Interactions of Gliocladium virens with Rhizoctonia solani and Pythium ultimum in Non-sterile Potting Medium

Interactions of Gliocladium virens with Pythium ultimum and Rhizoctonia solani under simulated in vivo conditions were observed microscopically. Different types of propagules of the three fungi were paired on nitrocellulose membranes and incubated at 25°C in non-sterile potting medium in Petri dishes for 1-5 days. Alginate-wheat bran prill were used as carriers for G. virens. Prill inoculated with G. virens and pre-incubated in potting medium for 3-5 days before placement on membranes did not inhibit the germination of Pythium sporangia, but subsequent Pythium growth was markedly stunted and distorted, with some hyphal collapse and cytoplasmic leakage. G. virens had no visible effect on older Pythium mycelium. Two to 5 days' growth of G. virens caused cytoplasmic leakage of Rhizoctonia mycelium, prevented secondary branching of hyphae and occasionally coiled around Rhizoctonia hyphae. Prill that were newly colonized by G. virens, but not prill pre-incubated for 3 or 5 days, stimulated the growth of Pythium mycelium and sporangia, Rhizoctonia mycelium and unprimed monilioid cells, probably by supplying nutrients. The timing of the interactions and their specificity for the different pathogen propagules were consistent with the production of gliotoxin by G. virens. This view was supported by in vitro experiments, in which pathogen propagules were incubated in a range of concentrations of gliotoxin in potato dextrose broth. Pythium sporangia and mycelium were inhibited by 1 or 2 μmg ml^-1, but Rhizoctonia monilioid cells and mycelium required 3-5 μmg ml^-1 for inhibition. At the lowest effective concentrations the inhibition was sometimes reversible, but propagules were killed at high concentrations of gliotoxin.     

Baiting with Sclerotium rolfsii for selective isolation of Gliocladium virens from natural soil

A baiting technique for selective isolation of Gliocladium virens from natural soil was developed by mixing Sclerotium rolfsii —colonized sorghum grains with moist natural soil and incubating at 30 ± 5°C for 6–10 days. G. virens developed large, distinct colonies on the soil surface by colonizing S. rolfsii and was then easily isolated for use in biocontrol programmes. Trichoderma spp. were present in the soil but never developed conspicuous colonies on the soil surface, even when the soil was supplemented with large numbers of conidia.     

绿粘帚霉菌株发酵液抗真菌活性及稳定性测定

采用杯碟法测定绿粘帚霉（RCEF4099）菌株的发酵液对8种植物病原真菌的抗菌活性。结果表明RCEF4099菌株发酵液对5种供试病原真菌的抑菌圈直径在18mm以上。对菌株发酵液的稳定性测定结果表明菌株转接6代之前,活性稳定,从第7代开始其活性缓慢降低,第10代的抑菌圈直径仅比出发菌株减少了1．7mm。RCEF4099菌株发酵液有较好的热稳定性,发酵液加热到60％仍有较高活性。该菌株的发酵液对酸的稳定性较好,抑菌活性最强的pH值为4。     

Histopathological studies of sclerotia of Rhizoctonia solani parasitized by the EGFP transformant of Trichoderma virens

Aims:  The aim of the study was to investigate the antagonistic interactions of Trichoderma species against Rhizoctonia solani sclerotia by enhanced green fluorescence protein (EGFP)-tagged transformant of Trichoderma virens TY009.
Methods and Results:  An EGFP was used as a report gene for transforming T. virens strain, and a stable EGFP transformant GF5 was obtained with the mycoparasitic activity against sclerotia of R. solani . Observation of parasitized sclerotia by fluorescence microscopy showed hyphae of transformant GF5 was able to invade into sclerotia and its colonization was mainly intercellar with uniformly distributed mycelium in sclerotia. The host cells were colonized, penetrated, and then the whole cells were replaced by transformant GF5 hyphae. Chlamydospores were seen after 10 days but mature ones after 20 days. Sclerotia became soft and decayed after 40 days but a few cells seemed not to be colonized completely.
Conclusions:  Trichoderma virens was able to parasitize sclerotia to make sclerotia soft and decayed, and its colonization was mainly intercellar in sclerotial tissues.
Significance and Impact of the Study:  This is first report of parasitism of sclerotia of R. solani by EGFP-tagged transformant, providing useful information for using T. virens as effective biocontrol agent.     

A new formulation system for the application of biocontrol fungi to soil

A new biocontrol formulation system was devised that does not require sterile conditions during preparation. It involves mixing vermiculite and powdered wheat bran with wet or dry fermentor biomass of Trichoderma spp. or Gliocladium virens, moistening with 0.05 N HCl, and drying the mixture. Before application to soil, the preparation (VBA‐FB) is activated by re‐moistening with 0.05 N HCl and incubated at room temperature for 2–3 days to stimulate development of young hyphae of the biocontrol fungus. Populations of biocontrol fungi proliferated to greater than 10⁷ colony‐forming units (cfu) per g of soil when activated VBA‐FB was added to soil. In soil artificially infested with Rhizoctonia solani, seven isolates of the 14 studied added as VBA‐FB reduced survival and 12 reduced saprophytic growth of the pathogen. Of these, two isolates of T. hamatum (TRI‐4, Tm‐23) and one of T. harzianum (Th‐87) were the most effective. Preparations formulated with either wet or dry biomass effectively reduced pathogen survival, but activated VBA‐FB was more effective than non‐activated VBA‐FB. Storage of VBA‐FB at 25°C for 24 weeks before activation reduced viability of isolates considerably more than storage at 5°C for 24 weeks. In addition, VBA‐FB stored at 5°C before activation more effectively reduced survival of R. solani than VBA‐FB stored at 25° C. Survival of R. solani was reduced by activated VBA‐FB applied to several soil types (sandy loam, sandy clay loam, clay). Some nitrogen fertilizers increased the efficacy of VBA‐FB preparations of several isolates.     

Stilbella aciculosa: A Potential Biocontrol Fungus against Rhizoctonia solani

A semi‐solid fermentation product of the potential biocontrol fungus Stilbella aciculosa was formulated on wheat bran: water (1:1, w/w) and incubated 5, 10 and 15 days before addition to soil infested with the pathogen Rhizoctonia solani. Generally, preparations did not reduce survival of the pathogen in infested beet seed but they did prevent saprophytic growth of the pathogen from beet seed into soil. The magnitude of reduction by the 15‐day‐old inoculum was greater than that by the 5‐day‐old inoculum. Ten‐day‐old bran preparations of S. aciculosa at rates of 0.5 and 1.0% (w/w) in soil prevented post‐emergence damping‐off of cotton, radish and sugar beet in the glasshouse and a rate of 1.0% gave stands similar to those in the non‐infested control soil. The antagonist, grown on perlite formulated with molasses, cornmeal, alfalfa tissue or corn stover, prevented damping‐off of cotton in a naturally infested soil. However, the stands were not as great as that in soil planted with pentachloronitrobenzene (PCNB)‐treated seed. Toxic metabolites, produced by S. aciculosa developing on various substrates, slightly inhibited the growth of R. solani in culture and induced cytoplasmic leakage of the pathogen mycelium.     

Evaluation of two Streptomyces spp. and compost for growth promotion and biocontrol potential against Rhizoctonia solani on pepper

The individual and synergistic potentials of two Streptomyces strains and compost for growth promotion and biocontrol against dumping off caused by Rhizoctonia solani on pepper were evaluated. Results showed that the compost can greatly enhance pepper growth while the combination of the two strains and compost was most efficient for the disease suppression.     

Biocontrol of Damping-off Diseases Caused by Rhizoctonia solani and Pythium ultimum with Alginate Prills of Gliocladium virens, Trichoderma hamatum and Various Food Bases

Alginate prills were formulated with the biomass of isolates of Gliocladium virens and Trichoderma spp. and various food bases (wheat bran, corn cobs, peanut hulls, soy fiber, castor pomace, cocoa hulls and chitin). Alginate prills with G. virens (Gl-21) biomass and all food bases except cocoa hull meal significantly reduced the damping-off of zinnia in a soil-less mix caused by Rhizoctonia solani and Pythium ultimum. The prills with bran, soy fiber, castor pomace or chitin resulted in stands similar to those in the non-infested control. In soil, prills with all the food bases and Thrichoderma hamatum (TRI-4) biomass controlled the damping-off of cotton caused by R. solani and gave stands comparable to, or better than, those in the non-infested control soil. Prills with all the food bases resulted in a proliferation of Gl-21 in a soil-less mix and of Gl-21 and TRI-4 in soil. Prills with food bases and TRI-4 biomass reduced the survival of R. solani in infested beet seed to less than 30%, with bran and chitin being the most effective food bases; prills with Gl-21 biomass and all food bases also reduced the survival of R. solani in beet seed, but not as much as did prills with TRI-4 biomass. In prills containing wheat bran, soy fiber or chitin, the biocontrol isolate Th-58 (T. harzianum) was almost as effective as TRI-4, but isolate Gl-3 (G. virens) was less effective. There was no significant interaction between the biocontrol fungus and the food base. The results suggest that the intrinsic properties of a selected fungus isolate are more important than some formulation variables in biocontrol.     

Molecular characterization of Rhizoctonia solani isolates the incitant of soybean root rot

Rhizoctonia solani isolates used in this investigation were identified as anastomosis-4 (AG-40), collected from different localities from Assiut governorate in Egypt. Pathogenicity test of seven isolates of R. solani was evaluated on soybean Giza 111 cultivar under greenhouse conditions. All tested isolates were able to infect soybean plants causing root rot with different degrees of severities, isolate No. 1, 2 and 3 showed significantly highest root rot severity, while isolate No. 5 gave the lowest percentage of root rot rating. The sodium dodecyl sulphate polyacrylamide gel electrophoresis patterns were used to compare three isolates of R. solani. There are no variations among R. solani isolates except a few exceptions according to their protein patterns. DNA markers obtained from all isolates showed genetic similarity among different isolates obtained from different geographical regions barring few exceptions. Correlation between DNA patterns of R. solani isolates and their virulence was detected, but no correlation with anastomosis groups (AG).     

Biocontrol of Rhizoctonia solani damping-off of sugar beet with native Streptomyces strains under field conditions

A 3-year study was conducted to evaluate the effectiveness of two disease-suppressive Streptomyces spp. to control sugar beet Rhizoctonia solani damping off under field conditions. Streptomyces seed treatments reduced seedling damping off in naturally (2005) and artificially (2006 and 2007) infested soils. All biocontrol agents provided better efficacy than Vitavax to control seedling damping-off. There were no significant differences among Streptomyces isolates. Isolate C increased plant stand by 19.5, 50.5 and 53.75% in 2005, 2006 and 2007, respectively. Evaluation of final harvest revealed that the root yield of the biocontrol agents increased compared to untreated control in these years.     

中国北方棉花主产区立枯丝核菌的融合群鉴定

从山东、河北、河南三省采集棉花立枯病样品和土壤200余份,经分离获得198个丝核菌Rhizoctonia solani分离物。菌丝融合测定及5.8S rDNA-ITS区序列分析结果表明,这些分离物分别属于多核丝核菌的AG4-HG-I和AG4-HG-III融合群以及双核丝核菌的AG-A、AG-F、AG-Fb融合群。其中AG4-HG-I是优势融合类群,占分离物总数的88.38%,其次是AG4-HG-III,占10.10%,AG-A、AG-F、AG-Fb各仅有1株。其中双核丝核菌AG-A、AG-F和AG-Fb融     

Trichoderma virens 106 inoculation stimulates defence enzyme activities and enhances phenolic levels in tomato plants leading to lowered Rhizoctonia solani infection

Interaction of tomato roots with Trichoderma virens TRS106 provided protection against Rhizoctonia solani-induced disease. In tomato, plants inoculated with R. solani disease symptoms were observed on the roots as brown, necrotic lesions. These symptoms were limited on plants treated with TRS106 and inoculated with R. solani. It was shown that TRS106 did not directly inhibit Rhizoctonia growth in in vitro test. The tested Trichoderma isolate stimulated systemic defence responses in tomato plants, by activating defence enzymes including guaiacol peroxidase (GPX), syringaldazine peroxidase (SPX) and phenylalanine ammonia lyase (PAL). Simultaneously, it enhanced accumulation of phenolics and hydrogen peroxide (H₂O₂) accompanied by decrease in lipid peroxidation in the leaves. HPLC analysis indicated remarkable increases in the concentrations of 22 phenolics in the leaves of Trichoderma-treated tomato, both uninoculated and inoculated with R. solani. Some of the phenolics were present in a free form, the others were accumulated in a bound form as glycosylated conjugates belonging to phenylpropanoids, hydroxybenzoic and cinnamic acid derivatives and flavonoids. Several of the detected phenolics: ferulic and salicylic acids, pyrocatechol and hesperetin were strongly toxic to R. solani in plate tests. The systemic mobilisation of phenolic metabolism might be an element of tomato defence response positively involved in biocontrol of R. solani by TRS106. Based on the results, T. virens TRS106 may have potential to develop a new biofungicide for integrated management of R. solani-induced disease.     

Variations in culture morphology and pathogenicity among protoplast-regenerated strains of Rhizoctonia solani

Abstract Protoplast-regenerated cultures derived from mycelia of cereal-infecting field isolates of Rhizoctonia solani exhibited major variations in cultural morphology and in pathogenicity. Each field isplate yielded three of four distinct morphological types of protoplast cultures. The presence of the new morphological phenotypes was attributed to the selection of homokaryons arising from protoplasts with single nuclei. Highly pathogenic field isolates produced protoplast cultures with higher virulence than those from weakly virulent pathogenic isolates, and homokaryotic strains were generally less pathogenic than the parental field isolate.     

Extruded Granular Formulation with Biomass of Biocontrol Gliocladium virens and Trichoderma spp. to Reduce Damping-off of Eggplant Caused by Rhizoctonia solani and Saprophytic Growth of the Pathogen in Soil-less Mix

Extruded granular formulations containing rice flour, gluten, Pyrax, vermiculite, canola oil, and fermentor-produced biomass of isolates of Gliocladium virens (Gl-3, Gl-21 and Gl-32), Trichoderma hamatum (TRI-4 and 31-3), T. harzianum (Th-32 and Th-87) and T. viride (Tv-101) were evaluated for their effect on the reduction of eggplant damping-off caused by Rhizoctonia solani, reduction of pathogen inoculum and proliferation of the isolates in a soil-less mix. Granules with all isolates except 31-3 significantly (P < 0.01) reduced damping-off, and granules with Gl-3, Gl-21, Gl-32, TRI-4 and Th-87 yielded stands comparable to that (90%) of the non-infested control. Granules with isolates Gl-21 and TRI-4 were the most effective in the reduction of saprophytic growth of R. solani, and there was a significant inverse correlation (r ² = - 0.82) between eggplant stand and saprophytic growth of the pathogen over all treatments. Isolate propagules proliferated to about 10⁷ colony-forming units (CFU) g^?1 of soil-less mix after a 6-week incubation, but there was no correlation between the number of CFU and eggplant stand or saprophytic growth reduction of the pathogen. Granules with Gl-21 and TRI-4 amended to pathogen-infested soil-less mix at a rate as low as 0.06% significantly (P < 0.05) reduced damping-off and pathogen saprophytic growth, and a rate of 0.25% of Gl-21 granules resulted in an eggplant stand comparable to that of the non-infested control. There was no significant correlation between the rate of granule amendment and the proliferation of Gl-21 and TRI-4. Granules of Gl-21 and TRI-4 also significantly prevented the spread of R. solani in flats of eggplant seedlings when the biocontrol granules were applied to the soil-less mix 1 day before the pathogen inoculum.     

In vitro antagonists of Rhizoctonia solani tested on lettuce: rhizosphere competence, biocontrol efficiency and rhizosphere microbial community response

The rhizosphere competence of 15 in vitro antagonists of Rhizoctonia solani was determined 4 weeks after sowing inoculated lettuce seeds into nonsterile soil. Based on the colonization ability determined by selective plating, eight strains were selected for growth chamber experiments to determine their efficacy in controlling bottom rot caused by R. solani on lettuce. Although in the first experiment all antagonists colonized the rhizosphere of lettuce with CFU counts above 2 × 10⁶ g⁻¹ of root fresh weight, only four isolates significantly reduced disease severity. In subsequent experiments involving these four antagonists, only Pseudomonas jessenii RU47 showed effective and consistent disease suppression. Plate counts and denaturing gradient gel electrophoresis (DGGE) fingerprints of Pseudomonas -specific gacA genes amplified from total community DNA confirmed that RU47 established as the dominant Pseudomonas population in the rhizosphere of inoculated lettuce plants. Furthermore, the DGGE fingerprint revealed that R. solani AG1-IB inoculation severely affected the bacterial and fungal community structure in the rhizosphere of lettuce and that these effects were much less pronounced in the presence of RU47. Although the exact mechanism of antagonistic activity and the ecology of RU47 remain to be further explored, our results suggest that RU47 is a promising agent to control bottom rot of lettuce.     

两株棉花立枯病拮抗菌MH1和MH25的筛选与鉴定

从棉花根际分离了1277个细菌分离物, 以棉花立枯病病原真菌立枯丝核菌(Rhizoctonia solani Kuhn)为靶标菌, 通过平板对峙法获得25个具有拮抗性能的分离物, 其中MH1和MH25具有较强的拮抗性能, 且拮抗性能稳定, 具有较好的生防潜力。经过形态观察、生理生化特征分析及16S rDNA序列分析, MH1为短芽孢杆菌(Brevibacillus brevis), MH25为枯草芽孢杆菌(Bacillus subtilis)。MH1和MH25的16S rDNA序列在GenBank中注册号分别为: EF488102, EF488103。     

新疆北疆棉田立枯丝核菌菌丝融合群及其营养亲和群研究

从新疆北疆棉区采集了典型的棉花立枯病病苗及棉田土标样686份,按常规分离方法分离得到399个分离物,从中鉴定出272个纯化的立枯丝核菌(Rhizotonia solaniKühn)菌株,经玻片吉姆萨氏染剂(Gimsa′sstain)染色程序观察细胞核数目,全部测试菌株均属于多核。用标准菌株,通过载玻片菌丝融合实验测定,将纯化的272个菌株划归为3个菌丝融合群:AG-2、AG-4和AG-5,分别占总菌株的6.24%、84.2%和1.1%。另有23个菌株不与任何标准菌株融合,占8.46%,说明新疆北疆棉田立枯丝核菌的优势菌系是多核丝核菌的AG-4融合群。通过从10种不同配方的培养基中筛选的效果好的麦芽蛋白胨(MPDA)配方培养基(Ⅱ)进行对峙培养,以建立标准菌株,将纯化获得的272个丝核菌菌株,划分为6个不同的营养亲和群,研究说明新疆北疆地区棉田立枯丝核菌各菌丝融合群内确有不同程度的分化。