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.     

Chemical constituents of Stereum subtomentosum and two other birch-associated basidiomycetes: an interspecies comparative study

Metabolites of the wood-rotting fungus Stereum subtomentosum Pouzar (Basidiomycetes, order Russulales, family Stereaceae) occurring on birch (Betula pendula Roth) trees were phytochemically investigated for the first time. Three main metabolite chemotypes present in MeOH extracts of the fruit bodies, viz. steroids, fatty acids, and water-soluble sugars, were fractionated, isolated, and identified by 1D/2D NMR-spectroscopic analyses, NMR data comparisons, and chemical correlations combined with GC/MS experiments. Thirteen compounds including two 5 alpha,8 alpha-epidioxy steroids, alpha,alpha'-trehalose, D-arabinitol, D-mannitol, and saturated/unsaturated fatty acids, were identified. Differences among S. subtomentosum and two other birch-associated fungal species, Trametes versicolor (L.: Fr.) Pilát, and Piptoporus betulinus (Bull.: Fr.) P. Karst (Basidiomycetes, order Polyporales, family Polyporaceae) were evaluated as regards the richness and abundance relationships in metabolite profiling.     

Isolation of Microbial Antagonists for Biocontrol of Grey Mould Disease of Strawberries

A screening programme is described for the assessment of the potential of biocontrol agents to control grey mould of strawberries caused by Botrytis cinerea. Bacteria were isolated from strawberry fruits, leaves and flowers from a commercial field site and screened for antagonism towards B. cinerea using two in vitro and one in vivo screening techniques. From 559 microorganisms isolated, 108 inhibited pathogen growth on agar plates and 27 of these prevented spore germination on Cellophane membranes. The ability of these 27 isolates to inhibit infection of young strawberry leaves by B. cinerea on whole plants under glass was then tested. Seven isolates reduced grey mould development and were subsequently assessed in a field trial. Two isolates, one of Bacillus pumilus and one of Pseudomonas fluorescens, were as effective or more effective than standard dichlofluanid sprays and may therefore be of potential value as antagonists of B. cinerea.     

Trypsin-like proteinases and trypsin inhibitors in fruiting bodies of higher fungi

The activity of trypsin-like proteinases and trypsin inhibitors was measured in fruiting bodies of various species of basidial fungi (Basidiomycetes). Fruiting bodies of all fungi contained these enzymes, with the exceptions of polypore (Coriolus versicolor (Fr.) Karst) and hedgehog fungus (Hericium erinaceus (Fr.) Quel), belonging to the families Polyporaceae and Hericiaceae, respectively, in which the enzyme activities were barely detectable. The activity of trypsin-like proteinases was the highest in fruiting bodies of Boletaceae and Agaricaceae. Fruiting bodies of all fungi contained trypsin inhibitors. The highest activity of trypsin inhibitors was detected in basidiomycetes of the families Boletaceae, Agaricaceae, and Pleurotaceae, including Boletus castanus (Fr.) Karst, orange-cap boletus (Leccinum aurantiacum (Fr.) Sing), and brown-cap boletus (Leccinum melanum (Fr.) Karst).     

吉林省担子菌补记(六)

报道了6个吉林省新记录担子菌,即青绿湿伞[Hygrocybepsittacina(Schaeff.:Fr.)W櫣ｎｓｃｈｅ],梭柄金钱菌[Collybiafusipes (Bull.:Fr.)Qu啨ｌ.],黄褐盔孢菌[Galerinahelvoliceps(Berk .etCurt.)Sing .],内鬼笔(EndophallusyunnanensisZangetPetersen) ,粉托鬼笔[Phallushadriani(Vent.)Pers.]和蓖齿地星(GeastrumpectinatumPers .)。其中,内鬼笔属为吉林省新记录属,青绿湿伞、梭柄金钱菌、内鬼笔和篦齿地星为首次在我国东北地区发现。标本存放在吉林农业大学标本馆(HMJAU)内。     

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.     

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.     

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.     

番茄灰霉病生防链霉菌筛选及鉴定

【背景】由灰葡萄孢侵染所致的番茄灰霉病是一类重要的真菌病害,生物防治具有环境友好、病原菌不易产生抗药性等特点,是果蔬灰霉病绿色防控的有效措施。【目的】筛选对番茄灰霉病具有防病作用且能促进番茄种子发芽的广谱拮抗性链霉菌,并明确该菌株种级分类地位。【方法】采用琼脂块法筛选拮抗番茄灰霉病菌的链霉菌菌株,采用对峙培养法和生长速率法检测菌株T22抑菌谱,通过产胞外酶活性检测、离体叶片防效和种子发芽试验明确该菌株的防病促生相关特性,根据形态学特征、生理生化特性和分子生物学方法对该菌株进行种类鉴定。【结果】从分离的56株放线菌中筛选到14株对番茄灰霉病菌具有拮抗效果的放线菌菌株,其中链霉菌T22对番茄灰霉病菌抑制作用最强,且具有较广抑菌谱,同时菌株T22具有产生纤维素酶和几丁质酶的能力。菌株T22无菌发酵滤液对番茄灰霉病菌、桃褐腐病菌、黄瓜枯萎病菌抑菌率分别为84.6%、81.5%和79.1%;其无菌发酵滤液原液对番茄灰霉病离体防效为55.1%;100倍稀释液处理番茄种子,胚轴、胚根和种子活力指数分别增加15.1%、29.7%和43.9%。根据形态学特征、生理生化特性和多基因聚类分析将链霉菌T22鉴定为白黑链霉菌(Streptomycesalboniger)。【结论】白黑链霉菌T22具有较强的抗真菌、产胞外酶、防病和促生活性,在番茄灰霉病生物防治中具有较好的开发应用潜力。     

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。     

Trypsin-Like Proteinases and Trypsin Inhibitors in Fruiting Bodies of Higher Fungi

The activity of trypsin-like proteinases and trypsin inhibitors was measured in fruiting bodies of various species of basidial fungi (Basidiomycetes). Fruiting bodies of all fungi contained these enzymes, with the exceptions of polypore (Coriolus versicolor (Fr.) Karst) and hedgehog fungus (Hericium erinaceus (Fr.) Quel), belonging to the families Polyporaceae and Hericiaceae, respectively, in which the enzyme activities were barely detectable. The activity of trypsin-like proteinases was the highest in fruiting bodies of Boletaceae and Agaricaceae. Fruiting bodies of all fungi contained trypsin inhibitors. The highest activity of trypsin inhibitors was detected in basidiomycetes of the families Boletaceae, Agaricaceae, and Pleurotaceae, including Boletus castanus (Fr.) Karst, orange-cap boletus (Leccinum aurantiacum (Fr.) Sing), and brown-cap boletus (Leccinum melanum (Fr.) Karst).     

Epicoccum nigrum for biocontrol agents in vitro of plant fungal pathogens

Epicoccum nigrum strains were evaluated in vitro as potential biological agents for control of the growth of Fusarium avenaceum, F. graminearum, F. oxysporum and Botrytis cinerea. Individual biotic effects of five strains of E. nigrum on the various fungi were determined using the biotic series method elaborated for fungi, on potato dextrose agar (PDA - Difco) medium. Our results show that E. nigrum strains limited the growth of all isolates of Fusarium spp., but not of those of Botrytis cinerea.     

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.     

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.     

Screening of micro-organisms for decolorization of melanins produced by bluestain fungi

A total of 17 fungi and four bacteria were screened for their ability to decolorize melanin, using isolated extracellular melanin of the bluestain fungus Aureobasidium pullulans as substrate. On agar media, decolorization was observed by four fungal strains: Bjerkandera adusta VTT-D-99746, Galactomyces geotrichum VTT-D-84228, Trametes hirsuta VTT-D-95443 and Trametes versicolor VTT-D-99747. The four fungi were more efficient on nitrogen-limited medium than on complete medium. The melanin-decolorizing activity of G. geotrichum appeared to be located on the mycelium and could be liberated into the medium enzymatically.     

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.     

The importance of inoculum size for the competitive ability of wood decomposing fungi

Abstract Competition among wood decay fungi was studied with a technique using sectors of various sizes of 8 cm diameter wood discs. The sectors representing 8–92% of the discs were precolonized by Heterobasidion annosum (Fr.) Bref., Resinicium bicolor (Alb. & Schw. ex Fr.) Parm, Phanerochaete sanguinea (Fr.) Hjortstam and Coniophora sp. DC. ex Me'rat before they were combined pairwise in close contact on top of water agar in 9 cm Petri dishes. Discs were regularly inspected for mycelial overgrowth and after 10 weeks mycelia were reisolated. Competitive success, measured as the replacement of the opposing fungus, was generally greatest for mycelia with sectors representing 92% of a disc and smallest for 8% sectors. R bicolor was the most, and H. annosum the least, competitive of the species investigated. The results indicate that mycelial size could be one major factor influencing the competitive success in nature. However, when paired on nutrient agar, the results of the interactions between the four species did not correspond to those in wood discs     

Mode of antagonism of Brevibacillus brevis against Botrytis cinerea in vitro

AIMS: To assess the activity of Brevibacillus brevis (formerly Bacillus brevis) Nagano and the antibiotic it produces, gramicidin S, against the plant pathogen Botrytis cinerea. METHODS AND RESULTS: Germination and growth of Bot. cinerea were assessed in the presence of B. brevis or gramicidin S in liquid media, on solid media and on leaf sections of Chinese cabbage. Germination was 10-fold more sensitive to gramicidin S than growth. Inhibition of Bot. cinerea was greater in liquid media compared with on solid media. Activity of gramicidin S against Bot. cinerea on leaf sections was much lower than in vitro. In vitro inhibition of Bot. cinerea by B. brevis Nagano was similar to equivalent levels of gramicidin. CONCLUSIONS: Antibiosis, via gramicidin S, is the mode of antagonism exhibited by B. brevis Nagano against Bot. cinerea in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: The mode of antagonism of B. brevis against Bot. cinerea was elucidated. The differing activity of gramicidin S against Bot. Cinerea in vitro and on leaf sections indicates one mechanism by which biocontrol activity may differ between laboratory and field conditions.     

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.     

In vitro attachment of phylloplane yeasts to Botrytis cinerea, Rhizoctonia solani, and Sclerotinia homoeocarpa

The ability of yeasts to attach to hyphae or conidia of phytopathogenic fungi has been speculated to contribute to biocontrol activity on plant surfaces. Attachment of phylloplane yeasts to Botrytis cinerea, Rhizoctonia solani, and Sclerotinia homoeocarpa was determined using in vitro attachment assays. Yeasts were incubated for 2 d on potato dextrose agar (PDA) prior to experimentation. A total of 292 yeasts cultured on PDA were screened for their ability to attach to conidia of B. cinerea; 260 isolates (89.1%) attached to conidia forming large aggregates of cells, and 22 isolates (7.5%) weakly attached to conidia with 1 or 2 yeast cells attached to a few conidia. Ten yeasts (3.4%), including 8 isolates of Cryptococcus laurentii, 1 isolate of Cryptococcus flavescens, and an unidentified species of Cryptococcus, failed to attach to conidia. All non-attaching yeasts produced copious extracellular polysaccharide (EPS) on PDA. Seventeen yeast isolates did not attach to hyphal fragments of B. cinerea, R. solani, and S. homoeocarpa after a 1 h incubation, but attachment was observed after 24 h. Culture medium, but not culture age, significantly affected the attachment of yeast cells to conidia of B. cinerea. The 10 yeast isolates that did not attach to conidia when grown on agar did attach to conidia (20%-57% of conidia with attached yeast cells) when cultured in liquid medium. Attachment of the biocontrol yeast Rhodotorula glutinis PM4 to conidia of B. cinerea was significantly greater at 1 x 10(7) yeast cells x mL(-1) than at lower concentrations of yeast cells. The ability of yeast cells to attach to fungal conidia or hyphae appears to be a common phenotype among phylloplane yeasts.