Antifungal activity of extracellular metabolites produced by Sporothrix flocculosa

The antifungal properties of extracellular compounds produced by the epiphytic fungus Sporothrix flocculosa were bioassayed against phytopathogenie fungi on the basis of inhibition of spore germination, and mycelial growth and induction of cellular leakage. Following incubation in stationary culture, S. flocculosa released antifungal metabolites into the culture medium which were extractable with méthylene chloride. When separated by thin layer chromatography, extracted metabolites yielded a compound(s) at R_f0.65 which inhibited development of Cladosporium cucumerinum and several other phytopathogenic fungi. Treatment of Botrytis cinerea and Fusarium oxysporum f.sp. radicis‐lycopersici (FORL) with the same compound(s) greatly reduced spore germination and biomass growth of both fungi. Additionally, both B. cinerea and FORL leaked electrolytes and proteins when grown in presence of the metabolites. Observations under electron microscopy revealed that FORL reacted to the presence of S. flocculosa metabolites by retraction of the plasmalemma and rapid disintegration of the cytoplasm. These reactions were similar to the ones induced by conidia of S. flocculosa when applied on powdery mildew fungi. These results provide strong evidence of the production of antifungal compounds in vivo and of their role in the antagonistic properties of S. flocculosa.     

Novel fungitoxicity assays for inhibition of germination-associated adhesion of Botrytis cinerea and Puccinia recondita spores

Botrytis cinerea and Puccinia recondita spores adhere strongly to polystyrene microtiter plates coincident with germination. We developed assays for inhibition of spore adhesion in 96-well microtiter plates by using sulforhodamine B staining to quantify the adherent spores. In both organisms, fungicides that inhibited germination strongly inhibited spore adhesion, with 50% effective concentrations (EC(50)s) comparable to those for inhibition of germination. In contrast, fungicides that acted after germination in B. cinerea inhibited spore adhesion to microtiter plates only at concentrations much higher than their EC(50)s for inhibition of mycelial growth. Similarly, in P. recondita the ergosterol biosynthesis inhibitors myclobutanil and fenbuconazole acted after germination and did not inhibit spore adhesion. The assays provide a rapid, high-throughput alternative to traditional spore germination assays and may be applicable to other fungi.     

Evaluation of the effects of biological preparations on phytopathogenic fungi Didymella applanata and Botrytis cinerea

Fungicidal and fungistatic effects of biological preparations involving bacteria of the genera Pseudomonas and Bacillus and fungi of the genus Chaetomium on phytopathogenic fungi Didymella applanata and Botrytis cinerea were evalauated. All the biological preparations under study inhibited the growth of colonies of the fungi; however, the degree of the inhibition depended on the nature of each particular microorganism and the concentration of each particular preparation. The preparation containing Bacillus subtilis at a concentration of 0.2% effected maximum suppression of B. cinerea (the diameter of the colonies decreased sevenfold). The preparations containing bacteria of the genus Pseudomonas and fungi of the genus Chaetomium were most efficient in suppressing D. applanata. The preparations containing B. subtilis and Chaetomium spp. showed promise as agents against simultaneous development of spur blight and Botrytis blight.     

Mycoparasitism of Acremonium strictum BCP on Botrytis cinerea, the gray mold pathogen

A fungal strain BCP, which parasitizes Botrytis cinerea gray mold pathogen, was isolated and identified as Acremonium strictum. BCP strain overgrew the colonies of B. cinerea and caused severe lysis of the host hyphae. Frequent penetration and hyphal growth of A. strictum BCP inside the mycelia of B. cinerea were observed under light microscopy. In addition, some morphological abnormalities such as granulation and vacuolation of the cytoplasm were observed in mycelia and spores of B. cinerea. In dual culture test, A. strictum BCP strongly inhibited the mycelial growth of several plant pathogenic fungi as well as B. cinerea. To our knowledge, this is the first report on mycoparasitism of Acremonium species on B. cinerea.     

357细菌及其拮抗物质对植物病原真菌的抑制

Bacillus cereus357细菌及其分泌的拮抗物质对水稻纹枯病菌和草莓灰霉病菌有较好的拮抗作用。它们可抑制或杀死这两种病原菌的菌丝,可使菌丝枯萎和产生畸形。357细菌分泌的拮抗物质还可抑制草莓灰霉病菌孢子的萌发和孢子的形成。该拮抗物质对热、酸、碱等有较高的稳定性。     

酶法制备壳寡糖对番茄灰霉病病菌抑制机理初探

目的：研究酶降解法制备的壳寡糖对番茄灰霉病病菌的抑制机理。方法：测定壳寡糖对番茄灰霉病菌菌丝发育、孢子萌发的抑制作用以及对菌丝形态、细胞膜透性和对菌丝体可溶性蛋白质含量的影响。结果：当壳寡糖处理浓度为1.5mg/mL以上时,对菌丝生长的抑制率达70%以上,EC50为0.72mg/mL。当处理浓度为6mg/mL时,对分生孢子的抑制率显著优于其他浓度水平,达87.4%,EC50为1.48mg/mL。显微观察,浓度为0.8mg/mL的壳寡糖处理可诱导菌丝分枝增多、菌体分隔增加,分生孢子形成受到抑制。此外壳寡糖能够引起菌丝细胞膜透性发生变化,造成菌丝体内含物的渗漏。壳寡糖处理66 h后,菌丝体可溶性蛋白含量比对照降低了36.7%。结论：壳寡糖对番茄灰霉病病菌抑制机理的探究为研究壳寡糖这一新型生物农药的作用机制提供依据。     

Bacillus cereus B-02对Botrytis cinerea 拮抗机理的研究

采用电子显微镜(扫描、透射)和激光扫描共聚焦显微镜,从细胞形态学和生理学水平上研究蜡样芽孢杆菌Bacillus cereus B-02过滤液对灰葡萄孢菌Botrytis cinerea的拈抗机理.结果表明,处理菌丝表面形态受到严重破坏,发牛强烈变形;荫丝细胞核、线粒体和细胞壁等哑细胞结构发生了明显改变,细胞内出现大量无膜透明内含物,并产生较人液泡.此外,处理菌丝DNA、线粒体膜电位和活性氧荧光强度均低于对照组,且差异极显著;说明B-02菌株对病原真菌菌丝细胞DNA的合成、线粒体膜电位和活性氧水平有重要影响.     

Bacillus cereus B-02对Botrytis cinerea拮抗机理的研究

采用电子显微镜(扫描、透射)和激光扫描共聚焦显微镜,从细胞形态学和生理学水平上研究蜡样芽孢杆菌Bacillus cereus B-02过滤液对灰葡萄孢菌Botrytis cinerea的拮抗机理。结果表明,处理菌丝表面形态受到严重破坏,发生强烈变形;菌丝细胞核、线粒体和细胞壁等亚细胞结构发生了明显改变,细胞内出现大量无膜透明内含物,并产生较大液泡。此外,处理菌丝DNA、线粒体膜电位和活性氧荧光强度均低于对照组,且差异极显著;说明B-02菌株对病原真菌菌丝细胞DNA的合成、线粒体膜电位和活性氧水平有重要影响。     

Antagonistic effect of Erwinia herbicola on in vitro spore germination and germ tube elongation of Botrytis cinerea and Penicillium expansum

Interaction between Erwinia herbicola and Botrytis cinerea and Penicillium expansum was studied in liquid culture. The results show that the bacteria directly inhibited spore germination of both fungi, especially during the first hours of the paired cultivation. The distinct taxis of bacteria to spores and germ tubes was frequently followed by their lysis. It is likely that bacteria act also by competition for nutrients. The rate of antagonistic activity of the bacteria against both fungi depended on their concentration in the mixture. Formation of chlamydospore-like structures at the apical end of B. cinerea germ tube suggests induction of a defence mechanism of this fungus while in unfavourable conditions.     

Biological Control of Phytopathogenic Fungi by Fatty Acids

The aim of the present study was to evaluate the antifungal activity of fatty acids against phytopathogenic fungi. Two pot experiments were conducted by mixing palmitic and oleic acids in the soil in which poor plant growth was observed. In addition, the antifungal activities of nine fatty acids (butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, oleic acid, and linoleic acid) against four phytopathogenic fungi: Alternaria solani, Colletotrichum lagenarium, Fusarium oxysporum f. sp. Cucumerinum, and Fusarium oxysporum f. sp. lycopersici, were assessed by measuring mycelial growth and spore germination via Petri dish assay. The results of the pot experiments showed that the mixture of palmitic and oleic acids enhanced the growth of the seedlings of continuous-tomato and continuous-cucumber. Except for oleic acid, in the Petri dish assay, the fatty acids tested were observed to inhibit the mycelial growth of one or more tested fungi. In addition to the suppression of mycelial growth, butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, and palmitic acid showed an inhibitory effect against spore germination and the extent of inhibition varied with both the type of fatty acids, and the fungi. In particular, capric acid displayed strong inhibitory effect against C. lagenarium on the mycelial growth and spore germination. The saturated fatty acids, i.e. palmitic acids, showed stronger antifungal activity than the unsaturated fatty acids, i.e. oleic acid. It suggests that fatty acids might be applicable to exploring for alternative approaches to integrated control of phytopathogens.     

Study of antifungal activities of seven essential oils from some Iranian medicinal plants against various postharvest phytopathogenic fungi

The aim of this study was to find the antifungal activities of seven essential oils from some Iranian medicinal plants that have maximum (100%) inhibition effect on the mycelium growth of postharvest phytopathogenic fungi. Among 20 examined species belonging to three families, only 7 species could stop the mycelium growth of phytopathogenic fungi. The selected plants include Trachyspermum ammi, Zataria multiflora Boiss., Satureia hortensis, Caryophillum aromaticus, Menthe piperita, Cuminum cyminum L. and Carum carvi, and fungi include Aspergillus flavus, Botrytis cinerea, Penicillium italicum, Penicillium expansum, Penicillium commune, Rhizopus stolonifer and Rhizopus lyococcus. The results showed that the essential oil of these plants could stop the mycelium growth at 500 ppm, but could not completely inhibit the spore germination, however reduced the spore germination to 80–90%. Among the fungi Rhizopus stolonifer and Rhizopus lyococcus are more resistant to the inhibition effects of essential oils. Among the plants, Cuminum cyminum L. and Carum carvi were slightly weaker than other plants. Also except for Cuminum cyminum L. and Carum carvi, the essential oils of other plants had fungicide effect while these two plants in most cases had fugistatic effect. The results showed that these essential oils can be used as an effective alternative control method.     

枯草芽孢杆菌JA脂肽类及挥发性物质抑菌效应的研究

枯草芽孢杆菌JA产生的脂肽类抗生素对植物病原真菌有广谱抗性.将发酵液经过酸沉淀、甲醇抽提以及反相高效液相色谱等步骤,分离得到脂肽类抗生素的纯品.经IC50实验和抗菌谱测定,考察了脂肽类抗生素对多种植物病原菌的作用,确定了脂肽类抗生素的抗菌谱.深入研究表明,枯草芽孢杆菌JA还产生未知成分的挥发性抑菌物质,能够抑制灰霉病菌孢子的萌发和菌丝的生长.脂肽类抗生素和挥发性抑菌物质的协同作用,有助于提高枯草芽孢杆菌的生物防治效果.     

Disruption of the Eng18B ENGase gene in the fungal biocontrol agent Trichoderma atroviride affects growth, conidiation and antagonistic ability

The recently identified phylogenetic subgroup B5 of fungal glycoside hydrolase family 18 genes encodes enzymes with mannosyl glycoprotein endo-N-acetyl-β-D-glucosaminidase (ENGase)-type activity. Intracellular ENGase activity is associated with the endoplasmic reticulum associated protein degradation pathway (ERAD) of misfolded glycoproteins, although the biological relevance in filamentous fungi is not known. Trichoderma atroviride is a mycoparasitic fungus that is used for biological control of plant pathogenic fungi. The present work is a functional study of the T. atroviride B5-group gene Eng18B, with emphasis on its role in fungal growth and antagonism. A homology model of T. atroviride Eng18B structure predicts a typical glycoside hydrolase family 18 (αβ)(8) barrel architecture. Gene expression analysis shows that Eng18B is induced in dual cultures with the fungal plant pathogens Botrytis cinerea and Rhizoctonia solani, although a basal expression is observed in all growth conditions tested. Eng18B disruption strains had significantly reduced growth rates but higher conidiation rates compared to the wild-type strain. However, growth rates on abiotic stress media were significantly higher in Eng18B disruption strains compared to the wild-type strain. No difference in spore germination, germ-tube morphology or in hyphal branching was detected. Disruption strains produced less biomass in liquid cultures than the wild-type strain when grown with chitin as the sole carbon source. In addition, we determined that Eng18B is required for the antagonistic ability of T. atroviride against the grey mould fungus B. cinerea in dual cultures and that this reduction in antagonistic ability is partly connected to a secreted factor. The phenotypes were recovered by re-introduction of an intact Eng18B gene fragment in mutant strains. A putative role of Eng18B ENGase activity in the endoplasmic reticulum associated protein degradation pathway of endogenous glycoproteins in T. atroviride is discussed in relation to the observed phenotypes.     

Morphogenic effects of ramihyphin A in filamentous fungi

Ramihyphin A at subfungistatio concentrations stimulates ramification of hyphae of filamentous fungi. Stimulation of terminal ramification of hyphae that can be observed particularly in phytopathogenic fungi is most frequent. Hyphae ofMicrosporon canis, Trichophyton mentagrophytes, Blastomyces dermatitidis, Coccidioides immitis andHistoplasma capsulatum ramify intensively laterally. Stimulation of the lateral ramification was observed inMonilia fructigena, Penicillium marneffei andPenicillium chrysogenum. The antibiotic induces also formation of vesicular structures in phytopathogens. Due to the substantial ramification of hyphae, both terminal and lateral, the growth of colonies is interrupted. The addition of the antibiotic to a growing colony ofBotrytis cinerea induces dichotomic ramification of terminal hyphae after 3 h of growth. Lateral hyphae begin to grow later and further ramify dichotomically. Dense bundles of ramified hyphae are formed after 24 h due to the unbalanced ramification and the colony no longer increases its size.     

Ethylene requirement during germination of Botrytis cinerea spores

Application of 2,5-norbornadiene, a competitive inhibitor of ethylene, effectively inhibited the germination of Botrytis cinerea Pers. ex Fr. spores. The transfer of spores from 2,5-norbornadiene to air relieved inhibition by norbornadiene, indicating that its effects are non-toxic and reversible. Ethephon (2-chloroethylophosphonic acid), which stimulates spore germination of B. cinerea , does not affect germination in the presence of norbornadiene. However, ethephon appeared to be effective in relieving inhibition, when norbornadiene was removed from the atmosphere surrounding spores. The addition of ethylene to an atmosphere enriched with norbornadiene, counteracted the inhibition of spore germination. The inhibition of spore germination by 2,5-norbornadiene and the reversal of this effect by ethephon or ethylene, indicate that the action of ethylene is indispensable for germination of B. cinerea spores.     

Antifungal activity of Tagetes patula extracts on some phytopathogenic fungi: ultrastructural evidence on Pythium ultimum

Methanol extract, obtained from Tagetes patula plant, was assayed against three phytopathogenic fungi: Botrytis cinerea, Fusarium moniliforme and Pythium ultimum. The antifungal activity was tested both in the dark and in the light, using two different lighting systems. The data showed that the extract proved to have a dose-dependent activity on all the fungi with a marked difference between treatments in the light than in the dark. Good growth inhibition was observed in fungi only when these were treated with the highest dose of the extract and irradiated, whereas the same dose gave only a modest inhibition when the experiment was conducted in the dark. At 5 and 10 microg/ml in the dark, growth increased. The results indicated that the presence of a luminous source enhances the antifungal activity, with small differences between UV-A and solar spectrum light. SEM and TEM observations on Pythium ultimum revealed that the Tagetes patula extract induced alterations on cell fungal membranes with a photoactivation mechanism possibly involving the production of free radicals and leading to a premature aging of the mycelium.     

Production of lytic enzymes by<Emphasis Type="Italic">Trichoderma</Emphasis> spp. and their effect on the growth of phytopathogenic fungi

The production of β-1,3-glucanases and chitinases by three strains ofTrichoderma in submerged cultures was determined. The synthesis of enzymes was induced by cell wall biopolymers of phytopathogenic fungi (Botrytis cinerea, Fusarium culmorum andF. oxysporum). T. hamatum produced the highest β-1,3-glucanase activity; the most effective inducer of enzyme synthesis was the biomass ofF. oxysporum. All examined strains ofTrichoderma inhibited phytopathogen growth in biotic tests. The diffusion tests showed that the lytic enzymes take part in growth inhibition of phytopathogenic fungi.     

戊唑醇对小麦纹枯菌超微结构的影响

用光镜和电镜技术研究了新型三唑类杀菌剂戊唑醇对小麦纹枯病菌发育的组织学和超微结构的影响.光镜和电镜观察发现戊唑醇处理导致病菌发生一系列变化菌丝呈现念珠状异常膨大和缢缩,菌丝原生质体内液泡和电子致密体增多,菌丝细胞壁不规则加厚,部分菌丝的隔膜发育受阻成畸形,不能形成正常的桶孔隔膜和桶孔覆垫.有些菌丝原生质体内出现菌丝内套菌丝的现象.这些变化最终导致菌丝细胞解体死亡.     

Characterisation of acetaldehyde effects on Rhizopus stolonifer and Botrytis cinerea

Acetaldehyde (AA) vapour reduced the in vitro growth of the fruit-infecting fungi Botrytis cinerea and Rhizopus stolonifer, and decreased the respiration of R. stolonifer. Spore germination, germ tube elongation and sporulation were inhibited in both fungi. AA induced the leakage of electrolytes from B. cinerea and R. stolonifer membranes. The conductivity found in the fungal extracts was positively correlated with the AA concentration applied. The leakage of reducing sugars and amino acids suggest that cell membranes are irreversibly disrupted by AA as a first step in inhibition of the fungi in vitro.     

鄂西北产苍耳子甲醇粗提物抑菌活性及其清除DPPH能力的初步评价

采用生长速率法、孢子萌发法及DPPH自由基清除法,对产自鄂西北的野生植物苍耳子粗提物体外抑菌活性及抗氧化性进行了初步测定.结果表明,苍耳子甲醇粗提物对绿色木霉、黄瓜灰霉菌、黑曲霉、终极腐霉、尖镰孢菌黄瓜专化型五种病原真菌均有一定的抑制作用,其中无论是抑制菌丝生长还是孢子萌发,均对黑曲霉显示出了显著的抑制活性.实验结果也...