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.     

The Use of a Radioimmunosorbent Assay for Botrytis cinerea1

A radioimmunosorbent assay was developed for Botrytis cinerea which can be used to detect as little as 100 ng of Botrytis mycelium (dry weight). Of 22 fungi tested in the assay, all significant reaction was restricted to members of the Sclerotiniaceae. No reaction was obtained with representative isolates of Aspergillus, Alternaria or Peiticillium. Other Botrytis species gave the most reaction relative to B. cinerea. and related genera such as Monilinia and Sclerotinia yielded small reactions. The assay can be used to detect fi. cinerea infection of grape berries when as little as 0.1 % of homogenate from infected fruit is mixed with homogenate from sound fruit to simulate a minor infection. The assay of free run juice from various lots of grapes was highly correlated with visual assessments of infection in the field. Pursuant to various theories concerning bloom time infection, the assay was used to analyse the B. cinerea content of immature berry tips and floral debris; however, no evidence of such colonization was obtained.     

Antagonism of Trichoderma harzianum ETS 323 on Botrytis cinerea Mycelium in Culture Conditions

ABSTRACT Previous studies have shown that the extracellular proteins of Trichoderma harzianum ETS 323 grown in the presence of deactivated Botrytis cinerea in culture include a putative l-amino acid oxidase and have suggested the involvement of this enzyme in the antagonistic mechanism. Here, we hypothesized that the mycoparasitic process of Trichoderma spp. against B. cinerea involves two steps; that is, an initial hyphal coiling stage and a subsequent hyphal coiling stage, with different coiling rates. The two-step antagonism of T. harzianum ETS 323 against B. cinerea during the mycoparasitic process in culture was evaluated using a biexponential equation. In addition, an l-amino acid oxidase (Th-l-AAO) was identified from T. harzianum ETS 323. The secretion of Th-l-AAO was increased when T. harzianum ETS 323 was grown with deactivated hyphae of B. cinerea. Moreover, in vitro assays indicated that Th-l-AAO effectively inhibited B. cinerea hyphal growth, caused cytosolic vacuolization in the hyphae, and led to hyphal lysis. Th-l-AAO also showed disease control against the development of B. cinerea on postharvest apple fruit and tobacco leaves. Furthermore, an apoptosis-like response, including the generation of reactive oxygen species, was observed in B. cinerea after treatment with Th-l-AAO, suggesting that Th-l-AAO triggers programmed cell death in B. cinerea. This may be associated with the two-step antagonism of T. harzianum ETS 323 against B. cinerea.     

Antagonism of Nutrient-Activated Conidia of Trichoderma harzianum (atroviride) P1 Against Botrytis cinerea

ABSTRACT The effect of preliminary nutrient activation on the ability of conidia of the antagonist Trichoderma harzianum (atroviride) P1 to suppress Botrytis cinerea was investigated in laboratory, greenhouse, and field trials. Preliminary nutrient activation at 21 degrees C accelerated subsequent germination of the antagonist at temperatures from 9 to 21 degrees C; at >/=18 degrees C, the germination time of preactivated T. harzianum P1 conidia did not differ significantly from that of B. cinerea. When coinoculated with B. cinerea, concentrated inocula of preactivated but ungerminated T. harzianum P1 conidia reduced in vitro germination of the pathogen by >/=87% at 12 to 25 degrees C; initially quiescent conidia achieved this level of suppression only at 25 degrees C. Application of quiescent T. harzianum P1 conidia to detached strawberry flowers in moist chambers reduced infection by B. cinerea by >/=85% at 24 degrees C, but only by 35% at 12 degrees C. Preactivated conidia reduced infection by >/=60% at 12 degrees C. Both quiescent and preactivated conidia significantly reduced latent infection in greenhouse-grown strawberries at a mean temperature of 19 degrees C, whereas only preactivated conidia were effective in the field at a mean temperature of 14 degrees C on the day of treatment application. An antagonistic mechanism based on initiation of germination in sufficiently concentrated inocula suggests that at suboptimal temperatures the efficacy of Trichoderma antagonists might be improved by conidia activation prior to application.     

Infection of flowers and fruits of red raspberry by Botrytis cinerea

The frequency and site of symptomless infection of flower buds, flowers, and fruits of red raspberry by Botrytis cinerea was studied using surface-disinfestation and culture techniques. Flower buds were rarely infected but open flowers were rapidly colonized and necrotic stamens and styles were an important source of infection for the developing fruit. The receptacle tissue within the drupelet cluster cavity was mostly sterile. Dichlofluanid sprays reduced symptomless infection of fruits and were more effective when applied early. Post-harvest rot tests indicated that incidence of grey mould was not related to symptomless infection unless fruits were surface-disinfested before subjecting to the rotting test.     

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.     

猕猴桃采后病害鉴定和侵染规律研究

 猕猴桃果实在0℃贮藏4周后开始出现腐烂,贮藏19周,腐烂率高达50%以上。腐烂部位几乎都在果蒂和花端两点以外的果身上,极少数是蒂腐和花端腐烂。病原菌有7种,其中Botrytis cinerea,Penicillium sp.和Phomopsis sp.3种为优势菌。病原菌主要通过采收运输时造成的机械伤口侵染,未发现在果实过熟衰老前直接侵染。     

Effects of Diffuse Colonization of Grape Berries by Uncinula necator on Bunch Rots, Berry Microflora, and Juice and Wine Quality

ABSTRACT Production of grape (principally cultivars of Vitis vinifera) for high-quality wines requires a high level of suppression of powdery mildew (Uncinula necator syn. Erysiphe necator). Severe infection of either fruit or foliage has well-documented and deleterious effects upon crop and wine quality. We found that berries nearly immune to infection by U. necator due to the development of ontogenic resistance may still support diffuse and inconspicuous mildew colonies when inoculated approximately 3 weeks post-bloom. Fruit with diffuse mildew colonies appear to be healthy and free of powdery mildew in late-season vineyard assessments with the naked eye. Nonetheless, presence of these colonies on berries was associated with (i) elevated populations of spoilage microorganisms; (ii) increased evolution of volatile ethyl acetate, acetic acid, and ethanol; (iii) increased infestation by insects known to be attracted to the aforementioned volatiles; (iv) increased rotting by Botrytis cinerea; and (v) increased frequency of perceived defects in wines prepared from fruit supporting diffuse powdery mildew colonies. Prevention of diffuse infection requires extending fungicidal protection until fruit are fully resistant to infection. Despite a perceived lack of improvement in disease control due to the insidious nature of diffuse powdery mildew, potential deleterious effects upon crop and wine quality thereby would be avoided.     

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.     

番茄灰霉病在果实上的侵染部位及防治新技术

 通过发病规律及人工接种研究,明确了番茄灰霉病菌Botrytis cinerea初侵染番茄果实的主要部位为残留的花瓣处及柱头处,随后扩展到果蒂部、脐部,最后蔓延到果实的其它部位。据此,在国内外首次提出了一种防治番茄果实灰霉病的新方法,在番茄用2,4-D蘸花后7~15 d(幼果直径10~20 mm)摘除残留的花瓣及柱头,防效达80%以上,对番茄单果重无影响。     

Infection of plum and nectarine flowers by Botrytis cinerea

The ability of Botrytis cinerea to infect plum and nectarine flowers was studied in moist chambers and in the orchard. Within 36 h of inoculation, the pathogen penetrated and colonized the stamens, styles and petals on shoots placed in moist chambers, causing blossom blight. Similar lesion development was observed following inoculation with dry and wet conidia, Hyphae were usually distorted in stylar tissue, but grew normally in petals and filaments. Growth of the fungus through filaments into the sepals or floral tubes, or through the transmitting tissue of the style into the ovary, was never observed. Symptoms of blossom blight were not observed on inoculated shoots in the orchard. The floral tube, bearing the sepals and stamens, dehisced within 14 days of fruit set and infected floral parts did not remain attached to young developing fruit. No relation was found between post-harvest decay and flower infection. Losses following post-harvest decay might have been caused by direct penetration of ripening fruit and not by flower infection. The importance of infected floral parts as a source of secondary inoculum on ripening fruit is discussed.     

Characterization of a Pterostilbene Dehydrodimer Produced by Laccase of Botrytis cinerea

ABSTRACT In the interaction between grapevines and Botrytis cinerea, one of the main aspects of pathogenicity is fungal ability to degrade phytoalexins synthesized by the plant in response to infection. Laccase-like stilbene oxidase activity in liquid cultures of B. cinerea has been shown to be related to the decrease of phytoalexin concentrations. Recent research and results presented in this paper determined the chemical structure of a pterostilbene metabolite produced by B. cinerea. Study of degradation of pterostilbene that has just one free hydroxy phenyl group function allowed us to determine the oxidative dimerization process undergone by grapevine phytoalexins after B. cinerea infection. The phytopathological significance of this degradation process in the B. cinerea interaction has also been discussed.     

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.     

Induced systemic resistance in Trichoderma harzianum T39 biocontrol of Botrytis cinerea

Biocontrol of Botrytis cinerea with Trichoderma spp. is generally believed to result from direct interaction of the biocontrol agent with the pathogen or from a Trichoderma-induced change in environmental conditions that affects B. cinerea development. In this work we provide arguments for the participation of induced plant defence in T. harzianum T39 control of B. cinerea. In tomato, lettuce, pepper, bean and tobacco, T. harzianum T39 application at sites spatially separated from the B. cinerea inoculation resulted in a 25–100%percnt; reduction of grey mould symptoms, caused by a delay or suppression of spreading lesion formation. Given the spatial separation of both micro-organisms, this effect was attributed to the induction of systemic resistance by T. harzianum T39. The observation that in bean the effect of T. harzianum T39 was similar to that of the rhizobacterium Pseudomonas aeruginosa KMPCH, a reference strain for the induction of systemic resistance, confirmed this hypothesis. Since B. cinerea control on tobacco leaves sprayed with T. harzianum T39 was similar to the control on leaves from T. harzianum T39 soil-treated plants, induction of plant defence might also participate in biocontrol on treated leaves.     

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。     

Significance of leaf infection by Botrytis cinerea in stem rotting of tomatoes grown in non-heated greenhouses

The most serious symptom of Botrytis cinerea in tomatoes grown in greenhouses is stem rotting. Lesions on the stem may result from direct infection or from progression of the rot along infected leaves, until infection approaches the stem. In a set of experiments conducted in commercial greenhouses, an experimental greenhouse and growth chambers, the significance of the two types of stem infections was studied. In non-heated greenhouses most of the stem lesions originated from progression of the pathogen along infected petioles. The rate at which B. cinerea had progressed on infected petioles was 0.3–0.5 cm/day, an average of ca. 6 weeks was needed for a leaf infection to approach the stem. Application of Trichoderma harzianum T39 extended this time by 1–2 weeks and application of chemical fungicides by 3 weeks. Influence of the environment on the progression of B. cinerea along infected petioles was then determined. Within range of 5–30 °C, the higher the temperature, the more rapid was the rate of disease progression. The fungus progressed more rapidly on tomato petioles incubated at high vapour pressure deficit (VPD) rather than at low VPD. The source-sink relationship of the plant governed the rate of B. cinerea progression along the petioles as well: it was more rapid when the source was restricted (by shading) and slower when the sink was restricted (by removal of flowers and small fruits). The possibility that sanitation of infected leaflets would reduce the incidence of stem rotting was examined in two experiments. In plots not treated with a fungicide, the sanitation treatment substantially decreased the incidence of stem lesions and this treatment was as effective as weekly application of chemical fungicides.     

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.