枯草芽孢杆菌对几种灰霉病菌的抑制效果研究

分别研究了枯草芽孢杆菌(BacillussubtilisCohn)培养液、过滤液和灭活液对葡萄灰霉病菌(GB)、草莓灰霉病菌(SB)、辣椒灰霉病菌(PB)和番茄灰霉病菌(TB)菌丝生长的抑制作用。结果表明:枯草芽孢杆菌培养液对GB、SB、PB和TB都有很好的抑制作用。在菌液浓度达到10 5CFU/mL时,对4种灰霉病菌的抑制率均达到了10 0 % ;当浓度降低为10 4CFU/mL时,抑制率明显降抵。而菌液浓度为10 8CFU/mL时的过滤液,对GB、PB和TB的抑制率也均在5 0 %以上。灭活液对灰霉菌的抑制作用明显减弱,菌液浓度为10 8CFU/mL时,对PB、GB、TB和SB的抑制率分别为73.6 %、39.5 %、5 0 %和2 5 %。     

In vitro and in vivo antimicrobial activity of Xenorhabdus bovienii YL002 against Phytophthora capsici and Botrytis cinerea

Aims: Developing new bio‐agents to control plant disease is desirable. Entomopathogenic bacteria Xenorhabdus spp. have potential antimicrobial activity in agriculture. This work was conducted to evaluate the antimicrobial activity of Xenorhabdus bovienii YL002 on plant pathogenic fungi and oomycete in vitro and the efficiency of this strain to reduce the in vivo incidence of grey mould rot on tomato plants caused by Botrytis cinerea and leaf scorch on pepper plants caused by Phytophthora capsici. Methods and Results: The antimicrobial activity of X. bovienii YL002 was firstly determined on in vitro plant pathogenic fungi and oomycete and then on tomato fruits and plants infected with B. cinerea and pepper plants infected with P. capsici. The cell‐free filtrate of X. bovienii YL002 exhibited highest inhibition effects (>98%) on mycelia growth of P. capsici and B. cinerea. The 50% inhibition concentration (EC₅₀) of the methanol‐extracted bioactive compounds (methanol extract) of the cell‐free filtrate against P. capsici and B. cinerea were 164·83 and 42·16 μg ml^?1. The methanol extract also had a strong effect on the spore germination of P. capsici and B. cinerea, with a EC₅₀ of 70·38 and 69·33 μg ml^?1, respectively. At 1000 μg ml^?1, the methanol extract showed a therapeutic effect of 70·82% and a protective effect of 77·4% against B. cinerea on tomato plants compared with the control. The methanol extract also showed potent effect against P. capsici, with a therapeutic effect of 68·14% and a protective effect of 65·46% on pepper plants compared with the control. Conclusions: Xenorhabdus bovienii YL002 produces antimicrobial compounds with strong activity on plant pathogenic fungi and oomycete and has the potential for controlling grey mould rot of tomato plants and leaf scorch of pepper and could be useful in integrated control against diverse plant pathogenic fungi and oomycete. Significance and Impact of the Study: This study showed the potential that X. bovienii YL002 can be used to control the grey mould rot caused by B. cinerea on tomato plants and leaf scorch caused by P. capsici on pepper plants with the objective to reduce treatments with chemical fungicides.     

灰葡萄孢交配型基因的分析与检测

通过生物信息对灰葡萄孢的MAT1‐1‐1与MAT1‐2‐1氨基酸序列进行了系统进化与结构域保守氨基酸分析,表明灰葡萄孢的交配型蛋白与核盘菌的亲源关系最近,结构域氨基酸比对结果表明该基因具有保守氨基酸的一致性与部分氨基酸的相似性。应用PCR技术检测灰葡萄孢交配型基因MAT1‐1‐1与MAT1‐2‐1,结果表明各种植区交配型菌株所占比例有较大的差异,多数种植区灰葡萄孢同时存在MAT1‐1与MAT1‐2两种交配类型,快速检测灰葡萄孢的交配型等位基因对于灰葡萄孢种群结构分析非常有意义。     

Potential use and mode of action of the new strain Bacillus thuringiensis UM96 for the biological control of the grey mould phytopathogen Botrytis cinerea

The potential use of Bacillus thuringiensis UM96 as a biocontrol agent for the grey mould phytopathogen Botrytis cinerea was evaluated. In order to dissect the mode of action of this UM96 strain, we also examined the role of lytic activities in the antagonism. First, B. thuringiensis UM96 was characterised based on 16S rRNA and gyrA gene sequencing and phenotypic traits. Petri dish biocontrol assays demonstrated that when strain UM96 was inoculated 24 h previous to B. cinerea, the mycelial growth was inhibited by up to 70%. Test for lytic enzymes activities of cellulase and glucanase was negative. Chitinase was the only positive enzyme activity in two different culture media. PCR detection of the chiB gene was also positive. Chitinolytic supernatants, obtained from rich and minimal media supplemented with colloidal chitin as the sole carbon source, from B. thuringiensis UM96 showed a strong inhibitory effect of B. cinerea that was not observed with heat-treated supernatant. Interestingly, when the supernatant was supplemented with 100 µM allosamidin, a chitinase specific inhibitor, the antagonistic activity was suppressed significantly. A lack of chitinase activity was also observed in allosamidin-treated supernatants. Our pathogenic B. cinerea strain also exhibited susceptibility to pure Streptomyces griseus chitinase. Finally, the chitinolytic strain B. thuringiensis UM96 was able to protect Medicago truncatula plants in vitro from B. cinerea infection and significantly reduced the necrotic zones and root browning of the plants. Together, these results suggest a potential use of B. thuringiensis UM96 for the biological control of B. cinerea and a role for chitinases during the antagonism displayed.     

Brief in vitro study on Botrytis cinerea and Aspergillus carbonarius regarding growth and ochratoxin A

Aims: To evaluate the effect of Botrytis cinerea growth on ochratoxin A (OTA) production by Aspergillus carbonarius and degradation. Methods and Results: OTA‐producing A. carbonarius and B. cinerea were grown on grape‐like medium at 20°C for 7 days. Radii of colonies were daily recorded and OTA was analysed. In addition, each B. cinerea isolate was inoculated on grape‐like synthetic nutrient medium (SNM) paired with each A. carbonarius isolate at a distance of 45 mm. Botrytis cinerea isolates were also grown in OTA‐spiked SNM. Growth rates of B. cinerea and A. carbonarius were 20 and 7·5 mm day^?1, respectively. The growth of the colonies of each species stopped when they contacted each other in paired cultures. OTA production by A. carbonarius in the contact area was affected by B. cinerea, but no clear trend was observed. All B. cinerea isolates showed to degrade between 24·2% and 26·7% of OTA from spiked SNM. Conclusions: The ecological advantage of B. cinerea, in terms of growth rate, vs. OTA‐producing Aspergillus in some wine‐growing regions and its ability to degrade OTA may explain the low levels of this toxin in noble wines. Significance and Impact of the Study: At determinate conditions, the presence of B. cinerea in grapes with A. carbonarius may help in reducing OTA accumulation.     

抗灰葡萄孢霉菌乳酸菌的筛选

从80株乳酸菌中筛选出45株具有抗灰葡萄孢霉菌活性的乳酸菌菌株,其中10株具有较强抗灰葡萄孢霉菌活性。对这10株乳酸菌菌株的抗植物致病真菌谱进行了研究,这10株乳酸菌对番茄早疫病菌,甜瓜疫霉菌,甜瓜枯萎病菌,苹果炭疽病菌的生长均有较强的抑制作用。其中1株具有广谱抗植物致病真菌活性的乳酸菌菌株BX6-4为植物乳杆菌。经番茄离体叶片接种试验发现,植物乳杆菌BX6-4的发酵液能够在体外强烈地抑制灰葡萄孢霉菌的生长。     

2-epi-Botcinin A and 3-O-Acetylbotcineric Acid from Botrytis cinerea

Two metabolites, 2-epi-botcinin A and 3-O-acetylbotcineric acid, were isolated from Botrytis cinerea (AEM211). The former compound was new, and the latter was known but structurally revised by us. In a test for antifungal activity against Magnaporthe grisea, a pathogen of rice blast disease, 2-epi-botcinin A was 8 times less active than botcinin A (MIC 100 μM), and the MIC value for 3-O-acetylbotcineric acid being 100 μM.     

产漆酶灰色葡萄孢霉培养条件的研究

通过对培养灰色葡萄孢霉（Botrytis cinerea）不同培养基产漆酶酶活力大小的比较,筛选出了产漆酶灰色葡萄孢霉的最佳培养方法。结果表明：灰色葡萄孢霉在蛋白胨5g/L,酵母提取物20 g/L,蔗糖20 g/L,MgSO4 1.5 g/L,CuSO4 0.006 g/L的培养基中生长最好。最佳的培养条件为：pH值4.9,温度25℃,转速100 r/min。     

An immunological study of the induction of polygalacturonases in Botrytis cinerea

Abstract There was no consistent increase with time in the total activity of extracellular polygalacturonase (PG), relative to mycelial dry weight, when Botrytis cinerea was grown on a modified Czapek Dox liquid medium containing galacturonic acid or glucose. However, preparative isoelectric focusing identified a peak of activity between pH 3.7 and 6.3 only when the fungus was grown in the presence of galacturonic acid. This activity was attributed to the induction of exo-PGs (exo-PGI and/or exo-PGII). Antisera raised against one of two endo-PGs (endo-PGI) reacted positively and specifically with endo-PGI and endo-PGII on Western blots, and both of these isozymes were present in Western blots when the fungus was grown in the presence of glucose or galacturonic acid after 48 h in vitro. Plate-trapped antigen-ELISA confirmed that the endo-PGs were constitutively expressed.     

番茄灰霉病害及其微生物防治的研究进展

综述了番茄灰霉病的病害,并从国内外拮抗菌以及内生菌的筛选和利用等方面概述了番茄灰霉病微生物防治的研究进展,提出了目前番茄灰霉病微生物防治的问题及今后的应用前景。     

Differential Behaviour of Mycelial Growth of Several Botrytis cinerea Strains on either Patchoulol- or Globulol-amended Media

Botrydial and dihidrobotrydial are two characteristic metabolites of the phytopathogenic fungus Botrytis cinerea , which are involved in the development of necrotic lesions on grapevine and tobacco. Patchoulol and globulol, two natural products which are analogues to precursors of botrydial and dihidrobotrydial, were tested on 10 B. cinerea strains which were isolated from different hosts and varied in aggressiveness on grapevine leaves. Mycelial growth of all strains was prevented when they were grown on either patchoulol- or globulol-amended malt agar media (200  μ g/ μ l). Each strain displayed a specific response pattern to those products, according to the high variability previously described for this species. Furthermore, strains were different from one another with regard to their level of aggressiveness against leaves detached from sherry grapevine vineyards.     

Genetic and Phenotypic Diversity among Botrytis cinerea Isolates in Iran

Forty-four Botrytis cinerea isolates from different hosts and geographical regions were studied for colony morphology, mycelial growth rate at different temperatures, pathogenicity and molecular diversity. Botrytis cinerea isolates had temperature optima of 20–25°C and isolates showed variation in growth rate at different temperatures. Two morphological types were identified among tested isolates: mycelial and sclerotial. The pathogenicity of isolates was tested on grapevine leaves, and it was revealed that nine of 44 isolates were non-pathogenic and among them seven were of mycelial type. There was no correlation between mycelium growth rate and pathogenicity. Genetic diversity was investigated using nine arbitrary decaprimers. No relationship was found between molecular clusters and geographical region or sampling time; whereas isolates from the same plant host tended to cluster with each other. Seven of nine non-pathogenic isolates were separated from pathogenic ones.     

Hexanoic acid protects tomato plants against Botrytis cinerea by priming defence responses and reducing oxidative stress

Treatment with the resistance priming inducer hexanoic acid (Hx) protects tomato plants from Botrytis cinerea by activating defence responses. To investigate the molecular mechanisms underlying hexanoic acid‐induced resistance (Hx‐IR), we compared the expression profiles of three different conditions: Botrytis‐infected plants (Inf), Hx‐treated plants (Hx) and Hx‐treated + infected plants (Hx+Inf). The microarray analysis at 24 h post‐inoculation showed that Hx and Hx+Inf plants exhibited the differential expression and priming of many Botrytis‐induced genes. Interestingly, we found that the activation by Hx of other genes was not altered by the fungus at this time point. These genes may be considered to be specific targets of the Hx priming effect and may help to elucidate its mechanisms of action. It is noteworthy that, in Hx and Hx+Inf plants, there was up‐regulation of proteinase inhibitor genes, DNA‐binding factors, enzymes involved in plant hormone signalling and synthesis, and, remarkably, the genes involved in oxidative stress. Given the relevance of the oxidative burst occurring in plant–pathogen interactions, the effect of Hx on this process was studied in depth. We showed by specific staining that reactive oxygen species (ROS) accumulation in Hx+Inf plants was reduced and more restricted around infection sites. In addition, these plants showed higher ratios of reduced to oxidized glutathione and ascorbate, and normal levels of antioxidant activities. The results obtained indicate that Hx protects tomato plants from B. cinerea by regulating and priming Botrytis‐specific and non‐specific genes, preventing the harmful effects of oxidative stress produced by infection.     

番茄几种挥发性组分对番茄灰葡萄孢的抑制作用

采用孢子悬滴培养法,测定番茄几种挥发性组分对番茄灰葡萄孢(Botrytiscinerea)孢子萌发及菌丝生长的抑制作用。结果表明各挥发性组分对灰葡萄孢均有抑制作用,抑制率的大小依次为挥发醛、芳香族化合物和萜类化合物。10μmol/L的(E)-2-己烯醛和壬烯醛可完全抑制孢子萌发和菌丝生长,4μmol/L的丁子香酚和10~20μmol/L的水杨酸甲酯使病菌菌丝异常生长。本研究为番茄挥发性物质在其抗病防御反应中的作用提供理论依据。