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1.
In this study, the biocontrol ability of seven grapevine-associated bacteria, previously reported as efficient against Botrytis cinerea under in vitro conditions, was evaluated in two vineyard orchards with the susceptible cv. Chardonnay during four consecutive years (2002–2005). It was shown that the severity of disease on grapevine leaves and berries was reduced to different levels, depending on the bacterial strain and inoculation method. Drenching the plant soil with these bacteria revealed a systemic resistance to B. cinerea, even without renewal of treatment. Accordingly, this resistance was associated with a stimulation of some plant defense responses such as chitinase and β-1,3-glucanase activities in both leaves and berries. In leaves, chitinase activity increased before veraison (end-July) while β-1,3-glucanase reached its maximum activity at ripening (September). Reverse patterns were observed in berries, with β-1,3-glucanase peaking at full veraison (end-August) and chitinase at a later development stage. Highest activities were observed with Acinetobacter lwoffii PTA-113 and Pseudomonas fluorescens PTA-CT2 in leaves, and with A. lwoffii PTA-113 and Pantoea agglomerans PTA-AF1 in berries. These results have demonstrated an induced protection of grapevine against B. cinerea by selected bacteria under field conditions, and suggest that induced resistance could be related to a stimulation of plant defense reactions in a successive manner.  相似文献   

2.
Chitosan inhibited growth of Botrytis cinerea in liquid culture and suppressed grey mould on detached grapevine leaves and bunch rot in commercial winegrapes. Germination of B. cinerea was completely inhibited in malt extract broth containing chitosan at concentrations greater than 0·125 g L?1. However, treated conidia were able to infect detached Chardonnay leaves and pathogenicity was not affected, even after incubation for 24 h in chitosan at 10 g L?1. When added after conidial germination, chitosan inhibited B. cinerea growth and induced morphological changes suggestive of possible curative activity. The effective concentration of chitosan that reduced mycelial growth by 50% (EC50) was 0·06 g L?1. As a foliar treatment, chitosan protected detached Chardonnay leaves against B. cinerea and reduced lesion diameter by up to 85% compared with untreated controls. Peroxidase and phenylalanine ammonia‐lyase activities were also induced in treated leaves. In vineyard studies, Chardonnay winegrapes exhibited 7·4% botrytis bunch rot severity at harvest in 2007 after treatment with an integrated programme that included chitosan sprays from bunch closure until 2 weeks preharvest, compared with 15·5% in untreated controls and 5·9% with fungicide treatment. In the following season, botrytis bunch rot severity was 44% in untreated Chardonnay at harvest and the integrated programme (21%) was less effective than fungicides (13·8%). However, in Sauvignon blanc winegrapes, the integrated and the fungicide programme each reduced botrytis bunch rot severity to 4% and were significantly different from the untreated control (11·5%). This study provides evidence that suppression of botrytis in winegrapes by chitosan involves direct and indirect modes of action.  相似文献   

3.
Defense responses of grapevine towards Botrytis cinerea were investigated. The expression of genes coding for proteins involved in defense were studied: (a) phenylalanine ammonia-lyase (PAL) and stilbene synthase (StSy), (b) an acidic chitinase (VCH3) and a basic chitinase (VCHIT1b), and (c) a polygalacturonase inhibitor protein (PGIP). Since no PGIP was known in grapevine, a complete cDNA sequence was first characterized by PCR and RACE-PCR amplifications. RNAs isolated from infected leaves and infected berries were analysed by semi-quantitative and real-time RT-PCRs. In infected leaves, the expression of PAL, StSy, PGIP and VCH3 genes occurred 6hours post inoculation (hpi). Increase of VCHIT1b gene expression was delayed (24hpi). Maximum levels of induction of these genes were observed at 48hpi, except for the VCH3 gene (24hpi). Activation of these defense responses was not sufficient to stop B. cinerea spread. In berries, no VCH3 gene expression was detected. Maximum levels of induction were observed in stage 3 (loss of berry colour and abundant production of conidia) for the PAL and PGIP genes, and in stage 4 (shrivelled berry) for the StSy and VCHIT1b genes.  相似文献   

4.
A class III chitinase gene (CHI2) is induced in cucumber plants (Cucumis sativa L.) in response to infection by pathogenic microorganisms. Infection of Botrytis cinerea, causal agent of gray mold disease on cucumber, also induces CHI2 expression. To investigate whether CHI2 is involved in resistance to gray mold disease, transgenic cucumber plants were produced to overexpress the CHI2 gene. One line was analyzed in detail in terms of disease resistance. The transgenic cucumber plant (CC2) constitutively expressed CHI2 and reduced the symptoms of B. cinerea for 4 days after inoculation compared with nontransgenic plants. However, this inhibitory effect was not absolute, and CC2 eventually developed serious disease symptoms. Chitinase activity of the crude extract from CC2 leaves was higher than that from nontransgenic plants. A high-molecular-weight fraction containing CHI2 from CC2 leaves had fungistatic activity against B. cinerea. Interestingly, the low-molecular-weight fraction from CC2 leaves with CHI2 removed also had fungistatic activity against B. cinerea. Not only the introduced chitinase activity but also the endogenous defense reactions activated by overexpression of CHI2 may be involved in the enhanced gray mold disease resistance in CC2.  相似文献   

5.
The epidemiology of Botrytis cinerea was studied in five annual strawberry crops using waiting-bed transplants, a system widely adopted in the Netherlands. On dead leaves of transplants the incidence of B. cinerea varied from 26.7% to 52.6%, but the leaf area with potential sporulation was low (3.5–15.6%). During each crop cycle, the availability of necrotic leaf substrate for spore production of B. cinerea was generally low and varied between seasons and with the quality of transplants. B. cinerea sporulated on a maximum of 15.5 cm2 of leaf area per plant, measured as potential sporulation. The aerial concentration of B. cinerea conidia in untreated plots did not differ from the concentration in plots where all dead leaves had been removed, nor from the concentration at 25–50 m distance from the strawberry plots. B. cinerea incidence on flowers ranged from 5% to 96%, but no correlation was found with the potential spore production on necrotic leaves. Grey mould at harvest varied from 1.4% to 11.3% and was correlated with the average precipitation during the harvesting period but not with B. cinerea incidence on flowers. Post-harvest grey mould ranged from 2.1% to 32.6% and was correlated with petal colonisation by B. cinerea. The results suggest that in the annual cropping system with waiting-bed transplants, necrotic leaves are not a significant source of B. cinerea inoculum, unlike in other strawberry production systems. Therefore, control measures of grey mould in this annual system should focus on protection of flowers and young developing fruits, and not on the reduction of inoculum production on leaf debris.  相似文献   

6.
Two antagonistic yeast strains Metschnikowia pulcherrima MACH1 and Rhodotorula sp. PW34 were tested for their efficacy against Botrytis cinerea in vitro and in vivo on apples. Metschnikowia pulcherrima strain MACH1 showed higher inhibition of B. cinerea compared to the strain PW34 in vitro on potato dextrose broth. Further, yeast strain MACH1 showed higher efficacy in reducing grey mould on apples compared to PW34 and the untreated control. In addition, partially purified extracellular proteins from strain MACH1 showed an inhibition to B. cinerea in vitro. The antagonistic yeast strains were tested for their efficacy to produce chitinases in different liquid media, including apple juice, amended with or without cell wall preparations (CWP) of B. cinerea. The study showed a higher production of chitinases from M. pulcherrima strain MACH1 when compared to PW34. Interestingly, the strain MACH1 secreted higher chitinases in the presence of cell wall fractions of B. cinerea. For this reason, the chitinase gene of strain MACH1 was amplified using PCR reactions and the nucleotide sequence data showed high homology to chitinases of other yeast strains. The results of the current study show that M. pulcherrima strain MACH1 has the ability to secrete chitinases in different liquid media including apple juice, and the enzyme could be involved in the post-harvest biological control of B. cinerea.  相似文献   

7.
Botrytis cinerea, the grey mould agent, is one of the most important pathogens of grapevine, due to the great yield losses caused and the economic costs related to disease control. Ground cover plants are assumed to have a role in the complex epidemiology of the pathogen, even if no information on the genetic variability of the strains is available. In this study, a molecular epidemiology approach, based on the comparative analysis of the nucleotide sequences of multiple genes (ITS1-ITS2, G3PDH, NEP1, NEP2, BC-hch, and sdhB), was used to evaluate whether B. cinerea isolated from herbaceous species contributes to grey mould diffusion on grapevines. From 330 samples collected in two vineyards in Lombardy, Italy, 63 B. cinerea strains were isolated from tissues of grapevine with symptoms (50 strains) and spontaneous ground cover plants (13 strains). Capsella bursa-pastoris, Cardamine impatiens, Lamium purpureum, and Crepis tectorum were identified as novel B. cinerea hosts. Sequence analysis and phylogeny showed that the same genotypes were present on both grapevines and herbaceous plants, with no fitness (estimated from growth and sporulation on potato dextrose agar) or pathogenicity (on grapevine leaves and berries, and tomato leaves) penalties. This confirms that ground cover plants can be a source of inoculum for B. cinerea on grapevine. Moreover, phylogenetic analysis of the BC-hch gene allowed the identification of two genetically distinct clusters, characterized by vegetative incompatibility and different distributions of the mating types, fitness, and pathogenicity. Therefore, B. cinerea seems to be composed of two diverging subpopulations that do not differ for host specialization.  相似文献   

8.
Botrytis cinerea, which causes grey mould, is a major pathogen of many crops. On strawberry, isolates of Trichoderma spp. can effectively control B. cinerea, but frequent application is necessary. Bees can be used to disseminate biological control agents to the target crop. We tested the ability of honey bees to disseminate Trichoderma harzianum T39 to control B. cinerea in strawberry in the field during the winter in Israel over two consecutive seasons. We used the recently developed ‘Triwaks’ dispenser for loading the bees with the T. harzianum inoculum. During both years, grey mould developed in late January in untreated control plots; at low to medium disease levels it was partially controlled by fungicide treatment, and was best controlled in bee-visited plots. At high disease levels neither chemical nor biological control was effective. To assess the spatial distribution of inoculum by bees, we sampled flowers up to 200 m from the hives and found effective levels of T. harzianum even at 200 m. The approach used in this study provides an effective control of grey mould in strawberry in conditions of low to medium grey mould incidence.  相似文献   

9.
采用菌丝生长速率法和孢子萌发法，分别测定了烟草灰霉病菌对多菌灵、嘧霉胺、异菌脲和丙环唑的敏感性，同时通过离体叶片法评估了这4种杀菌剂对烟草灰霉病的保护和治疗作用。结果表明:4种杀菌剂对烟草灰霉病菌的菌丝生长和孢子萌发均表现出了不同程度的抑制活性，并对灰霉病同时具有保护和治疗作用。其中多菌灵对菌丝生长的抑制活性最强，EC50平均值为0.06 mg/L，其次为丙环唑、嘧霉胺和异菌脲，EC50平均值分别为0.36、0.53和0.60 mg/L；异菌脲和丙环唑对烟草灰霉病菌孢子萌发的抑制活性较强，EC50平均值分别为2.05和2.21 mg/L，其次为嘧霉胺和多菌灵，EC50平均值分别为10.56和131.23 mg/L。异菌脲和多菌灵对灰霉病的保护作用和治疗作用均最强，当药剂质量浓度为200 mg/L时，其对离体叶片的保护和治疗作用防效分别为100%、100%和98.3%、91.8%。研究结果可为烟草灰霉病的科学防治提供依据。  相似文献   

10.
A series of imidazole-1-carboxylates was prepared by reacting various alcohols with trichloromethyl chloroformate and imidazole or N,N'-carbonyl-diimidazole. They were tested for fungitoxic activity in vitro against two phytopathogenic fungi, Botrytis cinerea (grey mould) and Gibberella fujikuroi, and for preventive efficacy against grey mould on cucumber leaves. 1-(4-Substituted phenoxymethyl)-2,2-dimethylpropylimidazole-1-carboxylates showed excellent in-vitro activity against B. cinerea, and moderate activity against G. fujikuroi, and some of them also effectively controlled grey mould in vivo. A 1H-1,2,4-triazole derivative corresponding to an imidazole derivative did not have any activity, while a thiocarboxylate corresponding to an imidazole carboxylate showed excellent activity against both B. cinerea and G. fujikuroi.  相似文献   

11.
Phyllosphere yeasts antagonistic to the infective activity of Botrytis cinerea were isolated from leaves of greenhouse-grown tomatoes and evaluated in a detached leaf assay for their ability to suppress grey mould. Nine of 30 recovered yeast isolates were found to reduce a disease index by >90% when compared to an untreated control. In greenhouse experiments, the yeast isolate Rhodotorula glutinis Y-44 was the most efficient in controlling grey mould of tomato plants. In further experiments in greenhouse-grown tomato plants the effectiveness of R. glutinis Y-44 was compared with two commercial fungicides. It was demonstrated that R. glutinis Y-44 was as effective as fungicides in controlling the pathogen. Moreover, the population of R. glutinis Y-44 was monitored for 8 weeks after application on tomato plants. The isolate successfully colonized the plant surface, although the population decreased by 10-fold 8 weeks after application. Since B. cinerea is also a major post-harvest pathogen for tomato fruits, the ability of R.␣glutinis Y-44, to protect artificially infected wounded tomato fruits was also tested. It was shown that R.␣glutinis Y-44 was able to reduce by 50% the percentage of infected wounds compared to the untreated controls.  相似文献   

12.
BACKGROUND: The antifungal properties of chitosan and acibenzolar‐S‐methyl were evaluated to assess their potential for protecting grapes against Botrytis cinerea Pers.: Fr. isolated from Vitis vinifera L. The objectives were to determine the effects of these compounds on the in vitro development of B. cinerea and to assess their effectiveness at controlling grey mould on grapes stored at different temperatures. RESULTS: Both agents significantly inhibited the radial growth of this fungus species. The EC50 was 1.77 mg mL?1 for chitosan and 3.44 mg mL?1 for acibenzolar‐S‐methyl. In addition, single grapes treated with aqueous solutions of chitosan (1.0 and 2.5 mg mL?1) and acibenzolar‐S‐methyl (1.0 and 3.0 mg mL?1) were inoculated with B. cinerea and incubated at both 4 and 24 °C. After 4 days at 24 °C, all the concentrations of chitosan and acibenzolar‐S‐methyl significantly reduced B. cinerea growth. However, at 4 °C, significant differences were only observed between chitosan at 2.5 mg mL?1 and acibenzolar‐S‐methyl at both 1.0 and 3.0 mg mL?1 and the corresponding controls. After 3 days at 24 °C, the greatest reduction in lesion size was obtained in grapes pretreated with acibenzolar‐S‐methyl at 3.0 mg mL?1. Only the highest doses of these products significantly reduced the lesion diameters when grapes were stored for 3 days at 4 °C. CONCLUSIONS: Chitosan and acibenzolar‐S‐methyl could directly inhibit the growth of Botrytis cinerea in vitro and confer resistance on grapes against grey mould. Pretreatment with these compounds could be an alternative to traditional fungicides in post‐harvest disease control in grapes. Copyright © 2010 Society of Chemical Industry  相似文献   

13.
No cross-resistance was observed between pyrimethanil or cyprodinil and the fungicides benomyl, iprodione or carbendazim + diethofencarb. In vitro, both anilinopyrimidine fungicides were effective against strains of Botrytis cinerea resistant to benzimidazoles and/or dicarboximides and against a wild type strain insensitive to diethofencarb (EC50 values ranged between 0.03–0.19 and 0.006–0.054gml–1 for pyrimethanil and cyprodinil, respectively). Preventive applications of anilinopyrimidines completely protected young cucumber plants and fruits that were inoculated with all strains of B. cinerea. The effectiveness of pyrimethanil against grey mould was studied in greenhouse grown tomatoes in relation to (a) the type of infection and the progress of the disease on different plant parts and (b) the response of the naturally occurring B. cinerea population to the selection pressure caused by eight successive applications of this fungicide. Pyrimethanil effectively controlled grey mould on leaves, fruits and stems but did not significantly reduce the number of dead plants and fruits with 'ghost spot' symptoms. The selection pressure caused by the consecutive applications of pyrimethanil resulted in reduction of its effectiveness on leaves that became apparent after the sixth application. This was correlated with a shift of the B. cinerea population (not previously exposed to anilinopyrimidines) towards reduced sensitivity, probably due to the development of a low level of resistance (R L = 7.7). Pyrimethanil delayed the onset of the disease but it did not reduce the infection rate.  相似文献   

14.
Genes encoding an acidic wheat class IV chitinase (383), an acidic wheat β 1,3-glucanase (638) and a rice cationic peroxidase (POC1) were introduced into ‘Nantes Coreless’ carrot (Daucus carota) by Agrobacterium-mediated transformation. The genes were introduced singly or in various combinations followed by selection imposed by the herbicide phosphinothricin. Regenerated plantlets were screened for presence and expression of the three transgenes using PCR, Southern and Northern hybridisations. Eighteen transgenic lines expressing a single transgene and 2 lines each co-expressing 638/383 and 383/POC1 were assessed for resistance to the necrotrophic fungal pathogens Botrytis cinerea and Sclerotinia sclerotiorum. Percentage leaf area diseased was measured 4 and 7 days after inoculation (dai) and compared to non-transformed control plants. Six lines expressing β-1,3-glucanase 638 alone had no enhanced resistance to B. cinerea at 4 dai and only slight resistance to S. sclerotiorum; there was no effect at 7 dai. Two out of the six lines expressing 383 alone had enhanced tolerance to both pathogens with a 20–50% reduction in disease development at 7 dai. Two lines co-expressing 638/383 had slight reductions in disease by (10–20%) similar to that of the lines expressing chitinase 383 alone. Highest levels of disease resistance were seen in transgenic lines expressing POC1, alone or in combination with chitinase 383. Disease symptoms were slower to develop and symptoms were reduced by up to 90% for B. cinerea and 70% for S. sclerotiorum. The 383/POC1 co-expressing plants developed disease at levels similar to that of POC1 alone. Petioles of plants over-expressing POC1 had higher levels of lignin accumulation constitutively compared to control plants, which was greatly enhanced following inoculation with S. sclerotiorum. These results indicate that peroxidase over-expression can lead to significant disease reduction against necrotrophic pathogens in transgenic carrot plants.  相似文献   

15.
Enzymatic defense responses and protection against Phytophthora nicotianae were studied in tobacco plants treated with chitosan of different molecular weights (MW) and degrees of acetylation (DA). The concentration and mode of chitosan application affected enzymatic induction in tobacco leaves. β-1,3-Glucanase (EC 3.2.1.6) activity required 10 times the polymer concentration relative to the oligochitosan mixture to induce the highest activity above control when treated by foliar spray, indicating the influence of molecular weight in this response. PAL (EC 4.3.1.5) and POD (EC 1.11.1.6) activities increased above control as a result of the influence of polymer degree of acetylation when treatments were applied by foliar spray. A higher DA favored PAL activity, whereas a lower DA induced higher POD activity. Using an in vitro bioassay, it was found that the three chitosan compounds caused a reduction of the infection index of P. nicotianae in tobacco plants that was dependent on elicitor concentrations. There was a significant relationship between the reduction of the infection index and an increase in PAL activity when chitosan was applied by foliar spray and by substrate drench to the plant rhizosphere. These results demonstrate the influence of chitosan physico–chemical properties in plant-induced resistance and the relevance of particular responses in plant protection against pathogens.  相似文献   

16.
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  相似文献   

17.
Sixty isolates of saprophytic microorganisms were screened for their ability to reduce the severity of grey mould (Botrytis cinerea) infection and sporulation. Isolates of the bacteriaXanthomonas maltophilia, Bacillus pumilus, Lactobacillus sp., andPseudomonas sp. and the fungusGliocladium catenulatum reduced germination of conidia of the pathogen and controlled disease on bean and tomato plants. Their activity under growth room conditions was good, consistent, and similar to the activity of the known biocontrol agent,Trichoderma harzianum T39 (non-formulated). Although the tested isolates may for nutrients with the germinating conidia ofB. cinerea, resistance induced in the host by live or dead cells were also found to be involved. Inhibitory compounds were not detected on treated leaves. Sporulation ofB. cinerea after its establishment on leaves was also reduced by the above mentioned isolates and byPenicillium sp.,Arthrinium montagnei, Ar. phaeospermum, Sesquicillium candelabrum, Chaetomium globosum, Alternaria alternata, Ulocladium atrum, andT. viride. These sporulation-inhibiting fungi did not reduce the infection of leaves byB. cinerea. Most of these selected fungi and bacteria were capable of reducing lesion expansion.  相似文献   

18.
Three experimental trials were carried out in Northern Italy during 1985 and 1986 in order to control grey mould of grapevine (Botrytis cinerea) by using isolates of Trichoderma spp. resistant to several fungicides commonly sprayed against grapevine pathogens, alone or in alternation with benzimidazoles or dicarboximides, in vineyards where fungicide-resistant strains of B. cinerea are frequent. The antagonists alone partially controlled the pathogen on cv. Moscato ?Asti. In one case, the integration of chemical and biological control measures showed slightly better results than for the fungicide alone (for benomyl but not for vinclozolin), but further trials are needed to investigate the full potential for using fungicide-resistant Trichoderma in alternation with fungicides. Trichoderma spp. performed very poorly on cv. Barbera.  相似文献   

19.
The effect of a chitosan coating on antifungal activity and rate of respiration, chitinase and β, 1-3 glucanase activities with reference to papaya variety ‘Rathna’ was investigated. One percent chitosan, extracted from locally available prawn waste, was selected as the effective concentration to inhibit spore germination via a series of experiments on potato dextrose agar. Rate of respiration and the concentration of CO2 in the internal cavity of chitosan-treated and untreated papaya were tested via gas chromatography. Chitinase and β,1-3 glucanase activities were tested in peel samples using gel diffusion and spectrophotometric assays, respectively. Complete inhibition of spore germination was observed in-vitro at treatments of 1% chitosan and above. This concentration significantly (P < 0.05) reduced both disease incidence and severity on inoculated fresh papaya. Significant (P < 0.05) decrease was observed in rate of respiration while internal CO2 concentration of the fruit increased (P < 0.05) with the chitosan treatment. Chitinase and β,1-3 glucanase activities of papaya variety Rathna subjected to chitosan treatment were much higher than in the untreated control. Chitosan shows antifungal activity to the anthracnose disease causing fungus and stimulates the defense response on the papaya peel by increasing the chitinase and β,1-3 glucanase activities. The antifungal activity of chitosan could be attributed to the induction of elicitation activity due to these defense enzymes. It also forms a semi-permeable coating around the fruit and extends storage life of papaya by reducing the rate of respiration and delaying ripening.  相似文献   

20.
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