Diversity of Botryosphaeriaceae causing grapevine trunk diseases and their spatial distribution under different climatic conditions in Algeria

The family Botryosphaeriaceae is one of the most widespread and cosmopolitan endophytic group of fungi. Every year, species of this family cause severe damages on table and wine grape production, worldwide. However, this threat is still poorly known in Algeria. In this study, a large number of Botryosphaeriaceae-like isolates were obtained from symptomatic grapevines collected from eight regions with different ecological conditions, namely: Boumerdès, Médéa, Algiers, Tipaza, El Taref, Sidi Bel Abbes, Biskra and Adrar. The isolates were identified using DNA sequences of the translation elongation factor (tef1-α) and internal transcribed spacer (ITS) regions. Eleven species belonging to six genera, including Neofusicoccum parvum, N. algeriense, N. vitifusiforme, N. stellenboschiana, N. luteum, Diplodia seriata, D. olivarum, Lasiodiplodia theobromae, Dothiorella sarmentorum, Botryosphaeria dothidea and Neoscytalidium dimidiatum were identified. The spatial distribution of the Botryosphaeriaceae showed that D. seriata and N. stellenboschiana were the most widespread in the Algerian vineyards, while L. theobromae was recorded in the desert region of Biskra. Pathogenicity trials showed that all species were pathogenic on detached green shoots of grapevine, with N. parvum and L. theobromae being the most aggressive.

     

Genomic characterization of mating type loci and mating type distribution in two apparently asexual plantation tree pathogens

The Eucalyptus stem canker pathogens Teratosphaeria gauchensis and T. zuluensis (Capnodiales, Teratosphaeriaceae) are found in many tropical regions of the world where their hosts are cultivated for plantation forestry. Population genetic analyses have suggested that some populations undergo recombination, even though their sexual states have never been observed. Against this background, the aim of this study was to characterize the mating type (MAT) locus of these species and thus to better understand the basis of their diversity. Known Mycosphaerellaceae MAT genes were used to identify and investigate the MAT locus in the T. gauchensis and T. zuluensis genomes. Both species were found to be heterothallic and primers were designed to amplify the opposite MAT idiomorphs as well as conserved regions within the MAT1-1-1 and MAT1-2-1 genes. Each Teratosphaeria MAT idiomorph was defined by either the MAT1-1-1 or the MAT1-2-1 gene, and an idiomorph-specific hypothetical protein (MAT1-1-10 and MAT1-2-12). Populations of T. zuluensis from Asia and southern Africa were dominated by a single mating type, whereas the proportions of the different idiomorphs for T. gauchensis in South America and southern Africa were similar. There was no physical evidence of sexual reproduction for either species and it is argued that although recombination may be possible, it is unlikely to form an important part of their life cycles in diseased Eucalyptus plantations. Instead, continuous human-mediated multiple introductions of these species have probably resulted in the current genetic structure of their populations, which holds risk for future disease outbreaks and interspecific hybridization.     

新疆鹰嘴豆壳二孢疫病病菌Ascochyta rabiei交配型、致病力及其对3种杀菌剂的敏感性测定

新疆是我国鹰嘴豆主要生产地,然而随着鹰嘴豆种植面积的扩大,集约化种植模式的发展以及广泛从国内外引种,鹰嘴豆壳二孢疫病(Ascochyta blight)开始发生。由于该病害在新疆发生历史较短,相关的研究还甚少。本研究对新疆鹰嘴豆壳二孢疫病病菌Ascochyta rabiei的交配型、致病力及对3种杀菌剂敏感性进行测定,结果表明,A.rabiei的交配型MAT1-1和MAT1-2在新疆木垒地区的分离频率明显偏离1∶1,以MAT1-2为优势种群,但其致病力与MAT1-1无显著差异,而MAT1-1对30%醚菌酯EC的敏感性显著低于MAT1-2。     

Variation in populations of Phytophthora infestans in Finland and Norway: mating type, metalaxyl resistance and virulence phenotype

Phytophthora infestans was isolated from potato leaves and tubers collected from different parts of Finland in 1990–96 and Norway in 1993–96. Isolates were assessed for mating type, resistance to metalaxyl and virulence phenotype. In Finland 15% of 200 isolates tested and in Norway 25% of 642 tested were A2 mating type. Differences in the A1/A2 ratio were evident among regions and A2 was not found in northern areas. In Finland the frequency of metalaxyl-resistant isolates, among 1834 tested, decreased from 59% in 1990–95 to 2% in 1996. In Norway 59% of 491 isolates tested were resistant to metalaxyl in 1996. Among 269 Finnish and 105 Norwegian isolates, all known virulence genes were found in both countries and race 1.3.4.7.10.11 was the most common (resistance gene R9 was not included in the differential set). Oospores were observed in potato leaves from three locations in the southern part of Norway. The implications of the 'new' populations in the Nordic countries are discussed.     

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 .     

Genetic diversity of Grapevine rupestris stem pitting-associated virus isolates from Tunisian grapevine germplasm

Grapevine rupestris stem pitting-associated virus (GRSPaV) is one of the most widespread grapevine viruses and is transmitted mainly by grafting. GRSPaV presence was tested in 487 samples representative of the Tunisian grapevine germplasm (including autochthonous, table, wine, wild grape, and rootstock varieties) from different Tunisian regions. GRSPaV infection was detected in 51.3% of samples from different Tunisian regions, among which the table grapevine cultivars were the most commonly infected (68.7%). Genetic variability of GRSPaV isolates from wild and cultivated grapevines was assessed by sequencing the partial capsid protein (CP) gene of 19 Tunisian isolates and 1 Italian GRSPaV isolate from Sicily, and the partial RNA-dependent RNA polymerase (RdRp) gene of 13 Tunisian GRSPaV isolates. According to phylogenetic analysis of CP nucleotide sequences obtained in this study and sequences retrieved from GenBank, Tunisian isolates fell into four phylogenetic groups already described (I, II, III, and IV) and two new phylogenetic groups (VI and VIII). Phylogenetic analysis of the partial RdRp gene revealed that Tunisian isolates of GRSPaV are distributed into four phylogroups. This study highlights the importance of regular monitoring of GRSPaV infections in Tunisia, with special regard to those grapevine accessions employed in conservation and selection programmes. In particular, the presence of new GRSPaV genetic variants and infection of wild grapevines must be taken into account in order to choose a correct control strategy.     

Genetic diversity and pathotype determination of Colletotrichum sublineolum isolates causing anthracnose in sorghum

Amplified fragment length polymorphism (AFLP) based genetic diversity was analyzed for 232 Colletotrichum sublineolum isolates collected between 2002 and 2004 from three geographically distinct regions of Texas, and from Arkansas, Georgia, and Puerto Rico. Results revealed significant levels of polymorphism (59%) among the isolates. Even so, genetic similarity between isolates was high, ranging from 0.78 to 1.00. Clustering of similar isolates did not correlate with either geographic origin or year of collection. Pathotypes of 20 of the isolates were determined using 14 sorghum lines previously used in Brazil and the United States and 4 from Sudan. Seventeen new pathotypes were established from the 18 isolates that gave uniform and consistent reactions on all host differentials over 2?years of greenhouse testing. Differentials BTx378 and QL3 were resistant to all isolates while BTx623 and TAM428 were universally susceptible both years. Each of these lines had shown differential responses in prior studies indicating that the pathogen population has sufficient diversity to adapt rapidly to changes in resistant host lines deployed. When the 2-step pathotype classification scheme was used, the 18 isolates examined in this study were placed in four pathotype groups (A, C, D and G), which would further then be separated into ten distinct pathotypes. Common sets of differentials and a standardized nomenclature will allow for comparison to be made among pathotypes of C. sublineolum detected from different regions and also could help direct planting of appropriate sorghum lines and aid in the development of more durable forms of resistance.     

A characterization of grapevine trunk diseases in France from data generated by the National Grapevine Wood Diseases Survey

Farmers' field survey data sets represent extremely valuable information, often having a heterogeneous data format. A large number of methods are available to process this kind of information, which may be combined to address successive, connected hypotheses, with definite objectives. The National Grapevine Trunk Diseases Survey was established in France in order to monitor and analyze the importance and progress of several grapevine diseases, in particular Eutypa dieback and Esca decline. Here we report a first series of analyses pertaining to the years 2003 to 2005 to characterize the incidences of the two diseases, as well as grapevine mortality, in relation to grapevine age, crop management, cultivars, and growing regions. Information representing 256 individual vineyards indicated mean incidences of 2.23 and 3.25% for Eutypa dieback and Esca decline, respectively. A combination of approaches (hierarchical cluster analysis, multiple correspondence analyses, and binomial logistic regressions) indicated distinct patterns of variation in the incidences of the two diseases according to crop management practices; limited linkage of specific crop management practices with diseases, especially Esca decline; a shared contribution of both diseases to mortality; and a weak linkage of the (now banned) arsenite-based pesticides with reduced Esca decline symptoms, but no such association with grapevine mortality. Differences and complementarities of the analytical approaches are discussed, as well as the implications of these analyses on grapevine health.     

Genetic diversity in the 3' terminal 4.7-kb region of grapevine leafroll-associated virus 3

Grapevine leafroll-associated virus 3 (GLRaV-3; Ampelovirus, Closteroviridae), associated with grapevine leafroll disease, is an important pathogen found across all major grape-growing regions of the world. The genetic diversity of GLRaV-3 in Napa Valley, CA, was studied by sequencing 4.7 kb in the 3' terminal region of 50 isolates obtained from Vitis vinifera 'Merlot'. GLRaV-3 isolates were subdivided into four distinct phylogenetic clades. No evidence of positive selection was observed in the data set, although neutral selection (ratio of nonsynonymous to synonymous substitution rates = 1.1) was observed in one open reading frame (ORF 11, p4). Additionally, the four clades had variable degrees of overall nucleotide diversity. Moreover, no geographical structure among isolates was observed, and isolates belonging to different phylogenetic clades were found in distinct vineyards, with one exception. Considered with the evidence of purifying selection (i.e., against deleterious mutations), these data indicate that the population of GLRaV-3 in Napa Valley is not expanding and its effective population size is not increasing. Furthermore, research on the biological characterization of GLRaV-3 strains might provide valuable insights on the biology of this species that may have epidemiological relevance.     

甘肃省大麦条纹病菌遗传多样性及致病力差异

为了解甘肃省大麦条纹病病原菌Pyrenophora graminea的遗传多样性及致病力差异,运用RAPD分子标记技术对大麦条纹病菌不同菌株进行遗传多样性分析,并采用三明治法进行菌株致病力差异研究。结果表明:17个RAPD标记从45个菌株中扩增出126条带,平均每个标记7.41条带,遗传相似系数范围为0.468 3～0.984 1,平均值为0.830 8,当遗传相似系数为0.723 6时,可将供试菌株划分为4个类群,分别包含41、2、1和1个菌株;致病力测定结果显示菌株QWC较菌株QQ致病力强,两菌株除在品种‘甘啤2号’和‘GP-3’上无致病力外,在其他供试品种上致病力均存在差异。表明大麦条纹病菌不同菌株间存在遗传差异,且菌株QWC和菌株QQ存在致病力差异。     

Characterization and distribution of mating type genes in the dothistroma needle blight pathogens

ABSTRACT Dothistroma septosporum and D. pini are the two causal agents of Dothistroma needle blight of Pinus spp. in natural forests and plantations. Degenerate primers amplified portions of mating type genes (MAT1-1-1 and MAT1-2) and chromosome walking was applied to obtain the full-length genes in both species. The mating-type-specific primers designed in this study could distinguish between the morphologically similar D. pini and D. septosporum and between the different mating types of these species. Screening of isolates from global collections of D. septosporum showed that only MAT2 isolates are present in Australian and New Zealand collections, where only the asexual form of the fungus has been found. In contrast, both mating types of D. septosporum were present in collections from Canada and Europe, where the sexual state is known. Intriguingly, collections from South Africa and the United Kingdom, where the sexual state of the fungus is unknown, included both mating types. In D. pini, for which no teleomorph is known, both mating types were present in collections from the United States. These results provided new insights into the biology and global distribution of two of the world's most important pine pathogens and should facilitate management of the diseases caused by these fungi.     

Viruses and virus diseases of grapevine in Palestine

Surveys were carried out in vineyards in the main grapevine-growing areas of Palestine (Hebron, Bethlehem, Gaza, Jerusalem, Ramallah, Jenin, Jericho and Nablus) to assess the presence and incidence of virus and virus-like diseases. Leafroll symptoms were observed in Bethlehem, Ramallah and Jerusalem in native and imported cultivars, with higher rates in the red-fruited Shami, Beitoni and Smari. Rugose-wood symptoms were also observed in local and foreign cultivars, especially on grafted vines with a high incidence in Bethlehem. Fanleaf symptoms were rarely observed, while phytoplasma-induced symptoms were observed in Jenin, Jericho and Bethlehem on cvs Biadi, Superior Seedless and Beitoni. ELISA tests showed that 463 out of 566 (82%) tested vines were infected by at least one virus. GVA was the prevailing virus (66.1%), followed by GLRaV-1 (45.6%), GLRaV-3 (21.7%), GFkV (15.7%) and GLRaV-2 (8.3%). GVB and GFLV were also detected to a lesser extent, their incidence ranging between 3.7 and 1.2%, whereas GLRaV-7 was detected in a single vine of cv. Sultanina of foreign origin. Vineyards in the Bethlehem area were particularly badly damaged (97.5%), and some local cultivars were totally (Jandali, Marrawi and Shoyoukhi) or heavily infected (Zaini, Biadi and Shami). ELISA testing of 69 young rootstock mother plants showed a relatively high incidence of virus infection (20.3%). Vein necrosis and vein mosaic diseases were also ascertained on graft-inoculated 110R and Vitis riparia indicator plants, whereas no viruses other than GFLV were mechanically transmitted from about 200 vines onto inoculated herbaceous hosts.     

Viruses and virus diseases of grapevine in Tunisia

Mature canes were collected from vines in the main grapevine-growing areas in Tunisia (Cape Bon, Bizerte, Ben Arous), from commercial vineyards and mother-plant plots, to assess the presence of virus and virus-like diseases. Biological (mechanical transmission onto herbaceous hosts and grafting onto indicator woody plants) and serological detection (ELISA) methods were applied. ELISA showed that 96.4% of 669 vines tested were infected, most of them (88.1%) by at least two viruses. Grapevine leafroll-associated 3 closterovirus (GLRaV-3) was the most widespread virus (87.9%), followed by grapevine A vitiviras (GVA, 69.4%), grapevine fleck virus (GFkV, 51.9%), grapevine leafroll-associated 1 closterovirus (GLRaV-1, 36.8%), grapevine leafroll-associated 2 closterovirus (GLRaV-2, 19.1%), grapevine fan leaf nepovirus (GFLV, 18.2%) and grapevine B vitiviras (GVB, 14.8%). ELISA tests yielded negative results for grapevine leafroll-associated 7 closterovirus (GLRaV-7) and potato X potexvirus (PVX). The highest infections were found in Bizerte and Cape Bon regions (100 and 99.2%), and in vineyards aged over 20 years (98.5%) as compared with the younger ones (81.1%). Rootstocks in mother-plant plots were practically free from all the viruses tested (1 plant infected out of 81), whereas severe infections were found in Vitis vinifera mother plants (67.4% of 341 samples), in particular table grapes (92.6%) compared with wine grapes (47.9%). In these mother-plant plots, the prevailing viruses were GLRaV-3 (41.3%), followed by GFkV (36.7%), GVA (27.9%), GLRaV-1 (17%) and GLRaV-2 (15.2%). GFLV and GVB were far more limited (1.5 and 0.6%, respectively). The presence of vein necrosis and vein mosaic was ascertained by transmission onto 110R and Vitis riparia indicators, whereas only GFLV was mechanically transmitted onto herbaceous hosts (from about 20% of the samples).     

Viruses and virus diseases of grapevine in Egypt

Surveys for virus and virus-like diseases were carried out in commercial vineyards of the main grapevine-growing areas of Egypt along the river Nile and in recently reclaimed desert lands. The only symptoms observed and identified with reasonable confidence in the field were those of leafroll disease in red-berried cultivars. No virus was transmitted to herbaceous hosts by mechanical inoculation from glasshouse-forced cuttings of about 300 vines (40% of total samples). By contrast, ELISA tests showed that 78% of the assayed European vines (521 out of 664) were infected by one (29%) or more (49%) viruses. Grapevine virus A (GVA) was the most widespread virus (67.9% infection), followed by Grapevine leafroll-associated virus 3 (GLRaV-3) (55.9% infection). All the other viruses tested for were scarcely represented, i.e. Grapevine leafroll-associated virus 1 (GLRaV-1) 1.8% infection, Grapevine leafroll-associated virus 2 (GLRaV-2) 1.4% infection, Grapevine virus B (GVB) (0.6% infection) and Grapevine fleck virus (GFkV) (0.2% infection), or, like Grapevine fanleaf virus (GFLV), were totally absent. The infection rate of native cultivars (86%) was particularly heavy. 'Banaty Abiad' and 'Romy Ahmer', the two major Egyptian cultivars, had infection levels of 78% and 89%, respectively, and 'Fayoumy', the most important cultivar in the Fayoum area, had 96% infection. Totally infected were the tested samples of several minor native cultivars such as 'Farg El-Tair', 'Siwi Abiad', 'Ta'afi', 'Romy Abiad', 'Eswid El-Wady', 'Edkawy' and 'Bez El-Anza'. Slightly better was the sanitary situation of imported European grapevine cultivars (60% infection) and of American rootstocks (11.5% infection). In rootstocks, infection rate by GVA and GLRaV-3 was 5.5%, whereas GVB and GLRaV-1 were only sporadically detected.     

Viruses and virus diseases of grapevine in Lebanon

Grapevines were surveyed for the presence of virus and virus-like diseases in the main viticultural areas of Lebanon (Bekaa valley, Mount Lebanon, South and North Lebanon). Symptoms of rugose wood were observed in vines ofall cultivars and areas surveyed, whereas leafroll was observed only in some vineyards of the Bekaa valley and, to a lesser extent, in South Lebanon on cvs Tfaifihi, Cinsaut and Cardinal. Symptoms of fanleaf and of phytoplasma-induced yellows were also observed with low frequency in the Bekaa valley on wine-grape cultivars. ELISA tests showed that 53% of 1536 Vitis vinifera vines individually checked were infected by one or more viruses. Grapevine trichovirus A (GVA) was the prevailing virus (32.4%), followed by grapevine fleck virus (GFkV) (19.5%) and grapevine leafroll-associated closterovirus 3 (GLRaV-3) (12.4%). Grapevine leafroll-associated closterovirus 1 (GLRaV-l), grapevine trichovirus B (GVB) and grapevine fanleaf nepovirus (GFLV) were also detected to a lesser extent, their incidence ranging between 1.1 and 3.6%.     

Temporal spore dispersal patterns of grapevine trunk pathogens in South Africa

Trunk disease pathogens of grapevines, viz. Phaeomoniella chlamydospora, Eutypa lata and several species in Botryosphaeriaceae, Phaeoacremonium and Phomopsis are known to infect fresh pruning wounds by means of air-borne inoculum released after rainfall or prolonged periods of high relative humidity. Recent surveys have demonstrated that most or all of these pathogens are present in climatically diverse grape growing regions of South Africa. However, the factors controlling spore dispersal of these pathogens in vineyards were largely unknown. To address this question, spore trapping was done in a Chenin Blanc vineyard in the Stellenbosch area, South Africa, for 14 weeks during the grapevine pruning period from June to mid-September of 2004 and 2005. Hourly recordings of weather data were done by a weather station in the row adjacent to the spore trap. Spores of E. lata and Phomopsis and species in Botryosphaeriaceae were trapped throughout the trapping periods of 2004 and 2005, with higher levels of trapped spores recorded in 2005. The spores of all three pathogens were trapped during or after periods of rainfall and/or high relative humidity. In neither of the 2 years were spores of Pa. chlamydospora or Phaeoacremonium spp. trapped. Results indicated that spore event incidence, as well as the amount of spores released during a spore event of above-mentioned pathogens, were governed by rainfall, relative humidity, temperature and wind speed prior to and during the spore events.     

Genetic diversity,sensitivity to phenylamide fungicides and aggressiveness of Phytophthora ramorum on Camellia,Rhododendron and Viburnum plants in Spain

Phytophthora ramorum has been detected in official plant health surveys on Rhododendron, Viburnum and Camellia in ornamental nurseries in northern Spain since 2003. A collection of 94 isolates of P. ramorum was obtained from 2003 to 2008 from plants with symptoms at different geographical locations. Isolates were identified based on morphology and sequence of the rDNA ITS region. Mating type, genetic variation, sensitivity to phenylamide fungicides and aggressiveness of these isolates were determined. All isolates belonged to the A1 mating type, ruling out the possibility of genetic recombination. Seven microsatellite markers were used to study genetic diversity; three out of the seven microsatellite markers were polymorphic within the Spanish population of P. ramorum. This study confirms that all Spanish isolates of P. ramorum belonged to the EU1 lineage. Twelve intralineage genotypes were detected, five that are unique to Spain (EU1MG38, EU1MG41, EU1MG37, EU1MG39 and EU1MG40) and seven that are also present in at least one other European country (EU1MG1, EU1MG29, EU1MG22, EU1MG13, EU1MG2, EU1MG18 and EU1MG26). Genotypes EU1MG37, EU1MG39 and EU1MG40 were isolated from Rhododendron from one region; EU1MG38 and EU1MG41 were isolated from Camellia from two different regions. Isolates of genotype EU1MG38 were resistant to metalaxyl and mefenoxam. The level of genetic diversity within the Spanish population of P. ramorum is limited and indicates a relatively recent clonal expansion.     

Genetic diversity of Orobanche cumana populations from Spain assessed using SSR markers

Orobanche cumana (sunflower broomrape) is found in Spain as an allochthonous species parasitising exclusively sunflower. For many years, it was distributed in the Guadalquivir Valley and Cuenca province, but in recent years, it has spread to new areas. The objective of this research was to study genetic diversity of O. cumana populations from Spain using robust co‐dominant molecular markers. Cluster analysis on a set of 50 populations using 15 microsatellite markers revealed the existence of two distant gene pools, one in Cuenca province and another one in the Guadalquivir Valley. Within each gene pool, both inter‐ and intrapopulation variability were extremely low. This population structure probably reflects a founder effect, with the two genetically distant gene pools deriving from separate introduction events. Different races occurred within the same gene pool, suggesting that current races might have evolved through mutation from a common genetic background. Most of the populations from new areas were identical to the populations from the Guadalquivir Valley. Only a few populations showed larger intrapopulation variation. In these cases, our results suggested the co‐existence of both gene pools within the same population, as well as the occurrence of genetic recombination between them. Genetic recombination between distant gene pools is an important mechanism for creating new variation, which might also have an effect on race evolution. These results will contribute to the establishment of improved crop breeding and management strategies for O. cumana control.