Nutrition and nitrogen-fixation in Malaysian

Pterocarpus indicus Willd.

By

LOK ENG HAI

Dip. (Forestry), BSc. (Forestry),

MSc. (Plant Science)

Thesis submitted in fulfillment of the requirements for the

degree of the Doctor of Philosophy

School of Biological Sciences & Biotechnology

Faculty of Sustainability, Environmental and Life Sciences

Murdoch University,

Perth, Western Australia

June 2011

II

DECLARATION

I declare that this thesis is my own account of my research and contains as its main

content work which has not previously been submitted for a degree at any tertiary

education institution.

……………… ……………. Lok Eng Hai

III

Abstract

Pterocarpus indicus is a promising tropical woody legume for the

establishment of forest plantations in Malaysia. Woody legumes that form

symbiotic relationships with nitrogen-fixing bacteria also play an important

role for forest restoration on degraded land. Although P. indicus has been

widely planted as an amenity tree in SE Asia, its silvicultural requirements

have not been determined. There are no recommendations for fertilizer or for

inoculation with nitrogen-fixing bacteria. This thesis explores the phosphorus

(P) and nitrogen (N) requirements of seedlings and identifies a range of

nitrogen-fixing bacteria capable of forming root nodules under glasshouse

conditions.

Four glasshouse experiments were undertaken on two soil types: Yalanbee

sandy gravel (YB) and yellow sand (YS) to determine the P and N

concentration ranges in the foliage of deficient and healthy plants and to define

critical nutrient concentrations for the diagnosis of deficiency. There was a

narrow range in rates of P fertilizer, supplied as aerophos, Ca(H2PO4)2.H2O,

between deficiency and toxicity in both soil types. The relationship between

yield and P concentration in the youngest fully expanded leaf (YFEL) enabled

critical P concentrations for the diagnosis of deficiency (0.17%) and toxicity

(0.41%) to be determined at 90% maximum yield from linear regressions fitted

to the data. The foliar P concentration ranges for deficiency and toxicity were

similar to other nitrogen-fixing trees. Only plants in YS responded to inorganic

N fertilizer, and soil analysis suggested that seedlings may take up ammonium-

N in preference to nitrate-N at luxury supply. A critical concentration for the

IV

diagnosis of N deficiency was not able to be determined due to the lack of data

points. Plants with adequate N fertilizer had YFEL N concentrations of 2-3.5%

dry weight.

To determine whether P. indicus is more sensitive to luxury soil P supply than

other fast-growing legume trees, the P response of P. indicus in YB was

compared with three other woody legumes, Pterocarpus macrocarpus, Acacia

mangium and Sesbania formosa. The sensitivity of P. indicus to high P was

confirmed and the response shown to be similar to P. macrocarpus. Both

species showed severe symptoms of P toxicity, namely leaf necrosis and

stunted growth. In contrast, shoot and root yields of A. mangium and S. formosa

were not reduced at luxury P supply and yield x fertilizer relationships were

able to be fitted to the Mitscherlich model. Critical P concentrations for the

diagnosis of P deficiency in A. mangium and S. formosa, derived using the

Mitscherlich model, were estimated to be 0.2-0.3% dry weight for the YFELs.

Two glasshouse inoculation trials were carried out using diverse strains of root

nodule bacteria in order to identify strains suitable for inoculation in the

nursery. There were eight strains from Bradyrhizobium, five from Rhizobium,

three from Sinorhizobium and two Mesorhizobium strains. P. indicus formed

nodules with strains from Bradyrhizobium, Rhizobium, Sinorhizobium and

Mesorhizobium, which suggests it is a promiscuous host. Nodules formed

were globose, single and of aeschynomenoid type. In the first trial,

Bradyrhizobium strain WSM 2096, promoted shoot growth while in the second

trial, Bradyrhizobium strain WSM 3712 promoted shoot growth. Growth

V

stimulation was similar to the uninoculated control supplied with inorganic N

as KNO3 but was inferior to plants given (NH4)2SO4.

The response of P. indicus to low soil P in inoculated and uninoculated plants

was studied in a pot trial comprising two P treatments (nil, just adequate).

Plants were grown for 3 and 6 weeks. At nil fertilizer P, uninoculated P.

indicus seedlings had higher total root length and root dry weight than those

with adequate P. Inoculation with WSM 3712 suppressed root growth relative

to uninoculated plants.

Information gathered in this thesis has application for the production of

planting stock in forest nurseries. Firstly, care needs to be taken to ensure that

rates of P supplied as hard or liquid fertilizer are not in the range likely to cause

toxicity. Secondly, if any starter inorganic N fertilizer is to be used then it

should be supplied either as ammonium-N or as urea. Thirdly, more research is

required in order to identify effective strains of rhizobia for widespread

commercial application. Fourthly, the critical foliar P concentrations identified

for the diagnosis of P deficiency and P toxicity can be used to help interpret

foliar analysis of seedlings in the future.

VI

Publications arising from this research

Lok E.H., O’Hara, G.W. and Dell, B. 2006. Nodulation of the legume Pterocarpus

indicus Willd. by diverse strains of rhizobia. Journal of Tropical Forest Science

18(3): 188-194.

It is anticipated that further publications will arise from this work.

VII

List of abbreviations

ANOVA Analysis of variance

CRS Centre for Rhizobium Studies at Murdoch University

DBH Diameter at breast height (1.3 m)

DMRT Duncan’s New Multiple Range test

FRIM Forest Research Institute Malaysia

INRA Institute national de la Recherche Agronomique, France

N Nitrogen

n Number of samples

P Phosphorus

PCR Polymerase chain reaction

R Rhizobium

R2

Correlated regression coefficients

RCBD Randomized complete block design

S.E. Standard error

Sp. Species (singular; spp., plural)

T Temperature ( oC)

WA Western Australia

WSM Western Australia Soil Microbiology

YB Yalanbee soil

YFEL Youngest fully expanded leaf

YMA Yeast mannitol agar medium

YS Yellow sand

VIII

Acknowledgement

Foremost, I would like to express my sincere gratitude to Prof. Dr. Bernie Dell, for

his supervision, guidance, directing my thoughts to develop this thesis, endless

patience, constant encouragement, support and many critical comments throughout

the course of this study.

My immense gratitude and thanks also to Associate Professor Dr. Graham O’Hara

for his helpful suggestions, valuable guidance, generous assistance and critical

comments. These studies would not have been possible without his consistent advice,

guidance, kind encouragement and interest in the progress of the study. I am also

thankful and grateful to all my supervisors for their undivided support and assistance

extended to me and my family during our memorable stay in Australia.

This postgraduate study was made possible through the Malaysian Government

scholarship generously offered by the Ministry of Science, Technology and

Innovation (MOSTI) and Public Service Department (PSD) for which it is greatly

acknowledged. My special thanks to Dato’ Dr. Abdul Latif Mohmod, Director

General of Forest Research Institute Malaysia (FRIM) and Mrs. Rahmah Bt. Wan

Raof, Human Resources and Development (FRIM) for granting a study leave and

their support to pursue this study. Similarly, I am thankful to the Murdoch University

for the financial support in granting me a Murdoch University Studentship during the

final stage of my study.

Special thanks are due to Professor John Howieson and his team from the Centre for

Rhizobium Studies (CRS), Murdoch University for his permission to use the

Centre’s facilities, technical help and useful advice at the Centre’s laboratory. I

IX

would also like to thank Mr. Gordon Thomson for his superb histology works and for

sharing his microscopic laboratory facilities. My sincere thanks are also due to Mr.

Kim Tan, Max Dawson and Kose’ Minetto for their companionship and assistance

during my study at the glasshouse. A big thanks to Associate Professor Dr. Mike

Calver for his invaluable advice on statistical analysis during the process of the

study.

I am greatly appreciative to Ben Bradshaw, Bill Dunstan, Paul Baba and Alan Chen

YingLong for their kind assistance and companionship throughout the course of my

study. To my close friend, Carlos Raphael, thanks for ever willing helps, always

reminding and inviting me to eat regularly and the coffee break hour together

especially during the final stage of my writing.

I would like to extend my deepest gratitude and appreciation to my wife, Doris Loh

Lee Fah and my children, Vince Lok Xia Ren and Jessica Lok Xia Shin for their

love, prayers and sacrifices. Thanks also to my sisters and brother who never missed

calling to ask on my well-being. Last but not least, I would also like to dedicate this

work to my late father and mother for their love and sacrifices, who never lost faith

on me.

X

Table of Contents

Declaration II

Abstract III

Publications arising from this thesis VI

List of Abbreviations VII

Acknowledgements VIII

Chapter 1 Introduction, literature review and thesis aims

1.1 Introduction 1

1.2 History of planted forests in Peninsular Malaysia 2

1.3 Pterocarpus and its background 3

1.4 Nutrition and symbiotic requirements of tropical woody legumes 14

1.5 Principles of plant analysis 18

1.6 Alternative approaches for determining nutrient disorders 21

1.7 N-fixing trees and root nodule bacteria 23

1.7 Conclusions 24

1.8 Aims of the thesis 25

Chapter 2 Diagnosis of phosphorus and nitrogen disorders in Pterocarpus

indicus seedlings

2.1 Introduction 28

2.2 Materials and methods 29

2.3 Results 35

2.4 Discussion 43

Chapter 3 The phosphorus response of Pterocarpus indicus seedlings in a

lateritic soil compared to three tropical tree legumes

3.1 Introduction 54

3.2 Materials and methods 56

3.3 Results 59

3.4 Discussion 74

XI

Chapter 4 Nodulation of Pterocarpus indicus by diverse strains of root nodule

bacteria

4.1 Introduction 77

4.2 Materials and methods 78

4.3 Results 83

4.4 Discussion 88

Chapter 5 Effective rhizobia for nodulation and nitrogen fixation on Pterocarpus

indicus seedlings

5.1 Introduction 92

5.2 Materials and methods 93

5.3 Results 94

5.4 Discussion 99

Chapter 6 Effects of phosphorus and root nodule bacteria on root growth in

Pterocarpus indicus seedlings

6.1 Introduction 104

6.2 Materials and methods 105

6.3 Results 107

6.4 Discussion 114

Chapter 7 General discussion 119

Appendices 127

References 133

1

Chapter 1

Introduction, literature review and thesis aims

1.1 Introduction

Plantations of timber and multiple-purpose tree species play an important role in

restoring soil productivity and ecosystem stability. They may also enhance biological

diversity for degraded tropical lands. The rapid population growth in developing

countries, especially in the tropics and sub-tropics, has resulted in many social and

economic problems that cause unabated need for food, fodder, energy and wood. It is

estimated that about 13 million ha/year of the tropical forests and woodlands are

being destroyed or seriously degraded through agricultural expansion, logging, land

clearing for development and fuel wood collection (Landly, 1982; Parrotta, 1996;

Wan Razali, 2003; FAO, 2005).

Tropical deforestation is often associated with the conversion of forest lands to

agricultural uses mostly to replace land that has lost productivity due to unsuitable

farming practices, shifting cultivation and illegal timber harvesting, among others

(Whitmore 1984; Harwood et al., 1993; Lamb, 1994). This is also true in Malaysia

where the major causes of deforestation and forest degradation are over-logging and

the conversion of forests to agricultural plantations (Appanah and Ismail, 1996;

Nambiar and Brown, 1997; Appanah and Razak, 1998). Currently, most of the

remaining forests are confined to the fragile hilly parts of the country and the vital

watershed areas (Baharuddin and Chin, 1986; Bordeleau and Prevost, 1994).

2

Plantations have now been recognized as integral parts of sustainable forest

management and complementary to natural forests (Darus and Radhuan, 1990). In

order to overcome the anticipated shortage of raw materials for the wood, pulp and

paper industries in Southeast Asia, plantations of both indigenous and fast-growing

exotic species are being widely exploited and much basic research is needed to

underpin these developments. Among the popular species used in the region,

including Malaysia, are Acacia mangium, Azadirachta excelsa, Dyera costulata,

Endospermum malaccensis, Eucalyptus spp., Gmelina arborea, Hevea brasiliensis,

Khaya ivorensis, Neolamarkia cadamba, Octomeles sumatrana, Paraserianthes

falcataria, Swietenia macrophylla and Tectona grandis. Other promising indigenous

species, such as dipterocarps and Pterocarpus indicus are also being considered

(FRIM in Focus, 2002; Krishnapillay and Appanah, 2002; MTIB, 2007). The later

species P. indicus is the subject of this thesis.

1.2 History of planted forests in Peninsular Malaysia

Planting of timber trees in Peninsular Malaysia started with the commercial planting

of Hevea brasiliensis (rubber) for latex in the late 1800s. A brief history of artificial

regeneration of rubber and forest plantation in Peninsular Malaysia has been

described by Appanah and Weinland (1993). By the end of 1999, a total of 1,700 ha.

model rubber plantations had been established at several locations in Peninsular

Malaysia. These include some of the promising clones tested like RRIM623,

RRIM900s series, PM10, PB235, PB260 and the newly launched RRIM2000s

clones. Some of these clones are expected to yield between 1,500-3,000 kg of latex

annually and with wood volume of between 0.75 to 1.3 m3 per tree after 15 years.

These figures are about 300% higher as compared to the older clones that are

currently being used.

3

Meanwhile, the search for other fast-growing hardwood species continues. Several

potential exotics and indigenous species have been identified for short-rotation tree

crops to produce general utility timber (Darus et al., 1990). In 1982, the

Compensatory Forest Plantation Project (CFPP) was launched in the states of

Pahang, Johor, Selangor and Negri Sembilan with a projected target of 188,000 ha.

However, due to the widespread occurrence of heart rot disease in Acacia mangium,

only about 50% of the targeted area was achieved by the end of 1999 (Hashim et al.,

1996). Heart rot has led to increased interest in other forest species (Forest

Department Annual Report, 2001; MTIB, 2007). Furthermore, data obtained in a

survey throughout Peninsular Malaysia indicate that there has recently been a

considerable increase in the area of forest plantations of 75,672 ha resulting from

some of the recommendations made by FRIM in 1996 and 1997 (Krishnapillay and

Ong, 2003).

1.3 Pterocarpus and its background

The genus Pterocarpus, a close relative of Dalbergia, belongs to the family

Leguminosae-subfamily Papilionoideae (Fagaceae) and is the largest family of

flowering plants (Polhill, 1981). It produces many beautiful, highly decorative

timbers that are rank among the finest luxury timbers. Among them is Pterocarpus

indicus Willd. which is locally known as narra in the Philippines, sonokembang or

angsana in Indonesia, pradoo or padauk in Thailand, rosewood in Papua New

Guinea, nanara in Vanuatu, liki in Solomon Islands and sena or angsana in Malaysia,

Brunei and Singapore (Corner, 1988). It has the status of National Tree in the

Philippines and is a favourite timber for the manufacture of fine, high quality

furniture. Due to its scarcity and very limited timber supply from planted trees, many

native stands are now fast disappearing. In Singapore, it was part of the country‟s

4

garden city planting program with many avenues graced by this attractive species,

and in Malaysia it has been used as a shade tree for over 200 years (Wong, 1982;

Corner, 1988; Ng, 1992). More recently, this species has been planted along roads in

West Kalimantan, Indonesia (Setiadi, 1995). The first extensive planting of the

species was recorded in 1802 by the British in Penang and earlier in Malacca in 1778

(Wee and Corlett, 1986). Since then, systematic planting has been carried out

especially in Singapore, and in some of the states of Malacca and Penang. The

National Academy of Sciences (NAS, 1979) concluded that despite their commercial

importance, Pterocarpus species had been almost entirely overlooked by science and

that research on all Pterocarpus species, including P. indicus, was important (Ang,

1988; Munyanziza and Oldeman, 1994; Valencia and Umali-Garcia, 1994; Desmet et

al., 1996; Effendi et al., 1996).

1.3.1 Biology and silviculture of Pterocarpus indicus

a) Natural distribution and habitat of Pterocarpus

The genus Pterocarpus is pantropical and comprises of 20 species with 11 in tropical

western Africa, 5 in the Indo-Pacific region and 4 in tropical South America (Rojo,

1977). Most of these species are large trees with the African and Asian species

producing high-quality wood. Two main species of Pterocarpus dominate in

Southeast Asia and some Pacific islands, namely a) Pterocarpus indicus that is native

to the Philippines, Malaysia, Indonesia, Vanuatu, Solomon Islands and Papua New

Guinea and b) Pterocarpus macrocarpus which is native to Myanmar, Thailand,

Laos, Cambodia and Vietnam. There are nine species which are commercially

valuable while six other species are listed as threatened for being over-exploited.

These include P. indicus and P. santalinus. P. santalinus is sometimes called red

5

sandalwood of India and its international trade is listed under International Union for

Conservation of Nature and Natural Resources (IUCN, 2006) as Red List of

Threatened Species except for P. santalinus which is classified in Appendix II under

the Convention of International Trade in Endangered Species (CITES) (Rojo, 1977;

Oldfield et al., 1998) (Table 1.1). The species showed that it may only be traded

internationally if accompanied by appropriate permits.

Due to its wide geographical distribution, there is considerable morphological and

ecological variation in P. indicus. It is possible that trees planted at any given locality

tend to be uniform because of extensive local clonal propagation (Rojo, 1977; Ng,

1992). However, due to over-exploitation and selected felling in Peninsular

Malaysia, P. indicus is no longer naturally available except for some relic trees that

are confined to a few states of Penang, Melaka, Port Dickson (Negri Sembilan),

Rompin (Pahang) and Taiping (Perak) (Ridley, 1922; NAS, 1979; Wong, 1982;

Wong, 1983; Corner, 1988; Soerianegara and Kartawinata, 1985).

In general, P. indicus is found at low to medium altitudes (up to 750 m) but seems to

thrive up to 1300 m in primary and secondary forests. It grows on a variety of soil

types from fertile agricultural to rocky soils and along inundated river banks, swamps

and lagoons (Allen and Allen, 1981; Corner, 1988; Ng, 1992). The actual habitat, the

associated tree species, occurrence, and soil conditions required in which it can grow

well have not been studied in Malaysia in contrast to Pterocarpus macrocarpus in

Thailand (Dhanmanonda, 1992; Liengsiri et al., 1998).

b) Botanical description

Pterocarpus indicus is a large, deciduous tree growing up to 33 m tall with a

maximum diameter of 7.5 m at breast height. Leaves are shed during the monsoon or

6

hot dry season (February to April). The bole is usually short with small thick

buttresses and a spreading crown of drooping twigs and foliage. Large buttresses,

however, sometimes occur and have a well developed near-surface lateral rooting.

Crowns are large and bear many long branches that are at first ascending but

eventually arch over and sometimes droop at the ends. Trees with long, willowy,

drooping branches are particularly conspicuous and attractive for playgrounds and

roadside planting (Figure 1.1). Open-grown trees tend to be buttressed and branching

with poor bole form. However, the bole form may improve if trees are planted with

close spacing and with other silvicultural treatments such as pruning and fertilization

(Ng Lian Teck, pers. comm.).

Table 1.1 Commercial Pterocarpus species, distribution, common name and uses

Species/Region Distribution Local name Use

Asia: 5 species

dalbergiodes South Asia Andaman

padauk

Highly sought hardwood

indicus South East Asia Narra, sena,

angsana

Classic furniture

macrocarpus Myanmar and

Thailand

Burma padauk Luxury timber, furniture

marsupium India and Sri Lanka - Important after teak

santalinus India Red

sandalwood

Fragrant wood, substitute

for Santalum album

Africa: 4 species

angolensis South Africa Muninga Furniture and decorative

erinaceus Senegal - Furniture and decorative

osun and soyauxii Nigeria to Zaire African padauk Furniture and decorative

Source: Modified from Rojo (1977)

The bark is smooth, pale grey on young trees, later becoming rough, dark brownish

grey and longitudinally cracked about 1 cm thick. The inner bark is yellowish, tinged

with pink and when cut, the bark exudes a bright, dark red resin which is believed to

have medicinal values (Burkill, 1935). The leaves are compound-pinnate with 6-12

alternate leaflets. The leaflets range from 7 x 3.5 to 11 x 5.5 cm, ovate to elliptic in

7

Figure 1.1 Mature trees of Pterocarpus indicus that are commonly planted at (a) an

urban playground and (b, c) along roadsides for recreational and amenity purposes

in Peninsular Malaysia.

(a)

(b)

(c)

8

shape with pronounced acuminate tips. In areas with seasonal climate, P. indicus is

noted for its gregarious flowering as this is stimulated by a sudden drop in

temperature caused by a heavy downpour on a hot day (Holttum, 1954; Wong, 1982;

Corner, 1988). This synchronous flowering ensures good cross-pollination. The

flowers are yellow, fragrant and borne in large axillary panicles. The fruit is a

compressed, indehiscent samara, disk-like or sometimes falcate, broadly winged with

a thickened central, usually woody or corky, seed-bearing central portion containing

1-2 seeds (Figure 1.2 a, b). The seed is kidney-shaped, with a smooth to undulating,

brown to blackish seed coat (Figure 1.2c). It takes about 3-4 months to mature.

Unlike many legumes, the fruit is indehiscent and is dispersed both by wind and

water. A good description of the species is given by Claveria (1952), Assidao and

Nastor (1961), Corner (1988), Ng (1992), Appanah and Weinland (1993).

c) Wood properties and utilization

The sapwood is light yellowish brown to grey and generally quite narrow. The

heartwood is bright yellowish red to golden brown. The heartwood is lustrous when

first exposed but ages to a dull yellowish hue. The wood is classified as moderately

heavy, hard, strong and durable (550-900 kg/m3 at 15% moisture). Texture is

moderately fine to moderately coarse with grain sometimes wavy and interlocked.

The wood is ranked as the finest for furniture, paneling, cabinet work, carvings and

tool handles while other known uses include decorative sliced veneer, interior wall

paneling, feature flooring (including strip and parquet), musical instruments, boat

building and specialized joinery (Eddowes, 1977).

In the Philippines, the export of narra wood has declined substantially from 3 million

kg in 1985 to 2.3 million kg (57% processed) and 430,000 kg (processed) in 1986

9

and 1987, respectively. However, a total ban on the felling of P. indicus has been

imposed in the Philippines due to decline in the resource. The species has now been

classified as vulnerable in CITES, Appendix 2. Following this ban, countries like

Papua New Guinea, the Solomon Islands and Vanuatu have also banned round-wood

for export of this species. However, processed wood is allowed for export. The price

for sawn boards was estimated to be about US $600-800/m3 FOB (Evans, 2000;

Grace, 2000; FAO, 2001).

1.3.2 Silvicultural characteristics

a) Seed germination

Germination is epigeous with one fruit producing one to three seeds. There are about

2450 seeds available in every 100 g of seeds. Seed is preferred over dewinged,

scarified fruit for sowing because of the shorter germination time (1-2 weeks versus

>2 months) and a more uniform and higher germination rate (>85%). An optimum

temperature range of 25 to 35 oC is recommended for both P. indicus and P.

macrocarpus (Hundley, 1956; Liengsiri and Hellum, 1988; Coles and Boyle, 1999)

but in the Forest Research Institute of Malaysia (FRIM), seeds of P. indicus sowed at

this temperature range can give a germination rate of between 75-85% without any

pre-treatment (Nor Asma, pers. comm.).

b) Natural regeneration

It has been suggested that P. indicus behaves like a pioneer species and grows best

in the open although some shade is required in the early stage of establishment (Ng,

1992). This also applies to other species of Pterocarpus such as P. macrocarpus in

Thailand (Piewluang et al., 1997). For instance, an initial study by Piewluang (1996)

showed that germination, survival and height growth of P. macrocarpus seemed to

10

improve as conditions become more open and less competitive. As a rule, Troup

(1921) indicates that natural regeneration is reported to be good only in rather dry,

open forest but is scant or absent in more moist forest where the vegetation cover is

dense. However, this appears to be anecdotal as no inventory data are available. In

Myanmar, natural regeneration of P. macrocarpus was reported to be poor and this

may be due to the tough fibrous outer covering of the fruit and the hard testa as

germination was only 40-50 % (U Saw Yan, 1993).

c) Artificial regeneration

Early attempts at establishing plantations of Pterocarpus had mixed success. For

example, Troup (1921) reported that attempts to establish a P. macrocarpus

plantation by direct sowing pods or planting nursery-raised seedlings have generally

been unsuccessful. The establishment of a 60 years old P. indicus trial in FRIM also

failed to perform well due to high mortality (Ang, 1988). This failure may be due to

a lack of understanding of the silvicultural requirements of the species including site

preference. While many of the early trials were poorly managed, it became evident

that young seedlings were not competitive with Imperata or other tall grasses

suggesting that regular weeding activities are necessary.

d) Vegetative propagation

Vegetative propagation by cuttings is generally a preferred way for ornamental and

roadside plantings because growth of seedlings is slow and there is considerable

variation in vigour (Wong, 1982). Clonal material through vegetative cuttings also

provides a mean of perpetuating desirable characters such as clear, straight boles

suitable for timber production. P. indicus is also unique among the big timber trees

as it has the capacity to root easily in different soil types and rooting capacity is not

11

lost with age (Claveria, 1952; Assidao and Nastor, 1961; Allen and Allen, 1981;

Wong, 1982). In urban and some reforestation activities, cuttings are used to produce

„instant shade‟ and fast-growing trees when transplanted with a high survival rate of

90% in the Philippines (Claveria, 1952; Assidao and Cerna, 1960; Sardina, 1961;

Wong, 1982). Hence, to ensure that cuttings propagate easily, stem size of more than

3 m long and 5 to 10 cm diameter is normally used. Cuttings should be procured

from a straight, healthy branch that comprises several nodes with preference for

young woody branches (Browne, 1955; Wong, 1982).

e) Growth and yield

There is very little published data on the growth and yield of P. indicus and these are

summarized in Table 1.2. It has been suggested (Evans, 1982) that early maintenance

of P. indicus is necessary to improve plant growth in the field and that initial

inorganic fertilizer application using 50 gm of 15:15:15 (NPK) is recommended one

month after planting, followed by 100 g after every six months. Pruning also plays an

important role in the silvicultural treatment of P. indicus as undesirable branches

should be removed to prevent knot formation which may affect growth and wood

quality (Evans, 1982).

f) Pests and diseases

P. indicus is a fairly disease and pest resistant tree species. A problem occurred in the

period 1875 to 1925 which was attributed to leafhopper transmission of a fungal

pathogen (Corner, 1988). Other related pests include the leaf miner, Neolithocollettis

pentadesma which in 1990 caused severe leaf defoliation to trees planted in the open

12

Figure 1.2 Fruits and seeds of Pterocarpus indicus: a) green and b) dried winged

fruits and c) extracted mature seeds (scale bar shows size of seed).

(a)

(b)

(c)

13

and along roadsides throughout Peninsular Malaysia (Ahmad et al., 1996) and

Crematopsyche pendulla that caused severe damage to leaves resulting in necrosis

and holes (Ahmad, 1993).

Table 1.2 Growth data of Pterocarpus indicus recorded for isolated individuals or

in plantations.

Note: GBHOB = Girth at breast height over bark and DBHOB = Diameter at breast

height over bark . * unknown age. Source: (Lok, 1996).

1.3.3 Gaps in knowledge

There is an urgent need for studies on the nutrition requirements of P. indicus

primarily on phosphorus (P) supply, given that the species will be introduced to

either poor or marginal soils in Peninsular Malaysia. There is also a need to explore

the symbiotic relationship with nitrogen (N)-fixing bacteria given that the conversion

of secondary forests into plantations is likely to result in a loss of soil microbial

diversity. Constraints due to P and N are also likely to be exacerbated on severely

compacted sites such as skid trails and log landings where all vegetation and top soil

Age

(yr)

Total height (m) GHOB (m) DBHOB (cm) Author

5 8 0.3 - NAS (1979)

60 7.1 0.5 - Ang (1988)

100 27 4.6 - Wong (1982)

* 36.4 3.6 12.6 Browne (1955)

* - 1.0-1.6 2.4-4.4 Nwoboshi (1982)

* 15.2 - - Troup (1921)

* 30.3 - - Corner (1988)

* - > 4 - Foxworthy (1927)

14

has been removed. If P. indicus plantations are to be set up, good nursery and

silvicultural practices will have to be developed for the planting stock and nutrition

could be a key factor in their success. Again, there is little information on nursery

and silvicultural practices for P. indicus.

1.4 Nutrition and symbiotic requirements of tropical woody legumes

a) Role of phosphorus and nitrogen on tree growth

Most tropical forest plantations are likely to be established on either poor or marginal

sites where essential nutrients in soils for tree growth are low or poorly available

(Holford, 1997; Evans and Turnbull, 2004). For example, P has been identified as

one of the major limitations to tree growth on reforestation sites but little information

is available on the response of tropical plantation timber species to P fertilizers

(Gobbina et al., 1989; Goncalves et al., 1997; Tiessen, 1998). In the tropics,

deficiencies of many macro and micronutrients can result from soil leaching

(Stoorvogel et al., 1993; Goncalves et al., 1997; Sanchez et al., 1997). Phosphorus

and nitrogen have been identified as the main limiting macronutrients that affect

plant growth, particularly biomass accumulation (Begon et al., 1990; Shepherd et al.,

1996). Deficiencies in P and N may also lead to poor bole form, reduced microbial

activity and increased susceptibility to pests and disease during the nursery stage and

in young plants after out-planting (Kadeba, 1978; Kanowski et al., 1991;

McLaughlin, 1996; Norton and Firestone, 1996; Otsamo, 1996; Goncalves et al.,

1997; Kaye and Hart, 1997; Dell et al., 2001; Xu et al., 2001 ). Hence, a critically

short supply in these nutrients can lead to the failure to sustain tree growth. Not

surprisingly, fertilization is a consideration in many industrial tree estates in SE Asia.

An example of the responses that can be achieved with the addition of fertilizer is a

15

study carried out in Papua New Guinea on some indigenous tree species in a

degraded grassland using NPK (17:5:22) at 500 kg/ha (Table 1.3).

Historically, the use of fertilizer in forestry has been less emphasized than in

agriculture due to the slow growth of trees, long gestation period, uncertain

economic returns and use of species that are adapted well to low P sites (Evans,

1992). Applications of P are sometimes necessary to ensure early growth rates on

some soils such as with conifers (Ballard, 1974) (see also Table 1.3), or to shorten

harvest rotations (Evans, 1992). In south China, P fertilization has been regarded as

essential to ensure growth for fast-growing eucalypts in plantations (Wang and Zhou,

1996; Xu, 2001).

Most P fertilizer fixed by soil components is converted into forms which are not

readily available to plants (Grove and Keraitis, 1976; Subba Rao, 1982). Several

biological methods of enhancing nutrient uptake may reduce the amount of inorganic

fertilizer required for optimum growth. Legumes are known to have a high P

requirement for optimum growth, nodule formation and nitrogen fixation (Mosse et

al., 1976). Besides direct uptake of these nutrients, beneficial soil symbionts such as

arbuscular mycorrhizal fungi (AMF) and rhizobia (root nodule bacteria) can play an

important role in P and N cycling (Craswell et al., 1986; Brundrett, 1991). The

contributions derived from these symbioses by plants can also contribute to minimize

the use of fertilizers (Barea and Azcon-Aguilar, 1983). Dual symbioses are also

known to significantly increase the uptake of nutrients and improve the production of

seeds compared to plants that are singly treated with either one of the symbionts

(Barea and Azcon-Aguilar, 1983; Ikram et al., 1987; Tian et al., 2003).

16

For N, the largest contribution of biological N2-fixation in land plants is derived from

the symbiosis between legumes and root nodule bacteria (Burris and Roberts, 1993).

A significant proportion of these are tropical tree species whose relationship with

root nodule bacteria continue to be widely studied (e.g. Allen and Allen, 1981;

Moreira et al., 1992; Oyaizu et al., 1993; Odee et al., 1995; Sprent, 1995; Sprent and

Parsons, 2004; Sprent et al., 2010). Nitrogen-fixing bacteria make N directly

available to plants as NH3 in symbiotic associations using the nitrogenase enzyme in

bacteroids of root nodules (Bergersen et al., 1988; Giller and Wilson, 1991).

Table 1.3 Comparisons between height increment for P in unfertilized and fertilized

four-years old stands in Kunjingini, Papua New Guinea (Extracted from Lamb,

1975).

Species

Height increment without

fertilizer (m)

Year 1 Year 4

Height increment with

fertilizer (m)

Year 1 Year 4

Acacia auriculiformis 1.77 3.99 2.68 9.66

Eucalyptus deglupta 0.79 1.49 3.67 7.65

Eucalyptus tereticornis 0.94 2.04 2.77 4.78

Intsia bujuga 0.58 0.36 0.49 0.97

Pterocarpus indicus 0.67 0.73 1.31 1.61

Tectona grandis 0.21 dead 2.60 3.04

Terminalia brassii 0.52 1.13 2.50 4.69

Terminalia complanata 0.40 dead 0.76 1.34

b) Nutrient functions

Phosphorus

Phosphorus is essential for plant growth as it is involved in most metabolic

processes. It is a constituent of nucleic acids, phospholipids, phosphoproteins,

phosphate esters, dinucleotides and adenosine triphoshate. It is required for the

storage and transfer of energy, photosynthesis, electron transport processes,

17

regulation of some enzymes such as the synthesis of sugars and starch and the

transport of carbohydrates. Since P is phloem mobile, purple symptoms of P

deficiency are normally first observed in old leaves (Dell et al., 2001).

Nitrogen

Nitrogen is a constituent of amino acids, amides, amines, N bases, nucleic acids,

alkaloids, chlorophyll and many coenzymes. It is also a structural component of cell

walls in plants. The main symptom of N deficiency is leaf chlorosis which is due to

reduced chlorophyll formation or enhanced senescence. Symptoms typically first

appear in mature leaves and then spread to the whole canopy. In young eucalypt

seedlings, the symptoms usually occur in young leaves (Dell et al., 2001). Since

amino acids are also precursors of the polypeptide chains of proteins, N also

influences many enzyme reactions. Inorganic N is absorbed by roots as nitrate or

ammonium forms depending on the preference for some species and iron availability

(Marschner, 1995). For example, E. globulus seedlings utilize ammonium more

effectively than nitrate and are susceptible to nitrate toxicity when moderate to high

levels of nitrate-containing fertilizer are used (Shedley et al., 1995).

c) Diagnosis by symptoms

Where symptoms are specific, diagnosis by visual symptoms is a fast and simple

indicator for nutrient status in plants. Often, diagnosis of nutrient disorders by visual

assessment is applied together with soil analysis and plant tissue analysis due to

insufficient specific symptoms being expressed and other limitations. Details of

commonly encountered limitations have been described by Ulrich and Hills (1967),

Dell and Robinson (1993), Dell and Malajczuk (1994), Grundon et al. (1997),

Uchida (2000) and Wong (2005).

18

1.5 Principles of plant analysis

Limitation in using visual symptoms to diagnose nutrient deficiencies has resulted in

plant analysis being the preferred means of delineating the deficiency and sufficiency

levels of plants. This is because concentrations of nutrients in plant tissue are

determined by the combined effects of all the processes affecting the supply of

nutrients in soil and uptake by roots.

Principles were originally defined by Ulrich (1952) and Ulrich and Hills (1967)

using field crops but they were later applied to woody plants (Chapman, 1967;

Brown, 1970; Ballard, 1980; Dow and Roberts, 1982; Lambert, 1984; Mead, 1984;

Weeman and Wells, 1990; Paudyal and Mjid, 1992; Shedley et al., 1995; Timmer,

1997; Orlander, 1998; Dell et al., 2001; Landis et al., 2004). Using the relationship

between nutrient concentration and plant yield, commonly shoot biomass (Smith,

1986), critical concentrations have been established for many economic field crop

species, but is less certain for timber species. The generalized relationship between

plant yield and nutrient concentration is shown in Figure 1.3 which shows four

zones:

1. Deficiency zone where with small changes in plant nutrient concentration,

plant growth increases sharply. The slight increase in nutrient concentration

may be attributed to the fact that for each additional increment of nutrient

absorbed by the plant, there is a corresponding increment of additional

growth;

2. Marginally deficiency zone where the increase in yield slows as the nutrient

concentration increases;

19

3. Adequate-luxury supply zone where an increase in nutrient markedly

increases the nutrient concentration in the plant but has no effect on its

growth, this suggests that the added nutrient is no longer the factor that limits

growth; and

4. Toxicity zone where an increase in nutrient supply increases nutrient

concentration in the plant to a toxic level causing a decline in plant growth.

One variation in the general calibration curve is known as the “Piper-Steenbjerg”

effect or “C-shaped” curve (Piper, 1942; Steenbjerg, 1951) which results when the

addition of a limiting nutrient to a severely deficient plant increases yield but

decreases nutrient concentration (Figure 1.3, lower). Plant analysis therefore, has

very limited value for diagnostic purposes unless the “C-shaped curve” can be

avoided. Ulrich and Hills (1967) suggested that this effect can be minimized by

sampling when early visible symptoms first appear. Alternatively, the “Piper-

Steenbjerg” effect may sometimes be avoided by sampling specific parts for nutrient

analysis. For example, by contrast with the whole shoot, young leaves of Cu-

deficient wheat, subterranean clover and peanut showed no “Piper-Steenbjerg” effect

(Robson, 1977; Loneragan et al., 1980; Reuter et al., 1981).

The diagnosis of nutrient deficiencies from plant analysis is based on the concept of

“critical nutrient concentration”. Ulrich (1952) has suggested that the critical

concentration of nutrient deficiency should be defined as the nutrient concentration

that is just deficient for maximum growth, adequate for maximum growth or the

concentration separating the zone of deficiency from the zone of adequacy and this

concentration is often taken at 5 or 10% below the maximum yield. He also

concluded that plant parts should be chosen in the narrow transition zone between

deficiency and luxury concentrations. Others have suggested that the critical

20

concentration should be in a range rather than a single value (Follet, 1965; Dow and

Roberts, 1982).

The relationships shown in Figure 1.3 are usually determined by growing plants with

a range of nutrient levels of the element being examined with all other essential

elements being adequate. For annual species, experiments may be undertaken in the

field or in pot culture. For long-lived tree species, it is much more difficult to obtain

field data to plot the relationship in Figure 1.3, and for this reason effort has

generally been placed on seedlings or cuttings. Whether plants can grown in the field

or in a greenhouse, harvest and chemical analyses are carried out, when there are

deficiency symptoms at the lower treatment or visible growth response is evident.

Figure 1.3 Relationships between nutrient concentration in plant tissues and growth

(Upper: Smith and Loneragan 1997; Lower: Ulrich and Hills, 1967).

21

The most common procedures for deriving the critical concentration of a given

nutrient have been the hand-fitted curve, the Mitscherlich equation (Ware et al.,

1982), the graphical and the statistical Cate Nelson procedures (Cate and Nelson,

1971), and the Smith and Dolby two phase linear model (Smith and Dolby, 1977).

However, the selection of a defined point on a hand-fitted curve has been the most

common method for deriving a critical nutrient concentration in early studies (Smith,

1986). The two main criticisms of the latter method are the subjective assessment

involved in the location of the curve through the points especially in the marginal

zone and the arbitrary use of 90% maximum yield to determine the corresponding

critical concentration.

1.6 Alternative approaches for determining nutrient disorders

Although plant analysis is the common method for nutrient deficiency diagnosis in

plant tissues, other methods have been applied with varying success. These are

briefly described below.

a) Comparison of healthy and unhealthy trees

Comparing nutrient concentrations in healthy and unhealthy plant tissues is a quick

and common way for preliminary assessment of nutrient disorders. Difficulties may

arise in the following situations: i) changes in severity of nutrient deficiencies with

plant age, ii) presence of symptoms caused by non nutritional factors, iii) similarity

of some symptoms associated with different nutrients, and iv) multiple effects from

multiple nutrient disorders. The disadvantage of using unhealthy tissue is that

symptoms may only appear when the nutrient supply to roots has markedly reduced

growth or caused stem malformation (Lambert, 1984; Mead, 1984; Dell, 1996;

22

Uchida, 2000; Dell et al., 2001). Detail on the sampling of plant tissues has been

described by Dell et al. (2001) for the diagnosis of nutrient disorders in eucalypts.

b) Nutrient ratios

This concept called Diagnostic and Recommendation Integrated System (DRIS) was

first developed by Beaufils (1957; 1971; 1973) and is described by Walworth and

Sumner (1987). The method involves measurement of the ratio of two or more

nutrients within a plant tissue and comparison with a predetermined range where

plant growth will not be limited. For example, using this method Schaffer (1988)

found that a combination of both manganese (Mn) and iron (Fe) are responsible for

the decline in mango yield.

Unlike critical nutrient concentrations, DRIS is a predictive rather than diagnostic

technique and thus may not be universally useful for plant analysis in forestry

practices (Smith and Loneragan, 1997). DRIS has been applied to identify yield-

limiting nutrients, sensitivity and long-term variations of different doses of

fertilization practices in fruit and other cash crops such as corn, mango, maize,

pineapple, sugarcane and sweet potato (Beaufils, 1971; Elwali and Gascho, 1983).

c) Biochemical tests

Biochemical techniques are more commonly applied in agricultural and horticultural

crops for nutrient-status of plants but have little application so far in forestry.

However, biochemical tests for P and Cu in eucalypts have been suggested (Dell,

1996). Biochemical tests are mostly based on the activity of enzymes which require

essential elements for normal functioning. For example, phosphatase activity in plant

tissues of E. diversicolor seedlings was shown to be a more useful indicator of P

status than foliar P concentrations in a glasshouse study (O‟Connell and Grove,

23

1987) and a lignin stain has been used to distinguish between Cu-deficient and Cu-

adequate E. maculata trees (Dell and Bywaters, 1989; Dell, 1994).

Unfortunately, none of these tests are presently being used commercially in forestry.

Biochemical assays may be better than nutrient analysis for predicting the nutrient

status of a plant where significant amounts of nutrients are held in physiologically

inactive forms (Leece, 1976) but with the lack of specificity and complexity in their

measurements they are seen as disadvantages.

1.7 N-fixing trees and root nodule bacteria

Quite a number of commercial timber species are N-fixing trees and these include

genera like Acacia, Casuarina, Dalbergia, Paraserianthes and Pterocarpus.

However, despite their abundance, diversity and economic importance as prized

timber species, few have been examined for their ability to nodulate and fix

atmospheric N symbiotically with root nodule bacteria (Allen and Allen, 1981; de

Faria et al. 1989; Wester and Hogberg, 1989; Sprent, 2006). Not only do some of

these species have potential for hardwood plantations but they could play an

important role as pioneer species in the rehabilitation of degraded and over-exploited

forests (Sprent, 2001). Generally, plantations of N-fixing trees are known to have

higher rates of nutrient cycling than plantations of non-fixing trees but little is known

of the long-term influence on soil properties (Binkley et al., 2000).

The N-fixing symbionts normally gain access to carbon substrates, nutrients and

protection from desiccation in return for fixing atmospheric N which is transferred to

the host plant for further processing into amino acids and proteins (Long, 1996;

Waters et al., 1998). This symbiotic relationship is at an advantage where N is a

limiting nutrient for plant growth, such as in many tropical ecosystems. Tropical

24

woody legumes, especially in the sub-family Papilionceae, are capable of nodulating

with local soil root nodule bacteria but little is known about the specificity, symbiotic

relationships, soil type preference or the capacity to fix N, especially in P. indicus

and Dalbergia spp. (Allen and Allen, 1981; de Faria, 1989; Moreira et al., 1998;

Roggy and Prevost, 1999; Sprent and Parsons, 2000; Sprent, 2001; Diabate et al.,

2005). According to de Faria et al. (1989) and Sprent (1995), only 57% and 20% of

the genera and species respectively in the tropical legumes have been examined but

poorly studied.

Planting of tropical woody legumes is normally integrated with agro-forestry

practices (Brewbaker, 1983). They are used as nurse or shade trees and in land

reclamation (Hegazi et al., 1994). Favoured species include Acacia mangium,

Leucaena leucocephala, Paraserianthes falcataria, Parkia javanica and Tamarindus

indica (Hashim et al.,1996). However, there is still a lack of knowledge regarding

tropical woody legumes. Attention is being focused on their role in nutrient cycling,

in their usefulness in reforestation projects, potential uses in wood and non-wood

products, and capacity to become invaders of new habitats such as Australian acacias

in South Africa and in some Asian regions (Allen and Allen, 1981; Brewbaker et al.,

1982; de Faria et al., 1987; Wilson and Tilman, 1991; Witkowski, 1991a, 1991b;

Berger, 1993; Streeter, 1994; Sprent, 1995; Unkovich et al., 1997; Dart, 1998;

Chang and Handley, 2000).

1.8 Conclusions

There is an urgent need for studies on the nutrition requirements of P. indicus

primarily on phosphorus supply, given that most forest plantation trees will be

introduced on rather poor and marginal soils in Peninsular Malaysia. There is also a

25

need to explore the symbiotic relationship between N-fixing bacteria given that the

conversion of secondary forests into plantations is likely to result in a loss of soil

microbial diversity (Hau and Corlett, 2003). More severe impacts are likely on most

high impacted sites such as skid trails and log landings where all vegetation and most

of the top soil have been depleted. If P. indicus plantations are to be set up, good

nursery practices will have to be developed for the planting stock and nutrition could

be a key factor. Again, there is little information on nursery practices for P. indicus.

1.9 Aims of the thesis

Pterocarpus indicus is a promising tree legume species with fast growth rates,

desirable characteristics for plantation forestry and an important tree for high quality

timber. However, plantations of this species are in their infancy although serious

attention has been given to the species as a result of over-exploitation in its natural

habitat. The silviculture requirements of P. indicus, including fertilizer requirements

and foliar nutrient standards of seedlings at the nursery stage or at field

establishment, are poorly understood. The capacity to form an effective symbiotic

relationship with root nodule bacteria and mycorrhizal fungi are also highly

recommended given the likelihood that most forest plantations in the tropics are

likely to be established on sites with silvicultural constraints. Hence, the overall aim

of this study is to provide a scientific basis for diagnosing nutrient deficiency in

order to recommend on fertilizer use and application of effective N-fixing symbionts

so as to optimize nursery operations and productivity of Pterocarpus indicus after

out-planting.

26

The specific objectives are:

a) Examine the sensitivity of P. indicus to changes in external P supply and identify

foliar nutrient concentrations that define the P status of seedlings under nursery

conditions;

b) Identify effective strains of root nodule bacteria for nodulation and N-fixing

capabilities;

c) Document responses in P. indicus roots at low external P supply; and

d) Provide recommendations on management of P and inoculation of P. indicus in

tropical nurseries.

Figure 1.4 shows the structure of the thesis in relation to these objectives.

27

Figure 1.4 shows the structure of the thesis in relation to these objectives.

Identifying

limiting

nutrients

Changes in root

morphology

Major nutrient constraints on growth,

diagnosis and symbiosis effects with root

nodule bacteria in nitrogen-fixing Pterocarpus

indicus (Chapter 1)

Identifying

limiting nutrients

Lack of information

on effective root

nodule bacteria

Identify effective root nodule

bacteria for nodulation and

nitrogen-fixation

(Chapter 5)

General discussion and recommendations (Chapter 7)

Diagnosis of P and N

deficiencies using

plant analysis

(Chapter 2)

Root nodule

bacteria for

nodulation

(Chapter 4)

Changes in root

morphology due to P

supply and root

nodule bacteria

(Chapter 6)

Comparison of P response to

3 other leguminous tree

species

(Chapter 3)

28

Chapter 2

Diagnosis of phosphorus and nitrogen disorders in

Pterocarpus indicus seedlings

2.1 Introduction

Pterocarpus indicus, is a potential tree species for forest plantation establishment in

Peninsular Malaysia (Soerianegara and Lemmens, 1993; FRIM 2002). It is expected

that constraints on macronutrient soil fertility may limit productivity (Holford, 1997;

Evans and Turnbull, 2004). Deficiency of phosphorus (P) or nitrogen (N) can

severely restrict tree growth rates, leading to poor bole form and may increase

susceptibility of trees to pests and diseases (Kadeba, 1978; Kanowski et al., 1991;

Otsamo, 1996; Goncacalves et al., 1997). Hence, in order to achieve satisfactory tree

growth these nutrients must be adequately supplied. In addition, appropriate

silvicultural operations and good forest management practices are necessary to

maintain soil fertility, reduce soil erosion and conserve water through subsequent

rotations so that the productive potential of the site can be maintained. Appropriate

forest plantation management techniques also favour the maintenance of biological

diversity (Troup, 1921; Bunyavejchewin, 1983; Sprent et al., 1989; Moreira et al.,

1992; Marschner, 1995; Odee et al., 1995; Oldfield et al., 1998; Sylla et al., 1998).

Analysis of foliar nutrient concentrations could provide information on the

nutritional status and requirements for long term survival and optimal growth of P.

indicus in plantations. Although the relationship between foliar nutrients and growth

has been broadly studied and successfully applied to pines and eucalypts (Lambert,

1984; Turner and Lambert, 1986; Drechsel and Zech, 1991; Dell et al., 2001), it has

29

not been widely applied to many tropical hardwood species. These relationships are

generally derived from fertilizer rates trials which are tedious to undertake. So far,

such trials are lacking for P. indicus.

Hence, glasshouse trials were undertaken with P. indicus with the following

objectives:

a) to determine the nutrient concentration ranges in the foliage of deficient

and healthy plants;

b) where possible to define critical concentrations for diagnosis of N and P

deficiencies; and

c) to define fertilizer responses in order to establish suitable rates of N and P

for experiments on root nodulation.

The data obtained could provide useful information for nursery managers and serve

as a guide for identifying some macronutrient constraints in seedlings following out-

planting in the field.

2.2 Materials and methods

2.2.1 Experimental design

Separate P and N pot trials were carried out under glasshouse conditions on two soil

types: Yalanbee sandy loam (YB) and a yellow sand (YS). A randomized complete

block design (RCBD) was used for each pot trial, consisting of ten treatments and

four replicate pots. Four germinated seedlings of P. indicus were planted per pot and

later thinned to three per pot to prevent self shading. The P treatments for the YB

trial were 0, 5, 10, 20, 40, 80, 160, 320, 640 and 1280 mg P kg-1

soil, hereafter

designated as P0, P5, …and P1280 and for YS were 0, 2, 4, 8, 16, 32, 64, 128, 256

and 512 mg P kg-1

sand, hereafter designated as P0, P2, …and P512. Phosphorus was

30

supplied as aerophos, Ca(H2PO4)2.H2O (from Albright and Wilson Australian Ltd).

The N treatments for the YB trial were 0, 5, 10, 15, 20, 25, 30, 35, 40, 45 mg N kg-1

soil, and hereafter designated as N0, N5, …and N45 and for YS were 0, 5, 10, 20, 30,

40, 50, 60, 70 and 80 mg N kg-1

sand, hereafter designated as N0, N5, …and N80.

Nitrogen was supplied as NH4NO3. The P and N trials on YB were carried out from

October, 2002 to January, 2003 (Spring-Summer) and the P and N trials on YS were

undertaken from July-October, 2003 (Winter-Spring), in an evaporatively cooled

glasshouse at Murdoch University, Western Australia. The annual mean temperatures

for both trials are given in Appendix I.

2.2.2 Seed and plant material

Seeds were obtained from large trees in the campus ground of the Forest Research

Institute of Malaysia (FRIM), Kuala Lumpur, Malaysia. Seeds were surface sterilized

in 70% (v/v) ethanol for 60 seconds, followed by 45 seconds in freshly prepared 4%

(v/v) sodium hypochlorite, and then rinsed in sterile distilled water before sowing.

The sterilized seeds were sown in plastic trays 29 cm wide x 35 cm length,

containing yellow sand which had been sterilized by autoclaving for 20 minutes at

121oC. Four germinated seedlings of P. indicus at the two-leaf stage were then

transferred into each treatment pot after about ten days.

2.2.3 Soil information

The YB soil is a typical laterite soil occurring on the Darling Range in Western

Australia. Generally, these laterite soils are infertile, have poor water holding

capacity, and strongly adsorb phosphate (Seddon, 1972). Crops here require inputs of

N and P fertilizers to produce commercial returns and in some places these can

induce secondary deficiencies of micronutrients such as Zn. The native vegetation is

31

a dry sclerophyll, open eucalypt forest. Soils in the region are deep sandy gravels

with the top soil classified as sand to sandy loam and the subsoil as sand to clayey

sand. Soil which had never been fertilized was collected at 0-20 cm depth from the

Allandale Research Farm Station in Wundowie which is about 63 km east of Perth

(31o47’S, 116

o22’E) and immediately north of the Great Eastern Highway. The soil

was sieved through a 4 x 4 mm stainless steel field mesh and mixed thoroughly

(Figure 2.1). The yellow sand was obtained from under virgin banksia woodland on

the Swan Coastal Plain near Gnangara north of Perth (31o46 ’S, 115

o51’E). Chemical

properties of the two virgin soil types are given in Table 2.1. These soils have

previously been used in studies on eucalypt nutrition and mycorrhizal responses

(Brundrett et al., 1994; McLaughlin, 1996; Karopoulos, 1999; Asher et al., 2002).

2.2.4 Background on climate for Perth, Western Australia

Perth is subjected to a Mediterranean climate, distinctively characterised by cool, wet

winters and very hot, dry summers. Heavy rainfall with gusty wind is typical in

winter. Approximately 80-95% of the annual average rainfall of 869 mm falls

between the months of May to October (Gerritse and Schofield, 1989).

2.2.5 Soil preparation and nutrient treatments

All trial soil was steam pasteurized twice at 90 oC for one hour to kill any pathogenic

or symbiotic organisms (Landis et al., 1989), allowed to cool for 24 hours, and re-

steamed. Similar pot culture trials have also been described by Brundrett et al.

(1996). Many soils in south-western Australia are infested with Phytophthora. The

pasteurized soil was then spread thinly over clean plastic and air-dried for five days.

Three kilograms dry soil was transferred into each non-draining plastic pot (150 cm

diameter x 17.5 mm height) lined with a clear plastic bag.

32

Figure 2.1 Yalanbee soil collection at the Allandale Research Station, Wundowie,

Western Australia. a1-a2) Sites where soil was collected to 20 cm depth, b1-b2)

Digging and collecting into gunny sacks, c) Soil that has been sieved and air-dried

before being potted.

(a1) (a2)

(b1) (b2)

(c)

33

Basal nutrients were applied to the surface of soil in solution (mg/kg soil): YB-51

Ca, 20 P, 20 N, 52 K, 7.4 Mg, 3 Mn, 2.3 Cu, 3 Zn, 0.1 Co, 0.6 Mo, 0.6 Na, and YS-

31 Ca, 48 P, 20 N, 90 K, 25 Mg, 15 Mn, 12.3 Cu, 13.8 Zn, 1 Co, 1.2 Mo, 3.3 Na.

When dry, the basal nutrients and treatments were mixed throughout by shaking the

soil in a plastic container for two minutes by hand. Additional N was supplied

fortnightly in an aqueous solution over 11 weeks to give a total of 99 mg N/pot. The

plants were not inoculated with N-fixing bacteria. The pots were watered by weight

to 10% field capacity and left to incubate for two days before planting (Figure 2.2).

After planting, pots were daily watered to field capacity.

2.2.6 Harvesting and plant analysis

The stem heights of the plants were taken before they were harvested at 10 weeks

old. Shoots were separated from the roots at one cm above the soil level and then

partitioned into the youngest fully expanded leaf (YFEL) and the rest of the shoot.

Roots were recovered by gently washing the soil over a 2 mm sieve and care was

taken to minimize loss of fine roots and root tips. Total root length was measured

using ASSESS software (Lamari, 2002). The plant parts were oven-dried at 70 oC for

at least 48 hours to constant weight. The dried YFELs were ground by hand in a

porcelain mortar and pestle. For P, the plant material was digested in concentrated

nitric acid (70% w/w) and H2O2 (30% w/w) using an open-vessel microwave oven

(CEM Mars 5) as described by Huang et al. (2004). All chemicals used were of

analytical grade. Total Kjeldahl N was determined following digestion of material in

concentrated sulphuric acid (98% w/w) as described by Zarcinas et al. (1987). Blank

solutions of sulphuric acid at 0, 0.5, 1, 2 and 4 mL together with a standard reference

material, Eucalypt 2000, were used in each digest batch to check for contamination.

34

Figure 2.2 Glasshouse trials using a) Yalanbee soil and b) yellow sand for the P-

growth response of Pterocarpus indicus seedlings. The surface of the yellow sand

was covered with white sterile polyvinyl chloride beads to reduce evaporation.

(a)

(b)

35

Total P and N were measured by induction coupled plasma spectroscopy. Combined

core soil samples of 200 g from each treatment were collected and sent to a

commercial laboratory (Westfarmers CSBP Limited) for analysis (Table 2.1).

Table 2.1 Mean soil chemical properties for the virgin Yalanbee soil and yellow sand

2.2.7 Statistical analysis

Data at harvest were analyzed using SPSS version 11.5. The effects of fertilizer

treatments on shoot dry weight, root biomass, root-whole plant biomass and root-

length ratio were subjected to analysis of variance (ANOVA). Means were compared

using Duncan’s New Multiple Range Test (DMRT) where ANOVA showed that

there was a significant difference between treatment means (P≤0.05). All data were

checked for normality before being analyzed and square root transformation was

carried out where appropriate to satisfy the homogeneity test by using Bartlett test at

F≤ 0.01.

A hand-fitted curve method was used for data analysis.

2.3 Results

2.3.1 Growth

a) Phosphorus

Property

Yalanbee soil Yellow sand

P (Colwell) (mg kg-1

) 20 4

Nitrate-N (mg kg-1

) 1 <1

Ammonium-N (mg kg-1

) 5 5

K (Colwell) (mg kg-1

) 60 20

S (mg kg-1

) 8.8 11.8

Organic carbon (%) 2.26 0.06

pH (H2O) 5.7 5.6

pH (CaCl2) 6.3 6.2

36

There was a significant shoot growth response of P. indicus to P fertilizer (P≤0.05).

Generally, growth of seedlings also increased with increasing P fertilizer treatments

in all soil types (Figures 2.3a and 2.4a) up to a maximum. There was a steep increase

at low P rates, a narrow optimum range and shoot growth at high P rates was

depressed. Maximum seedling growth occurred at lower rates of P in YS [(P128

(0.56 ± 0.04 g)] than in YB [P320 (3.60 ± 0.13 g)]. The stem height in YB plants was

about 50% higher than that in YS plants (Appendix II). Growth measured as shoot

dry weight, root biomass and total root length was severely depressed at low P rates

(P0-P32) in YS plants and at P0-P40 for YB plants. Withholding P reduced plant

yield by up to 390% and 330% in YS and YB, respectively.

Interestingly, both soil types shared a similar Colwell P concentration of 118 mg/kg

soil at maximum shoot dry weight (Figures 2.3b and 2.4b) even though the YB had a

greater buffering capacity at higher fertilizer rates and plant toxicity symptoms were

less severe (Figure not shown). Soil analysis showed that Colwell P was lower in YS

than YB at low fertilizer rates (Table 2.1).

b) Nitrogen

There was a significant growth response (P≤0.05) in YS to N fertilizer. As there was

no response in growth of P. indicus to N fertilizer in YB, this experiment is not

discussed further here. In YS, shoot dry weight increased linearly with N rates in the

deficiency range (N0-N5) then remained constant (N10-N80) (Figure 2.5). Soil

analysis suggests that P. indicus may have had a preference for ammonium-N over

nitrate-N at luxury N supply (Table 2.2). Concentrations of NH4+

remained low at 1

mg/kg across all N treatments whereas soil NO3-

concentrations increased at high

(50-80) N rates.

37

0 200 400 600 800 1000 1200 1400

P treatment (mg P/kg soil)

0

1

2

3

4

5

Sh

oo

t d

ry w

eig

ht

(g/p

ot)

(a)

0 100 200 300 400 500

Colwell phosphorus (mg/kg soil)

0

1

2

3

4

5

Sh

oo

t d

ry w

eig

ht

(g/p

ot)

(b)

Figure 2.3 Effects of P fertilizer (a) on shoot dry weight of Pterocarpus indicus and

(b) soil available P (Colwell) in Yalanbee soil. Data are means of 4 replicates with

standard error bars.

38

0 100 200 300 400 500 600

P treatment (mg P/kg soil)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Sh

oot

dry

weig

ht

(g/p

ot)

(c)

0 100 200 300 400 500

Colwell phosphorus (mg/kg soil)

0.0

0.2

0.4

0.6

0.8

Sh

oot

dry

weig

ht

(g/p

ot)

(d)

Figure 2.4 Effects of P fertilizer (a) on shoot dry weight of Pterocarpus indicus and

(b) soil available P (Colwell) in yellow sand. Data are means of 4 replicates with

standard error bars.

(a)

(b)

39

2.3.2 Foliar nutrient concentrations and plant symptoms

a) Phosphorus

The P concentration in the YFEL increased linearly with increase in P fertilizer from

0 to 19.2 mg/g (at P0-P512) in the YS plants and 0 to 5.5 mg/g (at P0-P1280) in YB

plants (Figure 2.6). Symptoms of P deficiency and toxicity are described in Table 2.3

along with P concentration ranges in the corresponding YFELs. The foliar contents

on different treatments are as in Appendix III.

Table 2.2 Mean properties of the soils at harvest for the P trial in Yalanbee (YB) and

yellow sand (YS) and the N trial in yellow sand.

1. YB-Phosphorus 0 5 10 20 40 80 160 320 640 1280

Colwell P (mg kg-1

) 6 9 12 14 24 45 85 118 298 458

Nitrate-N (mg kg-1

) 51 59 60 51 40 42 54 52 67 66

Ammonium-N (mg kg-1

) 71 79 82 61 46 49 51 51 57 80

pH (H2O) 5.8 5.9 5.8 5.8 5.9 5.8 5.7 5.7 5.8 5.9

pH (CaCl2) 5.3 5.4 5.3 5.2 5.3 5.2 5.2 5.3 5.3 5.4

2. YS-Phosphorus 0 2 4 8 16 32 64 128 256 512

Colwell P (mg kg-1

) 1 1 2 1 5 13 29 70 137 305

Nitrate-N (mg kg-1

) 48 51 47 41 56 43 35 30 32 27

Ammonium-N (mg kg-1

) 19 13 10 11 9 10 8 7 15 22

pH (H2O) 5.9 5.8 5.8 5.6 5.5 5.5 5.8 6.1 6.0 6.5

pH (CaCl2) 5.1 5.3 5.3 5.0 5.1 5.3 5.3 5.6 5.4 6.1

3. YS-Nitrogen 0 5 10 20 30 40 50 60 70 80

Colwell P (mg kg-1

) 20 23 20 20 23 25 23 23 21 22

Nitrate-N (mg kg-1

) 1 1 1 1 3 7 14 17 19 37

Ammonium-N (mg kg-1

) 1 1 1 3 1 1 1 1 1 1

pH (H2O) 5.6 5.7 5.6 5.6 5.5 5.6 5.6 5.6 5.5 5.5

pH (CaCl2) 5.9 6.1 6.4 5.1 5.0 5.1 5.0 5.0 5.0 5.0

40

0 10 20 30 40 50 60 70 80 90

N treatment (mg N/kg soil)

0.0

0.1

0.2

0.3

0.4

0.5

Sh

oot

dry

weig

ht

(g/p

ot)

Figure 2.5 Effect of N fertilizer on shoot dry weight of Pterocarpus indicus in

yellow sand. Data are means of 4 replicates with standard error bars.

y = 0.0375x

R2 = 0.9498

y = 0.0043x

R2 = 0.8821

0

5

10

15

20

25

0 200 400 600 800 1000 1200 1400

Fertiliser P rates (mg/kg soil)

YF

EL

co

ncen

trati

on

(m

g/g

)

YS Yellow sand YB Yalanbee soil

Figure 2.6 Change in total P concentration in the youngest fully expanded leaf

(YFEL) of Pterocarpus indicus seedlings in response to P fertilizer. Points represent

means of four replicates. Functions with R2

values are fitted separately to individual

replicate data for the YS and YB experiments.

41

Generally, P. indicus seedlings grown in YS produced smaller leaves than those

grown in YB. This is likely to have been due to the lower glasshouse temperatures

and shorter days in winter when YS trial was setup as compared to summer in the

YB trial. However, YS plants showed early deficiency symptoms (Figure 2.7a) at

lower P fertilizer treatments (P0-P32) than in YB plants (P0-P80). These seedlings

had depressed shoot dry weight, reduced size of the older leaves, light green or

chlorotic foliage and short stems that were highly branched and usually rosette-like

in appearance (Table 2.3). There was also reduced root development (Figure 2.8).

The YS plants with high YFEL concentration (at P256-512) had depressed (P≤0.05)

growth (root biomass and total root length) (Appendix IV). Similarly, YB plants

grown at P640-1280 had high foliar P concentration and depressed (P≤0.05) shoot

dry weight (Figure 2.11). Symptoms are given in Figure 2.7c and Figure 2.9.

b) Nitrogen

Unlike for P in the P rates trials, there was no linear relationship between foliar N

concentration and fertilizer rate for plants grown in YS (Figure 2.10). From N0 to

N40, foliar N concentrations were low but increased four-fold at N50 and above.

These two groups correspond to deficient and adequate plants respectively. Low or

deficient seedlings showed stunted growth (Table 2.3) with smaller leaves that were

yellowish green (Figure 2.7d) and had reduced root length (Appendix 2.3). At the

highest N rate, leaves were small, flaccid and the tips were necrotic, suggesting N

toxicity.

2.3.3 Critical phosphorus concentrations

Critical P concentrations in the YFEL of P. indicus seedlings corresponding to 90%

of maximum shoot dry weight were derived from linear relationships fitted to the

42

Table 2.3 Related symptoms and nutrient concentrations in the youngest fully

expanded leaves of Pterocarpus indicus.

shoot dry weight and the foliar P data (Figure 2.11). The fitted lines were divided

into three zones, namely: 1) zone of deficiency, 2) zone of adequacy and 3) zone of

toxicity. Data points for seedlings with severe toxicity symptoms were omitted for

the line fitting exercise. The regression coefficients (R2) for deficiency, adequacy and

Element Description Symptom Concentration

(mg g-1

dry

weight)

P Deficient

Mildly

deficient

Adequate

Mildly

toxic

Toxic

Malformed, stunted growth with soft

small leaves, early leaf shedding,

stunted stems, short internodes and

malformed apical buds

Leaves light green

Dark green healthy leaves and taller

seedlings

Mature leaves with marginal and

interveinal chlorosis

Shorter, stunted stems and coppicing,

branches with rosette-like small

leaves, shoot dying back from apex,

old leaves pale yellow with necrotic

spots, poor root development

<0.48 - 0.90

0.97 - 1.49

2.42 - 3.56

3.57-4.64

>4.65

N Deficient

Stunted growth with leaves pale,

uniform yellow

<4.0

Mildly

Deficient

Adequate

Mature leaves with marginal and

interveinal chlorosis, with young

pale green leaves

All leaves were dark green

4.0-7.0

20-35

43

toxicity zones were 0.39, 0.70 and 0.99, respectively. The estimated P concentration

in the YFEL for the diagnosis of P deficiency at 90% maximum shoot yield, was

0.17% and for diagnosis of P toxicity was 0.41%. These critical P concentrations

were estimated from bivariate fits using JMP.SAS (Version 5).

2.3.4 Critical nitrogen concentration and yield

In the nitrogen trial in YS, due to the wide gap in N concentrations in the YFEL of

deficient and adequate plants, it was not possible to define a critical leaf

concentration for the diagnosis of deficiency or toxicity. No linear relationship

between foliar N concentration and fertilizer rate for plants growth in YS was

obtained and this was previously explained under subheading 2.3.2 b.

2.4 Discussion

There was a strong growth response of Pterocarpus indicus to fertilizer P in the two

soils. The soils were selected because they had low concentrations of available P and

differ in P adsorption capacity (Schoknecht, 1997). Historically in the region, acute P

deficiency occurs when the land is first cleared for agriculture or plantation forestry.

Usually, when P fertilizers are applied the water-soluble P component reacts rapidly

with the surfaces of soil constituents (by adsorption to iron and aluminium oxides)

and with cations in soil solution (e.g. by precipitation with ions of calcium, iron and

aluminium) (Barrow, 1980) restricting P available for absorption by plant roots or

mycorrhizal fungi. As fertilizer P increased, there was an increase in Colwell P

(Colwell, 1963) and plant growth. This sodium bicarbonate soil test is widely used in

Western Australia and has been applied to prescribe fertilizer P requirements

44

Figure 2.7 Leaf symptoms observed in Pterocarpus indicus seedlings in response to

soil P in Yalanbee soil and soil N in yellow sand. Upper left to right: (a) changes in

leaf size with yellowish small malformed leaf for P10, healthy green P-adequate for

P320 leaves and chlorosis reduced size leaves for P1280 and (b) Lower left to right:

N-deficient leaves showing stunted growth and uniform pale yellow leaves in N5

treatment, mildly N-deficient N30 pale yellow leaves in N30 and healthy uniform

dark green leaves in N70.

(a)

(b)

P10 P320 P1280

N5

N30

N70

45

Figure 2.8 P deficient symptoms in Pterocarpus indicus seedlings at a) P0 treatment

showing stunted stems, b) adequate P160 treatment with tall green seedlings, c) (i) P-

deficient plant, (ii-iii) just adequate-P plants and iv) plant with P-toxicity – note the

stunted root growth.

(a)

(b)

(c)

(i) (ii)

(iii)

(iv)

46

Figure 2.9 P toxicity symptoms in Pterocarpus indicus at P640 and P1280

treatments. (a) mature leaves with interveinal chlorosis, (b) leaves with necrotic spots

and (c) stunted plants showing coppicing and rosette-like small leaves.

(b)

(a)

(c)

47

0

5

10

15

20

25

30

35

40

0 20 40 60 80 100

Fertiliser, N rates (mg/kg soil)

N C

on

cen

trati

on

(m

g/g

)

Figure 2.10 Relationship between N fertilizer rates and N concentration in the

youngest fully expanded leaf for Pterocarpus indicus grown in yellow sand.

y = 63.622x - 13.231

R2 = 0.3969

y = -0.4131x + 100.8

R2 = 0.7007

y = -5.7174x + 114

R2 = 0.9965

0

20

40

60

80

100

120

0 5 10 15 20Total P Concentration (mg/g)

Maxim

um

shoot

dry

weig

ht

(%)

Deficiency range Adequate range Toxicity range

Figure 2.11 Relationship between foliar P concentration (mg/g) and total shoot dry

weight (%) for Pterocarpus indicus seedlings using the combined data from the two

P experiments. The fitted lines were obtained using JMP.SAS linear fit estimates.

The replicates with the highest yield = 100%.

48

(Bolland and Wilson, 1994). However, soil tests only provide crude estimates for

predicting yields of annual crops and pasture (Bolland et al., 1989). This is the main

reason why this thesis focuses on plant, rather than soil P status, for diagnosis of P

deficiency.

Compared to the growth response of other woody plants and field crops in these

soils, there was surprisingly a narrow response range between P deficiency and P

toxicity for P. indicus. Although P toxicity was anticipated at the high fertilizer rate

in the yellow sand due to its low buffering capacity (Schoknecht, 1997), it was not

expected in the Yalanbee soil where the P adsorption capacity is much higher

(Barrow, 1977). This raises the possibility that P. indicus is an unusual tree that may

be sensitive to levels of fertilizer P supply that would normally not adversely affect

the growth of other legume tree species with fast growth potential. This will be

explored further in Chapter 3.

There was no evidence that P. indicus can function better at low soil P than other

species. The foliar P concentration range in the most deficient plants was 0.05-0.09%

which was similar to juvenile leaves in Acacia auriculiformis (Zech, 1990).

Deficiency ranges for Acacia mangium and Casuarina equisetifolia seedlings

obtained in pot trials (Table 2.4) are somewhat higher but these values merely reflect

the extent of P deficiency obtained in different experiments with soils differing in P

status. However, the adequate P foliar concentration range in P. indicus (0.24-0.36%)

was higher than in A. auriculiformis (0.12-0.25%) (Zech, 1990) but was similar to

other nitrogen-fixing trees such as Acacia mangium (0.35-0.5%) and Casuarina

equisetifolia (>0.25%) (Walker et al., 1993). Overall, the adequate P concentration

range in most legume tree species is comparatively higher than in non nitrogen-

49

fixing tree species such as Eucalyptus urophylla (0.1-0.3%) (Dell and Robinson,

1993), Swietenia macrophylla (>0.07-0.09%) (Yao, 1981) and Hevea brasiliensis

(0.14-0.17%) (Yew and Pushparajah, 1984). The foliar deficiency concentration

range was lower than reported in fruit trees such as cherry (Prunus avium) (<0.09%)

(Leece, 1976), cashew (Anacardium occidentale) (<0.14%) (Marchal, 1987) and

hazel-nut (Corylus avellana) (<0.1%) (Righetti et al., 1988). It was also lower than

oil palm (Elaeis guineensis) (<0.19%) (Coulter, 1958). Other broad–leaved tropical

tree species reported to have low P concentrations in deficient plants include Cedrela

odorata (<0.10%), Eucalyptus camaldulensis (0.05-0.08%), Dalbergia sissoo

(<0.08%) and Gmelina arborea (0.05%) (Drechsel and Zech, 1991). Differences in

the spread of the deficiency and adequate concentration ranges measured may arise

due to differences in soil P buffering capacity, the plant part sampled, the age of the

plant when samples were taken, the developmental stage of growth (e.g. vegetative

reproductive), and differences between species and provenances as reported by Dell

et al. (1987), Grove (1990), Dreshsel and Zech (1991), Netera et al. (1992),

Palomarki and Holopainen (1994), Sanginga et al. (1994) and Robinson et al. (1997).

The P toxicity range for P. indicus was 0.47-1.68% and this range has been reported

in six years old Acacia mangium (>0.5%) (Amir et al., 1993), and in the young

mature leaves of five years old Hevea brasiliensis (>3.0%) in Malaysia. The toxicity

range in other examples of broad–leaved tropical and fruit trees species are Pinus

radiata (>0.8% in the young mature foliage in the field) (Humphreys and Truman,

1964), Prunus persica (>0.40%) (Leece, 1976); Malus domestica (>3.0%)

(Goldspink, 1983), Prunus armeniaca (>0.4%) (Robinson and Nicholas, 1983) and

citrus (>0.25%) (Lee and Mayer, 1993).

50

Table 2.4 Comparisons of P concentrations for nitrogen-fixing and non-nitrogen

fixing tree species in the tropical and subtropical regions (Boardman et al., 1997).

A) Nitrogen-fixing

trees:

P concentration range (%)

Deficient Adequate Toxic

*Growth

stage

#Plant

part

1. Acacia

auriculiformis

<0.034-0.064 >0.12-0.25 Juv YMP

2. Acacia mangium <0.20-0.35 >0.35-0.50 >0.35-0.50 Juv YMP

3. Casuarina

equisetifolia

<0.15 >0.25 Seedl YS

4. Pterocarpus indicus

(present study)

0.05-0.09 0.24-0.36 0.47-1.68 Seedl YFEL

B) Non nitrogen-fixing

trees:

5. Eucalyptus urophylla <0.05-0.09 >0.1-0.3 Juv YMF

6. Hevea brasiliensis <0.127-0.131 >0.137-0.170 Seedl YB

7. Swietenia

macrophylla

<0.08-0.12 >0.07-0.09 Seedl WS

See note in Table 2.5 below.

Table 2.5 Comparisons of N concentration for N-fixing and other non-leguminous

tree species in the tropical and subtropical regions (Boardman et al., 1997).

Note: *Growth stage: Juv = Juvenile; Seedl = seedlings.

#Plant parts: YMP =

youngest mature petiolar organ; YFEL = youngest fully expanded leaf; YS = young

shoot; YB/YS = youngest leaflet/young leaf blade; and WS = whole shoot.

A) Nitrogen-fixing trees: N concentration range (%)

Deficient Adequate Toxic

*Growth

stage

#Plant

part

1. Acacia auriculiformis 0.81-0.84 >1.68-2.81 Juv YMP

2. Acacia mangium 2.04-3.0 Juv YMP

3. Casuarina equisetifolia <1.4 <2.53 Seedl YS

4. Pterocarpus indicus

(present study)

2.0 >2.5 5.6 Seedl YFEL

B) Non nitrogen-fixing

trees:

5. Eucalyptus urophylla <0.86 >1.1-3.0 Juv YMF

6. Hevea brasiliensis <1.49 >1.61-2.26 Seedl YB

7. Swietenia macrophylla <1.09-1.67 Seedl WS

51

Whilst foliar concentration ranges are widely used to assist in making decisions on

fertilizer management in plantations, such ranges need to be calibrated in the field.

Although the lower end of the deficiency range and the upper end of the adequate

range can be expected to vary between sites differing in soil fertility, critical

concentrations for the diagnosis of deficiency should be independent of site and

fertilizer type for a particular plant part and plant age. Furthermore, plant tissue tests

are independent of fertilizer placement.

Linear fits were used to determine critical P concentrations for the diagnosis of

deficiency and toxicity of P. indicus using the relationships between foliar P

concentration and maximum shoot dry weight (Figure 2.7). The linear regression

lines were based and determined on the highest correlated regression coefficients

(R2). Examination of Figure 2.7 shows that the fit of the lines in the regions where

growth is just affected by deficiency or toxicity is imperfect, and may underestimate

true critical concentrations.

No N response was obtained in the YB soil even though the two soil types used had

similar levels of nitrate and ammonium-N at the start of the trial (Table 2.1).

However, the YB soil had 2.26% organic C compared to 0.06% in YS. It seems that

micro-organisms may have made organic N available for plant growth in the YB soil

and this is why there was no N response. It was decided to focus research more on N-

fixation and P fertilizer and not investigate the lack of N response.

There was an early response of P. indicus to N fertilizer in the yellow sand. Growth

was depressed further in the deficiency range, at 2% foliar N in six N treatments

(from N0 to N40). There was no possibility of defining the concentration ranges in

the toxicity zone (Table 2.5). P. indicus seedlings appeared to have some preference

52

for ammonium-N over nitrate-N. Ammonium-N was depleted more than nitrate-N in

the N trial at luxury rates of N supply. There was greater depletion of ammonium-N

in the P fertilizer trial in YS but not in YB (Table 2.2). The conclusion that P. indicus

may prefer ammonium-N over nitrate-N assumes that soil microbes did not

substantially affect the balance of soil inorganic N pools. Soil pH effects (Moore and

Keraitis, 1971) observed in some eucalypt seedling studies (Bougher et al., 1990;

Dell et al., 1991; Shedley et al., 1995) are unlikely to have influenced N uptake as

the pH of the YS was similar across all fertilizer treatments. Shedley et al. (1995)

undertook a trial on Eucalyptus globulus using similar N-free yellow sand (pH 6.0).

They showed that foliar N concentrations in the YFEL ranged from 1% for N-

deficient plants to 3.3% for N-adequate plants. These values are lower than those

obtained for N-deficient plants in P. indicus (2%). Dell et al. (1991) reported that

preference for ammonium in E. diversicolor seedlings diminished when the seedlings

become mycorrhizal but this was not studied for P. indicus.

Some symptoms of nutrient disorders have been described previously for P. indicus.

Srivastava (1979), grew P. indicus using river sand and reported an increase in the

number of branches under P deficiency resulting in rosette-like plants. This symptom

was only observed for high P treatments (Figure 2.7) in this study. He also observed

that buds did not develop in plants that were severely N deficient. However, no P and

N analyses were carried out and no detail was provided of the trial duration.

Where symptoms are severe, this provides an early indication on the P status of a

crop. However, symptoms in the mild deficiency/toxicity ranges are often difficult to

detect and can easily be confused with some other limiting nutrients or stress agents.

53

This is particularly the case for loss of chlorophyll which can also be a consequence

of S deficiency. Hence, the emphasis on foliar analysis is this thesis.

In conclusion, the P concentration ranges and the critical P concentration may be

useful for monitoring the P status of nursery stock and in young seedlings after out-

planting. Whether benefit can be obtained by inoculation with strains of root nodule

bacteria will be discussed in Chapter 4.

54

Chapter 3

The phosphorus response of Pterocarpus indicus seedlings in a

lateritic soil compared to three tropical tree legumes

3.1 Introduction

In the previous chapter, there was surprisingly a narrow range between P deficiency

and toxicity responses for Pterocarpus indicus in two soils that are naturally low in

P. Whilst P toxicity was anticipated at the high fertilizer rate in the yellow sand due

to the low buffering capacity of this soil, it was not expected in the Yalanbee soil due

to its high P adsorption capacity (Barrow, 1977). Soils with similar structural and

chemical properties from the Darling Plateau have been routinely used in Western

Australia to study the nutrient requirements of field crops and some tree species

(Barrow, 1977; Wallace et al., 1986; Burgess et al., 1990; Dell and Robinson, 1993;

Dell and Xu, 1995; Shedley et al., 1995; Lu et al., 1998; Cheng et al., 2002). For

example, Barrow (1977) grew seedlings of Eucalyptus diversicolor (karri) and

Trifolium subterraneum (subterranean clover) in a lateritic soil and obtained response

curves with broad plateaus to fertilizer P in the range of 250 to 1000 mg P/pot

(Figure 3.1a). Each pot contained 0.86 kg soil and P was added in solution as

potassium dihydrogen phosphate before planting. Similar responses were obtained

for E. marginata (jarrah), Corymbia calophylla (marri), Acacia pulchella (prickly

acacia) and Pinus radiata (radiata pine) in a second experiment (Barrow, 1977).

These response curves have also been reported by other workers, including E.

marginata (Dell et al., 1987); and subterranean clover and P. pinaster (Robson and

Gilkes, 1980). Clearly, these published accounts contrast sharply with the response

shown for Pterocarpus indicus in Figure 3.1B. Even in the yellow sand used in

55

Chapter 2, published shoot responses (e.g. Bougher et al., 1990) to fertilizer P reveal

a broad plateau where moderate increases in soil P do not alter shoot biomass.

This raises the possibility that P. indicus is more sensitive to luxury soil P supply

than other legume tree species with fast growth potential. Hence, in order to

investigate this hypothesis, this chapter compares the P response of P. indicus with

three other woody leguminous species, viz. Acacia mangium Willd., Sesbania

formosa F. Muell. and Pterocarpus macrocarpus Kurz. Acacia mangium was chosen

because it is widely grown in plantations and for rehabilitation of degraded soils in

tropical humid zones (Sim, 1987; Udarbe and Hepburn, 1987; Turnbull et al., 1998).

Pterocarpus macrocarpus is valued for its high quality timber (Troup, 1921;

Soerianegara and Lemmens, 1994) and S. formosa is used as a host species for the

Indian sandalwood (Santalum album) in plantations in northern Australia

(Radomiljac, 1998). The experiment was undertaken using Yalanbee soil because of

the broad plateau shown in Figure 3.1A over which the response of P. indicus to

fertilizer P could be compared. The soil was collected as close as possible to where

Yalanbee soil was sourced in Chapter 2.

0 200 400 600 800 1000 1200 1400

P treatment (mg P/kg soil)

0

1

2

3

4

5

To

tal sh

oo

t d

ry w

eig

ht

(g/p

ot)

Figure 3.1 Comparison of effects of P fertilizer applied to lateritic soils from

southwestern Australia on shoot dry weight of (a) Eucalyptus diversicolor (solid line)

and Trifolium subterraneum (dashed line) taken from Barrow (1977); and (b)

Pterocarpus indicus (Chapter 2).

(a) (b)

56

3.2 Materials and methods

3.2.1 Experimental design

A randomized complete block design (RCBD) consisting of four tropical tree

legumes (Acacia mangium, Sesbania formosa, Pterocarpus macrocarpus and

Pterocarpus indicus), seven P treatments and four replicate pots was used. Initially,

four seedlings per pot were planted but these were progressively thinned to two by

the end of the trial. The treatments used were 0, 40, 80, 160, 320, 640 and 1280 mg P

kg-1

soil, hereafter designated as P0, P40, P80, P160, P320, P640 and P1280. The

trial was carried out from June to September, 2005 in a heated glasshouse at

Murdoch University, Western Australia (Figure 3.2) with mean minimum and

maximum temperature (± S.E.) of 15.22 ± 0.19 and 27.88 ± 0.26 oC, respectively.

Seed was sourced as follows: A. mangium from a commercial nursery, Perth,

Western Australia; S. formosa from trees at Kununurra, Western Australia; P. indicus

from Forest Research Institute Malaysia and P. macrocarpus from Faculty of

Forestry, Kasetsart University, Thailand.

3.2.2 Seed treatment and germination

Prior to sowing, the seeds were treated as follows. Seeds of A. mangium and S.

formosa were immersed in rapidly boiling water and removed after about 60 seconds.

They were then surface dried and put onto moistened sterile filter paper in 20 cm

diameter plastic Petri plates. The plates were covered with aluminum foil and placed

in a controlled temperature room at 25 oC, in the dark for 4 days and 2 days in the

light at the same temperature for germination.

Seeds of P. indicus and P. macrocarpus were surface sterilized in 70% (v/v) ethanol

for 60 seconds, followed by 45 seconds in freshly prepared 4% (v/v) sodium

57

hypochlorite, and then rinsed in sterile distilled water (x6) before sowing in sterilized

plastic trays, dimensions of 315 (W) x 425 (L) x 120 (H) cm, containing autoclaved

yellow sand. The seeds for all four species were sowed at a depth of about 1 cm from

the soil surface. They were transplanted into the pots when two cotyledons had

emerged from the soil (Chapter 2). Watering was carried out twice daily to field

capacity (10% by weight).

Figure 3.2 Phosphorus pot trial of four leguminous tree species in the glasshouse.

3.2.3 Soil information and preparation

Soil at 0-20 cm depth was collected from the same site used in Chapter 2. Soil

preparation was subjected to the same procedure described in Chapter 2 except that it

was not pasteurized. The soil had been shown to be free of Phytophthora by other

workers (Brundrett et al., 1994). The chemical properties of the batch of Yalanbee

soil used are given in Table 3.1. Low levels of many nutrients in the base rock

granite combined with prolonged deep-weathering and crystallization of kaolinite

and sesquioxides had resulted in a soil of poor nutrient status. The physical and

58

chemical properties of the soil series have been described by McArthur and Bettenay

(1960), Gilkes et al. (1973), Barrow (1977) and Robson and Gilkes (1980).

Table 3.1 Soil chemical properties for the virgin Yalanbee soil.

The same basal nutrients were applied and the plants were managed according to the

procedures described in Chapter 2 except for the following variations (mg/pot):

K2SO4 468; MgSO4.7H2O 73 and CaSO4.2H2O 400. Nitrogen was supplied weekly in

aqueous solution for 11 weeks as (NH4)2SO4 to give a total of 99 mg N/pot. The

plants were not inoculated with N-fixing bacteria.

3.2.4 Harvesting and plant analysis

Harvesting was staggered due to the differences in growth rates between species. The

harvesting sequence was S. formosa: (15 weeks), A. mangium (17 weeks), P.

macrocarpus (19 weeks) and P. indicus (21 weeks) after planting. The objective was

to obtain growth curves in relation to P supply, not to compare absolute growth rates.

Prior to harvest, stem height and collar diameter were taken using a ruler and scale

caliper, respectively. The plants were separated into the youngest fully expanded leaf

(YFEL), rest of the shoot, stem and roots. Roots were recovered by washing the root

ball over a 2 mm sieve and care was taken to minimize loss of fine roots. Shoots and

roots were dried in an oven at 70 oC for 48 hours to a constant weight. The dried

Property

Value

P (Colwell) (mg kg-1

) 4

Nitrate-N (mg kg-1

) 2

Ammonium-N (mg kg-1

) 1

K (Colwell) (mg kg-1

) 61

S (mg kg-1

) 4.0

Organic carbon (%) 2.6

pH (H2O) 5.6

pH (CaCl2) 6.3

59

samples were ground in a stainless steel home electric grinder and digested in

concentrated nitric acid using an open-vessel microwave oven (CEM Mars 5,

manufactured by CEM Corp., USA) followed by elemental quantification using an

ICP-AES as described in Chapter 2 and by Huang et al. (2004). Standard eucalypt

reference material was used for each digestion batch.

3.2.5 Statistical analysis

Multivariate analysis of variance (P≤0.01 and P≤0.05) was used depending on the

homogeneity of variances to compare growth rate of the species. The phosphorus

fertilizer treatments, however, were also analyzed separately for each species using

ANOVA (SPSS version 17.0) and the means were examined using Duncan’s New

Multiple Range Test.

The effects of phosphorus fertilizer treatments on growth were fitted where possible

to a Mitscherlich model equation Y = a-be–cx

; where Y = is the plant yield at the

nutrient concentration X, a, is the estimated maximum yield, and b and c are

parameters to be estimated (Ware et al., 1982). A hand-fitted curve method was also

tested for data analysis.

3.3 Results

3.3.1 Effect of phosphorus treatment on growth

There was a significant species x P interaction for all dependent variables measured

(Table 3.2). For convenience, the measured and observed parameters are considered

under five headings below.

a) Height and diameter

Generally, growth was poor in all P0 plant for the four tree species. P. indicus grew

the slowest, reaching maximum height and diameter of 12.9 ± 1.5 cm and 3.6 ± 0.3

60

mm at P60, respectively, at age 21 weeks (Figure 3.3). A similar height growth of

12.7 ± 1.3 cm was also obtained for P. indicus in the previous trial (Chapter 2). The

P. macrocarpus was also slow growing, obtaining maximum height and diameter

growth (data not presented) of 16.1 ± 0.8 cm and 3.8 ± 0.4 mm at P80, respectively,

at age 19 weeks.

By contrast, both A. mangium and S. formosa were fast growing with S. formosa

reaching height and diameter growth of 27.2 ± 0.3 cm and 4.9 ± 0.1 mm at P320 after

15 weeks and A. mangium with 16.7 ± 0.3 cm, 4.0 ± 0.3 mm at P1280 after 17

weeks, respectively.

0

5

10

15

20

25

30

0 40 80 160 320 640 1280

P Treatment (g/kg)

Heig

ht

(cm

)

Acacia mangiumSesbaniaP. indicusP.macrocarpus

Figure 3.3 Height growth of four leguminous tree species at harvest. Each data point

is the mean of four replicates with standard error bars.

b) Shoot and root dry weight growth

The response of shoot and root dry weights to P fertilizer differed markedly between

species. There were two groups of responses, with A. mangium and S. formosa

forming one group, where shoot and root dry weight responses could be fitted using

the Mitscherlich model (Figures 3.4, 3.5). P. indicus and P. macrocarpus formed a

P treatment (mg/kg soil)

61

second group where there were steep declines in growth at higher P treatments and

these response surfaces could not be fitted to the Mitscherlich model (Figures 3.6,

3.7). Maximum yields for shoot and root dry weight of 0.70 ± 0.15 and 0.35 ± 0.11

g/seedling at P160 for P. indicus were obtained, respectively. These yields were

more than three times higher than those at P0. Growth was further depressed by more

than 45% of maximum yield when high P fertilizer treatments (>P640) were applied

(Figures 3.6a, 3.7a). The response of P. macrocarpus was quite similar to P. indicus

with maximum shoot and root dry weight occurring at P160 and there was a

maximum yield depression of more than 55% at high P treatments (Figures 3.7a, b).

Table 3.2 MANOVA of main factors and their interaction and dependent variables.

a) Factor Wilks’

Lambda

F df1 df2 Sig.

Species (1) 0.011 103.714 24 224 <0.001

Phosphorus (2) 0.081 5.309 48 383 <0.001

(1) x (2) 0.009 3.566 144 580 <0.001

b) Source Dependent variable df1 df2 F Sig.

Species

Height

Diameter

Shoot dry weight

Root dry weight

Root/shoot ratio

2266

128

36

16

0.99

3

3

3

3

3

214.020

216.653

210.184

138.736

22.779

<0.001

<0.001

<0.001

<0.001

<0.001

Phosphorus Height

Diameter

Shoot dry weight

Root dry weight

Root/shoot ratio

1139

27

17

5

0.26

6

6

6

6

6

53.774

22.819

47.500

23.615

3.040

<0.001

<0.001

<0.001

<0.001

<0.001

Species x

Phosphorus

Height

Diameter

Shoot dry weight

Root dry weight

Root/shoot ratio

545

12

9

4

0.57

18

18

18

18

18

8.570

3.501

9.085

5.173

2.165

<0.001

<0.001

<0.001

<0.001

<0.001

62

Sh

oo

t d

ry w

eig

ht

(g/s

ee

dlin

g)

0 200 400 600 800 1,000 1,200 1,400

0

0.3

0.6

0.9

1.2

1.5

1.8

2.1

2.4

2.7

P treatment (mg/kg soil)

Root

dry

weig

ht

(g/

seedlin

g)

0 200 400 600 800 1,000 1,200 1,400

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Figure 3.4 The relationship between P supply and a) shoot dry weight and b) root dry

weight of Acacia mangium. The fitted curve was obtained using the Mitscherlich

model.

Y = 1.97–1.70 exp (-0.03X)

R2 = 0.84

Y = 1.28-1.05 exp (-0.02X)

R2 = 0.82

(a)

(b)

63

Sh

oo

t d

ry w

eig

ht

(g/s

ee

dlin

g)

0 200 400 600 800 1,000 1,200 1,400

0.0

0.3

0.6

0.9

1.2

1.5

1.8

2.1

2.4

2.7

3.0

P treatment (mg/kg soil)

Ro

ot

dry

we

igh

t (g

/se

ed

ling

)

0 200 400 600 800 1,000 1,200 1,400

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

Figure 3.5 The relationship between P supply and a) shoot dry weight and b) root dry

weight of Sesbania formosa. The fitted curve was obtained using the Mitscherlich

model.

Y = 2.24-1.98 exp (-2.63X)

R2 = 0.83

Y = 1.32-1.09 exp (-1.68X)

R2 = 0.65

(a)

(b)

64

a

ab

ab

ab

ab

ab

c(a)

0 200 400 600 800 1000 1200 1400

P treatments (mg/kg soil)

0.0

0.2

0.4

0.6

0.8

Sh

oo

t d

ry w

eig

ht

(g/s

ee

dlin

g)

a

a

ab

b

abab

ab

(b)

0 200 400 600 800 1000 1200 1400

P treatment (mg/kg soil)

0.0

0.1

0.2

0.3

0.4

Roo

t d

ry w

eig

ht

(g/s

ee

dlin

g)

Figure 3.6 Effects of P supply on a) shoot dry weight and b) root dry weight of

Pterocarpus indicus. Data points with the same letter are not significantly different at

P≤0.05 using DMRT.

65

a

abc

bc

c

abc

abc

ab

(a)

0.2

0.4

0.6

0.8

1.0

1.2

Sh

oo

t d

ry w

eig

ht

(g/s

ee

dlin

g)

a

c

cc

c c

b

(b)

0 200 400 600 800 1000 1200 1400

P treatment (mg/kg soil)

0.1

0.2

0.3

0.4

0.5

0.6

Roo

t d

ry w

eig

ht

(g/s

ee

dlin

g)

Figure 3.7 Effects of P supply on a) shoot dry weight and b) root dry weight of

Pterocarpus macrocarpus. Data points with the same letter are not significantly

different at p≤0.05 using DMRT.

66

In contrast to P. indicus and P. macrocarpus, there was no reduction in either shoot

or root yields for A. mangium and S. formosa at high P rates (P640 and P1280). The

fitted Mitscherlich curves (Figures 3.4, 3.5) showed that the maximum shoot and root

yields occurred between P160 and P320 for both species. Although the R2

values

were reasonable, ranging from 0.65 to 0.84, the fits of the curves to data points in the

region between deficiency and adequacy were not perfect (Figures 3.4, 3.5).

c) Root-shoot ratio

Root-shoot dry weight ratios were higher in P0 plants of A. mangium and S. formosa

than those in plants supplied with adequate P due to adaptive response of the species.

By contrast, root-shoot ratios for P. indicus and P. macrocarpus did not alter with

change in external P supply. The change and adaptive response are explained in

subheading 3.4.2. Root-shoot ratios in P0 for A. mangium and S. formosa were twice

those of the two Pterocarpus species (Table 3.3).

Table 3.3 Effect of P treatment on the relative root/shoot ratio of four

Leguminous tree species.

*Columns with the same letter are not significantly different using Duncan’s

New Multiple Range Test at P≤0.01.

P mg/kg A. mangium

S. formosa

P. indicus

P. macrocarpus

0 0.94c* 0.80b 0.48a 0.46a

40 0.61ab 0.50a 0.44a 0.58a

80 0.69ab 0.60ab 0.43a 0.59a

160 0.67ab 0.46a 0.46a 0.50a

320 0.61ab 0.54a 0.48a 0.48a

640 0.78bc 0.64ab 0.35a 0.56a

1280 0.57a 0.66ab 0.42a 0.48a

67

d) Symptoms

Both Pterocarpus species shared similar visible symptoms (Figures 3.8, 3.9). At low

P rates (P0 and P40), P-deficient plants were short, stunted and leaves were pale,

light green. At high P treatments (P640 and P1280), most of the plants suffered from

P toxicity and showed tip burn, spot necrosis in the lamina of older leaves, shorter

internodes and there were rosette-like leaves starting to develop as coppice.

Symptoms of P deficiency also occurred in A. mangium and S. formosa at P0. The P

deficient plants were short, stunted and leaves were pale, light green. These P

deficiency symptoms were similar to both Pterocarpus species. However, no

symptoms of P toxicity were expressed at high P rates in P640 and P1280, although

these plants had dark green leaves (Figure 3.11).

e) Relationship between shoot yield and foliar P concentration

In P. indicus and P. macrocarpus, shoot dry weight increased with increase in P

concentrations in the YFEL and then declined (Figure 3.12). Due to the small

number of data points obtained for P-adequate plants, no function was able to be

fitted to the data. Hence, critical values for the diagnosis of either efficiency or

toxicity could not be obtained.

Although there were large gaps in the deficiency range data for the relationship

between yield and P concentration in the YFEL for A. mangium and S. formosa

plants, it was possible to fit the Mitscherlich model to the data (Figure 3.13).

Tentative critical values for diagnosis of P deficiencies, at 10% below the plateaus,

were estimated to be 2-3 mg P/g dry weight for the two species.

68

Figure 3.8 Symptoms of P supply for Pterocarpus indicus seedlings. Left to right: a)

height growth variations from low to high P, and b) symptoms of leaves at low, toxic

and c) adequate P supply.

(a)

(b)

Low

Toxic

Adequate

69

Figure 3.9 Height and foliar symptoms in Pterocarpus indicus seedlings. a) Small

plant size at deficient P (P40), b) Smaller and shorter seedlings at increased P (P320)

supply, and c) stunted stem, yellowish and smaller leaves at high P (P640).

(b)

(c)

(a)

70

Figure 3.10 Symptoms of P supply for Pterocarpus macrocarpus seedlings. Left to

right: a) Seedlings at low (P0) and high P (P1280) supply, b) Leaves at P0 and P40,

c) All leaves at high P (P1280) showing necrotic tissue.

(a)

(b)

(c)

71

Figure 3.11 Symptoms associated with P supply for Acacia mangium (a,b) and

Sesbania formosa (c) seedlings. a) Left to right: P0, P80, P160, P320 and P1280, b)

Leaves from P0, P40, P80, P320 and P1280, c) P0, P40 and P320. Generally, leaves

were smaller in low P and larger in high P supply. No P toxicity symptom was

observed for both species.

(a)

(b)

(c)

72

0

20

40

60

80

100

0 2 4 6 8 10

Shoot dry

weig

ht (%

)

0

20

40

60

80

100

0 2 4 6 8 10 12 14 16

P concentration (mg/g)

Shoot dry

weig

ht (%

)

Figure 3.12 The relationship between P concentration in the youngest fully expanded

leaves at maximum yield of (a) Pterocarpus indicus and (b) Pterocarpus

macrocarpus and shoot dry weight (% maximum yield). Each data point is the mean

of 4 replicates.

(a)

P concentration (mg/g)

P concentration (mg/g)

(b)

73

Y = 75.67-223.90 exp (-1.11x)

R2 = 0.82

Shoot

dry

weig

ht

(%)

0 2 4 6 8 10 12 14 16

0

10

20

30

40

50

60

70

80

90

100

P concentration (mg/g)

Sho

ot

dry

we

igh

t (%

)

0 2 4 6 8 10 12 14 16

0

10

20

30

40

50

60

70

80

90

100

Figure 3.13 The relationship between P concentration in the youngest fully expanded

leaves of (a) Acacia mangium and (b) Sesbania formosa and shoot dry weight (%

maximum yield). The fitted lines were obtained using the Mitscherlich model. Each

data point is the mean of the four replicates.

(a)

(b)

Y = 80.11-125.72 exp (-1.04x)

R2 = 0.80

74

3.4 Discussion

3.4.1 Growth response to application of phosphorus

The main finding from this study is that the response of Pterocarpus indicus to P

differs markedly from those of Acacia mangium and Sesbania formosa. The similar

response of P. macrocarpus to P. indicus further suggests that Pterocarpus may be

very sensitive to P fertilizer, even in soil that has a high capacity to adsorb

phosphate. The repeatability of the results for P. indicus with Chapter 2 confirmed

that the toxicity response is real for this species when external P is high and the plant

started to exhibit foliar symptoms of P toxicity.

In A. mangium and S. formosa, the response to fertilizer was not greatly different

from those obtained previously by other workers for subterranean clover and

eucalypts (Figure 3.1) where no reduction in either shoot or root yields were obtained

at luxury supply and these relationships were able to be fitted with the Mitscherlich

model (Lamb, 1976; Barrow, 1977). Similar Mitscherlich-type responses have also

been reported in crops such as wheat (Triticum aestivum), lupin (Lupinus

angustifolius), Sesbania spp., Calliandra and Cedrela odorata (Bolland, 1992; Elliot

et al., 1997; Webb et al., 2000).

3.4.2 Root-shoot ratio

The increased allocation of dry matter to roots under P-deficiency stress in A.

mangium and S. formosa is a typical and important adaptive response that has been

observed in many woody plants and field crops including Hakea, Triticum, Medicago

and Lupinus (Maschner, 1995; Raghotama, 1999; Lamont, 2003; Shane et al., 2004;

Lambers et al., 2006). Adaptive responses to P deficiency can involve accumulation

of P in roots and stems that can then be used when shoot growth predominates (Dell,

1987; McCully, 1994; Raaimakers and Lambers, 1996). For instance, roots usually

75

behave as the preferential site for P storage in H. sericea and the capacity for storage

also can exceed the P concentration in leaves when the latter increases to values

where toxicity symptoms start to appear (Konowski et al., 1991; Sousa et al., 2007).

There may be other root adaptive responses to P deficiency and these include an

increase in mass ratio, root hair length, root hair density and specialized responses

such as formation of ‘proteoid’ or ‘cluster roots’ (Purnell, 1960; Dinkelaker et al.,

1995; Kirk and Du, 1997). Many plants also respond to low P with increased

exudation of carboxylates that play an important role in nutrient mobilization from

inorganic sources in the soil e.g., addition of carboxylates to soil such as citrate has

been shown to mobilize nutrients such as P and Fe (Jones and Darrah, 1994; Lambers

et al., 2002).

By contrast, the lack of root biomass response in the two Pterocarpus species is

unusual and this suggests that these plants may rely on other mechanisms for

facilitating P fertilizer uptake at low external supply since they appear not to depend

on adaptive responses such as increase in root length to access soil P (Jecke and Pate,

1995). However, root systems in P. indicus have not being studied in any detail even

though they contribute significantly to the ecophysiological function of the plant

system for water and nutrient uptake in the field (Van de Driessche, 1984;

Keerthisinghe et al., 1998; Gazal and Kubiske, 2004; Gazal et al., 2004). This will be

discussed further in Chapter 6.

3.4.3 Critical phosphorus concentration for the diagnosis of phosphorus deficiency

Tentative critical values for the diagnosis of P deficiency and P toxicity in Chapter 2

for P. indicus were not able to be confirmed in the present experiment due to the

limited data set for plants grown with adequate P. Furthermore, the tentative critical

76

values obtained from the fit of the Mitscherlich model to the data for A. mangium

and S. formosa are likely to have overestimated the true values, even though the R2

values were 0.8. There are two reasons for this conclusion. The first is the gap for

plants in the range from minor to serious deficiency. The second is that the fitted

maximum yields from the Mitscherlich model were less than 80% of the observed

maximum yield.

The critical P concentration for diagnosis of deficiency in Acacia mangium and

Sesbania formosa obtained in this study was 0.2-0.3%. This range is higher than

some critical P concentrations for diagnosis of deficiency reported for acacias and

some other woody plants. Examples are A. mangium (0.2-0.35%) in the youngest

mature phyllode in a sand pot trial (Sun et al. (1992), A. auriculiformis (0.03-0.06%)

in the youngest mature phyllode in the field (Zech, 1990), Swietenia macrophylla

(0.08-0.12%) in the whole shoot in the field (Yao, 1981), Eucalyptus camaldulensis

(0.05-0.08%) in the mature leaves in the field (Zech, 1990), Anacardium occidentale

(<0.14%) in the mature leaves in the field (Richards, 1992), Actinidia chinensis

(<0.13%) in the first leaf above the fruit in the field (Cresswell, 1989) and Prunus

persica (<0.09%) obtained from the field (Nicholas and Robinson, 1977; Cripps and

Goldspink, 1983).

In conclusion, this chapter shows that the response of P. indicus to P fertilizer is

similar to that in P. macrocarpus seedlings and differs from A. mangium and S.

formosa. It also shows that, by comparison to A. mangium and S. formosa, both

Pterocarpus species are sensitive to luxury levels of P fertilizer and readily exhibit

symptoms. Chapter 6 will investigate further the root adaptive response of P. indicus

seedlings to soil P supply.

77

Chapter 4

Nodulation of Pterocarpus indicus by diverse

strains of root nodule bacteria

4.1 Introduction

Pterocarpus indicus is a tropical legume with potential for commercial forest

plantations (Chapter 1). The ability of some legume tree species to nodulate

effectively has generated interest in providing a new avenue for forestry application

practices. These include maintaining forest ecosystem fertility, land reclamation, soil

stabilization and revegetation programs (Dreyfus and Dommergues, 1981; Faria et

al., 1987; Wilson and Tilman, 1991; Pate et al., 1993; Chang and Handley, 2000;

Sprent and Parsons, 2000; Perez-Fernandez and Lamont, 2003; Fougnies, 2007).

Legumes are often used for managing agricultural ecosystems in order to improve

their organic fertility, N economy or farming system flexibility (Andrew and

Kamprath, 1978; Reeves and Ewing, 1993; Brockwell et al., 1995; Sprent, 2001;

Howieson et al., 2004). Often, optimum performance of the N-fixing symbiosis

depends upon pre-selection of both symbiotic partners for adaptation to the target

environment which in turn present a challenge to root nodule bacteria nodulation and

survival (Chatel and Parker, 1973; Howieson and Ballard, 2004). In order to

investigate symbiotic partners for P. indicus, this chapter was undertaken as a

prerequisite to determine the effective strains for nodulation in P. indicus.

Studies on nodulation in legume crops are reported to be closely associated with

Rhizobium and Bradyrhizobium (Dreyfus et al., 1979) but this information is still

lacking in woody legumes where host-strain relations are unknown. Trees that

78

nodulate effectively with fast-growing root nodule bacteria are recognized as being

specific for nodulation (Gibson et al. 1982; Duhoux and Dommergues, 1985)

whereas species that nodulate with Bradyrhizobium are less specific but tend to be

promiscuous in nature. P. indicus is of no exception and this has been reported to be

a promiscuous host tree. For example, the African P. indicus introduced in Brazil

were found to nodulate with Bradyrhizobium in Brazilian soils (Moreira et al. 1992;

de Faria and de Lima, 1998) in the field. Similar observations have been recorded in

Laguna, Philippines (Oyaizu, 1993) with B. japonicum and another Bradyrhizobium

sp. and in Thailand (Manassila et al., 2007). No further studies have been carried out

reported and there is still a lack of understanding on the specificity, symbiotic

relationships and capacity to fix nitrogen in P. indicus (Lim, 1976; Allen and Allen,

1981; Oyaizu et al., 1993; de Faria and de Lima, 1998; Sprent, 2001).

Inoculation of tree legumes, using effective strains of root nodule bacteria (or

“rhizobia”), can be useful in commercial forest nurseries where increased N2-fixation

capability promotes sustained growth after out-planting (Turk and Keyser, 1992;

Martin-Laurent et al., 1999; Perez-Fernandez and Lamont, 2003). Since N deficiency

is a major fertility constraint in agriculture and forestry in the tropics (Dakora and

Keya, 1997; Kass et al., 1997; Leblanc et al., 2005), there is an urgent need to

exploit N2-fixing tree legumes for sustained productivity. The aim of this study

therefore, was to investigate the ability of Malaysian P. indicus to form nodules with

diverse strains of root nodule bacteria and to identify effective strains with potential

for application as inocula in the nursery and field (Chapter 5).

4.2 Materials and methods

4.2.1 Experimental design

79

A complete randomized block design was used with 18 strains of rhizobia (Table

4.1) and two control treatments (C1-uninoculated without added inorganic N and C2-

uninoculated with plus inorganic N, supplied as KNO3). There were four replicates

per treatment. Each replicate was a single pot (15 cm diameter x 17.5 cm depth)

containing three P. indicus seedlings. The trial was carried out between September

2003 and December 2003 in an evaporative-cooled glasshouse at Murdoch

University, Western Australia. The mean minimum and maximum temperatures (±

S.E.) were 18 ± 2 oC and 30 ± 3

oC, respectively.

4.2.2 Plant and soil material

Seeds were obtained from large trees in the campus ground of the Forest Research

Institute of Malaysia (FRIM), Kuala Lumpur, Malaysia as in Chapters 2 and 3.

Before sowing, the seeds were surface sterilized in 70% (v/v) ethanol for 60 seconds,

followed by 45 seconds in freshly prepared 4% (w/v) sodium hypochlorite, followed

by six rinses in sterile distilled water. Seeds were sown in plastic trays (365 cm

length x 315 cm width x 120 cm height) at a depth of about 1 cm from the surface of

the sterilized yellow sand. The sand was autoclaved for 20 minutes at 121oC and left

to dry for about 48 hours prior to sowing. After one week, seedlings with two open

cotyledons and 5 cm radicles were transplanted into the pots. The pots had been

soaked overnight in 4% (w/v) sodium hypochlorite, rinsed with sterile deionised

water, lined with a sterile paper towel and filled in a 3 kg pot N-free sterile yellow

sand at pH 6.0 (Chapter 2). The physical and chemical properties of the soil are as

described by Bettenay et al. (1960); Bougher et al. (1990) and Brundrett et al.

(1996).

80

To remove traces of inorganic nitrogen, the sand-filled pots were flushed twice with

two litres of boiling water and allowed to drain to field capacity. These pots were

then covered with sterile cling plastic sheeting until ready for use. After transplanting

and inoculation, all the pots were given a pulse of 20 ml aqueous solution containing

KNO3 (5 g/L) and the soil surface was covered with white sterile polyvinyl chloride

beads to reduce evaporation and to prevent contamination. Pots were watered with

deionised water as required, and weekly with sterile complete nutrient solution

(minus N) (Howieson et al., 1995). The nutrient solution contained (g/L): 0.31

MgSO4.7H2O, 0.21 KH2PO4, 0.44 K2SO4, 0.06 FeEDTA, 0.05 CaSO4 and trace

elements (mg/L) 0.116 H3BO4, 0.0045 Na2MoO4.2H2O, 0.134 ZnSO4.7H2O, 0.01

MnSO4.H2O, 0.03 CoSO4.H2O and 0.03 CuSO4.5H2O. The nitrogen-fed control was

supplied weekly with 5 ml of KNO3 (5 g/L). Water and nutrients were supplied

through a sterile white PVC tube inserted in the centre of each pot and covered with

a plastic cap.

4.2.3 Root nodule bacteria strains

The strains of root nodule bacteria used in this study are listed in Table 4.1. Strains

WSM 3712 were isolated from nodules of P. indicus collected in 2002. These

nodules were collected from a potted seedling in FRIM’s nursery and were stored

over silica gel. No description on the nodules was provided. Five nodules were taken

randomly for isolation irrespective of the size and colour, left to soak in distilled

water for four hours, surfaced sterilized by immersion in 70% (v/v) ethanol for 60

seconds, followed by 45 seconds in freshly prepared 4% (v/v) sodium hypochlorite

and by six rinses of sterile distilled water (Vincent, 1970; Beck et al., 1993). The

nodule was crushed in a drop of sterile water and the contents transferred using a

81

sterile wire loop onto yeast mannitol agar medium (YMA) as described by Howieson

et al. (1988) (Appendix VI).

Sixteen strains of WSM and TAL, obtained from the Centre for Rhizobium Studies

(CRS) at Murdoch University, Western Australia were recovered from vacuum-dried

ampoules using the CRS method (Howieson et al., 1988). The Rhizobium strain,

R602 (R. gallicum), was provided by the Institute National de la Recherche

Agronomique, INRA (France).

Table 4.1 Details of root nodule bacteria used in the study

Strain No. Species Host plant **

Source

TAL 643 Bradyrhizobium sp. Canavalia

gladiata

RRIM

TAL 648 Bradyrhizobium sp. Psophocarpus

tetragonolobus

RRIM

TAL 651 Bradyrhizobium sp. Calopogonium

mucunoides

RRIM

TAL 656 Bradyrhizobium sp. Pachyrhizus

erosus

RRIM

*R 602 Rhizobium gallicum Phaseolus

vulgaris

INRA

WSM 2096 Bradyrhizobium elkanii Glycine max CRS

WSM 2097 Bradyrhizobium japonicum Glycine max CRS WSM 2098 Bradyrhizobium liaoningense Glycine max CRS WSM 2100 Mesorhizobium ciceri Cicer arietinum CRS WSM 2105 Rhizobium galegae Galega sp. CRS WSM 2106 Rhizobium hainanense Desmodium sp. CRS WSM 2108 Rhizobium leguminosarum Pisum sativum CRS WSM 2110 Rhizobium tropici Phaeseolus

vulgaris

CRS

WSM 2114 Sinorhizobium meliloti Medicago sativa CRS WSM 2115 Sinorhizobium saheli Acacia senegal CRS WSM 2116 Sinorhizobium terangae Acacia senegal CRS WSM 2117 Mesorhizobium loti Lotus edulis CRS WSM 3712

Bradyrhizobium sp. Pterocarpus

indicus

FRIM

Source: *

Strain obtained from INRA, **

CRS = Centre for Rhizobium Studies, INRA =

Institute National de la Recherche Agronomique (France), FRIM = Forest Research

Institute of Malaysia and RRIM = Rubber Research Institute of Malaysia.

82

4.2.4 Inoculation

The bacteria were initially grown at 28 oC on YMA plates to produce sufficient

culture material for inoculation. The fast-growing strains of Rhizobium and

Sinorhizobium were incubated for 3-4 days, the medium-growing strains of

Mesorhizobium for 4-5 days and the slow-growing strains of Bradyrhizobium for 7-

10 days (Cowan, 1974; Howieson et al., 1988; O’hara et al., 2000). These colonies

were then washed from the Petri plates using sterile sucrose solution 1% (w/v) and a

suspension of 10 ml was used to inoculate each pot. Seedlings were inoculated two

days after transplanting using a sterile syringe to distribute the suspension evenly

onto the soil surface around each seedling.

4.2.5 Harvest

Plants were harvested after three months (15 weeks) when there was a visible

difference and stability in plant shoot and height growth between treatments. Roots

were extracted from the sand with running water and nodulation was assessed based

on the number of nodules, size, color and position on the roots (Howieson et al.,

1995). The shoots were separated and oven-dried at 70 oC for 48 hours or till

constant weight. Nodules used for microscopic studies were fixed overnight at 4 oC

in gluteraldhyde (3% v/v) and in phosphate (0.025) buffer before being dehydrated

and embedded in Spurr’s resin (Spurr, 1969). Thin sections, 1 µm in thickness, were

obtained using glass knives on a Reichert-Jung 2050 microtome. They were stained

in 1% methylene blue plus 1% Azur II in 1% sodium tetraborate (Richardson et al.,

1960). Observations on nodules were made using stereo (Olympus SZX 12) and

compound (Olympus BX 51) microscopes.

83

4.2.6 Identification of root nodule bacteria strain with primer, RPO1

The polymerase chain reaction (PCR) method was used to differentiate five

inoculants and nodule isolates strains comprising the four genera in WSM 3712,

WSM 2096, R602, WSM 2114 and WSM 2100. This method has been previously

used for differentiating and identifying rhizobia (Welsh and McClelland, 1990;

Williams et al., 1990; Richardson et al., 1995; Hebb et al., 1998; Nandasena, 2004).

The technique was used to confirm the nodules had developed with the inoculants

organisms and there had not been contamination in the glasshouse.

4.2.7 Statistical analysis

The data were subjected to analysis of variance (ANOVA) using the program SPSS

version 17.0. Data transformation was carried out where necessary before Duncan’s

New Multiple Range Test was used as a post hoc test to identify differences between

means at P ≤ 0.05.

4.3 Results

4.3.1 Nodulation in Pterocarpus indicus

The uninoculated controls remained nodule free indicating that soil sterilization was

successful and there was no contamination problem in the glasshouse. Nodules were

present on roots of P. indicus inoculated with strains from all four genera,

Bradyrhizobium, Mesorhizobium, Sinorhizobium and Rhizobium after three months.

Ten strains (TAL 643, TAL 651, R602, WSM 2096, WSM 2098, WSM 2100, WSM

2106, WSM 2110, WSM 2114 and WSM 3712) formed nodules (Table 4.1) whereas

eight strains (TAL 648, TAL 656, WSM 2097, WSM 2105, WSM 2108, WSM 2115,

WSM 2116 and WSM 2117) did not nodulate. P. indicus inoculated with four slow-

84

growing Bradyrhizobium strains (WSM 3712, TAL 643, WSM 2096 and TAL 651)

formed greater mean number of nodules per seedling whereas nodulated plants using

medium-growing strains (Mesorhizobium) and fast-growing (Rhizobium and

Sinorhizobium) formed low number of nodules (Table 4.2). Strain WSM 3712, which

was collected from the host plant, was identified as a Bradyrhizobium sp. based on

its growth rate on YMA.

4.3.2 Nodule location and size

Plants inoculated with Bradyrhizobium (WSM 3712, WSM 2096, TAL 643) formed

nodules on both the tap and main lateral roots near the root collar (Table 4.2, Figure

4.1). Nodules formed with WSM 3712 and WSM 2096 were larger in size and pink

internally, whereas nodules derived from other strains were smaller in size and white

internally (not illustrated in figures). There were also larger number and bigger

nodule sizes observed near the root collar and tap root than those of lateral roots

(Figure 4.1). P. indicus nodules were mostly globose, smooth, internally

aeschynomenoid in shape, having determinate growth in oblate form and containing

central infected tissues with few or no uninfected cells (astragaloid in the

terminology of Corby 1971, 1988; Brown and Walsh, 1994) (Figure 4.2).

4.3.3 Shoot dry weight

Plants nodulated with Bradyrhizobium elkanii (WSM 2096) and Bradyrhizobium sp.

(WSM 3712) were observed to have dark green shoots compared to the uninoculated

control (C1) plants without added inorganic N. However, only WSM 2096 had

significantly (P≤ 0.05) increased shoot dry weight at the time of harvest (Figure 4.3).

This increased in shoot dry weight was similar to the plants in the uninoculated

85

control (C2) that were supplied with inorganic N. The shoot dry weight obtained was

about three times greater than the control (C1) without inorganic N (Figure 4.3).

Table 4.2. Mean nodule number, nodulated seedlings, nodule distribution, nodule

size and nodule colour in Pterocarpus indicus seedlings for ten treatments.

** Nodule distribution (%) at: A = 0-3 cm; B = 3-5 cm and C = >5 cm from the root

collar for all four replicates, #Size (%):

L = large: > 5mm, M = medium: 3-5 mm and

S = small: < 3mm in diameter for all four replicates (Howieson et al., 1995).

Strain

Mean number of

nodules per

nodulated

seedling (± S.E.)

Nodulated

seedlings

(%)

**Nodule

distribution1

(%)

A B C

#Nodule size2

(%)

L M S

Nodule

colour

WSM 3712 25.3±6.3 83 93 7 - - 18 82 pink

TAL 643 17.3±5.0 83 57 28 15 - 19 81 white

WSM 2096 19.0±9.0 42 38 46 16 16 42 42 pink

TAL 651 16 8 100 - - - - 100 white

WSM 2106 3 8 100 - - - 100 - white

WSM 2114 2 8 100 - - - 100 - white

WSM 2100 1 8 100 - - - - 100 white

R 602 1 8 100 - - - - 100 white

WSM 2098 1.0±0.0 8 100 - - - - 100 white

WSM 2110 1 8 100 - - - - 100 white

86

Figure 4.1 Pterocarpus indicus seedlings nodulated with WSM 2096 at 15 weeks.

Left to right: a) control (C2) treatment, nodulated with WSM 2096 and control (C1)

seedlings, b) the location of nodules on 1) the tap root and 2) the lateral roots, and (c)

nodules at the root collar.

(a) (b)

(c)

1

1

2

87

Figure 4.2 Morphology and anatomy of Pterocarpus indicus nodules formed with

Bradyrhizobium elkanii (WSM 2096). (a) typical morphology of aeschynomenoid

nodules (Scale bar = 1 mm), (b) transverse section of 3 month old nodule. The darker

central stained region (X) shows infected tissues with determinate growth.

b

b

a

a a

aa a

a

a a a

C1 (

-N)

TA

L 6

51

WS

M 2

110

R 6

02

WS

M 2

100

TA

L 6

43

WS

M 2

098

WS

M 2

106

WS

M 2

114

WS

M 3

712

WS

M 2

096

C2 (

+N

)

Strain

0.0

0.1

0.2

0.3

0.4

Sh

oo

t D

ry W

eig

ht

(g)

a

b

b

Figure 4.3 Mean shoot dry weight of Pterocarpus indicus seedlings. Bars with the

same letter are not significantly different at P ≤ 0.05.

Shoot

dry

wei

ght

(g/

seed

ling

X

(a) (b)

88

4.3.4 Identification of strains by PCR

The five inoculant strains comprising species from Bradyrhizobium (WSM 3712,

WSM 2096), Rhizobium (R602), Sinorhizobium (WSM 2114) and Mesorhizobium

(WSM 2100) were differentiated with amplified fragment length from each other

using primer RPO1 (Figure 4.4). The strains isolated from the root nodule bacteria

had the same gel band patterns as the inoculant organisms (Figure 4.4).

bp L 1 2 3 4 5 6 L 1 2 3 4 5 6

Figure 4.4 PCR amplification profiles for detecting strains obtained from the a)

inoculants and b) nodule isolates (corresponding in lanes 1 to 6) for P. Indicus.

Strains left to right (1) WSM 3712, (2) WSM 2096, (3) R 602, (4) WSM 2114, (5)

WSM 2100 and 6) TAL 643. The standard ladder size in lane L is 1 kb.

4.4 Discussion

Knowledge of root nodule bacteria associated with P. indicus is limited. A

significant finding from this study was that P. indicus can be nodulated by diverse

strains of rhizobia from all four genera, namely Bradyrhizobium, Rhizobium,

Sinorhizobium and Mesorhizobium. Nodulation of Pterocapus indicus from slow-

(a) (b) 12,216

4072

3054

2036

89

growing Bradyrhizobium has been previously reported (Lim, 1976; Allen and Allen,

1981; de Faria et al., 1989; Oyaizu et al., 1993; Parker, 2004; Rasolomampianina et

al., 2005) but this is the first report of nodulation for P. indicus by Mesorhizobium,

Rhizobium and Sinorhizobium. This study also showed that P. indicus was able to

form root nodules with seven species of root nodule bacteria. This result provides

strong evidence to support the suggestion by Sprent and Parsons (2000) that

nodulating species of Pterocarpus, such as P. indicus, are promiscuous. It also

suggests that symbiotic promiscuity may be a benefit for legumes growing in natural

ecosystems but could also present problems for inoculation with improved strains in

the nursery or field. When there is competition for nodulation, more than one root

nodule bacteria genotype resident within the soil may be capable of nodulating the

legume (Amarger, 1981; Thies et al., 1991). This may then manifest over the year of

establishment of the legume in the field despite seed inoculation (Barran and

Bromfield, 1997; Denton et al., 2002) or may develop progressively as inoculant

strains become displaced by naturalised root nodule bacteria in seasons subsequent to

inoculation (Streeter, 1994; Evans and Turnbull, 2004). However, competition for

nodulation is often intra-specific, that is between strains of a single species of root

nodule bacteria, but there are examples of different species being able to co-nodulate

a specific legume genotype. For example, Pheseolus vulgaris may be nodulated by at

least five species of root nodule bacteria and in this case, inter-specific competition is

possible (Amarger et al., 1997).

Previous observations on nodulation in P. indicus were associated with the ability of

P. indicus to nodulate but little has been reported concerning the effectiveness of

these symbiotic relationships for increasing productivity of the host (Ssali and Keya,

1980; de Faria, 1989; Sprent, 1995; Moreira et al., 1998 and Sprent and Parsons

90

2000). Only one of the 10 strains used, Bradyrhizobium elkanii (WSM 2096) was

assumed to be partially effective for N2-fixation in P. indicus due to the visible pink

nodules and significantly increased shoot dry weight under soil limiting conditions.

Nodule effectiveness is discussed further in Chapter 5.

The nodules of P. indicus were found to be globose, single and of aeschynomenoid

type and this was similar to earlier reports of Higashi et al. (1987), Corby (1988),

Lim (1976), Allen and Allen (1981) and Sprent (2001). Similar nodule types are also

reported in other woody legumes such as Albizia paraserianthes and Acacia

auriculiformis (Corby, 1988, Lim, 1976; Allen and Allen, 1981 and Sprent, 2001).

Further inoculation studies with a wide range of strains of local root nodule bacteria,

including those isolated from taxonomically related genera such as Dalbergia, are

required so that these effective strains can be developed for potential use as

commercial inocula for Pterocarpus. Whether woody legumes require inoculation in

the nursery depends on the availability of compatible and effective root nodule

bacteria in the soils where they are to be out planted. This should be investigated

with any new plantation tree legume. The current study indicates that P. indicus is a

promiscuous host with the ability to form symbioses with both slow and fast growing

root nodule bacteria. However, the results show that this tree legume may have a

narrow range of effective root bacteria for forming a highly effective N2-fixing

symbiosis. Further work is required to quantify the role of these strains and their

effectiveness in N2-fixation. This also includes identifying more effective strains for

nursery use and to establish whether there are any indigeneous strains in silvicultural

sites in Malaysia that could promote efficient N2-fixation in this tree species.

91

In conclusion, this study showed that P. indicus can be nodulated by a diverse range

of root nodule bacteria and that it is a promiscuous host species. The ability to

nodulate however, does not necessary indicate these strains are effective for N2-

fixation as only one strain, isolated from P. indicus as WSM 2096, promoted growth

and that further studies are required.

92

Chapter 5

Growth response to nodulation and nitrogen-fixation in

Pterocarpus indicus seedlings

5.1 Introduction

In the previous chapter, a diverse range of root nodule bacteria were able to nodulate P.

indicus but only one promoted growth. The fact that nodulation can be variable and is

affected by many edaphic and climatic factors, raises the need for local nodulation

studies. These factors include soil pH (Cheng et al., 2002), water availability (Streeter,

2003) and availability of soil nutrients especially phosphorus (Manhart and Wong, 1980;

Van Kessel and Roskoski, 1981; Sprent, 2001) which are environmental factors known

to affect nodulation and nitrogen fixation in legumes. The possible preference of

ammonium-N over nitrate-N for P. indicus, reported in Chapter 2, prompted this repeat

study to use two inorganic N sources, namely, KNO3 and (NH4)2SO4. The failure to find

nodules in the field does not conclusively indicate the nodulation status of a species and

nodulation should be confirmed by inoculation tests (Chapter 4). Though nodule

inspections are relatively simple, they are technically important for exploitation and

sustainable management of nitrogen-fixing legumes.

The overall nodulation pattern in P. indicus showed a promiscuous response to

inoculation in Chapter 4. Comments on the effectiveness of nodules were based on the

internal colouration of the nodules at harvest (also indicated by Odee, 1997), shoot dry

weight and number of nodules produced (Bergersen, 1980). The number of nodules

93

produced in the plants does not necessarily indicate that they are effective. The aim of

this chapter is to confirm and investigate the nodulation pattern and ability of P. indicus

to fix nitrogen.

5.2 Materials and methods

5.2.1 Experimental design

A complete randomized block design consisting of ten strains of root nodule bacteria,

three control treatments and four replicate pots were used. The strains used were R602,

TAL 643, TAL 651, WSM 2096, WSM 2098, WSM 2100, WSM 2106, WSM 2110,

WSM 2114, WSM 3712 (Table 4.1, Chapter 4). The control treatments were: C1 (non-

inoculated and nil inorganic nitrogen), C2 (non-inoculated with inorganic N, KNO3) and

C3 (non-inoculated and with inorganic N, (NH4)2SO4. The seedlings were grown for 13

weeks from 28th March, 2005 to 30th June, 2005 in an evaporative-cooled glasshouse at

Murdoch University, Western Australia. The mean minimum and maximum

temperatures (± S.E.) were 18.6 ± 0.3 oC and 27.3 ± 0.2

oC, respectively. The procedures

used were as described in Chapter 4 except where stated below.

5.2.2 Soil preparation, inoculation and enumeration of root nodule bacteria

A sand mixture consisting of 1:1 mix of yellow sand: washed white river sand was used.

The basal nutrients were supplied as in Chapter 4 except for a reduced concentration

(mg/pot) of: K2SO4 450; MgSO4.7H2O 12.5and CaSO4.2H2O 250. Nodulation in terms

of mean number of nodules per seedling, total nodule number, % plants nodulated and

nodule location, size and colour were recorded at harvest. Other measurements taken

94

were plant height, shoot dry weight, root dry weight and total nitrogen concentrations in

the shoot and root without nodules.

5.2.3 Statistical analysis

Data were analyzed using the SPSS Statistical Analysis (version 14.0) to perform a one-

way ANOVA between the strains, shoot and root dry weight. Means were compared

using Duncan‟s New Multiple Range Test (DMRT) where the ANOVA showed there

were significant differences between means at P≤0.05.

5.3 Results

5.3.1 Nodulation

There was a significant effect (P≤0.05) of inoculation on nodulation. WSM 3712

(Bradyrhizobium sp.) formed the most nodules with a mean total of 48 per seedling

(Figure 5.1). TAL 643 was intermediate with 23 nodules per seedling. WSM 2110 and

WSM 2096 had elevated number of nodules but were not significantly different from the

remaining six inoculant treatments which had fewer than 10 nodules per seedling

(Figure 5.1). Nodulation was inconsistent among some replicates of some strains.

Bradyrhizobium strain, WSM 3712, TAL 643 and WSM 2096 were the only inoculated

treatments where all plants in all replicate pots formed nodules (Table 5.2). In WSM

2106, only 17% of the plants became nodulated. There was no nodule formed in the

control treatments.

5.3.2 Nodule location

Root nodules were mostly located on the tap and main lateral roots of the plants in the

collar region (Figure 5.2). The location of nodules has been described and used for

95

studies of agricultural crops (Howieson et al., 1995). Generally, nodules formed by the P.

indicus plants were abundant in the collar region and sparse in the lateral roots (Table

WS

M 3

71

2

TA

L 6

43

WS

M 2

11

0

WS

M 2

09

6

R 6

02

WS

M 2

09

8

WS

M 2

11

4

WS

M 2

10

6

TA

L 6

51

WS

M 2

10

0

Strains

0

10

20

30

40

50

No.

of

no

du

le p

er

se

ed

ling

c

b

abab

aa a

a a a

Figure 5.1 Mean numbers of nodules produced by Pterocarpus indicus using ten strains

of root nodule bacteria. Data are means of 4 replicates. Data with the same letters are not

significantly different at P≤0.05 using DMRT.

5.2). Large nodules formed in plants inoculated with WSM 2106, WSM 2114 and WSM

2098. Smaller nodules occurred in plants inoculated with WSM 2106, TAL 651 and

WSM 2100, and these were confined to the root collar region. The latter plants were

considered to be ineffectively nodulated.

5.3.3 Plant growth

Height

Strain

No.

of

nodule

s per

see

dli

ng

96

There was a significant increase (P≤0.05) in plant height for WSM 3712, TAL 643 and

R 602 (Table 5.3). Height increments in these plants were 20% higher than the

uninoculated control plants that were not supplied with inorganic N (C1) and were

Table 5.2 Total nodule number, % nodulated plants, nodule location, size and nodule

cross-section colour in Pterocarpus indicus.

*For all four replicates,

** Nodule distribution (%) at: A = 0-3 cm; B = 3-5 cm and C

= >5 cm from the root collar for all four replicates, #Size (%):

L = large: > 5mm, M =

medium: 3-5 mm and S = small: < 3mm in diameter

similar to plants supplied with nitrate-N (C2). However, height growth for all other

strains was inferior to the uninoculated plants supplied with nitrate and ammonium-N

(C2 and C3), respectively.

Shoot dry weight

There was a significant (P≤0.05) increase in shoot dry weight for WSM 3712 and TAL

643 (Table 5.3), being 200% and 140% higher, respectively, than the uninoculated

plants without inorganic N (C1).

Strain *Total

number of

nodules

% nodulated

plants (n=12)

**Nodule

distribution (%)

A B C

#Nodule

size (%)

L M S

Colour in

section

WSM 3712 378 100 75 14 11 4 21 75 pink

TAL 643 190 100 76 22 2 3 11 86 pink

WSM 2096 103 100 66 10 24 1 16 83 light pink

WSM 2110 92 42 53 32 15 - 11 89 white

R 602 28 50 86 7 7 4 42 54 white

WSM 2098 27 58 63 7 30 11 63 26 white

WSM 2114 19 50 100 - - 11 42 47 white

WSM 2106 8 17 63 37 - 12 12 76 white

TAL 651 10 33 60 20 20 - 60 40 white

WSM 2100 8 25 100 - - - 62 38 white

97

Figures 5.2 Nodule distribution for Pterocarpus indicus in strain WSM 3712 along the

tap root (a, b) 0-3 cm from the root collar; and (c,d) on the main lateral roots in strain

WSM 2096.

(a) (b)

(c) (d)

98

Table 5.3 Mean stem height, shoot and root dry weight of Pterocarpus indicus

inoculated with 10 strains of rhizobium or uninoculated and supplied with or without

inorganic N.

*1 = uninoculated and without N, *

2 = uninoculated with KNO3, *

3 = uninoculated with

(NH4)2 SO4, #Weight for root dry weight not included with root nodules. Data are means

of 4 replicates. Data with the same letters are not significantly different at P≤0.05 using

DMRT.

Shoot dry weight of the above two inoculated treatments was similar to the C2 plants.

Plants inoculated with WSM 3712 and TAL 643 had uniformly dark green leaves

whereas the non-inoculated controls in C2 and C3 had medium green leaves (Figure 5.3).

Leaves in all other inoculated plants remained light green and were similar to the

uninoculated control without inorganic N (C1).

Strains Stem height

(cm)/seedling

Shoot dry

weight (g)/pot

#Root dry weight

(g)/seedling

WSM 3712 7.39 ± 1.40b 0.45 ± 0.10c 0.23 ± 0.05cd

TAL 643 7.61 ± 0.68b 0.36 ± 0.05bc 0.18 ± 0.04bcd

WSM 2096 6.88 ± 0.31ab 0.28 ± 0.03ab 0.19 ± 0.02bcd

WSM 2110 6.58 ± 0.38ab 0.26 ± 0.01ab 0.18 ± 0.03bcd

R 602 7.57 ± 0.43b 0.27 ± 0.01ab 0.18 ± 0.03bcd

WSM 2098 6.57 ± 0.34ab 0.23 ± 0.01ab 0.15 ± 0.03abc

WSM 2114 6.57 ± 0.10ab 0.25 ± 0.03ab 0.20 ± 0.01cd

WSM 2106 5.98 ± 0.34a 0.17 ± 0.02a 0.16 ± 0.02abc

TAL 651 7.16 ± 0.38ab 0.25 ± 0.02ab 0.15 ± 0.01abc

WSM 2100 6.15 ± 0.35a 0.24 ± 0.02ab 0.10 ± 0.02ab

*1C1 6.18 ± 0.27a 0.15 ± 0.01a 0.08 ± 0.01a

*2C2 7.60 ± 0.29b 0.37 ± 0.09bc 0.15 ± 0.04abc

*3C3 13.11 ± 0.45c 0.78 ± 0.07d 0.27 ± 0.04d

99

Root dry weight

There was a significant (P≤0.05) increase in root dry weight for WSM 3712, TAL 643,

WSM 2096, WSM 2110, R602 and WSM 2114 (Table 5.3), being about 16-20% higher

than the uninoculated plants in C1. The root dry weight for all other strains was similar

to the controls in C1 and C2 but was inferior to the C3 which produced more root dry

weight (Table 5.3).

5.3.4 Total nitrogen content in shoot, root and whole plants

There was a significant increase (P≤0.05) in nitrogen content in the shoot of seedlings

inoculated with WSM 3712 (Table 5.4). This content was twice that of the C1 control,

and in other inoculated seedlings, and not different from the C2 control. However, the

shoot N content for WSM 3712 seedlings was four times lower than the C3 control

(Table 5.4). There was no significant difference in N content in the other uninoculated

seedlings (Table 5.4). The C3 control plants had the highest shoot and root N contents of

all treatments. Related N concentrations in the shoot and root are given in Appendix IV.

5.4 Discussion

The absence of nodules in non-inoculated control treatments (C1, C2 and C3) showed

that soil sterilization was successful and that contamination was not present. The study

confirmed the nodulating ability of the ten strains used earlier (Chapter 4) on P. indicus

and that P. indicus is a „promiscuous‟ host.

Interestingly, earlier the most effective strains for nodulation were slow-growing

Bradyrhizobium WSM 3712 and WSM 2096. Due to the fact that many “rhizobia” were

100

Figures 5.3 Effects of inorganic nitrogen and inoculation treatments on height and shoot

growth of Pterocarpus indicus plants at 13 weeks. From left to right: (a) Controls (C1,

C2, C3); b) Controls (C1, C2, C3) and WSM 3712; and (c) Front row: left to right

Controls (C2, C1) with strains TAL 651, WSM 2106 Rear row: left to right and Control

C3 with strains WSM 3712, TAL 643 and R 602.

(a)

(b)

(c)

101

Table 5.4 Total N content (mg/seedling) present in the shoot, root and total root+shoot

of Pterocarpus indicus, inoculated with 10 strains of root nodule bacteria and control

treatments that were or supplied with or without inorganic N.

C2*- N supplied as KNO3; C3

*- N supplied as (NH4)2SO4. Data are means of four

replicates. Data with the same letters are not significantly different at P≤0.05 using

DMRT.

able to nodulate P. indicus (Chapter 4 and 5) but only a few were effective, much more

research is required in commercial nurseries. None of the inoculated plants achieved the

same growth as plants supplied with adequate levels of inorganic N supplied as

(NH4)2SO4. The greater response to (NH4)2SO4 than KNO3 confirms the observation in

Chapter 3 that P. indicus is better able to utilize NH4-N for growth than NO3-N.

Treatment Shoot Root Total root+shoot

C1

(control minus N)

2.87±0.213a 1.24±0.09a 0.87±0.06a

WSM 3712 7.78±1.67c 2.19±0.30a 4.98±1.95a

TAL 643 5.50±1.04ab 2.06±0.16a 5.26±1.81a

WSM2096 5.06±0.40ab 2.03±0.15a 3.56±0.51a

R 602 4.53±0.10a 1.93±0.48a 2.82±0.82a

WSM2100 3.98±0.39a 1.88±0.30a 2.64±0.19a

TAL 651 3.52±0.33a 1.63±0.26a 2.44±0.26a

WSM 2110 4.05±0.27a 1.23±0.41a 2.22±0.19a

WSM2114 3.83±0.33a 1.47±0.11a 2.09±0.11a

WSM 2098 4.14±0.18a 1.81±0.38a 1.75±0.33a

WSM 2106 3.25±0.46a 1.17±0.29a 1.49±0.28a

C2*

(control plus N)

16.60±3.15c 3.43±0.72b 5.98±2.20a

C3*

(control plus N)

30.08±1.30d 5.69±0.58c 35.33±4.54b

102

Similar nodulation trends for slow-growing Bradyrhizobium spp. have been observed in

pot trials with related genera such as in Dalbergia sisso (De Faria, 1987; Bala et al.,

1990). Bradyrhizobium is a slow-growing genus that belongs to the non-specialized

cowpea group which is prevalent in many tropical soils (Dreyfus and Dommergues,

1981). There are some incompleted studies for Bradyrhizobium on P.indicus in the field

in Singapore and Brazil (Lim, 1976; Allen and Allen, 1981; Oyaizu et al., 1993; Sprent

and Parsons, 2000; Rasolomampianina et al., 2005) but nothing for Sinorhizobium and

Mesorhizobium. Further studies are required to understand the symbiotic relationships

under local conditions. Nodulation ability has been reported to vary among and within

plant species under different locations and environmental conditions. An example is

nodulation by P. indicus introduced into Brazil (Moreira et al., 1992; de Faria, 1998;

Perret et al., 2000). Furthermore, others include Acacia mangium in Australia (Lawrie,

1981) and Leucaena species in Fujian Province of China (Zou et al., 1998).

Unfavourable conditions such as soil pH, water, nitrogen and phosphorus levels (Chee et

al., 1989; Zahran, 1999) may alter symbiosis in the field.

Although Pterocarpus erinaceus and P. lucens was reported to be nodulated by

Bradyrhizobium elkanii (WSM 2096) and Bradyrhizobium japonicum (WSM 2097)

(Sylla et al., 1998), in this thesis however, they were ineffective in growth promotion

under N-limiting conditions. Longer trials are required to establish whether these

bacteria would be effective in the field. Because Bradyrhizobium (WSM 3712) was

originally isolated from P. indicus, it was anticipated that it would be a strong performer.

However, in pot trials it was only partially effective. In this and the previous study

(Chapter 4), it was not possible to quantify N-fixation. This was not done due to the

103

variations in nodules size even though the plants were supplied with adequate levels of

inorganic N, the fact that the majority of plants had ineffective nodules and the short

duration in which the pot trials were carried out. The presence of pink nodules indicates

functional nodules but not the rate of N fixation. The high N content for WSM 3712

plants suggest effective N fixation.

Many methods have been applied to quantify N in woody legumes. For example,

nitrogen-fixing abilities have been assessed using the acetylene-reduction assay, as

described by Hardy et al. (1968) and used by Ng and Hau (2009). Other approaches that

can be used to quantify N in woody legumes are the N balance method that estimates the

net increase in total N of a plant-soil system, 15

N natural abundance, 15

N isotope dilution

and ureide methods which aims to separate plant N into the fraction taken up from the

soil (Unkovich et al., 2008). These methods are also being used to quantify N2 fixation

in crop and pasture plants (e.g Chalk, 1985; Peoples and Herridge, 1990).

In conclusion, further trials are recommended using a broad collection of Bradyrizobium

from under forest plantations in Malaysia. These should be evaluated for N-fixation

capacity under both nursery and field conditions and compared to the best two

performers identified in this and an earlier chapter.

104

Chapter 6

Effects of phosphorus and root nodule bacteria on

root growth in Pterocarpus indicus seedlings

6.1 Introduction

The sensitivity of Pterocarpus to P fertilizer (Chapters 2 and 3) raises the question as to how

well this species is adapted to soils of low P status or with high P fixation. Phosphate

presence in the soil is regarded as one of the primary constraints for tree legume

development due to its immobilization in all but sandy soil types (Kadiata et al., 1996;

Franco and de Faria, 1997; Palmer and Young, 2000). However, no detailed research has

been undertaken on P. indicus or other taxa in this genus regarding root development and P

acquisition. In general, the root system of plants contributes significantly to

ecophysiological function for anchorage, uptake of water and nutrients, storage of

carbohydrates and synthesis of growth regulators (Gazal et al., 2004). Many plants adapt to

low soil P availability by increasing their ability to acquire P (Vadex et al., 1995;

Raghothama, 1999) and/or developing root architectures which maximize nutrient

acquisition (Ma et al., 2001; Waisel et al., 2002). These responses in P are normally

associated and enhanced through P uptake kinetics, production of cluster roots (such as in

Banksia and Lupinus species), differential root hair growth, mycorrhiza symbioses and/or

increase in root phosphatase activity (Reddell, 1993; Marschner, 1995; Gahoonia and

Nielsen, 1997; Bates and Lynch, 2000).

105

A common response in field and tree crop species is for increased allocation of biomass

below-ground in soils of low available P. This is one of the earliest responses observed in

Chapter 3 for Sesbania and Acacia but not for Pterocarpus. The objective of this chapter

was to quantify the morphological effect of early root and shoot development in response to

low soil P. It was hypothesised that the root system would show no adaptive changes to low

external P.

6.2 Materials and methods

6.2.1 Experimental design

A randomized complete factorial block design comprising two P treatments (0, 60 mg P/kg

soil, hereafter designated as P0 and P60, respectively), two rhizobia treatments (no

inoculum, inoculated with WSM 3712, hereafter designated as –R and +R, respectively) and

two harvests, hereafter designated as H1 and H2 for 3 and 6 weeks, respectively, and three

replicate pots was used. The P60 treatment was chosen because it was nearly optimum for

growth of plants in Chapter 2. The experiment was carried out during summer, from 4th

January-15th

February, 2006 in an evaporatively-cooled glasshouse. The mean minimum and

maximum (± S.E.) temperatures were 26.9 oC (± 0.3) and 29.2

oC (± 0.4), respectively.

6.2.2 Plant material, soil and basal nutrients

Seeds of P. indicus were treated and germinated in sterilized yellow sand as described in

Chapter 3. When two weeks of age, five seedlings were transplanted into yellow sand

(described in Chapter 2), that had been steam pasteurized to prevent contamination from

root nodule bacteria. Each pot contained 3 kg pot air-dried sand. The seedlings were thinned

after two weeks to three seedlings per pot to prevent self shading. Basal nutrients were

106

applied to all pots at half the dosage used in Chapter 3 due to the shorter growing period.

The total amount of nitrogen supplied was 9 mg N/pot for the duration of the trial. This rate

was chosen based on the results in Chapter 5.

6.2.3 Data collection

Shoot height was taken from the root collar to the shoot tip and tap root length from the root

collar to the root tip using a measuring ruler. The number of lateral roots and root orders

were designated on their presence from the tap root (Figure 6.1). Total root length was

determined using ASSESS: Image Analysis Software for Plant Disease Quantification, 2002

(Lamari, 2002).

The shoot was cut near the root collar of the stem and the root system washed over a 10 mm

sieve. Plant parts were dried in an oven at 70 oC for 48 hours to a constant weight. The

relative root hair density (%) was visually rated on the 1st lateral root along the tap root and

scores were given as: a) none, b) scarce and c) dense. Nodule number was recorded based on

the mean nodule number and % nodulated seedlings as described in Chapter 4.

6.2.4 Statistical analysis

Data were analyzed using the SPSS Statistical Analysis version 16 and SAS Program

version 6.12 to perform a one-way ANOVA. Means were compared using Duncan’s New

Multiple Range Test (DMRT) where the ANOVA showed there were significant differences

between means at P≤0.05.

107

Figure 6.1 Root system diversity: Lateral root order (1 and 2) from the root collar (A) of the

tap root for Pterocarpus indicus plants

6.3 Results

6.3.1 Shoot height, tap root length and relative root hair abundance

Generally, there was no difference in shoot height or tap root length across treatments at 3

and 6 weeks. The taproot obtained was almost three times longer than the shoot. Root hairs

were dense and comprising of about 89% along the taproot and lateral roots of the seedlings

throughout the duration of the study (Table 6.1).

6.3.2 Shoot and root dry weight

Shoot and root dry weights did not differ between treatments at 3 weeks (Figure 6.1a, c,

Table 6.2). By week 6, the plants inoculated with Bradyrhizobium had significantly (p<0.05)

less shoot dry weight than the uninoculated plants (Figure 6.2d). There was however, no

difference due to P treatments. The root response in dry weight was similar to that shown for

the shoot except that the P60-R plants had less biomass than the P0-R plants (Figure 6.2b).

1 2 2

1

1 2

2

A

108

6.3.3 Total root length

Total root length was significantly (P≤0.05) affected by P and R and there were 2-way and

3-way interactions (Table 6.2). Root length of P0-R plants was maximum at both H1 and H2

(Figure 6.3). Furthermore, inoculation with Bradyrhizobium (+R) appeared to suppress total

root length in P0 plants during early growth at week 3 three but had little effect in P60

plants.

Table 6.1 Shoot height, taproot length and relative root hair abundance of six weeks old

Pterocarpus indicus seedlings grown at two levels of phosphorus (P) fertiliser and without (-

R) or with Bradyrhizobium (+R). Symbols with the same letter are not significantly different

using Duncan’s New Multiple Range Test at P≤0.05. Values are means of three (3)

replicates with standard errors (S.E.) for two harvests.

* Means for two harvest **n = 72.

6.3.4 Nodulation

Nodule formation was observed at week 3. However, these nodulation were evaluated and

harvested at week 6 on all treated +R plants. There was a significant interaction between P x

R x H (Table 6.2). The plants that were inoculated with Bradyrhizobium and have adequate

P supply were also found to be necessary for high nodulation capability as these can

significantly increased nodule production by more than 60% (Figure 6.4).

Treatment

Shoot height (cm) ± S.E.

H1 H2

Tap Root length (cm) ± S.E.

H1 H2

Relative root hair

abundance (%)

Nil Few Dense

P0 - R 4.07±0.28 * 5.32±0.21 13.97±0.14b

* 14.40±0.35 - 11

** 89

P60 - R 4.07±0.20 5.47±0.22 13.57±0.14ab 14.63±0.22 - 22 78

P0 + R 3.83±0.20 5.05±0.26 13.13±0.18a 14.43±0.20 - 11 89

P60 + R 3.80±0.32 5.15±0.13 13.50±0.11ab 14.50±0.15 - 11 89

109

6.3.5 Lateral root number - orders 1 and 2

Phosphorus application significantly (P≤0.05) increased the total number of lateral roots in

order 1 at H1 and H2 and order 2 at H2 (Figure 6.5 a-c). There is also a significant effect of

lateral root (P≤0.05) on Bradyrhizobium (+R) treated plants on Order 1 and 2 (Table 6.3)

(Appendix 6.1). However, the lateral root orders 1 and 2 were significantly reduced in P

deficient plants where P was not added (P0) (Table 6.3).

Table 6.2 Analysis of variance (ANOVA) for effects of phosphorus (P), root nodule bacteria

(R) and harvest (H) on shoot and root length, shoot and root dry weight, lateral root orders 1

and 2, total root length and nodule number interactions on both harvests.

*Significant at P≤0.05, otherwise unless stated, ns = not significant

1Lateral root order 2 and nodule numbers were only obtained at H2.

Variable/

Contrast

Shoot

height

Root

length

Shoot

dry

weight

Root

dry

weight

Lateral

root

order 1

1Lateral

root

order 2

Total

root

length

Nodule

number

P 0.75ns 0.03* 0.54ns 0.22ns 0.00* 0.02* 0.00* 0.00*

R 0.13ns 0.00* 0.00* 0.00* 0.01* 0.00* 0.00* 0.00*

H 0.00* 0.00* 0.00* 0.00* 0.00* - 1.00 -

Rep 0.59ns 0.20ns 0.13ns 0.89ns 0.48ns 0.85ns 0.15ns 0.51ns

P x H 0.69ns 0.03* 0.12ns 0.16ns 0.42ns - 1.00 -

P x R 0.90ns 0.01 0.27ns 0.00 0.31ns 0.02 0.00* 0.00

R x H 0.91ns 0.71ns 0.00* 0.00* 0.24ns - 1.00 -

P x R x H 0.99ns 0.90ns 0.16ns 0.00* 0.68ns - 1.00 -

110

Figure 6.2 Effects of P treatment and inoculation with Bradyrhizobium on the root (a,b) and

shoot (c,d) dry weight in three (a,c) and six (b,d) weeks old Pterocarpus indicus seedlings.

Each data point is the mean of three replicates with standard error bars. Symbols with the

same letter are not significantly different using Duncan’s New Multiple Range Test at

P≤0.05.

a

a a

a

a

a a

(a)

(c)

(d) (b)

a

c

b

a ab

b

b

a a

111

Figure 6.3 Effects of P treatment and inoculation with Bradyrhizobium on total root length

of (a) three and (b) six week old Pterocarpus indicus seedlings. Each bar is the mean of

three replicates with standard error bars. Symbols with the same letter are not significantly

different using Duncan’s New Multiple Range Test at P≤0.05.

(a)

(b)

c

b

a ab

b

a a

a

112

Figure 6.4 Effects of P and inoculation with Bradyrhizobium on nodulation in the six week

old Pterocarpus indicus seedlings. Each data point is the mean of three replicates with

standard error bars. Symbols with the same letter are not significantly different using

Duncan’s New Multiple Range Test at P≤0.05.

Table 6.3 Effects on number of lateral root orders 1 and 2 in three (H1) and six (H2) weeks

old Pterocarpus indicus seedlings grown at two levels of phosphorus (P) fertiliser and with

(+R) and without Bradyrhizobium (-R). Symbols with the same letter are not significantly

different using Duncan’s New Multiple Range Test at P≤0.05. Values are means with

standard errors (S.E.) for two harvests.

Treatment Lateral root order 1 (L1) ± S.E.

H1 H2

Lateral root order 2 (L2) ± S.E.

H2

P0 - R 34.67±3.48ab 55.67±5.04a 46.00±6.11a

P60 - R 41.00±5.13a 69.33±4.10a 62.67±6.89a

P0 + R 25.67±1.86b 42.00±1.15b 25.00±4.04b

P60 + R 40.67±4.37a 59.33±5.36a 45.00±2.08a

a

a

b

a

b

(a)

b

113

Figure 6.5 Effects of P treatment and Bradyrhizobium on the first order lateral roots at three

(a) and six (b) weeks and second order lateral roots at six weeks (c) old Pterocarpus indicus

seedlings. Each data point is the mean of three replicates with standard error bars. Symbols

with the same letter are not significantly different using Duncan’s New Multiple Range Test

at P≤0.05.

a

b

a

b

a

b

a

b

(b)

(c)

a

b

a

b

(a)

114

6.4 Discussion

In P. indicus, root development was strongly affected by P supply. In the absence of

phosphatic fertilizer, total root length and total root dry weight were enhanced in

uninoculated plants. However, surprisingly P deficiency reduced the number of first order

lateral roots and did not seem to enhance root hairs in any visual way. Many plant species

like Arabidopsis thaliana are known to exhibit increased extension of root hairs per unit root

length under P starvation (Foehse and Jungk, 1983; Bates and Lynch, 1996; Terence and

Lynch, 2000; Zhang et al., 2003; Hermans et al., 2007; Sousa et al., 2007). This response

may be an important strategy that can enhance P acquisition by growing denser and longer

root hairs. Other components of root architecture, such as root elongation, were influenced

by the low P status of the soil. In A. thaliana, leaves responded to P deficiency by

accumulating sugars and starch in the leaves but this was not explored in P. indicus

(Cakmak et al., 1994; de Groot et al., 2003; Sanchez-Calderon et al., 2005). The other main

finding was that inoculation with Bradyrhizobium altered the plant’s response to fertilizer P.

For instance, neither root dry weight nor total root length were enhanced at low P in

nodulated plants. In addition, inoculated plants had lower biomass and less root length than

uninoculated plants. It is well known that early adaptive response for most plant species to

overcome P deficiency include enhanced internal remobilization of P through early leaf

senescence (Marschner, 1995), and an increased root to shoot ratio (Asher and Loneragon,

1967; Lambers et al. 1998) due to a greater proportion of the photosynthate being allocated

to root growth. Root morphology is important for the acquisition of P, a nutrient for which

supply to the plant depends on root exploration of soil and on diffusion to the root surface.

Many plants also respond to low P with increased exudation of carboxylates which play an

115

important role in nutrient mobilisation from inorganic sources in the soil (Gardner et al.

1983; Hinsinger, 1998; Jones, 1998). The addition of carboxylates, such as citrate, to soil

has been shown to mobilise nutrients such as P and Fe (Jones and Darrah, 1994; Lambers,

2002). Similarly, root-exuded citrate has been reported to enhance P uptake in Oryza sativa

(Kirk et al. 1999).

The mean root-shoot ratio at P0 , uninoculated plants was 0.45 at H1 and 0.48 at H2 (Table

6.4) and this was similar to uninoculated plants grown without P fertilizer in Chapters 2 and

3 (Table 6.4). However, at just adequate P (P60), the root-shoot ratio was lower in Chapter 2

than Chapter 6. The root-shoot ratio at H2 was the same as that observed in P80 plants in

Chapter 3. Differences in plants age may account for some of these differences but the

results obtained at 6 weeks in this chapter was different from older plants in Chapter 2. All

plants were grown in yellow sand with Colwell P of 4 mg/kg. Hence, other unexplained

factors must have contributed to the unrepeatability of the results between experiments. In

the Philippines, initial study on the root growth capacity or root regeneration at day 14,

showed that P. indicus transplants can response quickly to different levels of moisture

regimes within 7 -14 days, resulting in height, shoot, length of taproot, lateral root and root

total biomass increased but no further data were given (Russell, 1973; Gazal et al., 2004).

This immediate response to P. indicus was attributed to the ability of the tree species to

adapt to different environmental stress conditions. However, the nutrient status of the soil

was not studied.

There is an assumption that species with fine, extensive root systems have low external P

requirements for maximum growth and those with thick, small root systems generally have

116

Table 6.4 Comparison of mean root-shoot ratio for uninoculated Pterocarpus indicus

plants grown at low or just adequate P supply in yellow sand.

na = mean value not available

high external P requirements. It has been suggested that these intrinsic root characteristics

can be better determinants of P requirement than changes in root morphology under P

deficiency (Hill et al. 2006). Relative root hair presence in P. indicus did not increase with P

deficiency and this is contrast with plants such as Arabidopsis where root hairs tend to grow

longer and denser in response to low P availability (Foehse and Jungk, 1983; Bates and

Lynch, 1996; Ma et al. 2001). Root hairs are reported to be an important site for perception

of chemical signals with root nodule bacteria that may lead to nodule formation in legumes

(Marschner, 1995; Ridge and Emons, 2000; Jebara et al., 2005). These changes in root hairs,

such as increased extension, also enhance P uptake (Bates and Lynch, 2000; Svistoonoff et

al., 2007) by overcoming constraints imposed by the P-depletion zones around roots and

therefore P is taken up from a greater soil volume (Anghinoni and Barber, 1980; Misra et

al., 1988). Changes in root hairs probably involve auxins (Bates and Lynch, 1996; Nibau,

2008) as indole-3-acetic acid (IAA) promoted the formation of long root hairs in

Arabidopsis thaliana plants of adequate P status whereas the auxin inhibitor 2-(p-

chlorophenoxyl)-2-methylpropionic acid (CMPA) caused a reduction in root hair length in

plants grown under low P conditions (Peoples et al., 1995; Skene and James, 2000; Cahalan,

Treatment Chapter 2 Chapter 3 Chapter 6 H2

Age (weeks) 15 21 6

P0 0.402 0.440 0.476

P60

0.286 na 0.433

P80 na 0.432 na

117

2001; Sousa et al., 2007). The responses to P deficiency at week six showed that P. indicus

has an ability to enhance P uptake. This behaviour is similar to many crop plants. The

hypothesis that the root system of P. indicus would show no adaptive changes to low

external P is therefore not supported.

In this study, nodule formation was formed as early as week three but was only well

developed (pink nodules) in week six on plants that were inoculated with Bradyrhizobium in

P-sufficient plants. In earlier chapters, the period of early nodule formation was not studied.

Nodule number was significantly reduced due to P deficiency. This phenomenon was

observed in earlier trials on P. indicus where nodulation increased with sufficient P supply

(Chapters 4 and 5). Phosphorus had been reported to have an atypical pattern of effects on

nodule biomass and on host plant growth (Tang et al., 2001; Serraj, 2004). The deficiency of

P may limit nitrogen fixation through its effects on growth and survival of root nodule

bacteria, nodule formation, nodule function and host plant growth. For example, Robson et

al. (1981), Sessitsch et al. (2002), Howieson and Ballard (2004) observed that mineral

nitrogen (NH4)2NO3) increased the growth response of nodulated plants of subterranean

clover to P supply and suggested that P increased the symbiotic nitrogen-fixation through its

effect on host plant growth. In constrast, this relationship in soybean required high P supply

for optimal functioning than either the host plant growth or N assimilation in the plant (Gan

et al., 2004; Matiru and Dakora, 2004; Walch-Liu et al., 2006). For this, it has been

suggested that P deficiency affects subsequent nodule formation by restricting metabolite

supply from the host plant. In other studies, P deficiency has been demonstrated to decrease

nodule mass more than shoot growth, as well as nodule number and specific nitrogenase

activity of nodules in crops such as soybean (Singleton et al., 1985; Drevon and Hartwig,

118

1997); pea (Jakobsen, 1985), white clover (Hart, 1989) and Leucaena leucocephala

(Brandon et al., 1997). There are conflicting results that indicate P deficiency may or may

not affect nodule number per unit shoot mass (Ribet and Drevon, 1995; Vadez et al., 1996;

Drevon et al., 1997; Tang et al., 2001). However, such occurrences in nodulation capability

towards P deficiency may be associated with the legume species, genotype and experimental

conditions and as well as the inoculant strains. Interestingly, inoculated seedlings were

found to have lower biomass and less root length than the uninoculated plants. In some

plants, nodules which appeared to be a strong sink for P, with functions reliant on high P

requirements may have stored and act as a reservoir (Robson et al., 1981; Jakobson, 1985;

Hart, 1989; Vadex et al., 1995; Drevon and Hartwig, 1997).

In conclusion, this study showed that P deficiency affected root morphology resulting in

higher total root length and root dry weight in uninoculated plants but there was no marked

change in root hair morphology.

119

Chapter 7

General discussion

This is the first study to investigate nutritional requirements of Pterocarpus indicus.

Key findings were: i) seedling growth was depressed strongly in P-deficient soils

similar to most other plants but moderate rates of P fertilizer led to expression of P

toxicity symptoms; ii) foliar P concentration ranges for deficiency and toxicity were

similar to other nitrogen-fixing trees; iii) the relationship between yield and P

concentration in the youngest fully expanded leaf (YFEL) enabled critical P

concentrations to be determined for the diagnosis of deficiency (0.17% dry wt) and

toxicity (0.41% dry wt). Critical P concentrations exist for very few woody plant

species. These values can be used to help interpret foliar analysis of seedlings, and

young out-planted stock, in the future; and iv) uninoculated P. indicus seedlings

responded to P deficiency by increasing allocation of biomass to roots (increased

root length and root dry weight). Furthermore, glasshouse inoculation trials showed

that seedlings were able to form nodules with strains from Bradyrhizobium,

Rhizobium, Sinorhizobium and Mesorhizobium, suggesting P. indicus is a

promiscuous host. However, only two strains of Bradyrhizobium promoted shoot

growth, one of these was isolated from P. indicus in Malaysia and the other from

Glycine max in Africa. Another finding was that uninoculated seedlings grew better

when the N was supplied as ammonium-N than when supplied as nitrate-N.

Phosphorus, one of the main essential elements for plant growth, is often a limiting

nutrient in plantation soils. Hence addition of P is usually necessary to achieve good

120

establishment, survival and growth under adverse conditions (Ballard, 1974). The

application of fertilizer containing P can shorten forest plantation rotations especially

in areas where nutrients have been depleted following clearing of vegetation (Evans,

1992; Ndufa et al., 1999). Given that the growth of P. indicus seedlings was

markedly reduced in soils of low P status in this study, it is likely that they will

respond to fertilizer P when plantations become established on marginal soils in

Malaysia and elsewhere. This response is likely to be similar to P responses obtained

in other studies, such as for Acacia mangium (Nik Muhamad and Paudyal, 1999).

Many plants respond to a low availability of soil P by increasing exudation of

carboxylates which play an important role in nutrient mobilisation from inorganic

sources in soil (Gardner et al., 1983; Hinsinger, 1998). The addition of carboxylates

to soil, such as citrate, has been shown to mobilise P and Fe (Lambers et al., 2003;

Shane et al., 2004). Plant growth and P uptake of Lotus pedunculatus increased due

to P mobilisation from the action of exuded carboxylates on calcium phosphate

(Bolan et al., 1997). Similarly, P uptake by Oryza sativa was enhanced due to its

mobilisation by root-exuded citrate (Kirk et al., 1999).Very fast rates of carboxylate

exudation are exhibited by plants that produce cluster roots (Keerthisinghe et al.,

1998). The carboxylate composition of cluster root exudates from Banksia grandis

depends on soil type and the form of P in the soil (Lambers et al., 2002). When P

was supplied as aluminium phosphate, the major carboxylates were di- and

tricarboxylates. When P was supplied as iron phosphate, a mixture of mono-, di- and

tricaroxylates were exuded (Lambers et al., 2003).

121

Species differ in their ability to modify their root systems or in allocation of dry

matter below ground in response to low nutrient supply, but this may not be related

to their internal nutrient requirements or ability to take up nutrients from soil. For

example, Austrodanthonia richardsonii and Hordeum leporinum had a similar ability

to adjust to low P but had significantly different critical external P requirements and

growth rates under low P (Hill et al., 2006). In this study, Pterocarpus species were

sensitive to high P supply in contrast to some other leguminous tree species.

However, examination of the root morphology of P. indicus at low P supply did not

reveal any special adaptive features that might explain the response to adequate-

luxury soil P. For example, P. indicus did not develop cluster roots as occurs in some

lupins.

Symbiotic partnerships may alter the response of P. indicus to soil nutrients. For

example, it is possible that P. indicus may be able to adapt to infertile soils by

forming effective associations with mycorrhizal fungi but these were not examined in

this thesis. Furthermore, N-fixing rhizobia may alter uptake kinetics of P as, in white

clover (Hart, 1989) and soybean (Israel, 1987), nodules appeared to have a strategy

to regulate P influx which minimized P deficiency when the supply was low but

avoided excess when the supply was high. More studies are required to extend these

findings to tree legumes.

In field crops, the probability of developing symptoms of P toxicity increases as leaf

P concentrations exceed 1% dry wt (Marschner, 1995). What distinguish the

development of P toxicity in some members of the Proteaceae is not symptoms but

its occurrence at relatively low external P concentrations compared with those that

122

induce P toxicity in other species (Lambers et al., 2003; Shane et al., 2004). A

similar conclusion can be drawn for Pterocarpus. Some of the possible explanations

for the development of P toxicity in P. indicus, which require investigations are:

Interference with leaf water relations at high cellular P concentrations (Bhatti

and Loneragan, 1970). Inability to function with very low concentrations of

leaf P and capacity to translocate a large fraction of P from senescing leaves

before leaf shedding (Lambers et al., 1998).

Inability to reduce P-uptake rates at elevated, but still relatively low, external

P concentrations (Shane et al., 2003). These also resulted in the failure to

down regulate the uptake of high P in between the compartments of the roots

(Harrison and Helliwell, 1979; Raghothama, 1999; Hermans, 2006). In

addition to tolerating high P concentrations by transporting P between

compartments, roots may also down-regulate the uptake of P (Harrison and

Heliwell, 1989).

Failure to decrease the expression of genes encoding P transporters (Smith et

al., 2003). The possible existence of multiple Pi transporters genes with

different functional characteristics in plant cell membranes can result in

variation of internal P status or external concentration (Smith et al., 2003).

Plants have adapted a number of morphological and biochemical strategies to

reduce the depletion zone around roots and increase the concentration of Pi in

solution immediately adjacent to the sites of uptake. Some species also

develop highly branched root systems with more root apices that are efficient

in acquiring P. For instance, studies on Phaseolus have shown that P-

acquisition efficient genotypes have an altered geotropic response which

enables their lateral roots to grow out at angles that better explore the upper

layers of the soil (Lynch and Beebe, 1995).

Inability to store P in the roots and stems during early age of the plants and to

maintain low leaf P concentrations so as to prevent P-toxicity symptoms from

123

developing (Jeschke and Pate, 1995; Parks et al., 2000). A low P-absorption

capacity might be expected in slow-growing plants adapted to infertile soils

because: a) diffusion to the root surface is the major rate-limiting step in low-

P soils and this cannot be overcome by increased absorption capacity

(Nye,1977) and b) P demand to support growth is the major determinant of P-

absorption rate (Clarkson, 1985). In order, to temporarily avoid the toxic

effects of excessive P accumulation in the cytoplasm, P can be stored in

vacuoles, mostly as inorganic phosphate in higher plants (Raghothama,

1999).

Thus, in the future, much remains to be done in order to understand the P nutrition of

P. indicus including basic studies on the behaviour of P in the plant and identification

of storage pools of P as done previously with some tree species (Dell, 1987; Chen,

2005).

The ability of P. indicus to nodulate with diverse genera of root nodule bacteria

confirmed the promiscuous behaviour of the species. Two strains of Bradyrhizobium

were able to form functioning nodules but the work was not able to be repeated

between chapters 4 and 5. In the field, variability in nodulation in some situations

may be caused by the effect of nitrate on nodulation. Since legume symbioses do not

react uniformly to nitrate, some symbioses can delay nodulation and nodulation may

proceed when the soil reserves of nitrate-N become diminished (Howieson and

Ballard, 2004). However, this is unlikely to be the case in the pot trials as no starter

N fertilizer was used and the soil had very low soluble N levels. The duration of the

trials (3 months) should have allowed adequate time for nodulation to occur and

functionality to become established. Interestingly, the two most effective

Bradyrhizobium strains promoted growth in a commercial nursery (Figure 7.1).

From the research findings in this thesis, the following recommendations can be

made:

Firstly, care needs to be taken that rates of P supplied as hard or liquid

fertiliser are not in the range likely to cause toxicity in forest nurseries

124

producing P. indicus or in the field after outplanting. It is also essential to

identify fertiliser management practises that suit local soil conditions and

cultivation practices. Whether organic or inorganic fertilizers are used, P-

release characteristics of these should be checked and their appropriateness

for P. indicus determined. In the field, soft rock phosphate may be a safer

option particularly where fertilizer is applied close to the planting stock, such

as in the planting hole or heeled in at the time of planting. Most tropical

phosphate rock deposits are unreactive and require processing before use.

However, there are some lower-cost options to be considered include partial

acidulation, blending with soluble P sources or microbial solubilisation.

Figure 7.1 Four month old seedlings of Pterocarpus indicus in a commercial nursery

in tropical Western Australia. The two plants on the left are uninoculated, those in

the centre were inoculated with Bradyrhizobium WSM 2096 and those on the right

with WSM 3712.

125

The foliar nutrient standards, defined for P in chapter 2, can be used to inform

management decisions both for nursery stock and for seedlings after

outplanting in the first season of growth (Specht and Turner, 2006). The use

of foliar as opposed to soil analysis as indicators of site suitability has the

advantage that they are species-specific, and the concentrations present in the

leaf are the consequence of several factors, in particular, actual availability of

the element to that species given its root architecture, rate of growth and its

competitive environment (Specht and Turner, 2006). Leaves are considered

the most reliable of all plant parts and this is why foliar analysis was used in

this study on P. indicus. Foliar phosphorus has been shown in Eucalyptus and

Pinus to be positively correlated with growth (Lamb, 1977; Xu et al., 2002;

Palmer et al., 2005). Over time, critical concentrations for the diagnosis of P

deficiency can be defined in field rates trials once sites with P-limiting soils

have been identified (Simpson et al., 1998). These can then be used to

calibrate tree growth with suitable measures of soil P availability. At the same

time, foliar sampling guidelines can be established for trees of different age in

the field (Nik Muhamad et al., 1998)

The observation that P. indicus seedlings were possibly more able to use

ammonium-N than nitrate-N has ramifications for N fertilizer use. If any

starter inorganic N fertilizer is to be used in the nursery, then it should be

supplied either as ammonium-N or as urea. Also, the level of N needs to be

low so nodulation is not repressed. High levels of nitrate can be a potent

inhibitor to the plants for N2 fixation (People et al., 1995). If effective

nodulation occurs in the forest nursery, there should be no need to add

inorganic N fertilizer in the field.

Whilst the two Bradyrhizobium strains that promoted growth in pot trials

could be tried in a commercial nursery in Malaysia, more research is required

in order to identify effective strains of rhizobia for widespread commercial

application. It is important that inoculation be considered to improve the N

126

economy of commercial plantings (Howieson et al., 2000). Desirable strain

characters have been enunciated by Brockwell et al. (2005) as Table 12 on

page 73.

In the longer term, research should include development of more site specific

nutrient management practices for improving fertilizer use, considering factors such

as indigenous nutrient supply at each site. Preferably these should be done using

clonal lines of improved P. indicus.

127

Appendix I

Table 1. Glasshouse temperature for P and N trials

Year Minimum (oC) Maximum (

oC) Mean (

oC)

Mean S.E. Mean S.E. Mean S.E.

2002

Jan 21.69 0.199 32.10 0.235 26.90 0.169

Feb 21.43 0.149 30.57 0.327 26.00 0.206

Mar 21.24 0.169 29.93 0.424 25.59 0.234

Apr 19.71 0.205 27.96 0.416 23.84 0.191

May 18.55 0.196 26.34 0.901 22.45 0.509

Jun 17.31 0.198 22.50 0.518 19.90 0.301

Jul 17.63 0.316 22.04 0.304 19.83 0.236

Aug 18.14 0.280 23.48 0.214 20.81 0.205

Sep 17.93 0.460 28.34 0.502 23.14 0.354

Oct 19.10 0.487 28.31 0.427 23.71 0.362

Nov 19.62 0.333 32.54 0.601 26.08 0.344

Dec 20.88 0.501 33.88 0.581 27.38 0.496

2003

Jan 20.10 0.407 34.17 0.412 27.14 0.324

Feb 20.50 0.526 33.07 0.485 26.79 0.406

Mar 19.21 0.497 30.90 0.625 25.05 0.526

Apr 16.59 0.64 28.21 0.48 22.40 0.515

May 16.00 0.449 25.97 0.408 20.98 0.316

Jun 16.03 0.615 24.59 0.833 20.31 0.627

Jul 17.06 0.347 29.10 0.26 23.08 0.239

Aug 16.93 0.325 29.63 0.357 23.28 0.232

Sep 18.39 0.226 30.86 0.67 24.63 0.349

Oct 17.96 0.580 30.37 0.772 24.27 0.494

Nov 18.86 0.363 30.36 0.622 24.61 0.395

Dec 20.10 0.336 31.55 0.363 25.82 0.307

Note: Letters in bold/italic are the trials Chapter 2 and 4. Values are means with

standard errors (S.E.)

128

Appendix II

Table A1: Height measurement for 10 weeks old Pterocarpus indicus seedlings in

YB and YS Trials

YB trial

Treatments

Height (cm)±S.E YS trial

Treatments

Height (cm)±S.E

N0 11.625±1.143 N0 4.100±0.549

N5 12.625±0.987 N5 5.075±0.103

N10 12.500±1.486 N10 6.050±0.456

N15 11.700±1.798 N20 6.525±0.287

N20 10.125±0.427 N30 5.600±0.245

N25 10.500±1.041 N40 5.400±0.245

N30 11.050±2.438 N50 5.475±0.335

N35 11.750±0.924 N60 5.525±0.377

N40 12.125±0.554 N70 6.375±0.461

N45 12.625±1.179 N80 4.675±0.290

YB trial

Treatments

Height (cm)±S.E YS trial

Treatments

Height (cm)±S.E

P0 9.250±0.323 P0 5.425±0.397

P5 8.750±0.144 P2 5.525±0.206

P10 8.375±0.625 P4 5.500±0.453

P20 8.250±0.250 P8 5.700±0.123

P40 9.275±0.401 P16 5.600±0.212

P80 10.250±0.595 P32 5.325±0.197

P160 12.425±0.855 P64 7.800±0.337

P320 12.725±1.301 P128 6.175±0.527

P640 10.675±0.568 P256 5.200±0.498

P1280 12.200±0.363 P512 4.500±0.141

129

Appendix III

Table A2. Foliar analysis results for phosphorus and other micronutrients present

using Yalanbee soil (YB) and yellow sand (YS) trials

1(a)YB

P

mg/g

K

mg/g

Mg

mg/g

S

mg/g

Ca

mg/g

Na

mg/g

Fe

µg/g

Mn

µg /g

Zn

µg /g

Cu

µg /g

B

µg /g

P0 3.8 30.18 2.49 2.30 8.24 16.26 103.38 342.5 79.25 9.73 77.43

P5 4.2 33.47 2.98 2.01 7.49 20.40 124.33 281.90 95.90 11.33 111.3

P10 4.7 35.44 3.28 2.16 8.26 29.25 136.8 333.48 126.98 7.95 67.6

P20 5.9 37.60 2.12 2.11 6.49 37.91 102.83 255.55 99.85 7.93 64.88

P40 7.9 31.67 2.70 2.63 7.56 15.34 98.85 261.55 131.00 9.10 60.00

P80 8.6 28.49 2.42 2.93 8.28 10.12 108.33 291.63 108.33 9.23 60.48

P160 13.5 24.58 1.96 3.12 7.18 4.39 106.15 235.40 43.43 8.13 59.55

P320 34.0 24.93 1.96 3.09 9.75 5.98 101.10 306.53 53.43 9.50 70.58

P640 41.7 28.67 1.74 5.18 12.13 9.77 131.1 360.8 49.03 11.48 64.73

P1280 116.7 27.30 1.53 5.20 12.45 7.42 83.85 412.75 32.23 9.05 63.33

1(b)

YS

P0 0.70

P2 0.70

P4 0.79 35.21 2.28 3.75 7.73 0.71 148.95 398.40 34.50 15.25 23.55

P8 0.49

P16 0.80 32.81 1.97 3.19 7.49 1.38 115.25 459.10 37.80 14.10 22.90

P32 0.88

P64 1.34 28.01 2.69 3.11 7.94 0.60 68.20 288.30 39.45 13.70 20.00

P128 3.29 27.96 2.54 4.11 7.46 0.54 60.80 209.65 39.80 16.55 19.20

P256 10.15 39.72 1.67 5.17 11.73 1.04 104.90 282.35 28.00 21.75 19.30

P512 14.14

130

Appendix IV

Table A3. Effect of P supply on mean total shoot dry weight, root biomass,

root/whole plant ratio and total root length of Pterocarpus indicus seedlings.

Means followed by the same letter within a column are not significantly different at

P ≤ 0.05

YB

(mg P/kg

soil)

Shoot dry weight

(g/pot)

Root biomass

(g/pot)

Root/whole plant

(g/pot)

0 0.363 ±0.031f 0.401 ±0.042c 0.523 ±0.045ab

5 0.406 ±0.094f 0.431 ±0.036c 0.572 ±0.062a

10 0.339 ±0.039f 0.356 ±0.024c 0.516 ±0.034ab

20 0.278 ±0.043f 0.430 ±0.034c 0.542 ±0.068a

40 0.479 ±0.061f 0.482 ±0.049c 0.445 ±0.025abc

80 0.847 ±0.044e 0.623 ±0.018bc 0.425 ±0.010abc

160 2.241 ±0.027c 0.953 ±0.053a 0.306 ±0.009bcd

320 3.598 ±0.127a 0.860 ±0.208ab 0.140 ±0.038d

640 2.592 ±0.049b 0.558 ±0.065bc 0.184 ±0.024d

1280 1.818 ±0.023d 0.700 ±0.058abc 0.261 ±0.000cd

YS

(mg P/kg

soil)

Shoot dry weight

(g/pot)

Root biomass

(g/pot)

Root/whole

plant

Total root length

(cm/pot)

0 0.215 ±0.144bc 0.154 ±0.011bcd 0.402 ±0.018a 243.444 ±21.617abc

2 0.289 ±0.018bc 0.165 ±0.007bcd 0.363

±0.018ab

301.427 ±24.907abc

4 0.228 ±0.012bc 0.176 ±0.010abc 0.417 ±0.018a 250.567 ±14.859abc

8 0.240 ±0.018bc 0.179 ±0.017abc 0.428 ±0.018a 268.615 ±21.757abc

16 0.208 ±0.009bc 0.112 ±0.012cd 0.386 ±0.020a 187.960 ±16.635bc

32 0.261 ±0.021bc 0.163 ±0.016bcd 0.422 ±0.018a 236.048 ±33.519abc

64 0.508 ±0.035a 0.202 ±0.015ab 0.286 ±0.018b 363.826 ±29.478a

128 0.564 ±0.044a 0.251 ±0.014a 0.358

±0.018ab

329.108 ±44.454ab

256 0.310 ±0.039b 0.181 ±0.021ab 0.382 ±0.020a 258.898 ±34.346abc

512 0.172 ±0.016c 0.082 ±0.013d 0.430 ±0.021a 163.335 ±39.434c

131

Appendix V

Table A4. Growth media for preparing yeast mannitol agar (YMA) for

rhizobia (Based on recipe published by Howieson et al., 1988.

Inorganic nutrients Amount (g/L)

D-glucose 3g/l

Mannitol 2g/l

Yeast extract 1g/l

K2HPO4 0.5g/l

MgSO4.7H2O 0.2g/l

NaCl 0.1g/l

CaSO4. 2H2O 0.05g/l

NH4Cl 0.1g/l

Agar 15g/l

Note: 500mls of the agar solutions can prepare 20 Petri dish

132

Appendix VI

bc

c

bcabc

abcabc

abcabcab aba

d

d

C1(m

inu

s in

org

anic

N)

WS

M 3

712

WS

M 2

096

TA

L 6

43

R 6

02

WS

M 2

106

WS

M 2

100

TA

L 6

51

WS

M 2

110

WS

M 2

098

WS

M 2

114

C2 (

plu

s in

org

anic

N)

C3 (

plu

s in

org

anic

N)

Strain

0.0

1.0

2.0

3.0

4.0

5.0

% N

in

sho

ot

abc

d

cdbc

ab

abab

ab aba a

e

e

C1 (

min

us

ino

rgan

ic N

)

WS

M 3

712

TA

L 6

43

WS

M 2

100

WS

M 2

096

WS

M 2

098

R 6

02

WS

M 2

110

TA

L 6

51

WS

M 2

106

WS

M 2

114

C2 (

plu

s in

org

anc N

)

C3 (

plu

s in

org

anic

N)0.0

0.5

1.0

1.5

2.0

2.5

3.0

% N

in r

oot

c

c

a

b

ab

abab

abab

abab

ab ab

C1 (

min

us

inorg

an

ic N

)

WS

M 3

71

2

WS

M 2

10

0

TA

L 6

43

WS

M 2

09

6

WS

M 2

09

8

WS

M 2

10

6

R 6

02

WS

M 2

11

0

WS

M 2

11

4

TA

L 6

51

C2 (

plu

s in

org

anic

N)

C3 (

Plu

s in

org

an

ic N

)

Strain

0.0

0.9

1.8

2.7

3.6

4.5

% N

in to

tal pla

nt

Figure 1: Total N concentration (%) in a) shoot, b) root, c) total root+shoot of

Pterocarpus indicus seedlings. Data are means of 4 replicates. Data with the same

letter are not significantly different at P≤0.05 using DMRT.

(a) (b)

(c)

132

References

Ahmad, S.S. (1983) Ulat bungkus Crematopsyche pendulla Joannis (Lepidopter

psychidae) serangga perosak berbagai pokok di taman rekreasi. In 'Jalan

bicara seminar kebangsaan hutan, taman negara dan taman bandaran untuk

rekreasi.' Universiti Pertanian Malaysia, Serdang, Selangor, Malaysia. pp

359-365.

Ahmad, S.S., Sam, Y.L., Yaacob, A.W., Aida, M. (1996) Binomics of

Neolithocollettis pentadesma Meyrick, a leaf miner of angsana (Pterocarpus

indicus). The Malaysian Forester 59: 153-163.

Allen, O.N., Allen, E.K. (1981) The Leguminosae: A Source Book of

Characteristics, Uses and Nodulation. University of Wisconsin Press,

Madison, WI. 812 pp.

Amarger, N., Macheret, V., Laguerre, G. (1997) Rhizobium gallicum sp.nov. and

Rhizobium giardinii sp. nov. from Phaseolus vulgaris nodules. Journal of

Systematic Bacteriology 47: 996-1006.

Amir, H.M.S., Miller, H.G. (1991) Soil and foliar nutrient composition and their

influence on the accumulated basal area of the two Malaysian Tropical

Rainforest Reserves. Journal of Tropical Forest Science 4(2): 127-141.

Amir, H.M.S., Wan Rashidah, W.A.K. (1993) Growth differences, fertility status and

foliar deficiency levels of six years old Acacia mangium on Bris soils.

Journal of Tropical Forest Science 6: 230-238.

Andrew, C.S., Kamprath, E.J. (1978) Mineral Nutrition of Legumes in Tropical and

Subtropical Soils. Proceedings of a Workshop held at CSIRO Cunningham

Laboratory, Brisbane Australia-January 16-21, 1978. CSIRO, Melbourne.

415 pp.

Ang, L.H. (1988) A note on the growth of Pterocarpus indicus in a sixty-years old

plantations. Journal of Tropical Forest Science 1, 188-189.

Anghinoni, I., Barber, S.A. (1980) Phosphorus influx and growth characteristics of

corn roots as influenced by phosphorus supply. Agronomy Journal 72: 685-

688.

Anon (2000) Seed Leaflet. Denmark, Danish Forest Seed Centre.

Appanah, S., Abdul Razak, M.A. (1998) Planting high quality indigenous species in

Sarawak. What and where? Proceedings of planted forests in Sarawak-An

International Conference held on 16-17 February, 1998, Kuching, Sarawak,

Malaysia

133

Appanah, S., Ismail, H. 1996 Study on Baseline Information on Land in Malaysia

for Conversion into Forest Plantations. Malaysia Timber Council Report.

Kuala Lumpur. 118 pp.

Appanah, S., Weinland, G.W. (1993) Planting Quality Timber Trees in Peninsular

Malaysia-A Review. Malayan Forest Record. 221 pp.

Asher, C.J., Loneragan, J.F. (1967) Response of plants to phosphate concentration in

solution culture. I. Growth and phosphorus content. Journal of Soil Science

103: 225-233.

Assidao, F., Cerna, De La. (1960) The growth of young narra trees in plantation,

Camp 7 Minglanilla, Cebu. Philippines Journal of Forestry 16: 7-14.

Assidao, F., Nastor, M. (1961) Silvical characteristics of smooth narra (Pterocarpus

indicus Willd.). Philippines Journal of Forestry 17: 207-214.

Baharuddin, J., Chin, T.Y. (1986) Review of plantation experiences in Peninsular

Malaysia. In the Ninth Malaysian Forestry Conference, 13-20 October, 1986,

Kuching, Forestry Department, Sarawak, Malaysia.

Bala, N., Sharma, P.K., Lakshminarayana, K. (1990) Nodulation and nitrogen

fixation by salinity-tolerant rhizobia in symbiosis with tree legumes.

Agriculture Ecosystems and Environment 33: 33-46.

Ballard, R. (1974) Use of soil testing for predicting phosphate fertilizer requirements

of radiata pine at time of planting. New Zealand Journal of Forestry 4: 27-34.

Barea, J.M., Azcon-Aguilar, C. (1983) Mycorrhizas and their significance in

nodulating nitrogen fixing plants. Advances in Agronomy 36: 1-57.

Barrow, N.J. (1977) Phosphorus uptake and utilization by tree seedlings. Journal of

Botany 25: 571-584.

Bates, T.E. (1971) Factors affecting critical nutrient concentrations in plants and

their evaluation: A review. Soil Science 112: 116-130.

Bates, T.R., Lynch, J.P. (1996) Stimulation of root hair elongation in Arabidopsis

thaliana by low phosphorus availability. Plant, Cell and Environment 19:

529-538.

Bates, T.R., Lynch, J.P. (2000) The efficiency of Arabidopsis thaliana root hairs in

phosphorus acquisition. American Journal of Botany 87: 964-970.

Beaufils, E. R. (1971) Physiological diagnosis-a guide for improving maize

production based on principles developed for rubber trees. Fertilizer Society

of South Africa Journal 1:1-30.

134

Beck, D.P., Materon, L.A., Afandi, F. (1993) Practical Rhizobium-Legume

Technology Manual. International Centre for Agriculture Research in the Dry

Areas (ICARDA). Aleppo, Syria.

Begon, M., Harper, J.L., Townsend, C.R. (1990) Ecology: Individuals, Populations

and Communities. Blackwell Scientific Publications.

Bell, D.T. (1984) Foliar and twig macronutrients (N, P, K, Ca and Mg) in selected

species of Eucalyptus used in rehabilitation: Sources of variation. Plant and

Soil 81: 363-376.

Berger, J.I. (1993) Ecological restoration and non-indigenous plant species: a review.

Restoration Ecology 1: 74-82.

Bergersen, F.J. (1980) (Eds.). Methods for Evaluating Biological Nitrogen Fixation.

John Wiley and Sons. New York. 702 pp.

Bergersen, F.J., Peoples, M.B., Turner, G.L. (1988) Isotopic discriminations during

the accumulation of nitrogen by soybean. Australian Journal of Plant

Physiology 15: 407-420.

Bettenay, E., McArthur, W.M., Hingston, F.J. (1960) The Soil Associations of Part

of the Swan Coastal Plain, Western Australia. Australia Division of Soils and

Land Use Service, CSIRO, Perth.

Bhaskar, V. (2003) Root Hairs. The 'Gills' of Roots: Development, Structure and

Functions. Science Publishers, Inc. Enfield, New Hampshire, USA. 189 pp.

Bhatti, A.S., Loneragan, J.F. (1970) Phosphorus concentrations in wheat leaves in

relation to phosphorus toxicity. Agronomy Journal 62: 288-290.

Bhupal Raj., Prasad Rao, A. (2006) Identification of yield-limiting nutrients in

mango through DRIS indices. Communications in Soil Science and Plant

Analysis 37: 1761-1774.

Binkley, D., Giandina, C., Bashkin, M.A. (2000) Soil phosphorous pools and supply

under the influence of Eucalyptus saligna and nitrogen fixing Albizia

facaltaria. Forest Ecology and Management 128: 241–247.

Bougher, N.L., Grove, T.S., Malajczuk, N. (1990) Growth and phosphorus

acquisition of karri (Eucalyptus diversicolor F. Muell.) seedlings inoculated

with ectomycorrhizal fungi in relation to phosphorus supply. New Phytologist

114: 77-85.

Bordeleau, L.M., Prevost, D. (1994) Nodulation and nitrogen fixation in extreme

environments. Plant and Soil 161: 115-125.

135

Brandon, N.J., Shelton, H.M., Peck, D.M. (1997) Factors affecting the early growth

of Leucaena leucocophala 2. Importance of arbuscular mycorrhizal fungi,

grass competition and phosphorus application on yield and nodulation of

leucaena in pots. Australian Journal of Experimental Agriculture 37: 35-43.

Brewbaker, J.L. (1993) Tree improvement for agroforestry systems. In: Callaway,

M.B., Francis, C.A. (eds.). Crop Improvement for sustainable agriculture.

Chapter 8. University of Nebraska Press, Canada. pp. 132-156.

Brewbaker, J.L., van den Beldt, R., MacDicken, K. (1982) Nitrogen-fixing tree

resources: potentials and limitations. 'Biological Nitrogen Fixation'

Technology for Tropical Agriculture. Cali, Columbia. pp 413-425.

Brockwell, J., Searle, S.D., Jeavons, A.C., Waayers, M. (2005) Nitrogen Fixation in

Acacias: an Untapped Resource for Sustainable Plantations, Farm Forestry

and Land Reclamation. ACIAR Monograph 115, 132 pp.

Brown, J. R. (1970) Plant analysis. Missouri Agriculture Experiment. Station Bullen.

81 pp.

Brown, S.M., Walsh, K.B. (1994) Anatomy of the legume nodule cortex with respect

to nodule permeability. Australian Journal of Plant Physiology 21: 49-68.

Browne, F.G. (1955) Forest Trees of Sarawak and Brunei. Government Printing

Office, Kuching, Sarawak.

Brundrett, M. (1991) Mycorrhizas In Natural Ecosystems. Academic Press. London,

U.K.

Brundrett, M., Brougher, N., Dell, B., Grove, T., Malajczuk, N. (1996) Working with

Mycorrhizas in Forestry and Agriculture. Australian Centre for Inernational

Agricultural Research, Canberra, Australia.

Bunyavejchewin, S. (1983) Canopy structure of the dry dipterocarp forest in

Thailand. Thai Forestry Bulletin 14: 1-132.

Burgess, N.L., Grooves, T.S., Malajczuk, N. (1990) Growth and phosphorus

acquisition of karri (Eucalyptus diversicolor F. Muell) seedlings inoculated

with ectomycorrhizal fungi in relation to phosphorus supply. New Phytologist

114: 77-85.

Burkill, I.H. (1935) A Dictionary of the Economic Products of Malay Peninsula.

Volume II. Government of Straits Setttlements and Federated Malay States,

London, UK. pp 1858-1865.

Cate, R. B. Jr., Nelson, L. A. (1971) A simple statistical procedure for partitioning

soil test correlation data into two classes. Soil Science Society of American

Proceeding. 35: 658-659.

136

Chalk, P.M. (1985) Estimation of N2 fixation by isotope dilution; an appraisal of

techniques involving 2N enrichment and their applications. Soil Biology and

Biochemistry 31: 1901-1917.

Chang, S.X., Handley, L.L. (2000) Site history affects soil and plant 15N natural

abundances in forests of northern Vancouver Island, British Columbia.

Functional Ecology 14: 273-280.

Chapman, H.D. (ed.). 1966 Diagnostic criteria for plants and soil. University of

California, Division of Agriculture Science, Berkeley.

Chatel, D.L., Parker, C.A. (1973) Survival of field grown rhizobia over the dry

summer period in Western Australia. Soil Biology and Biochemistry 5: 415-

426.

Chee, C.W., Sundram, J., Date, R.A., Roughley, R.J. (1989) Nodulation of Leucaena

leucocephala in acid soils of Peninsular Malaysia. Tropical Grasslands 23:

171-178.

Cheng, Y., Watkin, E.L.J., O'Hara, G.W., Howieson, J.G. (2002) Medicago sativa

and Medicago murex differ in the nodulation response to soil acidity. Plant

and Soil 238: 31-39.

Clarkson, D.T. (1985) Factors affecting mineral nutrient acquisition by plants.

Annual Review of Plant Physiology and Plant Molecular Biology 36: 77-115.

Claveria, J.R. (1952) Notes on Narra (Pterocarpus spp.). Philippines Journal of

Forestry. 7: 31-40.

Coles, J.F. and Boyle, T.J.B. (1999) Pterocarpus macrocarpus-Genetics, Seed

Biology and Nursery Production. Center for International Forestry research

(CIFOR), Bogor, Indonesia.

Corby, H.D.I. (1971) The shape of leguminous nodules and the colour of leguminous

roots. Biological Nitrogen Fixation in Natural and Agricultural Habitats. In

Lie, T.A., Mulder, E.G. (eds.), Plant and Soil (special volume) 305.

Corby, H.D.I. (1988) Types of rhizobial nodules and their distribution among the

leguminosae. Kirkia 13: 53-123.

Coles, J.F, Boyle, T.J.B. (1999) (eds.) Pterocarpus macrocarpus – Genetics, Seed

Biology and Nursery Production. Center for Tropical Forestry, Bogor,

Indonesia. pp 1-19.

Corner, E.J.H. (1988) Wayside Trees of Malaysia. The Malayan Nature Society.

Kuala Lumpur, Malaysia.

Cowan, S.T. (1974) Manual for the Identification of Medical Bacteria. Cambridge

University Press, Cambridge.

137

Craswell, E.T., Loneragen, J.F., Keerati-Kasikorn, P. (1986) Mineral constraints to

food legume crop production in Asia. In: Wallis, E.S. and Byth, D.E. (eds.),

Food legume improvement for Asian farmng systems. ACIAR Proceeding

Series No. 18. Intra Pres, Melbourne, pp 99-111.

Dart, P. (1998) Nitrogen Fixation by Tropical Trees and Shrubs. Kluwer Academic

Press, The Netherlands.

Darus, H.A., Radhuan, S. (1999) Commercial forest plantations in Malaysia: Present

and Future. Proceedings of International Workshop BIO-REFOR, Nepal. pp

47-50.

Darus, H.A., Hashim, M.N., Ab Rasip, A.G., Lok, E.H. (1990) Khaya ivorensis and

Endospermum malacense as potential species for future reforestation

programme. Proceedings of the Conference on Malaysian Forestry and Forest

Products Research, October 3-4. Kuala Lumpur, Malaysia. In: S Appanah,

Ng, F.S.P. , Roslan, I. (eds.) pp 60-65.

de Faria, S.M., De Lima, H.C., Franco, A.A., Mucci, E.S.F., Sprent, J.I. (1987)

Nodulation of legume trees from south east Brazil. Plant and Soil 99: 347-

356.

de Faria, S.M., Lewis, G.P., Sprent, J.I., Sutherland, J.M. (1989) Occurrence of

nodulation in the Leguminosae. New Phytologist 111: 607-619.

de Faria, S.M., De Lima, H.C. (1998) Additional studies of the nodulation status of

legumes in Brazil. Plant and Soil 200: 185-192.

Dell, B., Baywaters, T. (1989) Copper deficiencyin young Eucalyptus maculate

plantations. Canadian Journal of Forest Research 19: 427-431.

Dell, B., Jones, S., Wilson, S.A. (1987) Phosphorus nutrition of jarrah (Eucalyptus

marginata) seedlings: Use of bark for diagnosing phosphorus deficiency.

Plant and Soil 97: 369-379.

Dell, B., Robinson, J.M. (1993) Symptoms of mineral nutrient deficiencies and the

nutrient concentration ranges in seedlings of Eucalyptus maculata Hook.

Plant and Soil 155/156: 255-261.

Dell, B., Malajczuk, N. (1994) Boron deficiency in eucalypt plantation in China.

Canadian Journal Forest Research 24: 2409-2416.

Dell, B., Xu, D. (1995) Diagnosis of zinc deficiency in seedlings of a tropical

eucalypt (Eucalyptus urophylla S.T. Blake). Plant and Soil 176: 329-332.

Dell, B. (1996) Diagnosis of nutrient deficiencies in eucalypts. In: Nutrition of

eucalypts. Attiwill, P.M. and Adams, M.A. (eds.). CSIRO, Melbourne,

Australia, pp 417-440.

138

Dell, B., Malajczuk, N., Xu, D., Grove, T.S. (2001) Nutrient Disorders in Plantation

Eucalypts. 2nd Edition. ACIAR Monograph 74. ACIAR, Canberra.

Denarie, J, Debelle, F., Rosenberg, C. (1992) Signalling and host range variation in

nodulation. Annual Review of Microbiology 46: 497-531.

Desmet, P.G., Shackleton, C.M., Robinson, E.R. (1996) The population dynamics

and life-history attributes of a Pterocarpus angolensis DC. Population in the

Northern Province, South Africa. South Africa Journal of Botany 62: 160-

166.

Dhanmanonda, P. (1992) The forest growth cycle in a dry dipterocarp forest. Thai

Journal of Forestry 11: 66-79.

Diabate, M., Munive, A., de Faria, S.M., Ba, A., Dreyfus, B., Galiana, A. (2005)

Occurrence of nodulation in unexplored leguminous trees native to the West

African tropical rainforest and inoculation response of native species useful

in reforestation. New Phytologist 166: 231-239.

Drechsel, P., Zech, W. (1991) Foliar nutrient level of broad-leaved tropical trees: A

tabular review. Plant and Soil 131: 29-36.

Drevon, J.J., Hartwig, U.A. (1997) Phosphorus deficiency increases the argon-

induced decline of nodule nitrogenise activity in soybean and alfalfa. Planta

201: 463-469.

Dreyfus, B.C., Dommergues, Y. (1981) Nodulation of Acacia species by fast and

slow-growing strains of Rhizobium. Applied and Environmental

Microbiology 41: 97-99.

Eddowes, P.J. (1977) XIV. Commercial timbers of Papua New Guinea, their

properties and uses. Forest Product Research Centre. Department of Primary

Industry, Port Moresby, Papua New Guinea.

Effendi, M., Rachmawati, I., Sinaga, M. (1996) Growth performance of redwood

(Pterocarpus indicus Willd.) in relation to volume of growth medium.

Buletin Penelitian Kehutanan, Kupang, Indonesia.

Elwali, A. M. O., Gascho, G.J. (1983) Sugarcane response to P, K, and DRIS

corrective treatments on Florida Histosols. Journal of Agronomy 75: 79-83.

Evans, J. (1982) Plantation Forestry in the Tropics. Clarendon Press, Oxford. 403 pp.

Evans, J. (1992) Plantation Forestry in the Tropics: Tree planting for industrial,

social, environmental and agroforestry purposes. 2nd

Edition. Oxford

University Press, New York. 424 pp.

139

Evans, J. (2000) Sustainability of productivity in successive rotations. Proceedings

of the International Conference on Timber Plantation Development, 7-9

November 2000, Manila, the Philippines, FAO-RAPA, Bangkok, Thailand.

pp 3321-3324.

Evans, J., Turnbull, J. (2004) Plantation Forestry in the Tropics: The role, silviculture

and use of planted forests for industrial, social, environmental and

agroforestry purposes. Third Edition. Oxford University Press, New York.

480 pp.

FAO (2001) Global Forest Resources Assessment 2000. FAO Forestry Paper 140.

Food and Agriculture Organization of the United Nations, Rome.

FAO (2005) Global Forest Resources Assessment 2005. 15 Key Findings. Food and

Agriculture Organization of the United Nations, Rome.

Fitter, A.H., Garbaye, J. (1993) Interactions between mycorrhizal fungi and other soil

organisms. Plant and Soil 159: 123-132.

Foehse, D., Jungk, A. (1983) Influence of phosphate and nitrate supply on root hair

formation of rape, spinach and tomato plants. Plant and Soil 74: 359-368.

Forest Department Annual Report, Peninsular Malaysia 2001.

Foxworthy, F.W., Garbaye, J. (1927) Commercial Timber Trees of Malay Peninsula.

Malayan Forest Record 3: 96-97.

Franco, A.A., de Faria, S.M. (1997) The contribution of nitrogen fixing tree legumes

to land reclaimation and sustainability in the tropics. Soil Biology and

Biochemistry 29(5/6): 897-904.

FRIM in Focus (2002) FRIM‟s Millenium Trees. Information newsletter published

by Forest Research Institute Malaysia (FRIM). July-September, 2002 Issue.

16 pp.

Gahoonia, T.S., Nielson, N.E. (1997) Variation in root hairs of barley cultivars

doubled soil phosphorus uptake. Euphytica 98: 177-182.

Gardner, W.K., Barber, D.A., Parbery, D.G. (1983) The acquisition of phosphorus by

Lupinus albus L. The probable mechanism by which phosphorus movement

in the soil/root interface is enhanced. Plant and Soil 70: 107-124.

Gazal, R.M., Blanche, C.A., Carandang, W.M. (2004) Root growth potential and

seedling morphological attributes of narra (Pterocarpus indicus Willd.)

transplants. Forest Ecology and Management 195: 259-266.

Giller, K.E., Wilson, K.J. (1991). Nitrogen Fixation in Tropical Cropping Systems.

CAB International, Wallingford, UK.

140

Gobbina, J., Kang, B.T., Atta-Krah, A.N. (1989) Effect of soil fertility on early

growth of Leucaena and Gliricidia in alley farms. Agroforestry Systems 8:

157-164.

Grace, K. (2000) Certification of plantations and plantation timber, Proceedings of

the International Conference on Timber Plantation Development, 7-9

November 2000, Manila, the Philippines. pp 297-308.

Grove, T.S. (1990) Twig and foliar nutrient concentrations in relation to nitrogen and

phosphorus supply in a eucalypt (Eucalyptus diversicolor F.Muell.) and an

understorey legume (Bossiaea laidlawiana Tovey and Morris). Plant and Soil

126(2): 265-275.

Groves, R.H., Keraitis, K. (1976) Survival of seedlings of three sclerophyll species at

high levels of phosphorus and nitrogen. Australian Journal of Botany 24:

681-690.

Grundon, N.J., Robson, A.D., Lambert, M.J., Snowball, K. (1997) Nutrient

deficiency and toxicity symptoms.' Plant Analysis: An Interpretation Manual.

Intra Press, Melbourne.

Hardy, R.W.F., Holsten, R.D., Jackson, E.K., Burn, R.C. (1968) The acetylene-

ethylene assay for N2 fixation: laboratory and field evaluation. Plant

Physiology 43: 1187-1207.

Harrison, A.F., Helliwell, D.R. (1979) A bioassay for comparing phosphorus

availability in soils. Journal of Applied Ecology 16: 497-505.

Hart, A.L. (1989) Distribution of phosphorus in nodulated white clover plants.

Journal of Plant Nutrition 12: 159-171.

Haselwandter, K., Bowen, G.D. (1996) Mycorrhizal relations in trees for

agroforestry and land rehabilitation. Forest Ecology and Management 81: 1-

17.

Hashim, M.N., Maziah, Z., Sheikh Ali, A. (1991) The incident of heart rot in Acacia

mangium Willd. plantations: A preliminary observation. pp. 54-60 In:

Appanah, S., Ng, F.S.P., Roslan, I. (eds.), International Conference of

Forestry and Forest Products Research. Forest Research Institute of

Malaysia, Kepong, Malaysia Institute of Malaysia, Kepong, Malaysia.

October 1-3, 1990.

Hau, B.C.H., Corlett, R.T. (2003) Factors affecting the early survival and growth of

native tree seedling planted on a degraded hillside grassland in Hong Kong,

China. Restoration Ecology 11(4): 483-488.

Hebb, D.M., Richardson, A.E., Reid, R., Brockwell, J. (1998) PCR as an ecological

tool to determine the establishment and persistence of Rhizobium strains

introduced into the field as seed inoculant. Australian Journal of Agriculture

Research 49: 923-934.

141

Hegazi, N.A., Fayez, M., Monib, M. (1994) (eds.) Nitrogen Fixation with Non-

Legumes. The Sixth International Symposium on Nitrogen Fixation with

Non-Legumes, Ismailia. Egypt, 6-10 September, 1993. The American

University in Cairo Press. 599 pp.

Hermans, C., Hammond, J.P., White, P.J., Verbruggen, N. (2006) How do plants

respond to nutrient shortage by biomass allocation? TRENDS in Plant

Science 11(12): 610-208.

Higashi, S., Abe, M., Reyes, G.D. (1987) Electron microscopic studies of the root

nodule of Pterocarpus indicus. Journal of General Applied Microbiology 33:

241-245.

Hill, J.O., Simpson, R.J., Moore, A.D., Chapman, D.E. (2006) Morphology and

response of roots of pasture species to phosphorus and nitrogen nutrition.

Plant and Soil 286: 7-19

Hinsinger, P. (1998) How do plant roots acquire mineral nutrients? Chemical

processes involved in the rhizosphere. Advanced Agronomy 64: 225-265.

Holford, I.C.R. (1997) Soil phosphorus: its measurement, and its uptake by plants.

Australian Journal of Soil Research 35: 227-239.

Holttum, R.E. (1954) Plant Life in Malaya. Longmans, Green and Company.

London.

Howieson, J.G., Ewing, M.A., D'Antuono, M.F. (1988) Selection of acid tolerance in

Rhizobium meliloti. Plant and Soil 105: 179-188.

Howieson, J.G., Loi, A., Carr, S.J. (1995) Biserulla pelecinus L.-legume pasture

species with potential for acid, duplex soils which is nodulated by unique

root-nodule bacteria. Australian Journal of Agriculture Research. 46: 997-

1009.

Howieson, J.G., O‟Hara, G.W., Carr, S.J. (2000) Changing roles for legumes in

Mediterranean agriculture: developments from an Australian perspective.

Field Crops Research 65: 107-122.

Howieson, J., Ballard, R. (2004) Optimising the legume symbiosis in stressful and

competitive environments within southern Australia-some contemporary

thoughts. Soil Biology and Biochemistry 36: 1261-1273.

Huang, L., Bell, R.W., Dell, B., Woodward, J. (2004) Rapid nitric acid digestion of

plant material with an open-vessel microwave system. Communications in

Soil Science and Plant Analysis 35: 427-440.

Humphreys, F.R., Truman, R.A. (1964) Aluminium and phosphorus requirements of

Pinus radiata. Plant and Soil 20: 131-134.

142

Hundley, H.G. (1956) Padauk, Pterocarpus macrocarpus Kurz. Burma Forester 7(1): 6-

38.

Israel, D.W. (1987) Investigation of the role of phosphorus in symbiotic dinitrogen

fixation. Plant Physiology 84: 835-840.

Ikram, A., Mahmud, A.W., Napi, D. (1987) Effects of P-fertilization and inoculation

by two vesicular-arbuscular mycorrhizal fungi on growth and nodulation of

Calopogonium caerulum. Plant and Soil 104: 195-207.

Jakobsen, I. (1985) The role of phosphorus in nitrogen fixation by young pea plants

(Pisum sativum). Physiologia Plantarum 64: 190-196.

Jechke, D.W., Pate, J.S. (1995) Mineral nutrition and transport in xylem and phloem

of Banksian prionotes (Proteaceae), a tree with dimorphic root morphology.

Journal of Experimental Botany 46: 895-905.

Jones, D.L. (1998) Organic acids in the rhizopheres- a critical review. Plant and Soil

205(1): 25-44.

Jones, D.I., Darrah, P.R. (1994) Role of root derived organic acids in the

mobilization of nutrients from the rhizosphere. Plant and Soil 166: 247-257.

Kadiata, B.D., Mulonggoy, K., Isirimah, N.O., Amakiri, M.A. (1996) Screening

woody and shrub legumes for growth, nodulation and nitrogen-fixation

potential in two constrasting soils. Agroforestry Systems 33: 137-152.

Keerthisinghe, G., Hocking, P.J., Ryan, P.R., Delhaize, E. (1998) Effect of

phosphorus supply on the formation and function of proteoid roots of white

lupin (Lupinus albus L.). Plant Cell and Environment 21: 467-478.

Kirk, G.J.D., Santos, E.E., Santos, M.B. (1999) Phosphate solubilization by organic

anion excretion from rice roots in aerobic soil: rates of excretion and

decomposition effects on rhizosphere pH and effects on phosphate solubility.

New Phytologist 142: 185-200.

Konowski, T., Kramer, P.J., Pallardy, S.G. (1991) The Physiological Ecology of

Woody Plants. Academic Press, San Diego.

Krishnapillay, B., Appanah, S. (2002) Forest Plantation Historical Background. pp.

3-11 In: Krishnapillay, B. (ed.), Manual for Forest Plantation Establishment

in Malaysia. Malayan Forest Records No.45, Forest Research Institute of

Malaysia, Kepong, Selangor.

Krishnapillay, B., Ong, T.H. (2003) Private sector forest plantation development in

Peninsular Malaysia. EC-FAO Partnership Programme on Information and

Analysis for Sustainable Forest Management: Linking National and

International Efforts in South Asia and Southeast Asia. Bangkok, Thailand.

36 pp.

143

Lamari,L. (2002) ASSESS:Image Analysis Software for Plant Disease

Quantification. The American Phytopathological Society, University of

Manitoba, Winnipeg, Canada.

Lamb, D. (1976) Variations in the foliar concentrations of macro and micro elements

in a fast-growing tropical eucalypt. Plant and Soil 45: 477-492.

Lamb, D. (1977) Relationship between growth and foliar nutrient concentrations in

Eucalyptus deglupta. Plant and Soil 47:495-508.

Lamb, D. (1994) Reforestation of degraded tropical forest lands in the Asia-Pacific

region. Journal of Tropical Forest Science 7(1): 1-7.

Lambers, H., Chaplin, F.S. III., Pons, T.L. (1998) Plant Physiological Ecology.

Springer-Verlag, New York.

Lambers, H, Juniper, D., Cawthray, G.R., Veneklaas, E.J., Martinex-Ferri, E. (2002)

The pattern of carboxylate exudation in Banksia grandis (Proteaceae) is

affected by the form of phosphate added to the soil. Plant and Soil 238: 111-

122.

Lambers, H., Cramer, M.D., Shane, M.W. Wouterlood, M., Poot, P., Veneklaas, E.J.

(2003) Structure and functioning of cluster roots and plant responses to

phosphate deficiency. Plant and Soil 248: ix-xix.

Lambers, H, Shane, M.W., Crammer, M.D., Pearse, S.J., Veneklaas, E.J. (2006) Root

structure and functioning for efficient acquisition of phosphorus: matching

morphological and physiological traits. Annual Botany 98: 693-713.

Lambert, M.J. (1984) The use of foliar analysis in fertilizer research. In: Grey, D.C.

Schonau, A.P.G., Schultz, C.J. (eds.), Proceedings of IUFRO Symposium on

Site Productivity of Fast Growing Plantations. Pretoria. Volume 1: 268-291.

South Africa Forestry Research Institute, Pretoria and Pietermaritzburg.

Lamont, B. (2003) Structure, ecology and physiology of root clusters - a review.

Plant and Soil 248: 1-19.

Landis, T.D., Tinus, R.W., McDonald, S.E., Barnett, J.P. (1989) The Biological

Component: Nursery Pests and Mycorrhizae. Vol. 5, The container tree

nursery manual. Agriculture Handbook 674. Washington, D.C., USA: US

Department of Agriculture, Forest Service. 171 pp.

Landly, J.P. (1982) Tropical forest resources. FAO Forest Paper 30. FAO, Rome.

Lawrie, A.C. (1981) Nitrogen fixation by native Australian legumes. Australian

Journal of Botany 29: 143-157.

Leece, D.R. (1976) Diagnosis of nutritional disorders of fruit trees by leaf and soil

analyses and biochemical indices. Journal of Australia Institute of

Agriculture Science 42: 3-19.

144

Lee, L.S., Mayer, D.G. (1993) Leaf and soil analysis data interpretation and fertiliser

recommendations for tree crops by a Fortran computer software package. In:

„Nutrition in Horticulture‟- Proceeding of a review workshop, Hunter, M.N.,

Eldershaw, V.J. (eds.) Gatton, 4-6 May. Department of Primary Indusries,

Brisbane.

Liengsiri, C, Boyle, T.J.B., Teh, F.C. (1998) Mating system of Pterocarpus

macrocarpus Kurz. in Thailand. Journal of Heredity 89: 216-221.

Liengsiri, C., Hellum, A. K. (1988) Effects of temperature on seed germination in

Pterocarpus macrocarpus. Journal of Seed Technology 12(1): 66-75.

Lim, G. (1976) Rhizobium and nodulation of legumes in Singapore. In 'Proceedings

of Symposium Somiplan'. Kuala Lumpur, Malaysia. In: Broughton, W.J.,

John, C.K., Rajarao, J.C. and Lim, B (eds.), University Malaya Press. pp 159-

175.

Lok, E.H. (1996) The Branching Behaviour and Silvicultural Potential of

Pterocarpus indicus Using Small Cuttings. M.Sc. thesis, Universiti Pertanian

Malaysia. 75 pp.

Lynch, J.P., Beebe, S.E. (1995) Adaptation of beans (Phaseolus vulgaris L.) to low

phosphorus availability. Hortscience 30(6): 1165-1171.

Ma, Z., Bielenberg, D.G., Brown, K.M., Lynch, J.P. (2001) Regulation of root hair

density by phosphorus availability in Arabidopsis thaliana. Plant,Cell and

Environment 24: 459-467.

Manhart, J.R., Wong, P.P. (1980) Nitrate effect on nitrogen fixation (acetylene

reduction): Activity of legume root nodules induced by Rhizobia with varied

nitrate reductase activities. Plant Physiology 65:502-505.

Marchal, J. (1987) Miscellaneous tropical. In “PlantAnalysis as a Guide to the

Nutrient Requirements of Temperate and Tropical Crops”. (Martin-Prevel, P.,

Gagnard, J. and Gautier (eds.) Lavousier Publishing Inc., New York. pp 440-

453.

Marschner, H. (1995) Mineral Nutrition of Higher Plants. Academic Press, London.

889 pp.

McArthur, W.M. (1991) Reference soils of south-western Australia. Department of

Agriculture, Perth, Western Australia.

McArthur, W.M., Bettenay, E. (1960) The development and distribution of the soils

on the Swan Coastal Plain, Western Australia. Commonwealth Scientific and

Industrial Research Organisation (CSIRO), Australia.

145

Mead, D.J. (1984) Diagnosis of nutrient deficiencies in plantations. In: Bowen G.D.,

Nambiar, E.K.S. (eds.), Nutrition of plantation forests. Academic Press, New

York. pp 259-291.

Misra, R.K., Alston, A.M., Dexter, A.R. (1988) Role of root hairs in phosphorus

depletion from a macro structured soil. Plant and Soil 107: 11-18.

Moreira, F.M.S., Haukka, K., Young, J.P.W., 1998. Biodiversity of rhizobia isolated

from a wide range of forest legumes in Brazil. Molecular Ecology 7: 889-

895.

Moreira, F.M.S., da Silva, M.F., de Faria, S.M. (1992) Occurrence of nodulation in

legume species in the Amazon region of Brazil. New Phytologist 121: 563-

570.

Mosse, B., Powell, C.L., Hayman, D.S. (1976) Plant growth response to vesicular-

arbuscular mycorrhiza. IX. Interaction between VA mycorrhiza, rock

phosphate and symbiotic nitrogen fixation. New Phytologist 76: 331-342.

MTIB (2007) Eight Selected Species For Forest Plantation Programme in Malaysia.

Malaysian Timber Industry Board (MTIB), Kuala Lumpur, Malaysia.

Munyanziza, E., Oldeman, R.A.A., 1994. Pterocarpus angolensis D.C.: field

survival strategies, growth, root pruning and fertilization in the nursery.

Fertiliser Research 40: 235-242.

Nambiar, E.K.S., Brown, A.G. (1997) (eds.) Management of Soil, Water and

Nutrients in tropical plantation forests. Australian Centre for International

Agricultural Research (ACIAR). ACIAR Monograph No. 43. Canberra,

Australia. 571 pp.

Nandasena, K.G. (2004) Rapid evolution of diversity in the root nodule bacteria of

Biserrula pelecinus L. PhD Thesis, Murdoch University, Murdoch, Western

Australia.

NAS, (1979) Tropical legumes: Resources for the future. National Academy of

Sciences, Washington.

Ndufa, J.K., Shepherd, K.D., Buresh, R.J., Jama, B. (1999) Nutrient uptake and

growth of young trees in a P-deficient soil: Tree species and phosphorus

effects. Forest Ecology and Management 122: 231-241.

Ng, F.S.P. (1992) Pterocarpus indicus- The majestic N-fixing tree. Nitrogen-Fixing

Tree Highlights (NFT), Hawaii, USA.

Nibau, C., Gibbss, D.J., Coates, J.C. (2008) Branching out in new direstions: the

control of root architecture by lateral root formation. New Phytologist 179

(3): 595-614.

146

Nik Muhamad M., Paudyal, B.K. (1999) Foliar sampling guidelines for different

aged Acacia mangium plantations in Peninsular Malaysia. Journal of Tropical

Forest Science 10: 431-437.

Norton, J.M., Firestone, M.K. (1996) N dynamics in the rhizosphere of Pinus

ponderosa seedlings. Soil Biology and Biochemistry 28: 351-362.

Nye, P.H. (1977) The rate-limiting step in plant nutrient absorption from soil. Soil

Science 123: 292-297.

Nwoboshi, L.C. (1982) Tropical Silviculture: Principles and Techniques.University

Press, Ibadan, Africa.

Odee, D.W., Sutherland, J.M., Kimiti, J.M., Sprent, J.I. (1995) Natural rhizobial

populations and nodulation status of woody legumes growing in diverse

Kenyan conditions. Plant and Soil 173: 211-224.

O' Hara, G.W., Tiwari, R.., Yates, R.., Dilworth, M., Howieson, J.G. (2000) Master

Class in Rhizobium Technology, 8-22 September, 2002 at Inia, Uruguay.

Centre for Rhizobium Studies, Murdoh University, Western University.

Old, K.M., Lee, S.S., Sharma, J.K. (1997) Diseases of Tropical Acacias. In:

Proceedings of International Workshop. Subanjeriji, South Sumatra,

Indonesia. CIFOR Special Publication 120 pp.

Oldfields, S., Lusty, C., MacKinven, A. (1998) The World Threatened Trees. World

Conservation Press, Cambridge, UK.

Orlander, B.J. (1998) Browsing damage by roe deer on Norway spruce seedlings

planted on clearcuts of different ages. II. Effect of seedling vigour. Forest

Ecology and Management 105 (1):295-302.

Oyaizu, H., Matsumoto, S., Minamisawa, K., Gamou, T. (1993) Distribution of

rhizobia in leguminous plants surveyed by phylogenetic identification.

Journal of General Applied Microbiology 39: 339-354.

Palmer, K.M., Young, J.P.W. (2000) Higher diversity of Rhizobium leguminosarum

biovar vicieae population in arable soils than in grass soils. Applied and

Environmental Microbiology 66: 2445-2450.

Palmer, D.J., Lowe, D.J., Payn, T.W., Hock, B.K., McLay, C.D.A., Kimberley, M.O.

(2005) Soil and foliar phosphorus as indicators of sustainability for Pinus

radiata plantation forestry in New Zealand. Forest Ecology and Management

220: 140-154.

Parks, S.E., Haigh, A.M., Creswell, G.C. (2000) Stem tissue phosphorus as an index

of the phosphorus status of Banksia ericifolia L. Plant and Soil 227: 59-65.

Parrotta, J.A. (1996) The role of plantation forests in rehabilitating degraded tropical

ecosystems. Agriculture, Ecosystems and Environment 41: 115-133.

147

Paudyal, B.K. and Majid, N.M. (1992) Foliar nutrient levels of different aged Acacia

mangium Willd. plantations in Kemasul Forest Reserve, Pahang, Peninsular

Malaysia. Tropical Ecology 2:34-40.

Peoples, M.B., Ladha, J.K., Herridge, D.F. (1995) Enhancing legume N2 fixation

through plant and soil management. Plant and Soil 174: 83-101.

Peoples, M.B., Herridge, D.F. (1990) Nitrogen fixation by legumes in tropical and

sub-tropical agriculture. Advances of Agronomy 44: 155-223.

Perez-Fernandex, M.A., Lamont, B.B. (2003) Nodulation and performance of exotic

and native legumes in Australian soils. Australian Journal of Botany 51: 543-

553.

Perret, X., Staehelin, C., Broughton, W.J. (2000) Molecular basis of symbiosis

promiscuity. Microbiology and Molecular Biology Reviews 64: 180-201.

Piewluang, C. (1996) Preliminary results of a study of natural regeneration in

Pterocarpus macrocarpus Kurz. Proceedings of 6th Silviculture Seminar.

Royal Forest Department, Udon Thani, Thailand.

Piewluang, C., Liengsiri, C., Boyle, T. (1997) Genetic variation of Pterocarpus

macrocarpus Kurz: variation in fruit and seed size and quality within and

among natural population. ASEAN Forest Tree Seed Centre, Muak Lek,

Thailand. pp 1-6

Polhill, R.M. (1981) Advances in Legume Systematics. Royal Botanic Gardens,

Kew, UK. pp 1-6.

Purnell, H.M. (1960) Studies of the family Proteaceae. I, Anatomy and morphology

of the roots of some Victorian species. Australian Journal of Botany 8: 35-50.

Quispel, A. (1974) (eds.) The Biology of Nitrogen Fixation. North Holland Research

Monographs Frontiers of Biology 33. North Holland Publishing Company,

Amsterdam. 769 pp.

Raaimakers, D., Lambers, H. (1996) Response to phosphorus supply of tropical tree

seedlings: a comparison between a pioneer species Tapirira obtusa and a

climax species Lecythis corrugata. New Phytologist 132: 97-102.

Radomiljac, A.M. (1998) The influence of pot host species, seedling age and

supplementary nursery nutrition on Santalum album Linn. (Indian

Sandalwood) plantation establishment within the Ord River Irrigation Area,

Western Australia. Forest Ecology and Management 102: 193-201.

Raghothama, K.G. (1999) Phosphate acquisition. Annual Review of Plant Physiology

and Plant Molecular Biology 50: 665-693.

148

Ramakrishna, A., Bailey, J.S., Kirchhof, G. (2009) A preliminary diagnosis and

recommendation integrated system (DRIS) model for diagnosing the nutrient

status of sweet potato (Ipomoea batatas). Plant Soil 316: 107-116.

Reddell, P. (1993) Soil Constraints to the Growth of Nitrogen-Fixiing Trees in

Tropical Environments. In: Symbioses in Nitrogen-Fixing Trees, Oxford and

IBH Publishing Co. Ltd., New Delhi, India.

Ribet, J., Drevon, J.J. (1995) Phosphorus deficiency increases the acetylene-induced

decline in nitrogenase activity in soybean (Glycine max (L.) Merr.) Journal of

Experimental Botany 46: 1479-1486.

Richardson, A.E., Viccars, L.A., Watson, J.M., Gibson, A.H. (1995) Differentiation

of Rhizobium strains using the polymerase chain reaction with random and

directed primers. Soil Biology and Biochemistry 27: 515-524.

Richardson, K.C., Jarret, L., Finke, E.H. (1960) Embedding in epoxy resins for ultra

thin sectioning in electron microscopy. Stain Technology 35: 313-323.

Ridge, R.W., Emons, A.M.C. (2000) Root Hairs: Cell and Molecular Biology.

Springer-Verlag, Tokyo. 336 pp.

Ridley, H.N. (1992) The Flora of the Malay Peninsula. Reeve, London.

Righetti, T.L., Alkoshab, O., Wilder, K. (1988) Diagnostic biases in DRIS

evaluations on sweet cherry and hazel nut. Communication Soil Science.

Plant Analysis 19: 1429-1447.

Robinson, J.B., Nicholas, P.R. (1983) Soils of Australia. Department of Agriculture,

South Australia. (Unpublished data).

Robson, A.D., Gilkes, R.J. (1980) Fertilizer responses (N,P,K,S and micro-nutrients)

on lateritic soils in south-western Australia- A review. International Seminar

on Laterization Processes. Oxford and IBM Publishing Co., Oxford, England.

pp 381-390.

Robson, J.A., O‟Hara, G.W., Abbot, L.K. (1981) Involvement of phosphorus in

nitrogen fixation by subterranean clover (Trifolium subterranean L.).

Australian Journal of Plant Physiology 8: 427-436.

Roggy, J.C., Prevost, M.F. (1999) Nitrogen-fixing legumes and silvigenesis in a rain

forest in French Guiana: A taxonomic and ecological approach. New

Phytologist 144: 283-294.

Rojo, J.P. (1977) Pantropic speciation of Pterocarpus (Leguminosae-Papilionaceae)

and the Malesia-Pacific species. Pterocarpus 3: 19-32.

Russell, E.W. (1973) Soil Conditions and Plant Growth. Longman Group Limited,

London. Tenth Edition. 849 pp.

149

Subba Rao, N.S. 1982 Biofertilizers. Advances in agricultural microbiology. In: N.S.

Subba Rao (ed.), Oxford and IBH, UK, Mohan Prilani, and New Delhi,

Butterworth and Co.

Salleh, Md Nor., Appanah, S. (1995). Reforestation of the Rubber Sector

Proceedings of the Rubber Grower‟s Conference held in Kuala Lumpur,

Malaysia. 17-19 July, 1995.

Sanchez, P.A., Shepherd, K.D., Soule, M.J., Place, F.M., Buresh, R.J., Izac, A.M.,

Uzo M.A., Kwesiga, F.R., Nideritu, C.G. and Woomer, P.L. (1997) (eds.),

Replenishing Soil Fertility in Africa, pp. 1-1-46. Soil Science Society of

America and American Society of Agronomy, Madison, Wisconsin, USA.

Sardina, A.C. (1961) The effect of length of narra cuttings on the percentage of

survival. Philippines Journal of Forestry 14: 59-66.

Seddon, R. (1972) Sense of Place. A Response to an Environment: the Swan Coastal

Plain.University of Western Australia, Perth, Western Australia.

Serraj, R. (2004) (Ed.) Symbiotic Nitrogen Fixation: Prospects for Enhanced

Application in Tropical Agriculture. Science Publisher, Inc. Enfield, USA.

367 pp.

Setiadi, Y. (1995) The status of reforestation in Indonesia. In 'Bio-Re/afforestation in

the Asia-Pacific Region.' Tokyo, Japan. K Suzuki, Sakurai, S., Ishii, K. and

Norisada, M. (eds.). pp 47-59

Shane, M.W., McCully, M.E., Lambers, H. (2004) Tissue and cellular phosphorus

storage during development of phosphorus toxicity in Hakea prostrata

(Proteaceae). Journal of Experimental Botany 55: 1033-1044.

Shedley, E., Dell, B., Grove, T.S. (1995) Diagnosis of nitrogen deficiency and

toxicity of Eucalyptus globulus seedlings by foliar analysis. Plant and Soil

177: 183-189.

Shepherd, K.D., Ohlsson, E., Okalebo, J.R., Ndufa, J.K. (1996) Potential impact of

agroforestry on soil nutrient balances at the farm scale in the East African

Highlands. Fertiliser Research 44:87-99.

Sim, B.L. (1987) Research on Acacia mangium in Sabah: A Review. In 'Australian

acacias in developing countries.' In: Turnbull, E.D. (ed.), Australian Centre

for International Agricultural Research (ACIAR), Canberra, Australia. pp.

164-166.

Singleton, P.W., Abel Magid, H.M,, Tavares, J.W. (1985) Effect of phosphorus on

the effectiveness of strains of Rhizobium japonicum. Soil Science Society of

America Journal 49: 613-616.

150

Skene, K.R., James, W.M. (2000) A comparison of the effects of auxin on cluster

root initiation and development in Grevillea robusta Cunn. Ex R.Br.

Proteaceae) and in the genus Lipinus (Leguminosae). Plant and Soil 219 (1-

2): 221-229.

Smith, F.W., Dolby, G.R. (1977) Derivation of diagnostic indices for assessing the

sulphur states of Panicum maximum var. trichoglume. Communications in

Soil Science and Plant Analysis 8: 221-240.

Smith, F.W., Loneragan, J.F. (1997) An Interpretation of Plant Analysis: Concepts

and Principles. CSIRO Publishing, Melbourne.

Smith, F.W., Mudge, S.R., Rae, A.L., Glassop, D. (2003) Phosphate transport in

plants. Plant and Soil 248: 71-83.

Soerianegara, I., Kartawinata, K. (1985) Silviculture management of the logged

natural dipterocarp forest in South-east Asia. In: Davidson, J., Tho Y.P.,

Bijileveld, M. (eds.). Future of Tropical Rain Forests in South-east Asia.

IUCN, Gland, Commission of Ecology Papers 10: 81-84.

Sousa, M.F., Facanha, A.R., Taveres, R.M., Lino-Neto, T., Geros, H. (2007)

Phosphate transport by proteid roots of Hakea sericea. Plant Science 173:

550-558.

Specht, A., Turner, J. (2006) Foliar nutrient concentrations in mixed-species

plantations of subtropical cabinet timber species and their potential as a

management tool. Forest Ecology and Management 233: 324-337.

Sprent, J.I. (1995) Legume trees and shrubs in the tropics: N2 fixation in perspective.

Soil Biology and Biochemistry. Soil Biology and Biochemistry 27: 401-407.

Sprent, J.I. (2001) Nodulation in Legumes. Royal Botanic Gardens: Kew, United

Kingdom.

Sprent, J.I., Odee, D.W., Dakora, F.D. (2010) African legumes: a vital but under-

utilised resource. Journal of Experimental Botany 61: 1257-1265.

Sprent, J.I., Parsons, R. (2000) Nitrogen fixation in legume and non-legume trees.

Field Crops Research 65: 183-196.

Sprent, J.I., Sutherland, J.M., De Faria, S.D. (1989) Structure and function of nodules

from woody legumes. In: Stirton, C.H. and Zarucchi, J.L. (Eds.), Advances in

Legumes Biology. Monograph of Systematic Botany, Missouri Botany

Garden 29: 559-578.

Spurr, A.R. (1969) A low viscosity epoxy resin embedding medium for electron

microscopy. Journal of Ultrastructure Research 26: 31.

Srivastava, P.B.L. (1979) Symptoms of deficiency in Pterocarpus indicus Willd.

seedlings. The Malaysian Forester 42: 130-137.

151

SSali, H., Keya, S.O. (1980) Nitrogen level and cultivar effects on nodulation,

dinitrogen fixation and yield grain legumes. II. Common bean cultivars. East

African Agriculture and Forestry Journal 45 (4): 277-283.

Stoorvogel, J.J., Smaling, E.M.A., Janssen, B.H. (1993) Calculating soil nutrient

balances in Africa at different scales. I. Supranational scale. Fertiliser

Research 35:237-250.

Streeter, J.G. (1994) Symbiotic N-fixation.' "Plant-environment interactions". Marcel

Dekker Publishere: New York.

Sun, J.S., Simpson, R.J., Sands, R. (1992) Nitrogenase activity of two genotypes of

Acacia mangium as affected by phosphorus nutrition. Plant and Soil 144: 51-

58.

Sylla, S.N., Lajudie, P., Ndoye, I., Neyra, A.T., Dreyfus, B. (1998) In 'Proceedings

of the Eighth Congress of the African Association for Biological Nitrogen

Fixation.' Capetown, South Africa. (Ed. Dakora, F.D.) pp 261-262.

Tang, C., Hinsinger, P., Drevon, J.J., Jaillard, B. (2001) Phosphorus deficiency

impairs early functioning and enhances proton release in roots of Medicago

truncatula, L. Annals of Botany 88: 131-138.

Thies, J.E., Singleton, P.W., Bohlool, B.B. (1991) Modeling symbiotic performance

of introduced rhizobia in the field bu uses of indices of population size and

nitrogen status of the soil. Applied and Environmental Microbiology 57: 29-

37.

Tian, C.J., He, X.Y., Zhong, Y., Chen, J.K. (2002) Effect of VA mycorrhizae and

Frankia dual inoculation on growth and nitrogen fixation of Hippophae

tibetana. Forest Ecology and Management 170: 307-312.

Tian, C.J., He., X.Y., Zhong, Y., Chen, J.K. (2003) Effect of inoculation with ecto-

and arbuscular mycorrhiza and Rhizobium on the growth and nitrogen

fixation by black locust, Robina pseudoacacia. New Forests 25: 125-131.

Timmer, V.R. (1997) Exponential nutrient loading: a new fertilization technique to

improve seedling performance on competitive sites. New Forests 13:279-299.

Troup, R.S. (1921) The Silviculture of Indian Trees. Volume 1. India International

Book Distributors, Dehra Dun, India.

Turk, D., Keyser, H.H. (1992) Rhizobia that nodulate tree legumes: specficity of the

host for nodulation and effectiveness. Canada Journal of Microbiology 38:

451-460.

Turnbull, J.W., Midgley, S.J., Cossalter, C. (1998) Tropical acacias planted in Asia:

An overview. In 'Recent developments in acacia planting.' In: JW Turnbull,

J.W., Crompton, H.R. and Pinyopusarerk, K. (eds.), Australian Centre for

International Agricultural Research (ACIAR), Canberra, Australia. pp 14-28.

152

Turner, J., Lambert, M.J. (1986) Nutritional and nutritional relationships of Pinus

radiata. Annual Review Ecology Systems 17: 325-350.

U, Saw Yan (1993) Genetic variation of Pterocarpus macrocarpus Kurz: variation in

fruit and seed size and quality in Myanmar. ASEAN Forest Tree Seed Centre,

Muak Lek, Thailand. pp 1-6

Uchida, R.S. (2000) Essential nutrients for plant growth: nutrient functions and

deficiency symptoms. College of Tropical Agriculture and Human Resources,

University of Hawaii, Manoa.

Udarbe, M.P., Hepburn, A.J. (1987) Development of Acacia mangium as a plantation

species in Sabah. In 'Australian acacias in developing countries.' JW

Turnbull, J.W. (ed.), Australian Centre for International Agriculture Research

(ACIAR), Canberra, Australia. pp 157-159.

Ulrich, A., Hills, F.J. (1967) Principles and Practices of Plant Analysis. In: Hardy,

G.W. (Ed.), Soil Testing and Plant Analysis, Part 2: Plant Analysis. Soil

Science Society of America, Madison. pp 11-24.

Unkovich, M.J., Pate, J.S., Sanford, P. (1997) Nitrogen fixation by annual legumes in

Australian mediterranean agriculture. Australian Journal of Agriculture

Research 48: 267-293.

Vadez, V., Lim, G., Durand, P., Diem, H.G. (1995) Comparative growth and

symbiotic performance of four Acacia mangium provenances from Papua

New Guinea in response to the supply of phosphorus at various

concentrations. Biology and Fertility of Soils 19: 60-64.

Vadez, V., Rodier, F., Payre, H. Drevon, J.J. (1996) Nodule permeability to O2 and

nitrogenase-linked respiration in bean genotypes varying in the tolerance of

N2 fixation to P deficiency. Plant Physiology and Biochemistry 34: 871-878.

Van den Driessche, R. (1984) Relationship between spacing and nitrogen fixation of

seedlings in the nursery, seedling mineral nutrition and out-planting

performance. Canadian Journal of Forest Research 14: 431-436.

Van Kessel, C., Roskoski, J.P. (1981) Nodulation and nitrogen fixation by Inga

jinicuil, a woody legume in coffee plantations. II Effects of soil nutrient on

nodulation and nitrogen fixation. Plant and Soil 59: 207-215.

Vincent, J.M. (1970) A Manual for the Practical Study of the Root Nodule Bacteria.

International Biological Program, Handbook 15, Blackwell Scientific

Publications, Oxford.

Waisel, Y., Eshel, A., Kafkafi, U. (2002) (Eds.) Plant Roots: The Hidden Half. Third

Edition. Marcel Dekker, Inc. New York. 1120 pp.

Walch-Liu, P., Ivanov, I.I., Filleur, S., Gan Y., Remans., Forde, B.G. (2006)

Nitrogen regulation of root branching. Annals of Botany 97: 875-881.

153

Wallace, I.M., Dell, B., Loneragan, J.F. (1986) Zince nutrition of Jarrah (Eucalyptus

marginata Donn ex Smith) seedlings. Australian Journal of Botany 34: 41-

51.

Walker, R.B., Chowdappa, P., Gessel, S.P. (1993) Major element deficiencies in

Casuarina equisetifolia. Fertilizer Research 34: 127-133.

Walworth, J., Summer, M.E. (1987) The diagnosis and recommendation integrated

system. Advances in Soil Science 6: 149-188.

Wan Razali, W.M. (2003) Failure of Tropical Management or Sustainable Forest

Management in Crisis? - R&D Perspective. Pp. 13-23 In: Azmy et al. (eds.),

International Conference of Forestry and Forest Products Research. Forest

Research Institute of Malaysia, Kepong, Malaysia. October 1-3, 2003.

Wang, H., Zhou, W. (1996) Fertilizer and eucalypt plantations in China. In: Attiwill,

P.M. and Adams, M.A. (eds.), Nutrition of eucalyptus. CSIRO, Melbourne,

Australia, pp 389-397.

Ware, G.O., Ohki, K., Moon, L.C. (1982) The Mitscherlich plant growth model for

determining critical nutrient deficiency levels. Journal of Agronomy 74: 88-

91.

Webb, M.J., Reddell, P., Grundon, N.J. (2001) A Visual Guide to Nutritional

Disorders of Tropical Timber Species: Swietenia macrophylla and Cedrela

odorata. ACIAR Monograph No. 16. Australian Centre for International

Agricultural Research, Canberra, Australia. 178 pp.

Welsh, J., McClelland, M. (1990) Fingerprinting genomes using PCR with arbitrary

primers. Nuclei Acid Research 18: 7213-7218.

Whitmore, T.C. (1984) Tropical Rain Forests of Far East. Second Edition.

ELBS/Oxford Press, Oxford.

Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A., Tingey, S.V. (1990)

DNA polymorphisms amplified by arbitrary primers are useful as genetic

markers. Nucleic Acids Research 18: 6531-6535.

Wilson, S.D., Tilman, D. (1991) Components of plant competition along an

experimental gradient of nitrogen availability. Ecology 72: 1050-1065.

Witkowski, E.T.F. (1991a) Effects of invasive alien acacias on nutrient cycling in the

coastal lowlands of the Cape Fynbos. Journal of Applied Ecology 28: 1-15.

Witkowski, E.T.F. (1991b) Growth and competition betweeen seedlings of Protea

repens L. and the alien invasive, Acacia saligna (Labill.) Wendl., in relation

to nutrient availability. Functional Ecology 4: 101-110.

154

Wong, T.M. (1983) Lesser known timber IX - Sena. Timber Digest No. 53. Forest

Research Institute, Kepong, Malaysia.

Wong, Y.K. (1982) Horticultural notes on the angsana (Pterocarpus indicus Willd.).

Gardens Bulletin of Singapore 34: 189-202.

Wong, M. (2005) Visual Symptoms of Plant Nutrient Deficiemncies in Nursery and

Landscape Plants. Soil and Crop Management : 1-3.

Xu, D. (2001) Managing nutrients to increase productivity of plantation eucalypts in

south China. PhD Thesis, Murdoch University, Perth Western Australia. 181

pp.

Xu, D., Dell, B., Malajczuk, N., Gong, M. 2001 Effects of P fertilisation and

ectomycorrhizal fungal inoculation on early growth of eucalypt plantations in

southern China. Plant and Soil 233: 45-57.

Xu, D., Dell, B., Malajczuk, N., Gong, M. 2002 Effects of fertilisation on

productivity and nutrient accumulation in a Eucalyptus grandis x E.

urophylla plantation in southern China. Forest Ecology and Management

161: 89-100.

Yao, C.E. (1981) Survival and growth of mahogany (Swietenia macrophylla King.)

seedlings under fertilized grassland condition. Sylvatrop Philippines Forest

Research Journal 6(4): 203-217.

Yew, F.K., Pushparajah, E. (1984) Plant tissue as indicators of soil nutrient

availability for Hevea: glasshouse evaluations. Journal of Rubber Research

Institute Malaysia 32(3): 171-181.

Zahran, H.H. (1999) Rhizobium-legume symbiosis and nitrogen fixation under

severe conditions and in an arid climate. Microbiology Molecular Biology

Revision 63(4): 968-989.

Zech, W. (1990) Mineral deficiencies in forest plantations of north-Luzon,

Philippines. Tropical Ecology 31: 22-31.

Zhang, Y.J., Lynch, J.P., Brown, K.M. (2003) Ethylene and phosphorus availability

have interacting yet distinct effects on root hair development. Journal of

Experimental Botany 54: 2351-2361.

Zou, X.L., Cai, K.Q., Huang, W.N. (1998) Hydrogenase activity in some species of

leguminous trees. China Journal of Tropical Crops 19(4): 71-76.