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.     

Inhibition of Methionine Biosynthesis in Botrytis cinerea by the Anilinopyrimidine Fungicide Pyrimethanil

When mycelium of Botrytis cinerea was treated with low concentrations of the anilinopyrimidine fungicide pyrimethanil the total amount of free amino acids increased. Qualitative variations were also induced: alanine, glutamine, lysine, glycine, histidine, asparagine, arginine, threonine and moreover, α-aminobutyrate and β-alanine were accumulated; cyst(e)ine, valine, leucine and citrulline were reduced. When mycelium of B. cinerea was incubated with Na₂[³⁵S]O₄, pyrimethanil at 1·5 μM induced a decrease of [³⁵S]methionine and simultaneously an increase of [³⁵S]cystathionine. These data indicate that the anilinopyrimidine fungicide pyrimethanil inhibits the biosynthesis of methionine and suggest that the primary target could be the cystathionine β-lyase. © 1997 SCI.     

武夷菌素对番茄灰霉菌的抑制作用及对番茄抗病性相关酶活性的影响

采用凹陷载玻片法测定了武夷菌素对番茄灰霉菌分生孢子萌发的抑制作用,在离体番茄叶片上测定了被武夷菌素处理后的番茄灰霉菌菌丝和分生孢子致病性的变化以及武夷菌素对番茄幼苗体内抗病性相关酶活性的影响。结果表明:武夷菌素对番茄灰霉菌的分生孢子有较强的抑制作用,其EC₅₀为14.1μg／mL。浓度为100μg／mL的武夷菌素可完全抑制孢子的萌发。武夷菌素能使灰霉菌菌丝和分生孢子的致病性明显下降,同时还能诱导番茄体内抗病性相关酶(SOD、POD、PPO、PAL)活性的增强,提高番茄幼苗的抗病性。     

Effects of certain herbicides on the fate of sporangiospores of Mucor piriformis and conidia of Botrytis cinerea and Penicillium expansum

The effects of several herbicides used in pome fruit orchards on the germination of spores and growth of mycelia of Botrytis cinerea, Mucor piriformis and Penicillium expansum in vitro and the survival of propagules of these fungi in soil were studied. Diuron in agar at 4–128 μg ml^?1 reduced germination of spores of B. cinerea and M. piriformis, and 2,4-D and paraquat at 32 μg ml^?1 similarly affected B. cinerea and P. expansum. Several herbicides at 128 μg ml^?1 in agar reduced growth of B. cinerea and M. piriformis but were ineffective against P. expansum. Propagule survival levels of the three fungi generally were lower in both autoclaved and non-autoclaved soil amended with herbicides than in non-amended soil. This effect was greatest in non-autoclaved soil, suggesting involvement of microbial antagonists. The most effective herbicides for reduction of fungal propagules in soil were 2,4-D, diuron, and paraquat.     

Detection of enzymatic activity and partial sequence of a chitinase gene in <Emphasis Type="Italic">Metschnikowia pulcherrima</Emphasis> strain MACH1 used as post-harvest biocontrol agent

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.     

The Interaction of Endochitinase, A Synthetic Peptide and Resveratrol in Controlling Fungi in vitro

The efficacy of two-way combinations of Trichoderma endochitinase (CHIT42), synthetic peptide ESF12 and resveratrol upon the control of growth of Botrytis cinerea and Penicillium digitatum were evaluated in vitro. CHIT42 had effective concentration for 50% growth inhibition (EC₅₀) values of 1.5 and 0.5 µM against B. cinerea and P. digitatum, respectively. CHIT42 also caused swelling, hyperbranching and bursting of P. digitatum at concentrations as low as 0.003 µM. The EC₅₀ values of ESF12 were 30.9 and 29.2 µM against B. cinerea and P. digitatum, respectively. Resveratrol had EC₅₀ values of 178.6 and 343.2 µM against B. cinerea and P. digitatum, respectively. Combinations of CHIT42 + ESF12, CHIT42 + resveratrol and ESF12 + resveratrol showed additive activity (interaction ratios between 0.5 and 1.5) against B. cinerea and P. digitatum.     

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。     

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     

Synthesis of 2-amino-6-oxocyclohexenylsulfonamides and their activity against Botrytis cinerea

BACKGROUND: With the objective of exploring the fungicidal activity of 2‐oxocyclohexylsulfonamides (2), a series of novel 2‐amino‐6‐oxocyclohexenylsulfonamides (6 to 23) were synthesised, and their fungicidal activities against Botrytis cinerea Pers. were evaluated in vitro and in vivo. RESULTS: The compounds were characterised by IR, ¹H NMR and elemental analysis. Bioassay results of mycelial growth showed that compounds 6 to 23 had a moderate antifungal activity against B. cinerea. N‐(2‐methylphenyl)‐2‐(2‐methylphenylamino)‐4,4‐dimethyl‐6‐oxocyclohexenylsulfonamide (13) and N‐(2‐chlorophenyl)‐2‐(2‐chlorophenylamino)‐6‐oxocyclohexenylsulfonamide (21) showed best antifungal activities, with EC₅₀ values of 8.05 and 10.56 µg mL^?1 respectively. Commercial fungicide procymidone provided an EC₅₀ value of 0.63 µg mL^?1. The conidial germination assay showed that most of compounds 6 to 23 possessed excellent inhibition of spore germination and germ‐tube elongation of conidia of B. cinerea. For in vivo control of B. cinerea colonising cucumber leaves, the compound N‐cyclohexyl‐2‐(cyclohexylamino)‐4,4‐dimethyl‐6‐oxocyclohexenylsulfonamide (19) showed a better control effect than the commercial fungicide procymidone. CONCLUSION: The present work demonstrated that 2‐amino‐6‐oxocyclohexenylsulfonamides can be used as possible new lead compounds for further developing novel fungicides against B. cinerea. Copyright © 2011 Society of Chemical Industry     

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。     

Adaptation to pyrrolnitrin in Botrytis cinerea and cost of resistance

The objective of this work was to estimate the risk of a decrease in the efficacy of biocontrol as a result of selection pressure exerted by biocontrol agents on Botrytis cinerea, focusing on pyrrolnitrin, an antibiotic identified in diverse biocontrol agents having an effect on B. cinerea. To evaluate a possible decrease in sensitivity to pyrrolnitrin, 10 successive generations of five isolates of B. cinerea were produced in vitro in the presence of a sublethal dose (10 μg L^?1) of the antibiotic. For one isolate, a significant reduction in the sensitivity to pyrrolnitrin at the fifth generation was observed with a resistance factor of c. 11. The production of 10 additional generations for four of these isolates, with increasing doses of pyrrolnitrin (100–4000 μg L^?1), resulted in the development of variants of B. cinerea with high levels of resistance to the antibiotic (RF > 1000) and a reduced sensitivity in vitro to a pyrrolnitrin‐producing bacterium. Reverse adaptation of resistant variants after 10 additional generations in the absence of selection pressure was not observed, suggesting stability of the resistance. Comparison of the pyrrolnitrin‐resistant generations and their sensitive parental isolates for mycelial growth, sporulation and aggressiveness on plant tissues revealed that the high level of resistance to pyrrolnitrin resulted in a high fitness cost. Mycelial growth was reduced between 1·7 to 3·6 times and sporulation reduced 3·8 to 6·6 times that of sensitive parental isolates. Similarly, aggressiveness was 7 to 10 and 3 to 10 times lower for resistant isolates on tomato and apple, respectively. This study provides evidence that a fungal plant pathogen is able to gradually build up resistance to an antibiotic produced by a biocontrol agent.     

The response of resistant and susceptible plants to diclofop-methyl

Wild oat (Avena fatua L.) plants sprayed at the 2-or 3-leaf stages of growth with diclotop-methyl developed chlorosis over the entire leaf blade of all leaves. The leaves became necfrotic ⁷days after spraying Shool growth was inhibited. In wheat (Triticum aesicum L cv.Waldron) discrete chlorotic areas developed only where the herbicide convicted the 2nd or 3rd leaf with no visible injury so new growth uf'ter treutment. Growth inhibition of susceptible oat (Avena sativa L. cv. Garry) was sensitive to placement of diclutop-methyl near the upica and meristematic sites of the plant. Chlorosis and necrosis were independent of herbicide placement. Selective herbicide placement induced chlorosis only or both chlorosis and growth inhibition Root growth in wild oat and barley (Hordeum rulgare L. cv. Dickson) was strongly inhibited by 1–0 μM diclofop-methyl. Wild oat shoots were killed when seedlings were root-treated with 10 μM diclofop-melhyl. The 100 μM rool treatment killed barley shoots but only stunted the growth of wheat shoots by approximately 50%. In root-ireated wheat plants the shoots were turgid and developed a light purple colour, whereas in foliar-treated plants the shoots developed discrete chlorotic areas.     

Phenotypic Differences Between vacuma and transposa subpopulations of Botrytis cinerea

One hundred and twenty-one single-spore strains of Botrytis cinerea isolated from Bordeaux vineyards were molecularly characterized as either transposa or vacuma, two subpopulations of B. cinerea distinguished by the presence of transposable elements. Forty-three vacuma and 68 transposa strains were distributed into two main classes (mycelial or sclerotial) by morphological phenotype according to the organ of origin. Strains isolated from overwintering sclerotia produced exclusively sclerotial colonies. The mycelial growth rate of 21 transposa and 13 vacuma strains was significantly influenced by agar-medium and temperature. The mycelial growth rate was significantly strain-dependent at favourable temperatures (15, 20 and 25 °C), but not at limiting ones (5 and 28 °C): vacuma strains showed the fastest growth rates. The strains of the two subpopulations were similar in virulence on both host species tested (Vitis vinifera and Nicotiana clevelandii). The grapevine leaves were significantly more susceptible to B. cinerea than those of tobacco. A significant negative correlation was established between virulence and mycelial growth rate. The epidemiological consequences concerning population structure of B. cinerea in vineyards are discussed.     

竹红菌甲素对番茄灰霉病菌的抑制作用

为研究竹红菌甲素(HA)对番茄灰霉病菌的抑制作用,分别采用菌丝生长速率法、电导率法、紫外分光光度法和电镜观察法研究了HA对番茄灰霉病菌抑制率以及其对电导率、蛋白质和核酸相对泄漏度、菌丝形态与超微结构的影响。结果表明:在黑暗条件下,HA对番茄灰霉病菌无明显抑制作用;但在12 000 lx光照条件下,抑菌作用显著,EC50值为17.16 mg/L,且随着HA浓度上升,抑菌率呈极显著增加趋势,40 mg/L下抑菌率达73.9%;番茄灰霉病菌细胞相对电导率、蛋白质与核酸相对泄漏度随HA浓度上升而明显升高;电镜观察可见,经34.32 mg/L的HA处理后,大多数菌丝出现畸形、膨大或缢缩等现象,细胞器几乎全部消失,细胞内出现大面积空腔,细胞遭到严重破坏。研究结果可为HA开发成绿色高效生物农药提供理论依据。     

Relationship between chemical structure and biological activity of triazole fungicides against Botrytis cinerea

The inhibitory activity of commercial and experimental triazole fungicides on the target enzyme, sterol 14α-demethylase (P450_14DM), was studied in a cell-free sterol synthesis assay of Botrytis cinerea Pers. ex Fr. In order to assess structure-activity relationships, the inhibitory activities of the compounds on radial growth of the fungus were tested as well. The EC₅₀ values (concentrations of fungicide inhibiting radial growth of B. cinerea on PDA by 50%) of all triazoles tested ranged between 10^?8 and 10^?5 m. IC₅₀ values (concentrations of fungicide inhibiting incorporation of [2-¹⁴C]mevalonate into C4-desmethyl sterols by 50%) generally ranged between 10^?9 and 10^?7 M and correlated with inhibition of radial mycelial growth. However, differences in IC₅₀ values did not reflect quantitatively the observed differences in EC₅₀ values, since the ratio between EC₅₀ and IC₅₀ increased with decreasing fungitoxicity. For a limited number of compounds the correlation between intrinsic inhibitory activity and fungitoxicity was low. Both in-vitro tests were used to investigate structure-activity relationships for stereoisomers of cyproconazole, SSF-109 and tebucona-zole. Fungitoxicity and the potency to inhibit cell-free C4-desmethyl sterol synthesis correlated for all stereoisomers tested. Mixtures of isomers of tebucona-zole or cyproconazole were slightly less active than the most potent isomer. The high activity of several commercial triazoles in both experiments implies that poor field performance of triazole fungicides against B. cinerea is due neither to insensitivity of the P450_14DM nor to low in-vitro sensitivity of the fungus.     

Failure to detect highly resistant strains in dicarboximide resistant field populations of Botrytis cinerea1

Field isolates of Botrytis cinerea with moderate levels of resistance to dicarboximide fungicides (ED50 1.0–4.9 μg ml^?1) and to dicloran were obtained from glasshouses where vinclozolin and iprodione failed to control grey mould. From sensitive and moderatcly-resistant cultures, laboratory isolates were selected on dicarboximide-amended medium, which were highly resistant to these fungicides (ED50 125->3000 μg ml^?1). Conidia of all the resistant isolates germinated well on media amended with 100 μg ml^?1 of the dicarboximides vinclozolin, iprodione, procymidone and myclozolin and with 5 μg ml^?1 of metomeclan. However, the spores of the moderately resistant isolates did not germinate on 100 μg ml^?1 metomeclan while the spores of the highly resistant isolates germinated well. Using media with 100 μg ml^?1 of metomeclan to distinguish between the two phenotypes, no highly resistant strain was detected among 312 resistant samples from five cucumber glasshouses with a high frequency of moderately resistant strains. From air-borne inoculum of five glasshouses with 100% resistant populations, 1604 colonies were recovered on vinclozolin-amended (100 μg ml^?1) medium and none on metomeclan-amended (100 μg ml^?1) medium. It is concluded that strains of B. cinerea highly resistant to dicarboximides are absent from field populations.     

The elicitor protein BcIEB1 and the derived peptide ieb35 provide long-term plant protection

BcIEB is a small protein secreted by the phytopathogenic fungus Botrytis cinerea that is recognized as a pathogen-associated molecular pattern (PAMP) by plants. This activity is mapped to a highly conserved region of 35 amino acids, the peptide ieb35. Moreover, it has been shown that the protein and the peptide induce systemic resistance to B. cinerea after their infiltration into tobacco leaves. In this work, the possible use of BcIBE1 or ieb35 as plant protective agents has been tested. Tobacco plants were sprayed, infiltrated, or treated at the seedling stage with the protein or the peptide, and plant susceptibility to pathogens with different lifestyles was then studied at various times after treatment. The results showed that both BcIEB1 and ieb35 caused a similar reduction in the lesion sizes caused by B. cinerea and in colonization by Pseudomonas syringae pv. tabaci (reduced by c. 30% and c. 40%, respectively), independently of the treatment type. In addition, the number of B. cinerea inoculations leading to successful infections was also reduced in plants infiltrated or sprayed with BcIEB1/ieb35. The defence-related genes PR1a, NPR1, and osmotin ap24 were all induced by the protein and the peptide. BcIEB1, and especially ieb35, may be considered as a potential environmentally friendly treatment to increase plant resistance to pathogens.     

Increased rhizosphere populations of Trichoderma asperellum strain T34 caused by secretion pattern of root exudates in tomato plants inoculated with Botrytis cinerea

Root exudates secreted from plants can modify rhizosphere microbiota by enhancing or inhibiting the growth of biological control agents (BCAs) and/or pathogens. Similarly, microorganisms can modify the secretion of plant root exudates. The aim of this study was to analyse the effect of a Botrytis cinerea leaf infection on the secretion of tomato root exudates and on the populations of the BCA Trichoderma asperellum strain T34 (T34). This study found that the secretion pattern of root exudates in tomato plants was influenced by B. cinerea infection in plant leaves. An increase in the levels of gluconic acid was observed, while levels of sucrose and inositol decreased. A decrease in the severity of B. cinerea by the induction of systemic resistance triggered by T34 was also observed. Tomato plants infected with B. cinerea maintained the populations of T34 in the roots, while populations of T34 decreased in plants not inoculated with the pathogen. Samples exposed to media containing gluconic acid (as the only carbon source or at the same concentration found in roots exudates) saw an increase in the in vitro growth of T34 compared to media without gluconic acid. In conclusion, a change in the secretion pattern of root exudates caused by B. cinerea, together with the enhanced growth of T34 in the presence of gluconic acid, indicates the existence of leaf to root communication. The result of this is enhanced populations of T34, and in turn induced disease resistance and a consequential reduction in disease severity.     

Effets de divers herbicides sur la croissance mycélienne de souches de Botrytis cinerea pers et de Penicillium expansum Link, sensibles ou résistantes à certains fongicides

Effects of various herbicides on mycelial growth of strains of Botrytis cinerea. Pers. and Pénicillium expansum Link, sensitive or resistant to certain fungicides Of seventy herbicides tested, thirty-eight were slightly toxic to B. cinerea (CI50, concentration giving 50% inhibition of the speed of mycelial growth, exceeding or equal to 100g of herbicide/ml of nutrient solution) and, of those, ten had no effect even at 1000 g/ml. The most active products (CI50 less than 10 g/ml) were Chlorthiamid, dichlobénil, nitrofen, propyzamide and phenolic derivatives (DNOC, ioxynil, PCP). In most cases P. expansum was less sensitive than B. cinerea except to endothal, propachlor, prynachlor and certain substituted ureas. Strains resistant to the benzimidazole fungicides (carbendazim, thiabendazole etc.) show increased sensitivity to certain carbamade herbicides (barban, chlorbufam etc.), this indicates the existence of a negative cross resistance between these groups of antimitotic pesticides. Strains resistant to the cyclic-imide fungicides (iprodionc, procymidone, vinclozolin) and to various aromatic compounds (biphenyl. chloroneb, dicloran etc.) may also be resistant to bipyridilium, dinitroanilinc and diphenylethcr herbicides, to chlorthamid. dichlobénil and oxadiazon. This cross resistance between pesticides with different biochemical modes of action has yet to be explained.