Plant diseases caused by phytopathogenic bacteria are the main cause of economic losses in the agricultural sector. Recently, an outbreak of bacterial leaf blight (BLB) occurred in the paddy fields of Sekinchan, Malaysia in October to December 2016. I would also like to thank all members in the laboratory for their help and encouragement during the last three months. especially my FYP teammate Ong Kher Nee who has made my FYP experience an unforgettable one.
CAUSING BACTERIAL LEAF BLOOD DISEASE IN PADDY PLANTS” was prepared by HAW XUE QI and submitted in partial fulfillment of the requirements for the degree of Bachelor of Science (Hons) Biomedical Sciences at Universiti Tunku Abdul Rahman. In addition, a 120-day variety will spend 60 days in the vegetative stage and 30 days in each reproductive stage and ripening stage when planted. spp.
BLB disease can be manifested by either leaf blight on mature plant or presented by kresek symptoms in the seedling stage (Agropedia, 2008; Mondal et al., 2011). In October to December 2016, BLB disease outbreaks causing severe losses to paddy farmers in the Sekinchan area have been reported (Figure 1.2). This is apparently the worst rice disease outbreak in the Sekinchan area in the last 30 years, with 90% of the 4,440 hectares of rice fields infected, causing yield losses between 50%-70%.
Moreover, the resistant cultivars were also not resilient varieties but rather susceptible to BLB disease (Muthiah, 2013; Lai and Chan, 2016).
Origin of cultivated rice
Bacterial leaf blight (BLB) disease
Rice is one of the most important food crops around the world, especially in the Asian countries. In Asia, BLB caused by Xanthomonas has been an important threat to rice production (Swings et al., 1990). In the Philippines, the susceptible rice crops experienced a yield loss of 22.5% in the wet season and 7.2% in the dry season, while the resistant rice crops experienced a smaller yield loss of about 9.5% and 1.8%, respectively (Savary et al., 2000).
In the year 1982 to 1994, there was an incidence of BLB outbreak reported in the rice fields of Peninsular Malaysia. In February 2014, another BLB outbreak caused by Xanthomonas was reported in the paddy fields of Padang Besar with an estimated yield loss of 60,000 metric tons (Utusan Online, 2014).
Apparatus and consumables
Preparation of culture media
Citrate basal medium
- MacConkey agar
- Nutrient agar
- Nutrient broth
- Oxidative fermentative (OF) medium
- Sulfide-indole-motility (SIM) medium
- Yeast dextrose chalk (YDC) agar
Nutrient agar was prepared by mixing 28 g of nutrient agar powder with dH2O to a final volume of 1 L. Nutrient broth was prepared by mixing 8 g of nutrient broth powder with dH2O to a final volume of 1 L. Medium was prepared by mixing 9.8 g of medium powder with dH2O to a final volume of 1 L.
SIM medium was prepared by mixing 30 g of SIM medium powder with dH2O to a final volume of 1 L. YDC agar was prepared by mixing 10 g of dextrose, 5 g of yeast extract, 10 g of calcium carbonate and 10 g of agar with dH2O to a final volume of 1 L .
Preparation of sterilized reagents
- Crystal violet, 0.1% w/v
- Glucose solution, 10% w/v
- Glycerol solution, 65% v/v
- Saline solution, 0.85% w/v
- Tris-acetate-EDTA buffer
Saline (0.85% w/v) was prepared by mixing 8.5 g of sodium chloride powder with dH2O to a final volume of 1 L.
List of bacterial strains that used as positive and negative controls in biochemical tests
Phenotypic observation of Pantoea strains .1 Preparation of master plates
Preparation of bacterial smear
The bacterial smear was first treated with 0.1% crystal violet for 1 minute, followed by rinsing with water. The bacteria are then treated with Lugol's iodine solution for 1 minute, washed for 30 seconds and decolorized with 95% ethanol. After that, the discolored bacteria were rinsed again with water and stained with safranin for 1 minute.
Biochemical tests
- Determination of PCR products by agarose gel electrophoresis Targeted PCR products were confirmed by electrophoresis on 2% w/v agarose
- Purification of PCR products
- DNA sequencing
- DNA sequence data analysis via BLASTn
The concentration of the purified PCR products was assessed with nanospectrophotometer as before (Section 3.9.1) and the concentration must be greater than 10 ng/uL to be sent for DNA sequencing. The purified PCR products together with forward and reverse primers in separated tubes were sent directly for DNA sequencing at First Base Laboratories Sdn Bhd Shah Alam Selangor, Malaysia. The DNA sequencing data of four housekeeping genes were first deposited in NCBI genebank to determine the identity and accession numbers for the Pantoea strains.
Local Alignment Search Tool Nucleotide (BLASTn) analysis, an open access database, which is available on the National Center for Biotechnology Information (NCBI) website with URL: https://blast.ncbi.nlm.
Long-term storage of bacterial strains
16S rRNA gene sequencing versus the API 20 NE system and the VITEK 2 ID-GNB card for identification of non-fermenting gram-negative bacteria in the clinical laboratory. Phylogeny and identification of Pantoea species associated with plants, humans and the natural environment based on multilocus sequence analysis (MLSA). Improved description of the genus Pantoea, description of four species from human clinical specimens, Pantoea septica sp.
Plant growth-promoting rhizobacteria mediate systemic-induced resistance in rice against bacterial leaf blight caused by Xanthomonas oryzae pv. An unusual stem necrosis of rice caused by Pantoea ananas and the first record of this pathogen in rice in Australia. Evolution of the gyrB gene and the molecular phylogeny of Enterobacteriaceae: a model molecule for molecular systematic studies.
Phylogeny and identification of Pantoea species and typing of Pantoea agglomerans strains by multilocus gene sequencing, Journal of Clinical Microbiology, 47 (2), p. Production of the antifungal compound pyrrolnitrin is regulated by quorum sensing in members of the Burkholderia cepacia complex. Reclassification of the causative agents of bacterial blight (Xanthomonas campestris pv. oryzae) and bacterial leaf spot (Xanthomonas campestris pv. oryzicola) of rice as pathogens of Xanthomonas oryzae (ex Isiyama 1922) sp.
Identification of the maize pathogen Pantoea stewartii by mass spectrometry of whole cell extracts and its detection with new PCR primers. Association of Pantoea ananatis and Pantoea agglomerans with leaf spot disease on ornamental plants of the Araceae family. Control of Penicillium digitatum on orange fruit combining Pantoea agglomerans with hot sodium bicarbonate dip.
