Erwinia mallotivora was isolated from papaya infected with dieback disease showing the typical symptoms of greasy, water-soaked lesions and spots on leaves. Phylogenetic analysis of 16S rRNA gene sequences showed that the strain belonged to the genus Erwinia and was united in a monophyletic group with E. mallotivora DSM 4565 (AJ233414). Earlier studies had indicated that the causal agent for this disease was E. papayae. However, our current studies, through Koch’s postulate, have confirmed that papaya dieback disease is caused by E. mallotivora. To our knowledge, this is the first new discovery of E. mallotivora as a causal agent of papaya dieback disease in Peninsular Malaysia. Previous reports have suggested that E. mallotivora causes leaf spot in Mallotus japonicus. However, this research confirms it also to be pathogenic to Carica papaya.
Erwinia mallotivora was isolated from papaya trees infected with dieback disease, which were planted at the Malaysian Agricultural Research and Development Institute (MARDI), Malaysia. Here, we report a draft genome sequence of E. mallotivora BT-MARDI, which offers an important source of information for understanding pathogen and host interaction during papaya dieback development.
Microbial proteases constitute one of the most important groups of industrially relevant enzymes. Proline iminopeptidases (PIPs) that specifically release amino-terminal proline from peptides are of major interest for applications in food biotechnology. Proline iminopeptidase has been extensively characterised in bacteria and filamentous fungi. However, no similar reports exist for yeasts. In this study, a protease gene from Glaciozyma antarctica designated as GaPIP was cloned and overexpressed in Escherichia coli. Sequence analyses of the gene revealed a 960 bp open reading frame encoding a 319 amino acid protein (35,406 Da). The purified recombinant GaPIP showed a specific activity of 3561 Umg−1 towards L-proline-p-nitroanilide, confirming its identity as a proline iminopeptidase. GaPIP is a cold-active enzyme with an optimum activity of 30 °C at pH 7.0. The enzyme is stable between pH 7.0 and 8.0 and able to retain its activity at 10–30 °C. Although GaPIP is a serine protease, only 25% inhibition by the serine protease inhibitor, phenylmethanesulfonylfluoride (PMSF) was recorded. This enzyme is strongly inhibited by the presence of EDTA, suggesting that it is a metalloenzyme. The dimeric structure of GaPIP was determined at a resolution of 2.4 Å. To date, GaPIP is the first characterised PIP from yeasts and the structure of GaPIP is the first structure for PIP from eukaryotes.
Sheath blight disease caused by Rhizoctonia solani is the most destructive diseases in rice cultivation. Development of transgenic indica rice MR219 through Agrobacterium tumefaciens strain EHA 105 harbouring the plasmid pCAMBIA 1305.2 with endo-beta-1,3-1,4-glucanase gene from Bacillus SP 289 is one of the strategies to engineer disease resistance. Four optimisation parameters were examined such as Agrobacterium culture cell density (0.1 to 1.0 based on OD600nm), callus immersion time in the Agrobacterium culture (30 to 120 minutes), duration of the subsequent drying time (15 to 120 minutes) and co-cultivation period (1 to 6 days). Hygromycin-resistant embryogenic calli developed after 8 to 10 weeks of transformation. Improved transformation rates were achieved when calli were incubated with an Agrobacterium suspension with a culture density of OD 600nm 0.2 for 30 mins, followed by 90 mins of drying time and co-cultivation for 3 days. PCR analysis of the transformants confirmed the presence of Bacillus SP 289 endo-beta-1,3-1,4-glucanase and hpt genes in the rice genome. The transgenic rice plants obtained in this study will be tested against sheath blight disease.
Blood disease bacterium A2 HR-MARDI was isolated from banana plants infected with banana blood disease and which were planted in Kuala Kangsar, Malaysia. Here, we report a draft genome sequence of blood disease bacterium A2 HR-MARDI, which could provide important information on the virulence mechanism of this pathogen.
A novel gene (CAS2) specifically expressed during appressorium formation was isolated from Colletotrichum gleosporioides using Differential Display RT-PCR. CAS2 comprises 368 deduced amino acid residues and is 50% identical to a hypothetical protein from Chaetomium globosum. ProtFun 2.2 server analysis predicted that Cas2 functions as a transport and binding protein. Based on putative transmembrane domain prediction software (HMMTOP), Cas2 protein is composed of five alpha-helical transmembrane domains with a very short external N-terminus tail and long internal C-terminus. ExPASy ScanProsite analysis showed the presence of integrin beta chain cysteine-rich domain, N-myristoylation site, EGF-like domain, 2Fe-2S ferredoxins, iron-sulfur binding region, VWFC domain, fungal hydrophobins signature, membrane lipoprotein lipid attachment site and Janus-faced atracotoxin (J-ACTX) family signature in CAS2 protein. Mutants with deleted CAS2 were not significantly different in terms of vegetative growth, conidiation, and appressoria production compared to wild type. However, the cas2 mutant produced multipolar germination, a feature which distinguishes it from wild type strain. Interestingly, the mutant is non-virulent to mango fruits, indicating that CAS2 may encode proteins that function as novel virulence factors in fungal pathogens.
Pi9 is one of the major blast resistance genes which encodes a nucleotide-binding site-leucine-rich repeat (NBS-LRR) domain-containing protein. This gene was observed to show resistance against many pathotypes of the blast pathogen in Malaysia. Resistance allele Pi9 from rice variety 75-1-127 had previously been cloned using map-based cloning strategy. The gene sequence was used to design specific primers to amplify susceptible Pi9 allele from MR219 rice variety prior to cloning. The resistance and susceptible allele of Pi9 were 8.587kb and 8.785kb in length respectively. Allele mining was carried out by comparing between the susceptible and resistance allele of Pi9. One potential InDels polymorphism at position 590bp and 920bp was identified. Primer named as Pi9_InDel was designed targeting this region in such a way that the resistance and susceptible genotypes yielded 327 bp and 438 bp amplicon respectively.
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