Interactions of Bacillus spp. and plants--with special reference to induced systemic resistance (ISR)

Biological control of soil-borne pathogens comprises the decrease of inoculum or of the disease producing activity of a pathogen through one or more mechanisms. Interest in biological control of soil-borne plant pathogens has increased considerably in the last few decades, because it may provide control of diseases that cannot or only partly be managed by other control strategies. Recent advances in microbial and molecular techniques have significantly contributed to new insights in underlying mechanisms by which introduced bacteria function. Colonization of plant roots is an essential step for both soil-borne pathogenic and beneficial rhizobacteria. Colonization patterns showed that rhizobacteria act as biocontrol agents or as growth-promoting bacteria form microcolonies or biofilms at preferred sites of root exudation. Such microcolonies are sites for bacteria to communicate with each other (quorum sensing) and to act in a coordinated manner. Elicitation of induced systemic resistance (ISR) by plant-associated bacteria was initially demonstrated using Pseudomonas spp. and other Gram-negative bacteria. Several strains of the species Bacillus amyloliquefaciens, B. subtilis, B. pasteurii, B. cereus, B. pumilus, B. mycoides, and B. sphaericus elicit significant reductions in the incidence or severity of various diseases on a diversity of hosts. Elicitation of ISR by these strains has been demonstrated in greenhouse or field trials on tomato, bell pepper, muskmelon, watermelon, sugar beet, tobacco, Arabidopsis sp., cucumber, loblolly pine, and two tropical crops (long cayenne pepper and green kuang futsoi). Protection resulting from ISR elicited by Bacillus spp. has been reported against leaf-spotting fungal and bacterial pathogens, systemic viruses, a crown-rotting fungal pathogen, root-knot nematodes, and a stem-blight fungal pathogen as well as damping-off, blue mold, and late blight diseases. This progress will lead to a more efficient use of these strains which is worthwhile approach to explore in context of biocontrol strategies.     

Einfluß der Behandlung von Rübensaatgut mit Rhizosphärebakterien auf den Befall durch Pilze der Gattung Pythium I. Antagonistische Wirkung verschiedener Bakterienisolate gegenüber Pythium spp.

Abstract The influence of a seed-dressing with rhizosphere bacteria on the infection of sugarbeet by fungi of the genus Pythium I. Antagonistic effect of different bacterial isolates towards Pythium spp.
Seed treatment with selectively isolated rhizosphere bacteria from the fluorescent pseudomonad group can protect sugar beet seedlings from damping-off caused by species of Pythium. The antagonistic rhizobacteria were equally effective in different soil substrates, both unsterilized and steam-sterilized. Antagonistic activity of an isolate was similar within seeds of a sugarbeet cultivar but different when different cultivars were compared. The number of bacteria adhering to the seed of eachcultivar which influenced the level of antagonism to Pythium infection, varied with seed morphology. A mixture of the three different isolates did not increase antagonistic activity when compared to the activity of the isolates individually.     

Compost-induced suppression of Pythium damping-off is mediated by fatty-acid-metabolizing seed-colonizing microbial communities

Leaf composts were studied for their suppressive effects on Pythium ultimum sporangium germination, cottonseed colonization, and the severity of Pythium damping-off of cotton. A focus of the work was to assess the role of fatty-acid-metabolizing microbial communities in disease suppression. Suppressiveness was expressed within the first few hours of seed germination as revealed by reduced P. ultimum sporangium germination, reduced seed colonization, and reduced damping-off in transplant experiments. These reductions were not observed when cottonseeds were sown in a conducive leaf compost. Microbial consortia recovered from the surface of cottonseeds during the first few hours of germination in suppressive compost (suppressive consortia) induced significant levels of damping-off suppression, whereas no suppression was induced by microbial consortia recovered from cottonseeds germinated in conducive compost (conducive consortia). Suppressive consortia rapidly metabolized linoleic acid, whereas conducive consortia did not. Furthermore, populations of fatty-acid-metabolizing bacteria and actinobacteria were higher in suppressive consortia than in conducive consortia. Individual bacterial isolates varied in their ability to metabolize linoleic acid and protect seedlings from damping-off. Results indicate that communities of compost-inhabiting microorganisms colonizing cottonseeds within the first few hours after sowing in a Pythium-suppressive compost play a major role in the suppression of P. ultimum sporangium germination, seed colonization, and damping-off. Results further indicate that fatty acid metabolism by these seed-colonizing bacterial consortia can explain the Pythium suppression observed.     

Effect of Storage on the Survival and Biocontrol Activity of Pythium oligandrum in Pelleted Sugar Beet Seed

Seeds of sugar beet were pelleted with oospores of Pythium oligandrum and stored for 6 years at 8 20IC. Mycelium of P. oligandrum grew from pelleted seed when plated on cornmeal agar (CMA) within 48 h from 100% of seeds stored for 0, 2 and 4 years, and from 93% of seeds stored for 6 years. The germination of oospores removed from pelleted seed immediately after pelleting was 30% on CMA after 18 h of incubation, but storage gradually reduced germination to only 16% after 48 h of incubation for oospores removed from seed stored for 6 years. The biocontrol activity of P. oligandrum -pelleted seed was also tested after 6 years of storage in mixes of soil naturally infested with Pythium spp. and Aphanomyces cochlioides , and sand. P. oligandrum -pelleted seed had no effect in reducing damping-off due to the combined effect of Pythium spp. and A. cochlioides in 5 and 1% (v/v) soil-sand mixtures. However, in the 1% (v/v) soil-sand mixture, P. oligandrum significantly reduced Pythium spp.-induced damping-off from 33 to 26%.     

Chitin-supplemented formulations improve biocontrol and plant growth promoting efficiency of Bacillus subtilis AF 1

Formulations of a chitinolytic biocontrol and a plant growth promoting Bacillus subtilis AF 1 were prepared in peat, in peat supplemented with either 0.5% chitin or Aspergillus niger mycelium, or in spent compost obtained from Agaricus bisporus cultivation and were evaluated for biocontrol of two fungal pathogens and plant growth promoting activities on pigeon pea and groundnut. A steady increase in cell numbers of introduced B. subtilis AF 1 was observed in all the formulations at 30 degrees C. The increase in cell numbers was about 5.0 log units. Peat or spent compost inoculated with physiologically active and dormant states of B. subtilis AF 1 showed different time period requirements to attain maximum cell numbers. The presence of chitin or A. niger (in peat) or A. bisporus (in spent compost) as supplement in the carrier material improved the multiplication of B. subtilis AF 1. When used as seed treatments, formulations of AF 1 in peat supplemented with chitin or chitin-containing materials showed better control of A. niger (causing crown rot of groundnut) and Fusarium udum (causing wilt of pigeon pea) than AF 1 culture alone, in both groundnut and pigeon pea. Bacillus subtilis AF 1 formulations promoted seed germination and biomass of both groundnut and pigeon pea even under pathogen pressure. Survival of AF 1 on fresh culture-treated and formulation product-treated plants was similar in pathogen-infested soil.     

Exploitation of gene(s) involved in 2,4-diacetylphloroglucinol biosynthesis to confer a new biocontrol capability to a Pseudomonas strain.

Tn5 mutagenesis and complementation analysis were used to clone a 6-kb genomic fragment required for biosynthesis of 2,4-diacetylphloroglucinol (Phl) from fluorescent Pseudomonas sp. strain F113. A recombinant plasmid, pCU203, containing this region partially complemented a Phl production-negative mutant (F113G22) derived from strain F113. When sugar beet seeds were sown into an unsterilized soil, in which sugar beet was subject to damping-off by Pythium ultimum, the emergence of sugar beet seeds inoculated with strain F113 was significantly greater than that of seeds inoculated with F113G22. Transfer of pCU203 into eight other Pseudomonas strains conferred the ability to synthesize Phl in only one of these strains, Pseudomonas sp. strain M114. Strain M114(pCU203) showed enhanced antagonism towards P. ultimum in vitro and significantly increased the emergence of sugar beet seeds in the same soil compared with emergence induced by the parent strain M114.     

Exploitation of gene(s) involved in 2,4-diacetylphloroglucinol biosynthesis to confer a new biocontrol capability to a Pseudomonas strain.

Tn5 mutagenesis and complementation analysis were used to clone a 6-kb genomic fragment required for biosynthesis of 2,4-diacetylphloroglucinol (Phl) from fluorescent Pseudomonas sp. strain F113. A recombinant plasmid, pCU203, containing this region partially complemented a Phl production-negative mutant (F113G22) derived from strain F113. When sugar beet seeds were sown into an unsterilized soil, in which sugar beet was subject to damping-off by Pythium ultimum, the emergence of sugar beet seeds inoculated with strain F113 was significantly greater than that of seeds inoculated with F113G22. Transfer of pCU203 into eight other Pseudomonas strains conferred the ability to synthesize Phl in only one of these strains, Pseudomonas sp. strain M114. Strain M114(pCU203) showed enhanced antagonism towards P. ultimum in vitro and significantly increased the emergence of sugar beet seeds in the same soil compared with emergence induced by the parent strain M114.     

Ecological basis for biocontrol of damping-off disease by Pseudomonas fluorescens 54/96

Pseudomonas fluorescens 54/96, originally isolated from the rhizosphere of sugar beet, has been shown to be commercially effective in field trials for the suppression of a number of fungal diseases of seedlings. In vitro and microcosm-based assays revealed that both the timing and method of application of bacteria were important for effective control of Pythium ultimum , the causative agent of damping-off disease. Following transposon mutagenesis (Tn 5lac ), mutants deficient for the suppression of Pythium ultimum infections of peas were isolated. Three major classes of insertional mutants of Ps. fluorescens 54/96 were identified which either inhibited sporulation, reduced mycelial growth or affected the regulation of bacterial metabolic activity. Evaluation of the metabolic capability of pathogen and antagonist revealed evidence for direct competition, as both the fungus and bacterium had similar sole carbon source nutrient utilization profiles. Further comparisons of the activity of the transposon mutants indicated that although the mechanisms of disease control were multifactorial, the most significant factor was the prevention of rapid spore germination in the presence of pea seeds.     

Pseudomonas fluorescens DR54 Reduces Sclerotia Formation,Biomass Development,and Disease Incidence of Rhizoctonia solani Causing Damping-Off in Sugar Beet

Effects of the biocontrol strain, Pseudomonas fluorescens DR54, on growth and disease development by Rhizoctonia solani causing damping-off in sugar beet were studied in soil microcosms and in pot experiments with natural, clay-type soil. In pot experiments with P. fluorescens DR54-treated seeds, significantly fewer Rhizoctonia-challenged seedlings showed damping-off symptoms than when not inoculated with the biocontrol agent. In the rhizosphere of P. fluorescens DR54 inoculated seeds, the bacterial inoculant was present in high numbers as shown by dilution plating and immunoblotting. By the ELISA antibody technique and direct microscopy of the fungal pathogen grown in soil microcosms, it was shown that the presence of P. fluorescens DR54 on the inoculated seeds had a strong inhibitory effect on development of both mycelium biomass and sclerotia formation by R. solani. In the field experiment, plant emergence was increased by treatment with P. fluorescens DR54 and the inoculant was found to be the dominating rhizosphere colonizing pseudomonad immediately after seedling emergence.     

Influence of Sugar Beet Seed Treatment with Pseudomonas fluorescens and Low Fungicide Doses on Infection with Pythium and Plant Yield and Quality

The effect of sugar beet seed inoculation with the bacterium Pseudomonas fluorescens and treatment with the fungicides Thiram 42‐S and Dithane S‐60 with and without seed inoculation aiming to control the root decay agents Pythium ultimum and Pythium debarianum was studied during a 2‐year trial on two soil types (Mollic Gleysols and Eutric Cambisols). The influence of the treatments on parameters of sugar beet yield and quality such as root yield, sugar content, sugar in molasses, sugar yield as well as percentage of the infected and decayed plants as a consequence of parasitic oomycete infestation will be described.     

Shelf-life of a Biocontrol Pseudomonas putida Applied to Sugar Beet Seeds Using Commercial Coatings

Pseudomonas putida 40RNF is a putative biological control agent (BCA) of Pythium damping-off of sugar beet. The survival of 40RNF during commercial seed treatment and its subsequent shelf-life (i.e. long-term viability and biocontrol activity) were assessed. Two methods were used to apply 40RNF to sugar beet seeds: incorporation into film-coats sprayed on to pre-pelleted seeds and incorporation into the pellet material prior to pelleting. Only 7.1% of applied 40RNF survived film-coating, but an initial concentration of 7 × 10⁸ ensured that 83.3% of a pre-determined target rate of 6 × 10⁷ |pellet was achieved. After 52 weeks of storage at 4°C,the numbers of 40RNF had declined by one to two orders of magnitude, with a decrease of approximately 50% in disease control. After 52 weeks at 18-20°C, 40RNF was below detectable limits (< 100|pellet), yet the biocontrol activity of the seed treatments was not reduced. The survival of 40RNF during incorporation into the pellet material was poor (< 0.2% of those applied, i.e. 5 × 10⁵ pellet). However, bacterial viability and biocontrol efficacy were maintained at 100% of the control value for 24 weeks when stored at 18-20°C. The results indicate that commercial seed treatments and the storage of pellets at ambient temperatures has potential for the introduction of bacterial BCAs into the spermosphere.     

Induction of plant defence compounds by Pseudomonas chlororaphis PA23 and Bacillus subtilis BSCBE4 in controlling damping-off of hot pepper caused by Pythium aphanidermatum

Bacillus subtilis strain BSCBE4 and Pseudomonas chlororaphis strain PA23 (=P. aureofaciens) were effective biocontrol agents against Pythium aphanidermatum, the causal agent of damping-off of hot pepper (Capsicum annum L.) in greenhouse vegetable production systems. Application of strains BSCBE4 and PA23 at the rate of 20 g kg^-1 of seed significantly increased the growth of hot pepper seedlings. The efficacies of various carriers in sustaining the population of these strains in storage were assessed. Both the antagonists survived up to 180 days of storage in peat and talc-based formulations. The two bacterial strains induced development of plant defence-related enzymes including phenylalanine ammonia lyase, peroxidase, polyphenol oxidase, phenol content, suppressed incidence of damping-off and increased growth of hot pepper seedlings.     

Potential biocontrol Bacillus sp. strains isolated by an improved method from vinegar waste compost exhibit antibiosis against fungal pathogens and promote growth of cucumbers

An in vitro antagonism test is a typical procedure for the selection of potential biocontrol strains. However, the traditional method of screening antagonistic bacteria in vitro is a time consuming method when conducting large-scale screening trials. In this study, an improved method for the selection of antagonistic bacteria in vitro from compost was established based on the traditional method. 21 Antagonistic bacteria out of 33 target strains isolated from vinegar waste compost using the improved method. The 16S rDNA gene showed the 21 strains all belonged to the Bacillus genus and 18 different types of fingerprints were obtained by enterobacterial repetitive inter-genic consensus (ERIC)-PCR. 18 Selected strains which had the unique fingerprints all exhibited broad-spectrum antagonism towards the tested fungi and at least two enzyme activities in vitro. Among them, majority of the isolates were siderophore producer, some of them showed nitrogen-fixing ability and small of them were IAA producer. Four out of five selected strains were found both to be effective in controlling wilt and damping-off disease and four strains showed strong growth-promoting activities for cucumber seedlings under greenhouse conditions. Thus, these results demonstrated that the improved method was an effective and rapid means to screen potential antagonistic microorganisms in vitro. The results also showed that Bacillus sp. strains in vinegar waste compost exhibited antibiosis against fungal pathogens and promoted the growth of cucumber seedlings.     

Strategy to select and assess antagonistic bacteria for biological control of Rhizoctonia solani Kühn

A screening strategy was developed to assess the potential of plant-associated bacteria to control diseases caused by Rhizoctonia solani Kühn. About 434 already characterized antagonistic bacterial strains isolated from diverse plant species and microenvironments were evaluated for biocontrol and plant growth promotion by a hierarchical combination of assays. Analyzing in vitro antagonism towards different Rhizoctonia isolates resulted in a selection of 20 potential biocontrol agents. The strains were characterized by their antagonistic mechanisms in vitro as well as their production of the plant growth hormone indole-3-acetic acid. The plant growth promoting effect by antagonistic bacteria was determined using a microtiter plate assay on the basis of lettuce seedlings. Lettuce and sugar beet as host plant were included in the biocontrol experiments in which the antagonistic effect of 17 bacterial isolates could be confirmed in vivo. Sequencing of the 16S rDNA gene and (or) fatty acid methyl ester gas chromatography was used to identify the antagonistic isolates. Molecular fingerprints of isolates obtained by BOX-polymerase chain reaction were compared to avoid further investigation with genetically very similar strains and to obtain unique molecular fingerprints for quality control and patent licensing. According to our strategy, an assessment scheme was developed and four interesting biological control agents, Pseudomonas reactans B3, Pseudomonas fluorescens B1, Serratia plymuthica B4, and Serratia odorifera B6, were found. While S. plymuthica B4 was the best candidate to biologically control Rhizoctonia in lettuce, P. reactans B3 was the best candidate to suppress the pathogen in sugar beet.     

Comparison of application methods to prolong the survival of potential biocontrol bacteria on stored sugar-beet seed

AIMS: To develop bacterial inoculation treatments on sugar-beet seed that will maintain a commercially acceptable degree of viability for a minimum of 4 months storage at ambient temperature. METHODS AND RESULTS: Single rifampicin-resistant (Rif(+)) strains of both Gram-positive and negative bacterial isolates (mostly pseudomonads) were applied in turn to sugar-beet seed in a comparative study by seed soaking, encapsulation in alginate, pelleting using an inoculated peat carrier or seed priming. The treated seed was assessed for bacterial survival over a time course by plating out homogenized samples onto a selective medium. Priming inoculation offered a significant improvement over all the other application strategies tested. After pelleting with fungicides and drying at 40 degrees C, Pseudomonas marginalis/putida P1W1 maintained populations of >6.6 log(10) CFU g(-1) seed during 4 months storage at 15 degrees C. Subsequent experiments verified a stabilized population under these storage conditions with commercial pellets at <7% moisture content. CONCLUSION: An inoculation method was established which allowed the survival on seed of a Gram-negative bacterium at ambient temperature with little loss in viability. SIGNIFICANCE AND IMPACT OF THE STUDY: This has promising implications for the delivery of beneficial bacteria, especially Gram-negative strains, on sugar beet.     

Effect of bacterial antagonists on lettuce: active biocontrol of Rhizoctonia solani and negligible, short-term effects on nontarget microorganisms

The aim of this study was to assess the biocontrol efficacy against Rhizoctonia solani of three bacterial antagonists introduced into naturally Rhizoctonia-infested lettuce fields and to analyse their impact on the indigenous plant-associated bacteria and fungi. Lettuce seedlings were inoculated with bacterial suspensions of two endophytic strains, Serratia plymuthica 3Re4-18 and Pseudomonas trivialis 3Re2-7, and with the rhizobacterium Pseudomonas fluorescens L13-6-12 7 days before and 5 days after planting in the field. Similar statistically significant biocontrol effects were observed for all applied bacterial antagonists compared with the uninoculated control. Single-strand conformation polymorphism analysis of 16S rRNA gene or ITS1 fragments revealed a highly diverse rhizosphere and a less diverse endophytic microbial community for lettuce. Representatives of several bacterial (Alpha-, Beta- and Gammaproteobacteria, Firmicutes, Bacteriodetes), fungal (Ascomycetes, Basidiomycetes) and protist (Oomycetes) groups were present inside or on lettuce plants. Surprisingly, given that lettuce is a vegetable that is eaten raw, species of genera such as Flavobacterium, Burkholderia, Staphylococcus, Cladosporium and Aspergillus, which contain potentially human pathogenic strains, were identified. Analysis of the indigenous bacterial and endophytic fungal populations revealed only negligible, short-term effects resulting from the bacterial treatments, and that they were more influenced by field site, plant growth stage and microenvironment.     

Suppression of sugar beet damping-off caused by Rhizoctonia solani using bacterial and fungal antagonists

Rhizoctonia damping-off caused by Rhizoctonia solani Kühn, is one of the most damaging sugar beet diseases. It causes serious economic damage wherever sugar beets are grown. Biological control is an efficient and environmentally friendly way to prevent damping-off disease. Suppression of damping-off disease caused by R. solani was carried out by four isolates of Bacillus subtilis (Ehrenberg) Cohn as well as three isolates of each of Trichoderma harzianum Rifai and Trichoderma hamatum (Bonord.) Bainier. The effect of Bacillus and Trichoderma isolates against R. solani was investigated in vitro and tested on sugar beet plants under greenhouse conditions. Isolates of Bacillus and Trichoderma were able to inhibit the growth of R. solani in dual culture. Furthermore, Trichoderma isolates gave high antagonistic effect than isolates of B. subtilis. Under greenhouse conditions, coating seeds by T. harzianum and B. subtilis separately, reduced seedling damping-off significantly. However, applications of T. harzianum increased the percentage of surviving plants more than B. subtilis in comparison to control. The obtained results indicate that T. harzianum and B. subtilis are very effective biocontrol agents that offer potential benefit in sugar beet damping-off and should be harnessed for further biocontrol applications.     

Grape marc compost: microbial studies and suppression of soil-borne mycosis in vegetable seedlings

Compost suppression of soil-borne diseases in horticultural crops has been attributed to the activities of antagonistic microorganisms. A great diversity of microorganisms, capable of suppressing pathogens naturally colonize compost. A large number of microbes appeared in microbiological analyses of grape marc compost. Most microorganisms were bacteria. Average percentages were 31% mesophilic and 28% thermophylic bacteria, 16% mesophilic actinomycetes and 20% thermophylic actinomycetes. Only a few mould and yeast morphologies were obtained, 4% and 1% respectively. Antagonist in vitro assays were performed with 432 microbial morphologies isolated from grape marc compost. The microbes isolated were extremely effective antagonists in in vitro assays against all the fungal pathogens tested. Seven microorganisms were selected for further bioassay with Rhizoctonia solani in radish, Fusarium oxysporum f. sp. radicis-cucumerinum in melon, and Phytophthora parasitica in tomato and two microorganisms with Pythium aphanidermatum in cucumber. Those experiments indicate that grape marc compost reduces the severity of Pythium damping-off in cucumber, but does not reduce the severity of Phytophthora root rot in tomato, Fusarium oxysporum f. sp. radicis-cucumerinum in melon and Rhizoctonia solani in radish. Better suppressive effects were not demonstrated by either compost or vermiculite amended with microbes selected from grape marc compost.     

Possible role of xanthobaccins produced by Stenotrophomonas sp. strain SB-K88 in suppression of sugar beet damping-off disease.

Three antifungal compounds, designated xanthobaccins A, B, and C, were isolated from the culture fluid of Stenotrophomonas sp. strain SB-K88, a rhizobacterium of sugar beet that suppresses damping-off disease. Production of xanthobaccin A in culture media was compared with the disease suppression activities of strain SB-K88 and less suppressive strains that were obtained by subculturing. Strain SB-K88 was applied to sugar beet seeds, and production of xanthobaccin A in the rhizosphere of seedlings was confirmed by using a test tube culture system under hydroponic culture conditions; 3 microg of xanthobaccin A was detected in the rhizosphere on a per-plant basis. Direct application of purified xanthobaccin A to seeds suppressed damping-off disease in soil naturally infested by Pythium spp. We suggest that xanthobaccin A produced by strain SB-K88 plays a key role in suppression of sugar beet damping-off disease.     

Impact of biocontrol Pseudomonas fluorescens CHA0 and a genetically modified derivative on the diversity of culturable fungi in the cucumber rhizosphere

Little is known about the effects of Pseudomonas biocontrol inoculants on nontarget rhizosphere fungi. This issue was addressed using the biocontrol agent Pseudomonas fluorescens CHA0-Rif, which produces the antimicrobial polyketides 2,4-diacetylphloroglucinol (Phl) and pyoluteorin (Plt) and protects cucumber from several fungal pathogens, including Pythium spp., as well as the genetically modified derivative CHA0-Rif(pME3424). Strain CHA0-Rif(pME3424) overproduces Phl and Plt and displays improved biocontrol efficacy compared with CHA0-Rif. Cucumber was grown repeatedly in the same soil, which was left uninoculated, was inoculated with CHA0-Rif or CHA0-Rif(pME3424), or was treated with the fungicide metalaxyl (Ridomil). Treatments were applied to soil at the start of each 32-day-long cucumber growth cycle, and their effects on the diversity of the rhizosphere populations of culturable fungi were assessed at the end of the first and fifth cycles. Over 11,000 colonies were studied and assigned to 105 fungal species (plus several sterile morphotypes). The most frequently isolated fungal species (mainly belonging to the genera Paecilomyces, Phialocephala, Fusarium, Gliocladium, Penicillium, Mortierella, Verticillium, Trichoderma, Staphylotrichum, Coniothyrium, Cylindrocarpon, Myrothecium, and Monocillium) were common in the four treatments, and no fungal species was totally suppressed or found exclusively following one particular treatment. However, in each of the two growth cycles studied, significant differences were found between treatments (e.g., between the control and the other treatments and/or between the two inoculation treatments) using discriminant analysis. Despite these differences in the composition and/or relative abundance of species in the fungal community, treatments had no effect on species diversity indices, and species abundance distributions fit the truncated lognormal function in most cases. In addition, the impact of treatments at the 32-day mark of either growth cycle was smaller than the effect of growing cucumber repeatedly in the same soil.