Degradation of stilbene-type phytoalexins in relation to the pathogenicity of Botrytis cinerea to grapevines

The ability of eight isolates of Botrytis cinerea to degrade the stilbene phytoalexins, resveratrol and pterostilbene, was compared with their pathogenicity to grapevines. All strains which degraded resveratrol and pterostilbene were highly or moderately pathogenic to in vitro cultures of grapevines ( Vitis rupestris ) after inoculation with agar disks containing mycelium, while those which were unable to degrade phytoalexins were non-pathogenic. In all cases, the hydroxystilbene-degrading activity was related to the presence of laccase activity in the culture filtrates, as shown by using syringaldazine as substrate. The role of laccase-mediated degradation of phytoalexins in relation to pathogenicity of B. cinerea to grapevines in discussed.     

Botrytis cinerea Infection in Grape Flowers: Defense Reaction, Latency, and Disease Expression

ABSTRACT Inflorescences of field-grown grapevines (Vitis vinifera L. cv. Gamay) were inoculated with a Botrytis cinerea conidia suspension or dried conidia at different stages during bloom in moist weather. Approximately 10% of the conidia germinated within 72 h, resulting in two to three times more latent infections than uninoculated controls in pea-size (7 mm in diameter) berries. In surface-sterilized pea-size berries, latent B. cinerea was present predominantly in the receptacle area. After veraison, latent B. cinerea also was found in the style and, in mature berries, latent colonies were distributed throughout the pulp. Inoculation at full bloom led to the highest disease severity (66%) at harvest, compared with 38% in controls. Stilbene stress metabolites in the flowers were measured by high-performance liquid chromatography. Resveratrol accumulated mainly after pre-bloom and full-bloom inoculation, but did not prevent infection. Piceid levels did not change following inoculation, while epsilon-viniferin was found in necrotic tissues only, and pterostilbene and alphaviniferin were not detected at all. B. cinerea conidia suspensions also were applied to various locations on flowers of pot-grown cvs. Pinot Noir and Chardonnay. Inoculation of the receptacle area, but not that of the stigma and ovary, resulted in latent infections. Stilbene synthesis was similar to the field results, with resveratrol accumulating mainly in the calyptra and receptacle area. Constitutive soluble phenolic compounds (mainly derivatives of quercetin and hydroxy-cinnamic acid) were present at high concentrations in the calyptra but at low levels in the receptacle area. These experiments confirmed bloom as a critical time for B. cinerea infection in grapes and suggest that the most likely site of infection is the receptacle area or cap scar exposed at anthesis. Stilbenes may have a limited role in inhibition of flower infection and latency in susceptible grape cultivars, and epsilon-viniferin may be a by-product rather than a deterrent of infection.     

Ultrastructural and Cytochemical Aspects of the Biological Control of Botrytis cinerea by Candida saitoana in Apple Fruit

ABSTRACT Biocontrol activity of Candida saitoana and its interaction with Botrytis cinerea in apple wounds were investigated. When cultured together, yeast attached to Botrytis sp. hyphal walls. In wounded apple tissue, C. saitoana restricted the proliferation of B. cinerea, multiplied, and suppressed disease caused by either B. cinerea or Penicillium expansum. In inoculated apple tissue without the yeast, fungal colonization caused an extensive degradation of host walls and altered cellulose labeling patterns. Hyphae in close proximity to the antagonistic yeast exhibited severe cytological injury, such as cell wall swelling and protoplasm degeneration. Colonization of the wound site by C. saitoana did not cause degradation of host cell walls. Host cell walls in close contact with C. saitoana cells and B. cinerea hyphae were well preserved and displayed an intense and regular cellulose labeling pattern. In addition to restricting fungal colonization, C. saitoana induced the formation of structural defense responses in apple tissue. The ability of C. saitoana to prevent the necrotrophic growth of the pathogen and stimulate structural defense responses may be the basis of its biocontrol activity.     

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.     

Influence of drought, salt stress and abscisic acid on the resistance of tomato to Botrytis cinerea and Oidium neolycopersici

Abiotic stress may affect plant response to pathogen attack through induced alterations in growth regulator and gene expression. Abscisic acid (ABA) mediates several plant responses to abiotic stress. The effects of drought, salt stress and ABA on the interaction of tomato ( Lycopersicon esculentum ) with the biotrophic fungus Oidium neolycopersici and the necrotrophic fungus Botrytis cinerea were investigated. Drought stress resulted in a twofold increase in endogenous ABA as well as a 50% reduction in B. cinerea infection and a significant suppression of O. neolycopersici on tomato cv. Moneymaker. Salt stress did not affect B. cinerea infection, but significantly reduced infection by O. neolycopersici , with no obvious increase in endogenous ABA. Compared with the wild type, the ABA-deficient sitiens mutant was more resistant to O. neolycopersici and B. cinerea . Exogenous ABA resulted in increased susceptibility of sitiens to both pathogens, but did not increase the basal susceptibility of wild-type plants. It is concluded that, in tomato, drought and salt stress stimulate different, but possibly overlapping, pathogen-defence pathways which may not necessarily involve ABA. Meanwhile, basal endogenous ABA levels suppress the resistance of tomato to O. neolycopersici and B. cinerea , but an ABA increase above the basal level, resulting from exogenous application, does not increase susceptibility to these pathogens.     

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.     

Virulence factors of Botrytis cinerea

Botrytis cinerea is responsible for gray mold disease in more than 200 host plant species. The infection of host plants is mediated by numerous extracellular enzymes, proteins and metabolites. Each of these compounds may play a role in different stages of the infection process. Cell wall-degrading enzymes may facilitate the penetration into the host surface, while toxins, oxalic acid and reactive oxygen species may contribute to killing of the host cells. Cell wall-degrading enzymes contribute to the conversion of host tissue into fungal biomass. On the other hand, B. cinerea infection induces biosynthesis of phytoalexins. Therefore, the ability to overcome a wide spectrum of phytoalexins contributes to the pathogenicity of the fungus with a broad host range. The cloning of the corresponding genes has facilitated studies on gene expression and targeted mutagenesis. This review gives an overview of the research performed on virulence factors that play the roles in pathogenesis.     

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.     

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.     

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.     

Infection of Commercial Hybrid Primula Seed by Botrytis cinerea and Latent Disease Spread Through the Plants

ABSTRACT Botrytis cinerea occurred commonly on cultivated Primula xpolyantha seed. The fungus was mostly on the outside of the seed but sometimes was present within the seed. The fungus frequently caused disease at maturity in plants grown from the seed, demonstrated by growing plants in a filtered airflow, isolated from other possible sources of infection. Young, commercially produced P. xpolyantha plants frequently had symptomless B. cinerea infections spread throughout the plants for up to 3 months, with symptoms appearing only at flowering. Single genetic individuals of B. cinerea, as determined by DNA fingerprinting, often were dispersed widely throughout an apparently healthy plant. Plants could, however, contain more than one isolate.     

酵母挥发性物质环辛四烯抑制灰霉菌的研究

为了探明生防酵母菌菌株C410产生的挥发性物质对灰霉菌的抑制作用机理,本文以挥发性物质的主要成分之一环辛四烯为研究对象,测定其对灰霉菌的糖、蛋白、DNA、RNA、细胞的形态及细胞膜通透性的影响。结果表明,在16.5μL/L环辛四烯的作用下,灰霉菌生长速率显著降低,总糖含量基本不变;还原糖含量显著降低;蛋白含量变化不显著;RNA的完整性显著降低。在49.5μL/L环辛四烯的作用下,灰霉菌的生长几乎完全停止,但细胞通透性不受影响,不导致细胞死亡。环辛四烯孵育下灰霉菌DNA分子没有被打断。由此推测还原糖和RNA代谢过程是环辛四烯作用于灰霉菌的重要靶标,抑制其生长及侵染。     

Improved resistance against Botrytis cinerea by grapevine-associated bacteria that induce a prime oxidative burst and phytoalexin production

Bacteria such as Pantoea agglomerans (Pa-AF2), Bacillus subtilis (Bs-271), Acinetobacter lwoffii (Al-113), and Pseudomonas fluorescens (Pf-CT2), originating from the vineyard, can induce defense responses and enhance resistance of grapevine against the fungal pathogen Botrytis cinerea. The perception of these bacteria by plant cells or tissues in relation to their activities remains unknown. In this study, we examined the relationships between the activity of each bacterium to induce or prime some defense responses, and its effectiveness to induce resistance in grapevine against B. cinerea. We showed that all selected bacteria are capable of inducing early oxidative burst and phytoalexin (trans-resveratrol and trans-ε-viniferin) production in grapevine cells and leaves. Pf-CT2 and Al-113 induced higher H(2)O(2) and trans-resveratrol accumulations, and were able to further prime plants for accelerated phytoalexin production after B. cinerea challenge. These two bacteria were also the most effective in inducing local and systemic resistance. A similar level of induced resistance was observed with live Pa-AF2 which also induced but not primed a greater accumulation of trans-resveratrol. However, Bs-271, which was less effective in inducing resistance, induced a lower trans-resveratrol synthesis, without priming activity. Treatment of grapevine cells with growing medium or crude extract of the bacteria quickly and strongly enhanced oxidative burst compared with the live bacteria. However, both treatments resulted in comparable amounts of phytoalexins and induced local and systemic resistance to B. cinerea as compared with those induced by living bacteria, with extracts from Pf-CT2 and Al-113 being the most effective. Together, these results indicate that induced resistance can be improved by treatment with bacteria or derived compounds which induced or primed plants for enhanced phytoalexin accumulation.     

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.     

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.     

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.     

喜树碱对番茄灰霉病菌的病理反应

以番茄灰霉病菌为供试菌种,采用电导率法、吸光度法和电镜分析法对经过喜树碱（CPT）处理的番茄灰霉病菌进行病理反应研究。试验结果表明经200 mg/L CPT溶液处理后,番茄灰霉病菌菌丝细胞膜的通透性增加了30.7%,细胞外蛋白质浓度提高了13.5%。番茄灰霉病菌经CPT溶液处理后,还原糖和可溶性蛋白的含量随着处理时间的增长先升高后降低最后趋于平缓,在0～1 h之间增长最快。CPT处理后的番茄灰霉病菌的菌丝畸形,出现膨大或缢缩,菌丝生长点缢缩,细胞中的空腔增多,线粒体增多且线粒体嵴变得模糊,细胞核有降解的现象。     

Chitosan oligomers and copper sulfate induce grapevine defense reactions and resistance to gray mold and downy mildew

ABSTRACT Chitosan (CHN), a deacetylated derivative of chitin, was shown to be efficient in promoting plant defense reactions. CHN oligomers of different molecular weight (MW) and degree of acetylation (DA) triggered an accumulation of phytoalexins, trans- and cis-resveratrol and their derivatives epsilon-viniferin and piceid, in grapevine leaves. Highest phytoalexin production was achieved within 48 h of incubation with CHN at 200 mug/ml with an MW of 1,500 and a DA of 20% (CHN1.5/20), while oligomers with greater MW were less efficient, indicating that a specific MW threshold could be required for phytoalexin response. Treatment of grapevine leaves by highly active CHN1.5/20 also led to marked induction of chitinase and beta-1,3-glucanase activities. CHN1.5/20 applied together with copper sulfate (CuSO(4)) strongly induced phytoalexin accumulation. CuSO(4) alone, especially at low concentrations also elicited a substantial production of phytoalexins in grapevine leaves. Evidence is also provided that CHN1.5/20 significantly reduced the infection of grapevine leaves by Botrytis cinerea and Plasmopara viticola, and in combination with CuSO(4) conferred protection against both pathogens.     

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.     

Evidence for oxidative detoxication of pterostilbene and resveratrol by a laccase-like stilbene oxidase produced by Botrytis cinerea

Botrytis cinerea produces a hydroxystilbene-degrading enzyme when grown on a pectin-containing medium. The enzyme, stilbene oxidase, has been identified as a laccasc. Stilbene oxidase oxidizes both pterostilbene and resveratrol to produce non-toxic products. The phytopathological significance of the degradation is discussed.