First Report of Soft Rot Disease Caused by Pectobacterium wasabiae on Sweet Potato, Tomato, and Eggplant in Malaysia.

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.

First Report of Marasmiellus palmivorus Causing Post-Emergence Damping Off on Coconut Seedlings in Malaysia.

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.

First Report of Tomato Gray Leaf Spot Disease Caused by Stemphylium solani in Malaysia.

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.

First Report of Alternaria tenuissima Causing Leaf Spot on Eggplant in Malaysia.

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.

First Report of Gray Leaf Spot on Pepper Caused by Stemphylium solani in Malaysia.

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.