Kamaruzaman Sijam

Selection and identification of non-pathogenic bacteria isolated from fermented pickles with antagonistic properties against two shrimp pathogens

In this study, potential probiotic strains were isolated from fermented pickles based on antagonistic activity against two shrimp pathogens (Vibrio harveyi and Vibrio parahaemolyticus). Two strains L10 and G1 were identified by biochemical tests, followed by16S ribosomal RNA gene sequence analysis as Bacillus subtilis, and characterized by PCR amplification of repetitive bacterial DNA elements (Rep-PCR). Subsequently, B. subtilis L10 and G1 strains were tested for antibacterial activity under different physical conditions, including culture medium, salinity, pH and temperature using the agar well diffusion assay. Among the different culture media, LB broth was the most suitable medium for antibacterial production. Both strains showed the highest level of antibacterial activity against two pathogens at 30 °C and 1.0% NaCl. Under the pH conditions, strain G1 showed the greatest activity against V. harveyi at pH 7.3–8.0 and against V. parahaemolyticus at pH 6.0–8.0, whereas strain L10 showed the greatest activity against two pathogens at pH 7.3. The cell-free supernatants of both strains were treated with four different enzymes in order to characterize the antibacterial substances against V. harveyi. The result showed considerable reduction of antibacterial activity for both strains, indicating the proteinaceous nature of the antibacterial substances. A wide range of tolerance to NaCl, pH and temperature was also recorded for both strains. In addition, both strains showed no virulence effect in juvenile shrimp Litopenaeus vannamei. On the basis of these results and safety of strains to L. vannamei, they may be considered for future challenge experiments in shrimp as a very promising alternative to the use of antibiotics.

Current advance methods for the identification of blast resistance genes in rice

Rice blast caused by Magnaporthe oryzae is one of the most devastating diseases of rice around the world and crop losses due to blast are considerably high. Many blast resistant rice varieties have been developed by classical plant breeding and adopted by farmers in various rice-growing countries. However, the variability in the pathogenicity of the blast fungus according to environment made blast disease a major concern for farmers, which remains a threat to the rice industry. With the utilization of molecular techniques, plant breeders have improved rice production systems and minimized yield losses. In this article, we have summarized the current advanced molecular techniques used for controlling blast disease. With the advent of new technologies like marker-assisted selection, molecular mapping, map-based cloning, marker-assisted backcrossing and allele mining, breeders have identified more than 100 Pi loci and 350 QTL in rice genome responsible for blast disease. These Pi genes and QTLs can be introgressed into a blast-susceptible cultivar through marker-assisted backcross breeding. These molecular techniques provide timesaving, environment friendly and labour-cost-saving ways to control blast disease. The knowledge of host-plant interactions in the frame of blast disease will lead to develop resistant varieties in the future.

Bacteria flora associated with different body parts of hatchery reared juvenilePenaeus monodon, tanks water and sediment

Bacteria flora of intestine and hepatopancreas, body surface and muscles of juvenilePenaeus monodon along with its rearing water and sediment was analyzed. Juvenile shrimp were reared in four tanks in the Hatchery complex, Department of Aquaculture, Faculty of Agriculture, University Putra Malaysia. Water quality parameters were measured every day. Samples were collected aseptically and homogenized before being inoculated in Tryptone Soy agar, Thiosulphate Citrate Bile Salt agar, MacConkey agar andPseudomonas-isolating agar. There was no significant difference between water quality parameters and shrimp body weight of replicate tanks. Total plate count for water and totalVibrio count for rearing water and digestive system were within previous reported ranges. Eight different genera were isolated in which 7 genera were identified. Gram negative bacteria were dominant (72%)Vibrio was the most dominant genera followed byShewanella andBurkholderia. Clavibacter followed byStaphylococcus were the most dominant gram positive bacteria. No coliform bacteria was detected in the shrimp body parts and rearing environment. Incidence ofShewanella in the digestive system was significantly higher than sediment, rearing water and muscles. This may be implied its ability to colonize in the digestive tract of juvenileP. monodon.

The potential of endomycorrhizal fungi in controlling tomato bacterial wilt Ralstonia solanacearum under glasshouse conditions

The impact of colonization by three mycorrhizal fungi on tomato bacterial wilt caused by Ralstonia solanaceraum was investigated. Three species of arbuscular mycorrhizal fungal (AMF) were tested ( Glomus mosseae , Scutellospora sp. and Gigaspora margarita ). Siginificant differences in tomato growth based on plant hieght was recorded between G. mosseae (125.25 cm) and all treatments. The combination of G. mosseae and R. solanacearum resulted in significantly taller tomato plants than G. margarita + R. solanacearum and Scutelospora sp.+ R. solanacearum . Shoot fresh and dry weight was higher in G. mosseae inoculated plants. No disease symptoms were observed in the combination treatment of G. mosseae and R. solanacearum . The plants treated with Scutellospora sp. showed low incidence of infection (105, 15%) at 15 and 20 days after inoculation, respectively. The combination of G. mosseae and R. solanacearum resulted in more increase in root morphology (root tips (434.75), root length (267.00 cm), root surface area (149.31 cm 2 ), root volume (3.77 cm 3 ), root fresh weight (4.75 g) and root dry weight (2.5 g). The treatment of G. mosseae + R. solanacearum was different significantly when compared to G. margarita and Scutellospora sp. + R. solanacearum treatments in all parameters considered. The highest number of AMF spores was recorded in G. mosseae treatment followed by Scutellospora sp. The concentration of N, P and K in G. mosseae + R. solanacearum treatment was significantly higher (N: 1.69; P: 0.51 and K: 1.65%) compared to G. margarita (N: 1.06 ; P: 0.11 and K: 1.02%) and Scutellospora sp., treatment (N: 1.48; P: 0.44 and K: 1.47%). Generally, the current findings has provided an evedance about the ability of AMF species to control bacterial wilt causal agents with significant differences between the species used. Keywords: Bio-control, wilt disease, tomato, Glomus mosseae

Fluorometric immunoassay for detecting the plant virus Citrus tristeza using carbon nanoparticles acting as quenchers and antibodies labeled with CdTe quantum dots

AbstractCadmium-telluride quantum dots (QDs) were conjugated to an antibody (Ab) against Citrus tristeza virus (CTV), while the coat protein (CP) of the CTV was immobilized on the surface of carbon nanoparticles (CNPs). Following immunobinding of the QD-Ab and the CP-loaded CNPs, the fluorescence of the CdTe QDs was quenched by the CNPs. This effect was exploited to design a detection assay for the CTV which was found more sensitive and specific than the existing enzyme linked immunosorbent assay (ELISA). The limit of detection was measured at about 220 ng⋅ mL‾1 of CTV. The Stern-Volmer plot of the CNPs-QD quencher pair showed a positive deviation from linearity which was ascribed to the presence of both static and dynamic quenching. Graphical abstractCadmium-telluride quantum dots (QDs) were conjugated to an antibody (Ab) against Citrus tristeza virus (CTV), while the coat protein (CP) of the CTV was immobilized on the surface of carbon nanoparticles (CNPs). Following immunobinding of the QD-Ab and the CP-loaded CNPs, the fluorescence of the CdTe QDs was quenched by the CNPs. This effect was exploited to design a detection assay for the CTV which was found more sensitive and specific than the existing enzyme linked immunosorbent assay (ELISA).

Introgression of Blast Resistance Genes (Putative Pi-b and Pi-kh) into Elite Rice Cultivar MR219 through Marker-Assisted Selection

Blast is the most common biotic stress leading to the reduction of rice yield in many rice-growing areas of the world, including Malaysia. Improvement of blast resistance of rice varieties cultivated in blast endemic areas is one of the most important objectives of rice breeding programs. In this study, the marker-assisted backcrossing strategy was applied to improve the blast resistance of the most popular Malaysian rice variety MR219 by introgressing blast resistance genes from the Pongsu Seribu 2 variety. Two blast resistance genes, Pi-b and Pi-kh, were pyramided into MR219. Foreground selection coupled with stringent phenotypic selection identified 15 plants homozygous for the Pi-b and Pi-kh genes, and background selection revealed more than 95% genome recovery of MR219 in advanced blast resistant lines. Phenotypic screening against blast disease indicated that advanced homozygous blast resistant lines were strongly resistant against pathotype P7.2 in the blast disease endemic areas. The morphological, yield, grain quality, and yield-contributing characteristics were significantly similar to those of MR219. The newly developed blast resistant improved lines will retain the high adoptability of MR219 by farmers. The present results will also play an important role in sustaining the rice production of Malaysia.

Effects of selected herbicides on soil microbial populations in oil palm plantation of Malaysia: A microcosm experiment

Herbicides are commonly used in Malaysia to control weeds in oil palm plantation. In addition to their impact on weeds, these herbicides are also affecting soil microorganisms which are responsible for numerous biological processes essential for crop production. In the present study, we assessed the impact of four commonly used herbicides (paraquat, glyphosate, glufosinate-ammonium and metsulfuron-methyl) on soil microbial populations in oil palm plantation. Our study showed that the herbicide treatments significantly inhibited the development of microbial populations in the soil, and the degree of inhibition closely related to the rates of their applications and varied with the types of herbicide. Paraquat caused the highest inhibitory effect to bacteria and actinomycetes, whereas fungi were most affected by glyphosate. Metsulfuron-methyl had least inhibitory effects to all the microbial populations. The highest inhibition (59.3%) for fungal population was observed at 6 DAT (days after treatment), whereas for the bacteria and actinomycetes (82.0 and 70.6%, respectively) were at 4 DAT. Increasing trend of inhibition on growth of microbial populations was observed from the initial effect until 6 DAT, followed by a drastic decrease of the inhibition at 10 DAT. No inhibition was observed at 20 DAT. The study suggests that the herbicide application to soil of oil palm plantation cause transient impacts on microbial population growth, when applied at recommended or even as high as double (2x) of the recommended field application rate.

First Report of Cabbage Soft Rot Caused by Pectobacterium carotovorum subsp. carotovorum in Malaysia

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 Kings 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.

Detection of Citrus tristeza virus by using fluorescence resonance energy transfer-based biosensor.

Due to the low titer or uneven distribution of Citrus tristeza virus (CTV) in field samples, detection of CTV by using conventional detection techniques may be difficult. Therefore, in the present work, the cadmium-telluride quantum dots (QDs) was conjugated with a specific antibody against coat protein (CP) of CTV, and the CP were immobilized on the surface of gold nanoparticles (AuNPs) to develop a specific and sensitive fluorescence resonance energy transfer (FRET)-based nanobiosensor for detecting CTV. The maximum FRET efficiency for the developed nano-biosensor was observed at 60% in AuNPs-CP/QDs-Ab ratio of 1:8.5. The designed system showed higher sensitivity and specificity over enzyme linked immunosorbent assay (ELISA) with a limit of detection of 0.13μgmL(-1) and 93% and 94% sensitivity and specificity, respectively. As designed sensor is rapid, sensitive, specific and efficient in detecting CTV, this could be envisioned for diagnostic applications, surveillance and plant certification program.

First Report of Spiroplasma citri (-Induced) Associated with Periwinkle Lethal Yellows in Southeast Asia

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.