This study was conducted to identify the causal organism of bark dieback disease of highbush blueberry (Vaccinium corymbosum L.) observed in Korea. Blueberry, a woody plant that is native to North America, belongs to the family Ericaceae and genus Vaccinium. Of the 400 species of blueberry in the world, most are distributed in the tropics of Malaysia and Southeast Asia. Highbush blueberry is abundantly grown in Canada and the United States and has become a popular commercial crop in Korea for products such as jam, wine, and sauce. Bark dieback disease of blueberry was found in Sunchang (<5% incidence), Jeollabuk-do, Korea in July 2009. Typical symptoms of the disease were blight and dieback on the stems with lesions extending along entire branches. Morphological examination revealed that the perithecia were of the globose type with a nipple, 155 to 490 (374.6) μm, and brown on the dead bark. Asci were bitunicate and clavate or cylindrical with dimensions of 63 to 125 × 16 to 20 μm and containing eight ascospores. Ascospores were of the long ovoid type with dimensions of 13.2 to 23.7 (17.98) × 25.4 to 41.1 (33.21) μm. From extracted genomic DNA, the internal transcribed spacer (ITS)-5.8S ribosomal DNA region was amplified with universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). A BLAST search of GenBank with the ITS sequence revealed that the Sunchang isolate (GenBank Accession No. HQ384217) had 99 to 100% sequence identity with the following Botryosphaeria dothidea accessions: FJ517657, AJ938005, FJ478129, FJ171723, and AJ938004. Phylogenetic analysis with the Sunchang isolate, B. dothidea strains, and related species revealed that the B. dothidea isolate and strains comprised a monophyletic group distinguished from other Botryosphaeria spp. including B. ribis, B. parva, B. protearum, B. lutea, B. australis, B. rhodina, B. obtuse, and B. stevensii (2). On the basis of morphological and molecular results, the isolate was identified as B. dothidea (Moug.) Ces. & De Not. A culture of B. dothidea isolate was grown on potato dextrose agar (PDA) for 10 days. A 5-mm plug was inoculated into stem wounds created with a No. 2 cork borer in 20 2-year-old disease-free blueberry plants grown in a greenhouse. Six plants inoculated with only PDA plugs served as noninoculated controls. The wounds were covered with Parafilm. After 3 months, the Parafilm was removed and black lesions were observed at the fungal inoculation sites, while no lesion was observed on the control plants. To complete Koch's postulates, the fungus was reisolated from the lesions and confirmed to be B. Dothidea (1). There is an urgent need to determine the spread of this disease in Korea, estimate the losses, and develop methods for reducing damage through biological and eco-friendly cultural control methods. References: (1) D. Jurc et al. Plant Pathol. 55:299, 2006. (2) B. Slippers et al. Mycologia 96:83, 2004.
Cabbage (Brassica oleracea L. var. capitata L.) is one of the most important vegetables cultivated in Pahang and Kelantan, Malaysia. Pectobacterium carotovorum can cause soft rot on a wide range of crops worldwide, especially in countries with warm and humid climates such as Malaysia. Cabbage with symptoms of soft rot from commercial fields were sampled and brought to the laboratory during the winter of 2010. Disease symptoms were a gray to pale brown discoloration and expanding water-soaked lesions on leaves. Several cabbage fields producing white cultivars were investigated and 27 samples were collected. Small pieces of leaf samples were immersed in 5 ml of saline solution (0.80% NaCl) for 20 min to disperse the bacterial cells. Fifty microliters of the resulting suspension was spread on nutrient agar (NA) and King's B medium and incubated at 30°C for 48 h. Purification of cultures was repeated twice on these media. Biochemical and phenotypical tests gave these results: gram negative, rod shaped, ability to grow under liquid paraffin (facultative anaerobe); oxidase negative; phosphatase negative; positive degradation of pectate; sensitive to erythromycin; negative to Keto-methyl glucoside utilization, indole production and reduction sugars from sucrose were negative; acid production from sorbitol and arabitol was negative and from melibiose, citrate, and raffinose was positive. Hypersensitivity reaction on tobacco leaf with the injection of 106 CFU/ml of bacterial suspension for all strains was positive. Four representative strains were able to cause soft rot using cabbage slices (three replications) inoculated with a bacterial suspension at 106 CFU/ml. Inoculated cabbage slices were incubated in a moist chamber at 80% relative humidity and disease symptoms occurred after 24 h. Cabbage slices inoculated with water as a control remained healthy. The bacteria reisolated from rotted cabbage slices on NA had P. carotovorum cultural characteristics and could cause soft rot in subsequent tests. PCR amplification with Y1 and Y2 primers (1), which are specific for P. carotovorum, produced a 434-bp band with 15 strains. PCR amplification of the 16S-23S rRNA intergenic transcribed spacer region (ITS) using G1 and L1 primers gave two main bands approximately 535 and 580 bp and one faint band approximately 740 bp when electrophoresed through a 1.5% agarose gel. The ITS-PCR products were digested with RsaI restriction enzyme. According to biochemical and physiological characterictics (2), PCR-based pel gene (1), and analysis by ITS-PCR and ITS-restriction fragment length polymorphism (3), all isolates were identified as P. carotovorum subsp. carotovorum. This pathogen has been reported from Thailand, Indonesia, and Singapore with whom Malaysia shares its boundaries. To our knowledge, this is the first report of P. carotovorum subsp. carotovorum in cabbage from Malaysia. References: (1) A. Darraas et al. Appl. Environ. Microbiol. 60:1437, 1994. (2) N. W. Schaad et al. Laboratory Guide for the Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society, St. Paul, 2001. (3) I. K. Toth et al. Appl. Environ. Microbiol. 67:4070, 2001.
Cosmos caudatus Kunth. (Asteraceae), commonly known as ulam raja, is widely grown as an herbal aromatic shrub. In Malaysia, its young leaves are popularly eaten raw as salad with other greens and have been reported to possess extremely high antioxidant properties, which may be partly responsible for some of its believed medicinal functions. In early 2010, a suspected powdery mildew was observed on ulam raja plants at the Agricultural Park of Universiti Putra Malaysia. Initially, individual, white, superficial colonies were small and almost circular. Later, they enlarged and coalesced to cover the whole abaxial leaf surface. With development of the disease, all green parts (leaves, stems, and petioles) became covered with a continuous mat of mildew, giving a dusty appearance. Newly emerged leaves rapidly became infected. Diseased leaves ultimately senesced and dried up, making them aesthetically unattractive and unmarketable. The pathogen produced conidia in short chains (four to six conidia) on erect conidiophores. Conidiophores were unbranched, cylindrical, 125 to 240 μm long, with a slightly swollen foot cell. Individual conidia were hyaline, ellipsoid, and 25 to 30 (27.5) × 15 to 20 (17.5) μm with fibrosin inclusions. Morphological descriptions were consistent with those described for Sphaerotheca fuliginea or S. fusca, which has lately been reclassified as Podosphaera fusca (1). From extracted genomic DNA of P. fusca UPM UR1, the internal transcribed spacer (ITS) region was amplified with ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). A BLAST search of GenBank with an ITS rDNA sequence of this fungus (GenBank Accession No. HQ589357) showed a maximum identity of 98% to the sequences of two P. fusca isolates (GenBank Accession Nos. AB525915.1 and AB525914.1). To satisfy Koch's postulates, the pathogenicity of fungal strain UPM UR1 was verified on 4-week-old plants. Inoculation was carried out by gently rubbing infected leaves onto healthy plants of C. caudatus. Ten pots of inoculated plants were kept under a plastic humid chamber and 10 pots of noninoculated plants, placed under another chamber, served as controls. After 48 h, the plants were then placed under natural conditions (25 to 28°C). Powdery mildew symptoms, similar to those on diseased field plants, appeared after 7 days on all inoculated plants. The white, superficial colonies enlarged and merged to cover large areas within 2 weeks. The infected leaf tissues became necrotic 6 to 8 days after the appearance of the first symptoms. Sporulation of P. fusca was observed on all infected leaves and stems. No symptoms were seen on the control plants. To our knowledge, this is the first report of P. fusca causing powdery mildew on C. caudatus in Malaysia. This pathogen has also been reported previously to be economically important on a number of other hosts. With ulam raja plants, more attention should be given to prevention and control measures to help manage this disease. Reference: (1) U. Braun and S. Takamatsu. Schlechtendalia 4:1, 2000.
Madagascar periwinkle, Catharanthus roseus (L.) G. Don, is a member of the Apocynaceae plant family that is native to Madagascar and produces dimeric terpenoid indole alkaloids that are used in the treatment of hypertension and cancer. Periwinkle as an indicator plant is highly susceptible to phytoplasmas and spiroplasma infection from different crops, and has been found to be naturally infected with spiroplasmas in Arizona, California, and the Mediterranean countries. In this study, surveys of suspected diseased periwinkles were conducted in various regions of Selangor State, Malaysia. Periwinkles showing rapid decline in the number and size of the flowers, premature abscission of buds and flowers, reduction in leaf size, chlorosis of the leaf tips and margins, general chlorosis, and stunting and dying plants were collected. These symptoms were widespread on periwinkle in this state. Diagnosis of the disease was based on symptomatology, grafting, serology (ELISA), PCR techniques, and cultivation. Tests for transmission by grafting were conducted using symptomatic periwinkle plants. Symptoms were induced on all eight graft-inoculated healthy periwinkles approximately 2 weeks after side grafting. Preliminary examination was performed by ELISA with Spiroplasma citri Saglio polyclonal antibody that was prepared against an Iranian S. citri isolate (H. Rahimian, unpublished data). Leaf extracts of all 24 symptomatic periwinkles gave positive ELISA reactions at OD405 readings ranging from 0.310 to 0.654 to the antibody against S. citri by the indirect ELISA method. Six healthy periwinkle leaves gave OD405 readings around 0.128. Total nucleic acids were extracted from 10 symptomatic and 5 asymptomatic plants (4). PCR using the ScR16F1/ScR16R1 primer pair designed to detect S. citri in carrot and P1/P7 and secA for1/rev3 primer pairs designed for identification of phytoplasmas were used to detect the causal agent (1-3). Amplification failed when the P1/P7 universal phytoplasma primer pair was used for diseased samples. However, the PCR assays resulted in products of 1,833 and 800 bp with ScR16F1/ScR16R1 and secA for1/rev3, respectively. Five of each ScR16F1/ScR16R1 and SecAfor1/SecArev3 products were cloned with the Topo TA cloning kit (Invitrogen, Carlsbad, CA), sequenced, and deposited as GenBank Accession Nos. HM015669 and FJ011099, respectively. Sequences for both genes indicated that S. citri was associated with the disease on periwinkle. ScR16F1/ScR16R1 products cloned from symptomatic periwinkles had 98% sequence identity with S. citri (GenBank Accession No. AM285316), while nucleotide sequences of SecAfor1/SecArev3 products had 88% sequence identity with S. citri GII3-3X (GenBank Accession No. AM285304). S. citri was cultivated from 10 S. citri-infected periwinkles using filtration and SP-4 media. Twenty culture tubes started to change culture medium color from red to yellow 1 month after cultivation. Helical and motile S. citri was observed in the dark-field microscope. To our knowledge, this is the first report on the presence and occurrence of S. citri in Southeast Asia and its association with lethal yellows on periwinkle in Malaysia. References: (1) J. Hodgetts et al. Int. J. Syst. Evol. Microbiol. 58:1826, 2008. (2) I.-M. Lee et al. Phytopathology 85:728, 1995. (3) I.-M. Lee et al. Plant Dis. 90:989, 2006. (4) Y.-P. Zhang et al. J. Virol. Methods. 71:45, 1998.
Cucumber (Cucumis sativus L.) is one of the most important vegetable fruits in Malaysia. Cucumber is principally grown in the states of Johor, Kelantan, and Perak. The broad host range Enterobacteriaceae pathogen, Pectobacterium carotovorum, can cause soft rot on stems or cucumber fruit. In Malaysia, cucumber is produced in a warm, humid climate, thus the plant is susceptible to attack by P. carotovorum at any time during production. In 2010, cucumber samples with wilted and chlorotic leaves, water-soaked lesions, and collapsed fruits were found in multiple fields. Small pieces of infected stems and fruit were immersed in 5 ml of saline solution (0.85% NaCl) for 20 min and then 50 μl of this suspension was spread onto nutrient agar (NA) and incubated at 27°C for 24 h. White-to-pale gray colonies with irregular margins were selected for analysis. For pathogenicity tests, cucumber fruits were surface sterilized by ethyl alcohol 70%, washed with sterilized distilled water, cut into small pieces, and inoculated with 20 μl of 108 CFU/ml suspensions of five representative strains. Cucumber plants were grown for 3 weeks in sterilized soil and their stems were inoculated with 20 μl of 108 CFU/ml of bacterial suspension. Inoculated samples and control (noninoculated) plants were placed in a growth chamber with 80 to 90% relative humidity at 27°C. Symptoms occurred on fruit slices and stems after 1 to 3 days and appeared the same as naturally infected samples, but the control samples remained healthy. Koch's postulates were fulfilled with the reisolation of cultures with the same characteristics as described earlier. Hypersensitivity reaction (HR) assays were done by infiltrating 108 CFU/ml of bacterial suspension into tobacco leaf epidermis and HR developed. All strains were subjected to biochemical and morphological assays, as well as molecular assessment. The strains were gram negative, facultative anaerobes, rod shaped, able to macerate potato slices and growth at 37°C; catalase positive; oxidase and phosphatase negative; able to degrade pectate; sensitive to erythromycin; negative for utilization of α-methyl glycoside, indole production, and reduction of sugars from sucrose; acid production from arabitol, sorbitol, and utilization of citrate were negative, but positive for raffinose and melibiose utilization. PCR amplification of the pel gene by Y1 and Y2 primers produced a 434-bp fragment on agarose gel 1% (1). Amplification of intergenic transcribed spacer region by G1 and L1 primers gave two main bands at approximately 535 and 580 bp on agarose gel 1.5%. The ITS-PCR products were digested with RsaI restriction enzyme (3). On the basis of biochemical and morphological characteristics, PCR-based pel gene and characterization of the ITS region, and digestion of the ITS-PCR products with RsaI restriction enzyme, all isolates were identified as P. carotovorum subsp. carotovorum. To our knowledge, this is the first report of soft rot caused by P. carotovorum subsp. carotovorum on cucumber from Malaysia. References: (1) A. Darraas et al. Appl. Environ. Microbiol. 60:1437, 1994. (2) N. W Schaad et al. Laboratory Guide for the Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society Press, St. Paul, 2001. (3) I. K. Toth et al. Appl. Environ. Microbiol. 67:4070, 2001.
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.
Indian spinach (Basella rubra L.) is a red stem species of Basella that is cultivated worldwide as an ornamental and the aerial parts are also consumed as a vegetable. In May of 2011, symptoms of damping-off were observed on approximately 10% of the plants at the stem base around the soil line of seedlings in a greenhouse in Homestead, FL. Lesions were initially water soaked, grayish to dark brown, irregular in shape, and sunken in appearance on large plants, causing the infected seedlings to collapse and eventually die. Symptomatic stem tissue was surface sterilized with 0.6% sodium hypochlorite, rinsed in sterile distilled water, air dried, and plated on potato dextrose agar (PDA). Plates were incubated at 25°C in darkness for 3 to 5 days. A fungus was isolated in all six isolations from symptomatic tissues on PDA. Fungal colonies on PDA were light gray to brown with abundant growth of mycelia, and the hyphae tended to branch at right angles when examined under a microscope. A septum was always present in the branch of hyphae near the originating point and a slight constriction at the branch was observed. Neither conidia nor conidiophores were found from the cultures on PDA. The characteristics of hyphae, especially the right angle branching of mycelia, indicate close similarity to those of Rhizoctonia solani (2,3). The internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1/ITS4 and sequenced (GenBank Accession No. JN545836). Subsequent database searches by the BLASTN program indicated that the resulting sequence had a 100% identity over 472 bp with the corresponding gene sequence of R. solani anastomosis group (AG) 4 (GenBank Accession No. JF701752.1), a fungal pathogen reported to cause damping-off on many crops. Pathogenicity was confirmed through inoculation of healthy India spinach plants with the hyphae of isolates. Four 4-week-old plants were inoculated with the isolates by placing a 5-mm PDA plug of mycelia at the stem base and covering with a thin layer of the soil. Another four plants treated with sterile PDA served as a control. After inoculation, the plants were covered with plastic bags for 24 h and maintained in a greenhouse with ambient conditions. Four days after inoculation, water-soaked, brown lesions, identical to the symptoms described above, were observed on the stem base of all inoculated plants, whereas no symptoms developed on the control plants. The fungus was isolated from affected stem samples, and the identity was confirmed by microscopic appearance of the hyphae and sequencing the ITS1/ITS4 intergenic spacer region, fulfilling Koch's postulates. This pathogenicity test was conducted twice. R. solani has been reported to cause damping-off of B. rubra in Ghana (1) and Malaysia (4). To our knowledge, this is the first report of damping-off caused by R. solani AG-4 on Indian spinach in Florida and the United States. With the increased interest in producing Asian vegetables for food and ornamental purposes, the occurrence of damping-off on Indian spinach needs to be taken into account when designing programs for disease management in Florida. References: (1) H. A. Dade. XXIX. Bull. Misc. Inform. 6:205, 1940. (2) J. R. Parmeter et al. Phytopathology 57:218, 1967. (3) B. Sneh et al. Identification of Rhizoctonia species. The American Phytopathological Society, St Paul, MN, 1991. (4) T. H. Williams and P. S. W. Liu. Phytopathol. Pap. 19:1, 1976.
In the last decade, rambutan (Nephelium lappaceum L., Sapindaceae) and pulasan (N. mutabile Blume) have been cultivated in Honduras to produce exotic fruits for export to North America (2). Recently, a disease was observed that produces dark brown to black fissured cankers from 1 to 3 cm long and 1 to 4 cm wide. The infected bark tissue becomes swollen with the middle region 3 to 8 mm thick. Symptoms appear when the trees are approximately 3 years old. As the trees mature, the cankers increase in size and weaken the branches, often resulting in breakage with the weight of the fruit causing substantial plant damage and fruit loss. In August 2010, fissured branch samples of rambutan and pulasan were collected from 6- to 8-year-old trees from the Humid Tropical Demonstrative Agroforestry Center in Honduras, Atlantida, La Masica (15°33'47.4″N, 87°05'2.5″W, elevation 106 m). A fungus associated with the cankers was identified as Dolabra nepheliae. It produces black, stipitate, elongate ascomata, 312 to 482 × 250 to 281 μm with broadly cylindric, bitunicate asci, 120 to 138 × 11.2 to 15.0 μm, and filiform, hyaline ascospores, 128 to 135 × 2.8 to 3.2 μm. Fungi from rambutan and pulasan were isolated on cornmeal agar plus 0.5% dextrose and antibiotics. On potato dextrose agar, the ascospores produced slow-growing colonies, 5 mm per week. In culture, isolates from both hosts produced pycnidia with elongated, slightly to strongly curved or S-shaped, hyaline conidia, 22.8 to 46.4 × 2.8 to 3.7 μm. This fungus was first reported on rambutan and pulasan from Malaysia (1,4), and later reported on rambutan and litchi in Hawaii and Puerto Rico (3). To our knowledge, this is the first report of D. nepheliae on pulasan and rambutan from Honduras. Specimens have been deposited at the U.S. National Fungus Collections (BPI 882442 on N. lappaceum and BPI 882443 on N. mutabile). Cultures were deposited at the Centraalbureau voor Schimmelcultures (CBS) as CBS 131490 on N. lappaceum and CBS 131491 on N. mutabile. Sequences of the internal transcribed spacer (ITS) region including ITS1, 5.8S, and ITS2 intergenic spacers were deposited in GenBank (Accession No. JQ004281 on N. lappaceum and Accession No. JQ004280 on N. mutabile). A BLAST search and pairwise comparison using the GenBank web server were used to compare ITS sequence data and recovered the following results: (i) CBS 131490 on N. lappaceum is 99% (538 of 544) identical to D. nepheliae CBS 123297 on Litchi chinensis from Puerto Rico; and (ii) CBS 131491 on N. mutabile is 99% (527 of 533) identical to the same strain of D. nepheliae. On the basis of the ITS sequence data, the isolates from Honduras were confirmed as the same species, D. nepheliae from Puerto Rico. Efforts to develop resistant germplasm and management strategies to control this disease have been initiated. References: (1) C. Booth and W. P. Ting. Trans. Brit. Mycol. Soc. 47:235, 1964. (2) T. Ramírez et al. Manual Para el Cultivo de Rambutan en Honduras. Fundación Hondureña de Investigación Agrícola. La Lima, Cortes, Honduras, 2003. (3) A. Y. Rossman et al. Plant Dis. 91:1685, 2007. (4) H. Zalasky et al. Can. J. Bot. 49:559, 1971.
Phalaenopsis orchids, originally from tropical Asia, are mainly planted in Thailand, Singapore, Malaysia, the Philippines, and Taiwan and have gained popularity from consumers all over the world. The cultivation area of Phalaenopsis orchids has been rising and large-scale bases have been established in mainland China, especially South China because of suitable environmental conditions. In September 2011, a soft rot of Phalaenopsis aphrodita was found in a Phalaenopsis planting base in Guangzhou with an incidence of ~15%. Infected plants initially showed water-soaked, pale-to-dark brown pinpoint spots on leaves that were sometimes surrounded by a yellow halo. Spots expanded rapidly with rising humidity and temperatures, and in a few days, severely extended over the blade with a light tan color and darker brown border. Lesions decayed with odorous fumes and tissues collapsed with inclusions exuding. The bacterium advanced to the stem and pedicle. Finally, leaves became papery dry and the pedicles lodged. Six diseased samples were collected, and bacteria were isolated from the edge of symptomatic tissues after sterilization in 0.3% NaOCl for 10 min, rinsing in sterile water three times, and placing on nutrient agar for culture. Twelve representative isolates were selected for further characterization. All strains were gram negative, grew at 37°C, were positive for indole production, and utilized malonate, glucose, and sucrose but not glucopyranoside, trehalose, or palatinose. Biolog identification (version 4.20.05, Hayward, CA) was performed and Pectobacterium chrysanthemi (SIM 0.868) was confirmed for the tested isolates (transfer to genus Dickeya). PCR was used to amplify the 16S rDNAgene with primers 27f and 1492r, dnaX gene with primers dnaXf and dnaXr (3), and gyrB gene with primers gyrBf (5'-GAAGGYAAAVTKCATCGTCAGG-3') and gyrB-r1 (5'-TCARATATCRATATTCGCYGCTTTC-3') designed on the basis of the published gyrB gene sequences of genus Dickeya. BLASTn was performed online, and phylogeny trees (100% bootstrap values) were created by means of MEGA 5.05 for these gene sequences, respectively. Results commonly showed that the representative tested strain, PA1, was most homologous to Dickeya dieffenbachiae with 98% identity for 16S rDNA(JN940859), 97% for dnaX (JN989971), and 96% for gyrB (JN971031). Thus, we recommend calling this isolate D. dieffenbachiae PA1. Pathogenicity tests were conducted by injecting 10 P. aphrodita seedlings with 100 μl of the bacterial suspension (1 × 108 CFU/ml) and another 10 were injected with 100 μl of sterile water as controls. Plants were inoculated in a greenhouse at 28 to 32°C and 90% relative humidity. Soft rot symptoms were observed after 2 days on the inoculated plants, but not on the control ones. The bacterium was isolated from the lesions and demonstrated identity to the inoculated plant by the 16S rDNA sequence comparison. Previously, similar diseases of P. amabilis were reported in Tangshan, Jiangsu, Zhejiang, and Wuhan and causal agents were identified as Erwinia spp. (2), Pseudomonas grimontii (1), E. chrysanthemi, and E. carotovora subsp. carovora (4). To our knowledge, this is the first report of D. dieffenbachiae causing soft rot disease on P. aphrodita in China. References: (1) X. L. Chu and B. Yang. Acta Phytopathol. Sin. 40:90, 2010. (2) Y. M. Li et al. J. Beijing Agric. Coll. 19:41, 2004. (3) M. Sławiak et al. Eur. J. Plant Pathol. 125:245, 2009. (4) Z. Y. Wu et al. J. Zhejiang For. Coll. 27:635, 2010.
Physic nut (Jatropha curcas L.) is an important biofuel crop worldwide. Although it has been reported to be resistant to pests and diseases (1), stem cankers have been observed on this plant at several locations in Peninsular Malaysia since early February 2008. Necrotic lesions on branches appear as scars with vascular discoloration in the tissue below the lesion. The affected area is brownish and sunken in appearance. Disease incidence of these symptomatic nonwoody plants can reach up to 80% in a plantation. Forty-eight samples of symptomatic branches collected from six locations (University Farm, Setiu, Gemenceh, Pulau Carey, Port Dickson, and Kuala Selangor) were surface sterilized in 10% bleach, rinsed twice with sterile distilled water, air dried on filter paper, and plated on water agar. After 4 days, fungal colonies on the agar were transferred to potato dextrose agar (PDA) and incubated at 25°C. Twenty-seven single-spore fungal cultures obtained from all locations produced white, aerial mycelium that became dull gray after a week in culture. Pycnidia from 30-day-old pure cultures produced dark brown, oval conidia that were two celled, thin walled, and oval shape with longitudinal striations. The average size of the conidia was 23.63 × 12.72 μm with a length/width ratio of 1.86. On the basis of conidial morphology, these cultures were identified as Lasiodiplodia theobromae. To confirm the identity of the isolates, the internal transcribed spacer (ITS) region was amplified with ITS1/ITS4 primers and sequenced. The sequences were deposited in GenBank (Accession Nos. HM466951, HM466953, HM466957, GU228527, HM466959, and GU219983). Sequences from the 27 isolates were 99 to 100% identical to two L. theobromae accessions in GenBank (Nos. HM008598 and HM999905). Hence, both morphological and molecular characteristics confirmed the isolates as L. theobromae. Pathogenicity tests were performed in the glasshouse with 2-month-old J. curcas seedlings. Each plant was wound inoculated by removing the bark on a branch to a depth of 2 mm with a 10-mm cork borer. Inoculation was conducted by inserting a 10-mm-diameter PDA plug of mycelium into the wound and wrapping the inoculation site with wetted, cotton wool and Parafilm. Control plants were treated with plugs of sterile PDA. Each isolate had four replicates and two controls. After 6 days of incubation, all inoculated plants produced sunken, necrotic lesions with vascular discoloration. Leaves were wilted and yellow above the point of inoculation on branches. The control plants remained symptomless. The pathogen was successfully reisolated from lesions on inoculated branches. L. theobromae has been reported to cause cankers and dieback in a wide range of hosts and is common in tropical and subtropical regions of the world (2,3). To our knowledge, this is the first report of stem canker associated with L. theobromae on J. curcas in Malaysia. References: (1) S. Chitra and S. K. Dhyani. Curr. Sci. 91:162, 2006. (2) S. Mohali et al. For. Pathol. 35:385, 2005. (3) E. Punithalingam. Page 519 in: CMI Descriptions of Pathogenic Fungi and Bacteria. Commonwealth Mycological Institute, Kew, Surrey, UK. 1976.
In June 2011, tomatoes (Solanum lycopersicum) in major growing areas of the Cameron Highlands and the Johor state in Malaysia were affected by a leaf spot disease. Disease incidence exceeded 80% in some severely infected regions. Symptoms on 50 observed plants initially appeared on leaves as small, brownish black specks, which later became grayish brown, angular lesions surrounded by a yellow border. As the lesions matured, the affected leaves dried up and became brittle and later developed cracks in the center of the lesions. A survey was performed in these growing areas and 27 isolates of the pathogen were isolated from the tomato leaves on potato carrot agar (PCA). The isolates were purified by the single spore technique and were transferred onto PCA and V8 agar media for conidiophore and conidia production under alternating light (8 hours per day) and darkness (16 hours per day) (4). Colonies on PCA and V8 agar exhibited grey mycelium and numerous conidia were formed at the terminal end of conidiophores. The conidiophores were up to 240 μm long. Conidia were oblong with 2 to 11 transverse and 1 to 6 longitudinal septa and were 24 to 69.6 μm long × 9.6 to 14.4 μm wide. The pathogen was identified as Stemphylium solani on the basis of morphological criteria (2). In addition, DNA was extracted and the internal transcribed spacer region (ITS) was amplified by universal primers ITS5 and ITS4 (1). The PCR product was purified by the commercial PCR purification kit and the purified PCR product sequenced. The resulting sequences were 100% identical to published S. solani sequences (GenBank Accestion Nos. AF203451 and HQ840713). The amplified ITS region was deposited with NCBI GenBank under Accession No. JQ657726. A representative isolate of the pathogen was inoculated on detached 45-day-old tomato leaves of Malaysian cultivar 152177-A for pathogenicity testing. One wounded and two nonwounded leaflets per leaf were used in this experiment. The leaves were wounded by applying pressure to leaf blades with the serrated edge of a forceps. A 20-μl drop of conidial suspension containing 105 conidia/ml was used to inoculate these leaves (3). The inoculated leaves were placed on moist filter paper in petri dishes and incubated for 48 h at 25°C. Control leaves were inoculated with sterilized distilled water. After 7 days, typical symptoms for S. solani similar to those observed in the farmers' fields developed on both wounded and nonwounded inoculated leaves, but not on noninoculated controls, and S. solani was consistently reisolated. To our knowledge, this is the first report of S. solani causing gray leaf spot of tomato in Malaysia. References: (1) M. P. S. Camara et al. Mycologia 94:660, 2002. (2) B. S. Kim et al. Plant Pathol. J. 15:348, 1999. (3) B. M. Pryor and T. J. Michailides. Phytopathology 92:406, 2002. (4) E. G. Simmons. CBS Biodiversity Series 6:775, 2007.
Symptoms of gray leaf spot were first observed in June 2011 on pepper (Capsicum annuum) plants cultivated in the Cameron Highlands and Johor State, the two main regions of pepper production in Malaysia (about 1,000 ha). Disease incidence exceeded 70% in severely infected fields and greenhouses. Symptoms initially appeared as tiny (average 1.3 mm in diameter), round, orange-brown spots on the leaves, with the center of each spot turning gray to white as the disease developed, and the margin of each spot remaining dark brown. A fungus was isolated consistently from the lesions using sections of symptomatic leaf tissue surface-sterilized in 1% NaOCl for 2 min, rinsed in sterile water, dried, and plated onto PDA and V8 agar media (3). After 7 days, the fungal colonies were gray, dematiaceous conidia had formed at the end of long conidiophores (19.2 to 33.6 × 12.0 to 21.6 μm), and the conidia typically had two to six transverse and one to four longitudinal septa. Fifteen isolates were identified as Stemphylium solani on the basis of morphological criteria described by Kim et al. (3). The universal primers ITS5 and ITS4 were used to amplify the internal transcribed spacer region (ITS1, 5.8, and ITS2) of ribosomal DNA (rDNA) of a representative isolate (2). A 570 bp fragment was amplified, purified, sequenced, and identified as S. solani using a BLAST search with 100% identity to the published ITS sequence of an S. solani isolate in GenBank (1). The sequence was deposited in GenBank (Accession No. JQ736024). Pathogenicity of the fungal isolate was tested by inoculating healthy pepper leaves of cv. 152177-A. A 20-μl drop of conidial suspension (105 spores/ml) was used to inoculate each of four detached, 45-day-old pepper leaves placed on moist filter papers in petri dishes (4). Four control leaves were inoculated similarly with sterilized, distilled water. The leaves were incubated at 25°C at 95% relative humidity for 7 days. Gray leaf spot symptoms similar to those observed on the original pepper plants began to develop on leaves inoculated with the fungus after 3 days, and S. solani was consistently reisolated from the leaves. Control leaves did not develop symptoms and the fungus was not reisolated from these leaves. Pathogenicity testing was repeated with the same results. To our knowledge, this is the first report of S. solani causing gray leaf spot on pepper in Malaysia. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) M. P. S. Camara et al. Mycologia 94:660, 2002. (3) B. S. Kim et al. Plant Pathol. J. 15:348, 1999. (4) B. M. Pryor and T. J. Michailides. Phytopathology 92:406, 2002.
A leaf spot on eggplant (Solanum melongena) was observed in major eggplant growing regions in Malaysia, including the Cameron Highlands and Johor State, during 2011. Disease incidence averaged approximately 30% in severely infected regions in about 150 ha of eggplant fields and greenhouses examined. Early symptoms consisted of small, circular, brown, necrotic spots uniformly distributed on leaves. The spots gradually enlarged and developed concentric rings. Eventually, the spots coalesced and caused extensive leaf senescence. A fungus was recovered consistently by plating surface-sterilized (1% NaOCl) sections of symptomatic leaf tissue onto potato dextrose agar (PDA). For conidial production, the fungus was grown on potato carrot agar (PCA) and V8 agar media under a 16-h/8-h dark/light photoperiod at 25°C (4). Fungal colonies were a dark olive color with loose, cottony mycelium. Simple conidiophores were ≤120 μm long and produced numerous conidia in long chains. Conidia averaged 20.0 × 7.5 μm and contained two to five transverse septa and the occasional longitudinal septum. Twelve isolates of the fungus were identified as Alternaria tenuissima on the basis of morphological characterization (4). Confirmation of the species identification was obtained by molecular characterization of the internal transcribed spacer (ITS) region of rDNA amplified from DNA extracted from a representative isolate using universal primers ITS4 and ITS5 (2). The 558 bp DNA band amplified was sent for direct sequencing. The sequence (GenBank Accession No. JQ736021) was subjected to BLAST analysis (1) and was 99% identical to published ITS rDNA sequences of isolates of A. tenuissima (GenBank Accession Nos. DQ323692 and AY154712). Pathogenicity tests were performed by inoculating four detached leaves from 45-day-old plants of the eggplant cv. 125066x with 20 μl drops (three drops/leaf) of a conidial suspension containing 105 conidia/ml in sterile distilled water. Four control leaves were inoculated with sterile water. Leaves inoculated with the fungus and those treated with sterile water were incubated in chambers at 25°C and 95% RH with a 12-h photoperiod/day (2). Leaf spot symptoms typical of those caused by A. tenuissima developed on leaves inoculated with the fungus 7 days after inoculation, and the fungus was consistently reisolated from these leaves. The control leaves remained asymptomatic and the pathogen was not reisolated from the leaves. The pathogenicity test was repeated with similar results. To our knowledge, this is the first report of A. tenuissima causing a leaf spot on eggplant in Malaysia. A. tenuissima has been reported to cause leaf spot and fruit rot on eggplant in India (3). References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) B. M. Pryor and T. J. Michailides. Phytopathology 92:406, 2002. (3) P. Raja et al. New Disease Rep. 12:31, 2005. (4) E. G. Simmons. Page 1 in: Alternaria Biology, Plant Diseases and Metabolites. J. Chelchowski and A. Visconti, eds. Elsevier, Amsterdam, 1992.
A DNA macroarray was previously developed to detect major fungal and oomycete pathogens of solanaceous crops. To provide a convenient alternative for researchers with no access to X-ray film-developing facilities, specific CCD cameras or Chemidoc XRS systems, a chromogenic detection method with sensitivity comparable with chemiluminescent detection, has been developed. A fungal (Stemphylium solani) and an oomycete (Phytophthora capsici) pathogen were used to develop the protocol using digoxigenin (DIG)-labeled targets. The internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (rDNA), including ITS1, 5.8S rDNA, and ITS2, was used as the target gene and polymerase chain reaction amplified as in the previous protocol. Various amounts of species-specific oligonucleotides on the array, quantities of DIG-labeled ITS amplicon, and hybridization temperatures were tested. The optimal conditions for hybridization were 55°C for 2 h using at least 10 pmol of each species-specific oligonucleotide and labeled target at 10 ng/ml of hybridization buffer. Incubation of the hybridized array with anti-DIG conjugated alkaline phosphatase substrates, NBT/BCIP, produced visible target signals between 1 and 3 h compared with 1 h in chemiluminescent detection. Samples from pure cultures, soil, and artificially inoculated plants were also used to compare the detection using chemiluminescent and chromogenic methods. Chromogenic detection was shown to yield similar results compared with chemiluminescent detection in regard to signal specificity, duration of hybridization between the array and targets, and cost, though it takes 1 to 2 h longer for the visualization process, thus providing a convenient alternative for researchers who lack darkroom facilities. To our knowledge, this is the first report of DNA macroarray detection of plant pathogens using a chromogenic method.
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.
In April and June 2010, coconut seedlings with symptoms of very slow growth, yellowing of leaves, and general abnormal leaf growth were observed in germination beds in Teluk Intan, Perak, Malaysia. The roots were soft, rotten, and brown, extending upward and downward from these lesions. Rhizomorphs and basidiocarps were produced on coconut seeds near the germination eye and identified as Marasmiellus palmivorus according description by Turner (2). Three isolates were obtained by plating surface sterilized symptomatic roots and basidiocarp on malt extract agar (MEA) amended with 85% lactic acid (1 ml added to 11 of the medium). To confirm the identity of the fungus, genomic DNA was extracted from mycelia and basidiocarps of isolates and the large subunit (LSU) region was amplified and sequenced using LR0R/LR7 primers (3). All isolates had identical LSU sequences (GenBank Accession No. JQ654233 to JQ654235). Sequences were identical to each other and 99% similar to a M. palmivorus sequence deposited in the NCBI database (Accession No. AY639434).To confirm pathogenicity, three isolates of M. palmivorus that were obtained from symptomatic plant tissue was inoculated onto seeds of Malaysian Red Dwarf variety. Each isolate was grown in 100 ml of malt extract broth in 250 ml Erlenmeyer flasks and incubated at 27 ± 2°C for 5 days on an orbital shaker (125 rpm). The resulting culture was passed through two layers of sterile cloth. Mycelial suspension was obtained by blending mycelia in 100 ml of sterile water. Seeds were sterilized by soaking in 10% v/v sodium hypochlorite in distilled water for 3 min. The seeds were then rinsed three times over running tap water. The calyx portion of the seed was removed and five holes were made around the germination eye. The seeds were inoculated by injecting 2 ml of suspension into each hole. The control seeds were inoculated with sterile distilled water only. The seeds were transferred to 40-cm diameter plastic pots containing a mixture of sand, soil, and peat in the ratio of 3:2:1, respectively, and steam treated at 100°C for 1.5 h. Pots were placed in the glasshouse with normal exposures to day-night cycles, temperatures of 29 ± 4°C, and high relative humidity (85 to 95%) achieved by spraying water twice daily. After 2 months, 75% of the inoculated seeds failed to germinate. It was speculated that the artificial inoculum was higher than under germination bed conditions. Rhizomorphs and basidiocarps were produced on husk seeds near the germination eye. Seedlings that emerged successfully developed symptoms similar to those observed in the germination bed. No symptoms developed in the noninoculated seeds and seedlings. At 80 days post inoculation, basidiocarps were observed emerging from three diseased seedlings near the germination eye. Three reisolations were made on MEA from root lesions surface sterilized. Pathogenicity tests and LSU sequence analyses indicated that M. palmivorus is the causal agent of the symptoms observed on coconut seedlings. M. palmivorus was first recorded on coconuts and oil palm in the 1920s (1) and attacks the fruit and the petiole on oil palm (2). To our knowledge, this is the first report of M. palmivorus causing post-emergence damping off on coconut seedlings. References: (1) K. G. Singh. A check-list of host and diseases in Malaysia. Ministry of Agriculture and Fisheries, Malaysia, 1973. (2) P. D. Turner. Oil palm diseases and disorders. Oxford University Press. 1981. (3) R. Vilgalys et al. J. Bacteriol. 172:4238, 1990.
In July 2011, a severe outbreak of pod and stem blight was observed on lima bean (Phaseolus lunatus L.) plants grown in the Cameron Highlands, located in Pahang State, Malaysia. Disease incidence varied from 33 to 75% in different fields. Pods and stems exhibited withered, light brown to reddish brown necrotic areas. Sub-circular and brown lesions were produced on the leaves. These lesions varied in size, often reaching a diameter of 1 to 2 cm. After tissue death, numerous pycnidia were observed on the surface of the pod or stem. The pycnidia diameter varied from 155 to 495 μm, averaging 265.45 μm, and on the surface of the pod or stem, pycnidia were often arranged concentrically or linearly, respectively. Pycnidiospores were hyaline, 1-celled, usually straight, and rarely, slightly curved. The α-spores varied from 5.5 to 9.0 × 2.5 to 4.0 μm; averaging 7.3 × 3.5 μm. The β-spores found either alone or with pycnidiospores in pycnidia were slender, hyaline, nonseptate, and straight or curved. Size varied from 15.8 to 38.0 × 1.3 to 2.1 μm; averaging 25.86 × 1.8 μm. The colony characteristics were recorded from pure cultures grown on potato dextrose agar plates, and incubated in darkness for 7 days at 25 °C, then exposed to 16/8 h light and dark periods at 25°C for a further 14 to 21 days. Morphological characteristics of the colonies and spores on PDA matched those described for P. phaseolorum var. sojae (2). Colonies were white, compact, with wavy mycelium and stromata with pycnidia that contained abundant β-spores. Sequence analysis of the ribosomal DNA internal transcribed spacer obtained from the Malaysian isolate FM1 (GenBank Accession No. JQ514150) using primers ITS5 and ITS4 (1) aligned with deposited sequences from GenBank confirmed identity and revealed 99% to 100% DNA similarity with P. phaseolorum strains (AY577815, AF001020, HM012819, JQ936148). The isolate FM1 was used for pathogenicity testing. Five non-infected detached leaves and pods of 4-week-old lima bean were surface sterilized and inoculated by placing 10 μl of conidial suspension (106 conidia ml-1) on the surface of leaves and pods using either the wound/drop or non-wound/drop method and distilled water used as control (3). The inoculated leaves and pods were incubated at 25 °C and 98% RH, and the experiment was performed twice. Disease reactions and symptoms were evaluated after inoculation. After one week, typical symptoms of pod and stem blight appeared with formation of pycnidia on the surface of the tissues, but not on non-inoculated controls. P. phaseolorum var. sojae was consistently reisolated from symptoms. To our knowledge, this is the first report of P. phaseolorum var. sojae causing pod and stem blight of lima bean in Malaysia. References: (1) R. Ford et al. Aust. Plant Pathol. 33:559, 2004. (2) G. L. Hartman et al. Compendium of Soybean Diseases. 4th ed. American Phytopathological Society, St. Paul, MN, 1999. (3) P. P. Than et al. Plant Pathol. 57:562, 2008.
In June 2011, lettuce (Lactuca sativa) plants cultivated in major lettuce growing areas in Malaysia, including the Pahang and Johor states, had extensive leaf spots. In severe cases, disease incidence was recorded more than 80%. Symptoms on 50 observed plants initially were as water soaked spots (1 to 2 mm in diameter) on leaves, and then became circular spots spreading over much of the leaves. In this research, main lettuce growing areas infected by the pathogen in the mentioned states were investigated and the pathogen was isolated onto potato dextrose agar (PDA). Colonies observed were greyish green to light brown. Single conidia were formed at the terminal end of conidiophores that were 28.8 to 40.8 μm long and 11.0 to 19.2 μm wide, and 2 to 7 transverse and 1 to 4 longitudinal septa. To produce conidia, the fungus was grown on potato carrot agar (PCA) and V8 juice agar media under 8-h/16-h light/dark photoperiod. Fourteen isolates were identified Stemphylium solani based on morphological criteria described by Kim et al. (1). To confirm morphological characterization, DNA of the fungus was extracted from mycelium and PCR was done using universal primers ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'), which amplified the internal transcribed spacer (ITS) region of rDNA (2). The sequencing result was subjected to BLAST analysis which was 99% identical to the other published sequences in the GenBank database (GenBank Accession Nos. AF203451 and HQ840713). The nucleotide sequence was deposited in GenBank under Accession No. JQ736022. Pathogenicity testing of representative isolate was done using 20 μl of conidial suspension with a concentration of 1 × 105/ml in droplets (three drops on each leaf) on four detached 45-day-old lettuce leaves cv. BBS012 (3). Fully expended leaves were placed on moist filter paper in petri dishes and were incubated in humid chambers at 25°C. The leaves inoculated with sterile water served as control. After 7 days, disease symptoms were observed, which were similar to those symptoms collected in infected fields and the fungus was reisolated and confirmed as S. solani based on morphological criteria (1) and molecular characterization (2). Control leaves remained healthy. Pathogenicity testing was completed twice. To our knowledge, this is the first report of S. solani on lettuce in Malaysia and it may become a serious problem because of its broad host range, variability in pathogenic isolates, and prolonged active phase of the disease cycle. Previous research has shown that S. solani is a causal agent of gray leaf spot on lettuce in China (4). References: (1) B. S. Kim et al. Plant Pathol. J. 20:85, 2004. (2) Y. R. Mehta et al. Current Microbiol. 44:323, 2002. (3) B. M. Pryor and T. J. Michailides. Phytopathology 92:406, 2002. (4) F. L. Tai. Sylloge Fungorum Sinicorum, Sci. Press, Acad. Sin., Peking, 1979.
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.
In August 2011, sweet potato (Ipomoea batatas), tomato (Solanum lycopersicum), and eggplant (S. melongena) crops from major growing areas of the Cameron highlands and Johor state in Malaysia were affected by a soft rot disease. Disease incidence exceeded 80, 75, and 65% in severely infected fields and greenhouses of sweet potato, tomato, and eggplant, respectively. The disease was characterized by dark and small water-soaked lesions or soft rot symptoms on sweet potato tubers, tomato stems, and eggplant fruits. In addition, extensive discoloration of vascular tissues, stem hollowness, and water-soaked, soft, dark green lesions that turned brown with age were observed on the stem of tomato and eggplant. A survey was performed in these growing areas and 22 isolates of the pathogen were obtained from sweet potato (12 isolates), tomato (6 isolates), and eggplant (4 isolates) on nutrient agar (NA) and eosin methylene blue (EMB) (4). The cultures were incubated at 27°C for 2 days and colonies that were emerald green on EMB or white to gray on NA were selected for further studies. All bacterial cultures isolated from the survey exhibited pectolytic ability on potato slices. These bacterial isolates were gram negative; rod shaped; N-acetylglucosaminyl transferase, gelatin liquefaction, and OPNG positive; and were also positive for acid production from D-galactose, lactosemelibiose, raffinose, citrate, and trehalose. They were negative for indol production, phosphatase activity, reducing substances from sucrose, and negative for acid production from maltose, sorbitol, inositol, inolin, melezitose, α-mathyl-D-glocoside, and D-arabitol. The bacteria did not grow on NA at 37°C. Based on these biochemical and morphological assays, the pathogen was identified as Pectobacterium wasabiae (2). In addition, DNA was extracted and PCR assay with two primers (16SF1 and 16SR1) was performed (4). Partial sequences of 16S rRNA (GenBank Accession Nos. JQ665714, JX494234, and JX513960) of sweet potato, tomato, and eggplant, respectively, exhibited a 99% identity with P. wasabiae strain SR91 (NR_026047 and NR_026047.1). A pathogenicity assay was carried out on sweet potato tubers (cv. Oren), tomato stems (cv. 152177-A), and eggplant fruits (cv. 125066x) with 4 randomly representative isolates obtained from each crop. Sweet potato tubers, tomato stems, and eggplant fruits (4 replications) were sanitized in 70% ethyl alcohol for 30 s, washed and rinsed in sterile distilled water, and needle punctured with a bacterial suspension at a concentration of 108 CFU/ml. Inoculated tubers, stems, and fruits were incubated in a moist chamber at 90 to 100% RH for 72 h at 25°C when lesions were measured. All inoculated tubers, stems, and fruits exhibited soft rot symptoms after 72 h similar to those observed in the fields and greenhouses and the same bacteria were consistently reisolated. Symptoms were not observed on controls. The pathogenicty test was repeated with similar results. P. wasabiae have been previously reported to cause soft rot on Japanese horseradish (3), and aerial stem rot on potato in New Zealand (4), the U.S. (2), and Iran (1). To our knowledge, this is the first report of sweet potato, tomato, and eggplant soft rot caused by P. wasabiae in Malaysia. References: (1) S. Baghaee-Ravari et al. Eur. J. Plant Pathol. 129:413, 2011. (2) S. De Boer and A. Kelman. Page 56 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria, 3rd ed. N. Schaad et al., eds. APS Press, St. Paul, 2001. (3) M. Goto et al. Int. J. Syst. Bacteriol. 37:130, 1987. (4) A. R. Pitman et al. Eur. J. Plant Pathol. 126:423, 2010.