability in

agricultural systems.

Therefore, the interest in alternatives for intensive application of high loads of nitrogen,

phosphate and kalium by fertilization is increasing. Beneficial micro-organisms are one of the

alternatives for sustainable agricultural management. These micro-organisms enable the

production of profitable yields by achieving the main requirements for sufficient nutrient

uptake from unpredictable and varying levels of soil nutrients in sustainable agricultural

systems. An important group of these beneficial micro-organisms include the symbiotic living

arbuscular mycorrhizal (AM) fungi that play a vital role in the acquisition of mineral nutrients

and water from soils stimulating plant development. Also, these beneficial symbiontic fungi

can suppress the development of below- and above ground plant pathogens, resulting in better

plant performance.

In the last few decades, the interest in sustainability is accompanied with the production and

application of the beneficial mycorrhizal fungi. Worldwide, companies are producing

mycorrhizal fungi in different formulas ranging from single AM fungi for specific markets to

mixed products for general markets. The application of AM fungi has enormous potential for

large-scale agricultural systems and can be beneficial in sustainable production of main crops

contributing to reduced input of chemical fertilizers and pesticides.

For successful application of AM fungi with economically profitable results, the environment

must be suitable for the development of AM fungal symbiosis. The European network COST

Action 870 focuses on the main environmental factors affecting the development of

mycorrhizal symbiosis. In this chapter, the importance of the environment is discussed in

relation to enhanced application of AM fungi.

Environmental factors

Thus far, the main focus of the majority of studies on mycorrhizal fungi has been on these

fungi themselves. In fact, mycorrhizal fungi have been centralized in the majority of the

research projects. Although, these projects provided us with a great increase of knowledge, a

limited number of studies addressed the effects of environmental factors on the development

of mycorrhizal fungi. For instance, the number of systematic studies relating the abundance

and diversity of AM fungi with the abiotic soil conditions is relatively low (Estaún et al.,

2002).

Considering that environmental factors have strong influence on the development of

organisms, I propose to pay more attention to the effects of the environment. For studies with

AM fungi, an environmental approach should be taken addressing the effects of

87

Dry olive mill residue, Jatropha and AM

Mediterranean countries produce annually about ten millions tonnes of olives which are used

for the production of olive oil. At the end of the process of olive oil extraction the so-called

dry olive mill residue (DOR) is generated (Vlyssides et al., 1998). DOR has phytotoxic

properties (Martín et al., 2002; Sampedro et al., 2005) and thus, constitutes a mayor

environmental problem in the main olive growing regions. However, when reducing its

phytotoxicity or when applying to plant showing an enhanced tolerance to DOR, due to its

content of organic matter and mineral nutrients, DOR might be employed for agronomic

purposes as a natural fertilizer (Bonanomi et al., 2006). Jatropha grow even under harshest

soil conditions and thus could be potential candidates to be used in combination with DOR.

We are interested whether DOR can be used to improve soil conditions for the cultivation of

Jatropha and how AM fungi affect the tolerance of Jatropha towards DOR.

Figure 2. Dry olive mill residue (DOR) is generated in the production of olive oil. Applied to

the soil due to its content of organic matter and mineral nutrients DOR can act as a natural

fertilizer by improving the soil quality and thus, can affect plant growth positively, resulting

in an enhanced production of biofuel.

References

Fairless D, 2007. The little shrub that could — maybe. Nature 449: 652-655

PPrroodduuccttiioonn ooff oolliivvee

ooiill

dry olive mill residue (DOR)

DOR application to soil Improvement of soil

characteristics

Improved plant growth (in combination with AMF)

EEnnhhaanncceedd bbiiooffuueell

pprroodduuccttiioonn

From Olive Oil to Biofuel

88

Juwarkar AA, Yadav SK, Kumar P, Singh SK, 2008. Effect of biosludge and biofertilizer

amendment on growth of Jatropha curcas in heavy metal contaminated soils. Environ Monit

Assess DOI 10.1007/s10661-007-0012-9.

Kumar GP, Yadav SK, Thawale PR, Singh SK, Juwarkar AA, 2008. Growth of Jatropha

curcas on heavy metal contaminated soil amended with industrial wastes and Azotobacter – A

greenhouse study. Bioresource Technology 99: 2078–2082

Martín J, Sampedro I, García-Romera I, García-Garrido JM, Ocampo JA, 2002. Arbuscular

mycorrhizal colonization and growth of soybean (Glycine maximum) and lettuce (Lactuca

sativa) and phytotoxic effects of olive mill residues. Soil Biol. Biochem. 34: 17691775.

Ragupathy S, Mahadevan A, 1993. Distribution of vesicular-arbuscular mycorrhizae in the

plants and rhizosphere soils of the tropical plains, Tamii Nadu, India. Mycorrhiza 3: 123-136.

Sampedro I, D‘Annibale A, Ocampo JA, Stazi SR, García-Romera I, 2005. Bioconversion of

olive-mill dry residue by Fusarium lateritum and subsequent impact on its phytotoxicity.

Chemosphere 60: 13931400.

Sharma N, 2007. Reclamation of ash ponds and cultivation of Jatropha curcas using

arbuscular mycorrhiza fungi as technology demonstration for biofuel production and

environmental cleaning in Chattisgarh state. Expert seminar on Jatropha curcas L. Agronomy

and genetics. 26-28 March 2007, Wageningen, the Netherlands, Published by

FACT Foundation.

Vlyssides AG, Loizidou M, Gimouhopoulos K, Zorpas A, 1998. Olive oil processing wastes

production and their characteristics in relation to olive oil extraction methods. Fresenius

Environ. Bull. 7: 308313.

Zhang Y, Wang Y, Jiang L, Xu Y, Wang Y, Lu D, Chen F, 2007. Aquaporin JcPIP2 is

Involved in Drought Responses in Jatropha curcas. Acta Biochimica et Biophysica Sinica 39:

787–794.

89

Challenges of AM applications in phytoremediation of post-industrial

wastes – case of Molinia caerulea

Ryszka P., Turnau K.

Institute of Environmental Sciences, Jagiellonian University, Gronostajowa 7, 30-387

Krakow, Poland

Abstract

Molinia caerulea was found in previous studies to be very useful in phytostabilisation of zinc

wastes. However, many factors might affect the success of this application. Wide range of

interactions between AMF-inoculated plants and other organisms should be considered as

introduced plants are hot spots of microbial diversity and activity. Here we present

preliminary results of investigations on M. caerulea vitality and interactions with Claviceps

purpurea, a fungus associated with graminoid plants.

Introduction

Mining and the activity of different industrial branches result in the deposition of large

amounts of wastes. Remediation and restoration of areas occupied by these materials are

usually a difficult task, due to extreme chemical and physical properties. Phytostabilisation is

often used to minimize mobility of potentially toxic wastes and threats to environment and

human health. However, creation of a stable plant cover is difficult. Selection of proper plants

and substratum amendments is considered as a crucial step. Growth and survival of

introduced plants can be improved by their symbionts. Mycorrhizal fungi were shown to be

very important organisms as they supply plants with phosphorus, nitrogen and other elements,

protect them against pathogens and reduce environmental stresses such as drought or heavy

metal toxicity (Turnau et al. 2006).

Our studies (Ryszka and Turnau 2007) shown that grasses occurring spontaneously on zinc

wastes should be considered for phytostabilisation strategies . Among them, Molinia caerulea

is a very promising plant. This plant showed high survival rates when planted without any

amendments into bare zinc wastes and ability to stabilize the substratum. All investigated

plants showed extensive colonisation by AMF fungi (Ryszka and Turnau 2007). However,

application of this species on larger scale needs to be optimised and require further

investigation. Multiplication of plant material from seeds, although difficult, will not exploit

local populations of M. caerulea on zinc wastes. Inoculation with native fungal strains

ensures best performance under harsh conditions and will not increase heavy metal content

within plant tissues. Beside AMF, diazotrophs – free-living nitrogen fixing bacteria

(Reinhold-Hurek and Hurek 1998) were reported from M. caerulea root system (Hamelin et

al. 2002), these microbes can increase amount of nitrogen available to plants. As introduced

plantlets can be considered as hot spots of microbial diversity and activity on bare zinc

wastes, their interactions with wide range of microorganisms might influence the

phytostabilisation effectiveness. The aim of the present research was to characterize the

interaction between M. caerulea, AMF and a parasitic fungus Claviceps purpurea.

Material and Methods

Field investigations were carried out on Trzebionka Mining Company zinc wastes (N

49°09'30", E19°25'10"). Detailed information on this site was given by Ryszka and Turnau

(2007). Molinia caerulea plants introduced during previous studies were investigated for the

presence of Claviceps purpurea ergots within inflorescences. Five classes of ergot frequency

90

were considered: 0 – no ergots; I – single ergot in the stalk; II – 2 or 3 ergots per stalk; III – 4

or 5 ergots; IV – more than 5 ergots per stalk. Intensity of leaf colour was also recorded.

Mycorrhizal colonization was estimated according to Trouvelot et al (1986)

(http://www2.dijon.inra.fr/mychintec/Mycocalc-prg/download.html). For this, roots were

collected, carefully washed and cleared in 10% KOH for 24 h at room temperature.

Subsequently, after careful washing in tap water, the roots were acidified for 1 h in 5% lactic

acid and stained for 24 h at room temperature in 0.05% aniline blue in lactic acid, in order to

visualize the fungal structures inside the roots. Material obtained this way was cut into 1 cm

pieces and mounted on slides in lactoglycerol. The following parameters were assessed in

collected root samples: frequency of mycorrhiza (F%), mycorrhizal intensity relative (M%)

and absolute (m%), arbuscule richness relative (A%) and absolute (a%).

Chlorophyll a fluorescence transients of intact leaves of M. caerulea were measured with a

Plant Efficiency Analyser (PEA) fluorimeter (Hansatech Instruments, GB). The transients,

induced by a red light of 600 W×m-2

, were recorded for 1 s, starting 50 µs after the onset of

illumination. The data was acquired every 10 µs for the first 2 ms and every 1 ms thereafter as

described by Strasser et al. (1995). Each transient was analysed according to the OJIP-test

(Strasser et al. 1995; Strasser et al. 2000). Performance index (PI) was calculated for the

characterization of PSII vitality and performance.

Results

Number of Claviceps purpurea ergots recorded in stalks of Molinia caerulea collected from

Trzebionka Mining Company zinc wastes varied from 0 to 10. Also, colour intensity of leaves

showed variation from pale green to dark green. The most intense leaf colour was noticed in

plants with higher (III–IV) number of ergots, whereas specimens with low number of ergots

were characterised by pale leaves (Table 1). Similar trend was observed in case of

photosystem II vitality. Plants with low number of ergots (0–I) showed lower performance

index than those with more infected stalks (Table 1), however no statistically significant

differences were found. A slight decrease in performance index was noticed in specimens

with medium number of ergots.

Similar observations were done in case of mycorrhizal colonization parameters. Presence of

arbuscular mycorrhiza was noticed in all investigated samples of M. caerulea. Most values

showed decrease in plants with medium (II) number of ergots per stalk, however non-infected

specimens showed slightly higher values of all parameters than infected ones (Table 1).

Among plants with different ergot frequency no statistically significant differences were

found between parameters describing mycorrhizal colonization. However, plants with highest

performance index showed slightly lower values of mycorrhizal colonization (Table 1).

Table 1. Mycorrhizal colonization parameters (F%, M%, m%, A%, a% – see methods for

explanation) and performance index (PI) of Molinia caerulea non-infected or infected by

Claviceps purpurea and growing on zinc industrial wastes.

Frequency of

ergots per stalk

(classes)

F% M% m% A% a% PI Colour of leaves

0 100.00 42.67 42.67 38.26 89.66 3.00 pale

I 96.39 37.85 36.10 28.90 69.84 2.51 yellow-green

II 77.16 15.60 22.47 11.56 76.98 3.50 light green

III 87.21 31.52 36.50 27.22 86.03 2.56 green

IV 90.09 34.04 35.10 26.39 76.01 4.87 dark green

91

Discussion

Analysis of chlorophyll a fluorescence is a useful method proven to show plant overall

performance and vitality (Strasser et al. 2000). Higher number of ergots in investigated

specimens of Molinia caerulea increased performance index and intensity of leaf colour, thus

suggesting a positive effect of presence of Claviceps purpurea within stalks. This fungus,

however, is usually regarded as a parasite, decreasing plant fitness by reduction of the number

of seeds. Seeds, however, are not so important in this particular case as this species multiplies

mostly by vegetative means while seeds need much more favourable conditions than that at

the industrial wastes. The influence of various endophytes of shoots on plant development is

broadly known. Up to our knowledge C. purpurea was never shown to stimulate plant

photosynthesis, especially at the end of vegetation period. The impact is especially strong

while the number of ergots was very high (that high frequency is mostly not observed under

the non-polluted condition in M. caerulea). On the contrary to such endophytes as

Neotyphodium/Epichloë, the mycelium of C. purpurea appears in the shoots only at the of

flower production and becomes the sink for energy while the formation of seeds starts.

Similar situation is in case of the leaf parasites that stimulate photosynthesis of the host in the

form of so called ―green islands‖. Under harsh environmental conditions such as drought, low

nutrient content, erosion and presence of heavy metals in soil any factor increasing plant

vitality should be regarded as positive for phytostabilization strategy. Frequency of C.

purpurea was not correlated with parameters of mycorrhizal colonization. Although, plants

with medium number of ergots showed lower values of these parameters than those with

either no or high number of ergots. Investigations of multiple plant-fungal interactions should

be carried out during optimisation of reclamation strategies for areas occupied by post-

industrial wastes.

Plants with highest performance index showed slightly lower values of mycorrhizal

colonization (Table 1). This may suggest lowering plant overall performance by native fungal

strains present in rhizosphere of investigated Molinia caerulea specimens. Evaluation of

native strains selected for further large-scale application of M. caerulea on zinc wastes can be

assisted by the use of chlorophyll a fluorescence.

References Hamelin J, Fromin N, Tarnawski S, Teyssier-Cuvelle S, Aragno M, 2002. nifH gene diversity

in the bacterial community associated with the rhizosphere of Molinia coerulea, an

oligonitrophilic perennial grass. Environ. Microbiol. 4: 477–481.

Reinhold-Hurek B, Hurek T, 1998. Life in grasses: diazotrophic endophytes. Trends

Microbiol. 6: 139–144.

Ryszka P, Turnau K, 2007. Arbuscular mycorrhiza of introduced and native grasses

colonizing zinc wastes: implications for restoration practices. Plant Soil 298: 219–229.

Strasser RJ, Srivastava A, Govindjee, 1995. Polyphasic chlorophyll a fluorescence transient in

plants and cyanobacteria. Photochem. Photobiol. 61: 32–42.

Strasser RJ, Srivastava A, Tsimilli-Michael M, 2000. The fluorescence transient as a tool to

characterise and screen photosynthetic samples. In: Yunus M, Pathre U, Mohanty P (eds),

Probing photosynthesis: mechanisms, regulation and adaptation. Taylor & Francis, London,

pp. 445–483.

Turnau K, Orlowska E, Ryszka P, Zubek S, Anielska T, Gawronski S, Jurkiewicz A, 2006.

Role of mycorrhizal fungi in phytoremediation and toxicity monitoring of heavy metal rich

industrial wastes in Southern Poland. In: Twardowska I, Allen HE, Häggblom MM (eds), Soil

and Water Pollution Monitoring, Protection and Remediation. Springer, Dordrecht, pp. 533–

551.

92

Role of arbuscular mycorrhiza in 137

CS uptake - the Chernobyl case

S. Dubchak1, J.W. Mietelski

2, K. Turnau

1,

1Institute of Environmental Sciences, Jagiellonian University; Gronostajowa str. 7, 30-387

Krakow, Poland; dubchak@eko.uj.edu.pl 2The Henryk Niewodniczański Institute of Nuclear Physics, Polish Academy of Sciences;

Radzikowskiego str. 152, 31-342 Krakow, Poland

Abstract

Arbuscular mycorrhizas of Plantago lanceolata collected from the Chernobyl exclusion zone

were investigated showing the presence of at least three morphotypes (Glomus tenuis,

Scutellospora sp. and G. clarum. The experiment to evaluate the influence of AM fungi on P.

lanceolata cultivated in soil collected from the area was carried out showing the attenuation

of the soil toxicity and decreased level of radioactive Cs uptake into the shoots.

Introduction

The accident of the Chernobyl nuclear power plant took place in 1986, when the reactor was

destroyed after two thermal explosions. Prolonged emission of the radioactive substances

from the damaged reactor led to the release of nearly of 3×1018

Bq (or 6700 ± 1000 kg) of

radionuclides. Up to 100 % of radioactive noble gases (133

Xe, 85

Kr etc); 20 to 30 % of

radioiodine (131

I); 12 to 15 % of 134,136,137

Cs and 3 to 4 % of less volatile radionuclide

(238,239,240,241,242

Pu, 241,243

Am, 242

Cm, 89,90

Sr, 95

Zr, 99

Mo, 103,106

Ru, 141,144

Ce, 154,155

Eu, 60

Co etc.)

were blew out into the atmosphere (IAEA, 1996). As a result, nearly 340 000 km2 mainly in

Europe were contaminated with long-lived isotopes to the levels exceeding 37 kBq/m2. Nearly

half of the released radionulides were deposited within a zone 30 km from Chernobyl, namely

1,0×1016

Bq of 137

Cs, 6,1×1015

Bq of 90

Sr and 3,3×1014

Bq of transuranium elements (U, Am,

Pu, Cm isotopes) (Kholosha et al. 1999). The principal part of 134,136,137

Cs (about 60-75 %)

within the 30-km Chernobyl zone was initially in the form of fuel particles. With time, the

importance of 137

Cs relative to 134

Cs increased due to its greater half-life. Presently, this

radionuclide largely contributes to the overall level of the external and internal exposure at

contaminated areas, especially beyond the exclusion zone. 137

Cs has been mainly retained in

the surface horizons of soils due to its reaction with clay and humic components and/or the

soil microflora (Thiry et al. 1993). Arbuscular mycorrhizal (AM) fungal strains may influence

the uptake of toxic elements by plants. So far not much is known about the impact of these

fungi on Cs uptake by plants and most of the studies focus on a few fungal species selected

among strains available in labs (Dupre de Balois et al. 2008).

The aims of the present research were to study: 1. The presence and diversity of AMF in

Plantago lanceolata roots collected from the Chernobyl exclusion zone; 2. to evaluate the

influence of AM fungi on P. lanceolata cultivated on soil collected from the area.

Methods

P. lanceolata roots and substratum were collected from three sites in an area within the 30 km

Chernobyl zone in June 2008. The substrata were characterized by 137

Cs activity ranging from

23320 ± 84 Bq·kg-1

to 4318 ± 12 Bq·kg-1

. To cultivate plants under experimental conditions

the substrata were heated twice for 1 hour at 100oC with a 48 hours interval in between. Two

weeks later the pH was adjusted to 6.5 with dolomite lime and the inoculum containing

propagules of G. intraradices was introduced. The seedlings of P. lanceolata were grown in

a growth chamber at 20°C, with a 12 h darkness period. Plant vitality was estimated after 12

weeks of growth using a Plant Efficiency Analyzer (PEA) fluorimeter (Hansatech

93

Instruments, UK) estimating chlorophyll a fluorescence transients of intact leaves. The data

were acquired as described by Strasser et al. (1995) and each transient was analyzed

according to the OJIP-test (Strasser et al. 2000). The plants were harvested. The plant roots

and shoots were separated, washed in deionized water, dried for 3-5 days at room

temperature, crushed and homogenized. Afterwards they were mixed with 5-10 ml mixture of

water with alcohol and sugar (sklad?). The suspension was evaporated to dryness at 55 0C and

the activity distribution of 137

Cs in shoots and roots was determined using a gamma-

spectrometer with semiconductor coaxial HPGe detector (efficiency 15 %) shielded by 10 cm

of lead. The analysis was carried out on 30 nonmycorrhizal plants and 120 mycorrhizal plants

at each radionuclide concentration of substrate.

For the estimation of mycorrhizal colonisation, the roots from field and laboratory

experiments were carefully washed with tap water, softened in 10% KOH for 24 hours,

washed in water, acidified in 5% lactic acid in water for 12–24 h, stained with 0.01% aniline

blue in lactic acid (to visualize AMF) for 24 h at room temperature and subsequently stained

for 24 h with 0.3 % Sudan IV in the solution of 75ml 95% ethanol and 25 ml of deionised

water (to visualize DSE). Roots were stored in lactoglycerol. Relative mycorrhizal root

length (M%), intensity of colonisation within individual mycorrhizal roots (m%), relative

arbuscular richness (A%) and arbuscule richness in root fragments where the arbuscules were

present (a%) were assessed using a light microscope

(http://www2.dijon.inra.fr/mychintec/Mycocalc-prg/download.html).

Results The analysis of the root material from sites within the Chernobyl exclusion zone revealed the

presence of three AM morphotypes. The most common was Glomus clarum forming distinct

thick-walled spores and mycelium, often with thicker cell walls. Only slightly less common

was Glomus tenuis forming mycelium of ca. 1-2 um thickness and vesicles of up to 3 um in

diameter. Much less frequent (10-20% of root fragments) was the morphotype that fits the

description of the genus Scutellospora with knobby auxiliary cells outside roots and

mycelium usually thicker than in the case of both other fungi, with uneven staining of the

hyphal walls in the form of sheath and typical hyphal projections. No statistical differences

were found concerning the total mycorrhizal colonization of field samples. A tendency

towards slightly lower frequency of AM was noticed in the case of the samples from the site

closest to the power station. In addition, the coarse AMF mycelium and the spores of G.

clarum were often overgrown inside the roots by DSE mycelium. Staining with Sudan IV

revealed the presence of DSE mycelium also within the cells where the arbuscules of AMF

were formed. Another modification of the hyphal cell wall was visible in roots collected from

two most central sampling sites with the highest radiocesium content. The modifications were

visible as thickenings of the cell walls that were strongly stained with the aniline blue. These

changes were not so common and well visible in the case of the samples from the lowest Cs

activity zone.

The pot experiment that was carried out on soil samples collected from the Chernobyl

exclusion zone showed that both mycorrhizal and nonmycorrhizal samples of P. lanceolata

were characterized by high transfer of radiocaesium to roots and shoots (Ktr >1). At the same

time, no statistically significant differences were revealed in the accumulation of 137

Cs

between roots of nonmycorrhizal and mycorrhizal plants grown both, in low Cs and high Cs

soil. The AM-colonized plants had statistically lower 137

Cs specific activity (Bq/kg) and

transfer factors in shoots of Plantago lanceolata cultivated both on soil with low and high Cs

content. As no statistically significant differences were found between plant biomasses of

mycorrhizal and nonmycorrhizal plants cultivated at two levels of radionuclide contamination,

the content of Cs per plant was significantly lower in shoots of plants cultivated at higher soil

94

radionuclide concentration. The survival of nonmycorrhizal plants was 3 times lower than

mycorrhizal plants. The photosynthetic activity measurements of P. lanceolata showed that

the nonmycorrhizal and mycorrhizal plants cultivated on substrata containing less

radionuclides had similar plant vitality index (PI). At the same time, on substratum from

higher radionuclide zones, the mycorrhizal plants had significantly higher PI than

nonmycorrhizal plants, although the mycorrhizal colonization of roots from soil with higher

radionuclide content was lower than from the soil of higher radionuclide content.

Discussion

In the present study P. lanceolata was used as a model mycorrhizal plant. Its usefulness as a

monitoring plant was shown several times in the past, as this plant forms mycorrhiza with a

broad range of AM fungi and can be found in diverse habitats. So far there were no published

studies concerning the mycorrhizal status of P. lanceolata in the vicinity of the Chernobyl

power station, as the access to this zone is highly limited. Our pilot studies showed that at

least three different AMF are present in this zone and form abundant mycorrhiza with well

developed arbuscules. Further studies will be carried out using molecular tools. Additinally,

two interesting phenomena were observed. The first concerns the abundant development of

DSE mycelium within the coarse mycelium and spores of AMF from the highly radioactive

soil of Chernobyl exclusion zone. While the presence of DSE within AMF spores can be often

seen in other areas, the colonization of the mycelium to such high extent was never seen so

far. The phenomenon was most frequent at the site closest to the power station. Further work

on these fungi might be of great interest. As shown by Dadachova et al. (2007) melanized

fungi are often dominating species in soils contaminated with radionuclides and it was

suggested that they even can grow faster due to the influence of ionising radiation on the

electronic properties of melanin. Additionally, light-pigmented (Paecilomyces lilacinus) and

melanin containing (Cladosporium cladosporioides) microfungi introduced together were

shown to improve the accumulation of radionuclides in aboveground plant biomass

(Zhdanova et al. 2003). So far, the effect of DSE colonizing the plant roots on radionuclide

accumulation within plant material is unknown. The second phenomenon concerns the

presence of hyphal wall thickenings. This phenomenon has not been published yet, but was

also observed in similar extend on industrial wastes rich in heavy metals (unpublished data).

According to the recent review by Dupre de Boulois et al. (2008) the ability of AM fungi to

transport radiocaesium into mycorrhizal plants remains uncertain or even controversial. In the

present research, the accumulation of Cs in shoots of mycorrhizal plants was lower than in

nomycorrhizal ones. As P. lanceolata is highly responsive to AMF, the differences could

result from altered uptake of many elements from the soil due to mycorrhizal fungi and

increased growth of roots and shoots of mycorrhizal plants. In the present case, the slight

increase of biomass is visible only in the case of higher radionuclide concentrations. The

effect of mycorrhiza is more clearly visible in terms of increased plant survival and reduced

radionuclide levels in plants grown on both substrata from Chernobyl exclusion zones,

visualized by the measurement of photosynthesis parameters.

In the present study, a G. intaradices strain was used for inoculation in the pot experiment.

This strain reacted visibly to the level of soil pollution by decreased values for mycorrhizal

colonization and arbuscule richness. This was not visible in the case of P. lanceolata roots

collected from field sites within the exclusion zone. The differences in fungal diversity seem

to be the reason. G. clarum is a good candidate to carry out further investigations as it is

easily cultivable and sporulating in laboratory conditions, and according to previous

experiences it can have diverse effects on the metal uptake by plants.

95

Acknowledgements We greatly acknowledge Dr. Anna Jurkiewicz (Aarhus University, DK)

for the linguistic comments on this manuscript.The studies were supported by Marie Curie

actions-EST EC Project MYCOREMED Grant No.20387.

References

Dadachova E, Bryan RA, Huang X., Moadel T, Schweitzer AD, Aisen P, Nosanchuk JD,

2007. Ionizing radiation changes the electronic properties of melanin and enhances the growth

of melanized fungi. PloS ONE (www.plosone.org) 5, e457: 1-13.

Dupre de Boulois H, Joner EJ, Leyval C, Jakobsen I, Chen BD, Ross P, Thiry Y, Rufyikiry G,

Delvaux B, Declerck S, 2008. Role and influence of mycorrhizal fungi on radiocesium

accumulation by plants. J. Environ. Radioactivity 99: 785-800.

International Atomic Energy Agency, 1996. One decade after Chernobyl: Environmental

impact assessment and further perspectives. IAEA Report No. J1-CN-63, Vienna, Austria,

422 p.

Kholosha V, Proskura M, Ivanov Yu, 1999. Radiation and ecological significance of natural

and technogenic objects of Exclusion zone. Bulletin of Ecological State of Exclusion Zone

and Zone of Unconditional (Obligatory) Resettlement, Chernobyl, Issue 13, pp.3-8.

Thiry Y, Myttenaere C, 1993. Behaviour of radiocaesium in forest multilayered soils. Journal

of Environmental Radioactivity, 18: 247-257.

Zhdanova N, Vasilevskaya A, Lashko T, Gerzabek MH, 2003. Effect of microscopic fungi on

the 137Cs accumulation by some plants growing on radionuclides contaminated soil. Pol. J.

Ecol. 51:37-44.

96

Assessment of the effects of pesticides of biological origin, biofumigation,

and soil solarization on arbuscular mycrorrhizal fungi and total fungal

diversity

Konstantinos Samourelis1, Ioannis Ipsilantis,

1 Dimitrios G. Karpouzas,

1 K. Papadopoulou

1

1University of Thessaly, Department of Biochemistry-Biotechnology, Ploutonos 26 and

Aiolou str., 41221 Larisa, Greece

Abstract Pesticides of biological origin, biofumigation, and soil solarization are tools for plant

protection in low imput agriculture. However, their effects on non target soil organisms, such

as arbuscular mycorrhizal fungi that may also be commercially used in such systems, are not

known. We will conduct pot experiments with tomato plants, inoculated or not with AMF,

that will be treated either with azadirachtin or Quillaja saponaria Molina extracts, or which

will be planted in potting media treated with soil solarization, or biofumigation. We will

measure plant shoot and root biomass, the percentage of AMF root length colonization and

spore numbers, total fungal length, and ergosterol content, and we will evaluate the treatment

effects to AMF and total fungal diversity using denaturing gradient gell electrophoresis

(DGGE).

97

Effects of Olive Mill Wastewaters on the Community and Function of

Arbuscular Mycorrhizal Fungi

Ioannis Ipsilantis,1 Dimitrios G. Karpouzas,

1 Constantinos Echaliotis,

2 K. Papadopoulou

1

1University of Thessaly, Department of Biochemistry-Biotechnology, Ploutonos 26 and Aiolou

str., 41221 Larisa, Greece 2 Agricultural University of Athens, Department of Natural Resources and Agricultural

Engineering, Iera Odos 75, 11851 Athens, Greece

Abstract

Olive mill wastewaters (OMW) pose a significant environmental concern for the

Mediterranean countries due to their large quantities produced during the olive oil extraction

process and their phenol related toxicity. We investigated the effects of OMW soil application

on the structure and function of the community of arbuscular mycorrhizal (AM) fungi. A

compartmentalized pot system with a 20 μm mesh net allowing fungal hyphae, but not roots,

to pass was used and Vicia faba L. with or without AM fungi was grown in one part (feeder

compartment) and OMW were applied in the other (receiver compartment). At 0, 10, and 30

days after OMW treatment (DAT) V. faba seedlings were planted in the receiver

compartment. At harvest shoot and root dry weights, soil hyphal length, and AM fungal root

colonization were recorded, and OMW effects on AM fungal diversity were studied by PCR-

DGGE fingerprinting. Planting directly after OMW application led to inhibition of plant

growth and AM fungal colonization, however with time there was either no difference or an

increase with OMW application. Soil hyphal length increased with OMW and decreased with

time to eventually no difference by day 30. Application of OMW had a significant impact on

structure of the AM fungal community in the roots of the plants in the receiver compartment

which were seeded immediately after OMW application. Effects of OMW were alleviated

partially or totally 10 and 30 days after OMW application . On going studies aim to identify

the phylogeny of the AM fungus associated with particular treatments as was revealed by the

DGGE fingerprints.

98

Development and evaluation of pure cultures of arbuscular mycorrhizal

fungi from Greek organic tomato farms

Konstantinos Antoniadis1, Ioannis Ipsilantis,

1 Dimitrios G. Karpouzas,

1 K. Papadopoulou

1

1University of Thessaly, Department of Biochemistry-Biotechnology, Ploutonos 26 and Aiolou

str., 41221 Larisa, Greece

Abstract

The limitations on fertilizer use in organic farming has rendered the use of arbuscular

mycorrhizal (AM) fungi for improvement of plant nutrition economically promising. We

hypothesize that organic farms harbor more effective AM fungi, and that native, rather than

foreign inoculum will be more effective for improving plant production. We used rhizosphere

soil from sampled certified organic tomato farms as inoculum, and we are developing pure

AM fungal cultures to determine their efficiency in pot studies with tomato.

Arbuscular mycorrhizae from Pancratium maritimum in three ecosystems

in South Peloponnese

K. Papadopoulou1, D. Nikopoulou

2, D. Nikopoulos

2, A. Alexopoulos

3

1University of Thessaly, Department of Biochemistry & Biotechnology, Larissa

2TEI of Kalamata

3Agricultural University of Athens, Laboratory of Vegetable Production, Athens

Abstract

Pancratium maritimum (Amaryllidaceae) is a threaten native sand dune plant in southern

Greece. We seasonal sampled soil and root from Pancratium maritimum and other sand dune

plants (Agropyrum junceum, Ammophila arenaria, Eryngium maritimum, Euphorbia

paralias, Echinops sp, Cyperus capitatus) for determination of arbuscular mycorrhizal fungal

spores and percentage of root length colonization. Three different ecosystems in Messinia

(Kaiafas and Foinikounda) and Lakonia (Elos) were selected. A seasonal pattern in

colonization and sporulation was observed, where root length colonization ranged on the

average 20-100% in P. maritimum and the number of spores was 2-40 g-1

soil, with a

maximum in July. All other plants were also colonized with A.junceum being the most

responsive and E. maritimum the least. There were no differences for AMF colonization

among the three ecosystems for P. maritimum while for all other plants no consistent pattern

was observed.

99

Effects of arbuscular mycorrhizal fungi on N, P and K uptake from

grapevine (Vitis Vinifera L.) in an organic and a conventional vineyard

Nikolaidou A.E.1,*

, Pavlatou-Ve A.K.2, Orfanoudakis M.

3, Kalbourtji K.L.

1 and Veresoglou

D.S. 1

1 Laboratory of Ecology and Environmental Protection,

2 Laboratory of Soil Science,

Faculty of Agriculture, 3 Laboratory of Forest Soils, Faculty of Forestry and Natural

Environment, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece *Email:

annikola@agro.auth.gr

Abstract

The effect of arbuscular mycorrhizal (AM) fungi on grapevine (Vitis vinifera L.) N, P and K

nutrition was studied in an organic and a conventional vineyard, during two growing seasons.

Benomyl application and control were used as treatments in a completely randomized design.

AM colonization enhanced N, P and K uptake at the organic vineyard, while during the first

growing season AM grapevine root colonization was higher at the organic than at the

conventional vineyard.

Symbiosis with arbuscular mycorrhizal (AM) fungi enhances N, P and K (Mäder et al. 2000)

acquisition of cultivated plants in agroecosystems with limited nutrient additions. The effect

of AM fungi on the uptake of N, P and K from grapevine (Vitis vinifera L.) was studied in an

organic (VOr) and a conventional (VCo) vineyard, during two growing seasons (2005-2006).

In April 2005, VOr and VCO had a soil content of 0.7 g and 0.8 g total N kg-1

, 15.0 mg and

55.1 mg Olsen P kg-1

and 0.9 meq and 1.6 meq exchangeable K+ 100 g

-1, respectively.

Benomyl was applied to the soil among eight neighbouring grapevines. Eight neighbouring

grapevines of an adjacent vineyard row were selected as control. In June 8th

, 2005 and in May

30th

, 2006, two composite samples per row of grapevine leaf blades were harvested, just 3

days after benomyl application. Nitrogen, P and K were determined. For root AM fungal

colonization, part of the grapevine roots was collected, washed, cleared in 2% KOH, stained

using trypan blue and examined for the presence or the absence of fungal structures (Koske

and Gemma, 1989). The experimental design was a completely randomized with two

replications, where the factors were the farming system (organic – conventional), inoculated

(AM) and non-inoculated (non-AM) grapevines and the growing season (2005 -2006).

In 2005, benomyl application suppressed AM fungi at both vineyards. AM grapevine root

colonization was higher and the network of hyphae was better organized at VOr than at VCo.

In 2006, benomyl application reduced AM fungi colonization only at VCo. AM grapevine

root colonization did not differ between the two vineyards. Leaf N concentration was

significantly higher in AM than non-AM grapevines in 2005 and at VOr than at VCo in 2006.

Leaf P concentration was significantly higher in AM than non-AM grapevines at VOr, in non-

AM than AM grapevines at VCo and at VCo than at VOr in non-AM grapevines. Leaf K

concentration was significantly higher in AM than non-AM grapevines at VOr and at VCo

than at VOr in non-AM grapevines.

References

Mäder P, Edenhofer S, Boller T, Wiemken A, Niggli U, 2000. Arbuscular mycorrhizae in a

long-term field trial comparing low-input (organic, biological) and high-input (conventional)

farming systems in a crop rotation. Biol Fertil Soils 31: 150-156.

Koske RE, Gemma JN, 1989. A modified procedure for staining roots to detect VA

mycorrhizas. Mycol Res 92: 486-505 Part 4.

100

Studying plant - AM interaction in carrot and chickpea. - Presentation of a

research approach

Vicente C. 1, Schneider C.

2, Tavares de Sousa M.

1 and Arnholdt-Schmitt B.

3*

Corresponding author: * email: eu_chair@uevora.pt 1 Instituto Nacional de Recursos Biológicos L-INIA Elvas (Ex-Estação de Melhoramento de

Plantas) Estrada Gil Vaz Apartado 6. 7350-951 Elvas, Portugal 2 Inoq GmbH, Solkau 2, 29465 Schnega, Germany

3EU Marie Curie Chair, ICAM, University of Évora, Portugal

Abstract

The interaction between plant and arbuscular mycorrhizal fungi (AMF) is initiated by the

recognition of plant signals, known as strigolactones, by AMF (Reinhardt, 2007). These

strigolactones are branching factors that induce hyphal branching by the activation of

mitochondria resulting in the beginning of gene expression and consequently morpho-

physiological changes at the pre-symbiotic stage of AMF (Akiyama et al. 2005; Besserer et

al. 2006). Previous knowledge of mitochondrial activation by root factors has been reported

by Tamasloukht et al., 2003. Tamasloukht and co-workers speculated that the exhibition of

low respiratory activity during the spores‘ germination in asymbiotic stage could be due to

alternative electron transports to minimize the carbon consumption. Additionally, in the pre-

symbiotic phase, specific root factors could activate their metabolism to a more efficient use

of carbon sources. The involvement of an alternative electron transport in the AMF

mitochondrial can be hypothetically interpreted as the function of the alternative oxidase

(AOX) pathway. AOX from fungi shares some properties with AOX from plants, yet fungal

AOX appears to have differing complex mechanisms for regulation and expression (Joseph-

Horne et al., 2001). We will present a research approach to the role of AOX in the plant-

arbuscular mycorrhiza interaction. The second phase of our work-plan will be designed to

understand if AOX has an important function in the pre-symbiotic and symbiotic stage of

AM. Daucus carota L. (carrot) will be used as plant model. Later, we plan to adapt and

optimize all experimental procedures for future application in important agronomical and

economical cultures, such as Cicer arietum L. (chickpea).

References Akiyama K, Matsuzaki K, and Hayashi H, 2005. Plant sesquiterpenes induce hyphal

branching in arbuscular mycorrhizal fungi. Nature 435: 824-827.

Besserer A, Puech-Pagés V, Kleter P, Gomez-Roldan V, Jauneau A, Roy S, Portals JC, Roux

C, Bécard G, and Séjalon-Delmas N, 2006. Strigolactones stimulate arbuscular mycorrhizal

fungi by activating mitochondria. PLOS Biology 4: 1239-1247.

Joseph-Horne T, Hollomon DW, and Wood PM, 2001. Fungal respiration: a fusion of

standard and alternative components. Biochimica et Biophysica Acta 504:179-195.

Reinhardt D, 2007. Programming good relations – development of the arbuscular mycorrhizal

symbiosis. Current Opinion in Plant Biology 10:98-105.

Tamasloukht MB, Séjalon-Delmas N, Kluever A, Jauneau A, Roux C, Bécard G, and Franken

P, 2003. Root factors induce mitochondrial-related gene expression and fungal respiration

during the development switch from asymbiosis to presymbiosis in the arbuscular

mycorrhizal fungus Gigaspora rosea. Plant Physiology 131:1468-1478.

101

The role of AM on Mediterranean grassland communities.

Alifragis D. 1 Orfanoudaki1s M.

1 Veresoglou D

2 . Mamolos A

2., Papaioannou A

1 .

1Forest Soil Lab AUTH

2 Ecology and Environmental Protection Lab

Corespondet authors e-mail dalifrag@for.auth.gr

Arstract

The spesific relationships among different plant species with AMF , how the AMF contributes

to the plant species richness and how this contributes to the plant biomass production. The

studies were focused in very low fertility soils. The studies were conducted in the AUTH

University Forest located in N. Greece. The results suggesting that plant monocultures and

mix cultures consisting of Agrostis capillaries, Poa pratensis, Cynodon dactylon, Plantago

lanceolata και Cichorium indybus was significant difference at the biomass plant production

in relation to the selective AMF inoculum applied. Plants in symbiosis with Glomus

intraradices (BEG 144) were with the highest aficasy on P uptake. Plantago lanceolata in

symbiosis with AMF was with the highest upteke on Mg and Ca, while Poa pratensis, Agrostis capillaries was with highest K. In synopsis the AMF species richnes contributes to

plant species richnes. The root colonization was differ at different seasons, and different

years.

102

DO HORTICULURAL PLANTS DEPEND ON MYCORRHIZAE IN

TERMS OF PHOSPHORUS AND ZINC UPTAKE İbrahim ORTAS1 and Çağdaş Akpınar.

1The University of Çukurova, Department of Soil Science, Adana- Turkey ABSTRACT

Nutrient deficiency especially P and Zn is a common nutritional problem for some crops production in Turkey. This problem results in the application of increasing amounts of several fertilizers sources. Mycorrhizal inoculation or indigenous potential of mycorrhizae in soil is a critical factor in crop production under low supply of Zn and P. Under semiarid conditions mycorrhizae contributes to overcoming mineral nutrient deficiencies seems to be a suitable agricultural strategies.

Under greenhouse condition mycorrhizal dependence was searched in term of Zinc (Zn) requirements. The effects of selected mycorrhizal inoculation and Zn applications on several plant such as citrus, maize, pepper, been has been were investigated in several Zn deficient soils. Soil was sterilized by autoclaving and plants were grown under greenhouse conditions. Also under the field experimenters several more plant were tested to search the effect on mycorrhizal inoculation on Zn uptake.

The effect of mycorrhizal inoculation on plant growth is changed by effectiveness of inoculum and time The results revealed that plants are strongly dependent on mycorrhizal infection. Although addition of Zn increased plant growth, but mycorrhizal dependence is much more depend on P nutrition.

INTRODUCTION

Mycorrhizal dependency for a representative plant species is a key factor for horticultural plants (Ortas et al., 2002). Mycorrhizal dependencies are high when plants were grown in low-P soils than plant growth in ill soil with P levels typical of highly productive agricultural soils (Gemma et al. 2002). Since it is known that some plants are strongly mycorrhizal dependent on P nutrition, but less is known about the mycorrhizal dependent on Zn nutrition. In the present study we attempted to study the role of mycorrhizal inoculation on growth of several plant speacies on a calcareous soil having both Zn and P deficiency. METHODS

Mycorrhizal dependency (MD) was determined by expressing the difference between the dry weight of the mycorrhizal plant and the dry weight of the nonmycorrhizal plant as a percentage of the dry weight of the mycorrhizal plant (Plenchette et al 1981). RESULTS AND DISCUSSION

The results obtained in Table 1, indicate that maize, pepper and kidney bean are mycorrizal-dependent plant at low P and Zn supply. Mycorrhizal inoculation in soil with P and Zn deficiency is a critical factor for MD in crop production as well as in P and Zn uptake (Ortas et al., 2002). Phosphorus treatments generally reduced mycorrhizal dependency, but Zn application did not lead to any difference. Beneficial effects were higher with G. etunicatum inoculation than with G. mosseae inoculation. In the present experiment although mycorrhizal inoculation increased plant Zn uptake, yet the plant was found to be much more mycorrhizal dependent on P nutrition (Ortas and Akpinar 2006). So far, all the experiments conducted have shown similar results (Ortas et al., 2002).

103

Table 1. Effect of mycorrhizal species and P and Zn interaction on mycorrhizal dependence for maize, pepper and bean plants P and Zn Supply

mg kg-1 soil Mycorrhizal Dependence (%)

G. etunicatum G. mosseae Maize Pepper Bean Maize Pepper BeanP0 83 74 49 83 82 43P1 Zn0 66 41 21 64 42 16P2 39 29 17 31 34 28P0 72 87 45 64 92 33P1 Zn1 50 9 16 49 15 20P2 34 6 16 19 18 17

Figure 1. Effect of P and Zn interaction on mycorrhizal dependency for citrus plant

Compared to non-inoculated control

plants, mycorrhizal inoculation increased dry matter of plants (Table 1). In well agreement with growth data, mycorrhizae inoculation enhanced P and Zn concentration of plants, especially under low supply of P and Zn. Mycorrhizae inoculated plants have high Zn content compared to the non-inoculated plants, it is clear that mycorrhizal inoculation helps plants to have more Zn uptake especially for citrus plants (Table 2). In generally, under the field experiment also mycorrhizae inoculated plants have high Zn content compare to non mycorrhizal plants.

From the present results (Figure 1), it seems MD was less affected by Zn supply than the P supply and since the non-inoculated plants did not respond to P and Zn supply, it can be said that citrus plants strongly depended on mycorrhizal infection. Since mycorrhizae inoculated plants have high Zn content compared to the non-inoculated plants, it is not clear whether mycorrhizal inoculation helps plants to have more Zn uptake or just as a results of higher plant growth (Ortas et al., 2002).

It seems that mycorrhizal dependence is an inherent characteristic for which plant nutrient requirement and uptake efficiency are important parameters, especially for P requirement. Considering the importance of mycorrhiza dependence for plant survival, it is of great interest to categorize species according to this characteristic.

REFERENCES Ortas, I., Ortakçı, D., Kaya, Z., Çınar, A. and Onelge N. (2002). Mycorrhizal dependency of

sour orange (Citrus aurantium L.) In term of phosphorus and zinc nutrition by different levels of phosphorus and zinc application. J. Plant Nutr. 25, 1263 – 1279.

Ortas, I and C. Akpinar, 2006. Response of kidney bean to arbuscular mycorrhizal inoculation and mycorrhizal dependency in P and Zn deficient soils. Acta Agriculturae Scandinavica, Section B - Plant Soil Science. 56;101 – 109.

Plenchette, C., Furlan, V., Fortin, J.A.(1981) Growth Stimulation of Apple Trees in Unsterilized Soil Under Field Conditions With VA Mycorrhiza Inoculation. Can. J. Bot. 59, 2003-2008.

Gemma, J.N.; Koske, R.E.; Habte, M. (2002) Mycorrhizal dependency of some endemic and endangered Hawaiian plant species. American Journal Of Botany. 89, 337-345.

Citrus Mycorrhiza Dependency

708090

100

P0 P1 P2

P and Zn Supply

Myc

orrh

iza

Dep

ende

ncy

(%) Zn0 Zn1 Zn2

104

The Effect of Mycorrhizae and Different P and Zn Rates Application On

Citrus (Citrus Sinensis L.) Growth and Nutrient Uptake Under Sterile and

Non-Sterile Soils Conditions Ibrahim ORTAS1 And Cagdas AKPINAR1

Department of Soil Science, Faculty of Agriculture, University of Çukurova, Adana, Turkey.

Abstract The soils are in the Çukurova Region (East Mediterranean Coast of Turkey) have high

clay content, pH and lime content and consequence crop production is limited. Accordingly

Zinc (Zn) and Phosphorus (P) deficiency are common problems in citrus plantation. In order

to evaluated the role of mycorrhizal inoculation on takedown the problem, the effect of the

mycorrhizae and P and Zn doses application on citrus growth (citrus sinensis L.) and nutrient

uptake were studied under sterile and non-sterile soils conditions. The experiment was set up

in the greenhouse conditions at the department of Soil Science, University of Çukurova,

Adana, Turkey. Glamus clarium was used as arbusculer mycorrhizae fungi at 1000 spores per

plant. Different P and Zn rates were applied beginning of the experiment. 0, 2.5, 5 mg kg-1 Zn,

0 and 200 mg kg-1 P were used as fertilizer in Çanakçı soils series (Typic xerofluvent).

Generally, plants grown in non-sterile soils were better than in sterile soils. In the both

soils (sterile, non-sterile) mycorrhizal inoculation increased plant dry matter and root

infection. The results show that the non-sterile soil significantly increased citrus dry matter

production, root infection % and P and Zn uptake. In non-inoculated and sterile soil, plant P

and Zn content significantly reduced compared to inoculated plants. However mycorrhizal

inoculation in sterile soil significantly increased plant biomass and nutrient uptake.