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.
Endophytic bacteria (Pseudomonas aeruginosa UPMP3 and Burkholderia cepacia UMPB3), isolated from within roots of oil palm (Elaeis guineensis Jacq.) were tested for their presymbiotic effects on two arbuscular mcorrhizal fungi, Glomus intraradices UT126 and Glomus clarum BR152B). These endophytic bacteria were also tested for antagonistic effects on Ganoderma boninense PER 71, a white wood rot fungal pathogen that causes a serious disease in oil palm. Spore germination and hyphal length of each arbuscular mycorrhizal fungal (AMF) pairing with endophytic bacteria was found to be significantly higher than spores plated in the absence of bacteria. Scanning electron microscopy (SEM) showed that the endophytic bacteria were scattered, resting or embedded on the surface hyaline layer or on the degraded walls of AMF spores, possibly feeding on the outer hyaline spore wall. The antagonistic effect of the endophytic bacteria was expressed as severe morphological abnormalities in the hyphal structures of G. boninense PER 71. The effects of the endophytic bacteria on G. boninense PER 71 hyphal structures were observed clearly under SEM. Severe inter-twisting, distortion, lysis and shriveling of the hyphal structures were observed. This study found that the effect of endophytic bacteria on G. intraradices UT126 and G. clarum BR152B resembled that of a mycorrhiza helper bacteria (MHB) association because the association significantly promoted AMF spore germination and hyphal length. However, the endophytic bacteria were extremely damaging to G. boninense PER 71.
Indian spinach (Basella rubra L.) is a red stem species of Basella that is cultivated worldwide as an ornamental and the aerial parts are also consumed as a vegetable. In May of 2011, symptoms of damping-off were observed on approximately 10% of the plants at the stem base around the soil line of seedlings in a greenhouse in Homestead, FL. Lesions were initially water soaked, grayish to dark brown, irregular in shape, and sunken in appearance on large plants, causing the infected seedlings to collapse and eventually die. Symptomatic stem tissue was surface sterilized with 0.6% sodium hypochlorite, rinsed in sterile distilled water, air dried, and plated on potato dextrose agar (PDA). Plates were incubated at 25°C in darkness for 3 to 5 days. A fungus was isolated in all six isolations from symptomatic tissues on PDA. Fungal colonies on PDA were light gray to brown with abundant growth of mycelia, and the hyphae tended to branch at right angles when examined under a microscope. A septum was always present in the branch of hyphae near the originating point and a slight constriction at the branch was observed. Neither conidia nor conidiophores were found from the cultures on PDA. The characteristics of hyphae, especially the right angle branching of mycelia, indicate close similarity to those of Rhizoctonia solani (2,3). The internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1/ITS4 and sequenced (GenBank Accession No. JN545836). Subsequent database searches by the BLASTN program indicated that the resulting sequence had a 100% identity over 472 bp with the corresponding gene sequence of R. solani anastomosis group (AG) 4 (GenBank Accession No. JF701752.1), a fungal pathogen reported to cause damping-off on many crops. Pathogenicity was confirmed through inoculation of healthy India spinach plants with the hyphae of isolates. Four 4-week-old plants were inoculated with the isolates by placing a 5-mm PDA plug of mycelia at the stem base and covering with a thin layer of the soil. Another four plants treated with sterile PDA served as a control. After inoculation, the plants were covered with plastic bags for 24 h and maintained in a greenhouse with ambient conditions. Four days after inoculation, water-soaked, brown lesions, identical to the symptoms described above, were observed on the stem base of all inoculated plants, whereas no symptoms developed on the control plants. The fungus was isolated from affected stem samples, and the identity was confirmed by microscopic appearance of the hyphae and sequencing the ITS1/ITS4 intergenic spacer region, fulfilling Koch's postulates. This pathogenicity test was conducted twice. R. solani has been reported to cause damping-off of B. rubra in Ghana (1) and Malaysia (4). To our knowledge, this is the first report of damping-off caused by R. solani AG-4 on Indian spinach in Florida and the United States. With the increased interest in producing Asian vegetables for food and ornamental purposes, the occurrence of damping-off on Indian spinach needs to be taken into account when designing programs for disease management in Florida. References: (1) H. A. Dade. XXIX. Bull. Misc. Inform. 6:205, 1940. (2) J. R. Parmeter et al. Phytopathology 57:218, 1967. (3) B. Sneh et al. Identification of Rhizoctonia species. The American Phytopathological Society, St Paul, MN, 1991. (4) T. H. Williams and P. S. W. Liu. Phytopathol. Pap. 19:1, 1976.
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.
Trichophyton rubrum is one of dermatophytes that penetrates keratinized tissues such as skin, hair and nail of human and animals. Recently, antifungal drugs such as imodazole and triazole was found to cause side effects, toxicity to patients and also not very efficient due to resistance to these drugs. As an alternative, some plants extract had been used to treat dermatophytes. This studies was done using Garlic extract (Allium sativum) to evaluate its effects on the growth of hypha of Trichophyton using Electron miscroscopy. Garlic had been known to posses antimicrobial, antiinflammatory, antithrombotic and antitumor activities. This studies found that garlic extract as low as 4 mg/mL inhibit the growth of hypha. Scanning electron microscopy studies revealed that hypha treated with garlic extract showed shrinkage, flat and cell wall demolition, similar to hypha treated with allicin (positive control) having rough surface, shrinkage and distortion. The tip of hypa became large after treatment with garlic extract. Transmission electron microscopy studies also found that hypha treated with allicin display cell wall thickening, local thickening, destruction of cytoplasmic content, mean while hypha treated with garlic extract exhibited cell wall thickening, disordered hyphal tip and desolution of cytoplasmic compartments and similar with hypha treated with allicin. These results showed that garlic extract and pure allicin could be use as an alternative to treat dermatophytes.
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.
The aims of the present study were to determine whether Allium sativum (garlic) extract has any effect on the morphology transformation of Candida albicans, and to investigate whether it could alter the gene expression level of SIR2, a morphogenetic control gene and SAP4, a gene encoding secreted aspartyl proteinase.
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).
Clitoria ternatea is known for its antimicrobial activity but the antifungal effects of leaf extract on growth and morphogenesis of Aspergillus niger have not been observed. The extract showed a favorable antifungal activity against A. niger with a minimum inhibition concentration 0.8 mg/mL and minimum fungicidal concentration 1.6 mg/mL, respectively. The leaf extract exhibited considerable antifungal activity against filamentous fungi in a dose-dependent manner with 0.4 mg/mL IC50 value on hyphal growth of A. niger. The main changes observed under scanning electron microscopy after C. ternatea extract treatment were loss of cytoplasm in fungal hyphae and the hyphal wall and its diameter became markedly thinner, distorted, and resulted in cell wall disruption. In addition, conidiophore alterations were also observed when A. niger was treated with C. ternatea leaf extract.
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.
Three novel endophytic streptomycetes have been isolated and characterized from plants with ethnobotanical uses on the Malay Peninsula including: Thottea grandiflora (family -Aristolochiaceae), Polyalthia spp. (family -Annonaceae), and Mapania sp. (family -Cyperaceae). Each isolate, as studied by scanning electron microscopy, has small hyphae, and produces typical barrel-shaped spores arising by hyphal fragmentation. Interestingly, although none has any detectable antibacterial killing properties, each has demonstrable killing activity against one or more pathogenic fungi including organisms such as Phytophthora erythroseptica, Pythium ultimum, Sclerotinia sclerotiorum, Mycosphaerella fijiensis and Rhizoctonia solani. Molecular biological studies on the rRNA gene sequence of each isolate revealed that it is distinct from all other genetic accessions of streptomyectes in GenBank, and each bears some genetic similarity to other streptomycetes. The bioactivity of each microbe was extractable in various organic solvents.
Fusarium oxysporum f.sp. cubense is the causal pathogen of wilt disease of banana. A cost-effective measure of control for this disease is still not available. Streptomyces violaceusniger strain G10 acts as an antifungal agent antagonistic towards many different phytopathogenic fungi, including different pathogenic races of the Fusarium wilt pathogen. In an attempt to understand the mode of action of this antagonist in nature, the interaction between S. violaceusniger strain G10 and F. oxysporum f.sp. cubense was first studied by paired incubation on agar plates. Evidence for the in vitro antibiosis of strain G10 was demonstrated by inhibition zones in the "cross-plug" assay plates. Microscopic observations showed lysis of hyphal ends in the inhibited fungal colonies. Culture of strain G10 in liquid media produces antifungal metabolites, which showed in vitro antagonistic effects against F. oxysporum f.sp. cubense such as swelling, distortion and excessive branching of hyphae, and inhibition of spore germination. An indirect method was used to show that antibiosis is one of the mechanisms of antagonism by which strain G10 acts against F. oxysporun f.sp. cubense in soil. This study suggests the potential of developing strain G10 for the biological control of Fusarium wilt disease of banana.
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.
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.
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.
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.
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.
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.
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.