additional experiences to elucidate the microbial component of soil suppressiveness towards...

Annals of Applied Biology (2005), 146:421–431

*Corresponding Author Email: [email protected]

© 2005 Association of Applied Biologists

421

Additional experiences to elucidate the microbial component of soil suppressiveness towards strawberry black root rot complex

L M MANICI∗, F CAPUTO and G BARUZZI

Research Institute for Industrial Crops (MiPAF), Via di Corticella 133, 40128 Bologna, Italy

Summary

Several studies were carried out to investigate the soil microbial components involved in suppressing strawberry black rot root which occurs throughout the Italian strawberry growing region. Quantitative and qualitative evaluation of fungi involved in black root rot were combined with several soil microbial parameters involved in soil suppressiveness towards black root rot agents. The fi rst survey, carried out in an intensively cultivated area of northern Italy, identifi ed Rhizoctonia spp. as the main root pathogen together with several typical weak pathogens belonging to the well-known black rot root complex of strawberry crop: Cylindrocarpon destructans, Fusarium oxysporum, F. solani, Pestalotia longiseta and others. The root colonisation frequency of strawberry plants increased strongly from autumn to spring at harvesting stage. Rhizoctonia spp. were the only pathogens which followed the rising trend of root colonisation with relative frequency; all the weak pathogens of strawberry black root rot complex did not vary their frequency. Only non-pathogenic fungi decreased from autumn to spring when at least 60% of colonising fungi were represented by Rhizoctonia. These data suggested that the late vegetative stage was the best time to record the soil inoculum of root rot agents in strawberry using root infection frequency as a parameter of soil health. A further study was performed in two fi elds, chosen for their common soil texture and pH, but with signifi cant differences in previous soil management: one (ALSIA) had been subjected to strawberry monoculture without organic input for several years; the other (CIF) has been managed according to a 4-year crop rotation and high organic input. In this study Pythium artifi cially inoculated was adopted as an indicator for the behaviour of saprophytically living pathogens in bulk soil. Pythium showed a sharp, different response after inoculation in bulk soil from the two soil systems evaluated. Pythium was suppressed only in the CIF fi eld where the highest levels of total fungi and fl uorescent bacteria and highest variability were observed. The suppressiveness conditions towards Pythium, observed in the CIF and absent in the ALSIA fi eld, corresponded with the root infection frequency recorded at the late vegetative stage on strawberry plants grown in the two fi elds: strawberry plants from the CIF fi eld showed lower root colonisation frequency and higher variability than that recorded on those coming from the ALSIA fi eld.

Key words: Biodiversity, Fragaria × ananassa Duch, Pythium, culturable bacteria, Rhizoctonia, soil health, total fungi

Introduction

Decline in strawberry (Fragaria × ananassa Duch.) yield caused by black root rot in Italy is the most frequent problem for strawberry production in this country (D’Ercole, 1970; Ciccarese & Cirulli, 1983). Black root rot is a worldwide disease that limits the yield of strawberry (Yuen et al., 1991; Wing et al., 1994) as well as vegetable crops (Davis & Nunez, 1999), fruit trees (Mai & Abawi, 1978; Mazzola, 1997), cereals (Bateman et al., 1998) and herbaceous crops (Vestberg, 1987). It is caused by a complex of several facultative saprophytic pathogens, also called non-lethal pathogens. The relative frequency of these pathogens and their role in inducing black root rot varies with the crop,

the environment and soil management techniques. In most cases, Rhizoctonia spp. and Pythium spp. are the most important agents. In strawberry, great variations in the relative frequency and role of those two pathogens in causing decline in yield have been reported (Watanabe, 1977; Xiao & Duniway, 1998; Martin, 1999). In all cases their occurrence is combined with several non-lethal pathogens like Cylindrocarpon spp., Fusarium oxysporum Schlecht., Fusarium spp. and several weak pathogens (Maas, 1984).

Van Bruggen & Grünwald (1996) defi ned the risk of root infection by plant pathogens as a soil quality parameter. Pankhurst et al. (1995) wrote that, among many soil biological variables measured, root pathogens can be considered as potential

L M MANICI ET AL.422

bio-indicators of soil fertility. This concept is particularly appropriate in the case of facultative saprophytic pathogens in soil. Furthermore, the behaviour of these pathogens in soil is strongly affected by microbial activity (Lumsden et al., 1983; Manici et al., 2003) and by variations in soil microbial communities (Mazzola, 2002). These two biological components of soil suppression (Grünwald et al., 2000) depend on organic matter content in the soil (Schnürer et al., 1985) and the soil management system (Bolton et al., 1985; Abawi & Widmer, 2000). Strawberry production in Italy is concentrated in a few main areas in the north and south. Integrated production (IP) is used in 80% of the total strawberry cultivated area in Emilia Romagna and 50% in the southern regions, while organic production accounts for 1% of the strawberry cultivated area in northern Italy and 4% to 5% in the south. In Italy the maximum life span of the strawberry crop does not exceed 1 year. The regional IP guidelines recommend 3 years as the minimum break between two consecutive strawberry crops. On the contrary, in traditional production there is a one year break with a winter cereal between two to three consecutive strawberry crop cycles. In that cropping system, periodic fumigation has been used widely in the past and is still applied, although the imminent phasing out of methyl bromide and the current restrictions on its use, together with the new focus on sustainable agriculture, have in recent years discouraged frequent soil fumigation and placed greater emphasis on improving soil fertility.

Following the new approach to soil management for strawberry production, a study of potential bio-indicators of soil fertility for this crop was performed. This study focused on root colonisation and on Pythium spp. as a possible bio-indicator. The pathogenicity of rot fungal pathogens of strawberry is variable (Xiao & Duniway, 1998). It is strongly affected by the crop growth stage and is enhanced by stress conditions mediated by abiotic factors such as water and temperature (Molot & Ferrière, 1989). The senescent strawberry roots are usually highly colonised by Rhizoctonia spp. (Elmer & La Mondia, 1999; Manici et al., 2000); therefore a survey of the strawberry root rot complex was performed at the late vegetative growth stage and at the mid-harvest stage using representative plant samples from fi elds with root rot decline. The aim was to fi nd the best strawberry plant growing stage at which feeding roots could be used to measure pathogen frequency and population composition.

This survey was combined with recordings of Pythium levels in soil from the fi elds where the strawberry samples were collected. Pythium is the saprophytic pathogen most sensitive to microbial activity (Chen et al., 1988a) and therefore it is

used as an indicator of soil suppression by several authors (Craft & Nelson, 1996; Grünwald et al., 2000). Using an artifi cial Pythium irregulare Buisman inoculum, in this study also allowed the potential value of this pathogen as a bio-indicator of soil sickness to be estimated. The fi nal aim of this study was to increase knowledge and fi nd the best method to increase soil fertility in low input strawberry farming as required by IP and organic management systems.

Materials and Methods

Two-year survey on strawberry root colonisationThere are several reports on strawberry black root

rot and the soil-borne fungi involved in this disease in Italy (D’Ercole, 1970; Ciccarese & Cirulli, 1983; Tamietti & Valmaggia, 1994). However, these reports have focused on the pathogenicity of fungi isolated from strawberry root tissues showing black root rot decline, symptoms which generally appear during the harvest stage. The aim of this study was to single out the best time when root colonisation frequency and fungal population composition could provide information on yield decline induced by black root rot. A survey was carried out in a sub-area of the Cesena area (latitude 44°), a strawberry growing region in the east Po Valley (northern Italy, Mediterranean area, between latitudes 30° and 45°, with a temperate sub-continental climate). That area is characterised by clay loam soil texture (pH 8.1–8.2) and fertility problems, due to the low content of organic matter occurring in this area (personal communication by Centro Ricerche Produzione Vegetale, Cesena). In 1997–1998 a survey was carried out on a 3000 m2 strawberry fi eld, named S. Giorgio (SG), that had been fumigated 8 years earlier and subsequently cultivated with strawberry mother plants for 7 years; in the previous year it had been left fallow. That cropping system, combined with a low organic matter content (OM%, 1.6) had induced yield decline described by farmers as ‘whole fertility decline’; black root rot was always observed.

In 1998–1999, six strawberry crops in private farms, within a 10 km radius around the fi rst fi eld trial, were chosen with the following criteria:

• fi elds never fumigated or not fumigated for at least 8 years, using a traditional soil management system;

• strawberry crops had been cultivated in the last 8 years adopting variable crop rotations with a winter cereal (strawberry–cereal–strawberry–cereal or strawberry–strawberry–cereal–strawberry).

In 1998–1999, an additional fi eld of about 3000 m2 in the experimental farm ‘Martorano 5’ (MT) where strawberries were grown in close rotation with cereals for the previous 10 years, was included

Soil suppressiveness towards strawberry black root rot complex 423

in the survey. Soil texture was silty clay loam with pH varying from 8 to 8.2 and OM% varied from 1.5% to 1.8%.

Plant samples were collected from each site at the two growing stages: late vegetative growth stage (late November–early December) and mid-harvest stage (middle April–May). Two feeding roots were taken from each strawberry plant, washed under running water, disinfected for 2 min in 1% sodium hypochlorite, and rinsed twice with sterile water. Three tissue segments (0.3 to 0.5 cm) were excised from the distal, middle and proximal parts of two feeding roots of each plant, regardless of rot symptoms. Root segments were placed on water agar (six per plate) and incubated for 3 to 4 days. In autumn and spring, plant samples were collected at random from each site as follows: 48 samples from S. Giorgio and Martorano sites, and 16 samples from each fi eld at six private farms. The relative colonisation frequency was recorded dividing the number of root segments from which fungal hyphae grew by the number of root segments incubated for each site (Table 1). Each colony was transferred to potato sucrose agar (PSA) amended with 200 mg mL–1 of streptomycin

sulphate. An additional number of root segments for each fi eld were placed on selective medium, cornmeal agar amended with pimaricin reduced to 4 mg L-1, 250 g L-1 ampicillin, 10 g L-1 rifampicin and 100 mg L-1 pentachloronitrobenzene (P

4ARP;

Jeffers & Martin, 1986), to record the presence of Phytophthora sp., although typical symptoms were not

observed.

Colonies were identifi ed on the basis

of macroscopic and microscopic morphological characteristics using the relevant taxonomic keys (Nelson et al., 1983; Maas, 1984; Burgess et al., 1988; Samson & van Reenen-Hoekstra, 1988; IMI, 1964–2003). Phoma spp. was identifi ed by the Centraalbureau voor Schimmelcultures (CBS) (Utrecht, the Netherlands). The isolates were identifi ed as Rhizoctonia on the basis of colony morphology and typical hypha branching. The number of nuclei within hyphal cells was determined on a representative number of isolates, using the clean slide technique (Kronland & Stanghellini, 1988).

Data on total root infection frequency and on the relative frequency of each fungal species were arc-sin transformed and subjected to one way analysis of variance for the difference between autumn and

Table 1. Root colonisation frequency and relative frequency of root colonising fungal species recorded in autumn (at the end of vegetative growing stage) and in spring (at middle harvesting stage), on plant samples from seven

strawberry crops in northern Italy

Season, year Site Root segments

(N)

Colonisation frequency

(%)

Relative frequency of root colonising fungi

Rhizoctonia spp.

(%)

F. oxysporum, F. solani (%)

Fusarium spp.

(%)a

Complex

(%)b

Other

(%)c

Autumn 1997 SG farm 288 33 43 13 4 8 32

Spring 1998 SG farm 288 89 57 30 1 7 2

Autumn 1998 MT farm 288 33 44 13 6 21 15

Spring 1999 MT farm 288 75 79 10 6 5 1

Autumn 1998 Six farms 576 (96 each farm)

35.7 4.8 26.0 2.7 30.2 36.3

Spring 1999 Six farms 576 (96 each farm)

63.5 58.8 10.7 1.3 16.3 13.5

Autumn Cesena area 1997–99 35 ± 7.5e 14 ± 6.6 23 ± 7.6 3 ± 1.3 26 ± 6.2 33 ± 6.6

Spring 68 ± 6.8 61 ± 8.4 13 ± 4.0 2 ± 1.1 13 ± 4.1 11 ± 3.5

P-valued Autumn–spring ≤ 0.01 ≤ 0.01 nsf ns ns ≤ 0.01

aFusarium spp.: F. equiseti, F. semitectum, F. compactumbComplex: Alternaria spp., Cylindrocarpon destructans, Phoma pomorum var. pomorum, Phoma pomorum var. calorpreferens, Phoma exigua var. exigua, Zythia fragariae, Pestalotia sp.cOther: Aspergillus sp., Mucor sp., Nigrospora sp., Penicillium sp., Rhizopus sp.dP-value obtained by analysis of variance of root infection frequencies in seven strawberry crops in autumn and the following springeStandard error; fNot signifi cant


spring using the Statgraphic Plus Program, version 2 (Manugistatic Inc, Rockville, MD).

Pythium spp. inoculum in strawberry fi eldsIn May 1997 and 1998, before strawberry crop

planting, six soil cores (6 cm diameter, 0–20 cm deep) were collected at random from the fi elds where the strawberry plants would be subsequently sampled. Samples of bulk soil from each fi eld were mixed by hand, air dried at room temperature for 5 days, then used for Pythium population recording.

Three soil sub-samples (25 g) from each fi eld were mixed with 225 mL of sterile water using a magnetic stirrer for 10 min to obtain a 10–1 diluted soil solution, and then analysed using the soil dilution plate method on selective media (Dhingra & Sinclair, 1986).

To isolate Pythium spp. from soil, 0.5 mL of 10–1 and 10–2 soil dilutions were spread on three plates of selective medium according to the modifi ed method of Jeffers & Martin (1986): cornmeal amended with 5 mg L–1 pimaricin, 250 mg L–1 ampicillin, 10 mg L-1 rifampicin, 100 mg L-1 of pentachloronitrobenzene and 1 g L–1 ox-gall (P

5ARP ). After incubating for

24 h at 18°C in the dark, the soil dilution was washed from the plate surfaces under running water, and the Pythium colonies were counted. Germinating propagules with typical Pythium morphology on P

5ARP + ox-gall were transferred to potato sucrose

agar (PSA) + 200 mg L–1 streptomycin sulphate to confi rm the Pythium spp. population recorded. Germinating propagules with the more frequent shape on selective media were transferred for identifi cation, on the basis of the Waterhouse (1967) key and description, (Waterhouse, 1968). Pythium identifi cation was performed or confi rmed by the Centraalbureau voor Schimmelcultures (Utrecht, the Netherlands). Pythium inoculum was reported as colony forming units (CFU) g–1 of air-dried soil. Three values for each site, each obtained by the mean of three plates where the soil dilution for each sub-sample had been incubated, were recorded.

Relationship among suppresiveness towards Pythium and towards root rot pathogens

Trials were performed in two strawberry fi elds located in two sites with a Mediterranean climate (between latitude 30° and 45°): Basilicata, south-east Italy, and the island of Sardinia, to the west of Italy. The experimental sites had a temperate climate. The fi rst fi eld was in the experimental centre (Az. Pantanello, Metaponto – Matera, latitude 40°) of the Extension Service of Basilicata Region (ALSIA). The fi eld had been fumigated 5 years earlier and then cultivated for 2 successive years with strawberry mother plants and left fallow the year before the trial. Soil texture (40% clay and 33% sand) was in the clay loam class (Schut, 1976),

pH was 7.4 and OM% was 0.63.The second fi eld, in the experimental centre (CIF,

Villasor – Cagliari, latitude 39°), of the Sardinian Extension Service (CIF), had not been fumigated. This fi eld had undergone a 4 year crop rotation, with periodic organic amendments, and strawberries had been cultivated 6 years previously. The soil texture of this fi eld was not the original one for this area, as the cultivation profi le had been changed by addition of soil from another site. The resulting soil texture (28.6% clay, 37.9% sand) was in the clay loam class (Schut, 1976), pH was 7.4 and OM% was 2.58.

Soil suppression of Pythium artifi cially inoculatedPythium irregulare cultures, isolated at our

Institute (International Mycological Institute Culture collection no. 368281), were maintained on potato dextrose agar (PDA) at 8 ± 2°C in the dark and periodically transferred. An oospore suspension was prepared following the method of Ayers & Lumsden (1975), starting from a 1-wk-old culture mat grown on V8 cholesterol broth, incubated in sterile water in the dark for 3 weeks at 18 ± 2°C. The oospores, suspended with a blender in sterile water, were counted using a haemocytometer and the concentration was diluted to obtain 1.2 × 105 oospores mL–1. This oospore suspension was stored in a refrigerator until inoculation time in full fi eld.

The experimental P. irregulare inoculum was placed in two experimental sites in early May 2000, on previously tilled soil. Samples of artifi cially inoculated soil were placed in cloth bags (500 g soil per bag) and buried 20 cm deep in three sites in each fi eld, along the diagonal of the fi eld. At each site, 2 kg soil samples, obtained by mixing by hand several soil samples collected at random from the fi eld, had been amended with 100 ml of 1.2 × 104 mL–1 P. irregulare oospore suspension. The original Pythium inoculum was recorded on soil samples collected at inoculation time in three sites following the method described above. The inoculum level in artifi cially inoculated soil samples was confi rmed by recording Pythium propagules the day after fi eld inoculation on additional inoculated soil samples maintained at 4–6°C until laboratory evaluation was done 2–4 days after full fi eld treatment. Inoculated soil samples were collected after 3, 6 and 12 weeks. Pythium inoculum was recorded following the same method as for the Pythium survey described above.

At the inoculum time, along the diagonal of the experimental fi eld near the Pythium inoculum sites, additional samples of bulk soil were collected to record total fungal population and bacterial population at each Pythium sampling using soil dilution plate methods as described below. Total population of fungi and fl uorescens bacteria was


evaluated at fi rst sampling time using soil dilution plate methods.

Total fungal population in bulk soilA 10–2 soil solution obtained from 25 g soil

samples was mixed with 49 ml of water agar + 3 g L–1 ox-gall and 200 mg L–1 streptomycin sulphate, distributed on fi ve Petri dishes (9 cm diameter) and incubated for 48 hours at 24 ± 2°C under natural light. Colonies were counted by visual

observation and expressed as CFU g–1 of air-dried soil. The composition of the fungal population was recorded at the fi rst sampling time and was recorded by visual observation under natural light of transparent agar disks including soil suspension. Each CFU shape observed was identifi ed with a symbol and a representative number (from 10 to 30) of colonies, corresponding to each symbol, was transferred to PDA + 200 mg L–1 of streptomycin sulphate on Petri dishes (50 mm diameter) to verify the composition of each cluster. The use of ox-gall, a fungistatic compound, in selective media and at a suitable dilution rate (10–3 soil dilution was used to confi rm what was observed at the 10–2 dilution used for counting CFU) gave the composition of the fungal population with satisfactory accuracy.

The data were handled as the mean of three plates for Pythium and fi ve for total fungi, for two soil sub-samples obtained in each of three experimental fi eld sites. Linear correlation coeffi cients between Pythium and total fungi were calculated for the whole trial and for each of the three following sampling times.

Fungal isolates with different morphologies were identifi ed on the basis of macroscopic and microscopic morphological characteristics using the relevant taxonomic keys (Nelson et al., 1983; Samson & van Reenen-Hoekstra, 1988).

Fluorescent bacterial population in bulk soilSoil suspension was prepared by adding 5 g of each

soil sub-sample to 45 mL of sterile distilled water and vortexing for 60 s. Serial soil dilutions (100 µL) were spread on King’s B media (KMB+) amended with ampicillin (40 mg L–1), cycloheximide (200 mg L–1) and chloramphenicol (13 mg L–1) (Simon & Ridge, 1974). Colonies were counted after 48 h of incubation at 26°C and expressed as CFU g–1 of soil.

The composition of the fl uorescent bacterial population was evaluated at the fi rst sampling time, and confi rmed by additional evaluations at subsequent sampling times. Colonies were clustered on the basis of morphological characteristics (shape, colour, size, etc.). A representative strain for each cluster was transferred to King’s B media (KMB+). The extraction of genomic DNA of each representative strain was carried out following the

extraction protocol used by Weidner et al. (1996). The difference in fl uorescent bacterial populations in the ALSIA and the CIF fi elds and the variations among the strains in each fi eld were evaluated using the amplifi ed ribosomal DNA restriction analysis (ARDRA) method.

ARDRA methodThe DNA coding for the 16S rRNA of each

isolate was amplifi ed with primers P0 (5΄-GAGAGTTTGATCCTGGCTCAG) and P6 (5΄-CTACGGCTACCTTGTTACGA). These primers were chosen on the basis of the conserved bacterial sequence as described by Grifoni et al. (1995). Amplifi cation was performed as previously described (Di Cello et al., 1997); each mixture contained 1 μL of DNA extract (100 ng), in 25 μl of Polymed Taq buffer containing 1.5 mM MgCl

2,

150 ng of each deoxynucleoside triphosphate at a concentration of 250 μM, and 0.5 U of Taq DNA polymerase (Invitrogen). The reaction mixtures were incubated in a thermocycler (Tgradient – Biometra) a 95°C for 1 min 30 s and then subjected to 35 cycles (95°C for 30 s), the annealing temperature for 30 s, and 72°C for 4 min. The annealing temperature was 57°C for the fi rst fi ve cycles, 55°C for the next fi ve cycles, and 50°C for the last 25 cycles. Finally, the mixtures were incubated at 72°C for 10 min and then at 60°C for 10 min. Five microlitres of each amplifi cation mixture were analysed by agarose (1% wt/vol) gel electrophoresis in Tris–borate EDTA (TBE) buffer.

Individual 5 µL aliquots of each PCR mixture, containing approximately 350 ng of amplifi ed 16S rDNA, were digested with 3 U of the restriction enzymes AluI, DdeI, HinfI, HaeIII or RsaI (GibcoBRL) in a total volume of 20 µL at 37ºC for 3 h. The enzymes AluI and RsaI were inactivated by heating the preparations to 65°C for 15 min. The reaction products were analysed by agarose (2.5% wt/vol) gel electrophoresis as above.

Banding patterns were visualised by staining with ethidium bromide and were scanned using GelDoc 1000 (BioRad). The banding patterns obtained with four enzymes were handled as haplotypic restriction fragment length polymorphism (RFLP) data and subjected to haplotype frequency estimation by Arlequin, version 2000, software (Schneider et al., 2000).

Root colonising fungi of strawberry plants from ALSIA and CIF fi elds

Strawberry plants were transplanted in late August, in the ALSIA fi eld, and in early September in the CIF fi eld. Strawberries were grown according to the Regional IP guidelines. In the fi rst half of December, plant samples were randomly collected from the two fi elds. In the ALSIA fi eld 84 plants


were collected, and in the CIF fi eld 64 plants. The root colonisation frequency of four feeding roots per plant was recorded as in the previous survey.

Results

Two year survey on strawberry root colonisationThe colonisation frequency on roots of strawberry

plants recorded in S. Giorgio fi eld was close to that observed in Martorano fi eld and in six strawberry crops at private farms in the Cesena area. Root colonisation frequency increased signifi cantly from late vegetative stage (35%) to harvesting stage (68%) of the strawberry plants (Table 1). Rhizoctonia spp. was the most frequent root colonising fungus at harvesting times, but colonisation frequency at late vegetative stage showed sharp difference varying from 44% at two experimental fi eld (SG farm and MT farm) to less than 10% in the six fi elds at private farms. Its relative frequency increased signifi cantly from autumn to spring; in all eight sites, it represents at least 60% of colonising fungi (Table 1).

To simplify the reading of relative frequency of colonising fungi (Table 1) the weak pathogens of strawberry roots were grouped as ‘Complex’: Alternaria spp., Cylindrocarpon destructans (Zinssm.) Scholten, Phoma pomorum von Thümen var. pomorum, Phoma pomorum var. calorpreferens Borema, De Gruyeter & Noordeloos, Phoma exigua Desmazières var. exigua, Zythia fragariae Laiback, Pestalotia sp., while the non-pathogenic saprophytic soil fungi were grouped as ‘Other’: Aspergillus spp., Mucor sp., Nigrospora sp., Penicillium sp., Rhizopus sp. The relative frequency of Fusarium oxysporum. emend Snyd. & Hans., Fusarium solani (Mart.), Fusarium spp. and ‘Complex’ pathogens remained unchanged, while non-pathogenic saprophytic soil fungi frequency decreased from autumn to spring, clearly replaced by Rhizoctonia in antagonistic competition for host root colonisation (Table 1).

The Rhizoctonia spp. population, showing a variable colony morphology with mostly brown colour and without sclerotia, was not easily ascribable to R. solani or R. fragariae using the nuclear state, therefore additional studies on Rhizoctonia spp. from strawberry plants from the Italian environment will be performed.

Pythium spp. in strawberry fi eldsPythium sp. was present in soil samples from

all the cultivated fi elds chosen in the Cesena area for the survey. The inoculum levels and predominant Pythium spp. are reported in Table 2. Pythium ultimum Trow. var. ultimum was the most widespread species found in the soil.

Relationship among suppressiveness towards

Pythium and towards root rot pathogens

Soil suppression of Pythium artifi cially inoculatedIn the ALSIA fi eld soil, populations of artifi cially

inoculated P. irregulare and the naturally present Pythium sylvaticum Campbell & Handrix (150 CFU g-1 soil) were static from the fi rst to last sampling (Fig. 1). This soil did not show any suppression of Pythium. On the other hand, in the CIF fi eld, P. irregulare was strongly suppressed to levels below 200 CFU g–1 soil from the fi rst sampling time, reaching a level of 100 CFU g–1 at the last sampling time (Fig. 2).

Table 2. Pythium spp. inoculum in bulk soil, recorded in June, before strawberry plant time, on 18 soil

cores

Site CFU g-–1

dry soilSoil

textureMost represented Pythium species

S. Giorgio 285 ± 12.7a

Silty clay loam

Pythium delienseMeurs, Pythium

ultimum. var. ultimum

Martorano 185 ± 16.5 Siltyclay loam

P. ultimum

Farms inCesena area

From 50 ± 6.3

to 180 ± 10.6

Siltyclay loam

Pythium spp. (P. ultimum

var. ultimum, P. irregulare

and Pythium spp.)

a Standard error.

Fig. 1. P. irregulare artifi cially inoculated variation in

ALSIA experimental fi eld. Bars represent Standard Error

Fig. 2. P. irregulare artifi cially inoculated variation in CIF experimental fi eld. Bars represent Standard Error.


Total fungal population in bulk soilIn the ALSIA fi eld, the total fungal population

remained below 10 000 (SE ± 918) CFU g–1 soil from the inoculum time to the last sampling time. The same inoculum level was also observed at strawberry planting time. Alternaria spp. and F. oxysporum represented more than 80% of the total fungal population recorded at all sampling times.

In the CIF fi eld, total fungi, starting from 13 000 CFU g–1 soil, increased gradually to 60 000 CFU g–1 soil two months after soil inoculation; the same level (65 000 CFU g–1 soil) was recorded on bulk soil at strawberry planting time in early September.

Total fungal population in CIF fi eld showed high variability: Fusarium genus was represented in approximately 60% of colonies transferred to growth media: F. equiseti Corda, F. compactum (Wollenw.) Gordon, F. graminearum Shwabe, F. oxysporum, F. avenaceum (Fr.) Sacc, F. semitectum Berk. & Rav., F. tricinctum (Corda) Sacc. Total fungal population was also represented by isolates belonging to genera the Penicillium, Aspergillus, Trichoderma and Mucor hiemalis Wehmer.

Pythium recorded in two experimental fi elds was negatively correlated to total fungi level. That negative correlation value increased from fi rst to last sampling time, giving a high and signifi cant correlation level (Table 3).

Fluorescent bacterial population in bulk soilThe fl uorescent bacterial population in bulk soil

had a decreasing trend from May to July in both

experimental fi elds. In the ALSIA fi eld, it decreased from 1.1 × 105 CFU g–1 soil at the fi rst sampling time to 1.3 × 103 CFU g-1 soil at the last sampling time, while in the CIF fi eld the bacterial population starting from the same level as ALSIA (1.4 × 105 CFU g–1 soil) showed a higher level (2.7 × 104 CFU g–1 soil) than the ALSIA fi eld at the last sampling time. The genetic composition of two populations completely differed and they did not have any common haplotype. The haplotype frequency estimation gave three haplotypes representing the fl uorescent bacterial population from ALSIA bulk soil: one haplotype was 75% of the recorded population, each of the other two haplotypes represented 12.5%. The fl uorescent bacterial population from CIF fi eld was represented by 10 haplotypes: one represented 50% of the population analysed, one 13.6%, while each of the other eight haplotypes represented 4.5% of the total bacterial population analysed.

Table 3. Linear correlation coeffi cient between total fungi and Pythium artifi cially inoculated in bulk soil,

in ALSIA and CIF fi elds

Pythium spp.

Whole trial time

Sampling time

Sample size 36 12 12 12

I II III

Total fungi –0.45a –0.21ns –046ns –0.87a

aP value ≤ 0.01

Table 4 - Relative composition of root colonising fungal species on strawberry plants from ALSIA and CIF fi eld, recorded at late vegetative growing stage

ALSIA fi eld(Root segments N. 1008

(Root colonisation frequency 67 %)

CIF Field(Root segments N. 768

(Root colonisation frequency 33%)

Relative frequency of fungal pathogens

(%)

Relative frequency of fungal pathogens

(%)

Rhizoctonia solani 43 Rhizoctonia solani 9

F. oxysporum & F. solani 2 F. oxysporum & F. solani 2

Fusarium spp. 3 Fusarium spp. 5

(F. equiseti, F. semitectum,

F. compactum)

(F. equiseti, F. semitectum,

F. compactum, F. sporotrichioides)

Complex 34 Complex 33

(Alternaria spp. (59%),

Zythia fragariae,

Cylindrocarpon destructans,

Pestalotia longiseta,

Phoma pomorum,

Phoma pomorum var. calorpreferens)

(Zythia fragariae,

Cylindrocarpon destructans,

Phoma pomorum)

Other 18 Other 51


Root colonising fungi of strawberry plants from the ALSIA and CIF fi elds

Strawberry plants from the ALSIA fi eld at the end of the vegetative growing stage showed rotted feeding roots. The fungal infection frequency, three months after transplant, was 67% on 1008 root segments. Most of the colonising fungi (82%) were agents of strawberry root rot: Rhizoctonia spp., F. solani, F. oxysporum and ‘Complex’ pathogens (Table 4). Rhizoctonia spp., 43% of root colonising fungi, was the most frequently isolated pathogen. Among the cluster of fungi called ‘Complex’ in that study, Alternaria was the most frequent followed by Phoma pomorum var. pomorum and Phoma pomorum var. calorpreferens, Phoma exigua var. exigua, Cylindrocarpon sp. Pestalotia longiseta Guba and Zythia fragariae (Table 4).

Strawberry plants from the CIF fi eld did not show any rot symptoms on feeding roots. Root colonisation frequency was 33% on 769 root explants. Strawberry root rot pathogens (Rhizoctonia spp., F. solani, F. oxysporum and Complex) represented 37% of total colonising fungi (Table 4). Rhizoctonia spp. frequency was less than 10% of all root colonising fungi while most of the colonising fungi (58%) were non-pathogenic saprophytic fungi such as Asperigillus spp., Penicillium spp., Mucor spp., Trichoderma spp., Rhizopus spp., Zophia rhizophilia Rabenh., sterile fungi (grouped as Other) and several Fusarium spp., including F. avenaceum, F. graminearum, F. tricinctum (Table 4).

In that survey, more than in that carried out in the Cesena area, Rhizoctonia strains showed mostly a white to pale colony morphology, as described for R. fragariae, but the variability of colony morphology observed among isolates and the inability to assess the number of nuclei within hyphal hypha cells did not allow isolated strains to be identifi ed as R. solani and R. fragariae.

Discussion

Yield decline occurring in intensive cropping systems is infl uenced by many physiological and nutritional factors. Data on strawberry crop health, handled independently of yield production, are therefore useful parameters to analyse along with several other soil microbial parameters to assess components involved in soil suppressiveness.

The results of previous surveys indicated that late vegetative growth stage is the optimum period to obtain root colonisation frequency data as a suitable qualitative and quantitative parameter of root rot fungal pathogens in existing cropping systems. This has been further confi rmed by the comparative study on two existing soil systems which shows signifi cant differences, emphasising how soil

management can affect soil suppressivenes and so soil health.

Frequency and population composition of root colonising fungi observed in the survey carried out in the Cesena area showed that the root colonising fungal population was quite constant and Rhizoctonia spp. was the most frequent colonising fungus. These results confi rmed the main role of Rhizoctonia spp. as the agent of black root rot agent in strawberry crops (Watanabe et al., 1977; Martin, 1988, 2000; Xiao & Duniway, 1998) and that the occurrence of this pathogen is always combined with several pathogens such as Cylindrocarpon sp., Fusarium oxysporum Schlecht., Fusarium spp. (Mena et al., 1975; Tamietti & Valmaggia, 1994; Pinkerton et al., 2002), and several other weak pathogens such as Phoma spp., Zithia fragariae, Alternaria sp. and others (Maas, 1984).

The Rhizoctonia spp. population showed high colony morphology variability as observed in other surveys (Botha et al., 2003). Given this variation in morphology, and the inadequacy of the hyphal nuclear cell state as the only tool to defi ne the binucleate state of R. fragariae, additional molecular (phylogenetic) studies on the Rhizoctonia spp. population isolated from strawberry crops in Italy will be performed to obtain a more precise taxonomic identifi cation.

Analysing the data of the fi rst survey, the need for a sharp difference in soil management was evident in order to perform a study able to characterise the direct (soil borne pathogen populations) and indirect (microbial populations) components of soil suppessiveness.

The ubiquitous occurrence of pathogenic Pythium species in strawberry fi elds of the Cesena area suggested the use of Pythium as target pathogen in the further studies performed with artifi cial inoculum in two existing cropping systems. Pythium spp. suppression is strongly affected by microbial activity (Chen et al., 1988b; Grünwald et al., 1997), and this pathogen has often been used as an indicator of soil suppressiveness in studies where microbial activity and bacterial population were expected to play a role on suppressiveness (Knudsen at al., 2002; Grünwald et al., 2000; Green & Jensen, 2000).

The lack of suppression towards Pythium spp., observed in the ALSIA fi eld, was further confi rmed by the highest colonisation level (67%) on roots of the plants grown in the same fi eld and the root rot severity observed at root sampling time. On the contrary, the suppressive effect towards P. irregulare, observed in the CIF fi eld, corresponded with the lower root colonisation frequency (33%) and root health observed in strawberry plants subsequently grown in that fi eld.

There are many microbial components of soil


suppression (Van Bruggen & Semenov, 2000; Whipps, 2001; Mazzola, 2002); in the current study, the experimental fi elds differed in the previous soil management and the organic matter content, but they had the same soil texture and pH, thus emphasising the role of several microbial parameters in root rot complex suppression. The negative correlation between total fungi (adopted as one of the indicators of soil microbial biomass) and Pythium (adopted as soil borne pathogen indicator of soil suppressiveness) in bulk soil, combined with the wide difference in root colonisation frequency observed in strawberry crops grown in experimental fi elds, supported the hypothesis that the microbial biomass is also one of the main components of suppressiveness for strawberry black root rot (Chen et al., 1988b; Craft & Nelson, 1996; Erhart et al., 1999). As regards the different total fungi amount in two experimental sites, given that seasonal fl uctuation is a well known behavior of soil fungi (Tojo et al., 1998; Manici et al., 2004), the large difference was evident comparing the variation range from May to September of total fungi in the ALSIA fi eld (from 8000 to 13 000 CFU g-1 ), with that observed in the CIF fi eld (from 13 000 to 60 000 CFU g-1).

The variability within the microbial population in bulk soil and root colonising fungi population, higher in the suppressive CIF fi eld than in the ALSIA fi eld, supported the hypothesis that microbial variability is another important component of soil suppressiveness in agricultural soils (Nitta, 1991; Mazzola & Gu, 2000; Gu & Mazzola, 2003).

The differences among fl uorescent Pseudomonas populations as determined in this study did not allow for a precise study of the role of a single bacterial strain in the suppression of non-lethal pathogens. However, the signifi cant difference in genetic composition of the two populations, and the greater variability observed within that coming from the suppressive fi eld (CIF), was an additional tool to show the difference between the two microbial populations.

Fungi are the largest component (Lynch & Panting, 1980) of the soil microbial biomass, a biotic component of soil quality (Kennedy & Papendick, 1995) strongly related to organic carbon content in agricultural soils (Anderson & Domsch, 1986). The importance of the fungal biomass for soil fertility has also been reported in recent studies by Laughlin & Stevens (2002) on grassland soils. The interaction among fungal biomass, suppressiveness, N availability and soil fertility is an aspect not well investigated until now; and will require further multidisciplinary investigation. The correspondence between total fungi population size and suppressiveness, found in this restricted study, fi ts well with that observed for suppressiveness towards the pathogens responsible for black root rot

agents of apples in both organic and conventional apple orchards (Manici et al., 2003).

In conclusion, the experiences described here show the potential of several microbial parameters to record the relative importance of soil borne pathogens as agents of black root rot, and how their control is affected by microbial conditions induced by soil management. In low-input farming systems soil management designed to increase the size and diversity of fungal and bacterial populations, combined with cropping practices reducing selection pressure towards soil borne pathogens by the strawberry crop, appear to be the main tools to enhance soil suppressiveness for this high economic value crop.

Acknowledgements

We are very grateful for the technical support of Dr A Di Stefano of ALSIA Experimental Farm, Azienda Pantanello, Matera and Dr M Muntoni of CIF Experimental Farm, Villasor Cagliari.

References

Abawi G S, Widmer T L. 2000. Impact of soil health management practices on soil-borne pathogens, nematodes and root diseases of vegetable crops. Applied Soil Ecology 15:37–47.

Anderson T H, Domsch K H. 1986. Carbon assimilation and microbial activity in soil. Zeitschrift für Pfl anzenernährung Bodenkunde 149:457–468.

Ayers W A, Lumsden R D. 1975. Factors affecting production and germination of oospores of three Pythium species. Phytopathology 65:1094–1100.

Bateman G L, Murray G, Gutteridge R J, Coşcun H. 1998. Effects of methods of straw disposal and depth cultivation on populations of Fusarium spp. in soil and on brown foot rot in continuous winter wheat. Annals of Applied Biology 132:35–47.

Bolton H Jr, Elliot L F, Papendick R I, Bezdicek D F. 1985. Soil microbial biomass and selected soil enzyme activity: effect of fertilization and cropping practices. Soil Biology and Biochemistry 17:297–302.

Botha A, Denman S, Lamprecht S C, Mazzola M, Crous P W. 2003. Characterisation and pathogenicity of Rhizoctonia isolates associated with black root rot of strawberries in the Western Cape Province, South Africa. Australasian Plant Pathology 32:195–201.

Burgess L W, Liddell C M, Summerell B A. 1988. Laboratory Manual for Fusarium Research. Sydney, Australia: Department of Plant Pathology and Entomology, University of Sydney.

Chen W, Hoitink H A J, Schmitthenner A F, Tuovinen O H. 1988a. The role of microbial activity in suppression of damping-off caused by Pythium ultimum. Phytopathology 78:314–322.

Chen W, Hoitink H A J, Madden L V. 1988b. Microbial activity and biomass in container media for predicting suppressiveness to damping-off caused by Pythium ultimum. Phytopathology 78:1447–1450.

Ciccarese F, Cirulli M. 1983. Osservazioni sui deperimenti dei fragoleti nell’Italia meridionale. Informatore Fitopatologico 33:59–62.

Craft C M, Nelson E B 1996. Microbial properties of composts


that suppress damping-off and root rot of creeping bentgrass caused by Pythium graminicola. Applied Environmental Microbiology 62:1550–1557.

Davis R M, Nunez J J. 1999. Infl uence of crop rotation on the incidence of Pythium- and Rhizoctonia-induced carrot root dieback. Plant Disease 83:146–148.

D’Ercole N. 1970. Sul deperimento progressivo con necrosi radicale della fragola. Atti 4° convegno Nazionale della fragola, Cesena (Italy), pp. 389–395.

Dhingra O D, Sinclair J B. 1985. Basic Plant Pathology Methods, pp. 179–189. Boca Raton, FL: CRC Press.

Di Cello F, Bevivino A, Chiarini L, Fani R, Paffetti D, Tabacchioni S, Dalmastri C. 1997. Biodiversity of Burkholderia cepacia population isolated from the maize rhizosphere at different plant growth stages. Applied and Environmental Microbiology 63:4485–4493.

Elmer W H, LaMondia J A. 1999. Infl uence of ammonium sulphate and rotation crops on strawberry black root rot. Plant Disease 83:119–123.

Erhart E, Burian K, Hartl W, Stich K. 1999. Suppression of Pythium ultimum by biowaste composts in relation to compost microbial biomass, activity and content of phenolic compounds. Journal of Phytopathology 147:299–305.

Green H, Jensen D F. 2000. Disease progression by active mycelial growth and biocontrol of Pythium ultimum var. ultimum studied using a rhizobox system. Phytopathology 90:1049–1055.

Grifoni A Bazzicalupo M, Di Serio C, Fancelli S, Fani R. 1995. Identifi cation of Azospirillum strains by restriction fragment length polymorphism of the 16S rDNA and of the histidine operon. FEMS Microbiology Letters 127:85–91.

Grünwald N J, Workneh F, Hu S, van Bruggen A H C. 1997. Comparison of an in vitro and a damping-off assay to test soils for suppressiveness to Pythium aphnidermatum. European Journal of Plant Pathology 103:55–63.

Grünwald N J, Hu S, van Bruggen A H C. 2000. Short-term cover crop decomposition in organic and conventional soils: soil microbial and nutrient cycling indicator variables associated with different levels of soil suppressiveness to Pythium aphanidermatum. European Journal of Plant Pathology 106:51–65.

Gu Y-H, Mazzola M. 2003. Modifi cation of fl uorescent pseudomonad community and control of apple replant disease induced in a wheat cultivar-specifi c manner. Applied Soil Ecology 24:57–72.

IMI. 196422003. Descriptions of fungi and bacteria. Sheet No. 1-1580. ISSN 0009-9716, Bakeham Lane, Egham, Surrey, UK : CABI Bioscience

Jeffers S, Martin S B. 1986. Comparison of two media selective for Phytophthora and Pythium species. Plant Disease 70:1038–1043.

Kennedy A C, Papendick R I. 1995. Microbial characteristics of soil quality. Journal of Soil and Water Conservation 50:243–248.

Knudsen I M B, Larseen K M, Jensen D F, Hockenhull J. 2002. Potential suppressiveness of different fi eld soils to Pythium dumping off of sugar beet. Applied Soil Ecology 21:119–129.

Kronland W C, Stanghellini M E. 1988. Clean slide technique for the observation of anastomosis and nuclear condition in Rhizoctonia solani. Phytopathology 78:820–822.

Laughlin R J, Stevens R J. 2002. Evidence for fungal dominance in denitrifi cation and codenitrifi cation in a grassland soil. Soil Science Society of America Journal 66:1540–1548.

Lumsden R D, Lewis J A, Papavizas G C. 1983. Effect of organic amendments on soilborne plant diseases and pathogen antagonists. In Environmentally Sound Agriculture, pp. 51-70. Ed. W Lockeretz. New York: Praeger Scientifi c.

Lynch J M, Panting L M. 1980. Variation in the size of soil

bio-mass. Soil Biology and Biochemistry 12:547–550.Maas J L. 1984. Compendium of Strawberry Diseases.

Beltsville, MD: APS Press. 138 pp.Mai W F, Abawi G S. 1978. Determining the cause and

extent of apple, cherry and pear disease under controlled conditions. Phytopathology 68:1540–1544.

Manici L M, Baruzzi G, Burzi P L. 2000. Indagine sulla colonizzazione di patogeni fungini su radici di piante fragola fresche (cime radicate) e frigoconservate. Conv. Naz “La fragola nel 2000”. Metaponto (MT), p. 63.

Manici L M, Ciavatta C, Kelderer M, Erschbaumer G. 2003. Replant problem in South Tyrol: role of fungal pathogens and microbial populations in conventional and organic apple orchards. Plant and Soil 256:315–324.

Manici L M, Caputo F, Babini V. 2004. Effect of green manure on Pythium spp. population and microbial communities in intensive cropping systems Plant and Soil 263:133–142.

Martin F N. 1988. Identifi cation, isolation frequency and pathogenicity of anastomosis groups of binucleate Rhizoctonia spp. from strawberry roots. Phytopathology 78:379–384.

Martin F N. 1999. Strawberry root rot and the recovery of Pythium and Rhizoctonia spp. In Proceedings of the Annual International Research Conference on Methyl Bromide Alternatives and Emission Reductions. Paper No. 6.

Martin F N. 2000. Rhizoctonia spp. recovered from strawberry roots in central coastal California. Phytopathology 90:345–353.

Mazzola M. 1997. Identifi cation and pathogenicity of Rhizoctonia spp. isolated from apple roots and orchard soil. Phytopathology 87:582–587.

Mazzola M. 2002. Mechanisms of natural soil suppressiveness to soilborne diseases. Antoine van Leeuwenhoek 81:557–564.

Mazzola M, Gu Y-H. 2000. Impact of wheat cultivation on microbial communities from replant soils and apple growth in greenhouse trials. Phytopathology 90:114–119.

Mena A J, Palacios de Garcia M E, Gonzàles M A. 1975. Root diseases of strawberry caused by Fusarium oxysporum Sch. F.s. fragariae Winks et Will and Rhizoctonia fragariae Husain et McKeen. Revista Agronomica del Noroesta Argentino 12:299–307.

Molot P M, Ferrière H. 1989. Susceptibility of strawberry cultivars to Rhizoctonia solani and R. fragariae as infl uenced by inoculation technique seasonal variations and physiological condition of the plants. Acta Horticulturae 265:535–540.

Nelson P E, Tousson T A, Marasas W F O. 1983. Fusarium Species. An Illustrated Manual for Identifi cation. Pennsylvania, PA: The Pennsylvania State University Press. 190 pp.

Nitta T. 1991. Diversity of root fungal fl oras: its implication on soil-borne diseases and crop growth. Japanese Agriculture Research Quarterly 25:6–11.

Pankhurst C E, Hawke B G, McDonald H J, Kirkby C A, Buckerfi eld J C, Michelsen P, O’Brien K A, Gupta V V S R, Doube B M. 1995. Evaluation of soil biological properties as potential bioindicators of soil health. Australian Journal of Experimental Agriculture 35:1015–1028.

Pinkerton J N, Ivors K L, Reeser P W, Bristow P R, Windom G E. 2002. The use of soil solarization for the management of soil-borne plant pathogens in strawberry and red raspberry production. Plant Disease 86:645–651.

Samson R A, van Reenen-Hoekstra E S. 1988. Introduction to Food-borne Pathogens. 3rd Edn. Baarn and Delft: Centraalbureau vor Schimmelcultures. 299 pp.

Schneider S, Roessli D, Escoffi er L. 2000. Arlequin ver. 2000. A Software for Population Genetic Data Analysis. Geneva, Switzerland: Genetic and Biometry Laboratory, University of Geneva.


Schnürer J, Clarholm M, Rosswall T. 1985. Microbial biomass and activity in an agricultural soil with different organic matter contents. Soil Biology and Biochemistry 17:611–618.

Schut P. 1976. Glossary of Terms in Soil Science. Publication 1459. Canada: Department of Agriculture. 44 pp.

Simon A, Ridge E H. 1974. The use of ampicillin in a simplifi ed selective medium for the isolation of fl uorescent pseudomonads. Journal of Applied Bacteriology 37:459–460.

Tamietti G, Valmaggia A. 1994. Il collasso della fragola in Piemonte. Informatore fi topatologico 44:55–58.

Tojo M, Abe T, Hasunama N, Minatodani M, Sato Y, Ohki ST, Osaki T. 1998. Seasonal population changes of Pythium spp. in bentgrass greens in Japan. Abs. 2.2.4. In Offered Papers at 7th International Congress of Plant Pathology, Edinburgh (UK), 9–16 August 1998.

van Bruggen A H C, Grünwald N J. 1996. Tests for risk assessment of root infection by plant pathogens. In Methods for Assessing Soil Quality, pp. 293–310. Eds J W Doran and A J Jones. Madison, WI: Soil Science Society of America.

van Bruggen A H C, Semenov A M. 2000. In search of biological indicators for soil health and disease suppression. Applied Soil Ecology 15:13–24.

Vestberg M. 1987. The effect of preceding crops on damping- off of sugar beet and some ecological properties of the fungus Pythium Pringsh. Journal of Agricultural Sciences of Finland 59:87–100.

Watanabe T. 1977. Fungi associated to strawberry roots in Japan. Transactions of the Mycological Society of Japan 18:251–256.

Watanabe T, Hashimoto K, Sato M. 1977. Pythium species associated with strawberry roots in Japan and their role in the strawberry stunt disease. Phytopathology 67:1324–1332.

Waterhouse G M. 1967. Key to Pythium Pringsheim. Mycological papers, No 109. Kew, Surrey: CAB International.

Waterhouse G M. 1968. The genus of Pythium Pringsheim. Mycological papers, No 110. Kew, Surrey: CAB International.

Weidner S, Arnold W, Pühler A. 1996. Diversity of uncultured microorganisms associated with the seagrass Halophila stipulacea estimated by restriction fragment length polymorphism analysis of PCR-amplifi ed 16S rRNA genes. Applied and Environmental Microbiology 62:766–771.

Wing K B, Pritts M P, Wilcox W F. 1994. Strawberry black root rot: a review. Advances in Strawberry Research 13:13–19.

Whipps J M. 2001. Microbial interaction and biocontrol in the rhizospere. Journal of Experimental Botany 52:487–511.

Xiao C L, Duniway J M. 1998. Frequency of isolation and pathogenicity of fungi on roots of strawberry in fumigated and nonfumigated soils. Phytopathology 88:S100.

Yuen G Y, Schroth M N, Weinhold A R, Hancock J G. 1991. Effect of soil fumigation with methyl bromide and chloropicrin on root health and yield of strawberry. Plant Disease 75:416–420.

(Revised version accepted 18 January 2005; Received 20 April 2004)

additional experiences to elucidate the microbial component of soil suppressiveness towards...

Documents