Structure and Expression In planta of Botrytis cinerea Ubiquitin Genes

To identify genes of the necrotrophic pathogenic fungus Botrytis cinerea that are expressed during infection of tomato leaves, a differential screening of a genomic library with radioactively labelled cDNA was performed. This resulted in the identification of a B. cinerea gene, denominated Bcubi4, which encodes a precursor protein consisting of four identical head-to-tail repeats of a 76aa ubiquitin unit. Subsequently a gene denominated Bcubi1CEP79, encoding a single ubiquitin unit joined to a Carboxyl Extension Protein of 79 amino acids, was isolated. The expression of the two ubiquitin genes was studied during pathogenesis of B. cinerea on tomato. Bcubi1CEP79, but not Bcubi4, mRNA was transiently induced at 16h after inoculation. The increased expression of the Bcubi1CEP79 gene at this stage of pathogenesis might be required for enhanced ribosomal biogenesis.     

球孢白僵菌定殖提高番茄对灰霉病抗性及其作用机理

为明确球孢白僵菌Beauveria bassiana定殖对番茄植株抗病性的影响及作用机理,采用灌根法将球孢白僵菌芽生孢子悬浮液接种于番茄植株内,并通过人工接种灰霉菌Botrytis cinerea评价球孢白僵菌定殖后番茄对灰霉病的抗性水平;检测灰霉菌胁迫下番茄植株不同位置叶片内球孢白僵菌的相对含量;测定番茄叶片内草酸氧化酶（oxalate oxidase,OXO）、几丁质酶（chitinase,CHI）和ATP合成酶（ATP synthase,atpA）3种抗病相关基因的表达量。结果表明,球孢白僵菌定殖能够提高番茄植株对灰霉病的抗性,接种灰霉菌第5天,番茄植株发病率、病斑直径和病情指数分别下降了61.6%、41.4%和26.4%;在灰霉菌胁迫下番茄植株内球孢白僵菌偏好于在病原菌感染位置定向聚集,并且引起植物抗病基因OXO、CHI和atpA的表达量上调。表明球孢白僵菌能通过内生定殖与植物互作提高植物抗病性,在植物病害生物防治领域有较大应用潜力。     

植物内生枯草芽孢杆菌Em7菌株对葡萄灰霉病菌的抑菌活性

通过室内皿内对峙抑菌试验、分生孢子萌发抑制试验、离体果实接种试验以及电镜技术,研究测定了分离自小麦根部的植物内生枯草芽孢杆菌Em7菌液对葡萄灰霉病菌Botrytiscinerea Pers.的抑制作用及抑菌机理。结果表明:用Em7菌液处理葡萄灰霉病菌后,在PDA培养基上形成了明显的抑菌圈,直径达2.81 cm;菌液对分生孢子萌发的抑制率达到88.65%;经Em7菌液处理后,离体果实病情指数明显低于空白对照,相对防治效果达到78.92%。电镜观察发现,处理组菌丝生长异常,体表凹凸不平,局部膨大成结或缢缩,分枝变多,菌丝体内液泡增多,细胞壁增厚,细胞膜透性发生变化。表明植物内生枯草芽孢杆菌Em7菌株对葡萄灰霉病菌有良好的抑制作用,并且可以有效控制葡萄灰霉病的发生。     

<Emphasis Type="Italic">Botrytis cinerea</Emphasis> induces senescence and is inhibited by autoregulated expression of the <Emphasis Type="Italic">IPT</Emphasis> gene

Botrytis cinerea is a non-specific, necrotrophic pathogen that attacks many plant species, including Arabidopsis and tomato. Since senescing leaves are particularly susceptible to infection by B. cinerea, we hypothesized that the fungus might induce senescence as part of its mode of action and that delaying leaf senescence might reduce the severity of B. cinerea infections. To examine these hypotheses, we followed the expression of Arabidopsis SAG12, a senescence-specific gene, upon infection with B. cinerea. Expression of SAG12 is induced by B. cinerea infection, indicating that B. cinerea induces senescence. The promoter of SAG12, as well as that of a second Arabidopsis senescence-associated gene, SAG13, whose expression is not specific to senescence, were previously analyzed in tomato plants and were found to be expressed in a similar manner in the two species. These promoters were previously used in tomato plants to drive the expression of isopentenyl transferase (IPT) from Agrobacterium to suppress leaf senescence (Swartzberg et al. in Plant Biology 8:579–586, 2006). In this study, we examined the expression of these promoters following infection of tomato plants with B. cinerea. Both promoters exhibit high expression levels upon B. cinerea infection of non-senescing leaves of tomato plants, supporting our conclusion that B. cinerea induces senescence as part of its mode of action. In contrast to B. cinerea, Trichoderma harzianum T39, a saprophytic fungus that is used as a biocontrol agent against B. cinerea, induces expression of SAG13 only. Expression of IPT, under the control of the SAG12 and SAG13 promoters in response to infection with B. cinerea resulted in suppression of B. cinerea-induced disease symptoms, substantiating our prediction that delaying leaf senescence might reduce susceptibility to B. cinerea. Contribution from the Agriculture Research Organization, The Volcani Center, Bet Dagan, Israel, No. 127/2006 series.     

玉米纹枯病菌全长cDNA文库的构建与鉴定

 以玉米纹枯病菌菌丝为材料,利用In-Fusion SMARTer^TM cDNA Lib Construction Kit构建玉米纹枯病菌全长cDNA文库。文库质量鉴定结果表明,文库滴度为1.2×10⁶pfu·mL^-1,重组率为99.2%,插入片段平均长度大于1.0 kb。随机挑取400个白色克隆进行测序,共获得329个高质量EST序列,经聚类拼接后得到250条unique EST,包括36条contigs和214条singletons。在GenBank 进行Blastn 与Blastx 同源比对,有227条EST 与已知核酸或蛋白有不同程度的同源性,占全部EST 的90.8%,其余23条无任何同源性,占9.2%。     

Subcelullar localization of proteins associated with <Emphasis Type="Italic">Prune dwarf virus</Emphasis> replication

The fungus Colletotrichum lindemuthianum is the causal agent of anthracnose, one of the most severe diseases of the common bean (Phaseolus vulgaris). The infection process begins with the adhesion of conidia to the plant’s surface. Appressoria are then formed, allowing penetration of the fungus. Next, the biotrophic phase begins, followed by the necrotrophic phase. Due to the peculiar nutrition mode of the fungus, including both of the previously mentioned stages, it is of great interest to determine which genes are involved in the transition between the two phases during the infection process. To determine this, suppression subtractive hybridization (SSH) was used in association with qRT-PCR in the present study. These methods allowed for the identification of genes that were differentially expressed at each developmental stage of the fungus in the plant. This is the first report on the use of the cited techniques to evaluate the infectious cycle of the fungus. A total of 175 sequences exhibited significant identity (e?≤?10^?5) with sequences present in the sequenced genomes of P. vulgaris and C. lindemuthianum; approximately 41 % of those were determined to belong to the fungus, and 59 % were determined to belong to the plant. Of the predicted sequences, 68 % were of unknown function or were not found in the databases. Among the analyzed expressed sequence tags (ESTs), sequences were found that encode proteins related to: primary and secondary metabolism; the transport of different compounds; the degradation/modification of proteins; cell regulation and signaling; cellular stress response; and the degradation of exogenous compounds. The obtained results allowed for the identification of sequences encoding proteins that are essential for the progression of anthracnose. Furthermore, it was possible to identify new genes, the functions of which have not yet been described, and even to identify unique genes of C. lindemuthianum that are involved in the pathogenicity and virulence of this fungus.     

Nested PCR‐RFLP is a high‐speed method to detect fungicide‐resistant Botrytis cinerea at an early growth stage of grapes

BACKGROUND: Grey mould caused by the fungus Botrytis cinerea Pers. ex Fr. is one of the major diseases in grapes. The use of fungicides is a simple strategy to protect grapes against B. cinerea disease. However, phenotypes exhibiting resistance to fungicides have been detected in B. cinerea populations. The variation of fungicide‐resistant B. cinerea isolates renders B. cinerea disease control difficult in grapevine fields. RESULTS: The authors have developed a nested polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) method to detect fungicide‐resistant B. cinerea isolates at an early growth stage of grapes in grapevine fields. The nested PCR‐RFLP method was carried out to detect benzimidazole‐, phenylcarbamate‐ and/or dicarboximide‐resistant B. cinerea isolates from grape berries and leaves at Eichorn–Lorenz growth stage 25 to 29. This method successfully detected fungicide‐resistant B. cinerea isolates at an early growth stage of grapes. In addition, only 8 h was required from tissue sampling to phenotyping of fungicide resistance of the isolates. CONCLUSION: It is proposed that the early diagnosis of fungicide‐resistant B. cinerea isolates would contribute to further improvement of integrated pest management against B. cinerea in grapevine fields, and that the nested PCR‐RFLP method is a high‐speed, sensitive and reliable tool for this purpose. Copyright © 2008 Society of Chemical Industry     

A Polymerase Chain Reaction (PCR) Assay for the Detection of Inoculum of Sclerotinia sclerotiorum

The development of a polymerase chain reaction (PCR) assay for the detection of inoculum of the plant pathogenic fungus Sclerotinia sclerotiorum is described. The PCR primers were designed using nuclear ribosomal DNA internal transcribed spacer sequences. Specific detection of DNA from S. sclerotiorum was possible even in the presence of a 40-fold excess of DNA from the closely related fungus Botrytis cinerea. PCR products were obtained from suspensions of untreated S. sclerotiorum ascospores alone, but DNA purification was required for detection in the presence of large numbers of B. cinerea conidiospores. Specific detection of inoculum of S. sclerotiorum was possible in field-based air-samples, using a Burkard spore trap, and from inoculated oilseed rape petals. The assay has potential for incorporation into a risk management system for S. sclerotiorum in oilseed rape crops.     

Resistance to Botrytis cinerea in Solanum lycopersicoides is Dominant in Hybrids with Tomato, and Involves induced Hyphal Death

Botrytis cinerea causes gray mold disease and affects hundreds of plant species, including tomato (Lycopersicon esculentum). The wild nightshade, Solanum lycopersicoides, is cross compatible with tomato and is more resistant to B. cinerea, thus representing a potential source for crop improvement. Tests involving droplet inoculation of detached leaves and spray inoculation of entire seedlings demonstrated that resistance to B. cinerea varies among S. lycopersicoides accessions, with S. lycopersicoides LA2951 being the most resistant accession tested. Expression of resistance in the intergeneric hybrid (L. esculentum cv. 'VF36' × S. lycopersicoides LA2951) suggested that resistance is at least partially dominant in tomato. A green fluorescent protein-tagged B. cinerea strain was used for confocal microscopic comparison of infection in leaves of S. lycopersicoides and tomato. Even though S. lycopersicoides supported spore germination, there was evidence for hyphal lysis and death 3 days after inoculation, at a time when lesions were expanding on susceptible tomato plants. The reduced frequency of B. cinerea lesion spread on S. lycopersicoides explains why this fungus produced fewer spores in this wild nightshade than in tomato.     

The Effect of Treatment With Ulocladium Atrum on Botrytis Cinerea-attack of Geranium (Pelargonium Zonale) Stock Plants and Cuttings

In two successive seasons, the effect of treatment of geranium stock plants with the competitive saprophytic fungus Ulocladium atrum as a biocontrol agent against Botrytis cinerea was compared to a fungicide treatment with Euparene M. B. cinerea incidence and severity on the stock plants, B. cinerea spore load in the air around stock plants and death of cuttings due to B. cinerea were scored. B. cinerea incidence and severity were much stronger in the second than the first experiment. This was quantitatively expressed by higher numbers of conidia of B. cinerea monitored in the second than the first year, both on necrotic (a maximum for the control of 27.5 × 10⁶ spores per sample - all necrotic leaves of five plants - in experiment 1 against 86 × 10⁶ in experiment 2) and green leaves, but numbers of conidia of B. cinerea recovered from the air were only slightly different. The death rate of cuttings was moderate in the first and extremely high in the second experiment. For the fungicide treatment, maximum sample values of 7% and 76% of 6-week old cuttings were killed in the first and the second experiment respectively. Treatment with U. atrum was effective in reducing all parameters studied. With the exception of the spore load of B. cinerea in the air and the success of cuttings, the effect of U. atrum varied from as good as the fungicide to half as effective. In the first trial, only Euparene M reduced spore load in the air, in the second trial only U. atrum consistently did so. In the first trial U. atrum reduced death of 4-week old cuttings, though less than fungicide (1.2, 20 and 38% killed with fungicide treatment, U. atrum treatment and control respectively). In the second trial only the fungicide reduced loss of cuttings. The impact of the data on the integration of U. atrum in a control system of B. cinerea in geranium is discussed.     

冬青卫矛内生放线菌YDG17菌株发酵液抑菌活性研究

从冬青卫矛植株中筛选得到一株内生链霉菌YDG17,其发酵液对多种植物病原真菌有较强的抑制作用。室内生物测定结果表明,YDG17菌株发酵液对供试的11种植物病原真菌菌丝生长均有一定的抑制作用,其中对小麦赤霉病菌Fusarium graminearum、番茄灰霉病菌Fulvia fulvum和番茄早疫病菌Alternaria solani的抑制作用最强,EC50值分别为259.98、336.13和100.72 mg/L;对供试的3种植物病原真菌孢子萌发也均有一定的抑制作用,其中对番茄灰霉病菌孢子萌发的抑制作用最强,EC50值为87.84 mg/L。离体子叶法测定结果表明,发酵液原液对番茄灰霉病的保护效果为97.62%,治疗效果为79.63%。盆栽试验结果表明,发酵液原液对番茄灰霉病的保护效果为71.34%,治疗效果为64.23%。捷克八溶剂系统纸色谱测定结果表明,该发酵液的主要活性物质为碱性水溶性抗生素。选用弱酸性离子交换树脂吸附法对YDG17发酵液活性成分进行了初步分离,并对其活性馏分进行ESI-MS/MS分析,表明其活性物质主要为链丝菌素类化合物,该类化合物为首次从植物内生菌中分离得到。     

海洋生境贝莱斯芽孢杆菌TCS001的鉴定及抑真菌活性

为研究海洋生境芽孢杆菌TCS001的分类地位和抑菌活性,通过形态和生理生化特征观察,并结合gyrA序列同源性分析对菌株进行了鉴定;通过平板对峙培养法测定了菌株TCS001对多种植物病原真菌的抑菌谱;采用菌丝生长速率法和凹玻片法,测定了不同浓度TCS001菌株发酵滤液对靶标菌黄瓜灰霉病菌Botrytis cinerea菌丝生长和孢子萌发的影响。结果显示:该菌株为贝莱斯芽孢杆菌Bacillus velezensis,其对6种供试病原菌均有一定的抑制效果,其中对黄瓜灰霉病菌的抑制率最高,达87.66%。不同稀释倍数下,TCS001发酵滤液对黄瓜灰霉病菌均有一定的抑制作用,其中,稀释5倍时对菌丝生长和孢子萌发的抑制率最高,分别为96.24%和98.05%,稀释20倍时抑制率也均达90%以上。形态学观察发现,TCS001发酵滤液可导致黄瓜灰霉病菌孢子萌发芽管中间或顶端膨大畸形。研究表明,海洋生境贝莱斯芽孢杆菌TCS001极具开发为微生物农药的潜能。     

Partial stem and leaf resistance against the fungal pathogen <Emphasis Type="Italic">Botrytis cinerea</Emphasis> in wild relatives of tomato

Tomato (Solanum lycopersicum) is one of many greenhouse crops that can be infected by the necrotrophic ascomycete Botrytis cinerea. Commercial cultivation of tomato is hampered by the lack of resistance. Quantitative resistance has been reported in wild tomato relatives, mostly based on leaf assays. We aimed to identify wild tomato relatives with resistance to B. cinerea based on quantitative assays both on leaves and stem segments, monitoring infection frequency and disease expansion rate as parameters. A quantitative tomato stem segment assay was developed. This stem assay and a previously described leaf assay were used to screen a collection of 22 Solanum accessions. Significant differences in disease parameters were observed among accessions. Resistance to B. cinerea was observed in a number of wild Solanum species, including accessions of S. chilense, S. habrochaites and S. neorickii, both in the leaf assay and the stem segment assay. A number of resistant and susceptible accessions were evaluated as adult plants under greenhouse conditions. The data obtained in greenhouse assays confirmed the leaf and stem disease data. The expression of several defence-related genes was studied in a subset of accessions. There was no apparent correlation between the expression levels of the genes tested and the quantitative resistance level to B. cinerea. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Mode of action of the anilino-pyrimidine fungicide pyrimethanil. 2. Effects on enzyme secretion in Botrytis cinerea

The effect of pyrimethanil on the levels of cell wall degrading enzymes secreted by Botrytis cinerea Pers. was investigated in diseased plant tissues and in liquid B. cinerea cultures. Total proteinase activity isolated from infected carrot slices which were treated with 5.0 μM pyrimethanil was decreased by 76%, 3 d after inoculation. Polygalacturonase, cellulase, proteinase and laccase activities were all decreased in the medium of three day-old cultures grown in the presence of pyrimethanil. The pyrimethanil concentrations resulting in 50% reduction in total enzyme activities (IC₅₀) were approximately 0.25 μM for polygalacturonase, cellulase and proteinase, and approximately 1.0 μM for laccase. No significant growth inhibition was observed at these pyrimethanil concentrations. Pyrimethanil did not inhibit the enzymes directly, nor did it inhibit the synthesis of cytosolic proteins. Therefore, it was proposed that the fungicide inhibits protein secretion at a post-translational stage in the secretory pathway. Large differences were found in the effects of pyrimethanil on the growth of B. cinerea in liquid cultures and on agar plates, depending on the composition of the medium. In liquid media containing cellulose and protein as carbon and nitrogen sources, growth inhibition occurred at 5.0 μM pyrimethanil, whilst no growth inhibition was observed with 50 μM pyrimethanil in malt extract. Similarly, growth occurred on potato/dextrose agar (PDA) at 0.5 μM pyrimethanil, but no growth was seen at this concentration on agars containing cellulose and protein. Thus it appears that pyrimethanil is most active in media where the fungus has to utilise extracellular enzymes to mobilise the nutrients it requires for growth.     

Genetic and phenotypic characterization of Botrytis calthae

Botrytis calthae is a necrotrophic plant pathogen, closely related to the ubiquitous broad host range fungus Botrytis cinerea, but highly host specific. Botrytis isolates from lesions of Caltha palustris grown at different locations were classified with genetic markers as either B. calthae or Botrytis pseudocinerea, or less frequently as B. cinerea. A PCR‐based identification of B. calthae was developed. Seven haplotypes of B. calthae could be distinguished. Compared to B. cinerea, mycelium growth of B. calthae was similar, but conidiation less abundant, and sclerotia formation was only partially repressed by light. Conidia of B. calthae germinated more slowly, and showed a highly acidic optimum (pH 2·5) compared to B. cinerea conidia (pH 5·3). All B. calthae isolates were sensitive to common anti‐Botrytis fungicides, but showed partial resistance to the succinate dehydrogenase inhibitors boscalid, fluopyram and carboxin. Infection experiments revealed a weak capability of B. calthae to induce necrotic lesions on plants that are hosts for B. cinerea. On C. palustris leaves, B. calthae induced similar lesions to B. cinerea. These data provide a basis for comparative molecular investigation of the physiology and host specificity of B. calthae and closely related Botrytis species.     

产香真菌GS-1菌株鉴定及其挥发性物质对番茄灰霉病的生防效果

为鉴定产香真菌GS-1菌株物种分类和评价其挥发性物质对番茄灰霉病的生防效果,通过形态学和ITS(internal transcribed spacer)序列分析对菌株GS-1进行物种鉴定,以对扣法测定其挥发物对番茄灰霉病菌Botrytis cinerea、水稻胡麻斑病菌Bipolaris oryzae、小麦赤霉病菌Fusarium graminearum、白菜黑斑病菌Alternaria brassicae和莴苣菌核病菌Sclerotinia sclerotiorum共5种植物病原真菌的熏蒸抑制活性,以果实针刺法测定挥发物对番茄灰霉病的抑制作用,并观察对其菌丝生长的影响。结果显示,菌株GS-1为普通瘤座孢Tubercularia vulgaris;其挥发物对5种植物病原真菌均有明显的熏蒸抑制作用,72 h的抑制率为58.05%~75.81%;其挥发物可造成番茄灰霉病菌菌丝畸形、分枝增多、原生质外渗等,对番茄果实灰霉病的防效72 h后达53.62%。表明产香真菌普通瘤座孢GS-1菌株挥发物对番茄灰霉病有良好的生防效果。     

Resistance profiles of Botrytis cinerea populations to several fungicide classes on greenhouse tomato and strawberry in Lebanon

Tomato and strawberry are the most important protected crops in Lebanon and are seriously affected by grey mould disease, caused by Botrytis cinerea. In the present study, the fungicide sensitivity assays revealed medium to high frequencies of B. cinerea isolates resistant to benzimidazoles, dicarboximides, and anilinopyrimidines on tomato and strawberry. Fludioxonil- and boscalid-resistant mutants were uncommonly found at generally low frequency on both crops. Resistance to fenhexamid was detected in only one site on tomato but in most sites on strawberry with high frequencies, and the occurrence of resistance to QoI fungicides was ascertained on both crops. The majority of the tested isolates (>90%) exhibited multiple fungicide resistance, and isolates resistant to the seven antibotrydial fungicide classes were detected on strawberry in three locations. A high level of resistance was shown by B. cinerea mutants resistant to boscalid, fenhexamid, and QoI fungicides, while two levels of moderate and high resistance to anilinopyrimidines were identified. Genetic analysis revealed point mutations in the target genes commonly associated with resistance in B. cinerea isolates, with all mutants resistant to dicarboximides, fenhexamid, boscalid, and QoI fungicides carrying single-nucleotide polymorphims in BcOS1 (I365S/N, Q369P, and N373S), Erg27 (F412V/I), SdhB (H272R/Y), and cytb (G143A) genes, respectively. The general incorrect use of fungicides has caused the development and spread of fungicide resistance as a widespread phenomenon on protected tomato and strawberry in Lebanon. The implementation of appropriate antiresistance strategies is highly recommended.