Trichoderma harzianum produces nonanoic acid, an inhibitor of spore germination and mycelial growth of two cacao pathogens

An isolate of Trichoderma harzianum Rifai from an infected cacao pod produces and secretes nonanoic (pelargonic) acid into a liquid culture medium. Nonanoic acid (NA) was very inhibitory to spore germination and mycelial growth of two cacao pathogens, Crinipellis perniciosa Stahel and Moniliophthora roreri Cif. H.C. Evans. It was highly active causing 75% inhibition of spore germination in an in vitro assay at a rate as low as 0.09 μM for M. roreri and 0.92 μM for C. perniciosa. Mycelial growth was comparatively less sensitive to inhibition, but still there was a 75% reduction in growth with 0.62 μM in M. roreri and 151 μM NA in C. perniciosa. In contrast, NA did not affect Trichoderma mycelial growth or spore germination at concentrations that were inhibitory to the pathogens. 6-pentyl-α-pyrone was also produced and secreted into the medium by T. harzianum, however; it was not antagonistic to the cacao pathogens. Although a number of metabolites produced by Trichoderma spp. have been identified in the past, this is the first report of NA production and secretion by any Trichoderma. The results suggest that NA may play a role in the successful use of some Trichoderma spp. isolates in the biocontrol of fungal diseases of plants.     

Transgenic cucumber expressing an endogenous class III chitinase gene has reduced symptoms from Botrytis cinerea

A class III chitinase gene (CHI2) is induced in cucumber plants (Cucumis sativa L.) in response to infection by pathogenic microorganisms. Infection of Botrytis cinerea, causal agent of gray mold disease on cucumber, also induces CHI2 expression. To investigate whether CHI2 is involved in resistance to gray mold disease, transgenic cucumber plants were produced to overexpress the CHI2 gene. One line was analyzed in detail in terms of disease resistance. The transgenic cucumber plant (CC2) constitutively expressed CHI2 and reduced the symptoms of B. cinerea for 4 days after inoculation compared with nontransgenic plants. However, this inhibitory effect was not absolute, and CC2 eventually developed serious disease symptoms. Chitinase activity of the crude extract from CC2 leaves was higher than that from nontransgenic plants. A high-molecular-weight fraction containing CHI2 from CC2 leaves had fungistatic activity against B. cinerea. Interestingly, the low-molecular-weight fraction from CC2 leaves with CHI2 removed also had fungistatic activity against B. cinerea. Not only the introduced chitinase activity but also the endogenous defense reactions activated by overexpression of CHI2 may be involved in the enhanced gray mold disease resistance in CC2.     

Synergism between potassium sorbate dips and brief exposure to high CO2 or O2 at curing temperature for the control of citrus postharvest green and blue molds

Synergistic effects and very effective control of citrus postharvest green and blue molds, caused by Penicillium digitatum and Penicillium italicum, respectively, were observed on artificially inoculated ‘Valencia’ oranges and ‘Clemenules’ and ‘Ortanique’ mandarins after a potassium sorbate (PS) treatment was followed by 2 days of storage in atmospheres of elevated CO₂ or O₂ at a curing temperature. A combined treatment consisting of 60-s dips in aqueous solutions of 3% PS heated to 62 °C was followed by 48-h exposure to air, 15 kPa CO₂ or 30 kPa O₂ at 33 °C. Control treatment was a 60-s water dip at 20 °C followed by 24-h exposure to air at 20 °C. Synergism was observed on citrus fruit either incubated at 20 °C for up to 22 days, simulating direct commercialization, or stored at 5 °C for up to 45 days, simulating commercial cold storage. This research offers potential new tools to the citrus industry for implementation of nonpolluting integrated postharvest disease management programs, especially devoted to high added value organic markets or export markets with zero residue tolerance.     

生防细菌复配对苹果主要病原真菌抑菌活性筛选及其稳定性

采用生长速率法测定4种生防细菌及其复配对5种苹果主要病原菌的抑制效果,并将筛选出的最佳复配生防细菌发酵液进行稳定性测定。结果表明,单一生防细菌发酵液中多粘类芽孢杆菌FS-1206对苹果树腐烂病菌的抑菌效果最好,为78.19%;当多粘类芽孢杆菌FS-1206与解淀粉芽孢杆菌TS-1203复配比例为 2∶1时,其对苹果树腐烂病菌的抑菌率高达86.64%,抑菌效果显著优于单一生防细菌发酵液。稳定性测定结果表明,复配生防细菌（FS-1206∶TS-1203= 2∶1）发酵液在紫外照射、高温和强酸强碱环境条件下,其抑菌活性均具有良好的稳定性。     

黑松针叶提取物抗霉菌活性的研究

采用滤纸片法,检测不同质量浓度的针叶提取物对霉菌的抑制圈大小,以研究黑松针叶提取物的抗霉菌活性。结果表明:黑松针叶提取物具有抗霉菌活性,但抑制效果与其质量浓度不成正比;3种提取物中,乙醇提取物的抗霉菌效果最显著,丙酮和水提取物的抗霉菌效果次之。其对黑曲霉、青霉、根霉起最佳抑制效果的质量浓度分别为6.25、25.01、2.5 g/L,其抑菌圈直径分别为14.8、16.61、2.6 mm。     

黄绿木霉菌对大豆根腐病镰刀菌的拮抗作用

利用室内对峙培养与发酵液处理病原菌研究了黄绿木霉(Trichoderma aureoviride)对大豆根腐病几种镰刀菌(Fusarium spp.)的拮抗作用,并利用盆栽试验研究了黄绿木霉对苗期大豆根腐病发生的影响.拮抗试验结果表明,黄绿木霉在与镰刀菌对峙培养中,菌丝交叉、接触与纠结,接触后镰刀菌菌丝被破坏;发酵产物只可延长镰刀菌孢子萌发的时间,但对孢子最终萌发率没有影响,发酵产物对菌丝生长速率的影响以山梨醇、葡萄糖及蔗糖作为碳源时,抑菌效果好,抑菌能力分别可达63%、51%、58%;盆栽试验证明黄绿木霉对大豆根腐病有较好的防治效果,防效可达73.2%,处理后幼苗株高与干重均高于未处理的对照.     

拮抗香蕉枯萎病菌的解淀粉芽孢杆菌LX1菌株的鉴定及其抗菌蛋白基因的克隆

收集海南不同盐碱地的土样进行拮抗香蕉枯萎病菌的微生物分离,对具有较强抑菌作用的菌株进行形态特征、生理生化和分子鉴定,并克隆其抗菌蛋白基因.结果分离到1株具有较强抑制香蕉枯萎病菌能力的细菌LXl,经鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens).抑菌实验发现LX1菌株的发酵上清液中粗蛋白有一定的抑菌作用,经过Sephacryl S-200HR柱层析、DEAE Sepharose Fast Flow离子交换层析分离纯化了其中抑菌作用最强的抗菌蛋白.质谱鉴定结果表明,抗菌蛋白与B.amyloliquefaciens FZB42内切葡聚糖酶同源性最高.根据质谱结果克隆了该抗菌蛋白的编码基因,该基因的核苷酸和氨基酸序列与B.amyloliquefaciens FZB42的内切葡聚糖酶的核苷酸和氨基酸序列同源性分别达99％和100％.拮抗香蕉枯萎病菌的芽孢杆菌LX1可作为潜在的防治香蕉枯萎病的的生防制剂,其抗菌蛋白基因也可通过遗传工程应用于香蕉枯萎病的防治.     

Soil volatile fungistasis and volatile fungistatic compounds

Fungistasis is a widespread phenomenon that can be mediated by soil microorganisms and volatile organic compounds (VOCs). The relationship between soil microorganisms and VOCs is still unclear, however, and many fungistatic compounds remain to be identified. We assessed the effects of soils (soil direct fungistasis) and VOCs produced by natural soils (soil volatile fungistasis) on the spore germination of several fungi. Both strong soil direct fungistasis and soil volatile fungistasis were observed in a wide range of soils. Soil fungistasis and VOC fungistasis were significantly correlated (P<0.001). The volatile fungistatic activity of soils stopped after autoclaving. Some VOCs were identified by using solid-phase microextraction-gas chromatography/mass spectrum. VOC composition and in vitro antagonism of relatively pure commercial compounds also were measured. Some VOCs, trimethylamine, 3-methyl-2-pentanone, dimethyl disulfide, methyl pyrazine, 2,5-dimethyl-pyrazine, benzaldehyde, N,N-dimethyloctylamine and nonadecane, were produced by various fungistatic soils. Moreover, antifungal activity test of above VOCs showed that trimethylamine, benzaldehyde, and N,N-dimethyloctylamine have strong antifungal activity even at low levels (4-12 mg l⁻¹).     

昆虫抗真菌肽基因的分泌型表达及活性鉴定

 【目的】黑腹果蝇(Drosophila melanogaster)抗真菌肽Drosomycin (Drs)及其同系物Drosomycin-like C (Drs-lC)和斑腹刺螠蝽(Podisus maculiverntris)抗真菌肽Thanatin对丝状真菌具有广谱高效的抑杀作用。实现抗真菌肽基因在酵母中的高效分泌型表达，对探讨利用转基因酵母的发酵液直接进行果蔬、食品和农产品防腐保鲜的生物技术研发有重要意义。【方法】将Drosomycin基因（Drs）及其同系物基因Drs-lC和Thanatin基因分别与酵母分泌型表达载体pPICZαA重组，构建成重组表达质粒pPICZαA-Drs、pPICZαA-Drs-lC和pPICZαA–Thanatin。利用电转化将重组质粒转化毕赤酵母GS115，经表型筛选和PCR鉴定获得的重组毕赤酵母转化子，在甲醇诱导下进行抗真菌肽的分泌表达。【结果】3种抗真菌肽基因的酵母表达产物对6个供试真菌中的5个有明显的抑制作用，Thanatin同时对供试细菌有抗菌活性。【结论】Drs、Drs-lC和Thanatin抗真菌肽基因成功地转化毕赤酵母，并实现其分泌型表达。     

黄粉虫幼虫抗真菌物质的诱导及其抗真菌活性研究

[目的]研究用真菌诱导黄粉虫幼虫表达抗菌物质及其对真菌的影响。[方法]用黄粉虫幼虫从土壤中诱捕真菌,培养出2种病原真菌作为诱导源分别对黄粉虫幼虫做诱导试验,每种试验分别进行对照、菌刺和菌饲3种处理,并于处理后1、2、3、4和5 d分别提取抗菌物质,用平板琼脂打孔扩散法测定其抑菌圈直径而确定其抗真菌活性。[结果]结果表明,黄粉虫幼虫受真菌诱导后抗真菌活性比对照都有不同程度的增加,同时不同真菌诱导表达样品对于相应的诱导真菌均表现明显的抗真菌活性。说明不同真菌诱导产生的抗真菌物质与诱导源有关,抗真菌物质为黄粉虫幼虫体内固有成分,诱导增加了抗真菌物质的表达量,同时刺激新的抗真菌物质的产生。[结论]用真菌直接诱导黄粉虫幼虫可以产生抗真菌物质。