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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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Validation of reference genes for the normalization of RT-qPCR gene expression in Bacillus siamensis 1021 grown in different culture media
(2022-01-01)
Nuwong, Warinya
;
Kittiwongwattana, Chokchai
Background and Objectives: House-keeping genes are generally selected as reference genes in gene expression analysis. However, some genes may not be stably expressed across all experimental conditions. Thus, this study aimed to validate seven house-keeping genes for gene expression analysis in Bacillus siamensis 1021. Materials and Methods: Strain 1021 was grown in potato dextrose broth, nutrient broth and mineral salt medium. Re-verse-transcription quantitative PCR was used to determine Cq values of seven reference genes including gyrA, gyrB, ssb and dnaB, rpsU, gat_Yqey and udp in these media. Expression stability of these genes was analyzed, using geNorm and Normfinder applications. The target gene ftsZ was used for assessment of the best candidate genes. Results: Based on geNorm and Normfinder, ssb was the most-stably expressed gene, while udp was the least-stably expressed gene. Pairwise variation indicated the combination of ssb, gyrA, gyrB and gatB_Yqey was suitable for the normalization of ftsZ expression. ftsZ expression in potato dextrose broth and mineral salt medium was higher than that in nutrient broth. In contrast, the normalization against udp resulted in an under-and overestimation of ftsZ expression in potato dextrose broth and mineral salt medium, respectively. Conclusion: The combination of ssb, gyrA, gyrB and gatB_Yqey was the best candidate for normalization of target gene expression in B. siamensis 1021 in these media. This study emphasized the significance of reference gene validation for gene expression analysis and provided a guideline for future gene expression studies in B. siamensis.
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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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β-glucosidase producing Bacillus isolated from Thua-nao, an indigenous fermented soybean food in Thailand
(2017-10-01)
Meannui, Nutwara
;
Riebroy, Siriporn
;
Tangwatcharin, Pussadee
;
Hong, Jeong Hwa
;
Sumpavapol, Punnanee
The characteristics of commercial dried flatten Thua-nao collected from Northern of Thailand, namely oven dried (LPO) and sun dried (LPS) samples from Lamphun, sun dried sample from Chiang Mai (CMS) and sun dried sample from Chiang Rai (CRS),were investigated. The variations of chemical and physical characteristics were found among these Thua-nao products. Seventeen isolates of spore-forming bacteria were obtained from such products. Among these, four isolates (LPO-2, LPO-4, LPS-2 and CMS-1) were identified as Bacillus subtilis group by API 50CHB kit. For cellulolytic enzyme production, CMS-1 exhibited the highest clear zone on tryptic soy agar supplemented with carboxymethyl cellulose (P<0.05). Contrastingly, LPO-2 had the highest β-glucosidase activity with the lowest clear zone of cellulolytic enzyme activity (P<0.05). From 16S rRNA gene sequencing result, LPO-2 could be identified as Bacillus sp. and closely related to B. subtilis group.
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Isolation and evaluation of biocontrol agents in controlling anthracnose disease of mango in Thailand
(2016-07-01)
Rungjindamai, Nattawut
The agricultural based economy is a core business in Thailand and food export is one of the main sources of income for the Thai population. However, pesticides are overused and misused. As a result there is an urgent need to reduce the use of synthetic chemicals. Biological control offers an alternative to the use of pesticides. Mango (Mangifera indica L.) is widely planted in Thailand and is one of the major cash crops for international export. However, mango suffers from various diseases especially anthracnose, a fungal disease caused by Colletotrichum gloeosporioides. One hundred and twelve isolates of epiphytic microbes were isolated from healthy leaves and fruits of mangoes; this included 93 and 19 isolates of epiphytic bacteria and yeasts, respectively. They were screened for bioactivity against a pathogenic strain of C. gloeosporioides isolated from diseased mangoes using a dual culture technique. Out of 112 isolates, eight isolates exhibited at least 60% inhibition. These isolates were further screened for their inhibition on mango using fruit inoculation. Two isolates reduced the lesion sizes caused by C. gloeosporioides compared to control treatment. These two isolates, based on phenotypical and biochemical tests, were identified as Bacillus sp. MB61 and Bacillus sp. LB72.
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A novel strategy to reduce overwintering inoculum of Monilinia laxa
(2014-10-02)
Rungjindamai, Nattawut
;
Jeffries, Peter
;
Xu, Xiang Ming
Brown rot on cherry and plum, caused by Monilinia laxa, is an important disease, for which overwintered mummified fruit is a significant inoculum source for infection of flowers and fruit in the spring. Experiments were conducted to assess the potential of applying plant protection products in winter and/or early spring to suppress sporulation on mummified fruit. Products tested included Indar 5 EW (a commercial fungicide, a.i. fenbuconazole), Aureobasidium pullulans Y126 [a candidate biocontrol agent (BCA)]), Bacillus sp. B91 (a candidate BCA) and Serenade (a commercially formulated BCA of Bacillus subtilis strain QST 713) in addition to control treated with tap water. Indar and A. pullulans Y126 significantly reduced sporulation when applied once in winter. Overall, a single treatment in early spring (February) was slightly more effective than single treatment in winter (November). Application of all products in both winter and early spring led to significant reduction in sporulation. Indar had the highest efficacy, reducing the number of spores from 9 × 105 to 5 × 103 per mummified plum when applied twice. Of the three BCAs applied on both occasions, A. pullulans Y126 had the highest efficacy, reducing the number of spores from 9 × 105 to 5 × 104 per mummified plum. There were no synergistic but additive effects between the two applications in winter and early spring based on the Bliss independence test. These results suggest that reducing overwintering inoculum in dormant season is effective and may be part of an integrated management strategy for brown rot on stone fruit.

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