New Insights into the Ecological Interaction Between Grape Berry Microorganisms and Drosophila Flies During the Development of Sour Rot

In this work, we studied the ecological interactions between grape berry microorganisms and Drosophila sp. flies involved in sour rot disease during grape ripening. After veráison the total microbial counts of grape berries affected by sour rot increased from about 2 log CFU/g of berries to more than 7 log CFU/g. Berry damage provoked a clear shift in yeast diversity from basidiomycetes to ascomycetous fermentative species. The latter were mostly Pichia terricola, Hanseniaspora uvarum, Candida zemplinina, and Zygoascus hellenicus. However, these species were not able to produce the metabolites characteristic of sour rot (gluconic and acetic acids) in inoculated berries. On the contrary, the acetic acid bacteria Gluconacetobacter saccharivorans produced high levels of these acids, mainly when berries were incubated in the presence of the insect Drosophila sp. Sour rot was not observed when grape bunches were physically separated from insects, even when berries were artificially injured. The wounds made in berry skin healed in the absence of insects, thus preventing the development of sour rot. Therefore, in the vineyard, the induction of sour rot depends on the contamination of wounded berries by a microbial consortium--yeasts and acetic acid bacteria--transported by drosophilid insects which disseminate sour rot among damaged berries. In the absence of these insects, plant defense mechanisms are effective and lead to skin healing, preventing disease spread. Thus, we showed that Drosophila sp. act as a vector for microorganisms associated with grape sour rot disease.     

Identification of Colletotrichum Species Causing Bitter Rot of Apple and Pear in Croatia

Symptoms of bitter rot were observed on apple and pear fruits in the field and in storage in Croatia between 2009 and 2011. Fifteen Colletotrichum isolates from apple and two from pear were collected and identified by sequencing of the ITS1 and ITS2 regions of the ribosomal DNA. Phylogenetic analysis revealed that ten isolates from apple and two isolates from pear could be identified as Colletotrichum fioriniae, five isolates from apple were clustered in Colletotrichum clavatum, while one isolate was in the Colletotrichum acutatum A7 group. All isolates caused typical bitter rot symptoms when inoculated on apple and pear fruits.     

Antifungal activity and biocontrol potential of Paenibacillus polymyxa HT16 against white rot pathogen (Coniella diplodiella Speq.) in table grapes

Paenibacillus polymyxa HT16 was evaluated for its ability to reduce white rot disease caused by Coniella diplodiella. HT16 showed significant inhibitory effects on C. diplodiella in vitro, reducing white rot disease in ‘Kyoho’ and ‘Thompson Seedless’ table grape varieties by more than 40% after inoculation. Liquid cultures were more effective than washed cell suspensions or cell-free supernatants, and a higher content of, and longer incubation time with, strain HT16 were more effective in reducing disease incidence. The spore germination rate and mycelial morphology of C. diplodiella were adversely affected by HT16. These results suggested that HT16 may be a promising biocontrol agent against pre-harvest and post-harvest decay in table grapes.     

Infection of Vitis vinifera (cv Chardonnay) Inflorescences by Colletotrichum acutatum and Greeneria uvicola

Detached Vitis vinifera (cv Chardonnay) inflorescences were inoculated with spore suspensions of either Colletotrichum acutatum or Greeneria uvicola at 25°C, and a combination of light microscopy, scanning electron microscopy and plating out of inoculated flowers on to dichloran rose bengal chloramphenicol agar was used to investigate the time frame for infection. Colletotrichum acutatum infection commenced within 2 h of inoculation, while infection by G. uvicola commenced between 12 and 18 h postinoculation. All parts of the flowers were infected by both fungi.     

Origin of (−)-Geosmin on Grapes: On the Complementary Action of Two Fungi, <Emphasis Type="Italic">Botrytis Cinerea</Emphasis> and <Emphasis Type="Italic">Penicillium Expansum</Emphasis>

One of the consequences of rot on grapes is the development of volatile compounds giving fungal, mouldy or earthy odours. Among these compounds, (−)-geosmin (trans-1,10-dimethyl-trans-9-decalol), a powerful aromatic compound with an earthy smell is a persistent defect in grape juice and wines made with at least partially rotten grapes. A microbiota analysis of rotten grapes containing (−)-geosmin was carried out on sites from four French regions from 1999 to 2002, to clarify the involvement in geosmin appearance of Streptomyces spp. and Penicillium spp., two types of microorganisms present on grape, that are known for their ability to produce geosmin. In earthy grapes, Botrytis cinerea was largely present. Different species of Streptomyces were also isolated, but their pH sensitivity was an extremely limiting parameter for their development on grape juice, grapes or stem, and consequently for their potentiality to generate geosmin in the vineyard. Penicillium expansum, producing geosmin on a model medium, was omnipresent. Penicillium carneum, which is also a geosmin producer, was represented by a single colony during the 4 years of this study. P. expansum alone was able to produce geosmin on a model medium but not on grapes. However, after 7 days’ pre-culture of some B. cinerea strains on grape juice, this juice became favourable to geosmin production by P. expansum. We demonstrated the necessary and complementary action of B. cinerea and P. expansum in geosmin production in grape juice and in crushed grape berries.     

A methyltransferase essential for the methoxypyrazine‐derived flavour of wine

Methoxypyrazines are a family of potent volatile compounds of diverse biological significance. They are used by insects and plants in chemical defence, are present in many vegetables and fruit and, in particular, impart herbaceous/green/vegetal sensory attributes to wines of certain varieties, including Cabernet Sauvignon. While pathways for methoxypyrazine biosynthesis have been postulated, none of the steps have been confirmed genetically. We have used the F₂ progeny of a cross between a rapid flowering grapevine dwarf mutant, which does not produce 3‐isobutyl‐2‐methoxypyrazine (IBMP), and Cabernet Sauvignon to identify the major locus responsible for accumulation of IBMP in unripe grape berries. Two candidate methyltransferase genes within the locus were identified and one was significantly associated with berry IBMP levels using association mapping. The enzyme encoded by this gene (VvOMT3) has high affinity for hydroxypyrazine precursors of methoxypyrazines. The gene is not expressed in the fruit of Pinot varieties, which lack IBMP, but is expressed in Cabernet Sauvignon at the time of accumulation of IBMP in the fruit. The results suggest that VvOMT3 is responsible for the final step in methoxypyrazine synthesis in grape berries and is the major determinant of IBMP production.     

Pierce's Disease of Grapevines in Taiwan: Isolation,Cultivation and Pathogenicity of Xylella fastidiosa

Characteristic symptoms of Pierce's disease (PD) in grapevines (Vitis vinifera L.) were observed in 2002 in the major grape production fields of central Taiwan. Disease severity in vineyards varied, and all investigated grape cultivars were affected. Diseased tissues were collected from fields for subsequent isolation and characterization of the causal agent of the disease (Xylella fastidiosa). Koch's postulates were fulfilled by artificially inoculating two purified PD bacteria to grape cultivars Kyoho, Honey Red and Golden Muscat. The inoculated plants developed typical leaf‐scorching symptoms, and similar disease severity developed in the three cultivars from which the bacterium was readily re‐isolated, proving that the leaf scorch of grapevines in Taiwan is caused by the fastidious X. fastidiosa. This confirmed PD of grapevines is also the first report from the Asian Continent. Phylogenetic analyses were performed by comparing the 16S rRNA gene and 16S‐23S rRNA internal transcribed spacer region (16S‐23S ITS) of 12 PD strains from Taiwan with the sequences of 13 X. fastidiosa strains from different hosts and different geographical areas. Results showed that the PD strains of Taiwan were closely related to the American X. fastidiosa grape strains but not to the pear strains of Taiwan, suggesting that the X. fastidiosa grape and pear strains of Taiwan may have evolved independently from each other.     

Occurrence of Charcoal Rot Caused by Macrophomina phaseolina,an Emerging Disease of Adzuki Bean in China

Adzuki bean (Vigna angularis) is an important legume crop in China. Soil‐borne charcoal rot caused by Macrophomina phaseolina (Tassi) Goid is an important and devastating disease of many crops including legumes. During late August and early September, 2014, symptoms similar to charcoal rot were observed on adzuki bean plants in Yulin City of Shanxi Province, and Fangshan County of Beijing, China. This study was conducted to determine the causal agent of the emerging disease on adzuki bean. Four fungal isolates were obtained and identified as M. phaseolina based on morphological and molecular characteristics, including species‐specific primer detection and sequences of internal transcribed spacer (ITS) of nuclear ribosomal DNA. The resulting sequences showed 99% identity with more than 60 M. phaseolina strains from diverse hosts. The virulence on adzuki bean was verified using pathogenicity tests, producing symptoms similar to those observed in the fields. To our knowledge, this is the first report of M. phaseolina causing charcoal rot on adzuki bean.     

The occurrence of sclerotium rot on Momordica charantia caused by Sclerotium rolfsii in Korea

Sclerotium rolfsii is one of the most destructive pathogens and thought to affect a broad range of plant hosts. In July 2014, an occurrence of sclerotium rot was observed on bitter melon (Momordica charantia L.) in Jinju, South Korea. The rot symptoms were most developed on stems and fruit near the soil line, and infected bitter melon plants withered and eventually died. White mycelial mats with numerous sclerotia were produced on diseased stems and fruit near the soil surface. Based on the morphological characteristics, pathogenicity tests, and DNA sequencing and phylogenetic analysis of the internal transcribed spacer (ITS) ribosomal RNA (rRNA) gene region, the causal fungus was identified as S. rolfsii Saccardo. This is the first report of sclerotium rot caused by S. rolfsii on bitter melon in Korea. The recent occurrence of sclerotium rot on bitter melon poses a potential threat to its production in Korea.     

Phyllosphere grapevine yeast Aureobasidium pullulans reduces Aspergillus carbonarius (sour rot) incidence in wine-producing vineyards in Greece

Phyllosphere yeasts were isolated from leaves of vine (Vitis vinifera L.) canes and evaluated in a detached berry assay for their ability to suppress Aspergillus carbonarius (Order: Eurotiales, Family: Trichocomaceae) growth. Seventeen of the 21 yeast isolates significantly reduced A. carbonarius growth, i.e. sour rot infection compared to untreated controls in laboratory tests. The most effective yeast isolate Aureobasidium pullulans (Order: Dothideales, Family: Dothioraceae), isolate Y-1, was field tested on two varieties of red grape, Grenache Rouge and Agiorgitiko located on the Island of Rhodes and in Corinthos County, Greece. It was demonstrated that A. pullulans Y-1 was as effective as the commercial fungicide fludioxonil + cyprodinil, in reducing sour rot infection, A. carbonarius presence on berries at harvest and ochratoxin A contamination in must.     

Shared and unique features of bacterial communities in native forest and vineyard phyllosphere

Phyllosphere bacteria have received little attention despite their important roles in shaping plant performance traits. In this study, we characterize the bacterial communities on leaves of native trees inhabiting sclerophyllous forests in central Chile, one of the world's biodiversity hotspots. Additionally, we provide profiles of bacterial communities on grape leaves and berries of organic and conventional vineyards. Results of 16S rRNA gene amplicon sequence analysis showed that 45% of OTUs were shared across forest leaves, grape leaves, and grape berries. Conventional management had higher number of OTUs shared with forest leaves than organic management. In addition, grape leaves subjected to conventional management had higher alpha diversity than those with organic management, while no significant effect of agricultural management was observed in grape berries. Indicator analysis showed that Bdellovibrio, Beijerinckia, and Spirosoma were typical for forest leaves, whereas Enhydrobacter, Delftia, Proteiniclasticum, Arsenicicoccus, and Alkaliphilus were typical for the vineyard phyllosphere. Regarding agricultural managements, Beijerinckia, Sedimentibacter, Nesterenkonia, Gluconobacter, Conexibacter, and Anaeromyxobacter were typical for conventional grape leaves, whereas no genus‐level indicator was found for organic vineyard leaves. These results provide new insights of the diversity patterns of the phyllosphere microbiome in native and cultivated lands and suggest that both of these microbiomes are connected and integrated systems.     

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.     

Rhizopus Stem Rot of Orobanche aegyptiaca caused by Rhizopus oryzae in China

Stem rot was recorded on Orobanche aegyptiaca in Shihezi City, Xinjiang Uygur Autonomous Region, China from 2010 to 2011. The pathogen was isolated repeatedly from the infected stems and was identified as Rhizopus oryzae based on morphology, cultural features and molecular analysis. Koch's postulates were supported by pathogenicity tests conducted on healthy plants grown on processing tomato and melon. To our knowledge, this paper is the first to report the occurrence of R. oryzae stem rot on O. aegyptiaca.     

不同颜色果袋对‘巨峰’葡萄果实中挥发性成分的影响

以‘巨峰’葡萄为试材,研究白袋、绿袋、红袋、蓝袋4种不同颜色果袋对葡萄成熟期果实中挥发性成分的影响,为葡萄专用果袋的研发提供理论依据.结果表明:不同颜色果袋可为葡萄果实的发育提供特定的光环境,不同套袋处理葡萄成熟期果实中的挥发性成分差异显著.‘巨峰’葡萄成熟期果实中检测到酯、醛、醇、酮类、萜烯类和芳香族化合物,对照、白袋、绿袋、红袋和蓝袋处理成熟期果实检测到的挥发性组分分别为33、37、38、32和34种.与对照相比,白袋处理乙酸乙酯、己酸乙酯等酯类物质含量降低,己醛、反式-2-己烯醛、癸醛含量增加;绿袋处理除3-己烯酸乙酯、反式-2-己烯酸乙酯、3-羟基丁酸乙酯、反式-2-己烯醛、(反,反)-2,4-己二烯醛、癸醛、苯乙醇外,其他共有组分的含量均出现降低;红袋处理乙酸乙酯、己酸乙酯等酯类物质含量降低,己醛、反式-2-己烯醛等的含量出现降低;蓝袋处理乙酸乙酯、己酸乙酯等主要酯类物质的含量未发生较大变化,己醛、反式-2-己烯醛等的含量出现升高.在非共有组分中,套袋处理中醇类组分的种类减少,萜烯类、芳香族化合物的种类增加.总体上,蓝袋处理果实中主要的‘果香型’酯类组分含量最高,白袋处理果实中主要的酯类组分及‘青草香型’的醛类组分含量较高,绿袋、红袋处理果实主要香气组分含量较低.     

Botrytis cinerea: Activité Antifongique dans les Jeunes Grappes de Vitis vinifera, Varieté Gamay

Botrytis cinerea: fungicidal activity in young grape berries of Vitis vinifera (Gamay) Extracts of young grape berries of Vitis vinifera (Gamay) inhibit the germination of conidia of Botrytis cinerea. This inhibitory activity is present in extracts obtained from clusters at bloom stage and is maintained until a developmental stage close to the maturity. These extracts ruptured the cellular membranes.     

Refining the biological factors affecting virulence of Botryosphaeriaceae on grapevines

Botryosphaeriaceae isolates of six species were assessed for their potential to infect grapevine tissues other than their tissues of isolation, primarily to determine sources of inocula that could contribute to bunch rot. Pathogenicity tests were conducted in vitro on berries and wood and in vivo on dormant buds of cultivars Chardonnay and Shiraz in glasshouse and field experiments. Tissue specificity and variation in virulence for different isolates was assessed. All isolates were able to infect and cause symptoms on detached 1‐year‐old canes and mature berries. Virulence was not affected by origin tissue and varied between isolates and within species. Inoculation of dormant buds did not affect bud burst or further development of shoots and fruit, however, a small number of Botryosphaeriaceae were reisolated from bunches at later growth stages. We conclude that Botryosphaeriaceae species are important pathogens of both the vegetative tissues and wood of grapevines. Grapevine wood infected with Botryosphaeriaceae could act as a source of inoculum for reproductive and vegetative tissue. Equally, the vegetative and reproductive tissues can also act as inoculum sources for wood infection. Therefore, all sources of inocula should be taken into consideration when developing management strategies for Botryosphaeria bunch rot and Botryosphaeria canker diseases.     

Contents of <Emphasis Type="Italic">Trans</Emphasis>-resveratrol in Korean Grape Cultivars,including “Kyoho”

Concentrations of trans-resveratrol were analyzed in 36 Korean-grown grape cultivars, including cv. Kyoho. This cultivar has large irregular berries and a deep-black skin and accounts for >15% of all grapevines grown in Korea. The content of trans-resveratrol is an important quality parameter, and its pattern of variability increased gradually until the time of harvest. Its distribution also fluctuated significantly among leaves, seeds, and exocarps, with the highest amount (1,477 μg g⁻¹ dry weight) occurring in the leaves. Among all cultivars evaluated here, trans-resveratrol contents were significantly higher in “Cheongsoo” and “High Bailey,” their levels being 50% above the overall mean (i.e., 2.0-mg g⁻¹ dry weight), whereas 14 cultivars had contents that were 10% less than that mean value     

Prevalence,geographic distribution and phylogenetic relationships among cryptic species of Plasmopara viticola in grape‐producing regions of Georgia and Florida,USA

Previous phylogenetic studies of the grape downy mildew pathogen, Plasmopara viticola, revealed five cryptic species in eastern North America that differed in their host range and geographic distribution. Preliminary comparative studies also documented differences in temperature responses during infection between certain cryptic species, indicating the biological relevance of knowing which cryptic species of the pathogen are present in a given region. However, limited information is available regarding the presence, prevalence and dynamics of cryptic species of P. viticola in the southeastern United States. Here, 301 P. viticola isolates obtained from cultivated grape species in five distinct grape‐growing regions of Georgia and Florida were subjected to cleaved amplified polymorphic sequence analysis and multilocus sequencing (internal transcribed spacer region of the rDNA, actin and β‐tubulin) to identify cryptic species and infer phylogenetic relationships. Three cryptic species, P. viticola clade aestivalis (Pva), clade vinifera (Pvv) and clade vulpina (Pvu), were identified in Georgia, whereas two, Pva and Pvv, were found in Florida; all three cryptic species are reported here for the first time in Georgia, whereas Pva is reported for the first time in Florida. Pva was the most prevalent cryptic species (72.1% of isolates) and was distributed widely from the North Georgia Mountains to Mid‐Florida, whereas Pvv (27.2%) and Pvu (0.7%) were found only in the Coastal Plain region of the two states. Interestingly, Pvu was obtained from French American hybrid Blanc du Bois and could be subcultured on Vitis vinifera Chardonnay, suggesting a broader host range than only the wild species Vitis vulpina reported previously.     

Epiphytic fungal community in Vitis vinifera of the Portuguese wine regions

In this work, fungi present in the grapevine's phyllosphere collected from the main demarcated wine regions of Portugal were identified, and their phylogenetic relationships were analysed. A total of 46 vine samples (leaves and berries) were collected from different parts of the country, being isolated a total of 117 fungal colonies that were identified to the genus level and sequenced in the following genetic regions: internal transcribed spacer region and 18S rRNA and β‐tubulin gene. Next, a phylogenetic tree reconstruction for each genetic region was built. The isolates retrieved from environmental samples belonged to the genera Alternaria (31%), Cladosporium (21%), Penicillium (19%), Aspergillus (7%) and Epicoccum (3%). No genetic signatures of exchange of genetic material were detected, and consequently, the reconstructed phylogenetic trees allowed to distinguish between these different species/genera. In the fungal composition of the Vitis vinifera phyllosphere, several potential pathogens were identified that can be associated with decreases in crop productivity. Knowledge of fungi identification and genetic diversity is pivotal for the development of more adequate crop management strategies. Furthermore, this information will provide guidelines for a more specific and wiser use of fungicides.

Significance and Impact of the Study

The knowledge on the composition of the phyllosphere microbial community is still limited, especially when fungi are concerned. These micro‐organisms not only play a crucial role in crop health and productivity but also interact with the winemaking process, determining the safety and quality of grape and grape‐derived products. The elucidation of the micro‐organisms present in the phyllosphere will have a notorious impact on plant breeding and protection programmes and disease management strategies, allowing a better control of pesticide applications.