Applicability of rep-PCR genomic fingerprinting to molecular discrimination of members of the genera Phaeoacremonium and Phaeomoniella

This study examined the applicability of rep-PCR genomic fingerprinting to molecular typing of Phaeoacremonium and Phaeomoniella species found on grapevines and associated with the esca syndrome. Fingerprints obtained by rep-PCR were compared with those obtained by microsatellite-primed PCR. The rep-PCR patterns clearly separated the species, and in some cases revealed intraspecific variability. Dendrograms revealed a clear genetic distance between Phaeomoniella chlamydospora and the three Phaeoacremonium species analysed. The results demonstrate that rep-PCR can be a complementary or alternative strategy to other molecular methods as a tool for identification of species and strains of these two genera.     

Identification and Spread of Fomitiporia punctata Associated with Wood Decay of Grapevine Showing Symptoms of Esca

ABSTRACT A full understanding of the pathology of esca, a chronic disease of grapevines, has been problematic, in part because the identity of the pathogen (or pathogens) has been difficult to determine. The wood decay symptoms of esca have been most often associated with Phellinus igniarius or Fomitiporia punctata. However, Koch's postulates have not been completely fulfilled because symptoms take many years to develop. The goal of this study was to determine the identity and mode of spread of basidiomycetes associated with wood decay in vines showing esca symptoms in Italian vineyards. Vineyards were intensively studied for the presence of basidiocarps, and mycelium was isolated from symptomatic vines. Fruiting bodies were identified by morphology, while mycelial isolates were identified by restriction fragment length polymorphism analysis of the internal transcribed spacer region of the nuclear ribosomal RNA gene cluster. Fomitiporia punctata fruiting bodies and mycelium were associated with approximately 50% of the vines showing esca symptoms in two vineyards; P. igniarius was not found in any samples. Fruiting bodies of F. punctata were found in five of six vineyards examined, but at low frequencies except in one vineyard. The diversity of somatic incompatibility types was very high; isolates from almost every vine had different somatic incompatibility types. With few exceptions, symptomatic and dead vines were not spatially aggregated within 12 vineyards. The combination of diverse somatic incompatibility types and lack of spatial aggregations are not consistent with the hypothesis that the disease is spread clonally through roots or by pruning tools. The correct identity of basidiomycetes associated with wood decay of vines with esca symptoms is important for understanding the epidemiology of this disease because F. punctata is found commonly on many woody hosts in Europe, which may represent a potential inoculum source for this disease.     

Immunological detection of Phaeoacremonium aleophilum,a fungal pathogen found in esca disease

Phaeoacremonium aleophilum is a fungal pathogen implicated in esca, a devastating disease affecting grapevines around the world. We have previously reported that the fungus secreted in its culture medium a variety of polypeptides, which allowed us to develop a serological method of detection. In this method, rabbit antibodies raised against the polypeptide fraction recognized secreted fungal proteins with high sensitivity (commonly 1 ng). These antibodies were specific since they cross-reacted with polypeptides secreted by various strains of Pm. aleophilum, but not with the proteins secreted by many other fungal pathogens implicated in other grapevine infections. Importantly, as shown by ELISA tests and immunolocalization, they did not cross-react with the secreted polypeptides of Eutypa lata (agent of Eutypa dieback), Neofusicoccum parvum (found in Black Dead Arm), and Phaeomoniella chlamydospora (also found in the fungal complex inducing esca). This serological approach permitted the development of a reliable dot-blot method to detect the presence in woody members of Pm. aleophilum in selectively infected trunk and canes. This method of diagnosis is rapid, easy to perform and non destructive for grapevines. Advantages and drawbacks of the method are discussed.     

Impact of temperature variations on toxic effects of the polypeptides secreted by Phaeoacremonium aleophilum

Wood-degrading diseases, in particular esca, Botryosphaeria dieback and Eutypa dieback, affect grapevines in vineyards around the world. The wood-inhabiting fungal pathogens degrade wood tissues in the colonization areas and induce, at distance, characteristic foliar symptoms. These effects have been previously partly correlated with the secretion of polypeptides (PFpal) presenting toxic effects on plant cells at various metabolic levels. These diseases are insidious and the symptoms can occur in mild or severe forms, showing differences from one year to another. Observations allowed to assume that variations in climatic conditions may intervene in this behavior. We showed here that a modification in the temperature regime modified the fungal development of Phaeoacremonium aleophilum and induced modifications of the intracellular structures, observed in particular in the nucleus, the vacuolar apparatus, the Golgi, the endoplasmic reticulum and also in the hyphal coat. Furthermore, changes in the pattern of the secreted polypeptides were observed as well quantitatively as qualitatively by electrophoretic analysis and immunolabeling by anti-PFpal antibodies. Subsequently, the different polypeptide fractions obtained from fungal cultures, either maintained at 20 °C or respectively transferred to colder (5 °C) and warmer (30 °C) temperatures, differentially affected the grapevine physiology as shown by the different effects induced on leaf anthocyanin synthesis, modification of proton secretion and lethality of cells in culture.     

Genetic diversity in populations of the fungi Phaeomoniella chlamydospora and Phaeoacremonium aleophilum on grapevine in France

Isolates of Phaeomoniella chlamydospora ( Phc ) and Phaeoacremonium aleophilum ( Pha ), two haploid, deuteromycetous fungi, were obtained from vines showing symptoms of esca disease in different localities in two French regions, and within a single vineyard in one of these regions. The population genetic structure was determined in both fungi using random amplified polymorphic DNA (RAPD) analysis. Populations of Phc showed similar levels of diversity at local and regional levels. The most frequent Phc haplotypes were found in every population, and the frequencies of positive alleles of markers were similar across populations. The hypothesis that recombination had occurred was rejected for the full set of samples, but not for the samples reduced to haplotypes, indicating that Phc may be a recombining species. Different features were identified in Pha populations. First, the southern population of Pha appeared more diverse than the south-western populations. Second, genetic differentiation was identified between Pha populations from southern and south-western regions for several RAPDs. Finally, in the southern population of Pha no evidence for recombination was obtained, even by reducing the sample to haplotypes. Within the single vineyard surveyed, several haplotypes of both fungi were recovered and randomly distributed. Thus different infection events appeared to have occurred on a low spatial scale. Data from this study showed that haplotypes of both fungi were distributed over long distances geographically, and that most of the vineyards surveyed were infested by more than one haplotype of Phc and Pha .     

Occurrence of <Emphasis Type="Italic">Phaeoacremonium</Emphasis> spp. and <Emphasis Type="Italic">Phaeomoniella chlamydospora</Emphasis> in grape propagation materials and young grapevines

During a 6-year study, grapevine propagation materials and young grapevines were analysed to evaluate the presence of internal wood discolouration and the occurrence of fungal species involved in Petri disease. The intensity of wood discolouration increased with the ageing of the plants. The maximum incidence of dark streaks was observed in the rootstock while necrosis originating from buds or nodes were notably present in the trunk and cordon of older vines. In contrast, the highest levels of brown-red halo symptoms, defined as discoloured areas around the pith, were recorded in the early growth stages. Phaeoacremonium spp. and Phaeomoniella chlamydospora were usually isolated from the rooted-grafts and the 3-year old plants, respectively. The number of infected grapevines increased with age. Most of the P. chlamydospora strains were isolated from dark streaks or dots, while Phaeoacremonium spp. were detected in brown-red halo symptoms and other symptomatic or asymptomatic wood. The greatest incidence of the two fungal taxa was recorded in the lower parts of the grapevine, including the roots and rootstock.     

Potential Inoculum Sources of Phaeomoniella chlamydospora in South African Grapevine Nurseries

Petri disease of grapevine is primarily caused by Phaeomoniella chlamydospora. This pathogen affects mostly young grapevines, but is also implicated in esca disease of older grapevines. Little is known about the disease cycle of this fungus. Infected propagation material was identified as a major means of dissemination of the pathogen. Recently, the pathogen was also detected from soil in South Africa and airborne conidia have been found in vineyards. The aim of this study was to use a molecular detection technique to test different samples collected from nurseries in South Africa at different nursery stages for the presence of P. chlamydospora. A one-tube nested-PCR technique was optimised for detecting P. chlamydospora in DNA extracted from soil, water, callusing medium and grapevine wood. The one-tube nested-PCR was sensitive enough to detect as little as 1 fg of P. chlamydospora genomic DNA from water and 10 fg from wood, callusing medium and soil. PCR analyses of the different nursery samples revealed the presence of several putative 360 bp P. chlamydospora specific bands. Subsequent sequence analyses and/or restriction enzyme digestions of all 360 bp PCR bands confirmed that all bands were P. chlamydospora specific, except for five bands obtained from callusing media and one from water. Phaeomoniella chlamydospora was positively detected in 25% of rootstock cane sections collected from mother blocks, 42% of rootstock cuttings and 16% of scion cuttings collected during grafting, 40% of water samples collected after pre-storage hydration, 67% of water samples collected during grafting, 8% of callusing medium samples and 17% of soil samples collected from mother blocks. These media can therefore be considered as possible inoculum sources of the pathogen during the nursery stages.     

Soft rot of root chicory in Hokkaido and its causal bacteria

In August 2010, bacterial soft rot was found on root chicory (Cichorium intybus var. sativum) in Hokkaido, Japan. Severely infected plants in fields were discolored, had wilted foliage, and black necrosis of petioles near the crown. Wilted leaves subsequently collapsed and died, forming a dry, brown or black rosette. The root and crown became partially or wholly soft-rotted. Slimy masses on infected areas of roots, turned dark brown or black. Gram-negative, rod-shaped, peritrichously flagellated, facultatively anaerobic bacteria were exclusively isolated from rotted roots, and typical symptoms were reproduced after inoculation with the strains. The bacteria were identified as Dickeya dianthicola, Pectobacterium carotovorum subsp. carotovorum, and Pectobacterium carotovorum subsp. odoriferum based on further bacteriological characterization and the sequence analysis of the malate dehydrogenase gene and 16S rRNA gene. These bacteria should be included with the previously reported Dickeya (=Erwinia) chrysanthemi in Saitama Prefecture, Japan, as causal pathogens of bacterial wilt of chicory.     

Genetic and virulence diversity, and mating type distribution of Togninia minima causing grapevine trunk diseases in Spain

Fifty eight single-spore Togninia minima (anamorph Phaeoacremonium aleophilum) isolates were recovered from grape rootstock wood of plants that showed symptoms of Petri disease and esca from 2001 to 2008 in Spain. These isolates were studied by means of mating type distribution, UP-PCR analysis, and virulence assays. Analysis of clone-corrected data sets showed equal frequencies of both mating types in the entire Spanish population, in the Ciudad Real region, at inter-vineyard and intra-vine spatial scales; while unequal mating type distribution was detected in Valencia and Zaragoza regions, at intra-vineyard and intra-vine spatial scales. This is the first study on distribution of T. minima mating types on spatial scales varying from vineyards to regions. A total of 49 polymorphic UP-PCR markers were obtained using seven UP-PCR primers. Four optimal clusters were inferred with Bayesian structure and multivariate analyses from the UP-PCR data. The high number of unique genotypes observed within the Spanish population, combined with a near-equal distribution of mating types, suggested that sexual reproduction probably does occur. However, based on allele distribution and frequency, each of the three subpopulations appeared to be evolving independently. Gene and genotype diversities across the subpopulations were similar and ranged from 0.24 to 0.27 and from 0.27 to 0.37, respectively. The detection of genetically identical isolates within and among subpopulations indicates that an asexual reproductive component should not be excluded. Contrast analysis among groups defined by UP-PCR analyses showed no significant differences in the virulence of T. minima isolates.     

Plectosphaerella rot of lettuce, coriander, and chervil caused by Plectosphaerella pauciseptata

Rot diseases of hydroponically cultured leaf lettuce, coriander, and chervil were found in Chiba Prefecture, Japan. Stems and petioles of the three diseased plants became brown or black with a soft rot, and vascular parts of the stems became discolored. Seemingly similar fungi were isolated from the diseased plants. Pathogenicity of these fungal isolates to each plant was demonstrated by inoculation tests. The fungi were identified as Plectosphaerella pauciseptata based on morphological and cultural characters. This identification was supported by phylogenetic analysis with rDNA ITS1-5.8S-ITS2 regions. The diseases were named Plectosphaerella rot (Plectosphaerella huhai-byo in Japanese) of lettuce, coriander, and chervil.     

Pathogenesis of Eutypa lata in grapevine: identification of virulence factors and biochemical characterization of cordon dieback

Eutypa lata is a vascular pathogen of woody plants. In the present study we (i) determined which component(s) of the cell wall polymers were degraded in naturally infected grapevines and in artificially inoculated grape wood blocks; (ii) compared the pattern of wood decay in the tolerant grape cv. Merlot versus the susceptible cv. Cabernet Sauvignon; and (iii) identified secondary metabolites and hydrolytic enzymes expressed by E. lata during wood degradation. Biochemical analyses and a cytochemical study indicated that glucose-rich polymers were primary targets of E. lata. Structural glucose and xylose of the hemicellulose fraction of the plant cell wall and starch were depleted in infected woods identically in both cultivars. Moreover, the more tolerant cv. Merlot always had more lignin in the wood than the susceptible cv. Cabernet Sauvignon, indicating that this polymer may play a role in disease resistance. In vitro assays demonstrated the production by E. lata of oxidases, glycosidases and starch degrading enzymes. Phytotoxic secondary metabolites were also produced but our data suggest that they may bind to the wood. Finally, we demonstrated that free glucose in liquid cultures repressed primary but not secondary metabolism.     

An environmentally sustainable approach for the management of <Emphasis Type="Italic">Phaeoacremonium minimum</Emphasis>, the main agent of wood diseases in <Emphasis Type="Italic">Actinidia deliciosa</Emphasis>

Among the fungi associated with the kiwi wood diseases, the vascular pathogen Phaeoacremonium minimum can infect plants already at nursery stage, without any external symptoms. At the moment, there are not effective control strategies. The effect of soil treatments applicable in organic agriculture was evaluated in two-years experiments on potted kiwi plants artificially inoculated with P. minimum. The soil treatments were based on commercial formulations of iron chelate, silicon, neem paste, Trichoderma afroharzianum strain T22, and cover cropping with selected perennial Poaceae. Cover cropping and iron chelate treatments enhanced the iron availability for the plants and significantly reduced wood necrosis caused by the pathogen. Both treatments also produced an increase of hairy root proliferation, so the plants were able to better cope with stress conditions. Laboratory assays showed the role of iron on the pathogen growth and its pathogenesis enzyme activities.     

Esca in young and mature vineyards,and molecular diagnosis of the associated fungi

Esca is one of the most important grapevine trunk diseases, and it can induce severe decline. In the past, the disease occurred mostly on mature vines, but today it is also present in young vineyards. The aim of this study was to investigate the incidence of esca in young (< 7 years old) and mature (> 11 years old) vineyards on cvs Montepulciano, Sangiovese, Verdicchio and Passerina located in the main viticultural areas of the Marche Region, central-eastern Italy. The average incidence of diseased plants was higher in mature (32.6%) than young (5.2%) vineyards, and Verdicchio and Passerina appeared to be the most sensitive among the cultivars considered, followed by Sangiovese and Montepulciano. The analysis of the spatial spread of esca carried out in two mature vineyards on cv. Verdicchio and a young vineyard on cv. Sangiovese showed a fluctuation in the numbers of infected plants over the three years of observation. The fungi associated with symptomatic plants were detected by classical and molecular tools. Isolation on agar media yielded colonies of Phaeomoniella chlamydospora (Pch), Botryosphaeria spp. (Bot), Fomitiporia mediterranea (Fomed) and, sporadically, Phaeoacremonium aleophilum (Pal). In samples from young plants, Bot and Pch were recurrent, while Pch and Fomed were found in mature vines and old rootstocks. Molecular detection with specific primer pairs for Pch, Pal, Fomed, and B. dothidea confirmed the data obtained using classical tools, and in some cases it was more sensitive. This study thus provides a further contribution to the association between causal agents and esca symptoms, and it confirms the importance of molecular tools for a sensitive detection of associated pathogens, which can also be present in propagative materials.     

Role of basidiomycete fungi in the grapevine trunk disease esca

Phaeomoniella chlamydospora and wood-rotting basidiomycete fungi, namely Fomitiporia spp., are known esca pathogens. However, the effect of their mixed infections and the sequence of infection on disease development is unclear. To determine the effects of single and co-inoculations on symptoms, potted Vitis vinifera ‘Crimson Seedless’ was inoculated with P. chlamydospora either alone or in combination with one of four basidiomycetes: Coprinellus radians, Fomitiporia langloisii, F. polymorpha and the novel species Tropicoporus texanus. Basidiomycetes were isolated from vines with foliar symptoms of esca in California and Texas. In sequential co-inoculations, the effects of different sequences of infection (P. chlamydospora first, basidiomycete 6 months later; and vice versa) were tested, compared to simultaneous co-inoculations. Plants inoculated with P. chlamydospora either alone or in combination with a basidiomycete (in any sequence) did not differ significantly in the length of black-streaking lesions. In plants inoculated only with a basidiomycete, the appearance of large brown lesions, coupled with the absence of this wood symptom from control plants, suggests that C. radians, F. langloisii and T. texanus are pathogenic. Foliar symptoms resembling those of esca in the field (marginal and/or interveinal scorching, combined with red and/or yellow discoloration) were statistically more frequent among plants inoculated with F. polymorpha or T. texanus, either simultaneously or following P. chlamydospora, compared to single inoculations. Sequential co-inoculations of a basidiomycete before or after P. chlamydospora were associated with similar lesion lengths, suggesting that basidiomycetes may not require infection by P. chlamydospora in order to extensively colonize the wood.     

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.     

Serological detection of grapevine virus a using antiserum to a nonstructural protein, the putative movement protein

ABSTRACT Grapevine virus A (GVA) is implicated in the etiology of the rugose wood disease. The coat protein (CP) and the putative movement protein (MP) genes of GVA were cloned and expressed in Escherichia coli and used to produce antisera. Both the CP and the MP were detected with their corresponding antisera in GVA-infected Nicotiana benthamiana. The MP was first detected at an early stage of the infection, 6 to 12 h after inoculation, and the CP was detected 2 to 3 days after inoculation. The CP and MP were detected by immunoblot analysis in rugose wood-affected grapevines. The MP could be detected in GVA-infected grapevines that tested negative for CP, both with CP antiserum and with a commercially available enzyme-linked immunosorbent assay kit. The study shows that detection of the nonstructural MP may be an effective means for serological detection of GVA infection in grapevines.     

弯孢菌引起的变色米初步研究

 本文报道了弯孢菌引起的变色米的症状,弯孢苗的种类及其对水稻的致病性。变色米的症状,因水稻品种、弯孢菌种类和接种方法而有差异,但一般在糙米上为浅至深色的褐色米;在稻谷上产生全褐型、半褐型、褐斑型、褐点型和退色型五种类型。后各地采集的变色谷(米)上分离到弯孢属9个种,即新月弯孢、膝曲弯孢、近缘弯孢、不正弯孢、中隔弯孢、棒状弯孢、苍白弯孢、画眉弯孢、假弯孢,其中6个种为国内首次报道。据在水稻孕穗至开花期人工接种结果,上述9个种对水稻均有致病性,并产生大量的变色谷(米)。     

AFLP markers reveal two genetic groups in the French population of the grapevine fungal pathogen <Emphasis Type="Italic">Phaeomoniella chlamydospora</Emphasis>

Phaeomoniella chlamydospora, (Chaetothyriales, Herpotrichiellaceae) is one of the main causal agents of Petri disease and esca on grapevines. We have used AFLP markers to study the population genetic structure of 74 isolates collected at different spatial scales: 56 isolates originated from vines with esca disease sampled from four French vineyards (Poitou-Charentes, Aquitaine, Languedoc-Roussillon, Alsace); 18 isolates were collected from a single plot (Aquitaine vineyard). Significant linkage disequilibrium indicated that P. chlamydospora populations are not panmictic, whereas the level of haplotypic diversity observed, 72 single multilocus haplotypes identified in total among the 74 isolates analysed, suggest that reproduction in this species may not be strictly clonal. Clustering analyses suggests the presence of two genetically differentiated but sympatric clusters of isolates. The level of differentiation between the two clusters is high (F _ST = 0.23) and significant at 13 out of the 21 loci analyzed. The most plausible explanation for this pattern of admixture is the coexistence in P. chlamydospora French populations of two predominant clonal lineages. Finally, the low level of spatial genetic differentiation in this study is consistent with the spread of this fungus through the transport of infected plant material by human activities.     

The spatiotemporal spread of esca disease in a Cabernet Sauvignon vineyard: a statistical analysis of field data

The occurrence and spread of plants showing esca symptoms were assessed in a vineyard located on the plains of a northern Italian wine‐growing region. Esca disease symptoms were assessed over 16 consecutive years, beginning one year after planting. The number of plants with symptoms was recorded over time, considering both vines with foliar symptoms in the year of assessment (manifest esca) and vines with foliar symptoms in previous years (hidden esca). The sum of manifest and hidden esca was indicated as cumulative esca. The first symptoms of esca appeared in the sixth year of cultivation, with the incidence of manifest esca increasing to approximately 3% nine years after planting. The number of cumulative plants with symptoms increased exponentially in the final period of observation. The aim of this work was to investigate the spatiotemporal spread of esca infection throughout the vineyard and to assess the distribution pattern of plants with symptoms using Bayesian spatiotemporal models. The research results seem to support a higher probability of infection along rows rather than among adjacent rows. This observation may have implications for the technical management of the vineyard.