Biological control using pathogenic microsporidia could be an alternative to chemical control of the diamondback moth (DBM) Plutella xylostella (Lepidoptera: Plutellidae). The microsporidium Nosema bombycis (NB) is one of the numerous pathogens that can be used in the Integrated Pest Management (IPM) of DBM. However, its pathogenicity or effectiveness can be influenced by various factors, particularly temperature. This study was therefore conducted to investigate the effect of temperature on NB infection of DBM larvae. Second-instar larvae at different doses (spore concentration: 0, 1×10²,1×10³,1×10⁴, and 1×10⁵) at 15°, 20°, 25°, 30° and 35°C and a relative humidity(RH) of 65% and light dark cycle (L:D) of 12∶12. Larval mortality was recorded at 24 h intervals until the larvae had either died or pupated. The results showed that the spore concentration had a significant negative effect on larval survival at all temperatures, although this effect was more pronounced (92%) at 35°C compared with that at 20 and 30°C (≃50%) and 25°C (26%). Histological observations showed that Nosema preferentially infected the adipose tissue and epithelial cells of the midgut, resulting in marked vacuolization of the cytoplasm. These findings suggest that Nosema damaged the midgut epithelial cells. Our results suggest that Nosema had a direct adverse effect on DBM, and could be utilized as an important biopesticide alternative to chemical insecticides in IPM.
To investigate the antifungal activity of conventional chitosan and chitosan-loaded nanoemulsions against anthracnose caused by Colletotrichum spp. isolated from different tropical fruits.
Matched MeSH terms: Spores, Fungal/drug effects; Spores, Fungal/growth & development
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.
Lipid biosynthesis produces glycerol, which is important in fueling turgor pressure necessary for germination and penetration of plant host by fungi. As the relationship between pathogenicity and the lipid biosynthetic pathway is not fully understood, we have elucidated the role of the fatty acid synthase beta subunit dehydratase (FAS1) gene in lipid biosynthesis. The FAS1 gene was silenced through homologous double crossover in Magnaporthe oryzae strain S6 to study the effect on lipid biosynthesis. The vegetative growth of Δfas1 mutants show the highest drop on oleic acid (between 10 and 50%), while the mycelial dry weight of mutants dropped significantly on all media. Conidiation of FAS1 mutants show a ~10- and ~5-fold reduction on oatmeal and Potato Dextrose Agar (PDA), respectively. Mutants formed mycelium that were mildly pigmented, indicating that the deletion of FAS1 may have affected melanin biosynthesis. Biochemical and gene expression studies concluded that the fatty acid degradation pathway might have been interrupted by FAS1 deletion. FAS1 mutants showed no enzyme activity on glucose or olive oil, suggesting that the mutants may lack functional peroxisomes and be defective in β-oxidation of fatty acids, hence explaining the reduced lipid deposits in the spores.
The rapid expansion of oil palm (OP) has led to its emergence as a commodity of strategic global importance. Palm oil is used extensively in food and as a precursor for biodiesel. The oil generates export earnings and bolsters the economy of many countries, particularly Indonesia and Malaysia. However, oil palms are prone to basal stem rot (BSR) caused by Ganoderma boninense which is the most threatening disease of OP. The current control measures for BSR management including cultural practices, mechanical and chemical treatment have not proved satisfactory. Alternative control measures to overcome the G. boninense problem are focused on the use of biological control agents and many potential bioagents were identified with little proven practical application. Planting OP varieties resistant to G. boninense could provide the ideal long-term solution to basal stem rot. The total resistance of palms to G. boninense has not yet been reported, and few examples of partial resistances have been observed. Importantly, basidiospores are now recognized as the method by which the disease is spread, and control methods require to be revaluated because of this phenomenon. Many methods developed to prevent the spread of the disease effectively are only tested at nursery levels and are only reported in national journals inhibiting the development of useful techniques globally. The initial procedures employed by the fungus to infect the OP require consideration in terms of the physiology of the growth of the fungus and its possible control. This review assesses critically the progress that has been made in BSR development and management in OP.
Studies on microsporidial infection mostly focus on immunodeficiency or immunosuppressive individuals. Therefore, this cross-sectional study describes the prevalence and risk factors of microsporidiosis among asymptomatic individuals in Malaysia.
Malaysian population widely consumes the cereal-based foods, oilseeds, nuts, and spices in their daily diet. Mycotoxigenic fungi are well known to invade food products under storage conditions and produce mycotoxins that have threat to human and animal health. Therefore, determining toxigenic fungi and aflatoxin B(1) (AFB1) in foods used for human consumption is of prime importance to develop suitable management strategies and to minimize risk. Ninety-five food products marketed in Penang, Malaysia were randomly collected from different supermarkets and were analyzed for presence of Aspergillus spp. by agar plate assay and AFB1 by enzyme-linked immunosorbent assay (ELISA). A. flavus was the dominant fungi in all foods followed by A. niger. Fifty-five A. flavus strains were tested for their ability to produce aflatoxins on rice grain substrate. Thirty-six (65.4%) strains out of 55 produced AFB1 ranging from 1700 to 4400 μg/kg and 17 strains (31%) produced AFB2 ranging from 620 to 1670 μg/kg. Natural occurrence of AFB1 could be detected in 72.6% food products ranging from 0.54 to 15.33 μg/kg with a mean of 1.95 μg/kg. Maximum AFB1 levels were detected in peanut products ranging from 1.47 to 15.33 μg/kg. AFB1 levels detected in all food products were below the Malaysian permissible limits (<35 μg/kg). Aspergillus spp. and AFB1 was not detected in any cookies tested. Although this survey was not comprehensive, it provides valuable information on aflatoxin levels in foods marketed in Malaysia.
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.
During an investigation of submerged leaves and twigs sampled from tropical peat swamp forests located in Peninsular Malaysia, an anamorphic fungus not attributable to a described genus was detected and isolated in pure culture. Conidial ontogeny was thoroughly studied and illustrated using both light and SEM, which revealed a unique conidial morphology. Analysis of partial nuLSU rDNA and ITS data revealed a phylogenetic position within the Xylariales (Ascomycota), but family affiliation remained unclear.
Leaf spot diseases are mainly caused by fungi including Fusarium. In the present study several species of Fusarium were isolated from the leaf spot lesion of mango (Mangifera indica L.) Based on morphological characteristics, TEF-1α sequences and phylogenetic analysis, five species were identified as F. proliferatum, F. semitectum, F. mangiferae, F. solani and F. chlamydosporum. Pathogenicity test indicated that representative isolates of F. proliferatum, F. semitectum and F. chlamydosporum were pathogenic on mango leaves causing leaf spot with low to moderate virulence. Nevertheless, abundance of spots on the leaf can disrupt photosynthesis which in turn reduced growth, and lead to susceptibility to infection by opportunistic pathogens due to weakening of the plant. Fusarium solani and F. mangiferae were non-pathogenic and it is possible that both species are saprophyte which associated with nutrient availability on the surface of the leaf through decaying leave tissues. The occurrence of Fusarium spp. on the leaf spot lesion and the effect from the disease needs to be considered when developing disease management method of mango cultivation as numerous spot on the leaves could effect the photosynthesis process and finally giving low yield and less quality of mango.
This study was conducted to evaluate the modification of the usual Gram-chromotrope staining technique developed in-house known as Gram-chromotrope Kinyoun (GCK) in comparison with the Weber Modified Trichrome (WMT) staining technique; as the reference technique. Two hundred and ninety fecal specimens received by the Microbiology Diagnostic Laboratory of Hospital Universiti Kebangsaan Malaysia were examined for the presence of microsporidial spores. The sensitivity and specificity of GCK compared to the reference technique were 98% and 98.3%, respectively. The positive and negative predictive values were 92.5% and 99.6%, respectively. The agreement between the reference technique and the GCK staining technique was statistically significant by Kappa statistics (K = 0.941, P < 0.001). It is concluded that the GCK staining technique has high sensitivity and specificity in the detection of microsporidial spores in fecal specimens. Hence, it is recommended to be used in the diagnosis of intestinal microsporidiosis.
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.
We report a case of an invasive infection with non-sporulating Chrysosporium species in a patient who was treated with chemotherapy for relapsed acute lymphoblastic leukemia. This patient presented with a persistent lobar pneumonia, skin lesions, and possible involvement of the central nervous system. The patient responded to treatment with amphotericin B and oral itraconazole.
Basal stem rot disease of oil palm caused by Ganoderma boninense is one of the most devastating diseases in oil palm
plantation resulting in low yield, loss of palm stands and shorter replanting cycle. To-date, there is no effective treatment
for Ganoderma infected palms. Control measures, either chemical or cultural approaches, show varying degrees of
effectiveness. The application of biological control agents which is environmental-friendly could be an attractive solution
to overcome the problem. Earlier, we had isolated a mycoparasite, Scytalidium parasiticum, from the basidiomata of
Ganoderma boninense. In vitro assay and nursery experiment showed that this fungus could suppress Ganoderma infection
and reduce disease severity. However, metabolites which might contribute to the antagonistic or mycoparasitic effect
remain unknown. In the current study, optimization of fungal sample processing, extraction, and analytical procedures
were conducted to obtain metabolites from the maize substrate colonized by mycoparasitic ascomycetous Scytalidium
parasiticum. This technique capable of producing sexual spores in sac-like organs. Untargeted metabolomics profiling
was carried out by using Liquid Chromatography Time of Flight Mass Spectrometry (LC-ToF-MS). We found that
S. parasiticum in both liquid- and solid-state cultivation gave higher metabolite when extracted with 60% methanol with
1% formic acid in combination with homogenisation methods such as ultrasonication and grinding. The findings from
this study are useful for optimisation of metabolite extraction from other fungi-Ganoderma-plant interactions.
Passiflora edulis Sims f. edulis, known as purple passion fruit, is a woody, perennial vine that is grown for its attractive two-part flower and its purple, edible fruit (4). In November 2009, passion fruit vines were collected during a regulatory nursery inspection in Santa Barbara County and submitted to the California Department of Food and Agriculture Plant Pest Diagnostics Laboratory. Nearly 100% of the plants inspected, all of which were approximately 1.25 m tall, appeared stunted, defoliated, and severely wilted. Dark brown vascular discoloration was present in the roots and lower stems of the plants. A pinkish violet Fusarium oxysporum colony containing chlamydospores, multiseptate macroconidia, and microconidia formed on monophialidic conidiophores was consistently isolated from roots and stems onto half-strength acidified potato dextrose agar (aPDA). All further experiments were done with an isolate obtained from a single conidium. A portion of the translation elongation factor gene (TEF-1α) was amplified and sequenced with primers ef1 and ef2 from our isolate (GenBank No. JF332039) (3). BLAST analysis of the 615-bp amplicon with the FUSARIUM-ID database showed 99% similarity with a F. oxysporum passion fruit isolate from Australia (NRRL 38273) (3). To confirm pathogenicity, washed roots of four-leaf stage seedlings approximately 10 cm tall were submerged in a conidial spore suspension (106 spores/ml) for 15 min. The conidial suspension was prepared by flooding 10-day-old cultures grown on aPDA medium with sterile distilled water. Seven seedlings were inoculated and planted in 10-cm2 pots and kept in a 25°C growth chamber with a 12-h photoperiod. Seven seedlings were mock inoculated with sterile water. After 3 weeks, four of the seven inoculated plants had leaves with yellow veins and discolored roots and had partially defoliated. Two of the four symptomatic plants also had brown stem cankers. F. oxysporum grew from the isolated roots and stems of all the inoculated plants. F. oxysporum did not grow from root and stem pieces from the water-dipped plants and the plants remained asymptomatic. Inoculations were repeated on plants approximately 15 cm tall with F. oxysporum growing from roots and stem pieces of all inoculated plants. Symptoms of yellow veins and root necrosis were not observed until 4 weeks after inoculation. Fusarium wilt caused by F. oxysporum f. sp. passiflorae is a significant disease of P. edulis f. edulis in Australia. The disease has also been reported in South Africa, Malaysia, Brazil, Panama, and Venezuela; but it is unclear as to whether the symptoms were caused by Fusarium wilt or Haematonectria canker (1). Banana poka (P. mollissima), P. ligularis, and P. foetida are also susceptible hosts (2). To our knowledge, this is the first report of Fusarium wilt caused by F. oxysporum f. sp. passiflorae on passion fruit in North America. Passion fruit is not commercially produced for consumption in California so the economic importance of this disease appears to be limited to nursery production and ornamental landscapes. The grower of the California nursery stated that the infected passion fruit plants had been propagated on site from seed. The source of inoculum at this nursery remains unknown. References: (1) I. H. Fischer and J. A. M. Rezende. Pest Tech. 2:1, 2008 (2) D. E. Garder. Plant. Dis. 73:476, 1989. (3) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004. (4) F. W. Martin et al. Econ. Bot. 24:333, 1970.
Soybean (Glycine max L.) is one of the most economically important crops in the world, and anthracnose is known to infect soybean in most countries. Colletotrichum truncatum is the common pathogen causing anthracnose of soybean. However, at least five species of Colletotrichum have been reported on soybean worldwide (2). In July 2010, anthracnose symptoms were observed on soybean in the experimental fields of the agriculture station in Ladang Dua, University Putra Malaysia located in Selangor state of Malaysia. Symptoms were initially observed on a few plants randomly within one field, but after 4 weeks, the disease was found in two additional fields scattered across an area of 1 km2. Pinkish-brown lesions were observed on the pods, and the formation of dark lesions on the leaves and stems was sometimes followed by stem girdling, dieback, and distorted growth. At later stages, numerous epidermal acervuli developed in the lesions, and mucilaginous conidial masses appeared during periods of high relative humidity. Conidia produced in acervuli were straight, cylindric, hyaline, and aseptate, with both ends rounded. Conidia measured (mean ± SD) 14.2 ± 0.6 × 3.6 ± 0.7 μm, and the L/W ratio was 3.95 μm. Six isolates of the fungus were obtained and identified as C. gloeosporioides on the basis of morphological characterization (3). The isolates were deposited in the University Putra of Malaysia Culture Collection (UPMCC). PDA cultures were white at first and subsequently became grayish to pink to reddish-brown. Amplification and sequence analysis of coding and none-coding regions of the ITS-rDNA (GenBank JX669450), actin (JX827430), β-tubulin (JX827454), histone (JX827448), chitin synthase (JX827436), and glyceraldehyde-3-phosphate dehydrogenase (JX827442) obtained from the representative isolate, CGM50, aligned with deposited sequences from GenBank and revealed 99 to 100% sequence identity with C. gloeosporioides strains (JX258757, JX009790, GQ849434, HM575301, JQ005413, and JX00948 from GenBank). One representative isolate, CGM50, was used for pathogenicity testing. Four non-infected detached leaves and pods of 24-day-old G. max var. Palmetto were surface-sterilized and inoculated by placing 10 μl of a conidial suspension (106 conidia ml-1) using either the wound/drop or non-wound/drop method (4), with 10 μl distilled water as a negative control. Leaves and pods were incubated at 25°C, 98% RH. The experiment was repeated twice. Five days after inoculation, the development of typical field symptoms, including acervuli formation, occurred on the leaves and pods of inoculated plants, but not on the negative controls. A fungus with the same colony and conidial morphology as CGM50 was recovered from the lesions on the inoculated leaves and pods. Anthracnose caused by C. gloeosporioides on soybean plants has been reported previously in different countries, but not in Malaysia (3). Geographically, the climate of Malaysia is highly conducive to maintain and cause outbreaks of anthracnose all year round; thus, the development of management recommendations will be inevitable for anthracnose control. To our knowledge, this is the first report of C. gloeosporioides causing anthracnose on soybean in Malaysia. References: (1) U. Damm et al. Fungal Diversity 39:45, 2009. (2) S. L. Chen et al. J. Phytopathol. 154:654, 2006. (3) B. C. Sutton. The Genus Glomerella and its Anamorph Colletotrichum. CAB International, Wallingford, UK, 1992. (4) P. P. Than et al. Plant Pathol. 57:562, 2008. ERRATUM: A correction was made to this Disease Note on May 19, 2014. The author N. Soleimani was added.
Banana is the second largest cultivated fruit crop in Malaysia, and is cultivated for both the domestic market and also for export. Anthranose is a well-known postharvest disease of banana and with high potential for damaging market value, as infection commonly occurs during storage. Anthracnose symptoms were observed on several varieties of banana such as mas, berangan, awak, nangka, and rastali in the states of Perak and Penang between August and October 2011. Approximately 80% of the fruits became infected with initial symptoms characterized as brown to black spots that later became sunken lesions with orange or salmon-colored conidial masses. Infected tissues (5 × 5 mm) were surface sterilized by dipping in 1% sodium hypochlorite (NaOCl) for 3 to 5 min, rinsed with sterile distilled water, and plated onto potato dextrose agar (PDA). Direct isolation was done by transferring the conidia from conidial masses using an inoculation loop and plating onto PDA. For both methods, the PDA plates were incubated at 27 ± 1°C with cycles of 12 h light and 12 h darkness. Visible growth of mycelium was observed after 4 to 5 days of incubation. Twenty isolates with conidial masses were recovered after 7 days of incubation. The isolates produced grayish white to grayish green and grey to moss dark green colony on PDA, pale orange conidial masses, and fusiform to cylindrical and hyaline conidia with an average size of 15 to 19 × 5 to 6 μm. Appresoria were ovate to obovate, dark brown, and 9 to 15 × 7 to 12 μm and setae were present, slightly swollen at the base, with a tapered apex, and brown. The cultural and morphological characteristics of the isolates were similar to those described for C. gleosporioides (1,2,3). All the C. gloeosporioides isolates were deposited in culture collection at Plant Pathology Lab, University Sains Malaysia. For confirmation of the identity of the isolates, ITS regions were sequenced using ITS4 and ITS5 primers. The isolates were deposited in GenBank with accessions JX163228, JX163231, JX163201, JX163230, JX163215, JX163223, JX163219, JX163202, JX163225, JX163222, JX163206, JX163218, JX163208, JX163209, JX163210, JX431560, JX163212, JX163213, JX431540, and JX431562. The resulting sequences showed 99% to 100% similarity with multiple C. gloeosporioides isolates in GenBank. Pathogenicity tests were conducted using mas, berangan, awak, nangka, and rastali bananas. Fruit surfaces were sterilized with 70% ethanol and wounded using a sterile scalpel. Two inoculation techniques were performed separately: mycelia plug and conidial suspension. Mycelial disc (5 mm) and a drop of 20 μl spore suspension (106 conidia/ml) were prepared from 7-day-old culture and placed on the fruit surface. The inoculated fruits were incubated at 27 ± 1°C for 10 days at 96.1% humidity. After 3 to 4 days of inoculation, brown to black spotted lesions were observed and coalesced to become black sunken lesions. Similar anthracnose symptoms were observed on all banana varieties tested. C. gloeosporioides was reisolated from the anthracnose lesions of all the inoculated fruit in which the cultural and morphological characteristics were the same as the original isolates. To our knowledge, this is the first report of C. gloeosporioides causing anthracnose of Musa spp. in Malaysia. References: (1) P. F. Cannon et al. Mycotaxon 104:189, 2008. (2) J. E. M. Mordue. Glomerella cingulata. CMI Description of Pathogenic Fungi and Bacteria, No. 315. CAB International,1971. (3) H. Prihastuti et al. Fungal Diversity 39:89, 2009.
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.
Mangosteen (Garcinia mangostana L.) is a tropical evergreen tree that produces one of the most prized tropical fruits, commonly known as the "Queen of the Fruits.″ Mangosteen has the potential to occupy a rapidly expanding niche market in Hawaii. In October 2009, a disease was observed that produced brown leaf spots and blotches surrounded by bright yellow halos at a mangosteen orchard located in Hakalau, Hawaii (19° 53' 49″ N, 155° 7' 35″ W). Recently transplanted 10+ year old trees were 95 to 100% infected. Pieces of infected leaves and stems were surface-sterilized, plated on potato dextrose agar (PDA), and incubated at 24°C ± 1°C for 21 days. The fungus growing on PDA was pale buff with sparse aerial mycelium and acervuli containing black, slimy spore masses. Single spore isolates were used for the morphological characteristics and molecular analysis. Conidia were 5-celled. Apical and basal cells were hyaline; the three median cells were umber to olivaceous. Conidia (n = 50) were 24.3 ± 0.2 × 7.5 ± 0.1 μm, with apical appendages, typically three, averaging 24.3 ± 0.4 μm long, and a basal appendage averaging 6.7 ± 0.2 μm long. DNA sequences were obtained from the β-tubulin gene and the internal transcribed spacer (ITS1 and ITS2) and 5.8S regions of the rDNA to confirm the identification. The morphological descriptions and measurements were similar to P. virgatula (Kleb.) Steyaert (1). Although sequence data of the ITS region (GenBank Accession No. JN542546) supports the identity of the fungus as P. virgatula, the taxonomy of this genus remains confused since there are only a few type cultures, so it is impossible to use sequences in GenBank to reliably clarify species names (2). To confirm pathogenicity, six leaves of two 3-year-old seedlings were inoculated. Seven-day-old cultures grown on 10% V8 agar at 24°C under continuous fluorescent lighting were used for inoculations. The inoculum consisted of spore suspensions in sterile distilled water adjusted to 6 × 105 conidia/ml. Using a fine haired paint brush, the inoculum was brushed onto the youngest leaves, while sterile distilled water was used as the control. The plants were incubated in a clear plastic bag placed on the laboratory bench at 24°C for 48 hours, then placed on a greenhouse bench and observed weekly for symptoms. After 14 days, leaf spots ranging in size from pinpoint to 5.4 mm in diameter with a distinctive yellow halo were present. Within 35 days, the leaf spots enlarged to leaf blotches ranging in size from 11.5 × 13.3 mm up to 28.3 × 34.6 mm with brown centers and a distinctive yellow halo identical to the field symptoms. A Pestalotiopsis sp. identical to that used to inoculate the seedlings was recovered from the leaf spots and blotches, confirming Koch's postulates. The experiment was repeated twice. Pestalotiopsis leaf blight has been reported in other countries growing mangosteen, including Thailand, Malaysia, and North Queensland, Australia (3). However, to our knowledge, this is the first report of a Pestalotiopsis sp. causing a disease on mangosteen in Hawaii. Although this disease is considered a minor problem in the literature (3), effective management practices should be established to avoid potential production losses. References: (1) E. F. Guba. Monograph of Pestalotia and Monochaetia. Harvard University Press, Cambridge, MA. 1961. (2) S. S. N. Maharachchikumbura et al. Fungal Div. 50:167, 2011. (3) R. C. Ploetz. Diseases of Tropical Fruit Crops. CABI Publishing. Wallingford, Oxfordshire, UK, 2003.