Dendrobium (Dendrobium candidum Wall. ex Lindl.) is a perennial herb in the Orchidaceae family. It has been used as traditional medicinal plant in China, Malaysia, Laos, and Thailand (2). Fungal disease is one of the most important factors affecting the development of Dendrobium production. During summer 2012, chocolate brown spots were observed on leaves of 2-year-old Dendrobium seedlings in a greenhouse in Hangzhou, Zhejiang Province, China, situated at 30.26°N and 120.19°E. Approximately 80% of the plants in each greenhouse were symptomatic. Diseased leaves exhibited irregular, chocolate brown, and necrotic lesions with a chlorotic halo, reaching 0.8 to 3.2 cm in diameter. Affected leaves began to senesce and withered in autumn, and all leaves of diseased plants fell off in the following spring. Symptomatic leaf tissues were cut into small pieces (4 to 5 mm long), surface-sterilized (immersed in 75% ethanol for 30 s, and then 1% sodium hypochlorite for 60 s), rinsed three times in sterilized distilled water, and then cultured on potato dextrose agar (PDA) amended with 30 mg/liter of kanamycin sulfate (dissolved in ddH2O). Petri plates were incubated in darkness at 25 ± 0.5°C, and a grey mycelium with a white border developed after 4 days. Fast-growing white mycelia were isolated from symptomatic leaf samples, and the mycelia became gray-brown with the onset of sporulation after 5 days. Conidia were unicellular, black, elliptical, and 11.4 to 14.3 μm (average 13.1 μm) in diameter. Based on these morphological and pathogenic characteristics, the isolates were tentatively identified as Nigrospora oryzae (1). Genomic DNA was extracted from a representative isolate F12-F, and a ~600-bp fragment was amplified and sequenced using the primers ITS1 and ITS4 (4). BLAST analysis showed that F12-F ITS sequence (Accession No. KF516962) had 99% similarity with the ITS sequence of an N. oryzae isolate (JQ863242.1). Healthy Dendrobium seedlings (4 months old) were used in pathogenicity tests under greenhouse conditions. Leaves were inoculated with mycelial plugs (5 mm in diameter) from a 5-day-old culture of strain F12-F, and sterile PDA plugs served as controls. Seedlings were covered with plastic bags for 5 days and maintained at 25 ± 0.5°C and 80 ± 5% relative humidity. Eight seedlings were used in each experiment, which was repeated three times. After 5 days, typical chocolate brown spots and black lesions were observed on inoculated leaves, whereas no symptoms developed on controls, which fulfilled Koch's postulates. This shows that N. oryzae can cause leaf spot of D. candidum. N. oryzae is a known pathogen for several hosts but has not been previously reported on any species of Dendrobium in China (3). To our knowledge, on the basis of literature, this is the first report of leaf spot of D. candidum caused by N. oryzae in China. References: (1) H. J. Hudson. Trans. Br. Mycol. Soc. 46:355, 1963. (2) Q. Jin et al. PLoS One. 8(4):e62352, 2013. (3) P. Sharma et al. J. Phytopathol. 161:439, 2013. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.
In the last decade, rambutan (Nephelium lappaceum L., Sapindaceae) and pulasan (N. mutabile Blume) have been cultivated in Honduras to produce exotic fruits for export to North America (2). Recently, a disease was observed that produces dark brown to black fissured cankers from 1 to 3 cm long and 1 to 4 cm wide. The infected bark tissue becomes swollen with the middle region 3 to 8 mm thick. Symptoms appear when the trees are approximately 3 years old. As the trees mature, the cankers increase in size and weaken the branches, often resulting in breakage with the weight of the fruit causing substantial plant damage and fruit loss. In August 2010, fissured branch samples of rambutan and pulasan were collected from 6- to 8-year-old trees from the Humid Tropical Demonstrative Agroforestry Center in Honduras, Atlantida, La Masica (15°33'47.4″N, 87°05'2.5″W, elevation 106 m). A fungus associated with the cankers was identified as Dolabra nepheliae. It produces black, stipitate, elongate ascomata, 312 to 482 × 250 to 281 μm with broadly cylindric, bitunicate asci, 120 to 138 × 11.2 to 15.0 μm, and filiform, hyaline ascospores, 128 to 135 × 2.8 to 3.2 μm. Fungi from rambutan and pulasan were isolated on cornmeal agar plus 0.5% dextrose and antibiotics. On potato dextrose agar, the ascospores produced slow-growing colonies, 5 mm per week. In culture, isolates from both hosts produced pycnidia with elongated, slightly to strongly curved or S-shaped, hyaline conidia, 22.8 to 46.4 × 2.8 to 3.7 μm. This fungus was first reported on rambutan and pulasan from Malaysia (1,4), and later reported on rambutan and litchi in Hawaii and Puerto Rico (3). To our knowledge, this is the first report of D. nepheliae on pulasan and rambutan from Honduras. Specimens have been deposited at the U.S. National Fungus Collections (BPI 882442 on N. lappaceum and BPI 882443 on N. mutabile). Cultures were deposited at the Centraalbureau voor Schimmelcultures (CBS) as CBS 131490 on N. lappaceum and CBS 131491 on N. mutabile. Sequences of the internal transcribed spacer (ITS) region including ITS1, 5.8S, and ITS2 intergenic spacers were deposited in GenBank (Accession No. JQ004281 on N. lappaceum and Accession No. JQ004280 on N. mutabile). A BLAST search and pairwise comparison using the GenBank web server were used to compare ITS sequence data and recovered the following results: (i) CBS 131490 on N. lappaceum is 99% (538 of 544) identical to D. nepheliae CBS 123297 on Litchi chinensis from Puerto Rico; and (ii) CBS 131491 on N. mutabile is 99% (527 of 533) identical to the same strain of D. nepheliae. On the basis of the ITS sequence data, the isolates from Honduras were confirmed as the same species, D. nepheliae from Puerto Rico. Efforts to develop resistant germplasm and management strategies to control this disease have been initiated. References: (1) C. Booth and W. P. Ting. Trans. Brit. Mycol. Soc. 47:235, 1964. (2) T. Ramírez et al. Manual Para el Cultivo de Rambutan en Honduras. Fundación Hondureña de Investigación Agrícola. La Lima, Cortes, Honduras, 2003. (3) A. Y. Rossman et al. Plant Dis. 91:1685, 2007. (4) H. Zalasky et al. Can. J. Bot. 49:559, 1971.
In June 2011, tomatoes (Solanum lycopersicum) in major growing areas of the Cameron Highlands and the Johor state in Malaysia were affected by a leaf spot disease. Disease incidence exceeded 80% in some severely infected regions. Symptoms on 50 observed plants initially appeared on leaves as small, brownish black specks, which later became grayish brown, angular lesions surrounded by a yellow border. As the lesions matured, the affected leaves dried up and became brittle and later developed cracks in the center of the lesions. A survey was performed in these growing areas and 27 isolates of the pathogen were isolated from the tomato leaves on potato carrot agar (PCA). The isolates were purified by the single spore technique and were transferred onto PCA and V8 agar media for conidiophore and conidia production under alternating light (8 hours per day) and darkness (16 hours per day) (4). Colonies on PCA and V8 agar exhibited grey mycelium and numerous conidia were formed at the terminal end of conidiophores. The conidiophores were up to 240 μm long. Conidia were oblong with 2 to 11 transverse and 1 to 6 longitudinal septa and were 24 to 69.6 μm long × 9.6 to 14.4 μm wide. The pathogen was identified as Stemphylium solani on the basis of morphological criteria (2). In addition, DNA was extracted and the internal transcribed spacer region (ITS) was amplified by universal primers ITS5 and ITS4 (1). The PCR product was purified by the commercial PCR purification kit and the purified PCR product sequenced. The resulting sequences were 100% identical to published S. solani sequences (GenBank Accestion Nos. AF203451 and HQ840713). The amplified ITS region was deposited with NCBI GenBank under Accession No. JQ657726. A representative isolate of the pathogen was inoculated on detached 45-day-old tomato leaves of Malaysian cultivar 152177-A for pathogenicity testing. One wounded and two nonwounded leaflets per leaf were used in this experiment. The leaves were wounded by applying pressure to leaf blades with the serrated edge of a forceps. A 20-μl drop of conidial suspension containing 105 conidia/ml was used to inoculate these leaves (3). The inoculated leaves were placed on moist filter paper in petri dishes and incubated for 48 h at 25°C. Control leaves were inoculated with sterilized distilled water. After 7 days, typical symptoms for S. solani similar to those observed in the farmers' fields developed on both wounded and nonwounded inoculated leaves, but not on noninoculated controls, and S. solani was consistently reisolated. To our knowledge, this is the first report of S. solani causing gray leaf spot of tomato in Malaysia. References: (1) M. P. S. Camara et al. Mycologia 94:660, 2002. (2) B. S. Kim et al. Plant Pathol. J. 15:348, 1999. (3) B. M. Pryor and T. J. Michailides. Phytopathology 92:406, 2002. (4) E. G. Simmons. CBS Biodiversity Series 6:775, 2007.
Endophytic fungi are those living inside the host plant without causing any apparent negative effect on the host plant. Two
isolates endophytic fungi from leaves and two isolates from root at Universiti Teknologi MARA (UiTM) Reserve Forest,
Negeri Sembilan were successfully isolated and identified by morphology and molecular characteristic. Samples were
surface sterilized and sub-cultured to obtain a pure culture. Characteristics of the isolates such as colony appearance,
mycelial texture, conidia/spores and pigmentation were studied to explore their morphology. Isolates were also subjected to
a PCR-based genotyping test. There were noticeable differences in morphological characteristics among the four isolates.
Microscopic analysis showed four isolates consist of septa and conidia/spores. The pigmentation result showed that
colony in A1leaf samples demonstrated an orange color on potato dextrose agar (PDA) media, colony in A1root demonstrate
a black texture in PDA media while hairy colonies in the others two isolates showed a white color on PDA media. Based on
molecular analyses the fungal genera showed 99-100% similarity with the related fungi recorded in the GenBank. Both
morphology and molecular sequencing of internal transcribed spacer (ITS) regions of endophytic fungi showed that three
isolates (A1root, C2leaf, and C3root) were grouped in Basidiomycota while one isolate (A1leaf) belonged to Ascomycota. The
endophyte funguses were identified as Daldinia sp. (A1leaf), Polyporales sp. (A1root,) Lentinus sp. (C2leaf,) and Rigidoporus
sp. (C3root). Overall, the new discoveries of isolated endophyte fungal have dyeing potential of fungal pigments which
offer a viable alternative to natural vegetable and harmful synthetic dyes.
Some Amanita specimens collected from Malaysia are critically investigated by morphological examination and molecular analysis of two gene fragments, the nuc rDNA partial 28S (28S) gene and the internal transcriber spacer (ITS1-5.8S-ITS2 = ITS) regions. Six phylogenetic species of Amanita section Caesareae are recognized among the studied collections. One of them is described as new, A. malayensis. Four of the phylogenetic species correspond with existing morphology-based taxa: A. aporema, A. javanica, A. princeps, and A. similis. The remaining species is not described because of the paucity of material. Detailed descriptions and the distribution of these southeastern Asian species are provided, along with a key to the species of section Caesareae from Malaysia.
Psidium guajava wilt is known from South Africa, Malaysia and Taiwan. The fungus causing this disease, Myxosporium psidii, forms dry chains of conidia on surfaces of pseudoparenchymatous sporodochia, which develop in blisters on bark. Similar sporodochia are characteristic of Nalanthamala madreeya, the type species of Nalanthamala. Nalanthamala, therefore, is the appropriate anamorph genus for Myxosporium psidii, while Myxosporium is a nomen nudum (based on M. croceum). For M. psidii the combination Nalanthamala psidii is proposed. Nalanthamala psidii, the palm pathogen Gliocladium (Penicillium) vermoesenii, another undescribed anamorphic species from palm, two species of Rubrinectria and the persimmon pathogen Acremonium diospyri are monophyletic and belong to the Nectriaceae (Hypocreales) based on partial nuclear large subunit ribosomal DNA (LSU rDNA) analyses. Rubrinectria, therefore, is the teleomorph of Nalanthamala, in which the anamorphs are classified as N. vermoesenii, N. diospyri or Nalanthamala sp. Nalanthamala squamicola, the only other Nalanthamala species, has affinities with the Bionectriaceae and is excluded from this group. Rubrinectria/Nalanthamala species form dimorphic conidiophores and conidia in culture. Fusiform, cylindrical, or allantoid conidia arise in colorless liquid heads on acremonium-like conidiophores; ovoidal conidia with somewhat truncated ends arise in long, persistent, dry chains on penicillate conidiophores. No penicillate but irregularly branched conidiophores were observed in N. diospyri. Conidia of N. psidii that are held in chains are shorter than those of N. madreeya, of which no living material is available. Nalanthamala psidii and N. diospyri are pathogenic specifically to their hosts. They form pale yellow to pale orange or brownish orange colonies, respectively, and more or less white conidial masses. Most strains of Rubrinectria sp., Nalanthamala sp. and N. vermoesenii originate from palm hosts, form mostly greenish or olive-brown colonies and white-to-salmon conidial masses. They form a monophyletic clade to which Nalanthamala psidii and N. diospyri are related based on analyses of the internal transcribed spacer regions and 5.8S rDNA (ITS rDNA), LSU rDNA, and partial beta-tubulin gene. Few polymorphic sites in the ITS rDNA and beta-tubulin gene indicate that Nalanthamala psidii comprises two lineages, one of which has been detected only in South Africa.
Control of nematode parasites of small ruminants in a wet, tropical environment using the nematophagous fungus, Duddingtonia flagrans, was assessed in this study. Two methods of fungal delivery were tested, namely as a daily feed supplement, or incorporated into feed blocks. Initially, pen trials were conducted with individually penned groups of sheep and goats at dose rates of 125,000 spores and 250,000 spores/kg live weight per day. At the lower dose rate this reduction was between 80 and 90% compared with the pre-treatment levels. At the higher dose rate, there was virtually complete suppression (>99% reduction) of larval recovery. Trials using the fungal feed blocks, showed that when animals were individually penned, they consumed only small amounts of the block (particularly goats), hence little effect on larval recovery in faecal cultures was observed. Grouping animals according to species and dose rate induced satisfactory block consumption and subsequent high levels of larval reduction in faecal cultures. These larval reductions were mirrored by the presence of fungus in faecal cultures. This work was followed by a small paddock trial, whereby three groups of sheep were fed either a feed supplement without fungal spores, supplement with spores, or offered fungal blocks. The dose rate of spores in the latter two groups was 500,000 spores/kg live weight per day. Egg counts were significantly reduced in the two fungal groups, compared with the control group and the latter required two salvage anthelmintic treatments to prevent mortality due to haemonchosis. Pasture larval numbers on the two fungal group plots were also much lower than on the control plot.
Three new species and one new variety of bioluminescent Mycena collected from Peninsular Malaysia are described herein. All new species belong to Mycena sect. Calodontes in what is known as the Mycena pura complex. Comprehensive descriptions, photographs, illustrations and comparisons with phenetically similar species are provided. Molecular sequences data from the nuclear internal transcribed spacers (ITS-1 and ITS-2, including the 5.8S rRNA) were used to infer relationships within sect. Calodontes. Axenic cultures were obtained to provide data on culture morphology. This is the first published photographic documentation of bioluminescent basidiomes of members of Mycena sect. Calodontes. Also, this addition brings the total known bioluminescent fungi to 77 species.
Mycena illuminans Henn. is described and re-evaluated based on recently collected material from peninsular Malaysia, providing comprehensive descriptions, illustrations and photographs. In addition to morphological data, axenic monokaryon and dikaryon cultures were established to provide data on culture morphology and the mating system of the species. Molecular sequences data from the nuclear large subunit (LSU) gene also are presented, confirming that M. illuminans is not a synonym of Mycena chlorophos.
A highly efficient and reproducible Agrobacterium-mediated transformation protocol for Ganoderma boninense was developed to facilitate observation of the early stage infection of basal stem rot (BSR). The method was proven amenable to different explants (basidiospore, protoplast, and mycelium) of G. boninense. The transformation efficiency was highest (62%) under a treatment combination of protoplast explant and Agrobacterium strain LBA4404, with successful expression of an hyg marker gene and gus-gfp fusion gene under the control of heterologous p416 glyceraldehyde 3-phosphate dehydrogenase promoter. Optimal transformation conditions included a 1:100 Agrobacterium/explant ratio, induction of Agrobacterium virulence genes in the presence of 250 μm acetosyringone, co-cultivation at 22°C for 2 days on nitrocellulose membrane overlaid on an induction medium, and regeneration of transformants on potato glucose agar prepared with 0.6 M sucrose and 20 mM phosphate buffer. Evaluated transformants were able to infect root tissues of oil palm plantlets with needle-like microhyphae during the penetration event. The availability of this model pathogen system for BSR may lead to a better understanding of the pathogenicity factors associated with G. boninense penetration into oil palm roots.
Abstract. The species identification of Enterocytozoon bieneusi and Encephalitozoon intestinalis is only possible using transmission electron microscopy (TEM), mo lecular techniques and immunofluorescence antibody assays (IFA). In this study, 50 positive and 50 negative fecal specimens for microsporidial spores using the Weber modified trichrome (WMT) staining technique were examined using IFA-MAbs. Of the 100 specimens examined, the microsporidial spores identified by IFA-MAbs were Enterocytozoon Bieneusi 42 (75%) Encephalitozoon intestinalis 7 (12.5%) and mixed infections 7 (12.5%). The sensitivity and specificity of IFA-MAbs in detecting microsporidial spores were 98% and 86%, respectively. The agreement between the WMT staining technique and IFA-MAbs was statistically significant by Kappa statistics (K = 0.840; p < 0.001). E. bieneusi was the commonest Microsporidia species isolated from the studied population; the presence of microsporidial spores detected by IFA-MAbs should be confirmed by other methods.
The Sfp-type 4'-phosphopantetheinyl transferase Ppt1 is required for activation of nonribosomal peptide synthetases, including α-aminoadipate reductase (AAR) for lysine biosynthesis and polyketide synthases, enzymes that biosynthesize peptide and polyketide secondary metabolites, respectively. Deletion of the PPT1 gene, from the maize pathogen Cochliobolus heterostrophus and the rice pathogen Cochliobolus miyabeanus, yielded strains that were significantly reduced in virulence to their hosts. In addition, ppt1 mutants of C. heterostrophus race T and Cochliobolus victoriae were unable to biosynthesize the host-selective toxins (HST) T-toxin and victorin, respectively, as judged by bioassays. Interestingly, ppt1 mutants of C. miyabeanus were shown to produce tenfold higher levels of the sesterterpene-type non-HST ophiobolin A, as compared with the wild-type strain. The ppt1 strains of all species were also reduced in tolerance to oxidative stress and iron depletion; both phenotypes are associated with inability to produce extracellular siderophores biosynthesized by the nonribosomal peptide synthetase Nps6. Colony surfaces were hydrophilic, a trait previously associated with absence of C. heterostrophus Nps4. Mutants were decreased in asexual sporulation and C. heterostrophus strains were female-sterile in sexual crosses; the latter phenotype was observed previously with mutants lacking Nps2, which produces an intracellular siderophore. As expected, mutants were albino, since they cannot produce the polyketide melanin and were auxotrophic for lysine because they lack an AAR.