Biochemical changes in infection droplets containing spores of Botrytis spp. Incubated in the seed cavities of pods of bean (Vicia faba L.)

Infection droplets containing spores of Botrytis cinerea become inhibitory to the growth of germ tubes of the fungus within 18 hr. of their incubation in bean pods. The inhibition is caused by an ether-soluble substance, which has been partially purified, and which counteracts the stimulatory effect of sucrose, glucose, fructose, galacturonic acid and several amino acids, which are also present in the infection droplets. Changes in concentration of these substances have been described in the first 24 hr. after placing infection droplets in pods. The only major difference between droplets containing B. fabae and B. cinerea concerns the nature of the ether-soluble substances produced. Following B. fabae infection a biologically inactive u.v. absorbing substance appears in high yield in place of the antifungal substance formed following B. cinerea infections.     

Characterization of antifungal glycoprotein in red-light-irradiated broadbean leaflets

 Red-light treatment of broadbean leaflets resulted in the production of antifungal substance(s) against Botrytis cinerea. The antifungal substance(s) was positively charged, as the antifungal constituent was removed by the cation exchanger CM cellulose. Treatment of infection droplets with glycosidases (α-mannosidase, β-galactosidase, β-glucosidase), glycol-specific reagent periodate (NaIO₄), and proteinase K completely eliminated antifungal activity, suggesting that both protein and carbohydrate are active components. The protein content of infection droplets was 0.148 mg/ml. The HPLC gel column analysis of infection droplets resulted in four fractions; all the fractions showed antifungal activity. Received: June 14, 2002 / Accepted: August 12, 2002 Correspondence to:Y. Honda     

Red-Light-Induced Resistance in Broad Bean (Vicia faba L.) to Leaf Spot Disease Caused by Alternaria tenuissima

The effect of different light wavelengths on the development of lesions induced by Alternaria tenuissima in broad bean leaves was investigated. Lesion development was completely suppressed in red‐light‐irradiated broad bean leaflets, irrespective of isolate or spore concentration. Pre‐treatment of leaflets with red light for 24 h before inoculation also suppressed lesion development. Alternaria tenuissima failed to produce infection hyphae in red‐light‐irradiated broad bean leaflets. These results indicate that disease suppression in broad bean leaflets is due to light‐induced resistance. Spore germination fluid (SGF) of A. tenuissima allowed non‐pathogenic Alternaria alternata to infect wounded and unwounded broad bean leaflets kept in the dark, results suggesting that SGF induced susceptibility. Red light suppressed susceptibility induced by A. tenuissima SGF; thus, lesion formation and development were suppressed when leaflets inoculated with the spores of A. alternata suspended in A. tenuissima SGF were kept under red light. From these results, we conclude that red light induced resistance in broad bean to leaf spot disease caused by A. tenuissima, and that SGF induced susceptibility of broad bean leaflets to a non‐pathogenic isolate of A. alternata.     

Characters of aerated compost tea from immature compost that limit colonization of bean leaflets by Botrytis cinerea

Aims: The aim was to produce and characterize an aerated compost tea (ACT) that suppressed growth of the plant pathogen Botrytis cinerea. Methods and Results: Three different open‐windrow composts were sampled weekly from the early secondary mesophilic stage until maturity. Each 10 kg of compost sample was extracted in 30 l of aerated water for 24, 48 or 72 h. Relative to water, all batches of ACT applied to detached bean leaflets reduced lesion development following single‐point inoculations of B. cinerea. There was a significant linear, inverse relationship between the internal windrow temperature of compost (≤51°C) used to prepare ACT and the extent of lesion development. Bacterial diversity in ACTs from one windrow was highest using compost sampled at 48°C. The compost weight‐to‐water volume ratios of 1 : 3, 1 : 10 or 1 : 30, using compost sampled from a fourth windrow at 50°C, also produced ACTs that reduced the growth of B. cinerea on bean leaflets. The ‘1 : 3’ ACT, and to a lesser degree the same ACT filtered to remove micro‐organisms, inhibited the germination of B. cinerea conidia. Conclusions: ACT produced using the methods reported here suppressed the growth of B. cinerea on bean leaflets, with an abundant and diverse microbial community likely to contribute to pathogen suppression. Significance and Impact of the Study: This is the first report of the use of immature compost to produce a pathogen‐suppressive ACT, suggesting that compost stage is an important production variable.     

The effect of pollen grains on infections caused by Botrytis cinerea Fr

The addition of pollen to spores of Botrytis cinerea Fr. in droplets of distilled water stimulates spore germination, growth of germ tubes and lesion development. Aqueous diffusate from pollen is as effective as pollen grains, and frozen pollen is more stimulatory than freshly collected pollen. The presence of pollen grains reduces considerably the number of spores needed to allow infection to occur. The lost germination ability and infectivity of old spores is restored by pollen. The stimulatory effect of the presence of pollen has been demonstrated both in vitro and on the surfaces of strawberry petals, strawberry fruits and broad bean leaves. Complete removal of the source of pollen, the anthers from strawberry fruits, markedly affected the speed and severity of infections of strawberry fruits. On broad bean leaves the addition of pollen grains to spores induced the development of spreading aggressive lesions. Preliminary work indicates that the effective principle in pollen is water-soluble, dialysable and heat-stable. Although glucose and fructose are important components of diffusate, neither glucose solution nor fructose solution nor a mixture of the two showed as marked effects as did pollen. Orange juice produces similar effects.     

The rates of fungal development and lesion formation in leaves of Vicia faba during infection by Botrytis cinerea and Botrytis fabae

Loss of the water droplet above inoculation sites during the first day after inoculation inhibited lesion formation by Botrytis cinerea and prevented the development of spreading lesions of B. fabae. With droplets present two general patterns of infection by B. cinerea were determined; in one, few or no symptoms were produced and in the other, limited lesions developed with marked browning of the inoculation site. Where few or no symptoms were produced, germination and germ-tube growth were inhibited on the leaf surface. B. cinerea was inhibited within the leaf at sites bearing limited lesions; invading hyphae were restricted to brown epidermal cells. Fungal growth on the leaf surface was greatest at sites with most browning beneath the droplet area. Variation in lesion development by B. cinerea could not be related to droplet position or leaf damage during normal preparation for inoculation. Plants differed in their susceptibility to lesion formation by B. cinerea. B. fabae, with droplet present, was not inhibited on the leaf surface and spread inter- and intra-cellularly beneath the inoculum drop and then into surrounding tissues. Delay in spread until the inoculation site was completely necrotic and colonized suggested that B. fabae is partially inhibited during the initial phase of infection. The rate of lesion spread varied in different plants and was most rapid in the youngest leaves.     

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。     

In vitro and in vivo antifungal activates of the essential oils of various plants against strawberry grey mould disease agent Botrytis cinerea

In order to investigate the effects of antifungal essential oils on postharvest decay and some quality factors of strawberry fruit, experiments were conducted under in vitro and in vivo conditions. The antifungal activates of essential oils obtained from fennel, anis, peppermint and cinnamon at concentrations 0, 200, 400, 600 and 800 μL L^?1 were investigated against Botrytis cinerea with four replications. In vitro results showed that the growth of B. cinerea was completely inhibited by fennel, cinnamon and anis essential oils at relatively low concentrations (400–800 μL L^?1). In vivo results showed that all the used essential oils at all applied concentrations caused an increase in the shelf life and inhibited of B. cinerea growth on strawberry fruits completely in comparison to the controls. The results of this study confirmed the antifungal effect of four essential oils in both in vitro and on fruit postharvest.     

Functional analysis of an extracellular catalase of Botrytis cinerea

There is evidence that the necrotrophic fungal pathogen Botrytis cinerea is exposed to oxidative processes within plant tissues. The pathogen itself also generates active oxygen species and H₂O₂ as pathogenicity factors. Our aim was to study how the pathogen may defend itself against cellular damage caused by the accumulation of H₂O₂ and the role of an extracellular catalase in its detoxification during the infection of tomato and bean plants by B. cinerea. Chloronaphthol staining followed by light microscopy showed that H₂O₂ accumulates in the infection zone in tomato and bean leaves. An extracellular catalase gene (denominated Bccat2) was cloned from B. cinerea. Exposure of mycelium to H₂O₂ in liquid culture resulted in increased Bccat2 mRNA levels in a concentration-dependent manner. Bccat2 mRNA was detected at early stages of tomato leaf infection, suggesting that B. cinerea experiences oxidative stress. Bccat2-deficient mutants were generated by transformation-mediated gene disruption. Mutants were more sensitive then the wild-type strain to H₂O₂in vitro, but they partly compensated for the absence of BcCAT2 by activating other protective mechanisms in the presence of H₂O₂. Bccat2-deficient mutants did not display a consistent reduction of virulence on bean and tomato leaves. Cerium chloride staining of infected leaf tissue for ultrastructural studies showed that Bccat2-deficient mutants were exposed to H₂O₂ comparably to the wild-type. The results suggest that B. cinerea is a robust pathogen adapted to growing in hostile oxidizing environments in host tissues.     

Splash dispersal of fungus spores and fungicides in the laboratory and greenhouse

A laboratory technique is described for the production of drops of simulated rain in which fungal spores were suspended. When such drops containing conidia of Botrytis fabae impacted on a target leaf the secondary droplets produced infections on receptor broad bean leaves. The capacity of fungicides applied to the target leaf to redistribute in secondary splash droplets was examined in terms of the infectivity of the spores in the droplets. The extent to which a copper fungicide reduced infection on the receptor leaves was related to the level and tenacity of the fungicide deposit on the target leaf. The effect of wetting agents on the redistribution of this fungicide could probably be explained by their influence on the tenacity of the initial deposit. In general the capacity of different fungicides to inhibit infection by the secondary droplets was related to the inherent toxicity of the fungicides to B. fabae. Implications of the dispersal of spores and fungicides by rain splash are briefly considered with reference to field conditions.     

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.     

Evidence for a constitutive cytoplasmic cutinase in ungerminated conidia of Botrytis cinerea Pers.: Fr.

Cytoplasmic soluble proteins from ungerminated conidia of Botrytis cinerea exhibited cutinase activity, while cell wall binding proteins lacked this activity. Cutinase activity in proteins extracted from cell walls and cytoplasm of ungerminated conidia of Botrytis cinerea was determined using p-nitrophenyl butyrate (PNB) and TLC analysis of products derived from hydrolysis of [³H]cutin. Treatment of conidia with indoxyl acetate, a substrate indicative of non-specific esterase and cutinase activity, also gave a positive reaction in the cytoplasm of ungerminated conidia. The possible role of a putative constitutive cutinase in the cytoplasm of conidia in the early stages of infection of plants by B. cinerea is discussed.     

Antifungal Activity of the Essential Oil of Basil (Ocimum basilicum)

The antifungal and fungicidal effects of two chemotypes of basil (Ocimum basilicum) oil and its major individual components were studied in a series of in vitro and in vivo experiments. Mycelial growth of the plant pathogenic fungus Botrytis fabae was reduced significantly by both the methyl chavicol chemotype oil and the linalol chemotype oil, and the major individual components of the oils all reduced fungal growth, with methyl chavicol, linalol, eugenol and eucalyptol reducing growth significantly. Combining the pure oil components in the same proportions as found in the whole oil led to very similar reductions in fungal growth, suggesting that the antifungal effects of the whole oils were due primarily to the major components. When the fungus was exposed to the oils in liquid culture, growth was reduced by concentrations considerably smaller than those used in the Petri dish studies. Botrytis fabae and the rust fungus Uromyces fabae were also controlled in vivo, with the whole oils of both chemotypes, as well as pure methyl chavicol and linalol, reducing infection of broad bean leaves significantly. Most effective control of fungal infection was achieved if the treatments were applied 3 h postinoculation.     

Studies on Biological Activity of Cyclic Imide Compounds

The structure-activity relationships of l-phenylpyrrolidine-2,5-dione derivatives were investigated on Sclerotinia sclerotiorum by the agar dilution method. In addition, several representative compounds were tested for antimicrobial spectra in vitro with 15 pathogenic microbes and for foliage protection activity in green house tests with rice blast, rice brown spot, rice sheath blight and kidney bean stem rot. It was found that 3,5-dihalo-substituents on the benzene moiety are essential to high antifungal activity against Sclerotinia sclerotiorum. Generally, l-(3′,5′-dihalophenyl)pyrrolidine-2,5-diones are active against Corticiaceae, Dematiaceae, Pleosporaceae and Sclerotiniaceae, especially active against Sclerotinia sclerotiorum and Botrytis cinerea (the conidia form of Sclerotinia fuckeliana). N-(3,5-Dichlorophenyl)itaconimide showed a peculiarly broad antimicrobial spectrum. In green house tests, these compounds showed high activity against rice brown spot, rice sheath blight and kidney bean stem rot. Results of green house tests on the above-mentioned diseases correlate fairly well with those of in vitro tests.     

Effect of extracts from in vitro-grown shoots of <Emphasis Type="Italic">Quillaja saponaria</Emphasis> Mol. on <Emphasis Type="Italic">Botrytis cinerea</Emphasis> Pers.

The ethanolic and aqueous extracts from in vitro shoots of Quillaja saponaria Mol. (Quillay) were studied for their antifungal activity against the phytopathogenic fungus Botrytis cinerea Pers. These extracts reduced conidial germination and mycelial growth of B. cinerea, ethanolic extracts being more active than aqueous extracts. In addition, the damage areas produced by this fungus on tomato leaves and strawberry fruits pre-treated with quillay extracts were diminished. The fungitoxic effect of in vitro-grown quillay extract was similar to those obtained with commercial fungicides of both natural (BC-1000) and synthetic (iprodione–dicarboximide) origin. On the other hand, the antifungal action of quillay extracts obtained from adult trees naturally grown was only slightly superior to the fungitoxic activity of the extract from in vitro plants. HPLC analysis of the extract showed that it contained saponins and some phenolic compounds such as chlorogenic, caffeic, vanillic, and salicylic acids, and scopoletin, which have been identified as antifungal agents on phytopathogenic fungi. The results obtained in this work, suggests that extracts of in vitro-grown quillay have an important protective effect against B. cinerea and support the use of an in vitro culture system as a biotechnological alternative to obtain environmental safe antifungal quillay extracts to control B. cinerea, contributing to the preservation of this indigenous Chilean species.     

Fungal Metabolism of the Prenylated Isoflavone Licoisoflavone A

Cultures of Aspergillus flavus and Botrytis cinerea have been found to rapidly convert the fungitoxic isoflavone licoisoflavone A [5,7,2′,4′-tetrahydroxy-3′-(3,3-dimethylallyl)isoflavone] into products with reduced antifungal activity, as judged from TLC plate bioassays against the growth of Cladosporium herbarum. Both fungi produced the same five metabolites, but often in greatly differing quantities. Using physico-chemical procedures, the metabolites were characterized as a glycol (2″,3″-dihydrodihydroxylicoisoflavone A), two dihydrofurano-isoflavones (one of which was identical with lupinisoflavone D found in Lupinus albus roots) and two hydroxydihydropyrano-isoflavones.     

The Saccharomyces cerevisiae chitinase,encoded by the CTS1-2 gene,confers antifungal activity against Botrytis cinerea to Transgenic tobacco

The Saccharomyces cerevisiae chitinase, encoded by the CTS1-2 gene has recently been confirmed by in vitro tests to possess antifungal abilities. In this study, the CTS1-2 gene has been evaluated for its in planta antifungal activity by constitutive overexpression in tobacco plants to assess its potential to increase the plant's defence against fungal pathogens. Transgenic tobacco plants, generated by Agrobacterium-mediated transformation, showed stable integration and inheritance of the transgene. Northern blot analyses conducted on the transgenic tobacco plants confirmed transgene expression. Leaf extracts from the transgenic lines inhibited Botrytis cinerea spore germination and hyphal growth by up to 70% in a quantitative in vitro assay, leading to severe physical damage on the hyphae. Several of the F₁ progeny lines were challenged with the fungal pathogen, B. cinerea, in a detached leaf infection assay, showing a decrease in susceptibility ranging from 50 to 70%. The plant lines that showed increased disease tolerance were also shown to have higher chitinase activities.     

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.