灰葡萄孢犬尿氨酸途径关键酶基因的鉴定

【背景】灰葡萄孢是一种重要的植物病原真菌,实验室前期明确了灰葡萄孢犬尿氨酸单加氧酶(kynurenine3-monooxygenase,KMO)基因BcKMO参与调控病菌的生长发育和致病力。犬尿氨酸单加氧酶(KMO)是犬尿氨酸途径的关键酶,但灰葡萄孢是否存在犬尿氨酸途径及其在病菌生长、发育和致病过程中的功能尚未见相关报道。【目的】鉴定灰葡萄孢犬尿氨酸途径中的关键酶基因,确定灰葡萄孢犬尿氨酸途径的存在,为阐明灰葡萄孢生长发育和致病力的分子机理奠定基础。【方法】利用生物信息学方法,对灰葡萄孢犬尿氨酸途径中犬尿氨酸酶(kynureninase,KYN)、吲哚-2,3-双加氧酶(indoleamine-2,3-dioxygenase,IDO)、犬尿氨酸氨基转移酶(kynurenine amino transferase,KAT)的编码基因进行分析;利用Real-time PCR技术,检测灰葡萄孢野生型BC22、BcKMO基因T-DNA插入突变体BCG183、恢复菌株BCG183/BcKMO中犬尿氨酸途径关键酶基因的表达水平;利用真菌犬尿氨酸酶KYN检测试剂盒,测定BcKMO突变体中犬尿氨酸酶(KYN)的含量。【结果】灰葡萄孢中含有2个犬尿氨酸氨基转移酶(KAT)的编码基因、3个吲哚-2,3-双加氧酶(IDO)的编码基因、10个犬尿氨酸氨基转移酶(KAT)的编码基因。灰葡萄孢KYN编码基因、IDO编码基因、KAT编码基因在突变体BCG183中的表达水平显著高于或低于在野生型和恢复菌株。突变体BCG183中犬尿氨酸酶(KYN)的含量显著低于野生型BC22和恢复菌株。【结论】灰葡萄孢中存在犬尿氨酸途径,灰葡萄孢BcKMO基因突变影响KYN、IDO和KAT编码基因的表达以及犬尿氨酸酶(KYN)的含量。     

Different products for biological control of Botrytis cinerea examined on wounded stem tissue of tomato plants

For the moment the agents that are used against Botrytis cinerea, in glasshouses were tomatoes are cultivated, are from chemical origin. For reducing the use of chemical agents in the future it is important to search for effective biological control agents against the fight of Botrytis cinerae. The following biological products Vital pasta, Vital gel and Elot-Vis were examined in there possibility to control Botrytis cinerea. Elot Vis was tested out in experiments that were carried out in climate chambers were leafs of 3 week old tomato plants were artificially infected with Botrytis cinerea spores. Also the biological products of Vital were first investigated in experiments that were carried out in climate chambers. In stead off leafs of tomato plants it were stem wounds of tomato plants who were treated with the pasta or the gel that was spread over the wounded surface after this has been inoculated with a suspension of conidia of Botrytis cinerae. The results of these first tests that were executed in the climate chambers were the circumstances for Botrytis cinerea were ideal seemed promising. In the next step these products were tested out on large scale in glasshouses. For each plant 5 wounds were created by removing the leafs, these wounds were or first treated with the Biological product and thereafter artificially infected with Botrytis cinerea spores to check out if these products can be used as a preventive agent or the wounds were first inoculated with a suspension of Botrytis cinerea spores and thereafter treated with the product. For the product Elot-Vis a few plants were totally sprayed with an Elot-Vis suspension before leafs were removed and the wounds were inoculated with conidia of Botrytis cinerea to check out if this product was able to activated the induced systemic resistance pathway. The experiments that were executed in glasshouses showed different percentages of succeeded Botrytis cinerea infections. This is probable due to the different weather conditions during both days that the experiments were executed. For the wounds that were treated with pasta it was difficult to distinguish wounds were Botrytis cinerea succeeded to infect the plant, because these wounds frequently didn't show any sign of infection on the surface but when the wounds with the pasta were cut open it was possible to see Botrytis cinerea infections inside the stem.     

生防细菌CNY-04筛选、鉴定及其对灰霉病菌的防效

【目的】为研究土壤细菌对蔬菜灰霉病的生防价值, 从辽宁、山东等地区的蔬菜种植基地采集土壤样本56份, 分离、筛选出对灰霉病具有稳定拮抗作用的细菌9株。【方法】采用平板对峙培养法进行初筛、复筛, 用抑菌圈法测定其抑菌效果, 并进行离体果实试验验证其对蔬菜灰霉病的防治效果, 通过形态学特征、生理生化特征及16S rRNA基因序列分析研究其分类地位。【结果】细菌CNY-04对蔬菜灰霉病的拮抗能力最强且遗传稳定, 抑菌圈直径达到34 mm; 初步鉴定该菌株为格氏沙雷菌(Serratia grimesii), 尚未见该菌在生防上的报道; CNY-04液体菌剂对离体番茄果实灰霉病的防效为69.23%, 50%多菌灵防效为75.39%, 24 h时接种CNY-04处理的番茄发病率为40.0%, 而48 h时接种处理的发病率为51.1%。【结论】CNY-04是一株较为理想的拮抗菌, 丰富了生防资源。     

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。     

农杆菌介导的灰葡萄孢T-DNA插入突变体库构建及插入位点分析

【目的】利用农杆菌(Agrobacterium tumefaciens)介导法对灰葡萄孢(Botrytis cinerea)进行转化,构建T-DNA插入突变体库,为从分子水平上认识灰葡萄孢的致病机制打下基础。【方法】以含有pCAMBIA 1390双元载体的农杆菌对灰葡萄孢进行转化,利用潮霉素进行筛选。对抗性稳定的转化子进行生物学和形态学观察,采用离体番茄叶片进行致病性测定。利用TAIL-PCR技术对突变体中T-DNA的旁侧序列进行克隆。【结果】得到了一些突变体,表现为生长速率减缓、产孢能力下降、致病力减弱等。克隆并分析了其中一个突变体中T-DNA插入的位置和旁侧序列。【结论】本实验建立了农杆菌介导的灰葡萄孢转化体系,构建了T-DNA插入的灰葡萄孢突变体库。用TAIL-PCR进行突变体中T-DNA旁侧序列的分析是可行的。     

Biocontrol agents of Botrytis cinerea tested in climate chambers by making artificial infection on tomato leafs

To reduce the use of chemical agents, that are causing damage to the environment, in the fight against Botrytis cinerea, different BCA's were tested for their possibility to control Botrytis cinerea in a biological way. In order to investigate the effectiveness of the different micro organisms and Elot-Vis, experiments were carried out in climate chambers with 5 weeks old tomato plants. Leafs on the plant were inoculated with drops of a suspension that contained spores of Botrytis cinerea. The possible antagonists that were tested in these experiments were Trichoderma harzianum (Trichodex), T. asperellum (Biofungus), T. hamatum (T382), Bacillus subtilis (Serenade and Phytovit) and Pseudomonas aeruginosa (7NSK2 and KMPCH). For all these different micro organisms the direct and the indirect influence on Botrytis cinerea was investigated. In tests where the direct influence of the antagonists was examined, the spores of the moulds or the bacteria were suspended together with spores of Botrytis cinerea and subsequently drops of this suspension were pippeted on the leafs. After a while by ideal circumstances for Botrytis cinerea the infections on the inoculated leafs were counted. For the indirect influence of the antagonists, also leafs of 5 weeks old tomato plants were inoculated with a suspension of Botrytis cinerea spores. The roots of the tomato plants that were used for testing the indirect influence were treated during there growth with a suspension of the antagonist to see if induced systemic resistance pathway (ISR) was activated. For testing the effectiveness of Elot-Vis, tomato plants were sprayed a few times with a solution of this product during their growth. Results of the climate chamber test of the plants that were treated with Elot-Vis, showed a reduction of Botrytis cinerea infections on the inoculated leafs. Biological control agents seem to be not always very effective against Botrytis cinerea. The biological control agents that are containing micro organisms are also depending on the circumstances of the environment for an optimal development. These conditions are not always that optimal to compete with Botrytis cinerea or other micro organisms that are present on the plant.     

Antifungal activity of valinomycin, a peptide antibiotic produced by Streptomyces sp. Strain M10 antagonistic to Botrytis cinerea

A strain of Streptomyces sp. (M10) antagonistic to Botrytis cinerea was isolated from orchard soil obtained from Jeju Island, Korea. An antifungal substance (CN1) was purified from the culture extracts of the strain, and then identified as valinomycin through extensive spectroscopic analyses. Valinomycin showed potent in vitro antifungal activity against Botrytis cinerea and also in vivo control efficacy against Botrytis blight development in cucumber plants. Overall, the disease control efficacy of valinomycin was similar to that of vinclozolin, a commercial fungicide. This study provides the first report on the disease control efficacy of valinomycin against Botrytis blight.     

根癌农杆菌介导的灰葡萄孢菌遗传转化研究

以pCAMBIA1300-N载体为骨架, 成功构建了以绿色荧光蛋白(gfp)为报告基因, 潮霉素(hph)为抗性筛选标记的载体pKPG, 并利用根癌农杆菌介导转化系统, 成功获得了能表达绿色荧光蛋白的重组灰葡萄孢菌。通过PCR检测转化子的绿色荧光蛋白基因和潮霉素抗性表达框, 观察菌丝和分生孢子的荧光表型, 以及gfp基因的Southern杂交验证, 结果表明：被测转化子基因组中均成功整合了目的基因片段。     

Direct fungicidal activities of C6-aldehydes are important constituents for defense responses in Arabidopsis against Botrytis cinerea

C6-aldehydes, such as (Z)-3-hexenal, (E)-2-hexenal, and n-hexanal, are volatile compounds formed by hydroperoxide lyase (HPL) and found in most terrestrial plants. They are fungicidal and bactericidal compounds, and are also signaling compounds to induce defense responses in plants. Transgenic plants having overexpressed or suppressed HPL activity (SH or ASH, respectively) showed lower or higher susceptibility against a necrotrophic fungal pathogen, Botrytis cinerea. In this study, we examined whether the modulated susceptibility was accountable to the direct fungicidal activity or to the signaling potency of C6-aldehydes. When wild-type Arabidopsis leaves were inoculated with B. cinerea, HPL expression was upregulated, and concomitantly, the amounts of C6-aldehydes increased. Higher amounts of C6-aldehydes found in inoculated SH plants inhibited growth of B. cinerea in vitro, while lower amounts found in ASH plants caused no inhibitory effect on the fungi. Thus, it was suggested that direct fungicidal activity of C6-aldehydes accounted for the modulated susceptibility. With SH plants higher amounts of camalexin could be found, but with the ASH plants no difference from wild-type plants could be found. Surplus amounts of C6-aldehydes could induce formation of camalexin as signaling compounds; however, this was not the case with wild-type and ASH plants. Accordingly, it could be assumed that direct fungicidal activity of C6-aldehydes were prominently responsible to the defense against B. cinerea but their signaling roles could be little responsible if any.     

Selective media for the specific isolation and enumeration of Botrytis cinerea conidia

AIMS: To develop selective media for the enumeration of Botrytis cinerea. METHODS AND RESULTS: Two new media, Botrytis Selective Medium (BSM) and Botrytis Spore Trap Medium (BSTM), were developed and compared with currently available media for the enumeration of B. cinerea conidia from the environment. The new Botrytis media proved advantageous over previous media because they were easier to prepare, had greater selectivity and allowed enumeration when a greater number of colony-forming units were present on individual plates. CONCLUSION: BSM and BSTM were shown to be suitable selective media for the enumeration of B. cinerea conidia from the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The media developed can be used to monitor the population of the pathogen B. cinerea and will allow detailed studies within the crop environments.     

Analysis of defensive responses activated by volatile allo-ocimene treatment in Arabidopsis thaliana

Since volatile allo-ocimene enhances resistance of Arabidopsis thaliana against Botrytis cinerea, we attempted to dissect the factors involved in this induced resistance. The penetration of B. cinerea hyphae into Arabidopsis epidermis and the growth of hyphae after penetration were suppressed on allo-ocimene-treated leaves. allo-Ocimene also induced lignification on cell walls and veins of the leaves. The treatment induced accumulation of antifungal substances including the Arabidopsis phytoalexin, camalexin. Induction of lignification and accumulation of camalexin elicited by B. cinerea infection on Arabidopsis leaves after treating with allo-ocimene was faster and more intense than that observed with the leaves that had not been treated with this volatile. This suggested that allo-ocimene could prime defensive responses in Arabidopsis. allo-Ocimene enhanced resistance against B. cinerea in an ethylene resistant mutant (etr1-1), a jasmonic acid resistant mutant (jar1-1) and a salicylic acid resistant mutant (npr1-1). Thus, it is suggested that a signaling pathway independent for ETR1, JAR1 and NPR1 was operative to induce the resistance. The series of responses observed after allo-ocimene-treatment was mostly similar to that observed after C6-aldehyde-treatment. The effect of C6-aldehyde-treatment has been largely accounted to the chemical reactivities of the compounds; however, from this result it can be suggested that resistance responses of Arabidopsis could be induced by the volatiles mostly independent on their reactivities and that a common signaling pathway unaffected by the reactivities of compound was activated by the volatiles.     

灰葡萄孢强除草菌株的筛选与活性物质的组分分析

灰葡萄孢产生的毒素具有较强的除草活性,开发成生物除草剂前景广阔。本试验以灰葡萄孢BC4为出发菌株,通过紫外线和化学诱变剂诱变分生孢子或菌丝体,获得了所产毒素除草活性比野生菌株显著增强的3个突变菌株。对各菌株产生的毒素提取液进行HPLC分析,发现获得的3个突变菌株中各个组分的含量均显著高于野生菌株,且遗传稳定。利用HPLC制备得到了4个组分,其中组分Ⅰ、Ⅳ对马唐和反枝苋具有很强的杀除活性,为灰葡萄孢的遗传改良及毒素源除草剂的工业化生产奠定了基础。     

Cloning and partial characterization of endopolygalacturonase genes from Botrytis cinerea

Botrytis cinerea is a plant-pathogenic fungus infecting over 200 different plant species. We use a molecular genetic approach to study the process of pectin degradation by the fungus. Recently, we described the cloning and characterization of an endopolygalacturonase (endoPG) gene from B. cinerea (Bcpg1) which is required for full virulence. Here we describe the cloning and characterization of five additional endoPG-encoding genes from B. cinerea SAS56. The identity at the amino acid level between the six endoPGs of B. cinerea varied from 34 to 73%. Phylogenetic analysis, by using a group of 35 related fungal endoPGs and as an outgroup one plant PG, resulted in the identification of five monophyletic groups of closely related proteins. The endoPG proteins from B. cinerea SAS56 could be assigned to three different monophyletic groups. DNA blot analysis revealed the presence of the complete endoPG gene family in other strains of B. cinerea, as well as in other Botrytis species. Differential gene expression of the gene family members was found in mycelium grown in liquid culture with either glucose or polygalacturonic acid as the carbon source.     

猪场沼液对蔬菜病原菌的抑制作用

用不同贮存时间的猪场沼液对7种蔬菜病原菌的抑制作用进行了研究。结果表明,新鲜猪场沼液原液对番茄灰霉病菌、番茄早疫病菌、辣椒疫病菌、黄瓜炭疽病菌和茄子灰霉病菌有较强的抑制作用,沼液贮存对番茄灰霉病菌、番茄早疫病菌和茄子灰霉病菌的抑制效果没有显著影响,但对辣椒疫病菌和黄瓜炭疽病菌的抑制效果影响显著;新鲜猪场沼液滤液对番茄灰霉病菌和茄子灰霉病菌有较强的抑制作用,沼液滤液贮存对番茄灰霉病菌和茄子灰霉病菌的抑菌效果没有显著影响。     

The symbiosis of Bacillus subtilis L-forms with Chinese cabbage seedlings inhibits conidial germination of Botrytis cinerea

AIMS: To establish whether germination of Botrytis cinerea was affected by the symbiosis of Bacillus subtilis L-form bacteria with Chinese cabbage. METHODS AND RESULTS: Germinating seeds of Chinese cabbage were co-cultivated with either L-forms of Bacillus subtilis or 5% (w/v) mannitol by soaking for 3 h. Seeds were then washed in sterile water, sown on a minimal medium and incubated in controlled conditions. L-form symbiosis was detected over a time course by ELISA. Conidial germination of Botrytis cinerea was significantly reduced on cotyledonous leaves of L-form-treated plants compared with controls. CONCLUSIONS: Symbiosis of B. subtilis L-form bacteria during seed germination of Chinese cabbage inhibits conidial germination in plants on subsequent exposure to Botrytis cinerea. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first account of plant symbiosis with L-form bacteria showing antagonism to a fungal plant pathogen. This has promising implications for the use of this L-form as a biocontrol agent.     

Botrytis fabiopsis, a new species causing chocolate spot of broad bean in central China

The current study was conducted to identify Botrytis spp. isolated from symptomatic broad bean plants grown in Hubei Province, China. Among 184 Botrytis strains, three distinct species, B. cinerea, B. fabae and a previously undescribed Botrytis sp., were identified based on morphology of colonies, sclerotia and conidia. The novel Botrytis sp. is described herein as a new species, Botrytis fabiopsis sp. nov. At 20 C B. fabiopsis grew on potato dextrose agar (PDA) at 12-13 mm d(-1), similar to B. fabae (13 mm d(-1)), but slower than B. cinerea (17-19 mm d(-1)). It formed pale gray colonies with short aerial mycelia and produced gray to black sclerotia in concentric rings on PDA. B. fabiopsis produced greater numbers of sclerotia than B. cinerea but fewer than B. fabae. Conidia produced by B. fabiopsis on broad bean leaves are hyaline to pale brown, elliptical to ovoid, wrinkled on the surface and are larger than conidia of B. fabae and B. cinerea. Phylogenetic analysis based on combined DNA sequence data of three nuclear genes (G3PDH, HSP60 and RPB2) showed that B. fabiopsis is closely related to B. galanthina, the causal agent of gray mold disease of Galanthus sp., but distantly related to B. fabae and B. cinerea. Sequence analysis of genes encoding necrosis and ethylene-inducing proteins (NEPs) indicated that B. fabiopsis is distinct from B. galanthina. Inoculation of broad bean leaves with conidia of B. fabiopsis caused typical chocolate spot symptoms with a similar disease severity to that caused by B. fabae but significantly greater than that caused by B. cinerea. This study suggests that B. fabiopsis is a new causal agent for chocolate spot of broad bean.     

DNA sequence analysis of herbarium specimens facilitates the revival of Botrytis mali, a postharvest pathogen of apple

The fungus Botrytis cinerea has been widely accepted as the species responsible for causing gray mold decay of apple, although a second species causing apple decay, B. mali, was reported in 1931. Botrytis mali was validly published in 1931, nevertheless it has always been considered a doubtful species. To study the relationship of Botrytis isolates causing gray mold on apple, DNA sequence analysis was employed. Twenty-eight Botrytis isolates consisting of 10 species were sampled, including two B. mali herbarium specimens from apple originally deposited in 1932. The DNA sequence analysis of the beta-tubulin and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) genes placed the isolates into groupings with defined species boundaries that generally reflected the morphologically based model for Botrytis classification. The B. cinerea isolates from apple and other host plants were placed in a single clade. The B. mali herbarium specimens however always fell well outside that clade. The DNA sequence analysis reported in this study support the initial work by Ruehle (1931) describing the apple pathogen B. mali as a unique species.     

Germling fusion via conidial anastomosis tubes in the grey mould Botrytis cinerea requires NADPH oxidase activity

In many filamentous ascomycete species, the early steps of colony development include fusion between germinating vegetative spores (conidial germlings). Often these fusion events are mediated by specialized hyphal structures, so-called conidial anastomosis tubes (CATs). Here, we show that germling fusion in the grey mould Botrytis cinerea is mediated by hyphal structures possessing the typical features of CATs. Formation of these structures is delayed when spores are germinating on complex media compared to growth on poor substrates. Fusion frequency is also influenced by the growth conditions of the precultures from which spores were obtained. During germination on hydrophobic plant surfaces, which induce pathogenic development, CAT formation is significantly suppressed. Screening of existing B. cinerea gene knockout mutants identified strains lacking the NADPH oxidase BcNoxA or the potential Nox regulator BcNoxR as fusion deficient, suggesting a potential role of reactive oxygen species (ROS) signalling in CAT formation and fusion.     

Bacillus isolates from the spermosphere of peas and dwarf French beans with antifungal activity against Botrytis cinerea and Pythium species

A range of isolation procedures including washing, sonication and incubation in nutrient broth were used separately and in combination to obtain potential bacterial antagonists to Botrytis cinerea and Pythium mamillatum from the testae and cotyledons of peas and dwarf French beans. Heat treatment was also used to bias this selection towards spore-forming bacteria. Ninety-two bacterial isolates were obtained, 72 of which were provisionally characterized as species of Bacillus . Four of these Bacillus isolates (B3, C1, D4 and J7) displayed distinct antagonism in vitro against Botrytis cinerea and P. mamillatum when screened using dual culture analysis. Further characterization of these antagonists using API 50CHB biochemical profiling identified isolate D4 as Bacillus polymyxa and isolates B3, C1 and J7 as strains of B. subtilis . In vitro screening techniques, using cell-free and heat-killed extracts of liquid cultures against Botrytis cinerea , demonstrated the production of antifungal compounds by these four Bacillus antagonists. With each isolate the antifungal activity was found not to be either exclusively spore-bound nor released entirely into the medium but present in both fractions. The antifungal compounds produced by these isolates were shown to be heat-stable. Their identification, production and release require further study for exploitation as biocontrol systems.