In vivo-Untersuchungen zur Entstehung und Funktion der Chlamydosporen von Botrytis cinerea Pers. am Wirt-Parasit-System Fuchsia hybrida – B. cinerea

In vivo-investigations on the formation and function of chlamydospores of Botrytis cinerea Pers. in the host-parasite-system Fuchsia hybrida–B. cinerea On naturally with Botrytis cinerea Pers. infected and artificially inoculated outdoor- and greenhouse-plants of Fuchsia hybrida the extra- and intramatrical formation of the B. cinerea- chlamydospores was investigated. The chlamydospores served 1. as structures of survival, which were tested with regard to their tolerance of drought, nutrient- and oxygen-deficiency, attack by bacteria and pH-requirements. 2. The chlamydospores represented dispersal units, which were capable of germination. 3. The chlamydospores could function as structures of infection, because after chlamydospore germination the outgrowing mycelium – either directly or after production of macroconidia – could serve as secondary inoculum and start new infections.     

Activité Fongicide du N-cetyl N,N,N-triméthylammonium Contre Botrytis cinerea Pers.

Fungicidal activity of N-cetyl N,N,N-trimethyl ammonium chloride to Botrytis cinerea Pers. Grey mould of grapes caused by Botrytis cinerea is the most important phytopatho-logical problem in swiss vineyards, since the appearance of resistant strains to dicarboxi-mides. Research for new chemicals is necessary. One compound, a cationic surfactant, the N-cetyl N,N,N-trimethyl ammonium chloride (CTAC), is very active in vitro against Botrytis cinerea. This product disturbs lethally the plasma membrane of conidia.     

Botrytis cinerea: Activité Antifongique dans les Jeunes Grappes de Vitis vinifera, Varieté Gamay

Botrytis cinerea: fungicidal activity in young grape berries of Vitis vinifera (Gamay) Extracts of young grape berries of Vitis vinifera (Gamay) inhibit the germination of conidia of Botrytis cinerea. This inhibitory activity is present in extracts obtained from clusters at bloom stage and is maintained until a developmental stage close to the maturity. These extracts ruptured the cellular membranes.     

Antagonistic effects of volatiles generated by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> on spore germination and hyphal growth of the plant pathogen, <Emphasis Type="Italic">Botrytis cinerea</Emphasis>

Botrytis cinerea is one of the most serious post-harvest pathogens of fruits and vegetables. Volatiles generated by Bacillus subtilis JA significantly inhibited both spore germination and elongation of germ tubes in Botrytis cinerea using a two-compartment agar-plate assay. The volatiles caused protoplasm retraction from the hyphal tips to the spores. Hua Chen and Xiang Xiao have contributed equally to this work.     

Effect of Gamma Irradiation on Strawberries as a Means of Extending Its Shelf-Life and Lethal Dose of Botrytis cinerea

Effectiveness of γ-irradiation as a protective means against post-harvest decay in ‘Donner’ strawberries produced in Gumma Pref., Japan, was investigated. In case when strawberries were kept at 23°C, 10-% infection due to saprophytic fungi, such as Botrytis cinerea, Rhizopus and Penicillium, was found 3 days after zero-kiloroentgen (kR) irradiation, 4 days after 100-kR irradiation, and 5 to 6 days after 200-kR irradiation. For the purpose of wrap-film, ordinary cellophane was found to be more favorable than water-proof cellophane at room temperature.

Main saprophytic fungus of ‘Donner’ berries was isolated and identified to be Botrytis cinerea Persoon. Lethal dose of B. cinerea was determined from its survival curve.     

Chitosan improves development,and protects<Emphasis Type="Italic"> Vitis vinifera</Emphasis> L. against<Emphasis Type="Italic"> Botrytis cinerea</Emphasis>

We evaluated the potential of chitosan both to stimulate plant development and to induce protection from Botrytis cinerea in Vitis vinifera L. plantlets. The presence of 1.75% (v/v) chitogel in the culture medium was the optimal concentration for in vitro grapevine plantlet growth, as determined by measurements on enhancement of root and shoot biomass. Photosynthesis and related parameters were also stimulated in chitogel-treated plantlets. Chitogel reduced the development of Botrytis cinerea and induced cytological alterations to the pathogen. When challenged with the fungus, a significant decrease in disease incidence was observed in plants growing on medium supplemented with chitogel. Furthermore, exogenous foliar applications of chitogel to plantlets growing on chitogel-free medium sensitized them so as to be protected against Botrytis cinerea attack. Our results indicate that chitogel can be used in the vineyard as a means to attain protection against Botrytis cinerea and that its application may counteract the wide use of chemical pesticides.Communicated by S. Gleddie     

嗜线虫致病杆菌抑制灰葡萄孢的效应

[背景] 灰葡萄孢（Botrytis cinerea）是引起葡萄采后病害的主要病原菌之一,严重影响葡萄的贮期和品质,给葡萄产业带来极大损失。利用拮抗微生物抑制采后病原菌生长已逐渐成为防治葡萄采后灰霉病的重要手段。[目的] 利用昆虫病原线虫共生细菌广谱高效的抑菌特性,从现有共生细菌资源中筛选对灰葡萄孢具有高拮抗作用的菌株,为葡萄采后灰霉病的抑制提供新的材料和研究方向。[方法] 通过平板对峙培养法和菌丝生长速率法分离筛选拮抗共生细菌,并对优选的高效拮抗共生细菌进行16S rRNA基因序列进化分析,采用扫描电镜观察其对灰葡萄孢菌丝生长的影响,利用损伤接种法对红地球葡萄防治效果进行验证。[结果] 初步分离筛选共获得9株拮抗菌,复筛与复测得到一株抑菌效果显著的共生细菌（命名为ALL）,经进化分析其为嗜线虫致病杆菌（Xenorhabdus nematophila）,其16S rRNA基因序列的Genbank登录号为MW488402,与菌株Xenorhabdus nematophi la NC116聚于同一分支,相似性达99.79%。扫描电镜观察该菌株导致灰葡萄孢菌丝扭曲变形、表面皱缩、失水塌陷,该菌株发酵（36 h）上清液浓度为1%时对灰葡萄孢菌丝抑制率达44.5%。在葡萄常温防效实验中,与对照组比较,ALL菌株发酵上清液对灰霉菌防治效果较好,3 d后防效为63.50%。[结论] 本研究应用昆虫病原线虫共生细菌生物防治葡萄贮期灰霉病,筛选出一株高效拮抗灰葡萄孢的昆虫病原线虫共生细菌,而且其上清液对灰葡萄孢具有良好的抑制效果,为生物防治贮期葡萄灰霉病提供了新的生物材料和相关研究基础。     

解淀粉芽孢杆菌B15抑菌物质对葡萄灰霉病灰葡萄孢的抑菌机理

【目的】利用荧光显微镜和激光共聚焦扫描显微镜技术初步探讨解淀粉芽孢杆菌(Bacillus amyloquefaciens)B15菌株发酵液中的抑菌混合物质伊枯草菌素A(iturin A)和芬芥素(fengycin)对葡萄灰霉病病原菌灰葡萄孢(Botrytis cinerea)的抑菌机理。【方法】采用琼脂稀释法讨论解淀粉芽孢杆菌B15发酵液对灰葡萄孢的抑菌活性。利用台盼蓝(trypan blue)染色、4′,6-二脒基-2-苯基吲哚(DAPI)、双氢罗丹明123(DHR123)、钙离子探针fluo-3/am和Annexin V-PI探针染色来观察解淀粉芽孢杆菌B15发酵液对灰葡萄孢细胞膜和菌丝形态、细胞核、活性氧、钙离子和磷脂酰丝氨酸层的影响。【结果】抑菌活性实验发现解淀粉芽孢杆菌B15发酵液对灰葡萄孢具有良好抑菌效果。荧光显微镜台盼蓝染色观察发现,经B15发酵液处理过的灰葡萄孢出现菌丝畸形、菌丝体粗大、尖端肿胀并被染成蓝色和明显的液泡化现象。同时未在处理组中观察到细胞内容物泄漏,说明处理组菌丝细胞膜未发生破损。该结果表明在此次试验中,B15发酵液中的抑菌有效物质不以破损细胞膜的方式直接导致灰葡萄孢的死亡。激光共聚焦显微镜观察结果发现,处理组的灰葡萄孢菌丝出现典型的细胞凋亡现象、染色质固缩、细胞核裂解、磷脂酰丝氨酸层外翻、活性氧和钙离子积累。【结论】该实验表明解淀粉芽孢杆菌B15发酵液以诱导细胞凋亡的形式来抑制灰葡萄孢菌丝的生长。     

Impact of <Emphasis Type="Italic">Quillaja saponaria</Emphasis> saponins on grapevine ecosystem organisms

The control of grapevine pathogens is a rising concern in Vitis vinifera culture. The current international trend is toward banning chemicals that are highly toxic to the environment and human workers, and adopting tighter regulations. We evaluated the impact of saponins on three kinds of organisms found in grapevine culture. The ectoparasitic nematode Xiphinema index, the parasitic fungus Botrytis cinerea and various yeast strains representative of the must fermentation population were incubated on synthetic media supplemented with variable concentrations of Quillaja saponaria saponins. Saponins induced reduction in the growth of B. cinerea and showed nematicide effects on X. index. The control of X. index and Botrytis cinerea is discussed in the context of the potential use of these chemicals as environmentally-friendly grapevine treatments. With Saccharomyces cerevisiae and other yeasts, saponins showed higher toxicity against S. cerevisiae strains isolated from wine or palm wine whereas laboratory strains or strains isolated from oak exhibited better resistance. This indicates that Q. saponaria saponins effects against yeast microflora should be assessed in the field before they can be considered an environmentally-safe new molecule against B. cinerea and X. index.     

Resistenz verschiedener Kultur- und Wildtomatenpflanzen (Lycopersicon spp.) gegenüber Botrytis cinerea Pers.

Resistance of different cultivated and wild tomato plants (Lycopersicon spp.) to Botrytis cinerea Pers. 20 provenances of different cultivated and wild tomato plants (Lycopersicon spp.) were screened for resistance to Botrytis cinerea Pers. using an in vitro-leaf necrosis test. The Botrytis resistance decreased with increasing age of the leaves corresponding to their insertion height (relative youth resistance respectively senescence susceptibility). The 6 B. cinerea-isolates used for inoculation differed significantly in virulence. With increasing inoculum age a virulence reduction of the various B. cinerea-isolates occurred. Within the investigated test plant collection 2 wild species –L. columbianum and L. hirsutum– proved to be resistant in each stage of development to all B. cinerea-isolates and additionally showed field resistance.     

Effect of cultural methods on leaf spot (<Emphasis Type="Italic">Mycosphaerella fragariae</Emphasis>) and gray mold (<Emphasis Type="Italic">Botrytis cinerea</Emphasis>) damage in strawberries

Damage of leaf spot, caused by Mycosphaerella fragariae and gray mold also called Botrytis fruit rot, caused by Botrytis cinerea, average fruit weight and yield were evaluated with regard to cultural methods over 2years. Leaf spot damage decreased significantly by around 90% due to leaf sanitation (removal of dead and leaf spot infected leaves in early spring) and by 50% due to plantation in a one-row-system instead of a two-row-system. When all leaves including the healthy green ones were removed in early spring, average fruit weight decreased significantly by 10%. Fruit sanitation – the third treatment – did not influence any of the measured parameters. Neither leaf sanitation nor fruit sanitation (removal of damaged fruits during harvest) reduced B. cinerea damage significant. Only the combination of a one-row-system, leaf sanitation and fruit sanitation almost halved (not significantly) B. cinerea damage in the first crop year compared to a two-row-system without leaf and fruit sanitation. B. cinerea damage correlated significantly and positively with the biomass of plants by R²= 0.47. According to this study and the cited literature it is suggested for humid Central European conditions to apply a one-row-system combined with leaf sanitation in early spring and fruit sanitation during harvest if fruit density is high, to reduce the risk of damages in larger dimension caused by M. fragariae and B. cinerea.     

Survival of Botrytis cinerea in Soil in South-Eastern Spain

The survival of Botrytis cinerea in sterile and unsterile soil at different temperatures and relative air humidities was investigated in south‐eastern Spain. Conidia survived only 7 days at 40^°C but, depending on relative humidity, for 30–90 days at 22^°C. High air humidity (95%) was needed to maintain soil humidity (8%) at a level that favoured conidial survival. Conidia survived better in sterile soil than in unsterile soil, probably because of the presence in the latter of soil microorganisms antagonistic to B. cinerea. Survival of conidia in environmental conditions simulating those in a greenhouse was less than 28 days. Results showed that B. cinerea conidia cannot survive over summer in south‐eastern Spain, and other primary sources of inocula are discussed.     

Transformation of Terpenoids in Grape Must by Botrytis cinerea

Experiments were done to investigate the volatile components in botrytized grape must and transformation of terpenoids in terpene-supplemented grape must by Botrytis cinerea. Twenty-eight compounds were identified in the volatile concentrate of botrytized must with a combined gas chromatograph-mass spectrometer. No terpenoids were detected in the concentrate. Linalool or terpinen-4-ol decreased a lot when Botrytis cinerea was cultured in the must with these terpenes for 15 days. In linalool-supplemented botrytized must 9 identified and 3 unidentified terpenes were found, while only geranial was detected in terpinen-4-ol-supplemented botrytized must. Botrytis cinerea did not produce terpenoid in grape must without terpenes, but transformed linalool added to grape must into some other monoterpenes.     

Endogene Hyphen bei Botrytis cinerea Pers.

Endohyphae in Botrytis cinerea Pers. Genesis, following differentiation and significance for pathogenesis The formation of endohyphae (secondary hyphae) in different cell types of Botrytis cinerea Pers. (6 isolates) is described. The following differentiation possibilities of these endohyphae within and—after egress—outside their cells of origin are presented. The influence of external factors (substrate, temperature, humidity, O₂-concentration) on these processes is considered. The significance of the endohyphae for pathogenesis is mainly due to their direct infectivity as well as the ability to develop normal mycelium.     

<Emphasis Type="Italic">Erwinia carotovora</Emphasis> elicitors and <Emphasis Type="Italic">Botrytis cinerea</Emphasis> activate defense responses in <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Background  

Vascular plants respond to pathogens by activating a diverse array of defense mechanisms. Studies with these plants have provided a wealth of information on pathogen recognition, signal transduction and the activation of defense responses. However, very little is known about the infection and defense responses of the bryophyte, Physcomitrella patens, to well-studied phytopathogens. The purpose of this study was to determine: i) whether two representative broad host range pathogens, Erwinia carotovora ssp. carotovora (E.c. carotovora) and Botrytis cinerea (B. cinerea), could infect Physcomitrella, and ii) whether B. cinerea, elicitors of a harpin (HrpN) producing E.c. carotovora strain (SCC1) or a HrpN-negative strain (SCC3193), could cause disease symptoms and induce defense responses in Physcomitrella.     

Secretion of beta-1,3-glucanases by the yeast Pichia membranifaciens and its possible role in the biocontrol of Botrytis cinerea causing grey mold disease of the grapevine

Pichia membranifaciens strain FY-101, isolated from grape skin, was found to be antagonistic to Botrytis cinerea, the causal organism of the grey mold disease of the grapevine. When grown together on solid as well as in liquid media, the yeast brings about the inhibition of Botrytis cinerea, which in turn loses its ability to produce the grey mold symptoms on the grapevine plantlets. The secretion of β-1,3-glucanases by P. membranifaciens is one of the possible mechanisms related to this antagonism. In vitro experiments confirm that this yeast can be used as a biological control organism against B. cinerea. An account of this antagonism and the production of β-1,3-glucanases by P. membranifaciens is given here. Received: 16 July 2001 / Accepted: 14 September 2001     

Dual Antagonism of Aldehydes and Epiphytic Bacteria from Strawberry Leaf Surfaces against the Pathogenic Fungus Botrytis cinerea in vitro

Dual culture experiments were conducted in vitro to evaluate the potential combined biological effect of epiphytic bacteria and plant volatiles formed during fatty acids degradation on the pathogenic fungus Botrytis cinerea. The aliphatic aldehydes hexanal, (E)-2-hexenal, (Z)-3-hexenal and (E)-2-nonenal showed an enhancing effect on the antagonistic interaction between the epiphytic bacteria Pseudomonas lurida, Pseudomonas rhizosphaerae, Pseudomonas parafulva, and Bacillus megaterium against the pathogenic fungus. The unsaturated aldehydes were found to be the most potent with the minimum effective concentration being 1 ppm. Increasing volatile concentrations led to the inhibition of Botrytis cinerea growth with concomitant increase of colony diameters of epiphytic bacteria. Especially (E)-2-nonenal showed a stronger inhibitory effect on different strains of the plant pathogenic fungus Botrytis cinerea than on the epiphytic bacteria. These results suggest that co-application of antagonistic bacteria with natural plant volatiles can enhance the effectiveness of the biocontrol agents against B. cinerea.     

Mycoviruses of Botrytis cinerea isolates from different hosts

Botrytis cinerea (teleomorph Botryotinia fuckeliana) is a necrotrophic plant pathogenic fungus that causes grey mould and enormous economic losses worldwide in different crops. Control of B. cinerea is difficult due to the appearance of fungicide‐resistant isolates, and the diversity in virulence due to genetic variability and, perhaps, the infection with mycoviruses or fungal viruses. The discovery of mycoviruses and their possible application as biocontrol agents, as well as their use as tools to study the plant–pathogen interaction, has encouraged their study in B. cinerea. Herein, we have analysed the occurrence of mycoviruses in Spanish B. cinerea isolates to approach a better understanding of the interactions among viruses, fungi and plants in this pathosystem. Fifty‐five percent of the B. cinerea isolates analysed contained double‐stranded RNA (dsRNA) elements, and the number of dsRNA elements, their relative concentration and size were variable among isolates. Some of these dsRNAs were related to the presence of virus like rod or isometric particles, and to cellular degeneration and malformed mitochondria. We have also demonstrated that a 3 kb dsRNA present in 55% of the isolates having dsRNA elements was a mycovirus genome. Partial sequence of that mycovirus presented high identity in nucleotide and amino acid sequence with Botrytis cinerea mitovirus 1 (BcMV1). Analysis of the genetic distance within Spanish BcMV1 sequences showed the existence of different isolates of this mitovirus inside the Spanish B. cinerea population analysed. This is the first report of the variability of dsRNA elements and the partial genome sequence of a mitovirus associated with Spanish B. cinerea isolates and the genetic diversity within Spanish isolates of BcMV1.     

Involvement of ethylene in the regulation of growth and development of the fungus Botrytis cinerea Pers. ex. Fr.

Application of ethephon slightly increased the growth of hyphae of Botrytis cinerea. A competitive inhibitor of ethylene binding, 2,5-norbornadiene (NBD), inhibited growth of hyphae and mycelium and retarded the development of Botrytis cinerea. Transfer of the mycelium from an atmosphere containing NBD to air relieved the inhibition, indicating that the NBD effects were non-toxic and reversible. Addition of exogenous ethylene to an atmosphere containing NBD (20 ml 1^-1) effectively reduced the inhibition. Inhibition due to 40 ml 1^–1 NBD was not relieved by ethylene at any of the concentrations tested; however, a positive effect of ethylene appeared following transfer of the mycelia to air. The results suggest that ethylene may be required for the growth and development of Botrytis cinerea.Abbreviations NBD 2,5-norbornadiene - ethephon 2-chloroethyl-phosphonic acid - PDA potato dextrose agar