Studies of some genes of Bacillus subtilis MN99  local strains against plant diseases

The objective of this study was the isolation and characterization of Bacillus subtilis local strains from the soil in Mongolia. These local strains of B. subtilis are showed to have high antagonistic activities against some plant pathogenic fungi and bacteria. Six strains of B. subtilis were isolated and characterized morphologically, physiologically and biochemically according the Bergey’s Manual of Systematic Bacteriology. In order to identify species of the isolated strains, we amplified and sequenced 16S rRNA gene, essential funtinal genes bmyB, spoVG and srfAA, which are related to antagonistic activity of these strains. The sequences were aligned using CLASTALW multiple sequence alignment tool. Phylogenetic tree was drawn according to Maximum likelihood”method and “Tamura-Nei” model using “MEGA-X version 10.2.6 program. Among all isolates of B. subtilis MN99 and 7/24 strains had higher antagonist activity against plant diseases. According to partial sequence of srfAA (620bp) gene of MN99, the local strain belongs to B. subtilis and partial sequence of bmyB (370bp), spoVG (22bp) gene of MN7/24 strain showed that the it belongs to B. atrophaeus species. All local strains of B. subtilis had bacillomycin synthesis gene, and B. subtilis MN99 strain had only surfactine synthesis gene, while did not have spore formation and hemolysis gene SpoVG.

Download Full-text

Molecular characterization of Nocardiopsis species from Didwana dry salt lake of Rajasthan, India

Journal of Applied and Natural Science ◽

10.31018/jans.v13i1.2574 ◽

2021 ◽

Vol 13 (1) ◽

pp. 396-401

Author(s):

Khushbu Parihar ◽

Alkesh Tak ◽

Praveen Gehlot ◽

Rakesh Pathak ◽

Sunil Kumar Singh

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Bioactive Compounds ◽

Salt Lake ◽

Bioactive Compound ◽

Rrna Gene ◽

Biochemical Tests ◽

Multiple Sequence ◽

Isolation And Characterization

The genus Nocardiopsis is well known to produce secondary metabolites especially antibacterial bioactive compound. Isolation and characterization of bioactive compounds producing novel isolates from unusual habitats are crucial. The present study was aimed to explore Didwana dry salt lake of Rajasthan state in India for the isolation and characterization of actinomycetes. The isolated actinomycetes isolates were characterized based on culture characteristics, biochemical tests and 16S rRNA gene sequencing. The 16S rRNA gene sequence analysis revealed that all the five isolates inhabiting soil of the said dry salt lake of Didwana, Rajasthan belonged to four species of Nocardiopsis viz., N. synnemataformans, N. potens, N. prasina and N. dassonvillei subsp. albirubida. The molecular identification based on 16S rRNA gene sequences was found accurate and robust. The phylogram generated through multiple sequence alignment of all the test isolates of Nocardiopsis revealed that the isolates aroused from a single branch and validated monophyletic association. The present study is the first report of exploring Nocardiopsis isolates from the dry salt lake. These characterized Nocardiopsis isolates isolated from Didwana dry salt lake habitat are novel stains and can be of significance in the detection and utilization of novel bioactive compounds.

Download Full-text

Chitinolytic Bacillus subtilis Ege-B-1.19 as a biocontrol agent against mycotoxigenic and phytopathogenic fungi

Turkish Journal of Biochemistry ◽

10.1515/tjb-2018-0144 ◽

2018 ◽

Vol 44 (3) ◽

pp. 323-331

Author(s):

Ozlem Oztopuz ◽

Nermin Sarigul ◽

Fakhra Liaqat ◽

Ro-Dong Park ◽

Rengin Eltem

Keyword(s):

Molecular Weight ◽

Bacillus Subtilis ◽

Biocontrol Agent ◽

Pathogenic Fungi ◽

Phytopathogenic Fungi ◽

Aspergillus Ochraceus ◽

Lytic Enzyme ◽

Rrna Gene ◽

Lytic Enzymes ◽

16S Rrna Gene Analysis

Abstract Background Biological control of pathogenic fungi is a possible alternate to the chemical control, which is harmful to humans and environment. Soil-borne Bacillus strains can be potential biocontrol agents and a source of lytic enzymes. Aim This study aimed to examine biocontrol potential and lytic enzyme activities of a soil isolate Bacillus subtilis Ege-B-1.19. Materials and methods Strain was identified by biochemical and 16S rRNA gene analysis and its biocontrol activity was investigated against Aspergillus niger EGE-K-213, Aspergillus foetidus EGE-K-211, Aspergillus ochraceus EGE-K-217, Fusarium solani KCTC6328, Rhizoctonia solani KACC40111 and Colletotrichum gloeosporioides KACC40689. Chitinase, chitosanase, N-acetyl-β-hexosaminidase and protease activities of B. subtilis Ege-B-1.19 were also determined. Chitosanase was purified using Sephadex G-150 column and its molecular weight was determined by SDS-PAGE. Chitooligosaccharides production using chitosanase was carried out and analysed by TLC and HPLC. Results Results depicted that B. subtilis Ege-B-1.19 has shown inhibitory effects against all the test fungi. Chitinase, chitosanase, N-acetyl-β-hexosaminidase and protease activities were determined as 2.7 U mL−1, 7.2 U mL−1, 6.2 U mL−1 and 12.2 U mL−1, respectively. Molecular weight of purified chitosanase was 44 kDa. Chitosanase hydrolysed chitosan to glucosamine (GlcN), dimers (GlcN)2 and trimers (GlcN)3. Conclusion Bacillus subtilis Ege-B-1.19 can be anticipated as useful biocontrol agent and its chitosanase can be utilized for enzymatic synthesis of chitooligosaccharides.

Download Full-text

ISOLATION AND SELECTION OF INDIGENOUS ANTIFUNGAL MICROORGANISMS AGAINST PATHOGENIC FUNGI OF PEPPER PLANT IN TAY NGUYEN

Vietnam Journal of Biotechnology ◽

10.15625/1811-4989/16/2/13452 ◽

2018 ◽

Vol 16 (2) ◽

pp. 385-392

Author(s):

Pham Thi Thuy Hoai ◽

Ton That Huu Dat ◽

Tran Thi Hong ◽

Nguyen Thi Kim Cuc ◽

Tran Dinh Man ◽

...

Keyword(s):

Fungal Pathogens ◽

Rapid Development ◽

Control Plant ◽

Morphological Characteristics ◽

Pathogenic Fungi ◽

Plant Diseases ◽

Rrna Gene ◽

Pepper Plant ◽

Bacterial Strains ◽

Genus Streptomyces

The pathogenic fungi often cause huge impacts on agricultural crops, and occupy over 80% of plant diseases. Fusarium oxysporum and Rhizoctonia solani are fungal pathogens that can lead to rapid development of plant diseases on important crops in Tay Nguyen (e.g., pepper, coffee, rubber, cashew). Therefore, the study of microorganisms with bioactivity against these pathogens is essential to control plant diseases. In this study, we isolated microorganisms from rhizospheres of pepper in Tay Nguyen and screened beneficial microbes against two pathogenic fungi using agar well diffusion assay. Obtained results showed that there are different about isolated microbial density between samples collected from diseased and healthy pepper. The bacterial population is higher in rhizosphere region of healthy pepper than in those of diseased plants. In contrast, fungal density is lower in rhizosphere region of healthy plants than in those of diseased ones. From isolation plates, we selected and purified 391 strains including 236 bacteria, 149 actinomycetes and 6 fungi for screening antifungal activity. Out of isolated microorganisms, 44 strains (36 bacteria, 6 actinomycetes, and 2 fungi) showed antagonistic activity against at least one of two pathogens (F. oxysporum and R. solani), of which 15 isolates showed activity against both fungi. Identification of isolates with highest activity using the 16S rRNA gene sequences showed bacterial strains belonged to different species Enterobacter ludwigii, Pseudomonas fulva, Bacillus subtilis, whereas 2 actinomycetes belonged to the genus Streptomyces: Streptomyces sp. and Streptomyces diastatochromogenes. Identification of the isolated fungus based on morphological characteristics and the 18S rRNA gene sequence revealed that this strain belonged to species Penicillium oxalicum. Our study revealed the potential of the indigenous microorganisms in preventing and controlling plant-pathogenic fungi.

Download Full-text

In vitro inhibition of plant pathogens by Bacillus subtilis and Enterobacter aerogenes and in vivo control of two postharvest cherry diseases

Canadian Journal of Microbiology ◽

10.1139/m86-178 ◽

1986 ◽

Vol 32 (12) ◽

pp. 963-967 ◽

Cited By ~ 50

Author(s):

R. S. Utkhede ◽

P. L. Sholberg

Keyword(s):

Bacillus Subtilis ◽

Verticillium Dahliae ◽

Enterobacter Aerogenes ◽

Brown Rot ◽

Pathogenic Fungi ◽

Pythium Ultimum ◽

Colletotrichum Lindemuthianum ◽

Plant Diseases ◽

Armillariella Mellea

Twenty-one isolates of the bacterium Bacillus subtilis and one of Enterobacter aerogenes were tested on agar for antagonism to Alternaria alternata, Armillariella mellea, Botrytis allii, Botrytis cinerea, Colletotrichum lindemuthianum, Monilinia fructicola, Penicillium expansum, Phytophthora cactorum, Pythium ultimum, Rhizoctonia solani, Sclerotinia sclerotiorum, Sclerotium cepivorum, Verticillium dahliae, and Venturia inequalis, causal organisms of many plant diseases. Enterobacter aerogenes was antagonisic to all of the pathogenic fungi tested except Verticillium dahliae and Armillariella mellea. Similarly, Bacillus subtilis was antagonistic to all of the pathogenic fungi tested except Pythium ultimum. When Enterobacter aerogenes and Bacillus subtilis were tested in vivo on cherry fruit for control of postharvest brown rot and alternaria rot, Enterobacter aerogenes was ineffective. Eleven isolates of Bacillus subtilis provided effective alternaria rot control and 15 isolates provided brown rot control which ranked with the best fungicide control.

Download Full-text

Isolation and Characterization of Probiotic Bacillus subtilis MKHJ 1-1 Possessing L-Asparaginase Activity

Applied Sciences ◽

10.3390/app11104466 ◽

2021 ◽

Vol 11 (10) ◽

pp. 4466

Author(s):

Hyeji Lim ◽

Sujin Oh ◽

Sungryul Yu ◽

Misook Kim

Keyword(s):

Bacillus Subtilis ◽

Bile Salt ◽

Shigella Flexneri ◽

Acid Resistance ◽

Rrna Gene ◽

Solution Ph ◽

Isolation And Characterization ◽

Gene Sequence Analysis ◽

Asparaginase Activity ◽

Bacillus Strains

The purpose of this study was to isolate functional Bacillus strains from Korean fermented soybeans and to evaluate their potential as probiotics. The L-asparaginase activity of MKHJ 1-1 was the highest among 162 Bacillus strains. This strain showed nonhemolysis and did not produce β-glucuronidase. Among the nine target bacteria, MKHJ 1-1 inhibited the growth of Escherichia coli, Pseudomonas aeruginosa, Shigella sonnei, Shigella flexneri, Klebsiella pneumoniae, Staphylococcus aureus, and Bacillus cereus. 16S rRNA gene sequence analysis resulted in MKHJ 1-1 identified as Bacillus subtilis subsp. stercoris D7XPN1. As a result of measuring the survival rate in 0.1% pepsin solution (pH 2.5) and 0.3% bile salt solution for 3 h, MKHJ 1-1 exhibited high acid resistance and was able to grow in the presence of bile salt. MKHJ 1-1 showed outstanding autoaggregation ability after 24 h. In addition, its coaggregation with pathogens was strong. Therefore, MKHJ 1-1 is a potential probiotic with L-asparaginase activity and without L-glutaminase activity, suggesting that it could be a new resource for use in the food and pharmaceutical industry.

Download Full-text

Differentiation Of Clarias Batrachus, C. Gariepinus And Heteropneustes Fossilis By Pcr-Sequencing Of Mitochondrial 16s Rrna Gene

Journal of the Asiatic Society of Bangladesh Science ◽

10.3329/jasbs.v41i1.46190 ◽

2015 ◽

Vol 41 (1) ◽

pp. 51-58

Author(s):

Mohammad Shamimul Alam ◽

Hawa Jahan ◽

Rowshan Ara Begum ◽

Reza M Shahjahan

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Fish Larva ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Valuable Insight ◽

Multiple Sequence ◽

Pcr Rflp ◽

Alternative Means

Heteropneustesfossilis, Clariasbatrachus and C. gariepinus are three major catfishes ofecological and economic importance. Identification of these fish species becomes aproblem when the usual external morphological features of the fish are lost or removed,such as in canned fish. Also, newly hatched fish larva is often difficult to identify. PCRsequencingprovides accurate alternative means of identification of individuals at specieslevel. So, 16S rRNA genes of three locally collected catfishes were sequenced after PCRamplification and compared with the same gene sequences available from othergeographical regions. Multiple sequence alignment of the 16S rRNA gene fragments ofthe catfish species has revealed polymorphic sites which can be used to differentiate thesethree species from one another and will provide valuable insight in choosing appropriaterestriction enzymes for PCR-RFLP based identification in future. Asiat. Soc. Bangladesh, Sci. 41(1): 51-58, June 2015

Download Full-text

Improved Production of Two Anti-Candida Lipopeptide Homologues Co- Produced by the Wild-Type Bacillus subtilis RLID 12.1 under Optimized Conditions

Current Pharmaceutical Biotechnology ◽

10.2174/1389201020666191205115008 ◽

2020 ◽

Vol 21 (5) ◽

pp. 438-450

Author(s):

Ramya Ramchandran ◽

Swetha Ramesh ◽

Anviksha A ◽

RamLal Thakur ◽

Arunaloke Chakrabarti ◽

...

Keyword(s):

Bacillus Subtilis ◽

Pathogenic Fungi ◽

Fold Increase ◽

Production Yield ◽

Yield Enhancement ◽

Bioactive Metabolites ◽

Candida Auris ◽

Media Formulation ◽

Static Conditions

Background:: Antifungal cyclic lipopeptides, bioactive metabolites produced by many species of the genus Bacillus, are promising alternatives to synthetic fungicides and antibiotics for the biocontrol of human pathogenic fungi. In a previous study, the co- production of five antifungal lipopeptides homologues (designated as AF1, AF2, AF3, AF4 and AF5) by the producer strain Bacillus subtilis RLID 12.1 using unoptimized medium was reported; though the two homologues AF3 and AF5 differed by 14 Da and in fatty acid chain length were found effective in antifungal action, the production/ yield rate of these two lipopeptides determined by High-Performance Liquid Chromatography was less in the unoptimized media. Methods:: In this study, the production/yield enhancement of the two compounds AF3 and AF5 was specifically targeted. Following the statistical optimization (Plackett-Burman and Box-Behnken designs) of media formulation, temperature and growth conditions, the production of AF3 and AF5 was improved by about 25.8- and 7.4-folds, respectively under static conditions. Results:: To boost the production of these two homologous lipopeptides in the optimized media, heat-inactivated Candida albicans cells were used as a supplement resulting in 34- and 14-fold increase of AF3 and AF5, respectively. Four clinical Candida auris isolates had AF3 and AF5 MICs (100 % inhibition) ranging between 4 and 16 μg/ml indicating the lipopeptide’s clinical potential. To determine the in vitro pharmacodynamic potential of AF3 and AF5, time-kill assays were conducted which showed that AF3 (at 4X and 8X concentrations) at 48h exhibited mean log reductions of 2.31 and 3.14 CFU/ml of C. albicans SC 5314, respectively whereas AF5 at 8X concentration showed a mean log reduction of 2.14 CFU/ml. Conclusion:: With the increasing threat of multidrug-resistant yeasts and fungi, these antifungal lipopeptides produced by optimized method promise to aid in the development of novel antifungal that targets disease-causing fungi with improved efficacy.

Download Full-text

Diversity and Biocontrol Potential of Cultivable Endophytic Bacteria Associated with Halophytes from the West Aral Sea Basin

Microorganisms ◽

10.3390/microorganisms9071448 ◽

2021 ◽

Vol 9 (7) ◽

pp. 1448

Author(s):

Lei Gao ◽

Jinbiao Ma ◽

Yonghong Liu ◽

Yin Huang ◽

Osama Abdalla Abdelshafy Mohamad ◽

...

Keyword(s):

Endophytic Bacteria ◽

Diversity Index ◽

Pathogenic Fungi ◽

Significant Inhibition ◽

Isolation Effect ◽

Aral Sea ◽

Rrna Gene ◽

Bacterial Isolates ◽

Aral Sea Basin ◽

Soil Borne Pathogens

Endophytes associated with halophytes may contribute to the host’s adaptation to adverse environmental conditions through improving their stress tolerance and protecting them from various soil-borne pathogens. In this study, the diversity and antifungal activity of endophytic bacteria associated with halophytic samples growing on the shore of the western Aral Sea in Uzbekistan were investigated. The endophytic bacteria were isolated from the nine halophytic samples by using the culture-dependent method and identified according to their 16S rRNA gene sequences. The screening of endophytic bacterial isolates with the ability to inhibit pathogenic fungi was completed by the plate confrontation method. A total of 289 endophytic bacterial isolates were isolated from the nine halophytes, and they belong to Firmicutes, Actinobacteria, and Proteobacteria. The predominant genera of the isolated endophytic bacteria were Bacillus, Staphylococcus, and Streptomyces, accounting for 38.5%, 24.7%, and 12.5% of the total number of isolates, respectively. The comparative analysis indicated that the isolation effect was better for the sample S8, with the highest diversity and richness indices. The diversity index of the sample S7 was the lowest, while the richness index of samples S5 and S6 was the lowest. By comparing the isolation effect of 12 different media, it was found that the M7 medium had the best performance for isolating endophytic bacteria associated with halophytes in the western Aral Sea Basin. In addition, the results showed that only a few isolates have the ability to produce ex-enzymes, and eight and four endophytic bacterial isolates exhibited significant inhibition to the growth of Valsa mali and Verticillium dahlia, respectively. The results of this study indicated that halophytes are an important source for the selection of microbes that may protect plant from soil-borne pathogens.

Download Full-text

The Role of Fungi in the Cocoa Production Chain and the Challenge of Climate Change

Journal of Fungi ◽

10.3390/jof7030202 ◽

2021 ◽

Vol 7 (3) ◽

pp. 202

Author(s):

Johannes Delgado-Ospina ◽

Junior Bernardo Molina-Hernández ◽

Clemencia Chaves-López ◽

Gianfranco Romanazzi ◽

Antonello Paparella

Keyword(s):

Climate Change ◽

Fungal Infections ◽

Close Association ◽

Pathogenic Fungi ◽

Fungal Species ◽

Mitigation Strategies ◽

Plant Diseases ◽

Production Chain ◽

Cocoa Production

Background: The role of fungi in cocoa crops is mainly associated with plant diseases and contamination of harvest with unwanted metabolites such as mycotoxins that can reach the final consumer. However, in recent years there has been interest in discovering other existing interactions in the environment that may be beneficial, such as antagonism, commensalism, and the production of specific enzymes, among others. Scope and approach: This review summarizes the different fungi species involved in cocoa production and the cocoa supply chain. In particular, it examines the presence of fungal species during cultivation, harvest, fermentation, drying, and storage, emphasizing the factors that possibly influence their prevalence in the different stages of production and the health risks associated with the production of mycotoxins in the light of recent literature. Key findings and conclusion: Fungi associated with the cocoa production chain have many different roles. They have evolved in a varied range of ecosystems in close association with plants and various habitats, affecting nearly all the cocoa chain steps. Reports of the isolation of 60 genera of fungi were found, of which only 19 were involved in several stages. Although endophytic fungi can help control some diseases caused by pathogenic fungi, climate change, with increased rain and temperatures, together with intensified exchanges, can favour most of these fungal infections, and the presence of highly aggressive new fungal genotypes increasing the concern of mycotoxin production. For this reason, mitigation strategies need to be determined to prevent the spread of disease-causing fungi and preserve beneficial ones.

Download Full-text

Isolation and Characterization of Lactic Acid Bacteria and Yeasts from Typical Bulgarian Sourdoughs

Microorganisms ◽

10.3390/microorganisms9071346 ◽

2021 ◽

Vol 9 (7) ◽

pp. 1346

Author(s):

Mariana Petkova ◽

Petya Stefanova ◽

Velitchka Gotcheva ◽

Angel Angelov

Keyword(s):

Lactic Acid ◽

Sequence Analysis ◽

Lactic Acid Bacteria ◽

Antimicrobial Properties ◽

Matter Content ◽

Starter Cultures ◽

Rrna Gene ◽

Dry Matter Content ◽

Isolation And Characterization ◽

Selection Of

Traditional sourdoughs in Bulgaria were almost extinct during the centralized food production system. However, a rapidly developing trend of sourdough revival in the country is setting the demand for increased production and use of commercial starter cultures. The selection of strains for such cultures is based on geographical specificity and beneficial technological properties. In this connection, the aim of this study was to isolate, identify and characterize lactic acid bacteria (LAB) and yeasts from typical Bulgarian sourdoughs for the selection of strains for commercial sourdough starter cultures. Twelve samples of typical Bulgarian sourdoughs were collected from different geographical locations. All samples were analyzed for pH, total titratable acidity and dry matter content. Enumeration of LAB and yeast was also carried out. Molecular identification by 16S rDNA sequence analysis was performed for 167 LAB isolates, and 106 yeast strains were identified by ITS1-5.8S-ITS2 rRNA gene partial sequence analysis. The LAB strains were characterized according to their amylolytic and proteolytic activity and acidification capacity, and 11 strains were selected for further testing of their antimicrobial properties. The strains with the most pronounced antibacterial and antifungal activity are listed as recommended candidates for the development of starter cultures for sourdoughs or other food products.

Download Full-text