Copper (Cu) tolerance was observed by endophytic fungi isolated from the carnivorous plant Nepenthes ampullaria (collected at an anthropogenically affected site, Kuching city; and a pristine site; Heart of Borneo). The fungal isolates, capable of tolerating Cu up to 1000 ppm (11 isolates in total), were identified through molecular method [internal transcribed spacer 4+5 (ITS4+5); ITS1+NL4; β-tubulin region using Bt2a + Bt2b], and all of them grouped with Diaporthe, Nigrospora, and Xylaria. A Cu biosorption study was then carried out using live and dead biomass of the 11 fungal isolates. The highest biosorption capacity of using live biomass was achieved by fungal isolates Xylaria sp. NA40 (73·26 ± 1·61 mg Cu per g biomass) and Diaporthe sp. NA41 (72·65 ± 2·23 mg Cu per g biomass), NA27 (59·81 ± 1·15 mg Cu per g biomass) and NA28 (56·85 ± 4·23 mg Cu per g biomass). The fungal isolate Diaporthe sp. NA41 also achieved the highest biosorption capacity of 59·33 ± 0·15 mg g-1 using dead biomass. The living biomass possessed a better biosorption capacity than the dead biomass (P
Two hundred and thirty soil samples from different localities were examined for the presence of geophilic keratinophilic fungi. Six species namely Microsporum gypseum--34 isolates, Chrysosporium keratinophilum--29, C. tropicum--20, Keratinophyton terreum--4, Trichophyton terrestre--8 and Chrysosporium species--3--were isolated. Most of these fungi were recovered from garden, field and river bank soil. The importance of these findings is briefly discussed.
Four new species of Stigmatomyces (Ascomycetes, Laboulbeniales, Stigmatomycetinae) parasitic on flies (Diptera) are described. These are S. benjaminii, parasitic on Spilochroa polita (Trixoscelididae) from Mexico, S. munarii, parasitic on Trixoscelis namibensis (Trixoscelididae) from Namibia, S. neurochaetae parasitic on Neurochaeta parviceps (Neurochaetidae) from Malaysia, and S. zaleae, parasitic on Zalea spp. (Tethinidae) from Australia. Both Trixoscelididae and Neurochaetidae are new host families for Laboulbeniales.
Neoscytalidium dimidiatum is an opportunistic fungus causing cutaneous infections mostly, which are difficult to treat due to antifungal resistance. In Malaysia, N. dimidiatum is associated with skin and nail infections, especially in the elderly. These infections may be mistaken for dermatophyte infections due to similar clinical appearance. In this study, Neoscytalidium isolates from cutaneous specimens, identified using morphological and molecular methods (28 Neoscytalidium dimidiatum and 1 Neoscytalidium sp.), were evaluated for susceptibility towards antifungal agents using the CLSI broth microdilution (M38-A2) and Etest methods. Amphotericin B, voriconazole, miconazole and clotrimazole showed high in vitro activity against all isolates with MIC ranging from 0.0313 to 1 µg/mL. Susceptibility towards fluconazole and itraconazole was noted in up to 10% of isolates, while ketoconazole was inactive against all isolates. Clinical breakpoints for antifungal drugs are not yet available for most filamentous fungi, including Neoscytalidium species. However, the results indicate that clinical isolates of N. dimidiatum in Malaysia were sensitive towards miconazole, clotrimazole, voriconazole and amphotericin B, in vitro.
Aquilaria malaccensis produces agarwood in response to wounding and fungal attack. However, information is limited regarding Aquilaria's interaction with its diverse fungal community. In this study, time-related changes of three natural fungal colonizers in two wounded wild A. malaccensis were tracked, beginning a few hours after wounding up to 12 months. Using species-specific primers derived from their nrITS sequences in quantitative real-time PCR (qPCR), we quantified the amount of Cunninghamella bainieri, Fusarium solani and Lasiodiplodia theobromae. Because time is a major factor affecting agarwood quantity and quality, 14 wood samples were collected at different time points, i.e., 0-18 h, 2-13 days, 2-18 weeks, and 6-12 months after wounding. qPCR data revealed that the abundance of the three species decreased over time. The fungi were detected in high numbers during the first few hours and days after wounding (40- to 25,000-fold higher levels compared with initial counts) and in low numbers (<1- to 3,200-fold higher than initially) many months later. Consistent with its role in defense response, the accumulation of secondary metabolites at the wounding site could have caused the decline in fungal abundance. Succession patterns of the two trees were not identical, indicating that fungal populations may have been affected by tree environment and wound microclimate. Our results are important for understanding the diversity of microbial community in wild Aquilaria species and their association to wound-induced agarwood formation. Fungi could be secondary triggers to agarwood production in situations where trees are wounded in attempt to induce agarwood.
Aureobasidium pullulans AY4 is an opportunistic pathogen that was isolated from the skin of an immunocompromised patient. We present here the draft genome of strain AY4, which reveals an abundance of genes relevant to bioindustrial applications, including biocontrol and biodegradation. Putative genes responsible for the pathogenicity of strain AY4 were also identified.
Pleosporales is the largest order in the fungal class Dothideomycetes. We report the 36,814,818-bp draft genome sequence and gene annotation of UM1110, a Pleosporales isolate associated with unclassified genera that is potentially a new fungal species. Analysis of the genome sequence led to the finding of genes associated with fungal adhesive proteins, secreted proteases, allergens, and pseudohyphal development.
Daldinia eschscholzii is an invasive endophyte that is most commonly found in plant tissues rich in secondary metabolites. We report the draft genome sequence of D. eschscholzii isolated from blood culture. The draft genome is 35,494,957 bp in length, with 42,898,665 reads, 61,449 contigs, and a G+C content of 46.8%. The genome was found to contain a high abundance of genes associated with plant cell wall degradation enzymes, mycotoxin production, and antifungal drug resistance.
Morphological features and Inter Simple Sequence Repeat (ISSR) polymorphism were employed to analyse 21 Corynespora cassiicola isolates obtained from a number of Hevea clones grown in rubber plantations in Malaysia. The C. cassiicola isolates used in this study were collected from several states in Malaysia from 1998 to 2005. The morphology of the isolates was characteristic of that previously described for C. cassiicola. Variations in colony and conidial morphology were observed not only among isolates but also within a single isolate with no inclination to either clonal or geographical origin of the isolates. ISSR analysis delineated the isolates into two distinct clusters. The dendrogram created from UPGMA analysis based on Nei and Li's coefficient (calculated from the binary matrix data of 106 amplified DNA bands generated from 8 ISSR primers) showed that cluster 1 encompasses 12 isolates from the states of Johor and Selangor (this cluster was further split into 2 sub clusters (1A, 1B), sub cluster 1B consists of a unique isolate, CKT05D); while cluster 2 comprises of 9 isolates that were obtained from the other states. Detached leaf assay performed on selected Hevea clones showed that the pathogenicity of representative isolates from cluster 1 (with the exception of CKT05D) resembled that of race 1; and isolates in cluster 2 showed pathogenicity similar to race 2 of the fungus that was previously identified in Malaysia. The isolate CKT05D from sub cluster 1B showed pathogenicity dissimilar to either race 1 or race 2.
This study examined the effect of host extract in the culture medium on anti-candidal activity of Phomopsis sp. ED2, previously isolated from the medicinal herb Orthosiphon stamineus Benth. Interestingly, upon addition of aqueous host extract to the culture medium, the ethyl acetate extract prepared from fermentative broth exhibited moderate anti-candidal activity in a disc diffusion assay. The minimal inhibitory concentration of this extract was 62.5 μg/ml and it only exhibited fungistatic activity against C. albicans. In the time-kill study, a 50% growth reduction of C. albicans was observed at 31.4 h for extract from the culture incorporating host extract. In the bioautography assay, only one single spot (Rf 0.59) developed from the extract exhibited anti-candidal activity. A spot with the a similar Rf was not detected for the crude extract from YES broth without host extract. This indicated that the terpenoid anti-candidal compound was only produced when the host extract was introduced into the medium. The study concluded that the incorporation of aqueous extract of the host plant into the culture medium significantly enhanced the anti-candidal activity of Phomopsis sp. ED2.
Garcinia is commonly found in Malaysia, but limited information is available regarding endophytic fungi associated with this plant. In this study, 24 endophytic fungi were successfully recovered from different parts of two Garcinia species. Characterization of endophytic fungi was performed based on the conserved internal transcribed spacer (ITS) region sequence analysis and the antimicrobial properties. Results revealed that fruits of the plant appeared to be the highest inhabitation site (38 %) as compared with others. Glomerella sp., Guignardia sp., and Phomopsis sp. appeared to be the predominant endophytic fungi group in Garcinia mangostana and Garcinia parvifolia. Phylogenetic relationships of the isolated endophytic fungi were estimated from the sequences of the ITS region. On the other hand, antibacterial screening showed 11 of the isolates possessed positive response towards pathogenic and nonpathogenic bacteria. However, there was no direct association between certain antibacterial properties with the specific genus observed.
Coevolution between hosts and pathogens generates strong selection pressures to maintain resistance and infectivity, respectively. Genomes of plant pathogens often encode major effect loci for the ability to successfully infect specific host genotypes. Hence, spatial heterogeneity in host genotypes coupled with abiotic factors could lead to locally adapted pathogen populations. However, the genetic basis of local adaptation is poorly understood. Rhynchosporium commune, the pathogen causing barley scald disease, interacts at least partially in a gene-for-gene manner with its host. We analyzed global field populations of 125 R. commune isolates to identify candidate genes for local adaptation. Whole genome sequencing data showed that the pathogen is subdivided into three genetic clusters associated with distinct geographic and climatic regions. Using haplotype-based selection scans applied independently to each genetic cluster, we found strong evidence for selective sweeps throughout the genome. Comparisons of loci under selection among clusters revealed little overlap, suggesting that ecological differences associated with each cluster led to variable selection regimes. The strongest signals of selection were found predominantly in the two clusters composed of isolates from Central Europe and Ethiopia. The strongest selective sweep regions encoded protein functions related to biotic and abiotic stress responses. Selective sweep regions were enriched in genes encoding functions in cellular localization, protein transport activity, and DNA damage responses. In contrast to the prevailing view that a small number of gene-for-gene interactions govern plant pathogen evolution, our analyses suggest that the evolutionary trajectory is largely determined by spatially heterogeneous biotic and abiotic selection pressures.
Despite the great importance of Aureobasidium pullulans in biotechnology, the fungus had emerged as an opportunistic human pathogen, especially among immunocompromised patients. Clinical detection of this rare human fungal pathogen presently relies on morphology diagnosis which may be misleading. Thus, a sensitive and accurate quantitative molecular assay for A. pullulans remains lacking. In this study, we presented the microscopy observations of A. pullulans that reveals the phenotypic plasticity of the fungus. A. pullulans-specific primers and molecular beacon probes were designed based on the fungal 18S ribosomal RNA (rRNA) gene. Comparison of two probes with varied quencher chemistry, namely BHQ-1 and Tamra, revealed high amplification efficiency of 104% and 108%, respectively. The optimized quantitative real-time PCR (qPCR) assays could detect and quantify up to 1 pg concentration of A. pullulans DNA. Both assays displayed satisfactory performance parameters at fast thermal cycling mode. The molecular assay has great potential as a molecular diagnosis tool for early detection of fungal infection caused by A. pullulans, which merits future study in clinical diagnosis.
Setosphaeria rostrata, a common plant pathogen causing leaf spot disease, affects a wide range of plant species, mainly grasses. Fungi were isolated from brown spots on rice leaves throughout Peninsular Malaysia, and 45 isolates were identified as Setosphaeria rostrata The isolates were then characterized using morphological and molecular approaches. The mating type was determined using PCR amplification of the mating type alleles, and isolates of opposite mating types were crossed to examine sexual reproduction. Based on nuclear ribosomal DNA ITS1-5.8S-ITS2 region (ITS) and beta-tubulin (BT2) sequences, two phylogenetic trees were constructed using the maximum likelihood method; S. rostrata was clustered in one well-supported clade. Pathogenicity tests showed that S. rostrata isolates are pathogenic, suggesting that it is the cause of the symptoms. Mating-type analyses indicated that three isolates carried the MAT1-1 allele, and the other 42 isolates carried MAT1-2 After isolates with opposite mating types were crossed on Sach's medium and incubated for 3 wk, six crosses produced pseudothecia that contained eight mature ascospores, and 12 other crosses produced numerous pseudothecia with no ascospores. To our knowledge, this is the first report on S. rostrata isolated from leaf spots on rice.
This paper reports on the vinca alkaloid produced by a novel Nigrospora sphaerica isolated from Catharanthus roseus. Through liquid chromatography-mass spectrometry (LCMS), only the crude mycelia extract of this fungus was positive for determination of vinblastine. This vinca alkaloid was then purified by using high-performance liquid chromatography (HPLC) and tested for cytotoxicity activity using MTT assays. The breast cell line cancer (MDA-MB 231) was treated with a purified vinblastine which was intracellulary produced by N. sphaerica. The purified vinblastine from extracted leaf of C. roseus was used as a standard comparison. A positive result with a value of half maximal inhibitory concentration (IC50 ) of > 32 μg ml-1 was observed compared with standard (IC50 ) of 350 μg ml-1 only. It showed that a vinblastine produced by N. sphaerica has a high cytotoxicity activity even though the concentration of vinblastine produced by this endophytic fungus was only 0.868 μg ml-1 .