Incidence, Aggressiveness and In Planta Interactions of Botrytis cinerea and other Filamentous Fungi Quiescent in Grape Berries and Dormant Buds in Central Washington State

Recovery of quiescent filamentous fungi from non‐symptomatic grape berries and dormant buds demonstrated dominance of Alternaria, Aureobasidium, Cladosporium, Ulocladium and other dematiaceous hyphomycetes. Up to 78% of berries contained fungi prior to harvest. Botrytis cinerea was recovered from 0.2 to 0.5% of surface‐disinfested berries just subsequent to fruit set, and 1.6–4.8% of surface‐disinfested, over‐wintered dormant buds. In laboratory inoculations of mature grape berries with strains of Alternaria, Aureobasidium, Cladosporium, Ulocladium and Botrytis, only the latter was aggressive in rotting berry fruits. Inoculations with B. cinerea alone and in combination with strains of Alternaria, Aureobasidium, Cladosporium and Ulocladium recovered from grape demonstrated that prior occupation of wound sites by the latter fungi resulted in reduced lesion size compared to inoculation with B. cinerea alone.     

Enhancement of in vitro growth and resistance to gray mould of Vitis vinifera co-cultured with plant growth-promoting rhizobacteria

The potential of a plant growth-promoting rhizobacterium, Pseudomonas sp. (strain PsJN), to stimulate the growth and enhancement of the resistance of grapevine (Vitis vinifera L.) transplants to gray mould caused by Botrytis cinerea has been investigated. In vitro inoculation of grapevine plantlets induced a significant plant growth promotion which made them more hardy and vigorous when compared to non-inoculated plantlets. This ability increased upon transplanting. When grown together with B. cinerea, the causal agent of gray mould, significant differences of aggressiveness were observed between the inoculated and non-inoculated plants. The presence of bacteria was accompanied by an induction of plant resistance to the pathogen. The beneficial effect from this plant-microbe association is being postulated.     

葡萄无核基因定位与作图的研究

以UBC 269484和GSLP1569的序列为支点,设计合成了包括UBC 269和GSLP1在内的9条引物,以葡萄 有核亲本红地球和无核亲本红光无核的DNA为模板,对这9个引物进行筛选,结果GSLP1、39970524 5号引物和 39970524 6号引物在无核亲本红光无核上扩增出了特异标记GSLP1569、39970524 5 564、39970524 6 1538和 39970524 6 1200。用这3个特异引物在红地球、红光无核、无核白和红地球×红光无核杂交组合F1代163株杂 种的DNA样上进行PCR扩增,结果4个特异标记在F1群体中与无核主效基因共分离。4个特异标记也出现于所 用组合中无核基因原始供给者无核白上。这些标记和葡萄无核主效基因相连锁。用QTXb17遗传作图软件,对葡 萄无核主效基因S定位与作图,当P=0.01时,LOD值在32.7～46.4之间,置信界限在0.2～9.9之间。这4个 特异标记和无核主效基因S处于在同一连锁群,位于无核主效基因S的两侧,覆盖基因组12.3cM。特异标记 39970524 5 564、GSLP1569、39970524 6 1538、39970524 6 1200距S基因的遗传距离分别为0.6cM、1.2cM、 4.9cM和11.1cM。     

病原侵染对采后葡萄激素水平的影响

外源ABA处理采后葡萄可显著促进乙烯释放和抑制IAA的生成,加重葡萄落粒。乙烯利对ABA促进作用不明显,但显著抑制IAA和GA3的生成。葡萄感病后组织内源激素水平发生了变化：ABA含量显著增加,乙烯释放率增大,IAA含量显著减少,这种变化趋势在果梗中尤为突出。     

Tracking cell wall changes in wine and table grapes undergoing Botrytis cinerea infection using glycan microarrays

Background and AimsThe necrotrophic fungus Botrytis cinerea infects a broad range of fruit crops including domesticated grapevine Vitis vinifera cultivars. Damage caused by this pathogen is severely detrimental to the table and wine grape industries and results in substantial crop losses worldwide. The apoplast and cell wall interface is an important setting where many plant–pathogen interactions take place and where some defence-related messenger molecules are generated. Limited studies have investigated changes in grape cell wall composition upon infection with B. cinerea, with much being inferred from studies on other fruit crops.MethodsIn this study, comprehensive microarray polymer profiling in combination with monosaccharide compositional analysis was applied for the first time to investigate cell wall compositional changes in the berries of wine (Sauvignon Blanc and Cabernet Sauvignon) and table (Dauphine and Barlinka) grape cultivars during Botrytis infection and tissue maceration. This was used in conjunction with scanning electron microscopy (SEM) and X-ray computed tomography (CT) to characterize infection progression.Key ResultsGrapes infected at veraison did not develop visible infection symptoms, whereas grapes inoculated at the post-veraison and ripe stages showed evidence of significant tissue degradation. The latter was characterized by a reduction in signals for pectin epitopes in the berry cell walls, implying the degradation of pectin polymers. The table grape cultivars showed more severe infection symptoms, and corresponding pectin depolymerization, compared with wine grape cultivars. In both grape types, hemicellulose layers were largely unaffected, as was the arabinogalactan protein content, whereas in moderate to severely infected table grape cultivars, evidence of extensin epitope deposition was present.ConclusionsSpecific changes in the grape cell wall compositional profiles appear to correlate with fungal disease susceptibility. Cell wall factors important in influencing resistance may include pectin methylesterification profiles, as well as extensin reorganization.     

Characterisation of the yeast Pichia membranifaciens and its possible use in the biological control of Botrytis cinerea, causing the grey mould disease of grapevine

Pichia membranifaciens strain FY-101, isolated from grape skins, was found to be antagonistic to Botrytis cinerea, the causal organism of the grey mould disease of the grapevine. When grown together on solid as well as liquid media, the yeast brings about the inhibition of this parasitic fungus, coagulation and leakage of its cytoplasm, and suppression of its ability to produce the characteristic grey mould symptoms on the grapevine plantlets. In vitro experiments confirm that this yeast can be used as a biological control organism against B. cinerea. An account of the molecular characterisation of P. membranifaciens (complete sequence of the ITS region of its ribosomal DNA, GenBank accession No. AF 270935), as well as the interaction between B. cinerea and the yeast, are given here.     

Co‐operational PCR Coupled with Dot Blot Hybridization for the Detection of Phaeomoniella chlamydospora on Infected Grapevine Wood

The technique consisting of the co‐operational PCR coupled with dot blot hybridization and posterior colorimetric visualization was developed for the detection of Phaeomoniella chlamydospora, one of the major pathogenic fungi involved in the Petri disease of grapevine. A partial region of the fungal rDNA including the internal transcribed spacer (ITS) region was amplified through co‐operational PCR for P. chlamydospora and 17 additional grapevine‐associated fungi included in the genera Botryosphaeria, Cryptovalsa, Cylindrocarpon, Dematophora, Diplodia, Dothiorella, Eutypa, Fomitiporia, Lasiodiplodia, Neofusicoccum, Phaeoacremonium, Phomopsis and Stereum, by using the primer pairs NSA3/NLC2 (external pair) and NSI1/NLB4 (inner pair). A specific probe (Pch2D) targeting the ITS2 region in the rDNA was developed for the detection of P. chlamydospora. Dot blot hybridizations carried out with the PCR products showed the specificity of the probe. Results indicated that Pch2D only hybridized with DNA amplicons of P. chlamydospora isolates, thus proving the specific detection of this fungus, while the 17 remaining species tested for the Pch2D probe resulted in negative results. Sensitivity of the technique was established below 0.1 pg of genomic DNA. This technique was further validated using artificially inoculated grapevine cuttings with P. chlamydospora. The efficacy of detection was established at 80% after two independent blind assays.     

Diapause dynamics, seasonal phenology, and pupal color dimorphism of Papilio polyxenes in southern Florida, USA

In the European grapevine moth, Lobesia botrana (Denis and Schiffermüller) (Lepidoptera: Tortricidae), it has recently been shown that volatiles emitted from the main host plant, grapevine, are attractive to adult females. Here, using wind tunnel experiments, we tested the attractiveness of various grapevine, Vitis vinifera L. (Vitaceae), plant parts at different phenological stages, including ripe berries infested with the pathogenic fungus Botrytis cinerea (Persoon: Fries) (Sclerotiniaceae) to laboratory‐reared virgin and mated male and female moths. We also tested the attractiveness of the non‐host plant, tansy [Tanacetum vulgare (L.) (Asteraceae)], as flowers and flower volatiles were previously shown to be attractive to L. botrana females in the field. Mated female moths were the only adults to exhibit upwind orientation to grape plant parts in the flight tunnel. The most attractive parts of the grapevine plants were leaves, flower buds, and ripe berries. No attraction was observed to flowers. This corresponds to the phenological stages available when females of the 2–3 generations during a year are present in the field. No attraction was observed to leaves and flowers of tansy.     

乙醇对‘木纳格’葡萄采后贮藏期间品质的影响

葡萄采收后分别用不同浓度乙醇处理放在低温下贮藏,测定其品质指标的结果表明：40％的乙醇能有效控制葡萄果实的腐烂,对果实可溶性固形物含量则无影响,还可减缓果实硬度和可滴定酸含量下降,以及抑制细胞膜透性的增加,从而提高果实的贮藏品质。     

葡萄蔗糖转化酶基因家族的生物信息学及响应逆境胁迫分析

蔗糖转化酶(invertase, INV)在植物生长发育和抵御胁迫中发挥着重要作用。研究从葡萄基因组数据库中鉴定出19个蔗糖转化酶基因,对基因结构和编码蛋白质的理化性质进行生物信息学分析,并利用qRT-PCR技术分析基因在不同激素和非生物胁迫条件下的表达特征,为进一步探索葡萄INV基因家族参与葡萄逆境响应提供了一定的理论依据。结果表明,(1)该基因家族编码蛋白的氨基酸长度在150～766 aa之间,理论等电点介于4.43～9.1之间,亚细胞定位预测发现其主要在细胞质中表达,此外液泡和细胞壁也存在部分基因表达;(2)共线性结果显示VvCINV与其他5个物种复制频率较高;(3)保守基序分析表明VvCwINV包含了所有的保守基序,且Glyco＿32和Glyco＿hydro＿100是VvINV基因主要结构域;(4)组织特异性表达分析发现多数基因在葡萄生长发育进程中都有表达;(5)qRT-PCR分析结果显示,VvINV基因家族在叶片中对激素处理和非生物胁迫的响应出现上调,VvCINV1在50 mg/L GA₃和10%PEG处理后上调表达极显著,VvCINV4在盐胁迫、ABA...     

灰葡萄孢交配型基因的分析与检测

通过生物信息对灰葡萄孢的MAT1‐1‐1与MAT1‐2‐1氨基酸序列进行了系统进化与结构域保守氨基酸分析,表明灰葡萄孢的交配型蛋白与核盘菌的亲源关系最近,结构域氨基酸比对结果表明该基因具有保守氨基酸的一致性与部分氨基酸的相似性。应用PCR技术检测灰葡萄孢交配型基因MAT1‐1‐1与MAT1‐2‐1,结果表明各种植区交配型菌株所占比例有较大的差异,多数种植区灰葡萄孢同时存在MAT1‐1与MAT1‐2两种交配类型,快速检测灰葡萄孢的交配型等位基因对于灰葡萄孢种群结构分析非常有意义。     

Brief in vitro study on Botrytis cinerea and Aspergillus carbonarius regarding growth and ochratoxin A

Aims: To evaluate the effect of Botrytis cinerea growth on ochratoxin A (OTA) production by Aspergillus carbonarius and degradation. Methods and Results: OTA‐producing A. carbonarius and B. cinerea were grown on grape‐like medium at 20°C for 7 days. Radii of colonies were daily recorded and OTA was analysed. In addition, each B. cinerea isolate was inoculated on grape‐like synthetic nutrient medium (SNM) paired with each A. carbonarius isolate at a distance of 45 mm. Botrytis cinerea isolates were also grown in OTA‐spiked SNM. Growth rates of B. cinerea and A. carbonarius were 20 and 7·5 mm day^?1, respectively. The growth of the colonies of each species stopped when they contacted each other in paired cultures. OTA production by A. carbonarius in the contact area was affected by B. cinerea, but no clear trend was observed. All B. cinerea isolates showed to degrade between 24·2% and 26·7% of OTA from spiked SNM. Conclusions: The ecological advantage of B. cinerea, in terms of growth rate, vs. OTA‐producing Aspergillus in some wine‐growing regions and its ability to degrade OTA may explain the low levels of this toxin in noble wines. Significance and Impact of the Study: At determinate conditions, the presence of B. cinerea in grapes with A. carbonarius may help in reducing OTA accumulation.     

Latency of Botrytis cinerea Pers.: Fr. and Biochemical Studies During Growth and Ripening of Two Grape Berry Cultivars, Respectively Susceptible and Resistant to Grey Mould

Latency and development of Botrytis cinerea were assessed under field conditions and after artificial inoculation of two grape varieties, Gamay (susceptible) and Gamaret (resistant). When the percentage of latent Botrytis was the same for both varieties, severity of visible grey mould remained very low in Gamaret berries, while Gamay clusters were destroyed by the disease to a high percentage. Some biochemical parameters were measured in berries, such as constitutive and induced anti‐fungal compounds, polymeric proanthocyanidins and lipid peroxidation products as markers of senescence. Differences were observed in polymeric proanthocyanidins (PPRA) of Gamaret compared with those of Gamay. Concentration and mean degree of polymerization (mDP) of PPRA were always higher in the berries of the resistant variety. The inhibitory effect of Gamaret PPRA on enzyme activity remained until harvest whereas Gamay PPRA lost their inhibitory activity at the beginning of véraison. Based on these results, resistance to B. cinerea seems to be linked to the maintainance of the fungus in its latent form in berries, mainly due to the ability of Gamaret PPRA to inhibit macerating fungal enzyme activities.     

葡萄胚胎发育与败育过程中胚珠的多胺含量变化

花后40~60d期间,种子败育型无核葡萄胚珠的3种多胺含量明显低于有核葡萄,据此认为无核葡萄胚珠中多胺含量急剧下降和较低的多胺水平可能是导致葡萄胚胎败育的重要原因。(Spd Spm)/Put和Spm/PAs比值变化与葡萄胚胎发育密切相关,较低的比值不利于胚胎的正常分化。     

灰霉病菌抗药位点及其分子检测方法研究进展

灰霉病由灰葡萄孢侵染所致,化学防治是目前最常用的治理方法,而随着杀菌剂的广泛使用,抗药菌株频繁出现。本文就近年来已研究报道的灰葡萄孢菌的抗药分子位点进行了系统总结,包括六大类杀菌剂,涉及5个基因;对灰霉病菌抗药位点的分子检测方法进行了综述,包括测序法、CAPS、ARMS、Tetra primer ARMS-PCR、AS real-time PCR、ASPPAA PCR和双杂交探针法,通过对不同检测方法进行比较分析,指出现有技术存在的问题并展望未来高通量的检测方法的发展方向。     

高温胁迫下不同光强对‘赤霞珠’葡萄PSII活性及恢复的影响

本文研究了高温与不同光强结合处理对‘赤霞珠’葡萄叶片PSII活性及恢复的影响。结果表明,高温黑暗处理（40℃,0μmaol·m-2．s-1）导致叶片PSII最大光化学效率（Fv/Fm）、反应中心吸收的光能用于电子传递的量子产额（ψEo）与单位反应中心光能的传递（ETo/RC）降低明显,且无恢复趋势,K点相对荧光（Vk）、单位反应中心光能的吸收（ABS／RC）与捕获（TRo／RC）显著升高。高温弱光处理（40℃,200μmol·m-2．s-1）后的叶片PSII活性明显恢复,ETo／RC降低明显,TRo/RC无显著变化。高温强光（40℃,1600μmol·m-2．S-1）处理导致单位面积有活性反应中心数量（RC/CSm）抑制程度最大,恢复程度较低。实验结果说明,高温处理下黑暗对葡萄PSII功能活性及恢复均会造成抑制,而弱光可以显著缓解高温对葡萄叶片的胁迫作用,并促进PSII的恢复,强光导致胁迫下的PSII功能抑制最明显。     

Symptoms, Wood Lesions and Fungi Associated with Esca in Organic Vineyards in Languedoc-Roussillon (France)

We analysed symptoms, wood lesions and fungi associated with esca in mature organic vineyards in the Languedoc‐Roussillon region. Most previous surveys concerning esca syndrome were conducted in conventionally managed vineyards in other regions. We first found that esca may be present at a very low level in vineyards that were not treated with sodium arsenite. Affected vines displayed three types of symptoms: leaves with interveinal necrosis, wilt of entire branches and these two symptoms in combination. During the 3‐year survey, not all affected vines displayed symptoms every year and the same vine rarely displayed the same symptom or combination of symptoms in successive years. The incidence of esca appeared to be correlated with the percentage of vines that died during the survey but no correlation was found with either mortality before the survey or with the age of the vineyard. Observation of cross‐sections of a total of 210 vines with esca symptoms and isolation of fungi from the wood lesions led to similar results to those obtained in conventionally managed vineyards. Four different lesions were identified: a white rot lesion, a brown lesion in a central position, a brown lesion in a sectorial position and a scattered black spotting in healthy wood. The most frequently observed fungal species were Fomitiporia mediterranea (Fom), Phaeomoniella chlamydospora, Phaeoacremonium aleophilum and Eutypa lata. The white rot lesion caused by Fom was generally accompanied by one or more other lesions in the same vine. Similarly, Fom was generally isolated with one or several other fungi. No relationship was detected between the expression of external symptoms and any specific lesion or fungus. Our study also showed that esca and Eutypa dieback are often superimposed in our region of sampling.