Siti Aisyah Alias

Improving ITS sequence data for identification of plant pathogenic fungi

SummaryPlant pathogenic fungi are a large and diverse assemblage of eukaryotes with substantial impacts on natural ecosystems and human endeavours. These taxa often have complex and poorly understood life cycles, lack observable, discriminatory morphological characters, and may not be amenable to in vitro culturing. As a result, species identification is frequently difficult. Molecular (DNA sequence) data have emerged as crucial information for the taxonomic identification of plant pathogenic fungi, with the nuclear ribosomal internal transcribed spacer (ITS) region being the most popular marker. However, international nucleotide sequence databases are accumulating numerous sequences of compromised or low-resolution taxonomic annotations and substandard technical quality, making their use in the molecular identification of plant pathogenic fungi problematic. Here we report on a concerted effort to identify high-quality reference sequences for various plant pathogenic fungi and to re-annotate incorrectly or insufficiently annotated public ITS sequences from these fungal lineages. A third objective was to enrich the sequences with geographical and ecological metadata. The results – a total of 31,954 changes – are incorporated in and made available through the UNITE database for molecular identification of fungi (http://unite.ut.ee), including standalone FASTA files of sequence data for local BLAST searches, use in the next-generation sequencing analysis platforms QIIME and mothur, and related applications. The present initiative is just a beginning to cover the wide spectrum of plant pathogenic fungi, and we invite all researchers with pertinent expertise to join the annotation effort.

Frequency of occurrence of fungi on wood in Malaysian mangroves

Over one hundred fungi have been reported on mangrove wood (Hyde and Jones, 1988; Jones and Kuthubutheen, 1989; Hyde and Jones, unpublished) from tropical and subtropical locations. A variety of factors affect the frequency of occurrence of these fungi, e.g . salinity, length of exposure of substrate, wood species and location within the mangrove. Two aspects are addressed in this paper: a) Comparison of the fungi present at three mangroves in Malaysia (Morib, Kuala Selangor, Port Dickson); and b) The fungi colonising specific mangrove wood, e.g. Avecennia marina, Bruguiera gymnorrhiza. Species diversity was greatest at Port Dickson with 63 fungi recorded while species composition varied from site to site. Common fungi at Morib and Kuala Selangor were Halocyphina villosa and Leptosphaeria australiensis along with Kallichroma tethys and Lulworthia grandispora at the latter site. None of these were common at Port Dickson, the dominant species at this site were Hypoxylon oceanicum and Massarina ramunculicola. A comparison of the fungi occurring on Avicennia marina and Bruguiera gymnorrhiza indicated that Halocyphina villosa was common on both timbers. Species diversity and abundance were greatest on Avicennia with the following fungi listed as common: Eutypa sp., Kallichroma tethys, Marinosphaera mangrovei, Phoma sp. and Julelia avicenniae.

Biodiversity and distribution of fungi associated with decomposing Nypa fruticans

Fungi associated with the decomposition of Nypa fruticans in Malaysia are under investigation. Forty-one fungi have been identified including 35 ascomycetes, four mitosporic fungi and two basidiomycetes. The distribution of intertidal fungi on palm structures including leaves, leaf veins, rachides, petiole bases, and inflorescences, and fungi on terrestrial parts have also been examined. No fungi were found on the leaf material, although several fungi were found on the leaf midribs, and possible reasons for this are given. Very few taxa developed on the inflorescences, but those that were present were abundant. The greatest density of fruiting structures occurred on the rachides, and the greatest diversity of fungal species occurred on the petiole bases. The terrestrial fungi differed from the intertidal fungi, although Linocarpon nipae occurred in both habitats. Reasons for the differences in fungal numbers and diversity on the various palm parts are discussed. The diversity of fungi at Morib mangrove was low when compared to previous studies on fungi on Nypa palm at Kampong Api Api in Brunei and in this study at Kuala Selangor mangrove in Malaysia.

Phyllosticta capitalensis, a widespread endophyte of plants

Phyllosticta capitalensis is an endophyte and weak plant pathogen with a worldwide distribution presently known from 70 plant families. This study isolated P. capitalensis from different host plants in northern Thailand, and determined their different life modes. Thirty strains of P. capitalensis were isolated as endophytes from 20 hosts. An additional 30 strains of P. capitalensis from other hosts and geographic locations were also obtained from established culture collections. Phylogenetic analysis using ITS, ACT and TEF gene data confirmed the identity of all isolates. Pathogenicity tests with five strains of P. capitalensis originating from different hosts were completed on their respective host plants. In all cases there was no infection of healthy leaves, indicating that this endophyte does not cause disease on healthy, unstressed host plants. That P. capitalensis is often isolated as an endophyte has important implications in fungal biology and plant health. Due to its endophytic nature, P. capitalensis is commonly found associated with lesions of plants, and often incorrectly identified as a species of quarantine importance, which again has implications for trade in agricultural and forestry production.

Extracellular hydrolase enzyme production by soil fungi from King George Island, Antarctica

Various microbial groups are well known to produce a range of extracellular enzymes and other secondary metabolites. However, the occurrence and importance of investment in such activities have received relatively limited attention in studies of Antarctic soil microbiota. In order to examine extracellular enzyme production in this chronically low-temperature environment, fungi were isolated from ornithogenic, pristine and human-impacted soils collected from the Fildes Peninsula, King George Island, Antarctica during the austral summer in February 2007. Twenty-eight isolates of psychrophilic and psychrotolerant fungi were obtained and screened at a culture temperature of 4°C for activity of extracellular hydrolase enzymes (amylase, cellulase, protease), using R2A agar plates supplemented with (a) starch for amylase activity, (b) carboxymethyl cellulose and trypan blue for cellulase activity or (c) skim milk for protease activity. Sixteen isolates showed activity for amylase, 23 for cellulase and 21 for protease. One isolate showed significant activity across all three enzyme types, and a further 10 isolates showed significant activity for at least two of the enzymes. No clear associations were apparent between the fungal taxa isolated and the type of source soil, or in the balance of production of different extracellular enzymes between the different soil habitats sampled. Investment in extracellular enzyme production is clearly an important element of the survival strategy of these fungi in maritime Antarctic soils.

Biodiversity of marine fungi in Malaysian mangroves

Abstract Senescent and decaying substrata were collected in selected Malaysian mangroves (prop roots, pneumatophores, overhanging branches and twigs of mangrove trees, driftwood) to study the diversity of marine mangrove fungi. One hundred and thirty-nine marine fungi were fully identified to specific level (115 Ascomycota, 2 Basidiomycota, 22 anamorphic taxa). Based on percentage of occurrence, the most common species were Lignincola laevis (16.8%), Verruculina enalia (13.3%), Trichocladium achrasporum (12.1%), Savoryella lignicola (11.8%), Dictyosporium pelagicum (11.2%), Halocyphina villosa (11.0%) and Lulworthia grandispora (10.9%). Among the samples, 84.2% supported an average number of 2.8 fungal species per sample. Of the 139 fungi identified in this study, circa 84% (117 species) are only found in the tropics/subtropics, mostly as mangrove species. Factors affecting the distribution and abundance of mangrove fungi are discussed, including: substrate specificity, sampling frequency and intensity, vertical distribution and geographical locations.

Linocarpon angustatum sp. nov., and Neolinocarpon nypicola sp. nov. from petioles of Nypa fruticans, and a list of fungi from aerial parts of this host

Two new ascomycetes,Linocarpon angustatum sp. nov. andNeolinocarpon nypicola sp. nov., are described from petioles ofNypa fruticans in Malaysia.Linocarpon angustatum differs from species in the genus in having needle-shaped ascospores.Neolinocarpon nypicola differs from species in the genus in having filiform ascospores which gradually taper towards the ends, and ascomata developing within well developed stromata. These new species are compared with existing species and illustrated with interference light micrographs. The fungi known from aerial parts ofNypa fruticans are listed.

Antimicrobial activities of marine fungi from Malaysia

Abstract In a systematic screening effort, extracts of marine fungi from Malaysia were investigated for antimicrobial activity and potentially active secondary metabolites. In preliminary experiments, the plug assay method was employed to screen 152 strains for antimicrobial activity. Of these, 82 exhibited biological activity, with most of the active strains (90.2%) having antibacterial activity, mainly towards Gram-positive bacteria, while only 9.6% had antifungal activity. On the basis of these results, five marine fungi, Fasciatispora nypae, Caryosporella rhizophorae, Melaspilea mangrovei, Leptosphaeria sp. and ascomycete strain 19 (NF) were selected for further investigation to confirm their biological activity by the disc diffusion assay method. The selected species had various degrees of activity against the test microorganisms, depending on culture conditions (stationary vs. shaking cultures) and incubation time (10–25 days). Only F. nypae showed a wider range of antifungal and antibacterial activity as compared to the remaining fungal strains under investigation. Therefore, bioactivity-guided fractionation was undertaken to isolate the active principles, resulting in the characterisation of 2,2,7-trimethyl-2H-chromen-5-ol (1) which had antimicrobial activity towards test microorganisms. The structure of 1, which previously had only been reported as a synthetic intermediate, but not as a natural product, was elucidated by mass spectrometry in conjunction with one- and two-dimensional NMR spectroscopy.

Cucullosporella mangrovei, ultrastructure of ascospores and their appendages

The ultrastructure ofCucullosporella mangrovei ascospores is described. Mature ascospores possess two wall layers, an outer electron-dense episporium and an innermost tripartite mesosporium. Episporial elaborations form electrondense spore wall ornamentations from which extend fibrils that may constitute a highly hydrated exosporium which was not visualised at either the scanning electron microscope or light microscope level. Ascospores possess a hamate appendage at each pole which unfolds in seawater to form a long thread. Ultrastructurally the polar appendage comprises folded fibro-granular electron-dense material and fine fibrils. The fibrils form a matrix around and within the fibro-granular appendage and around the entire unreleased ascospore. These fibrils have not been observed associated with the ascospore appendages in other species of the Halosphaeriales and are a discrete and new appendage component. The fibro-granular appendage and fibrils are bounded by the outer delimiting membrane which is absent around released ascospores. The nature of the spore appendage is compared with that of other marine and freshwater ascomycetes and the taxonomic assignment of the species is discussed.

Intertidal marine fungi from San Juan Island and comments on temperate water species

Summary Temperate marine fungi are a distinct ecological group and differ from those found in tropical habitats, with the exception of a few cosmopolitan species. Sixty-two marine fungi were collected at San Juan Island, a temperate site, in 1994. The most frequently collected species were: Halosphaeria appendiculata, Monodictyspelagica, Halosarpheia trullifera, Ceriosporopsis halima and a new ascomycete. Eight records of Capronia ciliomaris were made, all on coniferous bark (probably Pseudosuga sp.). The fungi collected are compared with those previously reported from San Juan Island. The biogeography of marine fungi is discussed.