DNA phylogeny, morphology and pathogenicity of Botryosphaeria species on grapevines

Several species of Botr yosphaeria are known to occur on grapevines, causing a wide range of disorders including bud mortality, dieback, brown wood streaking and bunch rot. In this study the 11 Botryosphaeria spp. associated with grapevines growing in various parts of the world, but primarily in South Africa, are distinguished based on morphology, DNA sequences (ITS-1, 5.8S, ITS-2 and EF1-α) and pathological data. Botryosphaeria australis, B. lutea, B. obtusa, B. parva, B. rhodina and a Diplodia sp. are confirmed from grapevines in South Africa, while Diplodia porosum, Fusicoccum viticlavatum and F. vitifusiforme are described as new. Although isolates of B. dothidea and B. stevensii are confirmed from grapevines in Portugal, neither of these species occurred in South Africa, nor were any isolates of B. ribis confirmed from grapevines. All grapevine isolates from Portugal, formerly presumed to be B. ribis, are identified as B. parva based on their EF1-α equence data. From artificial inoculations on grapevine shoots, we conclude that B. australis, B. parva, B. ribis and B. stevensii are more virulent than the other species studied. The Diplodia sp. collected from grapevine canes is morphologically similar but phylogenetically distinct from D. sarmentorum. Diplodia sarmentorum is confirmed as anamorph of Otthia spiraeae, the type species of the genus Otthia (Botryosphaeriaceae). A culture identified as O. spiraeae clustered within Botryosphaeria and thus is regarded as probable synonym. These findings confirm earlier suggestions that the generic concept of Botryosphaeria should be expanded to include genera with septate ascospores and Diplodia anamorphs.     

Two new species of Botryosphaeria with brown, 1-septate ascospores and Dothiorella anamorphs

Botryosphaeria sarmentorum sp. nov. and B. iberica sp. nov. are described and illustrated. These two species are unusual in this genus because of their brown, 1-septate ascospores. Phylogenetic analysis based on ITS and EF1-alpha sequences place them within the clade containing species with Fusicoccum anamorphs. The brown, 1-septate conidia, however, do not conform to Fusicoccum. Therefore phylogenetically and morphologically the anamorphs of these two species belong in a genus distinct from any of the currently accepted anamorph genera assigned to Botryosphaeria. Through a study of the type species of Dothiorella this genus is resurrected to accommodate anamorphs of Botryosphaeria with brown, 1-septate conidia. Botryosphaeria sarmentorum is shown to be the teleomorph of Diplodia sarmentorum, which in turn is transferred to Dothiorella. Otthia quercus is transferred to Botryosphaeria as B. quercicola nom. nov.     

A six locus phylogeny reveals high species diversity in Botryosphaeriaceae from California almond

Botryosphaeriaceae are important pathogens on a variety of woody hosts, including almond, a major crop in California. Almond is susceptible to Botryosphaeria dothidea that forms band cankers on almond trunks, and the same fungus was also isolated from cankers of the canopy. To study the diversity and host range of B. dothidea and allied species from almond we used 132 isolates from 36 plant hosts from five continents, including 45 strains from almond in California. Species were identified by comparison to 13 ex-type strains with phylogenetic analyses based on six loci, including the internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA gene repeat and portions of the coding genes elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase, heat shock protein, histone-3 and beta-tubulin. Seven species were found from almond: Botryosphaeria dothidea, Neofusicoccum parvum, Neof. mediterraneum, Neof. nonquaesitum, Diplodia seriata and Macrophomina phaseolina were identified from band cankers, and B. dothidea, Neof. mediterraneum, Neof. parvum and Dothiorella sarmentorum from canopy cankers. All were capable of inducing cankers on inoculated almond branches in the field. All species found on almond also occurred on other hosts, suggesting that infected vegetation adjacent to almond orchards could serve as source of inoculum of virulent almond strains. Of the 19 monophyletic groups obtained at the species level, 13 contained ex-type strains, five were morphologically similar to established species and one was morphologically distinct from its closest relatives, Neof. andinum and Neof. arbuti, as well as from the more than 190 described species of Fusicoccum and Neofusicoccum, and thus was described as the new species, Neof. nonquaesitum. Evidence for cryptic speciation was found in B. dothidea, Neof. ribis and Spencermartinsia viticola. Botryosphaeria dothidea and Neof. ribis comprised lineages that formed the morphologically distinct Dichomera anamorph not found in any other isolates recognized as B. dothidea and Neof. ribis. An S. viticola isolate from California was phylogenetically divergent and had conidia that differed morphologically from the type. Neofusicoccum parvum was diverse but lacked any morphological features correlating with molecular diversity. Phylogenetic analyses of combinations of datasets showed that pooled analyses of all six datasets resulted in the highest number of supported branches, suggesting that addition of more data might yet improve phylogenetic resolution.     

Phylogenetic and morphological re-evaluation of the Botryosphaeria species causing diseases of Mangifera indica

Species of Botryosphaeria are among the most serious pathogens that affect mango trees and fruit. Several species occur on mangoes, and these are identified mainly on the morphology of the anamorphs. Common taxa include Dothiorella dominicana, D. mangiferae (= Natrassia mangiferae), D. aromatica and an unidentified species, Dothiorella 'long'. The genus name Dothiorella, however, is acknowledged as a synonym of Diplodia. This study aimed to characterize and name the Botryosphaeria spp. associated with disease symptoms on mangoes. To achieve this isolates representing all four Dothiorella spp. mentioned above were compared with the anamorphs of known Botryosphaeria spp., based on conidial morphology and DNA sequence data. Two genomic regions were analyzed, namely the ITS rDNA and beta-tubulin regions. The morphological and molecular results confirmed that the fungi previously identified from mango as species of Dothiorella belong to Fusicoccum. Dothiorella dominicana isolates were identical to isolates of F. parvum (teleomorph = B. parva). A new epithet, namely F. mangiferum, is proposed for isolates previously treated as D. mangiferae or N. mangiferae. Isolates of D. aromatica were identified as F. aesculi (teleomorph = B. dothidea). A fourth Fusicoccum sp. also was identified as those isolates previously known as Dothiorella 'long'. A key is provided to distinguish these species based on anamorph morphology in culture. This study provides a basis for the identification of Botryosphaeria species from mango, which is important for disease control and to uphold quarantine regulations.     

Multiple gene sequences delimit Botryosphaeria australis sp. nov. from B. lutea

Botryosphaeria lutea (anamorph Fusicoccum luteum) most easily is distinguished from other Botryosphaeria spp. by a yellow pigment that is formed in young cultures. This fungus has been reported from a number of cultivated hosts in New Zealand and Portugal. During a survey of Botryosphaeria fungi that occur on native Acacia species in Australia, a yellow pigment was observed in some cultures. These isolates were morphologically similar to B. lutea, but the pigment differed slightly from the one formed by authentic B. lutea isolates. Preliminary data also revealed small differences in ITS rDNA sequence data. The aim of this study was to determine whether these small differences were indicative of separate species or merely variations within B. lutea. Anamorph, teleomorph and culture morphology were compared between B. lutea and Acacia isolates from Australia. Sequence data of two other genome regions, namely the β-tubulin and EF1-α gene and intron regions, were combined with ITS rDNA sequence data to determine the phylogenetic relationship between these isolates. Isolates of B. lutea and those from Australian Acacia species were not significantly different in spore morphology. The yellow pigment, however, was much more distinct in cultures of B. lutea than in cultures from Acacia. There were only a few base pair variations in each of the analyzed gene regions, but these variations were fixed in the two groups in all regions. By combining these data it was clear that B. lutea and the isolates from Acacia were distinct species, albeit very closely related. We, therefore, propose the new epithet B. australis for the fungus from Australia. Botryosphaeria australis also was isolated in this study from exotic Sequoiadendron trees in Australia. Re-analyses of GenBank data in this study showed that B. australis also occurs on other native Australian hosts, namely a Banksia sp. and a Eucalyptus sp., as well as a native Protea sp. in South Africa and on Pistachio in Italy. These records from GenBank have been identified previously as B. lutea. The common occurrence of B. australis on a variety of native hosts across Australia suggests that this fungus is native to this area.     

葡萄座腔菌属ITS-nrDNA的分子系统学分析

对收集的22株和GenBank下载的40株葡萄座腔菌Botryosphaeria ITS序列构建了MP(Maximum parsimony)进化树。在以Leucostoma为外群的情况下,来自我国(主要是陕西)的葡萄座腔菌树木溃疡病菌总体上可分为2个大群,BDGroup1与GenBank上报道的B.dothidea被聚在一起,BD Group2菌株全部来自我国关中咸阳,独立成群,与Botryosphaeria属其他种亲缘关系远。培养特征、分生孢子大小和致病性测定显示:来自我国的苹果轮纹病菌、苹果干腐病菌、梨轮纹病菌、桃树流胶病菌同B.dothidea亲缘关系近,在MP树中聚在一起(BD Group1)。ITS序列分析结果支持B.dothidea与B.berengeriana为同物异名的观点。B.ribis与B.parva亲缘关系近,而与B.dothidea亲缘关系远,支持B.ribis与B.dothidea为不同种的观点。     

枣树干腐病病原菌的鉴定

对山西省枣树上发生的一种新病害进行了调查和病菌分离与培养研究。通过形态学特征观察和rDNA ITS序列分析,证明该病菌为葡萄座腔菌Botryosphaeria dothidea的无性阶段Fusicoccum aesculi。     

Botryosphaeria viticola sp. nov. on grapevines: a new species with a Dothiorella anamorph

Botryosphaeria viticola sp. nov., isolated from pruned canes of Vitis vinifera in NE Spain, is described and illustrated. Phylogenetic analysis based on ITS and EF1-alpha sequences and morphological characters of both anamorph and teleomorph confirmed this taxon to be included within the group of Botryosphaeria species with Dothiorella anamorphs. It is related most closely to B. sarmentorum and B. iberica from which it differs in morphological characters of the teleomorph and DNA sequences.     

Morphology and phylogeny of <Emphasis Type="Italic">Botryosphaeria dothidea</Emphasis> causing fruit rot of olives

The taxonomic position of the causal agent of fruit rot of olives was determined from fresh collections of the fungus from central Greece. In culture it formed two types of conidia, namely fusiform, hyaline, aseptate conidia typical of the genus Fusicoccum, and dark-walled, ovoid, ellipsoid or fusiform, 1–2 septate conidia that are not typically observed in Fusicoccum. A phylogenetic analysis based on ITS and EF1- sequences placed the fungus within the same clade as Fusicoccum aesculi, which is the anamorph of Botryosphaeria dothidea, and the type of the genus Fusicoccum.     

Fusicoccum arbuti sp. nov. causing cankers on pacific madrone in western North America with notes on Fusicoccum dimidiatum, the correct name for Scytalidium dimidiatum and Nattrassia mangiferae

Pacific madrone (Arbutus menziesii) is a broadleaf evergreen tree native to western North America that has been in decline for the past 30 years. A fungus has been isolated and was verified as the cause of cankers on dying trees. It was determined to belong in the genus Fusicoccum, an asexual state of Botryosphaeria. This genus in both its sexual and asexual states commonly causes canker diseases of deciduous woody plants. Using morphological and molecular data the fungus causing cankers on Pacific madrone is characterized, described and illustrated as a new species of Fusicoccum, F. arbuti D.F. Farr & M. Elliott sp. nov. No sexual state is known for F. arbuti. Evidence from the literature, cultures and specimens suggests that F. arbuti, often mistakenly identified as Nattrassia mangiferae, has been causing madrone canker since at least 1968. Authentic isolates of Nattrassia mangiferae as the synanamorph Scytalidium dimidiatum were sequenced and determined to be different from Fusicoccum arbuti and to belong in Botryosphaeria/Fusicoccum. In addition to molecular sequence data, the morphology of the pycnidial and arthric conidial states of Nattrassia mangiferae/ Scytalidium dimidiatum resembles that of Fusicoccum. Therefore the correct name for Nattrassia mangiferae and its numerous synonyms (Dothiorella mangiferae, Torula dimidata, Scytilidium dimidiatum, Fusicoccum eucalypti, Hendersonula toruloidea, H. cypria, Exosporina fawcetii, H. agathidia, and S. lignicola) is Fusicoccum dimidiatum (Penz.) D.F. Farr, comb. nov.     

Evaluation of amplified ribosomal DNA restriction analysis as a method for the identification of Botryosphaeria species

The polymerase chain reaction was used to amplify a rDNA fragment containing the internal transcribed spacers (ITS1-5.8S-ITS2) and the D1/D2 variable domains of the 28S rDNA from 10 species of the genus Botryosphaeria (Fungi, Ascomycota). Restriction analysis of the amplicons with frequent-cutting endonucleases (amplified ribosomal DNA restriction analysis) allowed the definition of 12 rDNA haplotypes. Each of the rDNA haplotypes could be unambiguously assigned to a single Botryosphaeria species, thus allowing clear identification of all the species tested. Intraspecific polymorphism was very low and detected only in Botryosphaeria parva and Botryosphaeria dothidea. Cluster analysis of banding patterns of the isolates corresponded well with known species delineations. The method described in this paper provides a simple and rapid procedure for the differentiation and identification of Botryosphaeria isolates at the species level.     

Three new Lasiodiplodia spp. from the tropics, recognized based on DNA sequence comparisons and morphology

Botryosphaeria rhodina (anamorph Lasiodiplodia theobromae) is a common endophyte and opportunistic pathogen on more than 500 tree species in the tropics and subtropics. During routine disease surveys of plantations in Australia and Venezuela several isolates differing from L. theobromae were identified and subsequently characterized based upon morphology and ITS and EF1-alpha nucleotide sequences. These isolates grouped into three strongly supported clades related to but different from the known taxa, B. rhodina and L. gonubiensis, These have been described here as three new species L. venezuelensis sp. nov., L. crassispora sp. nov. and L. rubropurpurea sp. nov. The three could be distinguished easily from each other and the two described species of Lasiodiplodia, thus confirming phylogenetic separations. Furthermore all five Lasiodiplodia spp. now recognized separated from Diplodia spp. and Dothiorella spp. with 100% bootstrap support.     

Emendation of Dioszegia with redescription of Dioszegia hungarica and two new combinations,Dioszegia aurantiaca and Dioszegia crocea

During phylogenetic analyses of hymenomycetous yeasts based on 18S rDNA sequences, we found that Bullera armeniaca showed an extremely close phylogenetic relationship to Cryptococcus hungaricus. The analyses of internal transcribed spacer (ITS) regions of the two yeasts and the phylogenetically related species, Bullera aurantiaca and Bullera crocea, showed that B. armeniaca and C. hungaricus had identical sequences, indicating that these were conspecific. B. aurantiaca and B. crocea also showed high sequence similarity, 97.1% for ITS1, 100% for ITS2, and 98.7% for overall ITS regions. A DNA-DNA reassociation experiment revealed that B. armeniaca and C. hungaricus were conspecific and B. aurantiaca and B. crocea were two distinct species. These species occurred at a phylogenetically different lineage from that of Bulleromyces albus (anamorph: Bullera alba, type species of Bullera) and Filobasidiella neoformans (anamorph: Cryptococcus neoformans, neotype species of Cryptococcus). Based on these results, we emend the genus Dioszegia to include both ballistoconidium-forming and non-ballistoconidium-forming yeasts and redescribe the species Dioszegia hungarica. B. aurantiaca and B. crocea are also transferred to Dioszegia as Dioszegia aurantiaca comb. nov. and Dioszegia crocea comb. nov.     

引起蓝莓茎溃疡病的葡萄座腔菌属Botryosphaeria二新种

在我国华南地区发现了两个引起蓝莓茎溃疡病的Bofryosphaeria新种,即B.dolichospermatii和B.fujianensis,本研究对这两个物种进行了详细的形态学描述,并制作了图版.基于/TS、LSU、tef1-α 和TUB等4个基因片段的综合系统学分析,B.dolichospermatii和B.fu...     

Two new Ophiostoma species with Sporothrix anamorphs from Austria and Azerbaijan

The genus Ophiostoma includes numerous species of primarily insect-vectored, wood-staining fungi. Several anamorph genera that differ in their micronematous or macronematous conidiogenous cells have been associated with Ophiostoma species. Among the former group, Sporothrix is associated with many species and is characterized by conidiogenous cells that arise laterally or terminally from any place on the hyphae and produce nonseptate conidia on sympodially developing denticles. The purpose of this study was to characterize ophiostomatoid isolates with Sporothrix anamorphs recently collected in Austria and Azerbaijan. The isolates were characterized based on comparisons of rDNA and β-tubulin sequence data. Morphology, growth in culture, and sexual reproductive mode were also considered. Phylogenetic analyses of the combined sequence data showed that the isolates formed two distinct groups, one including isolates from Austria and the other isolates from Austria and Azerbaijan. Growth at 25 C and morphology revealed some differences between the two groups, and supported the view that they represent two new species, which we describe here as Ophiostoma fusiforme sp. nov. and Ophiostoma lunatum sp. nov. Both these groups phylogenetically were related to, but distinct from, Ophiostoma stenoceras.     

New molecular markers for fungal phylogenetics: two genes for species-level systematics in the Sordariomycetes (Ascomycota)

Although significant progress has been made resolving deep branches of the fungal tree of life, many fungal systematists are interested in species-level questions to both define species and assess fungal biodiversity. Fungal genome sequences are a useful resource to systematic biologists for developing new phylogenetic markers that better represent the whole genome. Here we report primers for two newly identified single-copy protein-coding genes, FG1093 and MS204, for use with ascomycetes. Although fungi were the focus of this study, this methodological approach could be easily applied to marker development for studies of other organisms. The tests used here to assess phylogenetic informativeness are computationally rapid, require only rudimentary datasets to evaluate existing or newly developed markers, and can be applied to other non-model organisms to assist in experimental design of phylogenetic studies. Phylogenetic utility of the markers was tested in two genera, Gnomoniopsis and Ophiognomonia (Gnomoniaceae, Diaporthales). The phylogenetic performance of β-tubulin, ITS, and tef-1α was compared with FG1093 and MS204. Phylogenies inferred from FG1093 and MS204 were largely in agreement with β-tubulin, ITS, and tef-1α although some topological conflict was observed. Resolution and support for branches differed based on the combination of markers used for each genus. Based on two independent tests of phylogenetic performance, FG1093 and MS204 were determined to be equal to or better than β-tubulin, ITS, and tef-1α in resolving species relationships. Differences were found in site-specific rate of evolution in all five markers. In addition, isolates from 15 orders and 22 families of Ascomycota were screened using primers for FG1093 and MS204 to demonstrate primer utility across a wide diversity of ascomycetes. The primer sets for the newly identified genes FG1093 and MS204 and methods used to develop them are useful additions to the ascomycete systematists' toolbox.     

Molecular analyses of Pythium irregulare isolates from grapevines in South Africa suggest a single variable species

The Pythium irregulare species complex is the most common and widespread Pythium spp. associated with grapevines in South Africa. This species complex has been subdivided into several morphological and phylogenetic species that are all highly similar at the sequence level [internal transcribed spacer (ITS) and cytochrome c oxidase (cox) regions]. The complex includes Pythium regulare and Pythium cylindrosporum, which are morphologically distinct, and P. irregulare sensu stricto (s.s.) and Pythium cryptoirregulare, which are morphologically similar. The aim of the current study was to determine whether 50 South African grapevine P. irregulare isolates represented more than one phylogenetically distinct species. The isolates were characterised using nuclear (ITS and β-tubulin) and mitochondrial (cox1 and cox2) gene region phylogenies and two isozyme loci [glucose-6-phosphate isomerase (Gpi) and malate dehydrogenase (Mdh-1)]. Some of the gene sequence data were difficult to interpret phylogenetically, since some isolates contained two or more polymorphic ITS copies within the same isolate (intra-isolate variation) that clustered into different ITS sub-clades, i.e. the P. irregulare s.s. and P. cryptoirregulare sub-clades. The molecular data furthermore only revealed the presence of one phylogenetic species, P. irregulare. Morphological analyses of a subset of the isolates confirmed that the isolates were P. irregulare, and further showed that the P. cylindrosporum ex-type strain formed typical P. irregulare oogonia, and not the previously reported distinct elongated oogonia. Some of the molecular analyses suggested the occurrence of outcrossing events and possibly the formation of aneuploids or polyploids since (i) the nuclear and mitochondrial gene data sets were incongruent, (ii) polymorphic ITS copies were present within the same isolate, (iii) heterozygosities were observed in the β-tubulin gene and Gpi and Mdh-1 loci in some isolates and (iv) more than two β-tubulin alleles were detected in some isolates. Altogether, the data suggest that P. irregulare, P. cryptoirregulare, P. cylindrosporum, and possibly P. regulare should be synonimised under the name P. irregulare.     

冬虫夏草及其相关类群的分子系统学分析

为了探明冬虫夏草及其相关类群的亲缘关系,以冬虫夏草、中国被毛孢及中华束丝孢(=冬虫夏草头孢=蝙蝠蛾多毛孢)共6株菌种作为内群和一株蛹草拟青霉作为外群进行了DNA随机多态型(RAPD)分析。此外,基于上述供试材料又在内群中增加了一株蝙蝠蛾拟青霉,并对内群和外群样品的nrDNA间隔区(ITS)碱基序列进行了测定;对于测定的8条序列连同来自GenBank中的4条相关序列进行了分子系统学分析。结果表明:中华束丝孢和中国被毛孢均系冬虫夏草菌的无性型。按照国际植物命名法规,中国被毛孢应为冬虫夏草菌无性型的正确名称。而蝙蝠蛾拟青霉为不同于冬虫夏草菌的另一种真菌;该名称由于不合格发表而不被国际植物命名法规所承认。