Characterization of Malaysian Pectobacterium spp. from vegetables using biochemical, molecular and phylogenetic methods.

In August 2011, vegetable crops showing symptoms of maceration and water soaked lesions on their tuber, leaf, and fruit were collected from four major vegetable growing states in Malaysia including Pahang, Johor, Melaka and Selangor. The majority of the causal organisms isolated from infected tissues (52 strains) were identified as Pectobacterium spp. based on PCR amplification of the pectate lyase (pel) gene and amplification of the 16S-23S rRNA (ITS) with G1 and L1 primers. Physiological and biochemical assays divided Malaysian Pectobacterium species into two main groups: Pectobacterium wasabiae and Pectobacterium carotovorum subsp carotovorum. Partial sequence of PCR product from reaction of putative Pectobacterium spp. with 16S rRNA confirmed the results obtained from physiological and biochemical assays used for identification of the bacterium. Application of specific primers such as Eca1F/Eca2r, Br1f/L1r, EXPCCF/EXPCCR, and also ITS-PCR following by RFLP by restriction enzyme (RsaI) successfully differentiated Malaysian P. wasabiae and P. carotovorum subsp carotovorum isolates from other species and subspecies of Pectobacterium. Phylogenetic analysis of Malaysian isolates with housekeeping genes (mdh, gapA) grouped Malaysian P. carotovorum subsp carotovorum and P. wasabiae in the same cluster with P. carotovorum subsp carotovorum (Ecc380) and P. wasabiae (SCRI488) respectively

Characterisation of Pectobacterium carotovorum causing soft rot on Kalanchoe gastonis-bonnierii in Malaysia

Soft rot disease can be found worldwide on fleshy storage tissues of fruits, vegetables and ornamentals. The soft rot Pectobacterium carotovorum subsp. carotovorum (Pcc) is an important pathogen of Kalanchoe spp. and other ornamental plants. The disease occurs on crops in the field, greenhouses and during transit, resulting great economic damages. The economic importance of crop loss by soft rot bacteria varies by severity of the disease and value of the crop. A destructive disease on Kalanchoe gastonis-bonnierii was observed in commercial ornamental plant greenhouses in Cameron highland and Melaka, Malaysia in 2011. Samples suspected to be infested with Pectobacterium spp. were brought to the laboratory. In pathogenicity test, a suspension of 106 CFU/ml of strains was able to cause soft rot on leaves and stems. A 434 bp banding pattern on 1% agarose gel was produced in polymerase chain reaction (PCR) amplification of pectate lyase encoding gene (Pel gene). PCR amplification of the intergenic transcribed spacer (ITS) (16S–23S rRNA) ITS region with G1 and L1 primers produced two main bands at about 540 and 570 bp. The ITS-PCR products were digested with RsaI restriction enzyme. For discrimination of the P. carotovorum subsp. carotovorum (Pcc) from P. carotovorum subsp. odoriferum (Pco), all isolates subjected to α-methyl glucoside test. All isolates were identified as Pcc based on phenotypic and molecular methods. This is the first report of soft rot disease caused by P. carotovorum subsp. carotovorum on K. gastonis-bonnierii, in Malaysia

Effect of different temperatures on the free amino acids, physico-chemical and microbial changes during storage of Barramundi (Lates calcarifer) fillets.

The effects of storage days and temperature on free amino acids, TVB-N, pH and microbial changes in Barramundi (Lates calcarifer) fillets kept at 0°C and 8°C were investigated for 20 days. At the end of the storage, significant differences were observed (p0.05) between two temperatures during the storage period were observed. Among two temperatures, the psychrophiles were initially 4.07 log CFU/g and exceeded the acceptable limit of 7 log CFU/g on the 12th and 8th day at 0°C and 8°C, respectively. Although, Total Plate Count (TPC) were initially 3.7 log CFU/g and exceeded the acceptable limit of 6 log CFU/g on the 12th day in the both storage temperatures. Histamine Forming Bacteria (HFB) was significantly (p<0.05) lower in Barramundi fillets storage at 0°C compared to the 8°C. Significant differences (p<0.05) between the concentrations of Total Volatile Base-Nitrogen (TVB-N) in fillets kept at 0°C and 8°C were observed

An outbreak of leaf spot caused by Stemphylium solani on eggplant in Malaysia.

In 2011, a severe gray leaf spot was observed on aubergine (Solanum melongena) in major aubergine growing areas in Malaysia, including the Pahang, Johor and Selangor states. Disease incidence was >70% in severely infected areas of approximately 150 ha of aubergine greenhouses and fields examined. Symptoms initially appeared as small (1-5 mm in 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. 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. 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. Further confirmation of the identification was obtained by molecular characterization. A BLAST search in the NCBI database revealed that the sequence was 99% identical with published ITS sequences for two isolates of Stemphylium solani (Accession Nos. AF203451 and HQ840713). This is thought to be the first report of Stemphylium solani on aubergine in Malaysia

First report of soft rot disease on pak choi (Brassica chinensis) caused by Dickeya dadantii subsp. dieffenbachiae in Malaysia

In June 2012, a soft rot disease of pak choi (Brassica chinensis) was observed in the vegetable-growing region of Cameron Highlands, in the Pahang state of Malaysia. Disease symptoms first appeared as small, water-soaked lesions at the base of petioles. The lesions enlarged rapidly and spread to the leaves, resulting in the collapse of the plant. Diseased samples were randomly collected from 12 infested fields. Soft rot incidence on pak choi varied from 38 to 56% in different commercial fields. The causal pathogen was identified as Dickeya dadantii subsp. dieffenbachiae based on morphological, biochemical, genetic (RFLP) and pathogenicity analyses. This is thought to be the first report of soft rot on pak choi caused by D. dadantii subsp. dieffenbachiae in Malaysia

Identification of Pithomyces chartarum causing leaf spot of cabbage in Malaysia

A leaf spot disease was observed on cabbage (Brassica oleracea L. var. capitata) affecting 80% of plants growing in greenhouses and fields in Serdang, Selangor, Malaysia. Symptomatic leaf samples were collected from infected plants and isolations made on agar medium. Single-spore isolates from resulting colonies were identified based on cultural and morphological characteristics as Pithomyces chartarum. Morphological identification was confirmed by sequence analysis of the internal transcribed spacer (ITS) regions 1 and 2, including 5.8S rDNA (ITS1-5.8S-ITS2). Pathogenicity tests indicated that P. chartarum causes leaf spot on cabbage. This is the first report of leaf spot caused by P. chartarum on cabbage in Malaysia

Analysis of genetic and virulence variability of Stemphylium lycopersici associated with leaf spot of vegetable crops

Stemphylium lycopersici (Enjoji) W. Yamam was initially described from tomato and has been reported to infect different hosts worldwide. Sequence analyses of the internal transcribed spacer (ITS) regions 1 and 2, including 5.8S rDNA (ITS-5.8S rDNA) and glyceraldehyde-3-phosphate dehydrogenase (gpd) gene, random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR), as well as virulence studies were conducted to analyze 46 S. lycopersici isolates. Stemphylium lycopersici isolates used in this study were obtained from diseased tomato (Solanum lycopersicum L.), eggplant (Solanum melongena L.), pepper (Capsicum annuum L.) and lettuce (Lactuca sativa L.) from major vegetable growing regions of Malaysia, including the three states of Pahang, Johor and Selangor between 2011 and 2012. Phylogenetic analysis of a combined dataset of the ITS-5.8S rDNA and gpd regions indicated that all isolates were clustered in the sub-cluster that comprised S. lycopersici, and were distinguished from other Stemphylium species. Cluster analyses using the UPGMA method for both RAPD and ISSR markers grouped S. lycopersici isolates into three main clusters with similarity index values of 67 and 68 %. The genetic diversity data confirmed that isolates of S. lycopersici are in concordance to host plants, and not geographical origin of the isolates. All S. lycopersici isolates were pathogenic on their original host plants and showed leaf spot symptoms; however, virulence variability was observed among the isolates. In cross-inoculation assays, the representative isolates were able to cause leaf spot symptoms on eggplant, pepper, lettuce and tomato, but not on cabbage

Alternaria capsicicola sp. nov., a new species causing leaf spot of pepper (Capsicum annuum) in Malaysia

A new species of Alternaria causing leaf spot of pepper (Capsicum annuum) obtained from the Cameron highlands, Pahang, Malaysia, was determined based on phylogenetic analyses, morphological characteristics, and pathogenicity assays. Phylogenetic analyses of combined dataset of the glyceraldehyde-3-phosphate dehydrogenase (gpd), Alternaria allergen a 1 (Alt a1) and calmodulin genes revealed that the new isolates clustered into a subclade distinct from the closely related Alternaria species A. tomato and A. burnsii. The solitary or short chains of conidia resemble those of A. burnsii. However, conidia with long beaks are morphologically similar to A. tomato. Hence, the pathogenic fungus is proposed as Alternaria capsicicola sp. nov. Pathogenicity assays indicated that A. capsicicola causes leaf spot on pepper

Characterization of Pectobacterium carotovorum and P. wasabiae and their potential control using antagonistic bacteria

In August 2011, vegetable crops showing symptoms of maceration and water soaked lesions on their tuber, leaf, and fruit were collected from four major vegetable growing states in Malaysia including Pahang, Johor, Melaka and Selangor. The majority of the causal organisms isolated from infected tissues (52 strains) were identified as Pectobacterium spp. based on PCR amplification of the pectate lyase (pel) gene and amplification of the 16S-23S rRNA (ITS) with G1 and L1 primers. Physiological and biochemical assays divided Malaysian Pectobacterium species into two main groups: Pectobacterium wasabiae and Pectobacterium carotovorum subsp carotovorum. Partial sequence of PCR product from reaction of putative Pectobacterium spp. with 16S rRNA confirmed the results obtained from physiological and biochemical assays used for identification of the bacterium. Application of specific primers such as Eca1F/Eca2r, Br1f/L1r, EXPCCF/EXPCCR, and also ITS-PCR following by RFLP by restriction enzyme (RsaI) successfully differentiated Malaysian P. wasabiae and P. carotovorum subsp carotovorum isolates from other species and subspecies of Pectobacterium. Phylogenetic analysis of Malaysian isolates with housekeeping genes (mdh, gapA) grouped Malaysian P. carotovorum subsp carotovorum and P. wasabiae in the same cluster with P. carotovorum subsp carotovorum (Ecc380) and P. wasabiae (SCRI488) respectively. In this study, some bacterial strains from the vegetable farm soil showed strong antagonistic activity against Pectobacterium carotovorum and Pectobacterium wasabiae in vitro on bell peppers, Chinese cabbage, cucumber, and tomato, and their cell-free filtered supernatants showed excellent biocontrol effect in controlling the potato maceration at room temperature. According to analysis of partial nucleotide sequences of 16S rRNA the isolates were identified as Pseudomonas chlororaphis, Pseudomonas fluorescens, Burkholderia cepacia, Bacillus subtilis, and Serratia liquefaciens. No significant differences in prevention of weight reduction by Pectobacterium spp. were observed between potato slices treated with antagonists andtreated with SH. Antibacterial agents isolated from Malaysian soils seem to be promising disinfectants to protect vegetables from soft rotting bacteria, and allow vegetables´ consumers and industries to reduce the amount of chemicals such as SH

First report of Pectobacterium wasabiae causing soft rot of cabbage in Malaysia.

Soft rot of cabbage (Brassica rapa) occurs sporadically in Malaysia, causing economic damage under the hot and wet Malaysian weather conditions that are suitable for disease development. In June 2011, 27 soft rotting bacteria were isolated from cabbage plants growing in the Cameron Highlands and Johor State in Malaysia where the economic losses exceeded 50% in severely infected fields and greenhouses. Five independent strains were initially identified as Pectobacterium wasabiae based on their inability to grow at 37°C, and elicit hypersensitive reaction (HR) on Nicotiana tabaccum and their ability to utilize raffinose and lactose. These bacterial strains were gram-negative, rod-shaped, N-acetylglucosaminyl transferase, gelatin liquefaction, and OPNG-positive and positive for acid production from D-galactose, lactosemelibiose, raffinose, citrate, and trehalose. All strains were negative for indole production, phosphatase activity, reducing sucrose, and negative for acid production from maltose, sorbitol, inositol, inolin, melezitose, α-methyl-D-glucoside, and D-arabitol. All the strains exhibited pectolytic activity on potato slices. PCR assays were conducted to distinguish P. wasabiae from P. carotovorum subsp. brasiliensis, P. atrosepticum, and other Pectobacterium species using primers Br1f/L1r (2), Eca1f/Eca2r (1), and EXPCCF/EXPCCR, respectively. DNA from strains did not yield the expected amplicon with the Br1f/L1r and Eca1f/Eca2r, whereas a 550-bp amplicon typical of DNA from P. wasabiae was produced with primers EXPCCF/EXPCCR. ITS-RFLP using the restriction enzyme, Rsa I, produced similar patterns for the Malaysian strains and the P. wasabiae type strain (SCRI488), but differentiated it from P. carotovora subsp. carotovora, P. atrosepticum, P. carotovorum subsp. brasiliensis, and Dickeya chrysanthemi type strains. BLAST analysis of the 16S rRNA DNA sequence (GenBank Accession No. KC445633) showed 99% identity to the 16S rRNA of Pw WPP163. Phylogenetic reconstruction using concatenated DNA sequences of mdh and gapA from P. wasabiae Cc6 (KC484657) and other related taxa (4) clustered Malaysian P. wasabiae strains with P. wasabiae SCRI488, readily distinguishing it from other closely related species of Pectobacterium. Pathogenicity assays were conducted on leaves and stems of four mature cabbage plants for each strain (var. oleifera) by injecting 10 μl of a bacterial suspension (108 CFU/ml) into either stems or leaves, and incubating them in a moist chamber at 80 to 90% relative humidity at 30°C. Water-soaked lesions similar to those observed in the fields and greenhouses were observed 72 h after injection and bacteria with similar characteristics were consistently reisolated. Symptoms were not observed on water-inoculated controls. The pathogenicity test was repeated with similar results. P. wasabiae was previously reported to cause soft rot of horseradish in Japan (3). However, to our knowledge, this is the first report of P. wasabiae infecting cabbage in Malaysia