A Reassessment of the Species Concept in Eutypa lata, the Causal Agent of Eutypa Dieback of Grapevine

ABSTRACT Eutypa dieback is a vascular disease of several cultivated crops and trees worldwide. The attribution of the name to the agent responsible for branch dieback is ambiguous. Pathogenicity of Eutypa sp. first was reported on apricot and the causal agent was named E. armeniacae. However, no morphological differences were reported with the previously described E. lata, and some authors considered both species synonymous. Others regarded them as distinct species on the basis of pathogenesis and molecular analysis. We further investigated the relatedness of both species by phylogenetic analyses of the internal transcribed spacer region and beta-tubulin gene. These analyses included several other taxa placed in the same family (Diatrypaceae), and yielded three groups. The isolates referred to as E. lata in previous work clustered with Diatrype stigma in one group. Isolates of E. armeniacae and E. lata clustered in a second group, supporting the synonymy of these species. The third group included other Eutypa spp. supporting the polyphyletic origin of this genus. Measurements of conidia length and secondary metabolite production of isolates supported the phylogenetic analyses. Secondary metabolites appeared to be a synapomorphic character shared by several taxa including E. lata, E. armeniacae, E. laevata, and E. petrakii var. petrakii.     

Detection of Eutypa lata and Eutypella vitis in Grapevine by Nested Multiplex Polymerase Chain Reaction

ABSTRACT Two fungi were isolated from grapevines in Michigan vineyards with Eutypa dieback symptoms: Eutypa lata and Eutypella vitis. These fungi are difficult to distinguish morphologically but are genetically distinct as determined by sequencing of the internal transcribed spacer (ITS) regions. The ITS regions of 25 Eutypa lata and 15 Eutypella vitis isolates were sequenced. Eutypa lata sequences were more variable than those of Eutypella vitis. Polymerase chain reaction (PCR) primers were designed for each species and evaluated against isolates of both fungi as well as 11 closely related Diatrypaceous fungi and 23 isolates of other fungi representing various pathogenic, saprophytic, and endophytic genera on grape and other small fruit crops. The primers were specific for their intended species. A nested multiplex PCR protocol was developed and used to successfully detect these fungi in wood samples from cankers with and without stromata from naturally infected vines as well as in artificially inoculated, potted canes. The primers developed in this study will assist in our abilities to diagnose and study the roles of Eutypa lata and Eutypella vitis in Eutypa dieback development.     

Molecular polymorphisms between populations of Pseudoperonospora cubensis from Greece and the Czech Republic and the phytopathological and phylogenetic implications

Molecular genetic polymorphisms within Pseudoperonospora cubensis isolates of different geographic origins were investigated to establish their phylogenetic relationships and to assess genetic variability between two distant pathogen populations. Thirty isolates originating from Greece (Crete; 15), the Czech Republic (13), the Netherlands (one) and France (one) were analysed by AFLP fingerprinting and ITS 5·8S rDNA sequence analysis. All isolates were obtained from cucumber ( Cucumis sativus ) plants showing typical downy mildew symptoms. Four AFLP primer combinations produced a total of 288 high-quality bands of which 45% were polymorphic, allowing isolates to be grouped into two separate clusters: one including the Central European (Czech Republic) and Western European (the Netherlands and France) and the other the Cretan isolates. Within each AFLP cluster there was some variation, which could be accounted for by geographic origin or pathogenicity. The two populations (Cretan vs. Central and Western European) exhibited a high degree of genetic isolation. There was no clear AFLP grouping of isolates on the basis of pathotypes. No variability was detected in the ITS1 region; however, ITS2 sequences grouped P. cubensis isolates in two subclusters: one with all investigated European and the other with Asian isolates. The two subclusters formed a larger P. cubensis cluster which was differentiated from the cluster of the neighbouring species Pseudoperonospora humuli . Within P. cubensis , AFLP fingerprints could resolve genetically isolated populations, even on small or medium geographic scales, while ITS2 sequence showed differences on a global scale, being only suitable for phylogenetic analyses.     

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.     

Genetic Relatedness of African and United States Populations of Cercospora zeae-maydis

Two taxonomically identical but genetically distinct sibling species, designated groups I and II, of Cercospora zeae-maydis cause gray leaf spot of maize in the United States. Isolates of the gray leaf spot pathogen from Africa were compared with isolates from the United States by amplified fragment length polymorphism (AFLP) analysis and restriction digests of internal transcribed spacer (ITS) regions and 5.8S ribosomal DNA (rDNA), as well as by morphological and cultural characteristics. The isolates from Africa were morphologically indistinguishable from the U.S. isolates in both groups, but like isolates of group II, they grew more slowly and failed to produce detectable amounts of cercosporin in culture. Analysis of restriction fragments from the ITS and rDNA regions digested with five endonucleases indicated that all of the African isolates shared the profile of the C. zeae-maydis group II population from the eastern United States and, thus, are distinct from the group I population, which is more prevalent in the United States and other parts of the world. Cluster analysis of 85 AFLP loci confirmed that the African and U.S. group II populations were conspecific (greater than 97% average similarity) with limited variability. Among all group II isolates, only 8 of 57 AFLP loci were polymorphic, and none was specific to either population. Thus, although gray leaf spot was reported in the United States several decades prior to the first record in Africa, the relative age of the two populations on their respective continents could not be ascertained with confidence. The absence of C. zeae-maydis group I in our samples from four countries in the major maize-producing region of Africa as well as the greater AFLP haplotype diversity found in the African group II population, however, suggest that Africa was the source of C. zeae-maydis group II in the United States. The overall paucity of AFLP variation in this sibling species further suggests that its origin is recent or that the ancestral population experienced a severe bottleneck prior to secondary migration.     

Sibling species of cercospora associated with gray leaf spot of maize

ABSTRACT Monoconidial isolates of the fungus causing gray leaf spot of maize were obtained from diseased leaves collected throughout the United States and analyzed for genetic variability at 111 amplified fragment length polymorphism (AFLP) loci. Cluster analysis revealed two very distinct groups of Cercospora zeae-maydis isolates. Both groups were found to be relatively uniform internally with an average genetic similarity among isolates of approximately 93 and 94%, respectively. The groups were separated from each other by a genetic distance of approximately 80%, a distance greater than that separating each group from the sorghum pathogen, C. sorghi (67 to 70%). Characteristics and dimensions of conidia and conid-iophores produced on infected plants or nutrient media were unreliable criteria for taxonomic differentiation of isolates composing the two groups of C. zeae-maydis. Nucleotide sequences of 5.8S ribosomal DNA (rDNA) and the internal transcribed spacer (ITS) regions were identical within each group but different between the two groups and different from C. sorghi. Restriction fragment length polymorphisms generated by digestion of the 5.8S rDNA and ITS regions with TaqI readily distinguished each group and C. sorghi. Isolates in one group were generally distributed throughout maize-producing regions of the United States; isolates in the other group were localized in the eastern third of the country. Both types were present in the same fields at some locations. The genetic distance based on AFLP profiles and different ITS nucleotide sequences between the two morphologically indistinguishable groups indicate that they are sibling species. Although it is unlikely that breeding for resistance to gray leaf spot will be confounded by local or regional variation in the pathogen, a vigilant approach is warranted, because two pathogenic species exist with unknown abilities to evolve new pathotypes.     

<Emphasis Type="Italic">Eutypa lata</Emphasis>, the causal agent of dieback in red currant (<Emphasis Type="Italic">Ribes rubrum</Emphasis>) and gooseberry (<Emphasis Type="Italic">R. uva-crispa</Emphasis>) in the Netherlands

Dieback of red currant (Ribes rubrum) and gooseberry (Ribes uva-crispa) is an increasing problem in commercial fields in the Netherlands. Field surveys were done in 2006–2007 and samples with dieback symptoms were analysed. In this study the causal agent was diagnosed as Eutypa lata, based on morphological characteristics and rDNA-ITS sequence data. The field surveys revealed the presence of the anamorph and teleomorph states of the fungus produced on dead infected currant wood. Eutypa lata is a vascular pathogen of many woody plants. Related fungi from the same family Diatrypaceae are difficult to distinguish from E. lata based on morphological features. The genetic variability of E. lata was compared by rDNA-ITS sequencing of isolates from different hosts and origins. Within the E. lata isolates little variability in the ITS sequences was observed. Phylogenetic analysis showed no clear subdivisions within the species. Eutypa lata strains isolated from the different hosts were closely related, indicating that there is no direct evidence for host specificity.     

广西地区葡萄黑痘病病原菌的分离与鉴定

采用常规组织分离法对广西南宁、河池两地的葡萄黑痘病菌进行分离、纯化,分别得到37和31株分离菌。经菌落形态观察及r DNA ITS序列分析,南宁37株分离菌为同一菌株,河池31株分离菌为同一菌株,以NN和HC分别代表两地菌株,对它们进行形态学、致病性鉴定及r DNA ITS区域序列分析。结果显示,两地菌株形态学与致病性存在较大差异,但都符合黑痘病菌生长形态。NN菌落为红棕色、近圆形,边缘光滑。菌落中心位置丘状凸起,表面有白色菌丝和透明粘稠的小液滴,周围边缘有较规则的褶皱。HC菌落呈浅橙色、近圆形,边缘光滑。菌落中心位置丘状凸起,表面有少量白色菌丝,无液滴,周围边缘有不规则褶皱隆起。人工接种葡萄后均能引起典型的黑痘病症状,NN致病性强于HC。使用r DNA ITS区域通用引物ITS1F/ITS4进行PCR扩增后,NN和HC分别得到1 124 bp和818 bp的片段。比对结果显示1 124 bp与Elsinoe ampelina(AY826763.1)序列覆盖率达92%,序列一致性达99%;818 bp与Elsinoe ampelina(AY826762.1)序列覆盖率达75%,序列一致性达99%。因此,NN和HC均是引起广西葡萄黑痘病的病原菌。     

Variation in susceptibility of grapevine pruning wound to infection by Eutypa lata in south-western France

Grapevine cultivars Cabernet Sauvignon (susceptible to Eutypa dieback), and Merlot (tolerant), were pruned three times during the dormant season (mid-December, mid-January and mid-February) and wounds on the 1-year-old canes were inoculated weekly with ascospores of Eutypa lata after pruning. No differences in susceptibility to infection were observed between cultivars, although in the vineyard they differed in symptom expression. Infection by E. lata varied with pruning date and the age of the pruning wound, and was higher and of longer duration with early pruning (December). At low temperature, infection of the pruning wounds by E. lata was increased, whereas the growth of other microorganisms was reduced. Moderate temperatures encouraged the growth of other microorganisms, notably Rhodotorula sp. This natural colonizer of grapevine pruning wounds was able to reduce the infection capacity of E. lata . It was more effective when inoculation with E. lata was carried out with low numbers of ascospores and when it was delayed until 14 days after application of the wound colonizer, infection being reduced by 95–100%.     

Genetic Diversity in Populations of Xanthomonas campestris pv. campestris in Cruciferous Weeds in Central Coastal California

ABSTRACT Xanthomonas campestris pv. campestris (X. campestris) infects a large number of cruciferous plants, including weeds. California has one of the largest and most diverse populations of wild cruciferous plants in the world. Although considerable information is available on the genetic diversity of X. campestris in commercial crop plants, nothing is known about the diversity in strains infecting weeds. To assess the genetic diversity among strains of X. campestris in weeds in noncultivated and cultivated areas, strains of the pathogen were isolated from populations of cruciferous weeds growing in coastal valley crop-production sites and from remote nonproduction sites along the California central coast. Results of fingerprinting over 68 strains using amplified fragment length polymorphism along with representative strains by sequence analysis showed the presence of seven genotypes. Genotypes A and B were limited to coastal sites; genotypes C, D, and E were from inland cultivated sites; and genotypes F and G were present in both coastal noncultivated and inland cultivated sites. Crop strains were grouped outside any weed strain group and were separated from the weed strains and other pathovars of X. campestris. These results revealed, for the first time, that strains of X. campestris present in noncultivated coastal weed populations generally were unique to a site and genetically distinct from strains present in populations of weeds in crop-production areas located nearby.     

Oidium neolycopersici: intraspecific variability inferred from amplified fragment length polymorphism analysis and relationship with closely related powdery mildew fungi infecting various plant species

Previous works indicated a considerable variation in the pathogenicity, virulence, and host range of Oidium neolycopersici isolates causing tomato powdery mildew epidemics in many parts of the world. In this study, rDNA internal transcribed spacer (ITS) sequences, and amplified fragment length polymorphism (AFLP) patterns were analyzed in 17 O. neolycopersici samples collected in Europe, North America, and Japan, including those which overcame some of the tomato major resistance genes. The ITS sequences were identical in all 10 samples tested and were also identical to ITS sequences of eight previously studied O. neolycopersici specimens. The AFLP analysis revealed a high genetic diversity in O. neolycopersici and indicated that all 17 samples represented different genotypes. This might suggest the existence of either a yet unrevealed sexual reproduction or other genetic mechanisms that maintain a high genetic variability in O. neolycopersici. No clear correlation was found between the virulence and the AFLP patterns of the O. neolycopersici isolates studied. The relationship between O. neolycopersici and powdery mildew anamorphs infecting Aquilegia vulgaris, Chelidonium majus, Passiflora caerulea, and Sedum alboroseum was also investigated. These anamorphs are morphologically indistinguishable from and phylogenetically closely related to O. neolycopersici. The cross-inoculation tests and the analyses of ITS sequences and AFLP patterns jointly indicated that the powdery mildew anamorphs collected from the above mentioned plant species all represent distinct, but closely related species according to the phylogenetic species recognition. All these species were pathogenic only to their original host plant species, except O. neolycopersici which infected S. alboroseum, tobacco, petunia, and Arabidopsis thaliana, in addition to tomato, in cross-inoculation tests. This is the first genome-wide study that investigates the relationships among powdery mildews that are closely related based on ITS sequences and morphology. The results indicate that morphologically indistinguishable powdery mildews that differed in only one to five single nucleotide positions in their ITS region are to be considered as different taxa with distinct host ranges.     

Characterization of Ribosomal DNA from Venturia inaequalis and Its Phylogenetic Relationship to rDNA from Other Tree-Fruit Venturia Species

ABSTRACT A portion of the 18S ribosomal DNA (rDNA) gene, the internal transcribed spacers (ITS1 and ITS2), and the 5.8S rDNA gene were polymerase chain reaction-amplified from strains and field populations of Venturia inaequalis and assessed for genetic variation. A previously reported optional group I intron in the 18S rDNA gene of V. inaequalis was detected in 75.0% of 92 strains collected worldwide and in 61.1 and 71.2% of 54 and 59 strains from two Michigan orchards, respectively. Sequence and restriction analysis of rDNA revealed four intron alleles, three of which were present both in worldwide strains and in each field population. Two ITS1 alleles were detected and found to be linked to specific intron alleles. The ITS1-5.8S-ITS2 sequences from V. asperata V. carpophila, V. cerasi, V. inaequalis, V. nashicola, V. pyrina, and Cladosporium caryigenum were compared using phylogenetic analysis. Strains of the Venturia species were placed in three distinct monophyletic groups in a phylogenetic tree. The first group comprised V. inaequalis; the second, V. pyrina and V. nashicola; and the third, V. cerasi, V. carpophila, and V. asperata. The described intron and ITS1 alleles in V. inaequalis provide genetic markers for subdividing populations of V. inaequalis, and the ITS1-5.8S-ITS2 sequences are valuable in determining the relationship of the species from tree-fruit crops with other Venturia species.     

Fish-isolated Naegleria strains and their phylogeny inferred from ITS and SSU rDNA sequences

Effort was made to identify Naegleria strains isolated from organs of fish, using phylogenetic analyses of SSU rDNA and ITS sequences. Eighteen fish-isolated strains studied enlarged substantially the so far available set of Naegleria strains characterized by both molecular markers. The phylogenetic analyses of separate and concatenated SSU rDNA and ITS sequences revealed phylogenetic relationships of strains under study; however, they failed to solve classification of fish-isolated strains into species. The sequence similarity of strain-representatives of Naegleria species as well as data obtained on intragenomic variation of ITS sequences discouraged the authors from the definition of new species. The results of the present study provide evidence of a need to re-evaluate the current practice of setting boundaries between species of the genus Naegleria. Sequences obtained in this study have been deposited in GenBank with accession numbers DQ768714-DQ768743.     

Morphological and molecular characterization of the sudden-death syndrome pathogen of soybean in Brazil

Brazilian Fusarium isolates causing soybean sudden death syndrome (SDS) were characterized by comparing them with other Fusarium isolates associated with soybean root rot, as well as F. solani f.sp. glycines isolates associated with the disease in the USA, using molecular (mitochondrial and nuclear rDNA), morphological, cultural and pathogenic characteristics. On the basis of pathogenicity data, and restriction fragment length polymorphism and sequence analysis of the rDNA internal transcribed spacer (ITS) regions, isolates formed a group distinct from nonSDS F. solani isolates, as well as other Fusarium species. ITS sequence analysis also revealed that Brazilian isolates were distinct from the majority of SDS pathogens from the USA ( Fusarium virguliforme ) and conformed to Fusarium tucumaniae .     

The influence of climate on foliar symptoms of eutypa dieback in grapevines

ABSTRACT Foliar symptoms of Eutypa dieback, caused by Eutypa lata, in grapevines, cv. Shiraz, varied from year to year in a 6-year study conducted in South Australia and, although trends were similar for vineyards within geographical regions, differences were observed between regions. We attempted to elucidate the causes underlying this variation and hypothesized that it was influenced by climatic factors. A number of possible relationships were identified between climate and symptom expression: (i) increased symptom expression was related to increased winter rainfall 18 months earlier, (ii) decreased disease incidence and prevalence were related to increased temperature in spring, and (iii) a reduction in disease incidence was related to both very high and very low rainfall in October. Theories for these relationships are proposed and require further investigation. A conceptual model was developed which requires validation and has the potential to predict the incidence of foliar symptoms of Eutypa dieback. Information from this study could lead to an improved integrated pest management system to suppress foliar symptoms and sustain productivity of vines infected with E. lata.     

Phylogenetic Relationships of Xylella fastidiosa Strains Isolated from Landscape Ornamentals in Southern California

ABSTRACT Xylella fastidiosa is an insect-borne, xylem-limited pathogenic bacterium that has been associated with a rise in incidence of diseased landscape ornamentals in southern California. The objective of this study was to genetically characterize strains isolated from ornamental hosts to understand their distribution and identity. Strains of X. fastidiosa isolated from ornamentals were characterized using a multiprimer polymerase chain reaction (PCR) system, random amplified polymorphic DNA (RAPD)-PCR, and sequence analysis of the 16S-23S rDNA intergenic spacer region (ISR). Based on RAPD-PCR and 16S-23S rDNA ISR, strains isolated from daylily, jacaranda, and magnolia clustered with members of X. fastidiosa subsp. sandyi and caused oleander leaf scorch but not Pierce's disease symptoms in glasshouse assays on oleander and grape, respectively. This demonstrated both that our groupings based on genetic characterization were valid and that strains of X. fastidiosa subsp. sandyi are present in hosts other than oleander. Strains isolated from Spanish broom, cherry, and one strain isolated from western redbud clustered with X. fastidiosa subsp. fastidiosa members. Strains isolated from purple-leafed plum, olive, peach, plum, sweetgum, maidenhair tree, crape myrtle, and another western redbud strain clustered with members of X. fastidiosa subsp. multiplex. All strains isolated from mulberry and one from heavenly bamboo formed a separate cluster that has not yet been defined as a subspecies.     

弯孢类炭疽菌rDNA ITS区的RFLP分析及分类研究

 应用核糖体DNA ITS区的RFLP分析对3 8个不同寄主来源的弯孢类炭疽菌菌株的遗传多样性和系统发育进行研究。结果表明:弯孢类炭疽菌的ITS扩增区(ITS₄~ITS₅)约为650 bp,无长度变异。5种内切酶(Alu I、Bsu RI、Hin 6 I、Hpa Ⅱ和Taq I)的酶切图谱在种内是相似或一致的,而种间差异较大。ITS-RFLP共迁带UPGMA聚类分析的结果表明:3 8个弯孢类炭疽菌菌株被聚为6个类群,群与群之间分界明显,表明种的分界相当明显。ITS-RFLP分析的结果还揭示了一些近似种的分类关系,如,按传统方法分别鉴定为Colletotrichum truncatum、C.circinans和C.capsici的许多菌株有相似或完全一致的内切酶酶切图谱,这些分子特征支持它们为同一个种。     

Molecular Characterisation of Alternaria linicola and its Detection in Linseed

Nucleotide sequences of the ribosomal DNA (rDNA) internal transcribed spacers (ITS) 1 and 2 and a 1068bp section of the beta-tubulin gene divided seven designated species of Alternaria into five taxa. Stemphylium botryosum formed a sixth closely related taxon. Isolates of A. linicola possessed an identical ITS sequence to one group of A. solani isolates, and two clusters of A. linicola isolates, revealed from beta-tubulin gene data to show minor variation, were as genetically similar to isolates of A. solani as they were to each other. We suggest, therefore, that A. linicola falls within the species A. solani. Similar results suggest that A. lini falls within the species A. alternata. RAPD analysis of the total genomic DNA from the Alternaria spp. concurred with the nucleotide sequence analyses. An oligonucleotide primer (ALP) was selected from the rDNA ITS1 region of A. linicola/A. solani. PCR with primers ALP and ITS4 (from a conserved region of the rDNA) amplified a c. 536bp fragment from isolates of A. linicola and A. solani but not from other Alternaria spp. nor from other fungi which may be associated with linseed. These primers amplified an identical fragment, confirmed by Southern hybridization, from DNA released from infected linseed seed and leaf tissues. These primers have the potential to be used also for the detection of A. solani in host tissues.     

Genetic variability within <Emphasis Type="Italic">Septoria carvi</Emphasis> Syd.- a pathogen of caraway <Emphasis Type="Italic">Carum carvi</Emphasis> L

Genetic variability within Septoria carvi isolates obtained from various organs of caraway cultivated in south-eastern and central Poland was studied using the RAPD-PCR technique. The tests were performed using randomly selected primers. The DNA profiles obtained using four primers proved useful in determining genetic variability among the genotypes of Septoria carvi isolates. The present study characterized the differences in the nucleotide sequence within the internal transcribed spacer region of rDNA (ITS1, 5.8S, ITS2) of selected S. carvi isolates and reference strains of Septoria spp. Moreover, eight isolates were sequenced for three loci: actin, calmodulin and translation elongation factor 1-alpha, and the obtained sequences were compared with the sequences of Septoria reference strains affecting other plants of the family Apiaceae. Phylogenetic analysis showed distinct differences of the tested isolates, which allowed to treat them Septoria carvi species affecting the above-ground organs of caraway Carum carvi L. This study is the first report on the genetic characteristics of the species S. carvi.