In September 1997, plants of Hibiscus manihot (locally called nambele) were observed on Vaitupu Island, Tuvalu, exhibiting an angular leaf mosaic and chlorosis that was not always clearly discernible. Electron microscopy of negatively stained sap from affected leaves revealed the presence of numerous isometric virus particles 28 nm in diameter. Poly-acrylamide gel electrophoresis of purified virus gave a single protein band of Mr 38,000 similar to that of the carmoviruses. Immunosorbent electron microscopy tests with antisera kindly provided by N. Spence showed the virus to be hibiscus chlorotic ringspot carmovirus (HCRSV) (1). This virus is also reported from El Salvador, the U.S., Australia, Thailand, Malaysia, Fiji, the Solomon Islands, and Vanuatu. It is not known how the virus reached Tuvalu but we suspect it was via infected cuttings, which were imported for the production of food supplements to combat acute deficiencies of vitamins A and C in the population. The virus is most likely to have been disseminated throughout the islands and atolls of Tuvalu through infected cuttings. Local spread within fields could occur through contaminated hands and cutting implements because of the ease with which the virus is mechanically transmitted. Reference: (1) H. E.Waterworth et al. Phytopathology 66:570, 1976.
Rambutan (Nephelium lappaceum L.) is a tropical fruit grown in Hawaii for the exotic fruit market. Fruit rot was observed periodically during 1998 and 1999 from two islands, Hawaii and Kauai, and severe fruit rot was observed during 2000 in orchards in Kurtistown and Papaikou on Hawaii. Symptoms were characterized by brown-to-black, water-soaked lesions on the fruit surface that progressed to blackening and drying of the pericarp, which often split and exposed the aril (flesh). In certain cultivars, immature, small green fruits were totally mummified. Rambutan trees with high incidence of fruit rot also showed symptoms of branch dieback and leaf spot. Lasmenia sp. Speg. sensu Sutton, identified by Centraalbureau voor Schimmelcultures (Baarn, the Netherlands), was isolated from infected fruit and necrotic leaves. Also associated with some of the fruit rot and dieback symptoms were Gliocephalotrichum simplex (J.A. Meyer) B. Wiley & E. Simmons, and G. bulbilium J.J. Ellis & Hesseltine. G. simplex was isolated from infected fruit, and G. bulbilium was isolated from discolored vascular tissues and infected fruit. Identification of species of Gliocephalotrichum was based on characteristics of conidiophores, sterile hairs, and chlamydospores (1,4). Culture characteristics were distinctive on potato dextrose agar (PDA), where the mycelium of G. bulbilium was light orange (peach) without reverse color, while G. simplex was golden-brown to grayish-yellow with dark brown reverse color. Both species produced a fruity odor after 6 days on PDA. In pathogenicity tests, healthy, washed rambutan fruits were wounded, inoculated with 30 μl of sterile distilled water (SDW) or a fungus spore suspension (105 to 106 spores per ml), and incubated in humidity chambers at room temperature (22°C) under continuous fluorescent light. Lasmenia sp. (strain KN-F99-1), G. simplex (strain KN-F2000-1), and G. bulbilium (strains KN-F2001-1 and KN-F2001-2) produced fruit rot symptoms on inoculated fruit and were reisolated from fruit with typical symptoms, fulfilling Koch's postulates. Controls (inoculated with SDW) had lower incidence or developed less severe symptoms than the fungus treatments. Inoculation tests were conducted at least twice. To our knowledge, this is the first report of Lasmenia sp. in Hawaii and the first report of the genus Gliocephalotrichum on rambutan in Hawaii. These pathogens are potentially economically important to rambutan in Hawaii. G. bulbilium has been reported previously on decaying wood of guava (Psidium guajava L.) in Hawaii (2), and the fungus causes field and postharvest rots of rambutan fruit in Thailand (3). References: (1) J. J. Ellis and C. W. Hesseltine. Bull. Torrey Bot. Club 89:21, 1962. (2) D. F. Farr et al. Fungi on Plants and Plant Products in the United States. The American Phytopathological Society, St. Paul, MN, 1989. (3) N. Visarathanonth and L. L. Ilag. Pages 51-57 in: Rambutan: Fruit Development, Postharvest Physiology and Marketing in ASEAN. ASEAN Food Handling Bureau, Kuala Lumpur, Malaysia, 1987. (4) B. J. Wiley and E. G. Simmons. Mycologia 63:575, 1971.
Sugarcane yellow leaf virus (SCYLV) was detected for the first time in 1996 in the Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD) sugarcane quarantine at Montpellier by reverse transcription-polymerase chain reaction (RT-PCR) in varieties from Brazil, Florida, Mauritius, and Réunion. Between 1997 and 2000, the virus was found by RT-PCR and/or tissue-blot immunoassay (TBIA) in additional varieties from Barbados, Cuba, Guadeloupe, Indonesia, Malaysia, Philippines, Puerto Rico, and Taiwan, suggesting a worldwide distribution of the pathogen. An excellent correlation was observed between results obtained for the two diagnostic techniques. However, even though only a few false negative results were obtained by either technique, both are now used to detect SCYLV in CIRAD's sugarcane quarantine in Montpellier. The pathogen was detected by TBIA or RT-PCR in all leaves of sugarcane foliage, but the highest percentage of infected vascular bundles was found in the top leaves. The long hot water treatment (soaking of cuttings in water at 25°C for 2 days and then at 50°C for 3 h) was ineffective in eliminating SCYLV from infected plants. Sugarcane varieties from various origins were grown in vitro by apical bud culture and apical meristem culture, and the latter proved to be the most effective method for producing SCYLV-free plants.
Production of tomato (Lycopersicon esculentum) in Bangladesh, Malaysia, Myanmar, Vietnam, and Laos has been severely affected by yellow leaf curl disease. Tomato leaf samples were collected from symptomatic tomato plants from farmers' fields in the five countries from 1997 to 1999. DNA was extracted from all samples, four from Vietnam, two each from Malaysia, Laos, and Myanmar, and seven from Bangladesh. Virus DNA was amplified by polymerase chain reaction (PCR) using the begomovirus-specific degenerate primer pair PAL1v 1978/PAR1c 715(1), which amplifies the top part of DNA A. All samples gave the expected 1.4-kb PCR product. The PCR product of one sample per country was cloned and sequenced. Based on the sequences of the 1.4-kb DNA products amplified by the first primer pair, specific primers were designed to complete each of the DNA A sequences. Computer-assisted sequence comparisons were performed with begomovirus sequences available in the laboratory at the Asian Vegetable Research and Development Center, Shanhua, Tainan, and in the GenBank sequence database. The five DNA species resembled DNA A of begomoviruses. For the detection of DNA B two degenerate primer pairs were used, DNABLC1/DNABLV2 and DNABLC2/DNABLV2 (DNABLC1: 5'-GTVAATGGRGTDCACTTCTG-3', DNABLC2: 5'-RGTDCACTT CTGYARGATGC-3', DNABLV2: 5'-GAGTAGTAGTGBAKGTTGCA-3'), which were specifically designed to amplify DNA B of Asian tomato geminiviruses. Only the virus associated with yellow leaf curl of tomato in Bangladesh was found to contain a DNA B component, which was detected with the DNABLC1/DNABLV2 primer pair. The DNA A sequence derived from the virus associated with tomato yellow leaf curl from Myanmar (GenBank Accession No. AF206674) showed highest sequence identity (94%) with tomato yellow leaf curl virus from Thailand (GenBank Accession No. X63015), suggesting that it is a closely related strain of this virus. The other four viruses were distinct begomoviruses, because their sequences shared less than 90% identity with known begomoviruses of tomato or other crops. The sequence derived from the virus associated with tomato yellow leaf curl from Vietnam (GenBank Accession No. AF264063) showed highest sequence identity (82%) with the virus associated with chili leaf curl from Malaysia (GenBank Accession No. AF414287), whereas the virus associated with yellow leaf curl symptoms in tomato in Bangladesh (GenBank Accession No. AF188481) had the highest sequence identity (88%) with a tobacco geminivirus from Yunnan, China (GenBank Accession No. AF240675). The sequence derived from the virus associated with tomato yellow leaf curl from Laos (GenBank Accession No. AF195782) had the highest sequence identity (88%) with the tomato begomovirus from Malaysia (GenBank Accession No. AF327436). This report provides further evidence of the great genetic diversity of tomato-infecting begomoviruses in Asia. Reference: M. R. Rojas et al. Plant Dis. 77:340, 1993.
Fusarium nygamai Burgess & Trimboli was first described in 1986 in Australia (1) and subsequently reported in Africa, China, Malaysia, Thailand, Puerto Rico, and the United States. F. nygamai has been reported on sorghum, millet, bean, cotton, and in soil where it exists as a colonizer of living plants or plant debris. F. nygamai was also reported as a pathogen of the witch-weed Striga hermonthica (Del.) Benth. To our knowledge, no reports are available on its pathogenicity on crops of economic importance. In a survey of species of Fusarium causing seedling blight and foot rot of rice (Oryza sativa L.) carried out in Sardinia (Oristano, S. Lucia), F. nygamai was isolated in association with other Fusarium species-F. moniliforme, F. proliferatum, F. oxysporum, F. solani, F. compactum, and F. equiseti. Infected seedlings exhibited a reddish brown cortical discoloration, which was more intense in older plants. The identification of F. nygamai was based on monoconidial cultures grown on carnation leaf-piece agar (CLA) (2). The shape of macroconidia, the formation of microconidia in short chains and false heads, and the presence of chlamydospores were used as the criteria for identification. Two pathogenicity tests comparing one isolate of F. nygamai with one isolate of F. moniliforme were conducted on rice cv. Arborio sown in artificially infested soil in a greenhouse at 22 to 25°C. The inoculum was prepared by growing both Fusarium species in cornmeal sand (1:30 wt/wt) at 25°C for 3 weeks. This inoculum was added to soil at 20 g per 500 ml of soil. Pre- and post-emergence damping-off was assessed. Both F. nygamai and F. moniliforme reduced the emergence of seedlings (33 to 59% and 25 to 50%, respectively, compared to uninoculated control). After 25 days, the seedlings in infested soil exhibited a browning of the basal leaf sheaths, which progressed to a leaf and stem necrosis. Foot rot symptoms caused by F. nygamai and F. moniliforme were similar, but seedlings infected by F. nygamai exhibited a more intense browning on the stem base and a significant reduction of plant height at the end of the experiment. Either F. nygamai or F. moniliforme were consistently isolated from symptomatic tissue from the respective treatments. References: (1) L. W. Burgess and D. Trimboli. Mycologia 78:223,1986. (2) N. L. Fisher et al. Phytopathology 72:151,1982.
Phytoplasmas (mycoplasmalike organisms, MLOs) associated with mitsuba (Japanese hone-wort) witches'-broom (JHW), garland chrysanthemum witches'-broom (GCW), eggplant dwarf (ED), tomato yellows (TY), marguerite yellows (MY), gentian witches'-broom (GW), and tsu-wabuki witches'-broom (TW) in Japan were investigated by polymerase chain reaction (PCR) amplification of DNA and restriction enzyme analysis of PCR products. The phytoplasmas could be separated into two groups, one containing strains JHW, GCW, ED, TY, and MY, and the other containing strains GW and TW, corresponding to two groups previously recognized on the basis of transmission by Macrosteles striifrons and Scleroracus flavopictus, respectively. The strains transmitted by M. striifrons were classified in 16S rRNA gene group 16SrI, which contains aster yellows and related phytoplasma strains. Strains GW and TW were classified in group 16SrIII, which contains phytoplasmas associated with peach X-disease, clover yellow edge, and related phytoplasmas. Digestion of amplified 16S rDNA with HpaII indicated that strains GW and TW were affiliated with subgroup 16SrIII-B, which contains clover yellow edge phytoplasma. All seven strains were distinguished from other phytoplasmas, including those associated with clover proliferation, ash yellows, elm yellows, and beet leafhopper-transmitted virescence in North America, and Malaysian periwinkle yellows and sweet potato witches'-broom in Asia.
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.
During March 2011 to June 2012, 50 banana plants of cultivar Musa × paradisiaca 'Horn' with Moko disease symptoms were randomly sampled in 12 different locations of 5 outbreak states in Peninsular Malaysia comprising Kedah, Selangor, Pahang, Negeri Sembilan, and Johor, with disease incidence exceeding 90% in some severely affected plantations. The disease symptoms observed in the infected plants included yellowing and wilting of the oldest leaves, which became necrotic, and eventually led to their dieback or collapse. The pulp of banana fruits also became discolored and exuded bacterial ooze. Vascular tissues in pseudostems were discolored. Fragments from symptomatic plant samples were excised and cultured on Kelman's-tetrazolium salt (TZC) medium. Twenty positive samples produced fluidal colonies that were either entirely white or white with pink centers after incubation for 24 to 48 h at 28°C on Kelman's-TZC medium and appeared as gram-negative rods after Gram staining. They were also positive for potassium hydroxide (KOH), Kovacs oxidase, and catalase tests, but negative for utilization of disaccharides and hexose alcohols, which are characteristics of biovar 1 Ralstonia solanacearum. For the pathogenicity test, 30 μl of 108 CFU/ml bacterial suspension of three selected virulent strains were injected into banana (Musa × paradisiaca 'Horn') leaves explants grown in plastic pots of 1,440 cm3 volume in a greenhouse, with temperature range from 26 to 35°C. Leaves that were infiltrated with sterile distilled water served as a negative control. Inoculations with all isolates were performed in three replications, as well as the uninoculated control leaves explants. The inoculated plants produced the same symptoms as observed on naturally diseased samples, whereas control plants remained asymptomatic. Strain cultures were re-isolated and possessed the morphological and biochemical characteristics as previously described. PCR amplification using race 2 R. solanacearum primers ISRso19-F (5'-TGGGAGAGGATGGCGGCTTT-3') and ISRso19-R (5'-TGACCCGCCTTTCGGTGTTT-3') (3) produced a 1,900-bp product from DNA of all bacterial strains. BLAST searches resulted that the sequences were 95 to 98% identical to published R. solanacearum strain race 2 insertion sequence ISRso19 (GenBank Accession No. AF450275). These genes were later deposited in GenBank (KC812051, KC812052, and KC812053). Phylotype-specific multiplex PCR (Pmx-PCR) and Musa-specific multiplex PCR (Mmx-PCR) were performed to identify the phylotype and sequevar of all isolates (4). Pmx-PCR showed that all isolates belonged to phylotype II, whereas Mmx-PCR showed that they belonged to phylotype II sequevar 4 displaying 351-bp amplicon. Although there were previously extensive studies on R. solanacearum associated with bacterial wilt disease of banana crops in Malaysia, none related to Moko disease has been reported (1,2). The result has a great importance to better understand and document R. solanacearum race 2 biovar 1, since banana has been identified as the second most important commercial fruit crop with a high economic value in Malaysia. References: (1) R. Khakvar et al. Plant Pathol. J. 7:162, 2008. (2) R. Khakvar et al. Am. J. Agri. Biol. Sci. 3:490, 2008. (3) Y. A. Lee and C. N. Khor. Plant Pathol. Bull. 12:57, 2003. (4) P. Prior et al. Pages 405-414 in: Bacterial Wilt Disease and the Ralstonia solanacearum Species Complex. The American Phytopathological Society, St. Paul, MN, 2005.
The foxtail palm (Wodyetia bifurcata), an Australian native species, is an adaptable and fast-growing landscape tree. The foxtail palm is most commonly used in landscaping in Malaysia. Coconut yellow decline (CYD) is the major disease of coconut associated with 16SrXIV phytoplasma group in Malaysia (1). Symptoms consistent with CYD, such as severe chlorosis, stunting, general decline, and death were observed in foxtail palms from the state of Selangor in Malaysia, indicating putative phytoplasma infection. Symptomatic trees loses their green and vivid appearance as a decorative and landscape ornament. To determine the presence of phytoplasma, samples were collected from the fronds of 12 symptomatic and four asymptomatic palms in September 2012, and total DNA was extracted using the CTAB method (3). Phytoplasma DNA was detected in eight symptomatic palms using nested PCR with universal phytoplasma 16S rDNA primer pairs, P1/P7 followed by R16F2n/R16R2 (2). Amplicons (1.2 kb in length) were generated from symptomatic foxtail palms but not from symptomless plants. Phytoplasma 16S rDNAs were cloned using a TOPO TA cloning kit (Invitrogen). Several white colonies from rDNA PCR products amplified from one sample with R16F2n/R16R2 were sequenced. Phytoplasma 16S rDNA gene sequences from single symptomatic foxtail palms showed 99% homology with a phytoplasma that causes Bermuda grass white leaf (AF248961) and coconut yellow decline (EU636906), which are both members of the 16SrXIV 'Candidatus Phytoplasma cynodontis' group. The sequences also showed 99% sequence identity with the onion yellows phytoplasma, OY-M strain, (NR074811), from the 'Candidatus Phytoplasma asteris' 16SrI-B subgroup. Sequences were deposited in the NCBI GenBank database (Accession Nos. KC751560 and KC751561). Restriction fragment length polymorphism (RFLP) analysis was done on nested PCR products produced with the primer pair R16F2n/R16R2. Amplified products were digested separately with AluI, HhaI, RsaI, and EcoRI restriction enzymes based on manufacturer's specifications. RFLP analysis of 16S rRNA gene sequences from symptomatic plants revealed two distinct profiles belonging to groups 16SrXIV and 16SrI with majority of the 16SrXIV group. RFLP results independently corroborated the findings from DNA sequencing. Additional virtual patterns were obtained by iPhyclassifier software (4). Actual and virtual patterns yielded identical profiles, similar to the reference patterns for the 16SrXIV-A and 16SrI-B subgroups. Both the sequence and RFLP results indicated that symptoms in infected foxtail palms were associated with two distinct phytoplasma species in Malaysia. These phytoplasmas, which are members of two different taxonomic groups, were found in symptomatic palms. Our results revealed that popular evergreen foxtail palms are susceptible to and severely affected by phytoplasma. To our knowledge, this is the first report of a mixed infection of a single host, Wodyetia bifurcata, by two different phytoplasma species, Candidatus Phytoplasma cynodontis and Candidatus Phytoplasma asteris, in Malaysia. References: (1) N. Nejat et al. Plant Pathol. 58:1152, 2009. (2) N. Nejat et al. Plant Pathol. J. 9:101, 2010. (3) Y. P. Zhang et al. J. Virol. Meth. 71:45, 1998. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.
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.
Hylocereus undatus widely grows in southern China. Some varieties are planted for their fruits, known as dragon fruits or Pitaya, while some varieties for their flowers known as Bawanghua. Fresh or dried flowers of Bawanghua are used as routine Chinese medicinal food. Since 2008, a serious anthracnose disease has led to great losses on Bawanghua flower production farms in the Baiyun district of Guangzhou city in China. Anthracnose symptoms on young stems of Bawanghua are reddish-brown, sunken lesions with pink masses of spores in the center. The lesions expand rapidly in the field or in storage, and may coalesce in the warm and wet environment in spring and summer in Guangzhou. Fewer flowers develop on infected stems than on healthy ones. The fungus overwinters in infected debris in the soil. The disease caused a loss of up to 50% on Bawanghua. Putative pathogenic fungi with whitish-orange colonies were isolated from a small piece of tissue (3 × 3 mm) cut from a lesion margin and cultured on potato dextrose agar in a growth chamber at 25°C, 80% RH. Dark colonies with acervuli bearing pinkish conidial masses formed 14 days later. Single celled conidia were 11 to 18 × 4 to 6 μm. Based on these morphological characteristics, the fungi were identified as Colletotrichum gloeosporioides (Penz.) Penz. & Sacc (2). To confirm this, DNA was extracted from isolate BWH1 and multilocus analyses were completed with DNA sequence data generated from partial ITS region of nrDNA, actin (ACT) and glutamine synthetase (GS) nucleotide sequences by PCR, with C. gloeosporioides specific primers as ITS4 (5'-TCCTCCGCTTATTGATATGC-3') / CgInt (5'-GGCCTCCCGCCTCCGGGCGG-3'), GS-F (5'-ATGGCCGAGTACATCTGG-3') / GS-R (5'-GAACCGTCGAAGTTCCAC-3') and actin-R (5'-ATGTGCAAGGCCGGTTTCGC-3') / actin-F (5'-TACGAGTCCTTCTGGCCCAT-3'). The sequence alignment results indicated that the obtained partial ITS sequence of 468 bp (GenBank Accession No. KF051997), actin sequence of 282 bp (KF712382), and GS sequence of 1,021 bp (KF719176) are 99%, 96%, and 95% identical to JQ676185.1 for partial ITS, FJ907430 for ACT, and FJ972589 for GS of C. gloeosporioides previously deposited, respectively. For testing its pathogenicity, 20 μl of conidia suspension (1 × 106 conidia/ml) using sterile distilled water (SDW) was inoculated into artificial wounds on six healthy young stems of Bawanghua using sterile fine-syringe needle. Meanwhile, 20 μl of SDW was inoculated on six healthy stems as a control. The inoculated stems were kept at 25°C, about 90% relative humidity. Three independent experiments were carried out. Reddish-brown lesions formed after 10 days, on 100% stems (18 in total) inoculated by C. gloeosporioides, while no lesion formed on any control. The pathogen was successfully re-isolated from the inoculated stem lesions on Bawanghua. Thus, Koch's postulates were fulfilled. Colletotrichum anthracnose has been reported on Pitaya in Japan (3), Malaysia (1) and in Brazil (4). To our knowledge, this is the first report of anthracnose disease caused by C. gloeosporioides on young stems of Bawanghua (H. undatus) in China. References: (1) M. Masyahit et al. Am. J. Appl. Sci. 6:902, 2009. (2) B. C. Sutton. Page 402 in: Colletotrichum Biology, Pathology and Control. J. A. Bailey and M. J. Jeger, eds. CAB International, Wallingford, UK, 1992. (3) S. Taba et al. Jpn. J. Phytopathol. 72:25, 2006. (4) L. M. Takahashi et al. Australas. Plant Dis. Notes 3:96, 2008.
At least nine Colletotrichum species, particularly Colletotrichum truncatum, have been recorded on legumes worldwide (1). In June 2010, samples of chickpea leaflets showing leaf spot disease symptoms were collected from experimental farms in Ladang Dua, Selangor state of Malaysia. Tan lesions with darker brown borders were observed on leaflets and were associated with premature leaf drop. Stem lesions initially appeared on the lower parts of stems and later progressed higher in the plant. Lesions often girdled the stem and caused severe dieback. Abundant acervuli developed in the lesions visible as black dots. Foliar lesions were removed, surface sterilized in 1% sodium hypochlorite for 2 min, rinsed twice with distilled water, dried on sterilized tissue paper, plated on PDA plates, and incubated at 25°C (3). Three isolates of the fungus were obtained and identified as C. truncatum on the basis of morphological characteristics (2). The isolates were deposited in the University Putra of Malaysia Culture Collection (UPMCC). Colony characteristics on PDA varied from greyish white to dark in color and exhibited mycelial growth with sparse acervuli. The isolates produced both sclerotia and setae in culture. Conidia (mean ± SD = 22 ± 0.83 × 3.6 ± 0.08 μm, L/W ratio = 6.1) produced in acervuli were falcate, hyaline, and aseptate, with tapering towards the acute and greatly curved apex. The conidial mass color varied from pale buff to saffron. Isolates produced simple to slightly lobed, mainly short clavate appressoria (mean ± SD = 9.60 ± 0.36 × 6.67 ± 0.29 μm, L/W ratio = 1.45). Amplification and sequence analysis of coding and none-coding regions of the ITS-rDNA (GenBank Accession JX971160), actin (JX975392), β-tubulin (KC109495), histone (KC109535), chitin synthase (KC109575), and glyceraldehyde-3-phosphate dehydrogenase (KC109615) obtained from the representative isolate, CTM37, aligned with deposited sequences from GenBank and revealed 99 to 100% sequence identity with C. truncatum strains (AJ301945, KC110827, GQ849442, GU228081, GU228359, and HM131501 from GenBank). Isolate CTM37 was used to test pathogenicity in the greenhouse. Five chickpea seeds of cultivar ILC-1929 were sown per pot in four replications. Ten days after seedling emergence, plants were inoculated with a spore suspension (concentration = 106 conidia ml-1) and check pots were sprayed with distilled water. After inoculation, the plants were covered with plastic bags for 48 h and kept at 28 to 33°C and >90% RH. After incubation, the plastic bags were removed and the plants were placed on greenhouse benches and monitored daily for symptom development (3). One week after inoculation, typical anthracnose symptoms developed on the leaves and stems of inoculated plants including acervuli formation, but not on the checks. A fungus with the same colony and conidial morphology as CTM37 was recovered from the lesions on the inoculated plants. The experiment was repeated twice. The ability to accurately diagnose Colletotrichum species is vital for the implementation of effective disease control and quarantine measures. We believe this is the first report of C. truncatum causing anthracnose on chickpea in Malaysia. References: (1) B. D. Gossen et al. Can. J. Plant Pathol. 31:65, 2009. (2) B. C. Sutton. The Genus Glomerella and its anamorph Colletotrichum. CAB International, Wallingford. UK. 1992. (3) 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.
In January 2011, branch samples were collected from langsat (Lansium domesticum Corr.), a fruit from Southeast Asia with an expanding niche market in Hawaii, exhibiting corky bark symptoms similar to that found on rambutan (Nephelium lappaceum) and litchi (Litchi chinensis) (3). The orchard, located along the Hamakua Coast of Hawaii Island, had 5- to 10-year-old trees, all with corky bark symptoms. As the trees matured, the cankers increased in size and covered the branches and racemes, often resulting in little to no fruit production. Scattered along the infected bark tissue were elongated, black ascomata present in the cracks. Ascomata were removed from the cracks using a scalpel blade, placed at the edge of a water agar petri dish and gently rolled along the agar surface to remove bark tissue and other debris. Individual ascomata were placed in 10-μl drops of 10% sodium hypochlorite on fresh water agar for 20 s, removed, and placed on potato dextrose agar petri dishes amended with 25 μg/ml streptomycin. The isolates were kept at 24°C under continuous fluorescent lighting. After 9 days, black pycnidia were present, which produced smooth, hyaline, linear to curved, filiform conidia, 4 to 6 septate (mostly 6), 31.8 to 70.1 × 2.0 to 2.8 μm. The morphological descriptions and measurements were similar to those reported for Dolabra nepheliae (3). The nucleotide sequence of the internal transcribed spacer (ITS) region including ITS1, 5.8S, and ITS2 intergenic spacers was determined for strain P11-1-1and a BLAST analysis of the sequence (GenBank Accession No. JX566449) revealed 99% similarity (586/587 bp) with the sequence of D. nepheliae strain BPI 882442 on N. lappaceum from Honduras. Based on morphology and ITS sequencing, the fungus associated with the cankers was identified as the same causal agent reported on rambutan and pulasan (N. mutabile) from Malaysia (1), and later reported on rambutan and litchi in Hawaii and Puerto Rico (3). Upon closer observations of the diseased samples, sections of corky bark contained at least two larval insects. The beetles were identified as Corticeus sp. (Coleoptera: Tenebrionidae) and Araecerus sp. (Coleoptera: Anthribidae) by the USDA-ARS Systematic Entomology Laboratory (Beltsville, MD). A corky bark disease on the trunk and larger limbs of mature langsat trees in Florida was thought to be caused by Cephalosporium sp. with larvae (Lepidoptera: Tineidae) feeding on the diseased tissue (2). It is not known the extent to which either of the beetle species is associated with L. domesticum in Hawaii or if they play a role in the bark disorder. To our knowledge, this is the first report of Dolabra nepheliae being found on langsat in Hawaii. Effective management practices should be established to avoid potential production losses or spreading the disease to alternative hosts. References: (1) C. Booth and W. P. Ting. Trans. Brit. Mycol. Soc. 47:235, 1964. (2) J. Morton. Langsat. In: Fruits of Warm Climates, p. 201-203. Julia F. Morton, Miami, FL, 1987. (3) A. Y. Rossman et al. Plant Dis. 91:1685, 2007.
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.
Plumeria spp., native to tropical America, are popular small trees grown widely in tropical areas of the world and as potted plants elsewhere. P. rubra and P. obtusa cultivars and hybrids are most common. A rust disease of a Plumeria sp. (likely P. rubra based on pointed leaf tips, leaves more than 18 cm (7 inches) long, and high rust susceptibility) was observed in November 2008 and again in June 2009 on homeowner plants in Baton Rouge, LA. A survey of five Baton Rouge retail nurseries in September 2009 revealed that 87% (90 of 103) of the plumeria plants were heavily infected with rust. Early symptoms included numerous 1-mm chlorotic spots on adaxial leaf surfaces followed by leaf chlorosis, necrosis, and abscission. Uredinia were numerous, mostly hypophyllous and yellowish orange. Urediniospores were catenulate, orange en masse, verrucose, globose, ovoid, ellipsoidal or angular, and measured 21.8 to 41.9 × 16.4 to 32.8 μm (average 29.4 × 22.6 μm). The rust was identified as Coleosporium plumeriae Pat. (= C. plumierae) (3). Teliospores were not found during this study. Pathogenicity tests were performed by spraying urediniospores (20,000/ml of deionized water) on three healthy Thai hybrid plumeria plants. Five leaves of each plant were misted with water and covered with plastic bags and three to five leaves were inoculated. Plants were held at 27°C for 27 h in a dew chamber and then moved outdoors. Typical rust symptoms and uredinia with urediniospores developed in 10 days on all inoculated leaves while noninoculated leaves remained healthy. Characteristics and spore measurements matched those of the rust from original infected plants. Additional plumeria rust inoculations were made to other Apocynaceae family members that included Allamanda cathartica, Catheranthus roseus (Madagascar periwinkle), Mandevilla splendens, Nerium oleander, and Vinca major. Catheranthus roseus was very susceptible to C. plumeriae with chlorotic leaf spots developing on the six inoculated plants after 8 days and uredinia with urediniospores appearing after 11 days. None of the other plant genera were susceptible to the rust. Plumeria rust was also observed on plumeria trees in urban landscapes in peninsular (Penang) and Bornean (Kota Kinabalu, Sabah) Malaysia in December 2007. To confirm identity, ~1,000 bp of nuclear rDNA 28S subunit from each (Lousiana, Penang, and Kota Kinabalu) was sequenced with rust-specific primers (1) and shared 100% identity (GenBank No. GU145555-6). Plumeria rust was first found on the island of Guadeloupe (3) and then spread to Central and South America. It has been known from Florida since 1960 under the synonym C. domingense (2), but has not been reported elsewhere in the continental United States. In more recent years, plumeria rust has spread to Hawaii, many Pacific islands, India, China, Taiwan, Thailand, Australia, and Nigeria (4). To our knowledge, this is the first report of plumeria rust from Louisiana and Malaysia and of susceptibility of another member of the Apocynaceae, Madagascar periwinkle, to C. plumeriae. Voucher material from Louisiana and Malaysia has been deposited in the Mycology Herbarium of Louisiana State University (LSUM). References: (1) M. C. Aime. Mycoscience 47:112, 2006. (2) Anonymous. Index of Plant Diseases in the United States. U.S. Dept. Agric. Handb. No. 165. Washington, D.C., 1960. (3) N. Patouillard. Bull. Soc. Mycol. Fr. 18:171, 1902. (4) C. To-Anun et al. Nat. Hist. J. Chulalongkorn Univ. 4:41, 2004.
Clausena lansium, also known as wampee (Clausena wampi), is a plant species native to China, Vietnam, the Philippines, Malaysia, and Indonesia, where it is widely cultivated, and also grown in India, Sri Lanka, Queensland, Florida, and Hawaii, but less frequently (3). The fruit can be consumed fresh or made into juice, jam, or succade. In summer to fall 2014, a soft rot disease was found in a wampee planting region in Yunan County, Guangdong Province, China. On Sept. 18, we collected diseased samples from a wampee orchard with about 20% disease incidence. The infected fruit initially showed pinpoint spots on the peel, water-soaked lesions, and light to dark brown discoloration. Spots expanded in 2 days, and tissues collapsed after 5 days. Severely affected fruit showed cracking or nonodorous decay. Five diseased samples were collected, and causal agents were isolated from symptomatic tissues 1 cm under the peel after surface sterilization in 0.3% NaOCl for 10 min and rinsing in sterile water three times. Tissues were placed on a Luria Bertani (LB) plate for culture. Ten representative isolates were selected for further characterization. No colony was isolated from healthy tissues. Colonies were round, smooth, with irregular edges, and produced a yellow pigment in culture. Biolog identification (Version 4.20.05) showed that all strains were gram negative, negative for indole production, and utilized glucose, maltose, trehalose, sucrose, D-lactose, and pectin but not sorbitol or gelatin. The isolates were identified as Pantoea agglomerans (SIM 0.69). Multilocus sequence analysis (MLSA) was conducted for rapid classification of the strains. Sequences of atpD, gyrB, infB, and rpoB were amplified using corresponding primers (2). All sequences of the 10 isolates were identical in each gene. BLASTn was performed, and maximum likelihood trees based on the concatenated nucleotide sequences of the four genes were constructed using MEGA6. Bootstrap values after 1,000 replicates were expressed as percentages. Results showed that the tested strain named CL1 was most homologous to P. anthophila, with 98% identity for atpD (KM521543), 100% for gyrB (KM521544), infB (KM521545), and rpoB (KM521546). The 16S rRNA sequence (KM521542) amplified by primers 27f and 1492r shared 99% identity with that of P. anthophila M19_2C (JN644500). P. anthophila was previously reclassified from P. agglomerans (3); therefore, we suggest naming this wampee pathogen P. anthophila. Subsequently, 10 wampee fruits were injected with 20 μl of bacterial suspension (1 × 108 CFU/ml) of strains CL1 and CL2, respectively, and another 10 were injected with 20 μl of LB medium as controls, all kept at 28°C for 4 days. Symptoms similar to those of natural infections were observed on inoculated fruits but not on the negative controls. Bacteria were isolated from diseased tissues and further identified as P. anthophila by gyrB sequencing. P. anthophila was reported to naturally infect balsam and marigold (1,2). To our knowledge, this is the first report of P. anthophila naturally causing soft rot disease and cracking on C. lansium (wampee). References: (1) C. Brady et al. Syst. Appl. Microbiol. 31:447, 2008. (2) C. Brady et al. Int. J. Syst. Evol. Microbiol. 59:2339, 2009. (3) J. Morton. Fruits of Warm Climates. Echo Point Books & Media, Miami, FL, 1987.
Bok choy (Brassica chinensis L.) is a temperate vegetable grown in the cool highland areas of Malaysia. In June 2010, vegetable growing areas of the Cameron Highlands, located in Pahang State, Malaysia, were surveyed for the prevalence of anthracnose disease caused by Colletotrichum species. Diseased samples were randomly collected from 12 infested fields. Anthracnose incidence on bok choy varied from 8 to 36% in different nursery fields. Disease symptoms initially appeared as small water-soaked spots scattered on the leaf petioles of young plants. As these spots increased in size, they developed irregular round spots that turned to sunken grayish brown lesions surrounded by brownish borders. When the lesions were numerous, leaves collapsed. Pale buff to salmon conidial mass and acervuli were observed on well-developed lesions. The acervuli diameter varied in size from 198 to 486 μm, averaging 278.5 μm. Morphological and cultural characteristics of the fungus were examined on potato dextrose agar incubated for 7 days at 25 ± 2°C under constant fluorescent light. Vegetative mycelia were hyaline, septate, branched, and 2 to 7 μm in diameter. The color of the fungal colonies was grayish brown. Conidia were hyaline, aseptate, falcate, apices acute, and 21.8 to 28.5 × 2.6 to 3.4 mm. Setae were pale brown to dark brown, 75 to 155 μm long, base cylindrical, and tapering towards the acute tip. Appressoria were solitary or in dense groups, light to dark brown, entire edge to lobed, roundish to clavate, 6.5 to 14 × 5.8 to 8.6 μm, averaging 9.2 × 6.8 μm, and had a L/W ratio of 1.35. Based on the keys outlined by Mordue 1971 (2) and Sutton 1980 (3), the characteristics of this fungus corresponded to Colletotrichum capsici. Sequence analysis of the ITS-rDNA obtained from the Malaysian strain CCM3 (GenBank Accession No. JQ685746) using primers ITS5 and ITS4 (1) when aligned with deposited sequences from GenBank revealed 99 to 100% sequence identity with C. capsici strains (DQ286158, JQ685754, DQ286156, GQ936210, and GQ369594). A representative strain CCM3 was used for pathogenicity testing. Four non-infected detached leaves of 2-week-old B. chinensis were surface-sterilized and inoculated by placing 10 μl of conidial suspension (106 conidia ml-1) using either the wound/drop or non-wound/drop method, and distilled water was used as a control (1). Leaves were incubated at 25°C, 98% RH. The experiment was repeated twice. Five days after inoculation, typical anthracnose symptoms with acervuli formation appeared on the surface of tissues inoculated with the spore suspension, but not on the water controls. A fungus with the characteristics of C. capsici was recovered from the lesions on the inoculated leaves. Anthracnose caused by C. capsici has been reported on different vegetable crops, but not on bok choy (3). To the best of our knowledge, this is the first report of C. capsici causing anthracnose on bok choy in Malaysia. References: (1) R. Ford et al. Aust. Plant Pathol. 33:559, 2004. (2) J. E. M. Mordue. CMI Description of Pathogenic Fungi and Bacteria. Commonwealth Mycol. Inst., Kew, UK. 1971. (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.
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.
Symptoms of water-soaked lesions and soft rot were first observed in June 2011 on bell pepper fruits (Capsicum annuum cv. Annuum) in the two main regions of pepper production in Malaysia (Cameron Highlands and Johor State). Economic losses exceeded 40% in severely infected fields and greenhouses with the estimated disease incidence of 70%. In pepper fruits damaged by insects, sunscald, or other factors, symptoms initially appeared in the peduncle and calyx tissues and entire fruits were turned into watery masses within 2 to 6 days. Fruits infected in the field tended to collapse and hang on the plant. When the contents leaked out, the outer skin of the fruit dried and remained attached to the plant. Field-grown transplants and infected soil were identified as probable sources of inocula. A total of 50 attached fruits were collected from 10 pepper fields and greenhouses located in the two growing regions. Tissue from the margins of water-soaked lesions was surface-sterilized in 1% NaOCl for 2 min, rinsed in sterile water, dried, and plated onto nutrient agar (NA) and eosin methylene blue agar (EMB) media (3). A similar bacterium was isolated from all samples. After 2 days, white to creamy bacterial colonies on NA and emerald green colonies on EMB developed. Five independent strains were subjected to further biochemical, molecular, and pathogenicity tests. Bacterial strains were gram-negative, motile rods, grew at 37°C, were facultatively anaerobic, oxidase-negative, phosphatase-negative, and catalase-positive. They degraded pectate, were sensitive to erythromycin, did not utilize Keto-methyl glucoside, were indole production-negative, and reduced sugars from sucrose (3). Acid production was negative from sorbitol and arabitol, but positive from melibiose and citrate. PCR amplification of the pel gene by Y1 and Y2 primers produced a 434-bp fragment (2). Amplification of the intergenic transcribed spacer (ITS) region by G1 and L1 primers (4) gave two amplicons ca. 550 and 580 bp long. The expected amplicon was not produced with any of the strains using primers Br1f/L1r and Eca1f/Eca2r (1), whereas a 550-bp PCR product, typical of Pectobacterium carotovorum subsp. carotovorum, was obtained with primers EXPCCF and EXPCCR (1). Based on biochemical and molecular characteristics, and analysis of PCR-RFLP of 16S-ITS-23R rRNA genes using Rsa I enzyme (4), all five bacterial strains were identified as P. carotovorum subsp. carotovorum. BLAST analysis of the 16S rRNA sequence (GenBank Accession No KC189032) showed 100% identity to the 16S rRNA of P. carotovorum subsp. carotovorum strain PPC192. For pathogenicity tests, four mature pepper fruits of cv. Annuum were inoculated by injecting 10 μl of a bacterial suspension (108 CFU/ml) into pericarps and the fruits were incubated in a moist chamber at 80 to 90% relative humidity and 30°C. After 72 h, water-soaked lesions similar to those observed in the fields and greenhouses were observed and bacteria with the same characteristics were consistently reisolated, thereby fulfilling Koch's postulates. Symptoms were not observed on water-inoculated controls. References: (1) S. Baghaee-Ravari et al. Eur. J. Plant Pathol. 129:413, 2001. (2) A. Darraas et al. Appl. Environ. Microbiol. 60:1437, 1994. (3) N. W Schaad et al. Laboratory Guide for the Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society Press, St Paul, MN, 2001. (4) I. K. Toth et al. Appl. Environ. Microbiol. 67:4070, 2001.