Virens Isolates Research Articles

Two Endopolygalacturonase Genes in Trichoderma virens: In Silico Characterization and Expression during Interaction with Plants

AbstractThe ability of Trichoderma spp. to antagonize most phytopathogenic fungi and to interact with plants by inducing their resistance and promoting their growth has been widely described. Endopolygalacturonases (endoPGs) produced by Trichoderma spp. can assist root penetration and play a pre‐eliciting role in induced systemic resistance (ISR), a beneficial effect detected in plants colonized by Trichoderma. In this study, two endoPG genes (Tvpg1 and Tvpg2) have been identified and partially characterized in a T. virens isolate, previously investigated for its antagonistic ability in several biological systems. An extensive in silico analysis showed relatively low sequence similarities (53%) between TvPG1 and TvPG2 proteins and underscored interesting phylogenetic relationships in the comparison of endoPG genes among fungal genomes. The phylogeny of endoPGs suggests a differentiation in action mechanisms associated with biological functions as one cluster shows only genes from plant‐interacting organisms. A gene expression analysis demonstrated a different regulation pattern for those genes. The Tvpg1 gene was induced both when the fungus was grown in liquid cultures supplemented with pectin or plant cell walls and when it was applied to tomato roots in a growth chamber. Expression times were comparable in both systems. On the contrary, the Tvpg2 gene displayed a constitutive expression in all conditions tested, including controls. The molecular communication between tomato roots and T. virens was investigated by checking the expression of a tomato PGIP (Lepgip1) in roots colonized by fungal mycelium. The Lepgip1 transcript was induced at times coincident with Tvpg1 expression, suggesting a correlation between the two gene products. This paper reports the results of first investigations of T. virens endoPG genes performed on a structural, phylogenetic and functional level.

Trichoderma from Aceh Sumatra reduce Phytophthora lesions on pods and cacao seedlings

The cocoa tree, Theobroma cacao L., suffers large yield losses in Aceh Indonesia due to the disease black pod rot, caused by Phytophthora spp. Despite having the largest area under cacao production in Sumatra, farmers in the Aceh region have low overall production because of losses to insect pests and black pod rot. Trichoderma spp. were isolated from the roots and leaves of cacao trees and screened as potential biological control agents. Isolates used in the study were Trichoderma asperellum isolates T2 and T4, Trichoderma longibrachiatum isolates T15 and T16, and Trichoderma virens isolates T1 and Tv. T1, T2, T4, and Tv completely colonized and destroyed Phytophthora tropicalis and Phytophthora palmivora mycelium in precolonized plate assays. All six isolates reduced P. tropicalis, but none reduced the growth of P. palmivora in dual plate assays. Phytophthora growth was suppressed on MIN media amended with sterile heat inactivated Trichoderma culture filtrates, with Tv best suppressing growth of both Phytophthora spp. T. virens isolate Tv was the only isolate observed coiling around P. tropicalis mycelium and disrupted the formation of P. palmivora sporangia. Of all six isolates, only Tv reduced P. palmivora lesion expansion in a detached pod assay, reducing severity by 71%. Tv also reduced P. palmivora infection on seedlings when applied aerially at 1×106 and 1×108conidia/ml, by 19% and 59%, respectively. T. virens isolate Tv is a mycoparasite, antagonizes Phytophthora in a dual plate assay, and shows antibiosis against Phytophthora spp., suggesting that multiple modes of action contribute to its ability to limit Phytophthora lesion expansion on cacao pods and seedlings.

Genetic analysis of the population structure of the rice false smut fungus, Villosiclava virens, in China using microsatellite markers mined from a genome assembly

Studies on population genetics of Villosiclava virens are limited because of the lack of polymorphic markers. Based on a draft genome sequence of isolate HWD‐2 produced in this study, 20 of 403 potential simple sequence repeats (SSR) loci showed polymorphisms in preliminary screening using eight diverse V. virens isolates. Among polymorphic loci, most of them with tetra‐ to hexanucleotide unit motifs showed higher levels of polymorphism than loci with smaller motifs. After testing with 20 polymorphic SSR markers, the 87 isolates of V. virens from eight populations in China showed a high level of genetic diversity, with each as a unique haplotype. This differs from some previous findings showing little to no genetic variation based on random amplified polymorphic DNA and amplified fragment length polymorphism analyses. Among eight populations from major rice production areas of China, the population from Guangxi province in south China showed the highest levels of polymorphism, which led to the speculation that it might be closer to the centre of origin of this pathogen. The northern, central and eastern populations (Jilin, Liaoning, Hubei, Hunan, Jiangxi and Zhejiang), when considered together as a group, showed significant molecular variation compared to the southern populations (Fujian and Guangxi) (ΦPT = 0·043, P = 0·037). A significant relationship (Mantel test, P = 0·027) but with low correlation (R2 = 0·23) was also found between geographic distance and genetic distance. The 20 polymorphic SSR primer pairs designed in this study provide a tool for further research on the population diversity of this emerging fungal pathogen of rice.

English

False smut of rice (Oryza sativa L.) caused by Ustilaginoidea virens (Cooke) Takahashi (teleomorph Villosiclava virens) is one of the most common fungal diseases of rice in China. The objectives of the research were: 1) to study the biology of Ustilaginoidea virens, 2) to develop suitable media for inoculum production of U. virens, and 3) to develop an artificial inoculation technique for the study of false smut resistance in rice. Seventy diseased rice samples were collected from seven districts of Guizhou province, China during 2007-2008 and 138 single-spore isolates of U. virens were isolated. Among two inoculation methods applied at 3 rice growth stages of rice variety Gangxiang 707 using spore suspension of U. virens isolate 2008-33-1, conidia injection at late booting stage produced the highest false smut incidence of 50.43%, while conidia spraying during the same stage gave only 34.75% disease incidence. Pathogenicity test of selected eight U. virens isolates on rice variety Zhongyou 177 using the conidia injection at late booting stage, showed that the isolates were different in their aggressiveness in which isolate 2008-11-1 gave the highest virulence score of 9 with the disease incidence of 81.66%, while isolate 2007-48-1 gave the lowest score of 5 with only 15.57% disease incidence. These eight U. virens isolates were further tested on six rice varieties using the same inoculation technique. Most of the rice varieties were either susceptible or moderately susceptible except Fengyouxiangzhan that was highly susceptible to U. virens isolate 2007 -79-1 and Nongfengyou 256 was moderately resistant. When the evaluation was done across all the U. virens isolates, Gangyou 827, Jixiangyou 830 and Fengyouxiangzhan were among the most susceptible varieties with disease incidence of 55.06, 54.79 and 53.50%, respectively, while Nongfengyou 256 varieties showed the lowest disease severity as 23.85%. For the pathogen strain, 2008-33-1 was the most aggressive giving 58.09% disease incidence while the fungal pathogen strain 2008-2-2 gave the lowest of 28.17%. Key words: False smut, rice, colony morphology, fungal characterization, Ustilaginoidea virens.

Genetically engineered Thompson Seedless grapevine plants designed for fungal tolerance: selection and characterization of the best performing individuals in a field trial.

The fungi Botrytis cinerea and Erysiphe necator are responsible for gray mold and powdery mildew diseases, respectively, which are among the most devastating diseases of grapes. Two endochitinase (ech42 and ech33) genes and one N-acetyl-β-D-hexosaminidase (nag70) gene from biocontrol agents related to Trichoderma spp. were used to develop a set of 103 genetically modified (GM) 'Thompson Seedless' lines (568 plants) that were established in open field in 2004 and evaluated for fungal tolerance starting in 2006. Statistical analyses were carried out considering transgene, explant origin, and plant response to both fungi in the field and in detached leaf assays. The results allowed for the selection of the 19 consistently most tolerant lines through two consecutive years (2007-2008 and 2008-2009 seasons). Plants from these lines were grafted onto the rootstock Harmony and established in the field in 2009 for further characterization. Transgene status was shown in most of these lines by Southern blot, real-time PCR, ELISA, and immunostrips; the most tolerant candidates expressed the ech42-nag70 double gene construct and the ech33 gene from a local Hypocrea virens isolate. B. cinerea growth assays in Petri dishes supplemented with berry juices extracted from the most tolerant individuals of the selected population was inhibited. These results demonstrate that improved fungal tolerance can be attributed to transgene expression and support the iterative molecular and physiological phenotyping in order to define selected individuals from a population of GM grapevines.

English

The study of morphology and bioefficacy of Trichoderma was undertaken to select the effective isolates against soil-borne pathogens. Fifty one (51) isolates (23 isolates of Trichoderma virens and 28 isolates of Trichoderma harzianum) were morphologically characterised based on the growth characteristics on PDA medium, the size and shape of phialides and conidia.These isolates were screened for bioefficacy against soil borne plant pathogens (Fusarium oxysporum, Rhizoctonia solani and Sclerotium rolfsii) based on their percent inhibition observed during dual culture, volatile and non-volatile methods. Eight T. virens isolates and 12 T. harzianum isolates were proven to be potential isolates against the soil-borne pathogens tested. No correlation was found between bioefficacy and morphology in both species isolates. Key words: Trichoderma virens, Trichoderma harzianum, morphology, dual culture, biocontrol and bioefficacy.

Genetic diversity analysis reveals that geographical environment plays a more important role than rice cultivar in Villosiclava virens population selection.

Rice false smut caused by Villosiclava virens is an economically important disease of grains worldwide. The genetic diversity of 153 isolates from six fields located in Wuhan (WH), Yichang Wangjia (YCW), Yichang Yaohe (YCY), Huanggang (HG), Yangxin (YX), and Jingzhou (JZ) in Hubei province of China were phylogenetically analyzed to evaluate the influence of environments and rice cultivars on the V. virens populations. Isolates (43) from Wuhan were from two rice cultivars, Wanxian 98 and Huajing 952, while most of the other isolates from fields YCW, YCY, HG, YX, and JZ originated from different rice cultivars with different genetic backgrounds. Genetic diversity of isolates was analyzed using random amplified polymorphic DNA (RAPD) and single-nucleotide polymorphisms (SNP). The isolates from the same cultivars in Wuhan tended to group together, indicating that the cultivars had an important impact on the fungal population. The 110 isolates from individual fields tended to cluster according to geographical origin. The values of Nei's gene diversity (H) and Shannon's information index (I) showed that the genetic diversity among isolates was higher between than within geographical populations. Furthermore, mean genetic distance between groups (0.006) was higher than mean genetic distance within groups (0.0048) according to MEGA 5.2. The pairwise population fixation index (FST) values also showed significant genetic differentiation between most populations. Higher genetic similarity of isolates from individual fields but different rice cultivars suggested that the geographical factor played a more important role in the selection of V. virens isolates than rice cultivars. This information could be used to improve the management strategy for rice false smut by adjusting the cultivation measures, such as controlling fertilizer, water, and planting density, in the rice field to change the microenvironment.

Genetic diversity and population structure of rice pathogen Ustilaginoidea virens in China.

Rice false smut caused by the fungal pathogen Ustilaginoidea virens is becoming a destructive disease throughout major rice-growing countries. Information about its genetic diversity and population structure is essential for rice breeding and efficient control of the disease. This study compared the genome sequences of two U . virens isolates. Three SNP-rich genomic regions were identified as molecular markers that could be used to analyze the genetic diversity and population structure of U . virens in China. A total of 56 multilocus sequence types (haplotypes) were identified out of 162 representative isolates from 15 provinces covering five major rice-growing areas in China. However, the phylogeny, based on sequences at individual SNP-rich regions, strongly conflicted with each other and there were significant genetic differences between different geographical populations. Gene flow between the different geographical populations and genetic differentiation within each geographical population were also detected. In addition, genetic recombination and genetic isolation resulting from geographic separation was also found.

Frequency distribution of sensitivity of Ustilaginoidea virens to four EBI fungicides, prochloraz, difenoconazole, propiconazole and tebuconazole, and their efficacy in controlling rice false smut in Anhui Province of China

False smut, caused by Ustilaginoidea virens, is an important emerging disease of rice (Oryza sativa L.) in China. Up to now, as most varieties with high yielding and good quality are susceptible or even highly susceptible to false smut in most rice-growing ecological regions, especially in Anhui Province, chemical control with fungicides would be an important measure for the control of this disease. The ergosterol biosynthesis inhibitor (EBI) fungicides, such as prochloraz, difenoconazole, propiconazole and tebuconazole, are extensively used in China for the control of rice diseases, such as rice sheath blight and rice blast. In this study, a total of 102 U. virens isolates (from Anhui Province of China) were tested for their sensitivity to these four EBI fungicides during the stage of mycelial growth. The EC50 ranges of values for prochloraz, difenoconazole, propiconazole and tebuconazole inhibiting mycelial growth of the 102 U. virens isolates were 0.04–0.75, 0.04–1.08, 0.04–0.38 and 0.03–0.57 μg ml−1, with the average EC50 values of 0.32 ± 0.08, 0.45 ± 0.08, 0.19 ± 0.03 and 0.21 ± 0.06 μg ml−1, respectively. These values suggested that the tested U. virens isolates were very sensitive to these four EBI fungicides. Results of field trials showed that two sprays of three of the fungicides exhibited greater control efficacy than a single spray for the control of rice false smut. Two sprays of each was better than a single spray for the control of rice sheath blight. Two sprays of 50% propiconazole EC at 300 g a.i. ha−1 gave the best control of rice false smut at both two sites during the two consecutive years, 2010 and 2011, with the control efficacy ranging from 71.5 to 74.3%. Sensitivity of the field U. virens isolates to EBI fungicides should be monitored. Mixtures, as well as alternation with other fungicides with different modes of action, should be tested.

The antagonistic activity of Trichoderma virens strain TvSUT10 against cassava stem rot in Thailand

In this current study, the beneficial filamentous fungi, Trichoderma virens , isolated from cassava field were investigated for antagonistic mode of action against Lasiodiplodia theobromae , the causal agent of cassava stem rot in Thailand. In vitro screening using the dual culture technique was undertaken to assess the potential of these Trichoderma isolates. Our results indicated that fifteen isolates of T. virens were collected from various areas of cassava field in Nakhon Ratchasima, Thailand. The T. virens isolate, TvSUT10, was the most effective isolate and inhibited L. theobromae mycelial growth by 84.12%, due to the antagonistic mechanism. Moreover, Trichoderma β-1,3-glucanase activity was determined, the result revealed that the highest activity was recorded in strain of T. virens TvSUT10 (25.7 U/ml). In addition, in the greenhouse experiment, the application of the TvSUT10 as a conidial suspension reduced the stem rot disease severity of cassava caused by 53%. The results indicated that the T. virens strain TvSUT10 has initial modes of action of biological control to protect cassava crop against L. theobromae infections in cassava. Key words : Trichoderma , cassava stem rot disease, growth inhibition, cassava, biocontrol.

Survey of Egg- and Cyst-parasitic Fungi of Potato Cyst Nematode in Indonesia

Twelve fungal isolates out of 123 isolates obtained from cysts and soils of potato cyst nematode (PCN)-infested fields in Central Java, Indonesia had parasitic abilities of over 50% on PCN eggs or females (cysts) in vitro pathogenicity tests. Cultural and morphological characters and DNA sequences of ribosomal genes in ITS region revealed that they were four isolates of Gliocladium (Trichoderma) virens, three isolates of Fusarium oxysporum, one of F. lateritium, one of Penicillium tritinum and two of Taralomyces spp. A hundred percent infections occurred in eggs or cysts by three fungal isolates G. virens, F. oxysporum and P. oxalicum, suggesting that these fungi may have a good potential for the PCN biocontrol. Especially, G. virens isolates, which occurred most frequently in the PCN-infested potato fields and are known to be highly adaptable to varying habitats, may be developed as reliable agents for controlling PCN with both egg- and cyst-parasitic capabilities and with high ecological adaptabilities.

CHARACTERISING THE PATHOGENICITY DIVERSITY OF USTILAGINOIDEA VIRENS IN HYBRID RICE IN CHINA

Fifty nine isolates of Ustilaginoidea virens, the cause of false smut of rice (Oryza sativa L.), were obtained from 46 rice hybrids in 14 counties in Sichuan, China during a survey conducted in 2006. Their pathogenicity was tested by inoculation on 3 rice hybrids, Gangyou182, Gangyou94- 11 and Yixiangyou2292, 6-9 days before heading in 2007. False smut was assessed 3 weeks after inoculation using a disease index (DI) based on symptom frequency on the panicles. The sporulation capacity of these isolates was also measured. The results showed that: (i) DIs were significantly different (P<0.01) both among pathogen isolates and rice hybrids, ranging from 0 to 98.52; (ii) there was a significant interaction between isolates and hybrids (P<0.01); (iii) significant differences in sporulation among the 59 isolates were found (P<0.01), but no relationship between sporulation and virulence on the 3 hybrids; (iv) variation in sporulation was observed among isolates originating from different counties and from the same county (P<0.05); (v) there were significant differences (P<0.01) between isolate groups from different host origin, female parents and male parents. Our results indicated a linkage between the pathogenicity of U. virens isolates and the resistance of rice hybrids. The 59 isolates could be classified into 6 groups based on their virulence to the tested rice hybrids. While variation in sporulation did not indicate host genotype-pathogen isolate interaction, pathogenicity data suggest specialisation, which is dependent on the site of origin of the isolates, the original host (rice hybrids) and the parentage of the original host.

Genetic Diversity of Rice False Smut Fungus, Ustilaginoidea virens and its Pronounced Differentiation of Populations in North China

AbstractRice false smut caused by Ustilaginoidea virens is an important constraint affecting rice yield and quality in Asia. In China, rice false smut is especially severe in the japonica rice‐growing areas in the North China. Nothing is known neither about the diversity of the pathogen in this region nor about the characteristic of its local population. In this study, 110 U. virens isolates sampled from Liaoning and Beijing of North China were analysed using amplified fragment length polymorphism (AFLP) markers to primarily understand the genetic diversity of this pathogen and its population characteristics. At the genetic distance of 0.32, all isolates were divided into two groups. Group A consists of two subgroups differentiated at the genetic distance of 0.55, subgroup 1 included all isolates from Liaoning with an average genetic similarity over 0.82 and subgroup 2 included 27 isolates from Beijing with similarity of 0.74. Group B consists of 28 isolates from Beijing having a diversity of 0.054. The isolates from the Liaoning province, where rice false smut has generated different take‐all epidemic for 20 years, showed a genetic diversity of 0.305, which was approximately equally distributed within and among populations. Whereas genetic diversity was 0.458 among isolates from Beijing, an extremely high level of genetic differentiation among 55 isolates was observed in this disease hotspot. Our results suggested that the populations among different locations where sexual stage of the pathogen was rare to be found within ecological region were similar, and the variation of this pathogen has mainly arisen via asexual mechanisms. The migration through human activities in breeding perhaps provides a means of transporting the pathogen from one region to another.

Biocontrol agents applied individually and in combination for suppression of soilborne diseases of cucumber

The soilborne pathogens Rhizoctonia solani, Pythium ultimum, and Meloidogyne incognita can cause severe economic losses to field- and greenhouse-grown cucumber. A collection of bacterial isolates and isolates GL3 and GL21 of Trichoderma virens were screened for suppression of diseases caused by these pathogens. T. virens isolates GL3 and GL21 provided the most effective suppression of damping-off caused by R. solani in greenhouse bioassays. Burkholderia ambifaria BC-F, B. cepacia BC-1, and Serratia marcescens N1-14 also provided significant suppression of R. solani relative to the pathogen check in some experiments. T. virens isolates GL3 and GL21 and S. marcescens isolates N1-6, N1-14, and N2-4 provided the most consistent and effective suppression of damping-off of cucumber caused by P. ultimum in growth chamber experiments. No microbial treatment containing individual or combined microbes significantly suppressed populations of M. incognita on cucumber or improved plant vigor in greenhouse bioassays. T. virens GL21 applied as a granular formulation, in combination with B. cepacia BC-1 or B. ambifaria BC-F applied as a seed treatment, significantly improved suppression of damping-off caused by R. solani over individual applications of these microbes in at least one experiment. Treatments combining B. cepacia BC-1, B. ambifaria BC-F, or S. marcescens isolates N1-14 or N2-4 with T. virens GL21 in R. solani biocontrol assays always resulted in plant stands that were similar or greater than treatments containing individual applications of these microbes. B. ambifaria BC-F combined with T. virens GL21 in seed treatments resulted in significantly improved suppression of damping-off caused by P. ultimum in two of three experiments. Populations of T. virens GL3 and GL21 were both substantially reduced after coincubation with B. cepacia BC-1, or S. marcescens isolates N1-14 or N2-4 for 10 to 12 d in cucumber rhizospheres. Populations of T. virens GL21 were slightly reduced after coincubation with B. ambifaria BC-F. Results presented here substantiate other studies reporting enhanced biocontrol performance with certain combinations of biocontrol agents. These results also indicate that antagonism among combinations of biocontrol agents can vary with the assay system employed.

Trichoderma harzianum T39 and T. virens DAR 74290 as potential biological control agents for Phytophthora erythroseptica

Trichoderma harzianum isolate T39 and T. virens isolate DAR 74290 were evaluated as potential biological agents for control of pink rot of potato and root and stem rot of tomato caused by Phytophthora erythroseptica. Cell-free metabolites of T. virens DAR 74290 completely inhibited growth of P. erythroseptica in vitro and appeared to be fungicidal. T. virens DAR 74290 and Trichodex, a commercial formulation of T. harzianum T39, were tested for their ability to protect potato and tomato plants from disease caused by P. erythroseptica in glasshouse experiments. Trichodex and T. virens DAR 74290, alone and combined, reduced disease severity in shoots and roots of potatoes 10 weeks after inoculation with the pathogen. The yield of potatoes from plants treated with P. erythroseptica and T. virens DAR 74290 (mean of 12.9 g fresh weight/pot) was significantly greater than in controls inoculated with the pathogen alone (mean of 2.1 g/pot). Treatment with Trichodex alone increased the yield of tubers compared to the uninoculated controls. T. virens DAR 74290 increased the survival of tomato seedlings inoculated with the pathogen, and both this isolate and Trichodex decreased the severity of disease on tomato.

Efficacy of Antagonists and Carbendazim against Dry Root Rot of Mungbean [ Vigna radiata (L.) Wilczek] Incited by Macrophomina phaseolina (Tassi.) Goid under Glasshouse Conditions

Bioeflicacy of carbendazim tolerant isolate of Trichoderma harzianum and exotic isolates of T. viride and T. virens were evaluated against dry root rot pathogen of mungbean incited by Macrophomina phaseolina under glasshouse conditions. T. harzianum when applied to the soil besides being effective against the disease, increased seed germination by 96 per cent, plant height by 35.5cm and total biomass by 2.53 g/plant compared to T. viride and T. virens isolates. In addition, soil application of T. hazianum @ 5 g/kg and seed treatment @ 10 8 conidia/ml was found effective against dry root rot pathogen. Integration of T. harzianum as soil application (5g/kg) + seed treatment (10 8 C/ml) with sub-lethal doses of carbendazim (0.02%) significantly reduced dry root rot incidence (95.3%) over soil treatment (91.5%) and seed treatment alone (86.5%).

Seed Treatment with a Fungal or a Bacterial Antagonist for Reducing Corn Damping-off Caused by Species of Pythium and Fusarium.

Bioassays were conducted under greenhouse conditions to test the efficacy of antagonists applied to corn (Zea mays) seed for protection against seed rot and seedling damping-off at 18 and 25°C in a field soil artificially infested with a combination of Pythium ultimum, P. arrhenomanes, and Fusarium graminearum. Biomass of Gliocladium virens isolates Gl-3 or Gl-21, Trichoderma viride isolate Tv-1, or peat-based slurry of Burkholderia cepacia isolates Bc-B, Bc-T, or Bc-1 was coated individually onto corn seeds in one test, and Gl-3 or Bc-B at four inoculum levels was used in another test. Seed treatments with most of the biocontrol agents, as well as with the fungicide captan, significantly (P ≤ 0.05) increased seedling stand, plant height and fresh weight, and decreased root rot severity compared with untreated seeds in pathogen-infested soil. Coating seeds with the biocontrol fungus G. virens isolate Gl-3 was the most effective treatment, resulting in greater (P ≤ 0.05) seedling stand, plant height, and fresh weight, and lower (P ≤ 0.05) severity of root rot than those parameters from seeds treated with captan or other antagonists at both temperatures. The results from the seeds treated with Gl-3 were similar to those of untreated seeds in noninfested soil. In treatments with Bc-1, Bc-T, Bc-B, or Tv-1, incubation temperature affected plant emergence, root rot severity, plant height, and fresh weight (P ≤ 0.01). Conversely, in seeds coated with Gl-3 or Gl-21, these parameters were similar at both temperatures. The minimum number of propagules needed per corn seed to obtain plant emergence comparable to that from captan-treated seeds was between 104 and 105 CFU for Gl-3 and >108 for Bc-B. When propagules of Gl-3 were applied at a rate >106 CFU per seed, seedling emergence was greater (P ≤ 0.05) than that from captan-treated seeds.

Influence of Pyrax/Biomass of Biocontrol Fungi on Snap Bean Damping-off Caused bySclerotium rolfsiiin the Field and on Germination of Sclerotia

A 2-year field study at Beltsville, MD, of soil artificially infested with sclerotia of Sclerotium rolfsii strain Sr-1 demonstrated the ability of fermentor-produced biomass of Gliocladium virens isolate Gl-3 in a powder formulation to prevent damping-off of snap beans caused by this pathogen. Plant stands were counted 11 and 35 days after planting. In addition, the CFU of Gl-3 per g of soil in the treatment plots were determined. Pyrax/biomass amended at rates of 15, 30, 60, and 120 g/1.1 m 2 plots to provide 0.6 to 6.6 x 104 CFU of Gl-3 per g of soil significantly increased plant stands after 35 days, compared with 7 and 19% stands in the pathogen-infested control soils for 1992 and 1993, respectively. In 1992, the stand increase was correlated (r 2 = 0.92) with increased rates of the preparation, such that 60 and 120 g of the Pyrax/biomass per plot resulted in stands comparable to those (>85%) in the noninfested control plots. In 1993, although there was no significant correlation (r 2 = 0.601) between rate of amendment and plant stand, all rates gave stands greater than that in the infested control but not as great as that in the noninfested control. Generally, soil populations of Gl-3 increased by 11 days with higher, but not lower, rates of Pyrax/biomass to about 105 CFU/g soil during both years. Population levels tended to decline after 35 days of plant growth, but generally remained higher than the amounts added. This population increase suggested establishment of Gl-3 in the soil. A study to determine the influence of Pyrax and biomass of various isolates of Trichoderma spp. and G. virens on the germination of sclerotia of two S. rolfsii isolates (Sr-1 and Sr-3) indicated considerable specificity. G. virens isolates were more effective in reducing sclerotial germination than were isolates of T. viride, T. hamatum, and T. harzianum. Moreover, isolate G1-3 was more effective that the other G. virens isolates. In addition, S. rolfsii isolate Sr-3, which produces larger and darker sclerotia than those of Sr-1, was less affected by the various isolates of Trichoderma spp. and G. virens than was Sr-1.

Characteristics of alginate pellets formulated with Trichoderma and Gliocladium and their effect on the proliferation of the fungi in soil

Pelletized formulations of wheat bran or kaolin clay in an alginate gel containing conidia, chlamydospores, or fermentor biomass (FB) of several isolates of the biocontrol fungi Trichoderma spp. and Gliocladium virens were prepared. The ability of fungal propagules within the pellets to proliferate in soil was determined. Higher population densities were obtained when alginate pellets added to soil contained chlamydospores rather than condia, and bran rather than kaolin as the bulking agent. The active ingredient in pellets prepared from FB was approximately 5% biomass by weight and contained many chlamydospores. Colony‐forming units (cfu) ranged from 106’to 1010/g of soil after soil amendment with FB pellets of 12 Trichoderma and G. virens isolates. Population densities were high during the first 3 weeks of incubation and declined only gradually during 9 weeks. Propagules in FB pellets were more viable at 5° than at 25°C. Viability at 25°C remained high (&gt; 70%) after 1 week, but declined to less than 10% after 24 weeks. Despite reduction in propagule viability in stored pellets, numbers of cfu formed after adding these pellets to soil were comparable with those formed from freshly prepared pellets.

Comparaison des aptitudes parasitaires de clones de Trichoderma vis-a-vis de quelques champignons a sclerotes

Nineteen monoconidial isolates (referred to as clones) of Trichoderma from different species aggregates, one isolate of Gliocladium virens, and one isolate of an Acrostalagmus sp. (that was naturally associated with sclerotia of Sclerotinia spp and Macrophomina phaseolina) were tested. They were incubated in controlled conditions, in sterile soil, with sclerotia of Corticium rolfsii, Sclerotinia minor, or S. sclerotiorum. At the end of appropriate periods of incubation (respectively 26, 20 and 8 days), the sclerotia were retrieved from soil and checked for invasion by the antagonist. Important differences between the parasitic ability of Trichoderma clones were noted. Clones from at least three different species ( T. aureoviride, T. hamatum, T. harzianum) exhibited a high antagonistic activity. Activity of the G. virens isolate was at the same level as the best clones of Trichoderma, whereas no parasitic tendencies were found in the isolate of Acrostalagmus sp., thus confirming previous results. A rather good correlation was found between the capacity of the clones for attacking C. rolfsii sclerotia and their ability to parasitize both Sclerotinia. In conclusion, it is proposed that a screening with only one of the sclerotial species would give clones efficient against all three, and possibly against related sclerotial types.