Mining new resources for grape resistance against Botryosphaeriaceae: a focus on Vitis vinifera subsp. sylvestris

Botryosphaeria dieback is an important grapevine trunk disease with global impact. Susceptibility differences between grape varieties manifest as different expression of canopy symptoms in the field. However, the cause of these dieback symptoms and their relation with wood necrosis remain only partially understood. As a first step towards future strategies for resistance breeding, wood necrosis was investigated over a large selection of the Vitaceae family members following artificial inoculation of the Botryosphaeriaceae fungi Neofusicoccum parvum and Diplodia seriata into woody internodes. Large variation of resistance levels was found, with good performance in several accessions from V. vinifera subsp. sylvestris, the ancestor of cultivated grapevine. To get insight into the mechanisms of this apparent resistance, expression of defence genes was studied in V. vinifera cv. Chardonnay, Gewürztraminer and different V. vinifera subsp. sylvestris genotypes, in both green and necrotic areas of inoculated woods. Resistance to Botryosphaeriaceae in V. vinifera subsp. sylvestris correlated with earlier and higher induction of some defence genes, both in green and necrotic wood. Moreover, leaves of several V. vinifera subsp. sylvestris accessions were also less susceptible to necrosis induced by treatment with a culture filtrate of Botryosphaeriaceae, compared to commercial cultivars of V. vinifera. The results show that V. vinifera subsp. sylvestris provides interesting genetic resources for breeding new varieties with enhanced resistance to botryosphaeria dieback.     

Characterization of Cytospora isolates from wood cankers of declining grapevine in North America,with the descriptions of two new Cytospora species

Cytospora species are ubiquitous pathogens of numerous woody plants, causing dieback and wood cankers in agronomic crops, timber trees and wildland trees (e.g. Prunus, Eucalyptus and Salix, respectively). Cytospora chrysosperma, C. cincta and C. leucostoma have been reported from grapevines in Iran showing symptoms of one or more recognized trunk diseases (esca, botryosphaeria‐, eutypa‐ and phomopsis diebacks); however, only C. chrysosperma was shown to be pathogenic to grapevine. To understand the potential role of Cytospora species in the grapevine trunk‐disease complex, 21 Cytospora isolates were examined that were recovered from dieback and wood cankers of Vitis vinifera and Vitis interspecific hybrids in seven northeastern U.S. states and two Canadian provinces. Phylogenetic analyses of ITS and translation elongation factor 1‐α identified two novel species: Cytospora vinacea sp. nov. and Cytospora viticola sp. nov. Differences in culture morphology and conidial dimensions also distinguished the species. When inoculated to the woody stems of potted V. vinifera ‘Thompson Seedless’ in the greenhouse, both species were pathogenic, based on development of wood lesions and fulfilment of Koch's postulates. Cytospora viticola was the most virulent based on lesion length at 12 months post‐inoculation. As cytospora canker shares some of the same general dieback‐type symptoms as botryosphaeria‐, eutypa‐ and phomopsis diebacks, it may be considered part of the grapevine trunk‐disease complex in eastern North America.     

Characterization of Botryosphaeriaceae species associated with diseased loquat (Eriobotrya japonica) in Spain

Loquat (Eriobotrya japonica) is an important subtropical fruit crop in Spain and other Mediterranean countries. In recent years, characteristic symptoms of branch canker and dieback have been observed in the main cultivated areas of loquat in Spain. The goal of this study was to identify and characterize the species of Botryosphaeriaceae associated with these symptoms. For this, 36 affected orchards were surveyed between 2010 and 2011 in six provinces of southeastern Spain. Eighty‐four isolates belonging to the family Botryosphaeriaceae were recovered from samples with symptoms. These isolates were characterized by means of phenotypical studies, DNA sequence analyses of the internal transcribed spacer (ITS) and part of the translation elongation factor 1‐α regions, and pathogenicity tests. Ten fungal species were identified: Diplodia malorum, Diplodia olivarum, Diplodia seriata, Diplodia pseudoseriata/Diplodia alatafructa, Diplodia sp., Dothiorella sarmentorum, Neofusicoccum mediterraneum, N. parvum, Spencermartinsia plurivora and S. viticola. In addition, Diplodia eriobotryicola and Dothiorella eriobotryae are newly described. The most frequent species isolated from cankers was D. seriata and, as far as is known, this is the first report of D. malorum, and species belonging to the complex D. pseudoseriata/D. alatafructa, in Spain. All species were pathogenic to 1‐year‐old loquat plants cv. Algerie, with Diplodia sp. and S. viticola as the most virulent.     

Detection of Botryosphaeriaceae species within grapevine woody tissues by nested PCR, with particular emphasis on the Neofusicoccum parvum/N. ribis complex

Several species of Botryosphaeriaceae and Phaeomoniella chlamydospora are common agents of grapevine decline worldwide. Currently, the use of culture independent PCR based techniques for detection of Botryosphaeriaceae within grapevine tissues has been limited to Botryosphaeria dothidea. In the present study, two Botryosphaeriaceae specific nested PCR assays were developed. One with a narrow target range, to detect Neofusicoccum parvum and the closely related species complex (Neofusicoccum parvum/N. ribis sensu Pavlic et al. Molecular Phylogenetics and Evolution 51:259–268, 2009) and another, with a wider range, to detect all 17 species of Botryosphaeriaceae which have been reported as potential wood pathogens of grapevine. The effectiveness of these assays was validated in vivo on naturally infected wood samples collected from standing vines and dormant grafted rooted cuttings commercialized in Italy by different nurseries in different years. All samples were also screened by means of a previously published nested PCR assay specific for Phaeomoniella chlamydospora. It was found that: 1) propagation material may play an important role as source of primary inoculum, not only of Phaeomoniella chlamydospora, as previously reported, but also for members of the Botryosphaeriaceae, among which Neofusicoccum parvum, Botryosphaeria dothidea and Diplodia seriata are the most common, and 2) multiple infections by different species belonging to Botryosphaeriaceae and/or Phaeomoniella chlamydospora occur frequently both in standing vines and propagation material. This last finding supports the hypothesis that at least some of the non-specific symptoms of grapevine decline may be due to the presence of different pathogens within host tissues.     

Production of phytotoxic metabolites by five species of Botryosphaeriaceae causing decline on grapevines, with special interest in the species Neofusicoccum luteum and N. parvum

In recent years an increasing number of species of Botryosphaeriaceae have been associated with grapevine decline worldwide. Five species isolated from declining grapevines in Spain (Botryosphaeria dothidea, Diplodia seriata, Dothiorella viticola, Neofusicoccum luteum and N. parvum) were checked for toxin production in liquid cultures. Cultural conditions for all fungi were adjusted to obtain optimal production of phytotoxic culture filtrates, by growing the fungi in steady liquid cultures of Czapek–Dox broth for different time intervals. Phytotoxicity of D. seriata and N. parvum reached a maximum after 14 days while the remaining species showed the highest phytotoxicity levels after 21 days in culture. All fungi produced hydrophilic high-molecular weight compounds with phytotoxic properties. In addition, N. luteum and N. parvum produced lipophilic low-molecular weight phytotoxins, not detected consistently among the remaining species. This led to a more exhaustive study on the phytotoxicity of N. luteum and N. parvum. Culture filtrates and corresponding extracts of both species were consistently highly phytotoxic in different assays. The gas-chromatography analysis of the acetylated O-methyl glycosides of the phytotoxic exopolysaccharides produced by N. parvum showed these substances to be composed mainly of glucose, mannose and galactose. Results suggest that phytotoxic metabolites could be involved in the virulence of both species in planta.     

Identification,potential inoculum sources and pathogenicity of botryosphaeriaceous species associated with grapevine dieback disease in New Zealand

This study investigated the prevalence and identity of botryosphaeriaceous dieback pathogens in necrotic grapevines tissues in New Zealand vineyards, and other woody hosts growing nearby. The presumptive identities of the isolates by conidial and cultural morphology were confirmed with ITS sequence data as Neofusicoccum australe, N. luteum, N. parvum and Diplodia seriata. They were isolated predominantly from necrotic stems of grapevine and other hosts, but also from leaves, flowers and wood debris of grapevines. Inoculation with conidia and mycelium of multiple isolates of each species onto excised and attached green shoots and trunks of five grapevine varieties, Cabernet sauvignon, Chardonnay, Pinot noir, Riesling, and Sauvignon blanc, showed that all varieties became infected to a similar extent. All species except D. seriata were pathogenic, irrespective of the host source, with N. luteum being the most and D. mutila the least pathogenic (P < 0.05). On trunks, N. parvum caused cankers and the other pathogenic species caused die-back when the inoculated vines became winter-dormant. Conidia were produced from green shoot lesions and die-back wood, which indicates potential inoculum sources for vineyard infection.     

Characterization of Botryosphaeriaceae from plantation‐grown Eucalyptus species in South China

The Botryosphaeriaceae is a species‐rich family that includes pathogens of a wide variety of trees, including Eucalyptus species. Symptoms typical of infection by the Botryosphaeriaceae have recently been observed in Eucalyptus plantations in South China. The aim of this study was to identify the Botryosphaeriaceae associated with these symptoms. Isolates were collected from branch cankers and senescent twigs of different Eucalyptus spp. All isolates resembling Botryosphaeriaceae were separated into groups based on conidial morphology. Initial identifications were made using PCR‐RFLP fingerprinting, by digesting the ITS region of the rDNA operon with the restriction enzymes CfoI and KspI. Furthermore, to distinguish isolates in the Neofusicoccum parvum/N. ribis complex, a locus (BotF15) previously shown to define these species, was amplified and restricted with CfoI. Selected isolates were then identified using comparisons of DNA sequence data for the ITS rDNA and translation elongation factor 1‐alpha (TEF‐1α) gene regions. Based on anamorph morphology and DNA sequence comparisons, five species were identified: Lasiodiplodia pseudotheobromae, L. theobromae, Neofusicoccum parvum, N. ribis sensu lato and one undescribed taxon, for which the name Fusicoccum fabicercianum sp. nov. is provided. Isolates of all species gave rise to lesions on the stems of an E. grandis clone in a glasshouse inoculation trial and on the stems of five Eucalyptus genotypes inoculated in the field, where L. pseudotheobromae and L. theobromae were most pathogenic. The five Eucalyptus genotypes differed in their susceptibility to the Botryosphaeriaceae species suggesting that breeding and selection offers opportunity for disease avoidance in the future.     

Characterization of Neofusicoccum parvum causing canker and dieback on Brachychiton species

Brachychiton species are planted in Italy as ornamental trees. A survey in a nursery in Sicily (Italy) revealed the presence of young B. acerifolius and B. populneus showing severe trunk cankers, massive gummosis from the bark cracks, wood discolouration, and twig dieback. Morphological characterization was based on conidia measurement, and molecular characterization was carried out performing phylogenetic analysis (Maximum Parsimony) based on multi-locus approach of partial ITS, EF-1α, and tub2. Identification of the causal agent resulted in Neofusicoccum parvum (Botryosphaeriaceae). Pathogenicity tests were conducted in order to fulfill the Koch’s postulates. Five and three-years-old potted Brachychiton plants were inoculated (indoor and outdoor) using mycelial plugs. All the inoculated plants showed severe symptoms similar of those observed in the nursery during the survey. Some of the inoculated plants were completely dead. Re-isolations fully confirmed the causal agent. For our knowledge, this is the first report worldwide of N. parvum attacking Brachychiton spp.

     

Susceptibility of grapevine pruning wounds to infection by Lasiodiplodia theobromae and Neofusicoccum parvum

The susceptibility of 1‐ and 2‐year‐old grapevine wood to botryosphaeria canker caused by Lasiodiplodia theobromae and Neofusicoccum parvum was evaluated in California in two seasons. In the 2007 dormant season, pruning‐wound susceptibility was highest when wounds were inoculated immediately after pruning in December (80% of pruning wounds were infected in Chardonnay for both fungal species and 75% and 98% in Cabernet Sauvignon for N. parvum and L. theobromae, respectively). In the 2008 dormant season, pruning‐wound susceptibility was highest in November in Chardonnay (86% and 93% for N. parvum and L. theobromae, respectively) and in December in Cabernet Sauvignon (71% and 75% for N. parvum and L. theobromae, respectively). The lowest infection rates (13–35%) were observed when vines were pruned and inoculated in March in both dormant seasons and for both cultivars. Susceptibility of pruning wounds did not differ significantly (P = 0·7612) between 1‐ and 2‐year‐old wood and consequently, pruning‐wound protection treatments should be applied to all wounds. In conclusion, grapevine pruning wounds were susceptible to infection by L. theobromae and N. parvum to varying extents throughout the dormant season in California (November–March), but, overall, susceptibility of pruning wounds was highest when inoculations were done immediately after pruning and decreased significantly as the interval between pruning and inoculation increased. Results of this study suggest that pruning grapevines in late winter (March) in California would significantly reduce the risk of infection by L. theobromae and N. parvum.     

Characterization of resistance mechanisms activated by Trichoderma harzianum T39 and benzothiadiazole to downy mildew in different grapevine cultivars

Downy mildew, caused by Plasmopara viticola, is one of the most destructive diseases of grapevine and is controlled with intense application of chemical fungicides. Treatment with Trichoderma harzianum T39 (T39) or benzothiadiazole‐7‐carbothioic acid S‐methyl ester (BTH) has been previously shown to activate grapevine resistance to downy mildew and reduce disease symptoms in the Pinot noir cultivar. However, enhancement of plant resistance can be affected by several factors, including plant genotype. In order to further extend the use of resistance inducers against downy mildew, the physiological and molecular properties of T39‐ and BTH‐activated resistance in different cultivars of table and wine grapes were characterized under greenhouse conditions. T39 treatment reduced downy mildew symptoms, but the degree of efficacy differed significantly among grapevine cultivars. However, efficacy of BTH‐activated resistance was consistently high in the different cultivars. Expression profiles of defence‐related genes differed among cultivars in response to resistance inducers and to pathogen inoculation. T39 treatment enhanced the expression of defence‐related genes in the responsive cultivars, before and after P. viticola inoculation. A positive correlation between the efficacy of T39 and the expression level of defence‐related genes was found in Primitivo and Pinot noir plants, while different genes or more complex processes were probably activated in Sugraone and Negroamaro. The data reported here suggest that the use of a responsive cultivar is particularly important to maximize the efficacy of resistance inducers and new natural inducers should be explored for the less responsive cultivars.     

Intraspecific variation in Diplodia seriata isolates occurring on grapevines in Spain

Variation of Diplodia seriata, a fungal species associated with botryosphaeria dieback of grapevine, was investigated with respect to its genetic, phenotypic and pathogenic characteristics. The inter‐simple sequence repeat (ISSR) technique was used to investigate the genetic diversity of 83 isolates of D. seriata. Five ISSR primers were able to provide reproducible and polymorphic DNA fingerprint patterns, thus showing a relevant genetic variability in the species. Analyses of ISSR data by different clustering methods grouped the isolates into two distinct clusters through the Bayesian and DAPC analyses. No relationships between either geographic or host origin of isolates and genetic clusters were observed. Several representative isolates from each genetic cluster were chosen for studying their conidial dimensions, in vitro mycelial growth, vegetative and mating compatibility, and pathogenicity on detached grapevine canes and potted vines. No significant differences in conidial dimensions were detected among the groups. Vegetative compatibility reactions were observed among isolates but this was not related with the genetic clustering. Production of sexual fruiting bodies in vegetative compatible crossings was not observed under the experimental conditions used in the study. All 14 isolates tested for pathogenicity were confirmed to be pathogenic according to the length of the necrotic lesions that they caused and their reisolation frequencies from the infected plant tissues. Differences in the length of necrosis were detected among isolates, thus revealing the existence of different virulence levels in the species.     

Pre‐infection processes of Botryosphaeriaceae spp.: adhesion of conidia to different substrata

Species of Botryosphaeriaceae are important wound pathogens of grapevines as causal agents of botryosphaeria dieback, but the behaviour of their conidia pre‐infection is unknown and may be important for disease development. Adhesion properties of conidia were investigated for Botryosphaeria dothidea, Neofusicoccum luteum and N. parvum on substrata with different affinities for water. Greatest adhesion on any surface was reached after 5 min for isolates N. luteum MM558, B. dothidea 007 and N. parvum G652 (53·1, 54·0 and 50·6%, respectively) and for N. luteum isolate CC445 after 20 min (61·4%). As conidia adhered well to all artificial substrata, it appeared as if the attachment process was nonspecific. Overall, surface wettability did not play a major role in the adhesion of conidia. Spore surface proteins appeared to play a role in the adhesion process because treatment of conidia of N. luteum MM558 with a protease completely prevented adhesion. Histochemical labelling of conidia and germlings with Coomassie brilliant blue (specific for proteins) was positive for all isolates, with a blue ‘halo’ often seen surrounding conidia or near the germ tube emergence point after incubation times conducive to germination. Alcian blue also stained material surrounding conidia after longer incubation times, which indicated that mucopolysaccharide and protein production may be involved in a second phase of adhesion.     

Botryosphaeriaceae species causing dieback on Annonaceae in Brazil

In Brazil, the Annonaceae species Annona muricata, A. squamosa, A. cherimola and atemoya (a hybrid of A. cherimola and A. squamosa) are cultivated in several regions, and produce fruits that are highly appreciated by consumers and are of great economic importance. Among the several diseases that can affect these crops, dieback is one of the most important, causing damage and, in the most severe cases, death of the plants. Due to the lack of suitable diagnostic studies up to now, this work aimed to identify the Botryosphaeriaceae species that cause dieback on Annonaceae in Brazil. Based on combined phylogenetic analyses of ITS, TEF-1α, TUB2 and RPB2, eight species of Botryosphaeriaceae were identified, namely Lasiodiplodia brasiliense, L. crassispora, L. hormozganensis, L. iraniensis, L. pseudotheobromae, L. subglobosa, L. theobromae and Pseudofusicoccum stromaticum. All species found in this study were pathogenic and caused symptoms of necrosis in stems and dieback. Thus, this study confirms species of Botryosphaeriaceae as causal agents of dieback on Annonaceae in Brazil.     

Detection of botryosphaeriaceous species in environmental samples using a multi‐species primer pair

Botryosphaeriaceous species are significant grapevine trunk pathogens worldwide, which can be difficult to identify to species level using conventional morphological methods. This study developed and optimized a quick, reliable molecular identification method that could facilitate investigations into the epidemiology of these diseases in vineyards. The multi‐species primers, BOT100F and BOT472R, amplified a 371–372 bp portion of the rRNA gene region from the six botryosphaeriaceous species commonly found in New Zealand vineyards. In silico analysis indicated that they would amplify DNA from six of the 12 lineages of the Botryosphaeriaceae, including all of the main species pathogenic to grapevines. A detection sensitivity of 1 and 0·1 pg DNA in standard and nested PCR, respectively, was achieved and this was calculated as equivalent to 2·5 conidia. Validation of the primers for environmental samples showed that their specificity was not compromized by the presence of competing DNA templates extracted from wood and soil. Single stranded conformational polymorphism (SSCP) analysis of the amplicons could resolve Neofusicoccum australe, N. luteum, Diplodia mutila and D. seriata, but did not differentiate between N. parvum and N. ribis. The optimized PCR‐SSCP was used to identify botryosphaeriaceous species present in rainwater traps collected over 1 year in a vineyard known to contain infected vines. It could detect multiple species in individual samples and demonstrated differences in the dispersal patterns of conidia from different species. Given the specificity and sensitivity of this method it could prove useful in epidemiology studies involving the numerous botryosphaeriaceous species that infect a wide range of host species.     

Development of isolate‐specific markers for Neofusicoccum parvum and N. luteum and their use to study rainwater splash dispersal in the vineyard

Unique bands were identified in single isolates of Neofusicoccum parvum and Neofusicoccum luteum using universally primed polymerase chain reaction (UP‐PCR) analysis of isolates obtained from grapevines and non‐grapevine hosts in New Zealand, Australia, South Africa and the USA. Primers were designed to amplify a 1550 bp portion of the 1573 bp marker band from N. parvum isolate B2141 and a 510 bp portion of the 524 bp marker band from N. luteum isolate G51a2. A PCR‐RFLP assay was developed to distinguish the N. parvum isolate B2141 from other N. parvum isolates, based on a polymorphism found in the marker band using the TaqI restriction endonuclease. For N. luteum isolate G51a2, the designed primers were specific at an annealing temperature of 63°C in the PCR. The sensitivity threshold of the N. parvum and N. luteum isolate‐specific markers was 50 pg and 5 pg, respectively, when used in standard PCR with purified genomic DNA. The sensitivity of the N. parvum isolate‐specific marker was increased to 0·5 pg by nested PCR. The specificity test of both isolate‐specific markers with six other Botryosphaeriaceae spp. showed that they were specific to their respective species and isolates. Both markers were able to detect the conidia of N. parvum and N. luteum marker isolates in rainwater samples collected at different distances from an inoculation point in the vineyard. The results showed that rain splash could disperse the conidia of both of these species up to 2 m from the inoculum point in a single rainfall event.     

Phylogeny,distribution and pathogenicity of Lasiodiplodia species associated with dieback of table grape in the main Brazilian exporting region

Botryosphaeria dieback is an important disease of table grape in the São Francisco Valley, the main Brazilian exporting region. The objectives of this study were to identify species of Lasiodiplodia associated with botryosphaeria dieback of table grapes in the São Francisco Valley, investigate the prevalence and distribution of the species in the region, and evaluate their pathogenicity and virulence in green shoots of table grape. A total of 112 Lasiodiplodia isolates were obtained from 14 vineyards, located in Casa Nova, Juazeiro and Petrolina. Fungal identifications were made using phylogenetic analysis based on partial sequences of translation elongation factor 1‐α (EF1‐α) and internal transcribed spacer (ITS) sequences, in combination with morphometric characteristics of conidia. Eight species of Lasiodiplodia were identified: L. brasiliense, L. crassispora, L. egyptiacae, L. euphorbicola, L. hormozganensis, L. jatrophicola, L. pseudotheobromae and L. theobromae. Except for L. crassispora, L. pseudotheobromae and L. theobromae, all the other species are reported for the first time on grapevine worldwide. The distribution of Lasiodiplodia species differed between the three table grape populations of São Francisco Valley. All Lasiodiplodia species isolated in this study were present in the population of Casa Nova and Lasiodiplodia theobromae was the most prevalent. All species of Lasiodiplodia were pathogenic on detached green shoots of grapevine, with L. brasiliense being the most virulent.     

Aetiology of branch dieback,panicle and shoot blight of pistachio associated with fungal trunk pathogens in southern Spain

Pistachio represents an emerging nut crop across the Mediterranean basin. In Spain, pistachio has been traditionally cultivated in marginal-dry areas with unfavourable climatic conditions for plant diseases. Consequently, little attention has been given to research on pistachio diseases until recently. Symptoms of branch dieback and cankers, and shoot and panicle blight have been recently observed in commercial pistachio orchards across southern Spain. In this study, 10 commercial pistachio orchards showing disease symptoms were surveyed between 2017 and 2018. Botryosphaeriaceae fungi were consistently isolated from affected shoots, among other fungal families with minor relevance. Representative isolates of each family were characterized based on colony and conidial morphology, optimum growth temperature, and the comparison of DNA sequence data (ITS, LSU, EF, TUB2, and ACT genomic regions). Detached and attached shoots, and attached panicles of pistachio cv. Kerman were inoculated with mycelial plugs or conidial suspensions to demonstrate the pathogenicity of the selected isolates. Botryosphaeria dothidea, Lasiodiplodia pseudotheobromae, Neofusicoccum mediterraneum, N. parvum, Diaporthe neotheicola, Diaporthe sp., Eutypa lata, Eutypa sp., Cytospora sp., and Phaeoacremonium minimum were identified. P. minimum had the highest optimum growth temperature (29.6 °C) and Cytospora sp. the lowest (21–22 °C). Botryosphaeriaceae isolates showed the largest lesions on inoculated shoots, with L. pseudotheobromae being the most aggressive, followed by Neofusicoccum species. Panicles inoculated with N. mediterraneum showed blight symptoms and canker formation 6 weeks after inoculation, without significant differences in aggressiveness between isolates. This work reports relevant information about this emerging disease in the novel Spanish pistachio-growing areas.     

Grapevine trunk diseases: complex and still poorly understood

This review presents an overview of eutypa dieback, esca and botryosphaeria dieback, the predominant grapevine trunk diseases worldwide. It covers their symptomatologies in the trunk, leaves and berries; the characteristics of the different fungal species associated with them; and host–pathogen interactions. Here, the host–pathogen relationship is defined at the cytological, physiological and molecular levels. Currently available experimental tools for studying these diseases, both in vitro and in the field, are discussed. Finally, a progress report on their control, which, since the ban of sodium arsenite, comprises chemical, biological and/or sanitation methods, is presented.     

Co‐infection by Botryosphaeriaceae and Ilyonectria spp. fungi during propagation causes decline of young grafted grapevines

Decline of newly planted, grafted grapevines is a serious viticultural problem worldwide. In the Riverina (New South Wales, Australia), characteristic symptoms include low fruit yields, very short shoots and severely stunted roots with black, sunken, necrotic lesions. To determine the cause, roots and wood tissue from affected plants in 20 vineyards (Vitis vinifera cv. Chardonnay grafted to V. champini cv. Ramsey rootstock) were assayed for microbial pathogens. Ilyonectria spp. (I. macrodidyma or I. liriodendra, producers of phytotoxin brefeldin A, BFA, and cause of black foot disease of grapevines) and Botryosphaeriaceae spp. (predominantly Diplodia seriata) were isolated from rootstocks of 100 and 95% of the plants, respectively. Togninia minima and Phaeomoniella chlamydospora (cause of grapevine Petri disease) were isolated from 13 and 7% of affected plants, respectively. All Ramsey rootstock stems of grafted plants sampled from a supplier nursery were infected with Ilyonectria spp. and D. seriata. Diplodia seriata, but not Ilyonectria spp., was also isolated from 25% of canes sampled from the rootstock source block. Root inoculation of potted, disease‐free Chardonnay plants with Ilyonectria isolates from diseased vineyards caused typical disease symptoms, while co‐inoculation with Botryosphaeriaceae spp. increased disease severity. This is the first study to show that a major cause of young grapevine decline can be sequential infection by Botryosphaeriaceae from rootstock cuttings and Ilyonectria spp. from nursery soil. Although the Petri disease fungi were less common in young declining grafted grapevines in the Riverina, they are likely to contribute to the decline of surviving plants as they mature.     

Cryptic species and population genetic structure of Plasmopara viticola in São Paulo State,Brazil

Downy mildew (Plasmopara viticola) is one of the most important diseases in grape-growing areas worldwide, including Brazil. To examine pathogen population biology and structure, P. viticola was sampled during the 2015/16 growing season from 516 lesions on nine grape cultivars in 11 locations in subtropical areas of São Paulo State, Brazil. For identification of cryptic species, a subsample of 130 isolates was subjected to cleaved amplified polymorphic sequence (CAPS) analysis, and for 91 of these isolates the ITS1 region was sequenced. These analyses suggest that the population of P. viticola in São Paulo State consists of a single cryptic species, P. viticola clade aestivalis. Seven microsatellite markers were used to determine the genetic structure of all 516 P. viticola isolates, identifying 23 alleles and 55 multilocus genotypes (MLGs). Among these MLGs, 34.5% were clonal and represented 93% of the isolates sampled. Four dominant genotypes were present in at least five different locations, corresponding to 65.7% of the isolates sampled. Genotypic diversity (Ĝ = 0.21–0.89) and clonal fraction (0.58–0.96) varied among locations (populations). Most populations showed significant deviation from Hardy–Weinberg expectations; in addition, excess of heterozygosity was verified for many loci. However, principal coordinate analysis revealed no clusters among locations and no significant isolation by distance was found, suggesting high levels of migration. The results indicate that downy mildew epidemics result from multiple clonal infections caused by a few genotypes of P. viticola, and reproduction of P. viticola in São Paulo State is predominantly asexual.