Development Of An Avirulent Pasteurella Multocida B:2 By Disruption Of The Aba392 Dna Fragment

Haemorrhagic septicaemia (HS) is an acute disease infecting cattle and buffaloes caused by Pasteurella multocida serotype B:2 which leads to great economic losses in countries with expanded animal industries such as Malaysia. This study was conducted to construct a mutant derived from the 921bp ABA392 virulence gene in order to make avirulent P. multocida serotype B:2, thus making this P. multocida B:2 mutant a potential candidate for a live-attenuated vaccine against HS. The detection of a fragment which is related to P. multocida B:2 pathogenicity, the 921bp ABA392 virulence gene was carried out using Polymerase Chain Reaction (PCR) assay. Two P. multocida isolates, namely PMTB and 3030, isolated from a HS outbreak were used for PCR amplification. The particular 921bp DNA fragment was found to be present in P. multocida B:2 genome as 803bp in size. Sequencing of the recombinant plasmids confirmed that the 803bp gene was 98% homologous to the reference sequence, the virulence 921bp ABA392 DNA fragment.Southern hybridization analysis revealed the 804bp of ABA392 gene was located at approximately 6kb position in the P. multocida B:2 genome. PCR performed towards the approximately 6kb DNA fragment produced an 803bp band which was confirmed to be the ABA392 virulence gene. The amplified PCR product was cloned and sequenced. Nucleotide sequences obtained were 98% identical to the reference strain, the 921bp ABA392 virulence gene. Pathogenicity test on the recombinant plasmids proved that the 804bp gene inserted within these plasmids still possessed the virulence properties of the 921bp ABA392 gene which may lead to HS disease in cattle and buffaloes. In an attempt to produce P. multocida B:2 mutants through allelic exchange, the 804bp of ABA392 gene which was disrupted with kanamycin cassette was cloned inside suicide plasmid pAKA19 through shotgun ligation technique. The desired 7kb product was transformed into several different E. coli (TOP 10, JM109, AS11Yλ and DH5α) hosts for preservation. Verification of the 7kb ligation product with digestion by PstI, HindIII and XhoI restriction enzymes revealed the exact sizes of kanamycin cassette (1.2kb), pAKA19 (5.0kb) and the disrupted 804bp of ABA392 virulence gene (804bp). An antibiotic sensitivity test on P. multocida B:2 and the respective E. coli strains were performed in order to select the donor and recipient strains for conjugation process. This test revealed that E. coli DH5α was suitable for the donor strain since it showed low resistance to streptomycin and P. multocida B:2 as the recipient strain for its high resistance towards streptomycin. Conjugation between donor and recipient strains was then achieved by plate-mating method. About 20 single colonies of positive transconjugants were picked and subcultured on BHI blood agar containing kanamycin for 5 days to encourage loss of pAKA19 plasmid and to enhance the allelic exchange between the ABA392::KmR insert with the native ABA392 gene on the recipient chromosome. No plasmid was observed which indicated the loss of suicide plasmid. Direct colony PCR was performed to detect the changes in the ABA392 gene of the parent strain, where a 2kb band was observed signifying that allelic exchange has taken place and the organism is now a P. multocida B:2 mutants. Parent strains of P. multocida B:2 were highly virulent and killed mice within 24 hours. Mice inoculated with P. multocida B:2 mutant survived. Direct smear from mice’s heart blood inoculated with the mutant showed the existence of bipolar organism which indicated the presence of P. multocida B:2. This result firmly suggests that the mutant, named as PMTBK was greatly attenuated and is thus a potential candidate organism for a live attenuated vaccine against HS

Distribution of Microbial Biomass, Carbon and Nitrogen in a Forest Reserves of Perak, Malaysia

Microbial biomass is the mass of living microorganisms in a particular ecosystem at a given time . Microbial biomass responds rapidly to different land use management techniques in at different location in forest reserves of Perak, Malaysi

Screening for active compounds in Rhus coriaria L. crude extract that inhibit the growth of Pseudomonas syringae and Ralstonia solanacearum

An experiment was performed to study the antibacterial activity of methanol, acetone, alcohol and aqueous extracts from the fruit of R. coriaria by disk diffusion assay in terms of minimum inhibitory concentrations (MIC), minimum bactericidal concentrations (MBC) and killing-time curve. The detection of the components was also fulfilled using Gas Chromatography–Mass Spectrometry (GC-MS) and also tested for their antibacterial activity. The tested bacteria were Pseudomonas syringae (Accession No. KJ858057), a tomato bacterial speck causal agent, and Ralstonia solanacearum (Accession No. KJ881159) causing tomato bacterial wilt. Furthermore, the inhibition criteria were made by different extracts of the sampled bacteria which were measured and compared with standard antibiotic (chloramphenicol). Aqueous extract displayed better outcomes against P. syringae and R. solanacearum as compared to chloramphenicol. According to the GC-MS test results, the aqueous extract was composed of 39 different phytocompounds, together with eight elements in the high peak region, namely Furfural, 1-Cyclopetene, 2,5 Furandione, Phloroglucinol, Succinic acid, Malic acid, P-Tolylacetic acid and Coumalic acid. Among these, it was discovered that 2,5 Furandione was the most important antibacterial element that is present in sumac. The results from the current study indicate that different extracts of R. coriaria contain a variety of antibacterial compounds which can potentially be used to produce an extensive range of herbal mixtures with anti-bacterial properties for controlling diseases in crops belonging to the Solanaceae family

Estimation of total phenolic acids, flavonoid compounds and antioxidant activity of Ficus deltoideavarieties and their HPLC profiles with different solvents

The aim of this study was to evaluate the effect of methanol and ethanol extraction on antioxidant activities, total polyphenol, phenolic acid and flavonoid content of different Ficus deltoideavarieties. Our findings revealed that fresh leaves of F. deltoideavar. kunstleri; FDK1 had the highest total polyphenol, phenolic acid, flavonoid and antioxidant activities compared to that of other varieties. Ethanol extraction of FDK1 showed the highest activity in total antioxidant (DPPH) (4.48 mg TE/g FW), polyphenol (1.13 mg GAE/g FW) and flavonoid content (6.81 mg RE/g FW) while methanol extraction showed the highest activity in total antioxidant (FRAP) (2.43 mg TE/g FW) and phenolic acid content (4.54 mg GAE/g FW). HPLC quantification in dried leaves of FDK1 found out rutin exhibited higher than naringin. The highest rutin and naringin was found in FDK1 and FDT2 (12.83 and 3.04 μg/g DW). These results demonstrate that extraction solvent and F. deltoideavariety influence the activity of total antioxidant, polyphenol, phenolic acid and flavonoid content

Phytoremediation studies on arsenic contaminated soils in Malaysia

Arsenic is a heavy metal that can exhibit both metallic and nonmetallic properties; concentrations in uncontaminated soil are generally in the ranges of 0.2 to 40 ppm while contaminated soils have been recorded to reach concentrations of up to 2500 ppm. Although arsenic can exist naturally, arsenic contamination occurs due to anthropogenic activities like the smelting of metals, vehicular emissions and the application of pesticides. Inorganic compounds of As can be very harmful to animals and human beings, effecting the nervous and cardiovascular system which eventually leads to death. As Malaysia faces increasing contamination problems, phytoremediation could prove to be savior as it is not only cheap, but also efficient. Cyperus rotundus, Imperata cylindrica, Ludwiga octovalvis, Lycpodium cernuum, Melastoma malabathricum, Mimosa pudica and Nelumbo nucifera are few plant species planted on soil contaminated with As

First report of Bacillus pumilus causing trunk bulges of rubber tree (Hevea brasiliensis) in Malaysia

The rubber industry is projected to increase to RM53 billion or USD1.3 billion of gross national income by 2020 in Malaysia (Malaysian Rubber Board 2009). Introductions of superclones with high yield potential such as RRIM 3001 are among the efforts to improve latex yield as well as timber yields. Superclone RRIM 3001, mostly cultivated by smallholders, yields about 3 tons of natural rubber latex per hectare per year. During September 2017, trunk bulging occurrences were observed in rubber trees (Hevea brasiliensis) RRIM 3001 superclone in Serdang, Selangor, Malaysia. These bulges appeared in different sizes and were similar to tumor-like bacteriosis on the whole trunk. Other symptoms were also observed, including depressed canker wounds with different sizes at the tapping zone and large bleeding lesions on the trunk and branches of the rubber trees. Symptomatic bark tissue of various rubber trees was collected at 1.52 m by using a sterile knife. The infected tissues were cut approximately at 0.5 × 0.5 cm, surface sterilized in 10% sodium hypochlorite, and washed two times in sterilized water. The tissues were mashed using pestle and mortar, shaken for 15 min in sterilized water, streaked onto nutrient agar (NA) medium, and incubated at 30°C for 24 h. From 15 putative strains, three potential strains (SM1, SM2, and SM3) were chosen for further characterization. Bacterial colonies were gram-positive, motile, endospore formers, catalase-positive and oxidase-negative, and produced a whitish pigment on NA medium. Polymerase chain reaction (PCR) amplification with 16S rRNA and Bsub-specific primers (Wattiau et al. 2001) was performed, and the products were sequenced. BLASTn analysis of both genes revealed that all strains were 99% identical to Bacillus pumilus strains M3 (GenBank accession no. MF461325) and PS23 (GenBank accession no. KP89557). The nucleotide sequences were later deposited in GenBank (accession nos. MH401100 to MH401102 for 16 rRNA gene and MH428001 to MH428003 for Bsub-specific gene). Phylogenetic analysis of Bsub-specific gene sequences showed all three strains were closely related to B. pumilus AGERI-PB1 reference strain in GenBank database with 99% similarity (accession no. LC385524). A pathogenicity test was carried out on 3-month-old superclone RRIM 3001 seedlings by inoculating 200 μl of 108 CFU/ml bacterial suspensions of all three strains to the upper part of the stem using a sterilized syringe. Parafilm was used to cover the inoculation sites. Six replicates were used for each treatment. Control seedlings were inoculated with sterilized water. Within 21 to 28 days postinoculation, rubber seedlings inoculated with B. pumilus strains produced symptoms such as necrosis on the leaves, cankers, exhibiting ooze, and tumor-like bacteriosis as observed naturally in the field. Control seedlings remained asymptomatic. Reisolation of the bacterium from the symptomatic tissues confirmed the presence of B. pumilus based on phenotypic characteristics and molecular characterization. To the best of our knowledge, this is the first official report of B. pumilus causing trunk bulges on rubber trees in Malaysia. Recent reports revealed that B. pumilus is pathogenic by causing disease to various type of plants including muskmelon (Song et al. 2018), ginger (Peng et al. 2013), and Scots pine (Kovaleva et al. 2015)

Genetic diversity of Ralstonia solanacearum phylotype II sequevar 4 strains associated with Moko disease of banana (Musa spp.) in Peninsular Malaysia

Moko disease caused by Ralstonia solanacearum (R. solanacearum) is a major disease affecting banana (Musa spp.) production. Although local reports suggested that this disease is widespread in Malaysia, molecular characterization of R. solanacearum strains associated with Moko disease in this country has not yet been done. During March 2011 to June 2012, 170 banana plants associated with Moko disease and the adjacent soil samples were collected in 12 different locations of five outbreak states in Peninsular Malaysia comprising Kedah, Selangor, Pahang, Negeri Sembilan and Johor, with disease incidence exceeding 80 % in some severely affected plantations. A total of 197 strains were identified as R. solanacearum-like colonies since they produced fluidal colonies that were white to pink coloration after incubation at 24 to 48 h at 29 °C on Kelman’s TZC agar medium, appeared as Gram-negative rods, and positive for potassium hydroxide (KOH), Kovacs oxidase, catalase and lipase activity on Tween 80 solution tests. Biovar tests disclosed that only 30 strains displayed characteristics of biovar 1 R. solanacearum associated with Moko disease, which was negative for utilization of disaccharides and hexose alcohols. Pathogenicity assay showed that these 30 strains were virulence towards Musa paradisiaca cv. Nipah explants with diverse degrees of virulence. Phylotype-specific multiplex PCR (Pmx-PCR) revealed all strains belonged to phylotype II displaying a 372 bp amplicon. Phylogenetic analyses of endoglucanase (egl) sequences clustered all 30 strains into phylotype II/4, together with the reference sequences strains from Peru (UW129, UW162 and UW163) and Colombia (UW070). Pooled rep-PCR fingerprinting method defined two major groups; cluster 1 (sub-group A and B) and cluster 2 (sub-group C), with 35 % average similarity coefficient within these two clusters. The sub-groups in cluster 1 were represented by strains from Kedah, Selangor, Negeri Sembilan and Johor; while cluster 2 sub-group was represented exclusively by strains of Pahang. To our knowledge, this is the first description of R. solanacearum phylotype II/4 in Malaysia and the Asian region. Our findings may expand constructive documentation and describe a better understanding on diversity of Malaysian R. solanacearum Moko-causing strains populations, thus will be useful for designing disease control strategies

Effects of different mulching materials and planting distance on selected soil properties of organic farms planted with Orthosiphon stamineus

A field study was carried out to determine the impact of mulching and planting distance on the growth of Orthosiphon stamineus, soil properties and also to observe the changes in pH and EC of soil in response to mulching and planting distance. The experiment was carried out at Ladang 16, Faculty of Agriculture UPM. Factorial Randomized Complete Block Design (RCBD) was incorporated with four replicates for each of the four treatments. The four treatments consisted of mulching, non-mulching, planting distance of 30 cm × 30 cm and planting distance of 45 cm × 45 cm. After eight weeks of planting, the plants were harvested while soil pH and EC were measured on a weekly basis throughout the planting period. Results showed that application of biochar and usage of mulching materials and suitable planting distance does helps to maintain the soil pH and electrical conductivity (EC) at the suitable range for crop growth. The level of the acidity of the soil is in the range of 5.3 to 6.61 which is considered appropriate for O. stamineus. As for influence of planting distance, it is best to give longer time for O. stamineus grows. It is recommended that more planting distance and types of mulching materials to be used to grow O. stamineus

Characterization of Ralstonia solanacearum race 2 biovar 1 associated with moko disease of banana in Peninsular Malaysia

Moko disease caused by Ralstonia solanacearum race 2 biovar 1 (R. solanacearum R2Bv1) is a major disease affecting banana (Musa spp.) production. Although local reports suggested that this disease is widespreading in Malaysia, characterization of R. solanacearum strains associated with Moko disease in this country has not been done. This study was conducted to isolate, identify and characterize R. solanacearum R2Bv1 of Moko-causing strains in Peninsular Malaysia. During March 2011 to June 2012, 170 banana plants associated with Moko disease and adjacent soil samples were collected in 12 different locations of five outbreak states in Peninsular Malaysia comprising Kedah, Selangor, Pahang, Negeri Sembilan and Johor with disease incidence exceeding 80 % in some severely affected plantations. All 197 isolates produced fluidal colonies that were white to pink coloration after incubation at 24 to 48 hours at 29 °C on Kelman‘s TZC agar medium and were divided into two defined colony type, the B and SFR types. These isolates appeared as Gram-negative rods after Gram-stain, and positive for potassium hydroxide (KOH), Kovacs oxidase, catalase and lipase activity on Tween 80 solution tests. In biovar determination, only 30 isolates displayed characteristics of biovar 1 R. solanacearum, which was negative for utilization of disaccharides and hexose alcohols. Tobacco hypersensitivity assay revealed all isolates elicited hypersensitive response (HR) at 12 h after infiltration, suggesting that they were of race 2. In preliminary pathogenicity study, all 30 isolates were virulence towards three Moko most affected local banana cultivars namely Musa paradisiaca cv. Nipah, Musa paradisiaca cv. Tanduk and Musa acuminata cv. Berangan cultivars with diverse degrees of virulence; highly virulent, moderately virulent and weakly virulent with isolate NS-N1 as the most virulent, while isolates Ked-KN4 and Ked-KN5 were classified as weakly virulent. Musa paradisiaca cv. Nipah (ABB triploid) significantly exhibited the highest degree of severity to R. solanacearum, followed by Musa paradisiaca cv. Tanduk (AAB triploid) and Musa acuminata cv. Berangan (AAA triploid). Moreover, statistical results revealed there were relationships between geographical origins of isolates and their severity, with the most and the lowest severity was related to isolates from Johor and Negeri Sembilan. This study represents the first evidence on the introduction of R. solanacearum biovar 1 associated with Moko disease of banana in Peninsular Malaysia. Partial 16S rDNA sequence analyses disclosed that all 30 isolates of R. solanacearum biovar 1 were clustered to the published R. solanacearum biovar 1 related to Moko-causing strains from the Philippines (MOD5 and R633) with 91 % Bayesian posterior probability support and completely different from Ralstonia syzygii (R. syzygii, S444E), blood disease bacterium (T520) and the outgroup strain, Xanthomonas spp. (55485). Meanwhile, phylogenetic analyses further demonstrated that all strains were grouped with 100% posterior probability support to the published R. solanacearum race 2 insertion sequence gene, ISRso19 (AF450275). Phylotype-specific multiplex PCR (Pmx-PCR) showed all strains belonged to phylotype II displaying a 372 bp amplicon. Phylogenetic analyses of endoglucanase (egl) sequences clustered all 30 strains into phylotype II/4, together with the reference sequences strains from Peru (UW129, UW162 and UW163) and Colombia (UW070). Bioinformatics analysis of pooled rep-PCR fingerprinting method defined two major groups; cluster 1 (sub-group A and B) and cluster 2 (sub-group C), with 35 % average similarity coefficient within these two clusters. The sub-groups in cluster 1 were represented by strains from Kedah, Selangor, Negeri Sembilan and Johor; while cluster 2 sub-group was represented exclusively by strains of Pahang. This is indeed the first time that genetic diversity of R. solanacearum R2Bv1 has been characterized in this country, where rep-PCR technique clearly distinguished clonal lineages of Moko-causing strains in Peninsular Malaysia. These findings provide constructive documentations on R. solanacearum R2Bv1 in Malaysia, since banana has been identified as the second most important commercial fruit crop with a high economic value in this country

Phylotype classification of Ralstonia solanacearum biovar 1 strains isolated from banana (Musa spp) in Malaysia

During 2011–2012, 15 bacterial isolates were obtained from wilting banana plants from seven locations in Malaysia. Characterisation of the Malaysian isolates was determined by biovar determination, pathogenicity test, phylotype-specific multiplex PCR (Pmx-PCR) and endoglucanase (egl) gene amplification. Based on the genotype, phenotype and pathogenic characteristics, all isolates were identified as Ralstonia solanacearum. Pmx- and egl-PCRs indicated that all isolates belong to phylotype II of Ralstonia species complex hierarchical classification. The neighbour joining phylogenetic tree of egl sequences also verified the results where the isolates were all clustered into phylotype II, together with the reference sequences strains, UW070 and UW162. Therefore, the results of our study may provide a better understanding on the taxonomy of R. solanacearum species occupying banana plantations in Malaysia. This study is indeed the first report of phylotype II classification of R. solanacearum biovar 1 strains isolated from banana plants in Malaysia