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.     

Termite pathogens: Effects of ingested Metarhizium,beauveria, and Gliocladium conidia on worker termites (Reticulitermes sp.)

Separate groups of subterranean termites (Reticulitermes sp.) were exposed to whole cultures of Beauveria bassiana, Gliocladium virens, or Metarhizium anisopliae. Individuals were removed after varying time intervals and hindgut contents were plated onto potato dextrose agar. Viable spores first appeared in the hindguts within 8 hr of exposure. Fungi reisolated from the hindguts of diseased termites were pathogenic of healthy termites. Histological examination showed that invasion of the hemocoel by M. anisopliae occurred exclusively through direct invasion of the integument ca. 24 hr after death. B. bassiana invaded, primarily through the alimentary tract, ca. 12 hr prior to termite death.     

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.     

Enhanced fungal resistance in transgenic cotton expressing an endochitinase gene from Trichoderma virens

Mycoparasitic fungi are proving to be rich sources of antifungal genes that can be utilized to genetically engineer important crops for resistance against fungal pathogens. We have transformed cotton and tobacco plants with a cDNA clone encoding a 42 kDa endochitinase from the mycoparasitic fungus, Trichoderma virens. Plants from 82 independently transformed callus lines of cotton were regenerated and analysed for transgene expression. Several primary transformants were identified with endochitinase activities that were significantly higher than the control values. Transgene integration and expression was confirmed by Southern and Northern blot analyses, respectively. The transgenic endochitinase activities were examined in the leaves of transgenic tobacco as well as in the leaves, roots, hypocotyls and seeds of transgenic cotton. Transgenic plants with elevated endochitinase activities also showed the expected 42 kDa endochitinase band in fluorescence, gel-based assays performed with the leaf extracts in both species. Homozygous T2 plants of the high endochitinase-expressing cotton lines were tested for disease resistance against a soil-borne pathogen, Rhizoctonia solani and a foliar pathogen, Alternaria alternata. Transgenic cotton plants showed significant resistance to both pathogens.     

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.     

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.     

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.     

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.     

胶霉毒素的研究进展

胶霉毒素(gliotoxin,GT)是一个分子量为326 Da的小分子化合物,其骨架是由非核糖体肽合成酶Gli P催化苯丙氨酸和丝氨酸缩合成的环二肽,属于表聚硫代哌嗪二酮类化合物,是一种重要的真菌次级代谢产物。体内外研究已经表明,GT对动植物产生多种效应,不仅具有免疫抑制功能和诱导细胞凋亡作用,在生物防治方面也具有潜在的应用价值。本文将对有关GT生物合成、诱导宿主效应机制及其潜在应用价值进行综述。     

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。     

Interpretation of variety × sowing date × sowing depth interaction for bean–Fusarium–Rhizoctonia pathosystem

Through this study, the sowing date?×?sowing depth?×?variety interaction for Rhizoctonia root rot (RRR) development was investigated in field trials over two growing seasons. Four-way Wald tests indicated that there was a lower RRR incidence in var. Red compared to var. White in the majority of experimental plots. At a 5?cm seeding depth, RRR incidence for plantings on 22 May and 5 June was often lower than that for 5 May date. A hierarchical cluster analysis grouped experimental plots using averages of RRR incidence, Fusarium root rot (FRR) index and the number of seeds per plant. Sowing depth influenced FRR, RRR and productivity more than variety and date factors. Comparison of clusters recognised lower disease and greater production levels for sowing on 22 May and 5 June at 5?cm depth. Thus, shallow seeding of bean varieties in late spring should be incorporated into FRR–RRR-control programmes.     

Brazilian isolates of Clonostachys rosea: colonization under different temperature and moisture conditions and temporal dynamics on strawberry leaves

Aims: In a research programme for managing diseases caused by Botryis cinerea, four isolates of the antagonistic fungus Clonostachys rosea (Cr) were obtained from different ecosystems in Brazil. We studied ecological requirements for the colonization of strawberry leaves by these isolates. Methods and Results: Temperature effects on both mycelial growth in vitro and leaf colonization by Cr were studied. At 10°C, growth on potato dextrose agar and colonization of leaf discs were poor. Optimum temperature for mycelial growth and leaf colonization was around 25°C. The isolates were applied to leaves which were exposed to 0–48 h intervals of moisture. They were also applied to leaves which remained from 0 to 36 h without wetness. All isolates efficiently colonized leaves, regardless of moisture interval or the delay to begin wetness. Although all isolates survived in green leaves of whole plants, colonization decreased throughout a 49‐day period. Conclusions: Brazilian isolates of Cr can establish and colonize strawberry leaves under a wide range of temperature and moisture conditions. Significance and Impact of the Study: It is expected that the Brazilian isolates of Cr will establish efficiently in strawberry leaves where they can compete with B. cinerea.     

Development of formulations based on Streptomyces rochei strain PTL2 spores for biocontrol of Rhizoctonia solani damping-off of tomato seedlings

Rhizoctonia solani is one of the most problematic soil-borne pathogenic fungi for several crop cultures worldwide. This study highlights the effectiveness of high-antagonistic Streptomyces rochei strain PTL2, isolated from root tissues of Panicum turgidum, in controlling the R. solani damping-off and growth promotion of tomato (cv. Marmande) seedlings. The isolate PTL2 was characterised for in vitro biocontrol and plant growth-promoting traits. It exhibited remarkable positive results in all trials, including production of hydrogen cyanide, siderophores, 1-aminocyclopropane-1-carboxylate deaminase and phytohormones, chitinolytic activity and inorganic phosphate solubilisation. PTL2 spores were formulated as wettable talcum powder, sodium alginate pellets and sodium alginate-clay pellets. Their abilities in the biocontrol of R. solani and plant growth promotion were investigated in autoclaved and non-autoclaved soils. Talcum powder and sodium alginate pellets significantly reduced the damping-off severity index compared to a positive control. The talcum powder exhibited the highest protective activity, reducing the disease incidence from 89.3% to 14.1%, whereas chemical seed treatment with Thiram^® provided a disease incidence of 16.7%. Furthermore, the talc-based powder formulation resulted in greatest increases in the root length, shoot length and dry weight of seedlings. The interesting biocontrol potential and growth enhancement of tomato seedlings open up promising perspectives for the possible application of talcum powder formulation based on PTL2 spores in crop improvement.     

棉立枯丝核菌(Rhizoctonia solani)中的一个dsDNA病毒

自从1962年Hollings在栽培蘑菇(Agaricus bisporus)中发现第一例真菌病毒以来,迄今已在100多种真菌中发现了病毒,多数含双链RNA基因组。1972年Bozarth报道在R.solani中发现病毒,但未报道该病毒的理化性质。1975年Finlker在R.solani的一个强致病力菌株中分离到双链RNA病毒,它含3个组分dsRNA。     

Genomic-assisted characterisation of Pseudomonas sp. strain Pf4, a potential biocontrol agent in hydroponics

In an attempt to select potential biocontrol agents against Pythium spp. and Rhizoctonia spp. root pathogens for use in soilless systems, 12 promising bacteria were selected for further investigations. Sequence analysis of the 16S rRNA gene revealed that three strains belonged to the genus Enterobacter, whereas nine strains belonged to the genus Pseudomonas. In in vitro assays, one strain of Pseudomonas sp., Pf4, closely related to Pseudomonas protegens (formerly Pseudomonas fluorescens), showed noteworthy antagonistic activity against two strains of Pythium aphanidermatum and two strains of Rhizoctonia solani AG 1-IB, with average inhibition of mycelial growth >80%. Strain Pf4 was used for in vivo treatments on lamb’s lettuce against R. solani root rot in small-scale hydroponics. Pf4-treated and untreated plants were daily monitored for symptom development and after two weeks of infection, a significant protective effect of Pf4 against root rot was recorded. The survival and population density of Pf4 on roots were also checked, demonstrating a density above the threshold value of 10⁵?CFU?g^?1 of root required for disease suppression. Known loci for the synthesis of antifungal metabolites, detected using PCR, and draft-genome sequencing of Pf4 demonstrated that Pseudomonas sp. Pf4 has the potential to produce an arsenal of secondary metabolites (plt, phl, ofa and fit-rzx gene clusters) very similar to that of the well-known biocontrol P. protegens strain Pf-5.