Endophytic Bacillus Subtilis Strain Research Articles

Rice sheath blight disease control by native endophytic Bacillus subtilis from Kuttanad, a Globally Important Agricultural Heritage System

ABSTRACT Kuttanad in India is known as ‘the rice bowl of Kerala’ and renowned for its unique rice cultivation system that is below sea level. However, rice production in this wetland ecosystem is seriously threatened by sheath blight disease caused by the fungus Rhizoctonia solani, which has resulted in a significant decline in grain quality and a 50% loss in yield. In the present study, we isolated two endophytic Bacillus subtilis strains, NIIST B616 and NIIST B627, from the roots of rice plants growing in the Kuttanad region and tested their efficacy in controlling sheath blight disease. We found that rice plants inoculated with a combination of both Bacillus strains and R. solani had a 64.71% lower disease incidence and a 31.02% higher harvest index than plants treated with R. solani alone. An analysis of defence-related enzymes and chemicals revealed that treatment with both isolates together greatly enhanced levels of l-phenylalanine ammonia-lyase (PAL; 2.8 fold), peroxidase (POX; 7.4 fold), polyphenol oxidase (PPO; 4.5 fold), and total phenol (8.5 fold) compared with plants treated solely with R. solani, indicating that these isolates significantly increased plant defence responses through induced systemic resistance (ISR), and this finding was validated through a split-root experiment. More detailed analysis using gas chromatography–tandem mass spectrometry (GC-MS/MS) and liquid chromatography–tandem mass spectrometry (LC-MS/MS) techniques revealed that cyclo-(Pro-Leu) and cyclo-(Pro-Phe) were the major components responsible for the antifungal properties of the two isolates and their capacity to trigger ISR in plants. Thus, the Bacillus strains and their metabolites may be deemed viable alternatives for controlling the prevalence of sheath blight disease in rice plants and augmenting rice production at unique locations such as Kuttanad.

Plant Growth Promotion and Biocontrol of Leaf Blight Caused by Nigrospora sphaerica on Passion Fruit by Endophytic Bacillus subtilis Strain GUCC4.

Passion fruit (Passiflora edulis Sims) is widely cultivated in tropic and sub-tropic regions for the production of fruit, flowers, cosmetics, and for pharmacological applications. Its high economic, nutritional, and medical values elicit the market demand, and the growing areas are rapidly increasing. Leaf blight caused by Nigrospora sphaerica is a new and emerging disease of passion fruit in Guizhou, in southwest China, where the unique karst mountainous landscape and climate conditions are considered potential areas of expansion for passion fruit production. Bacillus species are the most common biocontrol and plant-growth-promotion bacteria (PGPB) resources in agricultural systems. However, little is known about the endophytic existence of Bacillus spp. in the passion fruit phyllosphere as well as their potential as biocontrol agents and PGPB. In this study, 44 endophytic strains were isolated from 15 healthy passion fruit leaves, obtained from Guangxi province, China. Through purification and molecular identification, 42 of the isolates were ascribed to Bacillus species. Their inhibitory activity against N. sphaerica was tested in vitro. Eleven endophytic Bacillus spp. strains inhibited the pathogen by >65%. All of them produced biocontrol- and plant-growth-promotion-related metabolites, including indole-3-acetic acid (IAA), protease, cellulase, phosphatase, and solubilized phosphate. Furthermore, the plant growth promotion traits of the above 11 endophytic Bacillus strains were tested on passion fruit seedlings. One isolate, coded B. subtilis GUCC4, significantly increased passion fruit stem diameter, plant height, leaf length, leaf surface, fresh weight, and dry weight. In addition, B. subtilis GUCC4 reduced the proline content, which indicated its potential to positively regulate passion fruit biochemical properties and resulted in plant growth promotion effects. Finally, the biocontrol efficiencies of B. subtilis GUCC4 against N. sphaerica were determined in vivo under greenhouse conditions. Similarly to the fungicide mancozeb and to a commercial B. subtilis-based biofungicide, B. subtilis GUCC4 significantly reduced disease severity. These results suggest that B. subtilis GUCC4 has great potential as a biological control agent and as PGPB on passion fruit.

Endophytic Bacillus subtilis antagonize soil-borne fungal pathogens and suppress wilt complex disease in chickpea plants (Cicer arietinum L.).

The present study aimed to identify potential endophytic bacteria antagonistic against three soil-borne fungal pathogens, Rhizoctonia solani, Sclerotium rolfsii, and Fusarium oxysporum f.sp. ciceri causing root rot, collar rot, and fungal wilt diseases in chickpea plants, respectively. A total of 255 bacterial endophytes were isolated from the leaves, stems, and roots of seven different crop plants (chickpea, tomato, wheat, berseem, mustard, potato, and green pea). The dual culture-based screening for antifungal properties indicated that three endophytic isolates had strong inhibition (>50%) against all three pathogens tested. Based on morphological, biochemical, and molecular characterization, the selected isolates (TRO4, CLO5, and PLO3) were identified as different strains of Bacillus subtilis. The bacterial endophytes (TRO4 and CLO5) were positive for plant growth promoting (PGP) traits viz., ammonia, siderophore, and indole-3-acetic acid (IAA) production. The bio-efficacy of the endophytes (TRO4, CLO5, and PLO3) was tested by an in planta trial in chickpea pre-challenged with R. solani, S. rolfsii, and F. oxysporum f.sp. ciceri. The B. subtilis strains TRO4 and CLO5 were found to be effective in reducing percent disease incidence (p ≤ 0.05) and enhancing plant growth parameters. The different root parameters viz. root length (mm), surface area (cm2), root diameter (mm), and root volume (cm3) were significantly (p ≤ 0.05) increased in TRO4 and CLO5 inoculated chickpea plants. Confocal Scanning Laser Microscopy showed heavy colonization of bacteria in the roots of endophyte-inoculated chickpea plants. The inoculation of endophytic Bacillus subtilis strains TRO4 and CLO5 in chickpea plants through seed biopriming reduced the accumulation of superoxide, enhanced the plant defense enzymes, and induced the expression of Pathogenesis-Related (PR) genes. Semi-quantitative analysis of defense-related genes showed differential activation of PR genes (60srp and IFR) by endophyte inoculation. The results of the present study reveal the antagonistic potential of B. subtilis strains TRO4 and CLO5 against three major soil-borne fungal pathogens and their ability to suppress wilt complex disease in chickpea plants. This is the first report on the simultaneous suppression of three major soil-borne fungal pathogens causing wilt complex in chickpea plants by endophytic B. subtilis strains.

Endophytic Bacillus subtilis TR21 Improves Banana Plant Resistance to Fusarium oxysporum f. sp. cubense and Promotes Root Growth by Upregulating the Jasmonate and Brassinosteroid Biosynthesis Pathways.

The banana (Musa spp.) industry experiences dramatic annual losses from Fusarium wilt of banana disease, which is caused by the fungus Fusarium oxysporum f. sp. cubense (FOC). Pisang Awak banana 'Fenza No. 1' (Musa spp. cultivar Fenza No. 1), a major banana cultivar with high resistance to F. oxysporum f. sp. cubense race 4, is considered to be ideal for growth in problematic areas. However, 'Fenza No. 1' is still affected by F. oxysporum f. sp. cubense race 1 in the field. TR21 is an endophytic Bacillus subtilis strain isolated from orchids (Dendrobium sp.). Axillary spraying of banana plants with TR21 controls Fusarium wilt of banana, decreasing the growth period and increasing yields in the field. In this study, we established that TR21 increases root growth in different monocotyledonous plant species. By axillary inoculation, TR21 induced a similar transcriptomic change as that induced by F. oxysporum f. sp. cubense race 1 but also upregulated the biosynthetic pathways for the phytohormones brassinosteroid and jasmonic acid in 'Fenza No. 1' root tissues, indicating that TR21 increases Fusarium wilt of banana resistance, shortens growth period, and increases yield of banana by inducing specific transcriptional reprogramming and modulating phytohormone levels. These findings will contribute to the identification of candidate genes related to plant resistance against fungi in a nonmodel system and facilitate further study and exploitation of endophytic biocontrol agents.

Biodegradation of dibutyl phthalate by a novel endophytic Bacillus subtilis strain HB-T2 under in-vitro and in-vivo conditions

ABSTRACT The environmental prevalence and potential toxicity of dibutyl phthalate (DBP) motivate the attempt to develop feasible strategies to deal with DBP contamination. In this study, a strain of endphytic bacteria HB-T2 was isolated from sorrel roots and identified as Bacillus sp. by analysing its morphology, physiology, biochemistry and 16S rDNA sequence. The degradation efficiency of DBP by HB-T2 was almost identical under the temperature of 30∼40°C, but was significantly enhanced as the culture pH and inoculum size increases from 6.0 to 8.0, and 1% to 5% respectively. The degradation kinetics of DBP could be well described by the first-order kinetic model, with the degradation half-life ranging from 1.59 to 7.61 h when the initial concentrations of DBP were in the range of 5–20 mg/L. LC-MS analysis of the culture samples taken at varying intervals revealed monobutyl phthalate, phthalic acid and protocatechuic acid as the major metabolic intermediates during the degradation process. HB-T2 exhibited an excellent capability to degrade a wide range of phthalate esters (PAEs), especially butyl benzyl phthalate (BBP), dipentyl phthalate (DPP), and diisobutyl phthalate (DIBP). Inoculation of HB-T2 into Chinese cabbage (Brassica chinensis L.) growing in DBP-contaminated soils could significantly reduce the DBP levels in plant tissues and relieve the phytotoxic effects of DBP. Results of this study highlighted the great potential of this novel endophytic Bacillus subtilis strain HB-T2 for bioremediation of PAEs contamination and improvement of agricultural product safety by reducing PAEs accumulation in edible crops.

The Effect of Endophytic Bacteria Bacillus subtilis and Salicylic Acid on Some Resistance and Quality Traits of Stored Solanum tuberosum L. Tubers Infected with Fusarium Dry Rot.

The effect of endophytic Bacillus subtilis (strains 10-4, 26D) and their compositions with salicylic acid (SA) on some resistance and quality traits of stored potatoes infected with Fusarium dry rot were studied. The experiments were carried out on hydroponically grown Solanum tuberosum L. tubers that were infected before storage with Fusarium oxysporum and coated with B. subtilis 10-4, 26D with and without exogenous SA, and then stored for six months. It has been shown that 10-4, 26D, 10-4 + SA, and 26D + SA reduced in different levels (up to 30–50%) the incidence of F. oxysporum-caused dry rot (with the highest effect for 10-4 + SA). SA notably enhanced the positive effect of 10-4, while for 26D, such an effect was not observed. All of the tested treatments increased amylase (AMY) and AMY inhibitors activity in infected tubers, while decreased Fusarium-induced protease activity (except in the case of 10-4 + SA, which promoted a slight increase) was revealed. 10-4, 26D, and their compositions with SA decreased (in different degrees) the pathogen-caused lipid peroxidation, proline, and reducing sugars accumulation in potatoes after long-term storage. It was also discovered 10-4 and 26D, regardless of SA presence, decrease pathogen-induced glycoalkaloids α-Solanine and α-Chaconine accumulation and preserved increased levels of starch and total dry matter in infected stored potatoes. The findings indicate endophytic B. subtilis and its compositions with SA is a promising eco-friendly and bio-safe approach to cope with postharvest decays of potato during long-term storage; however, when developing preparations-compositions it should take into account the strain-dependent manner of B. subtilis action together with SA.

Biocontrol of the internalization of Salmonella enterica and Enterohaemorrhagic Escherichia coli in mung bean sprouts with an endophytic Bacillus subtilis

Internalization of Salmonella enterica and enterohaemorrhagic Escherichia coli (EHEC) in seed sprouts poses a health risk to consumers, and the conventional sanitization methods are not always effective to reduce this risk. This study initiated a biocontrol approach to limit the internalization using endophytic Bacillus subtilis strains, which were isolated from the inner tissue of mung bean seeds or lettuce stems. By using the deferred agar method, 12 strains of B. subtilis out of 94 putative Bacillus isolates displayed inhibitory activity against at least one of the pathogenic indicators, S. enterica Typhimurium ATCC 14028 and E. coli O157:H7 505B. Two B. subtilis isolates (LCA1 and M24) showed a broad inhibitory spectrum against multiple strains of S. enterica and EHEC, Staphylococcus aureus sp., Klebsiella pneumoniae ATCC 700603, and Listeria monocytogenes Scott A, while the laboratory B. subtilis strain 168 was only moderately inhibitory against L. monocytogenes. To facilitate the tracking of the three B. subtilis strains (LCA1, M24, and 168) in the mung bean sprouts, the three strains were genetically engineered to carry the chloramphenicol acetyltransferase (cat), generating the strains LCA1-cat, M24-cat, and 168-cat, respectively. Data of the study using the cat-tagged strains demonstrated that both the two vegetable-associated and the laboratory B. subtilis strains could internalize in mung bean sprouts during the sprouting, but the latter displayed about 1.2 lg CFU/g of seeds lower in internalization. Overall, the presence of the three B. subtilis strains could significantly reduce the internalization of S. enterica or EHEC cocktail in mung bean sprouts during the sprouting. Among them, LCA1 showed the greatest inhibition against the EHEC cocktails with a reduction of about 2.0lg CFU/g of seeds by the end of sprouting (day 5), while 168 had the smallest reduction at about 0.6lg CFU/g of seeds. In addition, the three strains demonstrated a similar inhibition against the S. enterica cocktails by a reduction of about 1.1–1.4lg CFU/g of seeds by day 5. Results of this study suggest that the source (native vs. alien) of B. subtilis isolates may not affect the efficacy of the inhibition, but it might be affected by the production of antimicrobial substance and/or nutrition/space competition. The results also indicate that strain LCA1 may be useful as a biocontrol agent to reduce Salmonella and EHEC contamination in seed sprouts.

Biocontrol potential of chenodeoxycholic acid (CDCA) and endophytic Bacillus subtilis strains against the most destructive grapevine pathogens

A total of 41 endophytic bacterial strains were assessed for antimicrobial activity against indicator organisms in a preliminary screening. Of these, two bacterial strains that showed a strong antimicrobial activity were selected and identified as Bacillus subtilis (Bs1 and Bs2). The activities of Bs1 and Bs2 strains and different bile acids were assessed against Botrytis cinerea and Plasmopara viticola grapevine pathogens using in vitro and in vivo methods. Bs1 and Bs2 strains strongly inhibited Botrytis cinerea growth in vitro. The cell-free filtrates of both strains also reduced Botrytis cinerea growth. Chenodeoxycholic acid (CDCA) inhibited Botrytis cinerea growth in potato dextrose agar medium and suppressed P. viticola development in grapevine leaf discs. Epifluorescence microscopy observations revealed an important restriction of P. viticola development at the vesicle stage. In addition, CDCA markedly reduced the ability of P. viticola sporangia to release zoospores in vitro, confirming the direct fungicidal effect of this bile acid.

Purification, characterization, and heterologous expression of an antifungal protein from the endophytic Bacillus subtilis strain Em7 and its activity against Sclerotinia sclerotiorum.

An antifungal protein exhibiting a high activity against Sclerotinia sclerotiorum in vivo was purified by ammonium sulfate precipitation, hydrophobic chromatography, and gel filtration chromatography from the culture filtrate of the endophytic Bacillus subtilis strain Em7. The protein was characterized as a β-1,3-1,4-glucanase according to amino acid analysis, and showed excellent properties in thermal stability and acid resistance. At the same time, the antifungal protein was cloned and heterologously expressed in Escherichia coli BL21. The recombinant protein was purified and showed similar enzymatic properties to the native protein, exhibiting strong inhibitory activity against S. sclerotiorum. This shows that the β-1,3-1,4-glucanase may play a very important role in B. subtilis Em7 biocontrol function. In addition, many physiochemical properties of the native and purified recombinant protein were compared, including the effect of pH, temperature, metal cations, substrate specificity, and kinetic parameters. All parameters were similar between the native and recombinant purified protein, indicating that the purified recombinant protein has potential for industrial applications.

Endophytic Bacillus subtilis strain E1R-J is a promising biocontrol agent for wheat powdery mildew.

In this study, the biocontrol efficacies of 14 endophytic bacterial strains were tested against Blumeria graminis f. sp. tritici (Bgt) in pot experiments under greenhouse conditions. Bacillus subtilis strain E1R-j significantly reduced disease index and exhibited the best control (90.97%). When different formulations of E1R-j were sprayed 24 h before Bgt inoculation, fermentation liquid without bacterial cell and crude protein suspension displayed the similar effects; and they reduced disease index more than bacterial cell suspension (109 cfu mL−1) and fermentation liquid without protein. The control effects were not significantly different between 1011 and 109 cfu mL−1 of bacterial cell suspension but were higher than 107 cfu mL−1. Further observations showed that conidial germination and appressorial formation of Bgt were retarded by spraying E1R-j 24 h before Bgt inoculation. Compared with the water check, conidial germination and appressorial formation were decreased by 43.3% and 42.7%, respectively. In the treatment with E1R-j, the number of houstoria significantly reduced and the speed of mycelial extension was slowed down in the wheat leaves. Scanning electron microscopy observation revealed that E1R-j significantly suppressed the conidial germination and caused rupture and deformation of germ tubes. On the surface of wheat leaves, mycelia and conidiophores became shrinking.

Biological control of wheat stripe rust by an endophytic Bacillus subtilis strain E1R-j in greenhouse and field trials

Endophytic Bacillus subtilis strain E1R-j, isolated from wheat roots, inhibits a wide range of microorganisms. In this study, we determined its inhibitory effect on urediniospore germination of Puccinia striiformis f. sp. tritici (Pst), the causal agent of wheat stripe rust and biocontrol effect on the rust development using bacterial cell suspension (BCS) and fermentation liquid with and without bacterial cells (FLBC and FL). Urediniospore germination was significantly inhibited by all three formulations in vitro. The maximum inhibition rate was 84.1% treated with the 10-fold dilution of original formulation of FLBC. Microscopic observation revealed that the bacterial treatments caused urediniospores and germ tubes ruptured, which resulted in protoplasm release and dysfunction of fungal structures. Urediniospore germination was also significantly inhibited by spraying E1R-j on leaf surface before inoculation. Formulations FLBC and FL were both better than formulation BCS. In a condition-controlled greenhouse, application of FLBC and FL 24 and 0 h before Pst inoculation displayed a protective mode in reducing disease severity, and the control efficacy was between 54.0% and 87.7%. For formulation BCS, only the application of 24 h before Pst inoculation significantly reduced rust severity with control efficacy 51.8%. The incidence of diseased leaves in the treatments of FLBC and FL were significantly reduced compared to the non-treated plots in the field experiments conducted in the season 2007–2008. The control efficacy was 51% and 43% for the two treatments, respectively. This study is helpful for further developing a biocontrol approach for managing stripe rust.

Involvement of phytohormones in the development of interaction between wheat seedlings and endophytic Bacillus subtilis strain 11BM

Wheat (Triticum aestivum L., cv. Kazakhstanskaya 10) seedling growth activation by the endophytic 11BM strain of the bacterium Bacillus subtilis Cohn and the involvement of phytohormones in this process were studied. At the concentration used, bacteria enhanced wheat root and shoot growth as compared to control. Biotests demonstrated auxin-, cytokinin-, and gibberellin-like activities of the endophyte, but the result depended on the way of experiment performing. When wheat seeds were treated with the spores of B. subtilis 11BM strain, the concentrations of IAA and ABA increased transiently in the roots and shoots of the seedlings. The involvement of IAA oxidase in the plant response to inoculation with bacteria was shown; it was accompanied by a decrease in enzyme activity, which occurred later than auxin accumulation. It was concluded that observed changes in the hormonal status of wheat plants under the influence of the endophytic bacterial strain may be one of the mechanisms for seedling growth stimulation.

Phosphate-Mobilizing Activity of the Endophytic Bacillus subtilis Strains and their Effect on wheat Roots Micorrhization Ratio

Phosphate-Mobilizing Activity of the Endophytic Bacillus subtilis Strains and their Effect on wheat Roots Micorrhization Ratio

Биологические особенности эндофитных штаммов Bacillus subtilis как перспективной основы новых биопрепаратов

Исследованы некоторые свойства новых эндофитных штаммов Bacillus subtilis 11В и 11ВМ. Показано, что одним из общих механизмов стимуляции роста разных видов растений обоими штаммами может быть повышение содержания фитогормона ИУК в растительных тканях.

Isolation and partial characterization of an antifungal protein from the endophytic Bacillus subtilis strain EDR4

An antifungal protein E2, from the culture filtrate of the endophytic Bacillus subtilis strain EDR4 of wheat with a high activity against numerous fungal species in vitro and take-all in wheat caused by Gaeumannomyces graminis var. tritici in vivo, was purified by (NH 4) 2SO 4 precipitation, hydrophobic-interaction chromatography, anion-exchange chromatography and polyacrylamide gel electrophoresis (PAGE). The molecular mass of the protein was about 377.0 kDa determined by gel permeation chromatography (GPC) using a Superdex 200 10/300 GL pre-packed column and the pI value of the protein detected by isoelectric focusing PAGE was 6.59. Following sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) the antifungal protein showed a band with a molecular mass of 39.1 kDa, which suggest that the native protein consists of multi-subunits. The amino acid sequences of three peptides from the antifungal protein were obtained by using a nano-ESI-MS/MS (Q-TOF2) System. The protein isolated may be regarded as a new protein according to amino acid sequences of three peptides. The purified protein exhibited inhibitory activity on mycelium growth of e.g. Fusarium graminearum, Macrophoma kuwatsukai, Rhizoctonia cerealis, Fusarium oxysporum f.sp. vasinfectum, Botrytis cinerea and G. graminis var. tritici ( Ggt). Scanning electron microscopy showed that hyphae of Ggt treated with the antifungal protein were severely deformed. The antifungal protein E2 exhibited ribonuclease and hemagglutinating activities as well as a trifle protease activity. However, no β-1,3-glucanase, β-1,4-glucanase, chitinase or protease inhibitory activities were detected.

Biological control against bacterial wilt and colonization of mulberry by an endophytic Bacillus subtilis strain

Forty-five bacterial isolates were collected from surface-sterilized leaves of mulberry (Morus alba L.). By screening their antagonistic activities against Ralstonia solanacearum in vitro, four isolates showed a remarkable inhibitory effect. The evaluation of the antagonistic strains against bacterial wilt of mulberry indicated that the strain Lu144 effectively reduced disease incidence. In the greenhouse, Lu144 displayed effective biological control against bacterial wilt of mulberry when it was applied to sterile or nonsterile soil before the infection by the pathogen. Based on bacteriological properties and 16S rRNA gene sequencing, Lu144 was identified as a strain of Bacillus subtilis. The endophytic population and infection process of Lu144 in mulberry seedlings was explored following recovery of the green fluorescent protein (GFP)-labeled Lu144 and examination of the labeled strain by confocal laser scanning microscopy. Interestingly, the infection of GFP-labeled Lu144 cells into the mulberry seedlings occurred through the cracks formed at the lateral root junctions and the zone of differentiation and elongation, and the cells were able to develop and transfer in mulberry and mainly in the intercellular spaces of different tissues. The population of the GFP-labeled Lu144 inoculant was larger and more stable in leaves than that in roots and stems.