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Biocontrol Potential, Genome and Nonribosomal Peptide Synthetase Gene Expression of Bacillus velezensis 2211
(2023-01-01)
Niemhom, Nantawan
;
Kittiwongwattana, Chokchai
Members of the genus Bacillus produced a diverse group of antimicrobial compounds. Here, we presented the antifungal activity and genome sequence analysis of Bacillus sp. 2211, a potential plant-growth-promoting bacterium. Bacterial supernatants from strain 2211 cultures in nutrient broth (NB) and potato dextrose broth (PDB) suppressed the mycelial growth of Pyricularia oryzae, Colletotrichum aenigma, Colletotrichum fructicola and Fusarium oxysporum. The supernatants were also able to suppress spore germination of these fungi, except for F. oxysporum. However, the supernatant from PDB displayed a significantly higher inhibition activity than NB. Additionally, the supernatant from PDB significantly reduced the disease severity caused by P. oryzae on rice seedlings. The genome of strain 2211 was sequenced. The highest digital DNA-DNA hybridization (80.1%) and average nucleotide identity (97.57%) levels indicated that strain 2211 was a member of the species Bacillus velezensis. The phylogenomic analysis showed that it clustered with B. velezensis NRRL B-41580T, B. velezensis KACC 13105 and B. velezensis subsp. plantarum FZB42T. The gene expression analysis showed the up-regulation of nonribosomal peptide synthetase (NRPS) genes bmyA, fenB and dhbE in PDB, compared to NB. This work demonstrated that the culture media affected the antagonistic activity of strain 2211 possibly through the modification of NRPS biosynthesis genes.
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Analysis of secondary metabolites and genes related to the pathogenicity of the rice blast fungus Pyricularia oryzae
(2022-09-01)
Pradapphai, P.
;
Kumlung, T.
;
Parinthawong, N.
Pyricularia oryzae causes rice blast disease and is one of the most significant plant pathogens worldwide. The rice blast fungus can produce a variety of phytotoxic secondary metabolites and has been found to be essential for host invasion. Two isolates, virulent isolate RBR55003 and non-virulent isolate CCO56003 were selected which based on their abilities to cause blast symptoms on rice leaves. Pyriculol and picolinic acid were detected in culture filtrate of both isolates during the exponential growth phase. Expression of genes involved in fungal pathogenicity and plant defense were analyzed. The expression of MoPks19 gene in isolate RBR55003 was higher than avirulent isolate CCO56003, indicating that MoPks19 may play a role in the pathogenesis of RBR55003. The expression of the OsWRKY30 gene that plays an important role in the regulation of a defense-related gene in rice was higher in CCO56003 inoculated than in RBR55003 inoculated rice sample, indicating the increased resistance of rice plants occurring in rice after non-virulent isolate infection, while the expression of the OsCPS4 gene involved in phytoalexin synthesis in rice was not different after isolate infections. Gene expression analysis revealed different responses according to plant-pathogen relationships.
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Correlation of Antifungal Activities and Nonribosomal Peptide Synthetase Gene Expression of Bacillus siamensis 1021
(2022-03-01)
Nuwong, Warinya
;
Kittiwongwattana, Chokchai
Members of the genus Bacillus were biocontrol agents against various phytopathogenic fungi. Several factors affected the regulation and production of nonribosomal peptides (NRPs) in Bacillus species. The aim of this study was to examine how culture media influenced the antagonistic activities and gene expression of Bacillus siamensis 1021. The bacterial supernatant from potato dextrose broth (PDB) exhibited higher inhibitory effects against mycelia of Pyricularia oryzae and Colletotrichum fructicola, when compared with those from nutrient broth (NB) and minimal salt medium (MSM). However, the biocontrol activities of these supernatants were not significantly different when they were tested against P. oryzae and C. fructicola conidia. Additionally, the bacterial supernatant from PDB significantly reduced the disease severity caused by P. oryzae in rice seedlings when compared with the water-treated control group. The genome of strain 1021 was sequenced, and biosynthetic gene clusters of nonribosomal peptides surfactin, fengycin and bacillibactin were identified. Effects of PDB, NB and MSM media on expression levels of core biosynthetic genes of surfactin (srfAA), fengycin (fenC) and bacillibactin (dhbE) gene clusters were determined by reverse-transcription quantitative PCR. fenC expression was significantly increased in PDB and corresponded with the antagonistic activities against fungal mycelia. Conversely, expression of the regulatory genes comA and codY in PDB were highly reduced in PDB, indicating their negative relation with fenC expression. This expression analysis indirectly suggested that fengycin was potentially the bioactive compound of B. siamensis 1021 against P. oryzae and C. fructicola.
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Medium Effect on Antagonistic Activity and Detection of Nonribosomal Peptide Synthetase Genes in Epiphytic Bacillus Strains
(2021-01-01)
Apimeteethamrong, Suchitra
;
Kittiwongwattana, Chokchai
The biosynthesis of non-ribosomal peptides (NRPs) in biocontrol bacteria was one of the major antagonistic mechanisms for their application in agriculture. Bacillus spp. 1021, 2211 and 3210 were previously shown to inhibit mycelial growth of the leaf blast fungus Pyricularia oryzae. Here, we aimed to further study the antagonistic mechanism in those three strains. Cell-free supernatants obtained from bacteria grown in potato dextrose broth (PDB) exhibited a higher degree of inhibition against P. oryzae when compared to those obtained from nutrient broth (NB). This indicated the effect of culture media in the production of extracellular antibiotic compounds by these strains. Phylogenetic analysis of their partial 16S rRNA gene sequences indicated a close relationship between the three strains and Bacillus siamensis KCTC13613T, Bacillus amyloliquefaciens DSM7T and Bacillus velezensis CR-502T. Complete genome sequences of these Bacillus species were analyzed on the antiSMASH server to identify the presence of NRP biosynthesis gene clusters. Non-degenerate primers were designed for the detection of the core biosynthesis genes for surfactin (srfAA), fengycin (fenC) and bacillibactin (dhbF). All three genes were amplified in strains 1021 and 2211, while only srfAA and dhbF were detected in strain 3210. Phylogenetic analysis of the deduced amino acid sequences indicated that the sequences of strain 1021 were distinct from those of strains 2211 and 3210. This result indirectly suggests the possibility of NRP production as the antagonistic mechanism of these three strains.
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Pathogenicity of Pyricularia oryzae on elite rice cultivars and geographical distribution of avirulence genes causing blast disease in Thailand
(2020-07-01)
Parinthawong, N.
;
Tansian, P.
Pyricularia oryzae is a plant pathogenic fungus causing rice blast disease. In Thailand, there is very little information about pathogenicity and avirulence gene of rice blast pathogen. Each of the 50 blast isolates obtained from blast disease samples collected from 19 provinces was investigated for avirulence gene on 31 near isogenic lines (NILs) and their pathotype on elite rice cultivars was determined using 24 Thai elite rice cultivars. Among the 50 isolates, Avr-Pita-2 was discovered mostly in isolates from the North (83%), Avr-Pikp and Avr-Pi7(t) were discovered in isolates from the North East (94%), while 8 avirulence genes including Avr-Pikp, Avr-Pikh, Avr-Pi1, Avr-Pi7(t), Avr-Piz, Avr-Pi12, Avr-Pi19 and Avr-Pi20 were discovered in isolates from the South (88%) and Avr-Pi9 was discovered in isolates from the Central region of Thailand (71%). Isolates BRM60001 that carried the most numbers of Avr gene was unable to infect any elite rice cultivars, while isolates that carried as less as 2 Avr genes could infect few elite rice cultivars. Isolate CRI59004.1 carried Avr-Pik (Avr-Pikh, Avr-Pikm, Avr-Pi7(t)), Avr-Pi9, Avr-Pish, Avr-Pib, Avr-Piz-5, Avr-Pita, Avr-Pita-2 and Avr-Pi19, could infect the greatest numbers of elite rice cultivars. The information regarding the distribution of Avr gene in P. oryzae will be useful for forecasting rice blast and disease resistance breeding programs.
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Investigation of rice blast resistant genes in Thai elite rice varieties (Oryza sativa L.) for improvement of broad-spectrum blast disease resistance variety
(2020-01-01)
Poonsin, R.
;
Parinthawong, N.
The broad-spectrum blast disease resistance rice Huai variety (GS19567) has been reported to contain more than one blast resistance genes which one of them was mapped on chromosome 11. The blast resistance genes was investigated using gene specific primers in Huai and other 40 Thai elite rice varieties. The results obtained from polymerase chain reaction revealed that all tested varieties contained at least one blast resistant gene. The most frequent finding resistant gene was Pib, while Pib was missing in Huai variety. Therefore, in order to enhance broad spectrum blast disease resistance in a sustaining blast resistance variety, the resistance gene Pib is recommened to be used as candidate gene in breeding program.
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Blast resistance evaluation and genetic inheritance of gene controlling leaf blast resistance in Dawk Pa-yawm Rai variety (GS23774)
(2020-01-01)
Janthasri, S.
;
Parinthawong, N.
The landrace rice Dawk Pa-yawm Rai variety was highly resistant to 18 blast isolates, moderately resistant to 5 and susceptible to 2 blast isolates. Therefore, Dawk Pa-yawm Rai variety was broad-spectrum resistance to many blast isolates and can be used as genetic resource in breeding programs for blast disease resistance. The genetic inheritance of blast resistance in F<inf>2</inf> population was identified. Seven hundred and thirty F<inf>2</inf> plants from a cross between KDML105 (recipient) and Dawk Pa-yawm Rai (donor) were generated. The disease assessment revealed 524 F<inf>2</inf> plants were resistant and 206 F<inf>2</inf> plants were susceptible. The segregation of resistance and susceptible phenotypes showed a goodness of fit to the ratio 3:1 (R:S). The result found is important factor and useful in mapping of blast resistant gene in Dawk Pa-yawm Rai variety.
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Classification and diversity assessment of Pyricularia oryzae based on leaf and neck blast pathogenesis on Khao Dawk Mali 105
(2020-01-01)
Pradapphai, P.
;
Parinthawong, N.
Forty isolates of P. oryzae were obtained from disease outbreak areas in various regions of Thailand. The fungi were divided into two groups; group 1 included 2 isolates which cause only neck blast with disease score of 7 – 9 and group 2 included 23 isolates which cause leaf and neck blast symptoms with disease scores of 5 – 6 and 7 – 9, respectively. The two groups of fungi were tested on 31 NILs. The avirulence genes including Avr-Pik (Avr-Pik, Avr-Pikm, Avr-Pikp), Avr-Pi1, Avr-Pi3, Avr-Pi5(t), Avr-Pi7(t), Avr-Pi9, Avr-Pi20, Avr-Pii and Avr-Pita were found in both groups whereas Avr-Pi11, Avr-Pi12, Avr-Pi19, Avr-Pia, Avr-Pib, Avr-Pikh, Avr-Piks, Avr-Pish, Avr-Pita, Avr-Piz, Avr-Piz-5 and Avr-Piz-t were detected only in the group 2. The results explained the relationship between fungi and the avirulence genes functioned at different stages of rice.
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Investigation of blast resistance genes in a broad-spectrum resistant indigenous rice, Yang Mawng variety
(2020-01-01)
Suksiri, W.
;
Parinthawong, N.
The indigenous rice Yang Mawng variety was highly resistant to 24 isolates and moderate resistant to 3 isolates of P. oryzae. The results confirmed the broad-spectrum resistance to blast disease of Yang Mawng variety. The pathogenicity test of 27 isolates of P. oryzae was conducted on 31 Near Isogenic Lines (NILs). The result showed that virulence index (VI) was valued from 0 to 0.50 and the most pathogenic isolate on NILs was UBN2010 13515. The investigation for 8 rice blast disease resistance genes of Yang Mawng variety was done using blast resistance gene specific primers and PCR technique. The result showed that 5 blast resistance genes including Pi9, Pigm(t), Pi54, Pib and Pita, were detectable. The information of this research is a preliminary data for study of resistance genes present in Yang Mawng variety.
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Diversity and plant growth promoting activities of rice epiphytic bacteria
(2019-01-01)
Aphimeteetamrong, Suchitra
;
Kittiwongwattana, Chokchai
Plant-associated bacteria have been known for their direct and indirect abilities to promote plant growth. Because of their benefits on plants, they are generally recognized as plant growth promoting bacteria (PGPB). In the present study, epiphytic bacteria were isolated from surfaces of roots, stems and leaves of rice plants that were grown in organic farms. Of 113 bacterial isolates, 68 and 45 isolates were stained Gram-positive and Gram-negative, respectively. Based on their cell shapes, 106 isolates were bacilli while seven isolates were cocci. The use of the 16S rRNA gene sequence analysis indicated that they were members of phyla Fimicutes (54.87%), Proteobacteria (38.05%) and Bacteroidetes ( 7. 08% ). All isolates were assessed for their abilities in nitrogen fixation, phosphate solubilization, siderophore production, auxin production and 1-aminocyclopropane-1-caboxylic acid (ACC) deaminase activity. The result showed that 91 isolates were tested positives for at least one activity. The antagonistic activity against the pathogenic fungus Pyricularia oryzae was also tested. Twenty-three isolates inhibited the growth of the fungal mycelia. The percentage of growth inhibition of these isolates against P. oryzae ranged from 73.10+3.24% to 100.00+0.00%. The protease and chitinase activities were found in 19 and 2 isolates, respectively. However, crude preparation of bioactive compounds from bacterial cultures yielded negative results. Based on the results obtained in this study, the surfaces of rice plants were colonized by various bacteria with potentials in plant growth promotion.

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