Resistance of Malus domestica fruit to Botrytis cinerea depends on endogenous ethylene biosynthesis

The plant hormone ethylene regulates fruit ripening, other developmental processes, and a subset of defense responses. Here, we show that 1-aminocyclopropane-1-carboxylic acid synthase (ACS)-silenced apple (Malus domestica) fruit that express a sense construct of ACS were more susceptible to Botrytis cinerea than untransformed apple, demonstrating that ethylene strengthens fruit resistance to B. cinerea infection. Because ethylene response factors (ERFs) are known to contribute to resistance against B. cinerea via the ethylene-signaling pathway, we cloned four ERF cDNAs from fruit of M. domestica: MdERF3, -4, -5, and -6. Expression of all four MdERF mRNAs was ethylene dependent and induced by wounding or by B. cinerea infection. B. cinerea infection suppressed rapid induction of wound-related MdERF expression. MdERF3 was the only mRNA induced by wounding and B. cinerea infection in ACS-suppressed apple fruit, although its induction was reduced compared with wild-type apple. Promoter regions of all four MdERF genes were cloned and putative cis-elements were identified in each promoter. Transient expression of MdERF3 in tobacco increased expression of the GCC-box containing gene chitinase 48.     

Activity of the Antifungal Protein from Aspergillus giganteus Against Botrytis cinerea

ABSTRACT Botrytis blight (gray mold), caused by Botrytis cinerea, is one of the most widely distributed diseases of ornamental plants. In geranium plants, gray mold is responsible for important losses in production. The mold Aspergillus giganteus is known to produce and secrete a basic low-molecular-weight protein, the antifungal protein (AFP). Here, the antifungal properties of the Aspergillus AFP against various B. cinerea isolates obtained from naturally infected geranium plants were investigated. AFP strongly inhibited mycelial growth as well as conidial germination of B. cinerea. Microscopic observations of fungal cultures treated with AFP revealed reduced hyphal elongation and swollen hyphal tips. Washout experiments in which B. cinerea was incubated with AFP for different periods of time and then washed away revealed a fungicidal activity of AFP. Application of AFP on geranium plants protected leaves against Botrytis infection. Cecropin A also was active against this pathogen. An additive effect against the fungus was observed when AFP was combined with cecropin A. These results are discussed in relation to the potential of the afp gene to enhance crop protection against B. cinerea diseases.     

The influence of Thrips obscuratus on infection and contamination of kiwifruit by Botrytis cinerea

Infestation of kiwifruit flowers by Thrips obscuratus , and inoculation with Botrytis cinerea , increased the amount of flower infection, external contamination and internal infection of fruit at harvest. Combination of these treatments was synergistic. The mechanisms of this role of T. obscuratus in the epidemiology of B. cinerea and development of stem-end rot after cool storage are discussed in relation to commercial production, harvesting and handling procedures. Significant reductions in inoculum available for fruit infection may be achievable by control of populations of T. obscuratus in orchards during flowering.     

Development of a polyclonal antibody-based immunoassay for the early detection of Sclerotinia sclerotiorum in rapeseed petals

A serological test has been developed that allows the early detection of infection of young petals by Sclerotinia sclerotiorum , an important pathogen of rapeseed. Two steps were required to obtain an antiserum sufficiently specific for S. sclerotiorum. Soluble mycelial extracts of S. sclerotiorum were used to produce the first generation polyclonal antiserum. This was not specific for S. sclerotiorum in double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and allowed the screening of cross-reacting fungal species such as Botrytis cinerea , a pathogen commonly present on rapeseed petals. Using a polyclonal anti- B. cinerea serum enabled the absorption, by serial cycles, of S. sclerotiorum antigens common to B. cinerea. Residual antigens were then used as immunogens for the production of two second generation antisera (S1 and S2) which were then tested by DAS-ELISA. Cross-reactions with B. cinerea decreased with purification cycles of the immunogen whereas Cross-reactions with some unrelated fungi slightly increased. S. sclerotiorum and B. cinerea were distinguishable using antiserum S2.     

Postharvest Biological Control of Botrytis cinerea on Kiwifruit by Volatiles of [Isabella] Grapes

ABSTRACT The potential of volatile substances emitted by 'Isabella' grapes (Vitis labrusca) to control gray mold (Botrytis cinerea) on 'Hayward' kiwifruit (Actinidia deliciosa) was studied. The closed Mariotte system was used as a bioassay method to analyze quantitatively the biological action of these volatiles on B. cinerea growth. In vivo experiments compared the effects of volatiles from 'Isabella' grapes versus volatiles from 'Roditis' grapes (V. vinifera) and a B. cinerea control on the growth and disease development of B. cinerea on kiwifruit. The effect of the volatiles on the growth of B. cinerea was tested at various temperatures and times of inoculation after the wounding of kiwifruit, as well as using various weights and developmental stages of the grapes. The 'Isabella' volatiles limited the incidence of infection by reducing both the inoculum density and the activity of the pathogen. The weight and developmental stage of the grapes were important in the degree of inhibitory action of the 'Isabella' volatiles. The inhibitory action was more pronounced at 21 degrees C irrespective of the inoculation time after wounding. The study shows the potential for successful biological control of B. cinerea on kiwifruit by volatiles from 'Isabella' grapes.     

Control of postharvest Botrytis fruit rot of strawberry by volatile organic compounds of Candida intermedia

A study was conducted to identify volatile organic compounds or volatiles produced by Candida intermedia strain C410 using gas chromatography-mass spectrometry, and to determine efficacy of the volatiles of C. intermedia in suppression of conidial germination and mycelial growth of Botrytis cinerea and control of Botrytis fruit rot of strawberry. Results showed that, among 49 volatiles (esters, alcohols, alkenes, alkanes, alkynes, organic acids, ketones, and aldehydes) identified from C. intermedia cultures on yeast extract peptone dextrose agar, two compounds, 1,3,5,7-cyclooctatetraene and 3-methyl-1-butanol, were the most abundant. Synthetic chemicals of 1,3,5,7-cyclooctatetraene; 3-methyl-1-butanol; 2-nonanone; pentanoic acid, 4-methyl-, ethyl ester; 3-methyl-1-butanol, acetate; acetic acid, pentyl ester; and hexanoic acid, ethyl ester were highly inhibitory to conidial germination and mycelial growth of B. cinerea. Inhibition of conidial germination and mycelial growth of B. cinerea by volatiles of C. intermedia was also observed. Meanwhile, results showed that incidence and severity of Botrytis fruit rot of strawberry was significantly (P < 0.01) reduced by exposure of the strawberry fruit to the volatiles from C. intermedia cultures or C. intermedia-infested strawberry fruit. These results suggest that the volatiles of C. intermedia C410 are promising biofumigants for control of Botrytis fruit rot of strawberry.     

Factors affecting symptom production by latent Botrytis cinerea in Primula×polyantha

Latent infection by Botrytis cinerea was frequently detected in young Primula × polyantha (horticultural hybrid polyanthus) plants. Genetically marked isolates were used to demonstrate that conidial inocula applied to young plants generally did not result in disease appearing on the leaves until plants flowered, regardless of when plants were inoculated. The severity of disease was proportional to the length of each day spent at 100% relative humidity during the latent period, regardless of time or form of inoculum. Plants with a prior B. cinerea infection were more susceptible to further infection and also showed symptoms of B. cinerea infection more quickly. Bacteria found to be associated with B. cinerea increased the likelihood of B. cinerea symptoms being seen on the plant, but did not directly assist infection.     

Development of a monoclonal antibody-based immunodetection assay for Botrytis cinerea

Three hybridoma cell lines secreting antibodies that specifically recognize Botrytis cinerea and B.fabae , but not B. allii, have been raised from splenocytes of mice immunized with a low molecular-weight fraction (30 kDa) from surface washings of B. cinerea. Antibodies from these cell lines have been used to develop an antigen-based elisa test that will detect B. cinerea in strawberries. This monoclonal antibody immunoassay detection assay should prove useful to both the cut-flower and wine industries. Supernatants from the three specific cell lines recognize mycelial fragments, saline extracts of mycelia and germinating conidia by both ELISA and immunofluorescence. Recognition of non-germinating spores is poor. Supernatants from the specific cell lines did not recognize other fungi normally involved in post-harvest spoilage of fruits and vegetables. Supernatants from KH4 gave the lowest background values with healthy tissue. Indirect evidence from heat, protease and periodate treatment of the antigens indicates that antibodies from all three specific cell lines are recognizing carbohydrate epitopes on a glycoprotein.     

The use of Trichoderma species to control strawberry fruit rots

The effect of temperature on the growth and antagonistic properties of Trichoderma species against Botrytis cinerea and Mucor mucedo (strawberry fruit pathogens) was studied. Five strongly antagonistic isolates were further used in field experiments. The incidence of pre-harvest rots caused by B. cinerea and the rate of post-harvest spoilage were similarly reduced when strawberry flowers were sprayed either with the fungicide dichlofluanid or with spores of selected Trichoderma isolates.     

Selective Isolation of Bacterial Antagonists of Botrytis cinerea

Most potential fungal antagonists have been identified only after exhaustive non-selective screening. Bacteria antagonistic to Botrytis cinerea were selected using a simple method based on techniques developed for trace enrichment studies, where a crude cell wall preparation from B. cinerea was used as a selective substrate and bacteria were isolated based on growth on cell wall agar media. Most of the 52 bacterial isolates that were obtained from the surface of organically grown strawberry fruit and could grow on B. cinerea wall media in culture showed some ability to inhibit the growth of the fungus. Potato dextrose agar seeded with B. cinerea spores (104ml–1) were challenged with each of these bacterial isolates at concentrations of 109 and 106 colony forming units ml–1. Eleven of the 52 isolates initially recovered, subsequently demonstrated strong antagonism in vitro and were selected for additional screening tests on strawberry fruit. All 11 isolates reduced grey mold rot incidence on fruit in storage. Three of the best isolates were tested in limited field trials, and also reduced grey mold rot on fruit under field conditions.     

出芽短梗霉菌株YW1防治储藏期草莓灰霉病的研究

本文研究了出芽短梗霉Aureobasidium pullulans菌株Yw1防治储藏期草莓果实灰霉病Botrytiscinerea的效果,并对其防病机制进行了初步探索。结果表明,菌株Ywl可以有效的控制储藏期草莓灰霉病的发生,发病率可由95．1％降至30．5％-55．6％,菌株Ywl产生的挥发性物质可以抑制灰葡萄孢菌丝生长,抑制率达到52．1％。去除菌体的上清液对灰霉菌无明显抑制活性,说明其抗菌作用机制可能是活菌的竞争作用。由此可见,菌株Ywl是1株有应用潜力的生防菌。     

Biological Control of Botrytis cinerea by Volatiles of 'Isabella' Grapes

ABSTRACT The effect of volatiles from cv. Isabella (Vitis labrusca) on the growth of Botrytis cinerea was tested in vitro and in situ, in the latter case on 'Roditis' grapes (V. vinifera), at various temperatures. The goal of the research was to determine whether the volatiles emitted by Isabella grapes could be effective biocontrol agents of Botrytis cinerea. The closed Mariotte system was used as a bioassay method to analyze quantitatively the biological action of these volatiles on fungal growth and disease development. The in vitro experiments revealed the inhibitory action of the Isabella volatiles on the sporulation and sclerotia formation of the fungus, as well as the stimulating action of the Roditis volatiles on the sporulation of the fungus. The in situ study confirmed the antifungal action of the Isabella volatiles as they reduced the inoculum and pathogenicity of B. cinerea. The antibiotic action was more pronounced at 21 degrees C. The study indicates that Isabella volatiles act as biocontrol agents of B. cinerea.     

The effectiveness of postharvest hot water dipping on the control of grey and black moulds in sweet red pepper (Capsicum annuum)

The effectiveness of hot water dipping on the control of grey mould, caused by Botrytis cinerea , and black mould, caused by Alternaria alternata on sweet red pepper quality was investigated. Dipping naturally infected or artificially inoculated fruit at 508C for 3 min completely inhibited, or significantly reduced, decay development caused by B. cinerea and A. alternata , respectively. Heat damage was observed on fruit dipped for 5 min at 50°C, or at 55°C for 1 min or longer. Damage appeared as cracks and pitting on the fruit surface. Spore germination and germ tube elongation in vitro was inversely related to the duration of exposure or to the range of temperature used. The ET₅₀ for spore germination for B. cinerea was 3.2, 1.5 and 0.8 min, and for A. alternata 8.8, 4.2 and 1.4 min, at 45, 50 and 55°C, respectively. The ET₅₀ for germ tube elongation for Botrytis was 2.6, 0.9 and 0.5 min, and for Alternaria , 7.2, 2.5 and 1.6 min, at 45, 50 and 55°C. The mode of action of hot water dips on decay of pepper appears to be by direct interaction with the fungi.     

Characterization of QoI resistance in Botrytis cinerea and identification of two types of mitochondrial cytochrome b gene

Botrytis cinerea field isolates collected in Japan were screened for resistance to Qo inhibitor fungicides (QoIs). Of the 198 isolates screened, six grew well on a medium containing azoxystrobin, a QoI, when salicylhydroxamic acid, an alternative oxidase inhibitor, was present. The resistance mutation in the cytochrome b gene ( cytb ) was characterized. All QoI-resistant isolates had the same mutation (GGT to G C T) in cytb that led to the substitution of glycine by alanine at position 143 of cytochrome b , which is known to confer QoI resistance in plant pathogens. To detect this mutation, a hybridization probe assay based on real-time PCR amplification and melting curve analysis was developed. Using DNA samples prepared from aubergines coinfected with QoI-resistant and QoI-sensitive B. cinerea isolates, two similar peak profiles with their corresponding melting temperatures were obtained. This result suggests that QoI-resistant and QoI-sensitive isolates may compete equally in terms of pathogenicity, and the assay may be used to assess the population ratio of mutant and wild-type isolates. However, the hybridization probe did not anneal to PCR products derived from the DNA samples of some QoI-sensitive isolates. Structural analysis of cytb revealed that B. cinerea field isolates could be classified into two groups: one with three introns and the other with an additional intron (Bcbi-143/144 intron) inserted between the 143rd and 144th codons. All 88 isolates possessing the Bcbi-143/144 intron were azoxystrobin-sensitive, suggesting that the QoI-resistant mutation at codon 143 in cytb prevents self-splicing of the Bcbi-143/144 intron, as proposed in some other plant pathogens.     

Evaluation of systemic fungicides for use in the glasshouse industry

The primary evaluation of ten fungicides for the control of cucumber powdery mildew (Sphaerotheca fuliginea), tomato leaf mould (Cladosporium fulvum) and tomato grey mould (Botrytis cinerea) indicated that nine of them were systemically active against one or more of the pathogens when applied as soil drenches. Benomyl, thiophanate, thiophanate-methyl, thiabendazole and Cela W52A showed activity against all three diseases, but dimethirimol, triarimol and dodemorph were more specific and mainly active against cucumber powdery mildew. An in vitro bioassay generally indicated that fungitoxicity was not translocated from sprayed to unsprayed leaves, and though toxicity to B. cinerea, following soil drenches of benomyl, thiophanate and thiophanate-methyl, was detected by bioassay in leaf sap and calyces it was not detected in fruit. Furthermore, extended evaluation of benomyl for the control of grey mould on cropping tomotoes showed that in the presence of abundant B. cinerea inoculum, benomyl soil drench treatment effectively controlled flower and leaf infection but not fruit ghost-spotting.     

Control of Postharvest Decay of Apple Fruit with Candida saitoana and Induction of Defense Responses

ABSTRACT The ability of Candida saitoana to induce systemic resistance in apple fruit against Botrytis cinerea was investigated. To separate the antagonistic activity of C. saitoana from its ability to induce resistance, the antagonist and the pathogen were applied in spatially separated wounds. In fresh apples, C. saitoana applied 0 or 24 h before inoculation with B. cinerea showed no effect on lesion development caused by B. cinerea. When applied 48 to 72 h preinoculation with B. cinerea, however, C. saitoana reduced lesion diameter by more than 50 and 70%, respectively, compared with wounding. C. saitoana had no effect on lesion development on stored apples, regardless of the lag period between yeast treatment and inoculation with B. cinerea. In addition to inducing systemic resistance, C. saitoana increased chitinase and beta-1,3-glucanase activities with a higher accumulation in fresh than in stored apples. In fresh apples, the onset of systemic resistance to B. cinerea coincided with the increase in chitinase and beta-1,3-glucanase activity in systemically protected tissue. These studies show that C. saitoana is capable of inducing systemic resistance in apple fruit and indirectly suggest that antifungal hydrolases are involved in the observed systemic protection.     

连续施药及室内药剂驯化对番茄灰葡萄孢对咯菌腈敏感性的影响

为了解连续施药对番茄灰葡萄孢对咯菌腈敏感性的影响,在温室条件下,从灰霉病发生初期开始,按推荐剂量(有效成分67.5 g/hm2)定期向番茄苗喷施咯菌腈,通过菌丝生长速率法检测灰葡萄孢对咯菌腈的敏感性。结果表明,在番茄1个生长季节中连续施药7次后,病菌对咯菌腈仍表现敏感,其EC50值在0.004 7～0.046 2 μg/mL之间。在含咯菌腈的PDA培养基上对30个灰葡萄孢敏感菌株进行药剂驯化7代后,共产生2个抗药性突变体(Rg-12和Rdz-28),其中Rg-12对咯菌腈的EC50值是其亲本菌株g-12的46倍,Rdz-28的EC50值大于500 μg/mL,2个抗性突变体在菌丝生长速率、产孢量和产菌核能力方面均显著低于其亲本菌株。推测灰葡萄孢可能不易对咯菌腈产生抗性,咯菌腈可作为防治番茄灰霉病的理想候选药剂。     

Biosuppression of Botrytis cinerea in grapes

There is increasing interest in the use of biological control agents (BCAs) and plant resistance stimulants to suppress botrytis bunch rot in grapes, caused by Botrytis cinerea . Numerous different filamentous fungi, bacteria and yeasts have been selected as potential BCAs for control of grey mould based upon demonstrated antagonism towards B. cinerea. Biological suppression of the pathogen arises via competition for nutrients and space, the production of inhibitory metabolites and/or parasitism. Preformed and inducible grapevine defence mechanisms also contribute to disease suppression by preventing or delaying pathogenic infection. Furthermore, various biotic and abiotic agents can stimulate grapevine defence mechanisms and so elevate resistance to B. cinerea infection. Biosuppression of B. cinerea in vineyards, using BCAs and resistance stimulants, has been inconsistent when compared with that observed in controlled glasshouse or laboratory conditions. This may be attributable, in part, to the innate variability of the field environment. Research to improve field efficacy has focused on formulation improvement, the use of BCA mixtures and combinational approaches involving BCAs and plant resistance stimulants with complementary modes of action.     

啶菌噁唑对番茄灰霉病菌的抑菌作用研究

采用生物测定方法研究了啶菌噁唑对番茄灰霉病菌Botrytis cinerea不同发育阶段的影响。结果表明:其对菌落扩展、芽管伸长、分生孢子产生和菌核形成均具有显著的抑制活性,其中啶菌噁唑对P-9菌株菌丝生长和芽管伸长的EC50值分别为0.193和0.154 μ g/mL,0.5 μ g/mL的啶菌噁唑对菌丝生长和芽管伸长的抑制率大于77%,对分生孢子和菌核萌发的抑制率分别低于10％和40%,表明药剂对分生孢子萌发无明显抑制作用。紫外吸收分析显示,啶菌 NFDA1 唑可破坏细胞的膜结构,促使菌体核酸和蛋白外渗。离体叶片毒力测定结果表明,120 μ g/mL的啶菌 NFDA1 唑可有效抑制番茄灰霉病病斑扩展,抑制率达83.74%。     

灰葡萄孢对腐霉利的抗性分子机制及快速检测技术

为明确灰葡萄孢Botrytis cinerea对腐霉利的抗性现状,于2017—2018年采用单孢分离法从浙江省5个地区的草莓大棚共分离获得200个菌株.通过区分剂量法测定了其对腐霉利的抗性,对抗药性菌株的分子机制进行了分析,并根据抗药性分子机制,建立了B.cinerea腐霉利高抗基因型的环介导等温扩增(loop-med...