Phylogeny and taxonomy of species in the Grosmannia serpens complex

Grosmannia serpens was first described from pine in Italy in 1936 and it has been recorded subsequently from many countries in both the northern and southern hemispheres. The fungus is vectored primarily by root-infesting bark beetles and has been reported to contribute to pine-root diseases in Italy and South Africa. The objective of this study was to consider the identity of a global collection of isolates not previously available and using DNA sequence-based comparisons not previously applied to most of these isolates. Phylogenetic analyses of the ITS2-LSU, actin, beta-tubulin, calmodulin and translation elongation factor-1 alpha sequences revealed that these morphologically similar isolates represent a complex of five cryptic species. Grosmannia serpens sensu stricto thus is redefined and comprises only isolates from Italy including the ex-type isolate. The ex-type isolate of Verticicladiella alacris was shown to be distinct from G. serpens, and a new holomorphic species, G. alacris, is described. The teleomorph state of G. alacris was obtained through mating studies in the laboratory, confirming that this species is heterothallic. Most of the available isolates, including those from South Africa, USA, France, Portugal and some from Spain, represent G. alacris. The remaining three taxa, known only in their anamorph states, are described as the new species Leptographium gibbsii for isolates from the UK, L. yamaokae for isolates from Japan and L. castellanum for isolates from Spain and the Dominican Republic.     

Leptographium tereforme sp. nov. and other Ophiostomatales isolated from the root-feeding bark beetle Hylurgus ligniperda in California

The redhaired pine bark beetle Hylurgus ligniperda (F.) is native to Europe but was discovered in Los Angeles, California, in 2003. This root-and stump-feeding beetle is a common vector of Ophiostomatales, which are potential tree pathogens or causes of blue stain of conifer sapwood. In this study Ophiostomatales were isolated on a cycloheximide-amended medium from 118 adult H. ligniperda collected from infested logs of Pinus halepensis and P. pinea at two sites in California. In total eight species of Ophiostomatales were identified and seven species that occasionally were isolated were unidentified. The most frequently isolated species were Ophiostoma ips and Grosmannia galeiforme, which were isolated respectively from 31% and 23% of the 118 beetles. The other species isolated included O. piceae (isolated from 9% of the beetles), O. querci (8%) and Leptographium tereforme sp. nov. (6%). Grosmannia huntii, L. serpens, three Sporothrix species, O. floccosum, O. stenoceras, two unidentified Hyalorhinocladiella sp. and a sterile fungus each were isolated from fewer then 5% of beetles. Most of the identified species already were known in USA and have been found in association with H. ligniperda in other countries. However the new species, L. tereforme, and G. galeiforme were recorded from USA for the first time, and this is the first report of L. serpens from western North America.     

Description of a Distinctive Aflatoxin-Producing Strain of <Emphasis Type="Italic">Aspergillus nomius</Emphasis> that Produces Submerged Sclerotia

A new distinctive strain of Aspergillus nomius that produces the potent mycotoxins, aflatoxins, is described from pistachio, pecan, and fig orchards in California. Similar to the typical strain of A. nomius (as represented by the ex-type), the O strain produced both B and G aflatoxins but not cyclopiazonic acid, had similar conidial ornamentation, and grew poorly at 42°C. Furthermore, previous published DNA sequence supports that the new strain is very closely related to the ex-type of A. nomius. However, the O strain differs from the ex-type in several morphological characters. The ex-type was initially described as producing “indeterminate sclerotia” that appear as large (up to 3 mm long) elongated sclerotia on surfaces of media. The O strain produces only small spherical sclerotia (mean diameter <0.3 mm) submerged in the medium. In addition, the O strain has predominantly uniseriate conidial heads, whereas the typical strain of A. nomius has predominantly biseriate heads. The O strain colony color on both Czapek solution agar and Czapek yeast extract agar was more yellowish than the ex-type of A. nomius and other common aflatoxin-producing fungi. Isolates of the O strain reported here from several orchards represent the first report of A. nomius in California.     

Diagnostic characterization of Penicillium palitans, P. commune and P. solitum

A total of 98 isolates of Penicillium commune and P. solitum were analysed and it was shown that these isolates produced three unique combinations of secondary metabolites. Penicillium commune produced cyclopiazonic acid, cyclopaldic acid and rugulovasine A and B; P. solitum produced compactin and a new group including the ex-type culture of P. palitans produced cyclopiazonic acid and fumigaclavine A. Isolates in all three groups were able to produce cyclopenin, cyclopenol and viridicatin. An optimal thin layer chromatography system to detect the secondary metabolites from P. commune, P. palitans and P. solitum was developed using an agar plug method. The results were confirmed by high performance liquid chromatography with diode array detection. The chemotaxonomic allocations were backed up by differences in conidial colour on Czapek yeast autolysate agar, reverse colour on yeast extract sucrose agar and origin of the isolates. Even though P. palitans previously has been considered synonymous with P. commune or P. solitum it was concluded that P. palitans is a distinct species.     

Genotypic variation in Penicillium chysogenum from indoor environments

We examined 198 isolates of P. chysogenum recovered from 109 houses in Wallaceburg, Ontario, and 25 culture collection isolates including seven ex-type strains. Multilocus genotypes were determined by heteroduplex mobility assay of regions spanning introns in acetyl co-enzyme A synthase, beta-tubulin, thioredoxin reductase and the internal transcribed spacer regions of the nuclear ribosomal subrepeat. Five unique multilocus haplotypes were revealed without evidence of recombination, indicating strictly clonal population structures. Phylogenetic analysis of allele sequences using maximum parsimony resolved three strongly supported lineages. The dominant clade included more than 90% of house isolates in addition to the notable laboratory contaminant isolated by Alexander Fleming in 1929 in Britain. A second clade contained more than 5% of house isolates clustered with the ex-type strains of P. chysogenum and P. notatum. Follow-up sampling of outdoor air in the locality failed to reveal P. chysogenum, confirming the rarity of this fungus in outdoor air.     

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.     

Genetic characterization of Raffaelea quercivora isolates collected from areas of oak wilt in Japan

This study was undertaken to improve understanding of the phylogenetic position of pathogenic fungi implicated in the oak wilt in Japan. Sequences were obtained from three regions of partial nuclear ribosomal DNA of 25 isolates of Raffaelea quercivora including an ex-type strain, all of which were collected from seven areas of disease outbreak. All the isolates formed one clear clade with high bootstrap values, distinctly delimited from the closest species, R. montetyi. These results indicate that the R. quercivora is phylogenetically a well-defined taxon.     

Oogonial biometry and phylogenetic analyses of the Pythium vexans species group from woody agricultural hosts in South Africa reveal distinct groups within this taxon

Pythium vexans fits into the internal transcribed spacer (ITS) clade K sensu Lévesque & De Cock (2004). Within clade K, P. vexans forms a distinct clade containing two enigmatic species, Pythium indigoferae and Pythium cucurbitacearum of which no ex-type strains are available. In South Africa, as well as in other regions of the world, P. vexans isolates are known to be heterogeneous in their ITS sequences and may consist of more than one species. This study aimed to investigate the diversity of South African P. vexans isolates, mainly from grapevines, but also citrus and apple using (i) phylogenetic analyses of the ITS, cytochrome c oxidase (cox) I, cox II, and β-tubulin regions and (ii) seven biometric oogonial parameters. Each of the phylogenies clustered P. vexans isolates into a single well-supported clade, distinct from other clade K species. The β-tubulin region was phylogenetically uninformative regarding the P. vexans group. The ITS phylogeny and combined cox I and II phylogenies, although each revealing several P. vexans subclades, were incongruent. One of the most striking incongruences was the presence of one cox subclade that contained two distinct ITS subclades (Ib and IV). Three groups (A-C) were subjectively identified among South African P. vexans isolates using (i) phylogenetic clades (ITS and cox), (ii) univariate analysis of oogonial diameters, and (iii) multivariate analyses of biometric oogonial parameters. Group A is considered to be P. vexans s. str. since it contained the P. vexans CBS reference strain from Van der Plaats-Niterink (1981). This group had significantly smaller oogonial diameters than group B and C isolates. Group B contained the isolates from ITS subclades Ib and IV, which formed a single cox subclade. The ITS subclade IV isolates were all sexually sterile or produced mainly abortive oospores, as opposed to the sexually fertile subclade Ib isolates, and may thus represent a distinct assemblage within group B. Although ITS subclade Ib included the P. indigoferae ex-type sequence, this group was considered to be P. vexans since South African isolates in this clade produced globose sporangia. Group C contained four apple isolates that were related to, but distinct from P. cucurbitacearum. Although P. vexans groups A-C might be distinct species, they are not described here as such due to (i) these groups only representing some of the known diversity in P. vexans, (ii) conflicting gene tree phylogenies preventing phylogenetic species identification, and (iii) sexually sterile isolates preventing the broad application of biometrical data.     

<Emphasis Type="Italic">Ophiostoma tsotsi</Emphasis> sp. nov., A Wound-infesting Fungus of Hardwood Trees in Africa

Polymorphic sequence-characterised marker assays from a recent diversity study on the Ascomycete fungus Ophiostoma quercus reported that some isolates from Africa were genetically distinct from O. quercus. In the present study, these African isolates were compared with authentic O. quercus isolates by evaluating morphological characters, growth in culture, mating compatibility and DNA sequence data. The isolates from Africa were morphologically similar to O. quercus, presenting Pesotum and Sporothrix synanamorphs in culture. Phylogenetic analyses of the ribosomal internal transcribed spacer regions 1 and 2, β-tubulin and translation elongation factor 1-α gene regions confirmed that the African group represents a distinct species within the hardwood lineage of the O. piceae complex, closely related to O. ulmi and O. himal-ulmi. Mating studies between O. quercus and the African isolates showed that isolates mated predominantly with those of their own group, although there were rare cases of fertile crosses between the groups. Isolates residing in the African lineage are described here as a new species, O. tsotsi sp. nov.     

Studies on Morinia: recognition of Morinia longiappendiculata sp. nov. as a new endophytic fungus, and a new circumscription of Morinia pestalozzioides

A new coelomycete, Morinia longiappendiculata sp. nov., isolated from living stems of four plant species in central Spain, is described. The distinctive morphological characteristics of this fungus are the production of conidia with long basal and apical appendages on filiform conidiogenous cells that contrasts with the short-appendaged conidia and cylindrical conidiogenic cells of the type species, M. pestalozzioides. Comparative sequence analysis of the ITS rDNA region and fragments of the translation elongation factor 1alpha, actin and chitin synthase 1 genes and the study of the HPLC profiles of the M. longiappendiculata and M. pestalozzioides isolates supported the recognition of the new species. Comparison of the ITS rDNA sequences of the Morinia isolates with GenBank sequences indicated that the genus belongs to the Amphisphaeriaceae with the highest similarity to Bartalinia and Truncatella. Bresadola's original definition of M. pestalozzioides is updated by adding information on conidiogenesis and molecular data. A lectotype and epitype are designated for the species. A study of bioactive metabolites revealed that M. pestalozzioides cultures produced moriniafungin, a novel sordarin analog with potent antifungal activity.     

Batch culture screening of marine microalgal nutrient removal from primary sewage effluent

A total of 102 marine microalgal species (including 66 isolates from a sewage outfall site in St Andrews Bay, Scotland) were screened for their ability to remove ammonium and phosphate from primary treated sewage. Primary effluent was diluted 1:1 with sterile seawater, to which the test alga was added for batch culture experiments under controlled environmental conditions of seven or two days duration. The results demonstrated that although microalgae vary in their treatment capabilities, some species were able to remove >90% of both the ammonium and phosphate after seven and even two days culture. All of these best-treating species were found to be unialgal after sequential culture, indicating their potential value for further screening under continuous culture conditions, and over half were isolates from the outfall site. The best-treating algal species showed a variety of growth responses to changes in salinity between 32 and 0, but all had similar growth responses to a series of sewage:seawater dilutions, suggesting that for some species, other properties of the diluted sewage had more effect on algal growth than salinity. These results demonstrate that a contact time of two days was adequate for >90% removal of ammonium and phosphate by some species of marine microalgae, and that limitations on their growth were not a result of the hyposalinity of the diluted sewage compared to seawater.     

Molecular characterization and pathogenicity of Colletotrichum sp. from guava

Guava (Psidium guajava) fruit is vulnerable to postharvest diseases, such as anthracnose. In the present study, molecular characterisation and pathogenicity of Colletotrichum associated with antharcnose disease of guava fruit were conducted. From anthracnose lesion of guava, 20 isolates were successfully recovered. Based on colony colours, conidia, appressoria and presence or absence of setae, and ITS regions and ß-tubulin gene sequences, the isolates were identified as Colletotrichum gloeosporioides. Phylogenetic analysis based on combined data-sets using neighbour-joining method showed that C. gloeosporioides isolates did not group with C. gloeosporioides epitype strain, and thus the isolates were referred to as C. gloeosporioides species complex or C. gloeosporioides sensu lato. Pathogenicity tests using wounded treatment showed that C. gloeosporioides isolates from guava were pathogenic causing anthracnose on the fruits. The present study showed that C. gloeosporioides sensu lato is the most common species causing antharcnose disease of guava fruit.     

普遍栽培灵芝种类的拉丁学名

本文对中国普遍栽培灵芝种类的拉丁学名进行了讨论,对学名的使用历史及存在的争议进行了回顾,认为中国普遍栽培的灵芝学名应为Ganoderma lingzhi,而不是G. sichuanense;四川灵芝G. sichuanense在形态与分子序列上均与灵芝G. lingzhi不同,是一个独立存在的物种,其后选的附加模式标本与主模式标本差异较大,且附加模式标本的地理位置与主模式标本的地理位置距离较远、海拔差异较大、生态习性也不同,四川灵芝G. sichuanense的附加模式标本代表的是与四川灵芝G. sichuanense不同的物种,应该被废除。     

PCR-RFLP of ITS rDNA for the rapid identification of Penicillium subgenus Biverticillium species

RFLP of ITS rDNA is proposed as a useful tool for molecular identification of the most common species of biverticillate penicillia. 60 isolates were analysed representing 13 species and 21 unique sequences were produced. The combination of five restriction enzymes was successful in separating 12 species. However, the variety Penicillium purpurogenum var. rubrisclerotium remained indistinguishable from Penicillium funiculosum. P. funiculosum appeared as the most confused species, being mis-identified with Penicillium miniolutum and Penicillium pinophilum, which were originally part of the species, and with P. purpurogenum perhaps because of the common production of red pigment. Penicillium variabile was difficult to investigate as introns were found on half of the isolates. Penicillium piceum, Penicillium rugulosum, Penicillium loliense, Penicillium erythromellis and P. purpurogenum were homogeneous from molecular and morphological positions and corresponded to a well circumscribed taxon. Furthermore, intraspecific variability was evidenced within P. pinophilum and P. funiculosum. The ex-type isolate of P. funiculosum produced a unique pattern. The method is sensitive, rapid and inexpensive and can be used for isolate identification of the biverticillate species. It is recommended particularly when many isolates have to be authentificated prior to analysis for phylogenetic assessment or population genetics.     

Systematics of Catenulifera (anamorphic Hyaloscyphaceae) with an assessment of the phylogenetic position of Phialophora hyalina

Catenulifera, typified by C. rhodogena (=Scopulariopsis rhodogena), was established to accommodate the anamorphs of Hyphodiscus (Ascomycota, Helotiales) and to delimit these taxa from members of Phialophora section Catenulatae. Catenulifera rhodogena has been inferred as monophyletic, but its relationship to ascomycetes with poorly differentiated phialidic anamorphs remains enigmatic. To test the hypothesis that C. rhodogena is closely related to morphologically similar species of Phialophora and to further explore the systematics of Catenulifera, we analyzed nuclear rDNA and β-tubulin gene sequences of isolates identified as C. rhodogena, Hyphodiscus hymeniophilus, P. brachyconia, P. brevicollaris and P. hyalina. ITS-LSU and ITS-LSU-β-tubulin phylogenies positioned all isolates except P. hyalina in a single, well-supported clade that consisted of three subclades. Subclade 1 included fungicolous isolates of C. rhodogena and H. hymeniophilus that did not fluoresce when exposed to long-wave UV light. Subclade 2 contained fungicolous and lignicolous strains of C. rhodogena that produced fluorescent colonies and possessed a 366bp indel in the LSU rRNA gene. Neither lineage encompassed the ex-type strains of Cistella rubescens (=H. hymeniophilus) or S. rhodogena, but the former isolate was inferred as sister to Subclade 2. Subclade 3 included all isolates of P. brachyconia, a species recognized here as C. brachyconia comb. nov. A fourth isolate of P. brachyconia that was extralimital to Subclade 3 is described as C. luxurians sp. nov. The positions of C. brevicollaris comb. nov., a species based on P. brevicollaris, and C. luxurians were not resolved in the ITS-LSU phylogeny. P. hyalina is not closely related to Catenulifera.     

Melobesia subplana Rosenvinge (Corallinales, Rhodophyta) is conspecific with Pneophyllum fragile Kützing

The epiphytic species Melobesia subplana erected by Rosenvinge in 1917, has for years been without a verified genus affiliation, because the holotype material did not possess crucial generic information, namely spore germination disc patterns. The species was based on Danish material and collection of new material at the type locality was carried out. Accordance of the holotype and the new material allowed an epitype to be established. Examination of the epitype showed an eight-celled spore germination disc, which verified an ealier suggestion of the species belonging to the genus Pneophyllym. The material, upon which Rosenvinge based M. subplana , was already identified by Foslie as Melobesia lejolisii Rosanoff. However, Rosenvinge found the Danish material to differ so much from M. lejolisii , that he chose to erect the new species. M. lejolisii is now considered homospecific with Pneophyllum fragile Kützing. Comparative studies of the anatomy and reproductive structures of M. subplana and P. fragile revealed concordancy between the two species, and M. subplana is thus considered conspecific with P. fragile.     

Genetic and phenotypic variation of Phytophthora crassamura isolates from California nurseries and restoration sites

Phenotypic and sequence data were used to characterize 28 isolates resembling Phytophthora megasperma from 14 host species in 2 plant production facilities and 10 restoration sites across the San Francisco Bay Area (California; USA). Size of the oogonia and DNA sequences (nuclear internal transcribed spacer (ITS) and mitochondrial cytochrome c oxidase subunit 1 (COX 1)) were compared, and sensitivity to mefenoxam and pathogenicity were measured. Based on ITS 61 % of isolates matched ex-type sequences of Phytophthora crassamura from Italy, and the remainder matched or were close to the P. megasperma ex-type. However, all California P. crassamura genotypes belonged to four unique COX 1 haplotype lineages isolated from both nurseries and restoration sites. Although lineages were sensitive to mefenoxam, a significant difference in sensitivity was identified, and all continued growth in-vitro. These results suggested previous mefenoxam exposure in plant production facilities resulting in tolerance. In conclusion, all evidence pointed to a nursery origin of novel P. crassamura lineages found in California restoration sites. In this study, COX 1 sequences and oogonia size provided information relevant to identify geographic and evolutionary intraspecific variation within P. crassamura, and was additionally used to track the spread of this species from nurseries into wildlands.     

Comparative study reveals unique features of the mycobiota in peat soils samples from Japan and Scotland

We isolated filamentous fungi from soil samples of peat layers in Aomori and Oita Prefectures in Japan and Perth and Kinross district in Scotland by a serial dilution plate technique. The mycobiota in each peat soil showed some common and characteristic features. The abundance of fungal isolates (CFU/g) from peat soil was low: about 1/3 to 1/30 compared with evergreen or coniferous forests or cultivated soil. Trichoderma or Mucorales species were scarcely observed; these fungi occupied only 3% of the total number of colonies. On the other hand, fungi such as Conioscypha and Tolypocladium that are normally isolated rather rarely were encountered at a comparatively high rate. Acremonium guillematii and Tolypocladium cylindrosporum were recorded for the first time in Japan. Sterile fungi occupied 50% of the total number of isolates. The low abundance of fast-growing fungi enabled us to pick slow-growing fungi up easily from the isolation medium. It is interesting that species not previously described in Japan, or scarcely reported, were isolated commonly from both Japanese and Scottish samples. A peat soil sample is therefore an attractive source of untapped microbial resources.