Biofilms are an important medical burden, notably for patients with orthopaedic device-related infections. When polymicrobial, these infections are more lethal and recalcitrant. Inter-kingdom biofilm infections are poorly understood and challenging to treat. Here, an in vitro three-species model including Staphylococcus aureus, Escherichia coli and Candida albicans was developed, to represent part of the diversity observed in orthopaedic infections or other clinical contexts. The importance of fungal hyphae for biofilm formation and virulence factor expression was explored. Two protocols were set up, allowing, or not, for hyphal formation. Culturable cells and biomass were characterised in both models, and biofilms were imaged in bright-field, confocal and electron microscopes. The expression of genes related to virulence, adhesion, exopolysaccharide synthesis and stress response was analysed in early-stage and mature biofilms. It was found that biofilms enriched in hyphae had larger biomass and showed higher expression levels of genes related to bacterial virulence or exopolysaccharides synthesis.
Rigidoporus microporus is the fungus accountable for the white root rot disease that is detrimental to the rubber tree, Hevea brasiliensis. The pathogenicity mechanism of R. microporus and the identity of the fungal proteins and metabolites involved during the infection process remain unclear. In this study, the protein and metabolite profiles of two R. microporus isolates, Segamat (SEG) and Ayer Molek (AM), were investigated during an in vitro interaction with H. brasiliensis. The isolates were used to inoculate H. brasiliensis clone RRIM 2025, and mycelia adhering to the roots of the plant were collected for analysis. Transmission electron microscope (TEM) images acquired confirms the hyphae attachment and colonization of the mycelia on the root of the H. brasiliensis clones after 4 days of inoculation. The protein samples were subjected to 2-DE analysis and analyzed using MALDI-ToF MS/MS, while the metabolites were extracted using methanol and analyzed using LC/MS-QTOF. Based on the differential analyses, upregulation of proteins that are essential for fungal evolution such as malate dehydrogenase, fructose 1,6-biphosphate aldolase, and glyceraldehyde-3-phosphate dehydrogenase hints an indirect role in fungal pathogenicity, while metabolomic analysis suggests an increase in acidic compounds which may lead to increased cell wall degrading enzyme activity. Bioinformatics analyses revealed that the carbohydrate and amino acid metabolisms were prominently affected in response to the fungal pathogenicity. In addition to that, other pathways that were significantly affected include "Protein Ubiquitination Pathway," Unfolded Protein Response," "HIFα Signaling," and "Sirtuin Signaling Pathway." The identification of responsive proteins and metabolites from this study promotes a better understanding of mechanisms underlying R. microporus pathogenesis and provides a list of potential biological markers for early recognition of the white root rot disease.
Metarhizium anisopliae (Metchnikoff) Sorokin, a pathogenic fungus to insects, infects the subterranean termite, Coptotermes curvignathus Holmgren, a devastating pest of plantation trees in the tropics. Electron microscopy and proteomics were used to investigate the infection and developmental process of M. anisopliae in C. curvignathus. Fungal infection was initiated by germ tube penetration through the host's cuticle as observed at 6 h post-inoculation (PI), after which it elongated into the host's integumental tissue. The colonization process continued as seen from dissemination of blastospores in the hemocoel at 96 h PI. At this time point, the emergent mycelia had mummified the host and forty-eight hours later, new conidia were dispersed on the termites' body surface. Meanwhile, hyphal bodies were observed in abundance in the intercellular space in the host's body. The proteomes of the pathogen and host were isolated separately using inoculated termite samples withdrawn at each PI-time point and analyzed in two-dimensional electrophoresis (2-DE) gels. Proteins expressed in termites showed evidence of being related to cell regulation and the immune response, while those expressed in M. anisopliae, to transportation and fungal virulence. This study provides new information on the interaction between termites and its entomopathogen, with potential utilization for developing future biopesticide to control the termite population.
The medicinal plant, Syzygium leucoxylon or commonly known as Obah found in North Borneo was considered as traditional medicine by local committee. Two new phenolics, leucoxenols A (1) and B (2) were isolated and identified as major secondary metabolites from the leaves of S. leucoxylon. Their chemical structures were elucidated based on spectroscopic data such as NMR and HRESIMS. Furthermore, these compounds were active against selected strains of fungi.
Candida albicans ATCC 14053 and Candida parapsilosis ATCC 22019 hyphal-wall protein 1 (HWP1) are involved in hyphae formation and pathogenesis. The transcriptional agglutinin-like sequence 3 (ALS3) genes in both species are responsible for the development of biofilm and colonization on tooth surfaces. Therefore, we investigated the expression of HWP1 and ALS3 quantitatively in C. albicans and C. parapsilosis and examined the biofilm structure upon exposure to various nicotine concentrations. In vitro, biofilms of Candida species were developed directly on slides using the Lab-Tek Chamber Slide System and visualized by confocal laser scanning microscopy. Quantitative real-time polymerase chain reaction was used to measure HWP1 and ALS3 expression in C. albicans ATCC 14053 and C. parapsilosis ATCC 22019. The results indicated that nicotine multiplied the number of yeast cells and increased the extracellular polysaccharides of Candida species. We also found that 1-2 mg/mL nicotine could enhance the formation of biofilm. The findings also revealed that the expression of HWP1 and ALS3 in Candida species were increased as the nicotine concentration increased. Therefore, nicotine influences the biofilm development of oral-associated C. albicans ATCC 14053 and C. parapsilosis ATCC 22019.
Introduction:Candida albicans is an opportunistic fungus that is associated with oral carcinogenesis. In addition, biofilm formation has been one of the important virulence factors of the yeast. Streptococcus salivarius K12 is an oral probiotic while Musa acuminata is a well-known prebiotic. The objective of this study is to investigate the effect of S. salivarius K12 and M. acuminata skin aqueous extract (synbiotic) on C. albicans with the hypothesis that S. salivariusK12 and M. acuminata inhibit C. albicans biofilm formation. Methods: To develop mono-species biofilm, C. albicans(ATCC MYA-4901 and cancer isolates, ALC2 and ALC3 strains) and S. salivarius K12 were standardised to 105 cells and 106 cells, respectively and grown in 96-well plate in nutrient broth (NB) or RPMI at 37 °C for 72 h. Polymicro-bial biofilms were developed by inoculating both microorganisms in the same well with similar cell number as in mono-species. To determine the effect of synbiotic, similar protocol was repeated by mixing with 800 mg mL-1 of M. acuminata skin extract and incubated at 37 °C for 72 h. The medium was replenished at every 24 h, aseptically. Finally, the biofilms were assessed using crystal violet assay and the optical density was measured at OD620nm. Results:C. albicans strain MYA-4901 and ALC3, when grown in polymicrobial with S. salivarius K12 in NB that is predominated by yeast-form C. albicans, exhibited decreased biofilms by 71.40±11.7% and 49.40±3.9%, respec-tively when compared to the expected biofilms. Meanwhile in RPMI, which C. albicans strain ATCC MYA-4901, ALC2 and ALC3 were predominated by hyphal-form showed decreased biofilms by 72.0±26.7%, 53.4±14.4% and 65.7±6.7%, respectively when compared to the expected biofilms. Conclusion:S. salivarius K12 and M. acuminata skin extract synbiotic inhibit biofilm formation of C. albicans yeast and hyphal forms thus supported the hypothesis of the present study.
Introduction:Candida spp. are most common opportunistic pathogenic yeast that inhabit human oral cavity, epider-mis, gastrointestinal tract, and vagina leading to candidiasis. The transition of this yeast from commensal to potent pathogen is facilitated by numbers of virulence factors including biofilm formation. While most reports on candidi-asis are associated with formation Candida albicans biofilms, however, non-albicans Candida species prevalence is of growing concern. Recently, the use of probiotics as antifungal and antibiofilm has gained an increasing attention. As such, we aim to evaluate the inhibitory effect of monomicrobial and polymicrobial of Streptococcus salivariuson six strains of NAC namely Candida dubliniensis, Candida glabrata, Candida krusei, Candida lusitanaei, Candida parapsilosis and Candida tropicalis. Methods: Antifungal activity of S. salivarius on NAC species was performed using well diffusion method on Mueller Hinton Agar (MHA) and the diameter of inhibition zone were assessed. For formation of monomicrobial biofilm, standardized cell suspensions of NAC species (1 x 105 cells/ml) and probiotic Streptococcus salivarius (1 x 106 cells/ml) were grown in RPMI or nutrient broth media at 37°C for 72 h. Meanwhile to study polymicrobial biofilm of both NAC and S. salivarius, similar protocol was employed by inoculating both microorganisms with a similar cell density as in monomicrobial. Finally, biofilm formation was assessed through quantification of total biomass by crystal violet (CV) assay and the absorbance of adherent biofilm was measured in triplicate at 620nm. Results: Antifungal susceptibility testing of S. salivarius on all six NAC species discerned no zone of inhibition. Furthermore, our results showed variability of monomicrobial and polymicrobial biofilm biomass between NAC species and growth medium. All six polymicrobial NB-grown and RPMI-grown exhibited decreased of the biofilm formation. C. parapsilosis co-cultured with S. salivarius in NB medium had shown lowest biofilm bio-mass by 75.51+_1.34% while in RPMI medium, C. lusitanaei demonstrated with most reduced biofilm biomass by 67.03+_5.19. Conclusion: Our study elucidated the antagonistic relationship between Streptococcus salivarius and non-albicans Candida by supressing the growth of polymicrobial biofilm and pseudohyphae/hyphae of NAC species.
BACKGROUND: Basal stem rot (BSR) caused by hemibiotroph Ganoderma boninense is a devastating disease resulting in a major loss to the oil palm industry. Since there is no physical symptom in oil palm at the early stage of G. boninense infection, characterisation of molecular defense responses in oil palm during early interaction with the fungus is of the utmost importance. Oil palm (Elaeis guineensis) seedlings were artificially infected with G. boninense inoculums and root samples were obtained following a time-course of 0, 3, 7, and 11 days-post-inoculation (d.p.i) for RNA sequencing (RNA-seq) and identification of differentially expressed genes (DEGs).
RESULTS: The host counter-attack was evidenced based on fungal hyphae and Ganoderma DNA observed at 3 d.p.i which became significantly reduced at 7 and 11 d.p.i. DEGs revealed upregulation of multifaceted defense related genes such as PR-protein (EgPR-1), protease inhibitor (EgBGIA), PRR protein (EgLYK3) chitinase (EgCht) and expansin (EgEXPB18) at 3 d.p.i and 7 d.p.i which dropped at 11 d.p.i. Later stage involved highly expressed transcription factors EgERF113 and EgMYC2 as potential regulators of necrotrophic defense at 11 d.p.i. The reactive oxygen species (ROS) elicitor: peroxidase (EgPER) and NADPH oxidase (EgRBOH) were upregulated and maintained throughout the treatment period. Growth and nutrient distribution were probably compromised through suppression of auxin signalling and iron uptake genes.
CONCLUSIONS: Based on the analysis of oil palm gene expression, it was deduced that the biotrophic phase of Ganoderma had possibly occurred at the early phase (3 until 7 d.p.i) before being challenged by the fungus via switching its lifestyle into the necrotrophic phase at later stage (11 d.p.i) and finally succumbed the host. Together, the findings suggest the dynamic defense process in oil palm and potential candidates that can serve as phase-specific biomarkers at the early stages of oil palm-G. boninense interaction.
The taxonomic position of a novel Amycolatopsis strain isolated from a high altitude Atacama Desert subsurface soil was established using a polyphasic approach. The strain, isolate H5T, was shown to have chemical properties typical of members of the genus Amycolatopsis such as meso-diaminopimelic acid as the diamino acid in the cell wall peptidoglycan, arabinose and galactose as diagnostic sugars and MK-9(H4) as the predominant isoprenologue. It also has cultural and morphological properties consistent with its classification in the genus, notably the formation of branching substrate hyphae which fragment into rod-like elements. 16S rRNA gene sequence analyses showed that the strain is closely related to the type strain of Amycolatopsis mediterranei but could be distinguished from this and other related Amycolatopsis strains using a broad range of phenotypic properties. It was separated readily from the type strain of Amycolatopsis balhymycina, its near phylogenetic neighbour, based on multi-locus sequence data, by low average nucleotide identity (92.9%) and in silico DNA/DNA relatedness values (51.3%) calculated from draft genome assemblies. Consequently, the strain is considered to represent a novel species of Amycolatopsis for which the name Amycolatopsis vastitatis sp. nov. is proposed. The type strain is H5T (= NCIMB 14970T = NRRL B-65279T).
Trichophyton rubrum is a common pathogenic fungal species that is responsible for causing infection on
human skin, hair and nail. The antifungal-resistant strains complicate the treatment regime.
Hydroxychavicol (HC) is one of the main compounds from Piper betel leaf that have antifungal potential and
its mechanism of action has not been studied yet. The objective of this preliminary study to determine the
antifungal properties of HC against T. rubrum using transmission electron microscope (TEM) on gross and
ultrastructure of T. rubrum hypha. T. rubrum was treated with HC and miconazole (MI) at concentrations of
1.25, 2.5, 5 and 10 mg/mL for 1, 3, 5 and 7 days continuously. Generally, fungi structures became more
severely damaged at increasing treatment duration. Microscopically, the fungi’s cell wall treated with HC
showed a rough surface, shrinkage and demolition similar to the MI treated group. The fungi organelles were
also demolished and disorganized. This study revealed that HC has the ability to inhibit T. rubrum growth
and has potential to be an antifungal agent for skin infections.
Candida wangnamkhiaoensis is a species clustered under the Hyphopichia clade has not ever been isolated from any clinical specimens. To the best of our knowledge, this is the first report of C. wangnamkhiaoensis associated with fungaemia in immunocompromised paediatric patient. The isolate was assigned a strain name as UZ1679/14, in which the identification was confirmed by a polymerase chain reaction-sequencing of the internal transcribed spacer (ITS) and large subunit (LSU) regions of the rRNA gene. Antifungal susceptibility pattern showed that the isolate was sensitive to anidulafungin, caspofungin, fluconazole and voriconazole. The patient clinically improved after the antifungal treatment with caspofungin.
This paper reported on the various filamentous fungi strains that were isolated from a wild grown Catharanthus roseus. Based on the morphological characteristics and molecular technique through a Polymerase Chain Reaction and DNA sequencing method using internal transcribed spacer (ITS), these fungi had been identified as a Colletotrichum sp., Macrophomina phaseolina, Nigrospora sphaerica and Fusarium solani. The ultrastructures of spores and hyphae were observed under a Scanning Electron Microscope. The hydrolytic enzyme test showed that all strains were positive in secreting cellulase. Colletotrichum sp. and F. solani strains also gave a positive result for amylase while only F. solani was capable to secrete protease. These fungi were putatively classified as endophytic fungi since they produced extracellular enzymes that allow them to penetrate plant cell walls and colonize with symbiotic properties.
Timber scaffold boards have been widely utilised in the offshore construction industry. However, technical specifications
and inspection procedure for the application of scaffold boards from a wood material were inadequate. In the development
of the standard requirements, evaluation of actual engineering practices could indicate the level of workplace safety. A
study was conducted to identify risk and safety measures concerning the use of timber scaffold boards in construction.
This article discusses on the occupational risks and ergonomics issues of the scaffolding application based on the physical
and mechanical conditions of tropical timber scaffold boards extracted from offshore oil and gas rigs. The boards were
exposed to a seasonal climate of East Coast monsoon of South China Sea between November and February. The scaffolding
application extended up to 20 m directly above the ocean surface and some boards were completely immersed below
the ocean surface particularly during elevated tidal waves. Some of the boards were installed as the on-deck platforms.
Forty scaffold boards fabricated from tropical timber species were evaluated. Physical characteristics of the boards
were described by natural and man-made factors. A three-point bending test was conducted to determine the maximum
load capacity of each board and the mode of fracture was evaluated. Timber identification test was conducted to identify
the groups of the timber being used. The microscopic observation confirmed the presence of hyphae which indicated the
biological deterioration has happened in some of the boards. Occupational risks were summarised based on the results
of the physical and mechanical assessments
The aim of this study was to investigate the relationship between expression of Candida albicans alcohol dehydrogenases (ADH) genes in archival formalin-fixed paraffin-embedded (FFPE) samples from biopsies of leukoplakia.
Rubber tree (Hevea brasiliensis) is an important crop in tropical regions of China. In October 2013, a new stem rot disease was found on cv. Yunyan77-4 at a rubber tree plantation in Hekou, Yunnan Province. There were about 100 plants, and diseased rubber trees accounted for 30% or less. Initially, brown-punctuate secretion appeared on the stem, which was 5 to 6 cm above the ground. Eventually, the secretion became black and no latex produced from the rubber tree bark. After removing the secretion, the diseased bark was brown putrescence, but the circumambient bark was normal. Upon peeling the surface bark, the inner bark and xylem had brown rot and was musty. The junction between health and disease was undulate. On the two most serious plants, parts of leaves on the crown were yellow, and the root near the diseased stem was dry and puce. The pathogen was isolated and designated HbFO01; the pathogenicity was established by following Koch's postulates. The pathogen was cultivated on a potato dextrose agar (PDA) plate at 28°C for 4 days. Ten plants of rubber tree cv. Yunyan77-4 were selected from a disease-free plantation in Haikou, Hainan Province, and the stem diameter was about 7 cm. The bark of five plants was peeled, and one mycelium disk with a diameter of 1 cm was inserted into the cut and covered again with the bark. The other five plants were treated with agar disks as controls. The inoculation site was kept moist for 2 days, and then the mycelium and agar disk were removed. On eighth day, symptoms similar to the original stem lesions were observed on stems of inoculated plants, while only scars formed on stems of control plants. The pathogen was re-isolated from the lesions of inoculated plants. On PDA plates, the pathogen colony was circular and white with tidy edges and rich aerial hyphae. Microscopic examination showed microconidia and chlamydospores were produced abundantly on PDA medium. The falciform macroconidia were only produced on lesions and were slightly curved, with a curved apical cell and foot shaped to pointed basal cell, usually 3-septate, 16.2 to 24.2 × 3.2 to 4.0 μm. Microconidia were produced in false heads, oval, 0-septate, 6.2 to 8.2 × 3.3 to 3.8 μm, and the phialide was cylindrical. Chlamydospores were oval, 6.4 to 7.2 × 3.1 to 3.8 μm, alone produced in hypha. Morphological characteristics of the specimen were similar to the descriptions for Fusarium oxysporum (2). Genomic DNA of this isolate was extracted with a CTAB protocol (4) from mycelium and used as a template for amplification of the internal transcribed spacer (ITS) region of rDNA with primer pair ITS1/ITS4 (1). The full length of this sequence is 503 nt (GenBank Accession No. KJ009335), which exactly matched several sequences (e.g., JF807394.1, JX897002.1, and HQ451888.1) of F. oxysporum. Williams and Liu had listed F. oxysporum as the economically important pathogen of Hevea in Asia (3), while this is, to our knowledge, the first report of stem rot caused by F. oxysporum on rubber tree in China. References: (1) D. E. L. Cooke et al. Fungal Genet. Biol. 30:17, 2000. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual, 2006. (3) T. H. Williams and P. S. W. Liu. A host list of plant diseases in Sabah, Malaysia, 1976. (4) J. R. Xu et al. Genetics 143:175, 1996.
Trichophyton rubrum (T. rubrum) is one of the most common dermatophytes worldwide. This fungus invaded skin appendages of humans and animals. Recently, resistance to antifungal drugs as well as appearance of side effects due to indication of these kinds of antibiotics has been reported. Besides, using some plant extracts have been indicated in herbal medicine as an alternative treatment of these fungal infections. The aim of this study was to investigate the effects of Garlic (Allium sativum) and pure allicin on the growth of hypha in T. rubrum using Electron miscroscopy.
The rice blast fungus Magnaporthe oryzae causes plant disease via specialised infection structures called appressoria. These dome-shaped cells are able to generate enormous internal pressure, which enables penetration of rice tissue by invasive hyphae. Previous studies have shown that mobilisation of lipid bodies and subsequent lipid metabolism are essential pre-requisites for successful appressorium-mediated plant infection, which requires autophagic recycling of the contents of germinated spores and germ tubes to the developing appressorium. Here, we set out to identify putative regulators of lipid metabolism in the rice blast fungus. We report the identification of FAR1 and FAR2, which encode highly conserved members of the Zn2-Cys6 family of transcriptional regulators. We generated Δfar1, Δfar2 and Δfar1Δfar2 double mutants in M. oryzae and show that these deletion mutants are deficient in growth on long chain fatty acids. In addition, Δfar2 mutants are also unable to grow on acetate and short chain fatty acids. FAR1 and FAR2 are necessary for differential expression of genes involved in fatty acid β-oxidation, acetyl-CoA translocation, peroxisomal biogenesis, and the glyoxylate cycle in response to the presence of lipids. Furthermore, FAR2 is necessary for expression of genes associated with acetyl-CoA synthesis. Interestingly, Δfar1, Δfar2 and Δfar1Δfar2 mutants show no observable delay or reduction in lipid body mobilisation during plant infection, suggesting that these transcriptional regulators control lipid substrate utilization by the fungus but not the mobilisation of intracellular lipid reserves during infection-related morphogenesis.
The mitogen-activated protein (MAP) kinase pathways has been implicated in the pathogenicity of various pathogenic fungi and plays important roles in regulating pathogenicity-related morphogenesis. This work describes the isolation and characterization of MAP kinase gene, Cgl-SLT2, from Colletotrichum gloeosporioides. A DNA sequence, including 1,633 bp of Cgl-SLT2 open-reading frame and its promoter and terminator regions, was isolated via DNA walking and cloned. To analyze gene function, a gene disruption cassette containing hygromycin-resistant gene was constructed, and Cgl-SLT2 was inactivated via gene deletion. Analysis on Cgl-slt2 mutant revealed a defect in vegetative growth and sporulation as compared to the wild-type strain. When grown under nutrient-limiting conditions, hyperbranched hyphal morphology was observed in the mutant. Conidia induction for germination on rubber wax-coated hard surfaces revealed no differences in the percentage of conidial germination between the wild-type and Cgl-slt2 mutant. However, the percentage of appressorium formation in the mutant was greatly reduced. Bipolar germination in the mutant was higher than in the wild-type at 8-h post-induction. A pathogenicity assay revealed that the mutant was unable to infect either wounded or unwounded mangoes. These results suggest that the Cgl-SLT2 MAP kinase is required for C. gloeosporioides conidiation, polarized growth, appressorium formation and pathogenicity.
Rambutan (Nephelium lappaceum L.) is among the tropical fruit grown in Malaysia and the demand for export rose in 2011. A fruit rot was observed between August and December 2011 from several areas in the states of Pulau Pinang and Perak, Malaysia. The symptoms initially appeared as light brown, water-soaked lesions that developed first in the pericarp and pulp, later enlarging and becoming dark brown. Greyish brown mycelia were observed on infected areas that turned yellowish at later stages of infection. Gliocephalotrichum bacillisporum was isolated from infected fruit by surface sterilization techniques. Conidia were mass-transferred onto potato dexstrose agar (PDA) plates and incubated at 27 ± 1°C. Tissue pieces (5 × 5 mm) excised from the margins between infected and healthy areas were then surface sterilized in 1% sodium hypochlorite for 3 to 5 min before being rinsed with distilled water, plated on PDA, and incubated at 27 ± 1°C for 7 days. Ten isolates of G. bacillisporum were obtained. Colonies on PDA were initially white before turning yellow with a feathery appearance. Microscopic characteristics on carnation leaf agar (CLA) consisted of hyaline conidia that were slightly ellipsoid to bacilliform with rounded apex ranging from 6.0 to 8.5 μm long and 2.0 to 2.5 μm wide. Conidiophores (70 to 130 μm long) were mostly single arising from large hypha approximately 13 to 16 μm. The conidiogenous structures were mostly quadriverticillate with dense, short, penicillate branches. The phialides were cylindrical and finger-like. Chlamydospores were present singly, in groups of 2 to 4, or in occasionally branched short chains and were brown in color with thick walls ranging from 11 to 13 μm. The cultural and morphological characteristics of G. bacillisporum isolates in the present study were very similar to previously published descriptions (1) except the conidiophores formed without sterile stipe extensions. All the G. bacillisporum isolates were deposited in culture collection at the Plant Pathology Lab, University Sains Malaysia, Penang. Molecular identification was accomplished from the ITS regions using ITS1 and ITS2 primers, and the β-tubulin gene using Bt2a and Bt2b primers (2). BLAST results from the ITS regions showed a 98 to 99% similarity with sequences of G. bacillisporum isolates reported in GenBank. Accession numbers of G. bacillisporum ITS regions: JX484850, JX484852, JX484853, JX484856, JX484858, JX484860, JX484862, JX484866, JX484867, and JX484868. The identity of G. bacillisporum isolates infecting rambutan was further confirmed by β-tubulin sequences (KC683909, KC683911, KC683912, KC683916, KC683919, KC683920, KC683923, KC683926, and KC683927), which showed 92 to 95% similarity with sequences of G. bacillisporum. Pathogenicity tests were also performed using mycelial plug (5 mm) and sprayed conidial suspensions (20 μl suspension of 106 conidia/ml) prepared from 7-day-old cultures. Inoculated fruits were incubated at 27 ± 1°C and after 10 days, similar rotting symptoms appeared on the fruit surface. The pathogen was reisolated from fruit rot lesions, thus fulfilling Koch's postulates, and tests were repeated twice. To our knowledge, this is the first report of G. bacillisporum causing fruit rot of rambutan (N. lappaceum L.) in Malaysia. References: (1) C. Decock et al. Mycologia 98:488, 2006. (2) N. L. Glass and G. C. Donaldson. Appl. Environ Microbiol. 61:1323, 1995.