Basal stem rot (BSR), caused by the Ganoderma fungus, is an infectious disease that affects oil palm (Elaeis guineensis) plantations. BSR leads to a significant economic loss and reductions in yields of up to Malaysian Ringgit (RM) 1.5 billion (US$400 million) yearly. By 2020, the disease may affect ∼1.7 million tonnes of fresh fruit bunches. The plants appear symptomless in the early stages of infection, although most plants die after they are infected. Thus, early, accurate, and nondestructive disease detection is crucial to control the impact of the disease on yields. Terrestrial laser scanning (TLS) is an active remote-sensing, noncontact, cost-effective, precise, and user-friendly method. Through high-resolution scanning of a tree's dimension and morphology, TLS offers an accurate indicator for health and development. This study proposes an efficient image processing technique using point clouds obtained from TLS ground input data. A total of 40 samples (10 samples for each severity level) of oil palm trees were collected from 9-year-old trees using a ground-based laser scanner. Each tree was scanned four times at a distance of 1.5 m. The recorded laser scans were synched and merged to create a cluster of point clouds. An overhead two-dimensional image of the oil palm tree canopy was used to analyze three canopy architectures in terms of the number of pixels inside the crown (crown pixel), the degree of angle between fronds (frond angle), and the number of fronds (frond number). The results show that the crown pixel, frond angle, and frond number are significantly related and that the BSR severity levels are highly correlated (R2 = 0.76, P < 0.0001; R2 = 0.96, P < 0.0001; and R2 = 0.97, P < 0.0001, respectively). Analysis of variance followed post hoc tests by Student-Newman-Keuls (Newman-Keuls) and Dunnett for frond number presented the best results and showed that all levels were significantly different at a 5% significance level. Therefore, the earliest stage that a Ganoderma infection could be detected was mildly infected (T1). For frond angle, all post hoc tests showed consistent results, and all levels were significantly separated except for T0 and T1. By using the crown pixel parameter, healthy trees (T0) were separated from unhealthy trees (moderate infection [T2] and severe infection [T3]), although there was still some overlap with T1. Thus, Ganoderma infection could be detected as early as the T2 level by using the crown pixel and the frond angle parameters. It is hard to differentiate between T0 and T1, because during mild infection, the symptoms are highly similar. Meanwhile, T2 and T3 were placed in the same group, because they showed the same trend. This study demonstrates that the TLS is useful for detecting low-level infection as early as T1 (mild severity). TLS proved beneficial in managing oil palm plantation disease.
Ganoderma boninense is a causal agent of basal stem rot (BSR) and is responsible for a significant portion of oil palm (Elaeis guineensis) losses, which can reach US$500 million a year in Southeast Asia. At the early stage of this disease, infected palms are symptomless, which imposes difficulties in detecting the disease. In spite of the availability of tissue and DNA sampling techniques, there is a particular need for replacing costly field data collection methods for detecting Ganoderma in its early stage with a technique derived from spectroscopic and imagery data. Therefore, this study was carried out to apply the artificial neural network (ANN) analysis technique for discriminating and classifying fungal infections in oil palm trees at an early stage using raw, first, and second derivative spectroradiometer datasets. These were acquired from 1,016 spectral signatures of foliar samples in four disease levels (T1: healthy, T2: mildly-infected, T3: moderately infected, and T4: severely infected). Most of the satisfactory results occurred in the visible range, especially in the green wavelength. The healthy oil palms and those which were infected by Ganoderma at an early stage (T2) were classified satisfactorily with an accuracy of 83.3%, and 100.0% in 540 to 550 nm, respectively, by ANN using first derivative spectral data. The results further indicated that the sensitive frond number modeled by ANN provided the highest accuracy of 100.0% for frond number 9 compared with frond 17. This study showed evidence that employment of ANN can predict the early infection of BSR disease on oil palm with a high degree of accuracy.
Sooty blotch and flyspeck (SBFS) is a fungal disease complex that can cause significant economic losses to apple growers by blemishing the fruit surface with dark-colored colonies. Little is known about the phenology of host infection for this diverse group of epiphytes. In 2009 and 2010, we investigated the timing of infection of apple fruit by SBFS species in six commercial apple orchards in Iowa. Five trees in each orchard received no fungicide sprays after fruit set. Within 3 weeks after fruit set, 60 apples per tree were covered with Japanese fruit bags to minimize inoculum deposition. Subsequently, a subsample of bagged apples was exposed for a single 2-week-long period and then rebagged for the remainder of the growing season. Experimental treatments included seven consecutive 2-week-long exposure periods; control treatments were apples that were either bagged or exposed for the entire season. After apples had been stored at 2°C for 6 weeks following harvest, all SBFS colonies on the apples were identified to species using a PCR-RFLP protocol. A total of 15 species were identified. For the seven most prevalent species, the number of infections per cm2 of fruit surface was greatest on apples that had been exposed early in the season. Two SBFS species, Peltaster fructicola and Colletogloeopsis-like FG2, differed significantly from each other in time required to attain 50% of the total number of colonies per apple, and analysis of variance indicated a significant interaction of SBFS taxon with exposure period. Our findings are the first evidence of species-specific patterns in timing of SBFS inoculum deposition and infection on apple fruit, and strengthen previous observations that most SBFS infections resulting in visible colonies at harvest develop from infections that occur early in the fruit development period. By defining taxon-specific phenological patterns of fruit infection, our findings, when combined with knowledge of region-specific patterns of taxon prevalence, provide a foundation for development of more efficient and cost-effective SBFS management tactics.
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.
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.
On May 9, 2013, symptoms reminiscent of bacterial leaf streak (BLS) caused by Xanthomonas oryzae pv. oryzicola were observed on rice plants at the panicle emergence stage at Musenyi, Gihanga, and Rugombo fields in Burundi. Affected leaves showed water-soaked translucent lesions and yellow-brown to black streaks, sometimes with visible exudates on leaf surfaces. Symptomatic leaves were ground in sterile water and the suspensions obtained were subjected to a multiplex PCR assay diagnostic for X. oryzae pathovars (3). Three DNA fragments (331, 691, and 945 bp) corresponding to X. oryzae pv. oryzicola were observed after agarose gel electrophoresis. Single bacterial colonies were then isolated from surface-sterilized, infected leaves after grinding in sterile water and plating of 10-fold dilutions of the cell suspension on semi-selective PSA medium (4). After incubation at 28°C for 5 days, each of four independent cultures yielded single yellow, mucoid Xanthomonas-like colonies (named Bur_1, Bur_2, Bur_6, and Bur_7) that resembled the positive control strain MAI10 (1). These strains originated from Musenyi (Bur_1), Gihanga (Bur_2), and Rugumbo (Bur_6 and Bur_7). Multiplex PCR assays on the four putative X. oryzae pv. oryzicola strains yielded the three diagnostic DNA fragments mentioned above. All strains were further analyzed by sequence analysis of portions of the gyrB gene using the universal primers gyrB1-F and gyrB1-R for PCR amplification (5). The 762-bp DNA fragment was identical to gyrB sequences from the Asian X. oryzae pv. oryzicola strains BLS256 (Philippines), ICMP 12013 (China), LMG 797 and NCPPB 2921 (both Malaysia), and from the African strain MAI3 (Mali) (2). The partial nucleotide sequence of the gyrB gene of Bur_1 was submitted to GenBank (Accession No. KJ801400). Pathogenicity tests were performed on greenhouse-grown 4-week-old rice plants of the cvs. Nipponbare, Azucena, IRBB 1, IRBB 2, IRBB 3, IRBB 7, FKR 14, PNA64F4-56, TCS 10, Gigante, and Adny 11. Bacterial cultures were grown overnight in PSA medium and re-suspended in sterile water (1 × 108 CFU/ml). Plants were inoculated with bacterial suspensions either by spraying or by leaf infiltration (1). For spray inoculation, four plants per accession and strain were used while three leaves per plant and four plants per accession and strain were inoculated by tissue infiltration. After 15 days of incubation in a BSL-3 containment facility (27 ± 1°C with a 12-h photoperiod), the spray-inoculated plants showed water-soaked lesions with yellow exudates identical to those seen in the field. For syringe-infiltrated leaves, the same symptoms were observed at the infiltrated leaf area. Re-isolation of bacteria from symptomatic leaves yielded colonies with the typical Xanthomonas morphology that were confirmed by multiplex PCR to be X. oryzae pv. oryzicola, thus fulfilling Koch's postulates. Bur_1 has been deposited in the Collection Française de Bactéries Phytopathogènes as strain CFBP 8170 ( http://www.angers-nantes.inra.fr/cfbp/ ). To our knowledge, this is the first report of X. oryzae pv. oryzicola causing bacterial leaf streak on rice in Burundi. Further surveys will help to assess its importance in the country. References: (1) C. Gonzalez et al., Mol. Plant Microbe Interact. 20:534, 2007. (2) A. Hajri et al. Mol. Plant Pathol. 13:288, 2012. (3) J. M. Lang et al. Plant Dis. 94:311, 2010. (4) L. Poulin et al. Plant Dis. 98:1423, 2014. (5) J. M. Young et al. Syst. Appl. Microbiol. 31:366, 2008.
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.
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.
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.
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.
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.
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.
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.
In 2011, a severe gray leaf spot was observed on eggplant (Solanum melongena) in major eggplant growing areas in Malaysia, including the Pahang, Johor, and Selangor states. Disease incidence was >70% in severely infected areas of about 150 ha of eggplant greenhouses and fields examined. Symptoms initially appeared as small (1 to 5 mm diameter), brownish-black specks with concentric circles on the lower leaves. The specks then coalesced and developed into greyish-brown, necrotic lesions, which also appeared on the upper leaves. Eventually, the leaves senesced and were shed. Tissue cut from the edges of leaf spots were surface-sterilized in 1% NaOCl for 2 min, rinsed in sterilized water, dried, and incubated on potato dextrose agar (PDA). Fungal colonies were greyish green to light brown, and produced a yellow pigment. Single, muriform, brown, oblong conidia formed at the terminal end of each conidiophore, were each 21.6 to 45.6 μm long and 11.5 to 21.6 μm wide, and contained 2 to 7 transverse and 1 to 4 longitudinal septa. The conidiophores were tan to light brown and ≤220 μm long. Based on these morphological criteria, 25 isolates of the fungus were identified as Stemphylium solani (1). To produce conidia in culture, 7-day-old single-conidial cultures were established on potato carrot agar (PCA) and V8 juice agar media under an 8-h/16-h light/dark photoperiod at 25°C (4). Further confirmation of the identification was obtained by molecular characterization in which fungal DNA was extracted and the internal transcribed spacer (ITS) region of ribosomal DNA amplified using primers ITS5 and ITS4 (2), followed by direct sequencing. A BLAST search in the NCBI database revealed that the sequence was 99% identical with published ITS sequences for two isolates of S. solani (Accession Nos. AF203451 and HQ840713). The amplified ITS region was deposited in GenBank (JQ736023). Pathogenicity testing of a representative isolate was performed on detached, 45-day-old eggplant leaves of the cv. 125066-X under laboratory conditions. Four fully expanded leaves (one wounded and two non-wounded leaflets/leaf) were placed on moist filter paper in petri dishes, and each leaflet inoculated with a 20-μl drop of a conidial suspension containing 1 × 105 conidia/ml in sterilized, distilled water (3). The leaves were wounded by applying pressure to leaf blades with the serrated edge of forceps. Four control leaves were inoculated similarly with sterilized, distilled water. Inoculated leaves were incubated in humid chambers at 25°C with 95% RH and a 12-h photoperiod. After 7 days, symptoms similar to those observed in the original fields developed on both wounded and non-wounded inoculated leaves, but not on control leaves, and S. solani was reisolated consistently from the symptoms using the same method as the original isolations. Control leaves remained asymptomatic and the fungus was not isolated from these leaves. The pathogenicity testing was repeated with similar results. To our knowledge, this is the first report of S. solani on eggplant in Malaysia. References: (1) B. S. Kim et al. Plant Pathol. J. 20:85, 2004. (2) Y. R. Mehta et al. Curr. Microbiol. 44:323, 2002. (3) B. M. Pryor and T. J. Michailides. Phytopathology 92:406, 2002. (4) E. G. Simmons. CBS Biodiv. Series 6:775, 2007.
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.
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.
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.