Enhanced control efficacy of Bacillus subtilis NM4 via integration of chlorothalonil on potato early blight caused by Alternaria solani.

Early blight caused by Alternaria solani is a common foliar disease of potato around the world, and serious infections result in reduced yields and marketability due to infected tubers. The major aim of this study is to figure out the synergistic effect between microorganism and fungicides and to evaluate the effectiveness of Bacillus subtilis NM4 in the control of early blight in potato. Based on its colonial morphology and a 16S rRNA analysis, a bacterial antagonist isolated from kimchi was identified as B. subtilis NM4 and it has strong antifungal and anti-oomycete activity against several phytopathogenic fungi and oomycetes. The culture filtrate of strain NM4 with the fungicide effectively suppressed the mycelial growth of A. solani, with the highest growth inhibition rate of 83.48%. Although exposure to culture filtrate prompted hyphal alterations in A. solani, including bulging, combining it with the fungicide caused more severe hyphal damage with continuous bulging. Surfactins and fengycins, two lipopeptide groups, were isolated and identified as the main compounds in two fractions using LC-ESI-MS. Although the surfactin-containing fraction failed to inhibit growth, the fengycin-containing fraction, alone and in combination with chlorothalonil, restricted mycelial development, producing severe hyphal deformations with formation of chlamydospores. A pot experiment combining strain NM4, applied as a broth culture, with fungicide, at half the recommended concentration, resulted in a significant reduction in potato early blight severity. Our results indicate the feasibility of an integrated approach for the management of early blight in potato that can reduce fungicide application rates, promoting a healthy ecosystem in agriculture.

Role of an antagonistic bacterium, Bacillus subtilis PE7, in growth promotion of netted melon (Cucumis melo L. var. reticulatus Naud.).

The aim of this study was to determine the plant growth-promoting effect of Bacillus subtilis PE7 on growth of melon plants. B. subtilis PE7 isolated from kimchi was identified based on colonial and microscopic morphology along with analyses of 16S rRNA and pycA gene sequences. Strain PE7 showed different levels of inhibition on phytopathogens and was able to grow at variable temperatures and pH values. Strain PE7 had the ability to produce siderophores, indole-3-acetic acid (IAA), ammonia, exopolysaccharides, and 1-aminocyclopropane-1-carboxylic acid deaminase, as well as solubilize insoluble phosphate and zinc. The IAA secretion of strain PE7 showed a concentration-dependent pattern based on the concentration of l-tryptophan supplemented in the fertilizer-based culture medium. The LC-MS analysis indicates the presence of IAA in the culture filtrate of strain PE7. Treatment of the B. subtilis PE7 culture containing different metabolites, mainly IAA, significantly promoted melon growth in terms of higher growth parameters and greater plant nutrient contents compared to treatments with the culture without IAA, fertilizer, and water. The cells of B. subtilis PE7 attached to and firmly colonized the roots of the bacterized melon plants. Based on our results, B. subtilis PE7 can be utilized as a potential microbial fertilizer to substitute chemical fertilizers in sustainable agriculture.

Butyl succinate-mediated control of Bacillus velezensis ce 100 for apple anthracnose caused by Colletotrichum gloeosporioides.

AIMS: Microbial biocontrol agents have become an effective option to mitigate the harmfulness of chemical pesticides in recent years. This study demonstrates the control efficacy of Bacillus velezensis CE 100 on the anthracnose causal agent, Colletotrichum gloeosporioides. METHODS AND RESULTS: In vitro antifungal assays revealed that the culture filtrate and volatile organic compounds of B. velezensis CE 100 strongly restricted the mycelial development of C. gloeosporioides. Moreover, a bioactive compound, butyl succinate, was isolated from the n-butanol crude extract of B. velezensis CE 100 (bce), and identified by liquid chromatography-electrospray ionization hybrid ion-trap and time-of-flight mass spectrometry (LC-ESI-QTOF-MS) and one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR). Treatment with purified butyl succinate at a concentration of 300 µg mL-1 strongly controlled conidial germination of C. gloeosporioides with an inhibition rate of 98.66%, whereas butyl succinate at a concentration of 400 µg mL-1 showed weak antifungal action on the mycelial growth of C. gloeosporioides with an inhibition rate of 31.25%. Scanning electron microscopy revealed that the morphologies of butyl succinate-treated hyphae and conidia of C. gloeosporioides were severely deformed with shriveled and wrinkled surfaces. Furthermore, butyl succinate was able to control carbendazim-resistant C. gloeosporioides, demonstrating that it could be a promising agent for the suppression of other carbendazim-resistant fungal pathogens. An in vivo biocontrol assay demonstrated that the strain ce 100 broth culture and butyl succinate showed higher control efficacy on apple anthracnose than bce. CONCLUSIONS: Our findings provide insight into the antifungal potential of B. velezensis  ce 100 and its butyl succinate for efficient control of phytopathogenic fungi, such as C. gloeosporiodes, in plant disease protection. This is the first study to demonstrate the antifungal potential of bacteria-derived butyl succinate for control of C. gloeosporioides.

Antifungal Potential of Bacillus velezensis CE 100 for the Control of Different Colletotrichum Species through Isolation of Active Dipeptide, Cyclo-(D-phenylalanyl-D-prolyl).

Colletotrichum species are important fungal pathogens causing anthracnose of tropical and subtropical fruit and vegetable crops. Dual culture assay indicated that Bacillus velezensis CE 100 was a strong antagonist against C. acutatum, C. coccodes, C. dematium, and C. gloeosporioides. The volatile organic compounds produced by B. velezensis CE 100 affected mycelial growth of Colletotrichum species tested in our study and caused twisted hyphal structures of all these fungal species. Chloroform crude compounds of B. velezensis CE 100 inhibited four Colletotrichum species in a concentration-dependent manner and induced severe damage in hyphal morphology of these fungal pathogens, including swelling, bulging, and multiple branching. Moreover, the active cyclic dipeptide, cyclo-(D-phenylalanyl-D-prolyl), was isolated from chloroform crude extract and identified by nuclear magnetic resonance (NMR) and mass spectrometry. The inhibitory effect of cyclo-(D-phenylalanyl-D-prolyl) on conidial germination of C. gloeosporioides occurred in a concentration-dependent manner. The conidial germination rate was completely inhibited by a concentration of 3 mg/mL of cyclo-(D-phenylalanyl-D-prolyl). Scanning electron micrographs revealed that the exposure to cyclic dipeptide resulted in seriously deformed hyphae and conidia with shriveled surfaces in dipeptide-treated C. gloeosporioides. Therefore, active dipeptide-producing B. velezensis CE 100 is a promising biocontrol agent for Colletotrichum species causing anthracnose.

Antifungal Activity of Bacillus velezensis CE 100 against Anthracnose Disease (Colletotrichum gloeosporioides) and Growth Promotion of Walnut (Juglans regia L.) Trees.

Walnut anthracnose caused by Colletotrichum gloeosporioides is a deleterious disease that severely affects the production of walnut (Juglans regia L.). The aim of this study was to assess the antifungal and growth promotion activities of Bacillus velezensis CE 100 as an alternative to chemical use in walnut production. The crude enzyme from B. velezensis CE 100 exhibited chitinase, protease, and ß-l,3-glucanase activity and degraded the cell wall of C. gloeosporioides, causing the inhibition of spore germination and mycelial growth by 99.3% and 33.6% at 100 µL/mL, respectively. The field application of B. velezensis CE 100 culture broth resulted in a 1.3-fold and 6.9-fold decrease in anthracnose disease severity compared to the conventional and control groups, respectively. Moreover, B. velezensis CE 100 produced indole-3-acetic acid (up to 1.4 µg/mL) and exhibited the potential for ammonium production and phosphate solubilization to enhance the availability of essential nutrients. Thus, field inoculation of B. velezensis CE 100 improved walnut root development, increased nutrient uptake, enhanced chlorophyll content, and consequently improved total biomass by 1.5-fold and 2.0-fold compared to the conventional and control groups, respectively. These results demonstrate that B. velezensis CE 100 is an effective biocontrol agent against anthracnose disease and a potential plant growth-promoting bacteria in walnut tree production.

Biological Control of Leaf Blight Disease Caused by Pestalotiopsis maculans and Growth Promotion of Quercus acutissima Carruth Container Seedlings Using Bacillus velezensis CE 100.

Leaf blight disease caused by Pestalotiopsismaculans lead to deleterious losses in the quality of forest container seedlings. The use of plant growth-promoting bacteria provides a promising strategy to simultaneously control diseases and enhance forest seedling production. This study investigated the biocontrol of leaf blight disease and growth promotion potential of Bacillus velezensis CE 100 in Quercus acutissima Carruth seedlings. B. velezensis CE 100 produced cell wall degrading enzymes, such as chitinase, ß-l,3-glucanase, and protease, which caused cell wall lysis and hyphae deformation of P. maculans, leading to mycelial growth inhibition by 54.94%. Inoculation of B. velezensis CE 100 suppressed P. maculans infection and increased seedling survival rate by 1.6-fold and 1.3-fold compared to chemical fertilizer and control, respectively. In addition, B. velezensis CE 100 produced indole-3-acetic acid, which improved root development and nutrient uptake compared to chemical fertilizer and control. Especially, inoculation with B. velezensis CE 100 increased the total nitrogen content of Q. acutissima seedlings, improved the chlorophyll index in the leaves, and increased seedling biomass by 1.3-fold and 2.2-fold compared to chemical fertilizer and control, respectively. Thus, B. velezensis CE 100 could be applied in the eco-friendly production of high-quality forest seedlings.

Antifungal compound, methyl hippurate from Bacillus velezensis CE 100 and its inhibitory effect on growth of Botrytis cinerea.

Botrytis cinerea, the causal agent of gray mold is one of the major devastating fungal pathogens that occurs in strawberry cultivation and leads to massive losses. Due to the rapid emergence of resistant strains in recent years, an ecofriendly disease management strategy needs to be developed to control this aggressive pathogen. Bacillus velezensis CE 100 exhibited strong antagonistic activity with 53.05% against B. cinerea by dual culture method. In the present study, 50% of culture filtrate supplemented into PDA medium absolutely inhibited mycelial growth of B. cinerea whereas the highest concentration (960 mg/L) of different crude extracts including ethyl acetate, chloroform, and n-butanol crude extracts of B. velezensis CE 100, strongly inhibited mycelial growth of B. cinerea with the highest inhibition of 79.26%, 70.21% and 69.59% respectively, resulting in severe damage to hyphal structures with bulging and swellings. Hence, the antifungal compound responsible was progressively separated from ethyl acetate crude extract using medium pressure liquid chromatography. The purified compound was identified as methyl hippurate by nuclear magnetic resonance and mass spectrometry. The inhibitory effect of methyl hippurate on both spore germination and mycelial growth of B. cinerea was revealed by its dose-dependent pattern. The spore germination rate was completely restricted at a concentration of 3 mg/mL of methyl hippurate whereas no mycelial growth was observed in agar medium supplemented with 4 mg/mL and 6 mg/mL of methyl hippurate by poisoned food method. Microscopic imaging revealed that the morphologies of spores were severely altered by long-time exposure to methyl hippurate at concentrations of 1 mg/mL, 2 mg/mL and 3 mg/mL and hyphae of B. cinerea were severely deformed by exposure to methyl hippurate at concentrations of 2 mg/mL, 4 mg/mL and 6 mg/mL. No significant inhibition on tomato seed germination was observed in treatments with methyl hippurate (2 mg/mL) for both 6 h and 12 h soaking period as compared to the controls. Based on these results, B. velezensis CE 100 could be considered a potential agent for development of environmentally friendly disease control strategies as a consequence of the synergetic interactions of diverse crude metabolites and methyl hippurate.

The Role of Lysobacter antibioticus HS124 on the Control of Fall Webworm (Hyphantria cunea Drury) and Growth Promotion of Canadian Poplar (Populus canadensis Moench) at Saemangeum Reclaimed Land in Korea.

Populus canadensis Moench forests established in Saemangeum-reclaimed land have been invaded by Hyphantria cunea Drury, causing defoliation and stunted growth. This study investigated the biocontrol potential of cuticle degrading chitinase and protease secreted by Lysobacter antibioticus HS124 against H. cunea larvae. In addition, L. antibioticus HS124 was examined for indole-3-acetic acid phytohormone production for plant growth promotion. To determine the larvicidal activity in the laboratory experiments, crude enzymes, bacteria culture, CY medium, and water (control) were sprayed on the larvae reared on natural diet in insect rearing dishes. Treatment with crude enzymes and bacteria culture caused 76.7% and 66.7% larvae mortality, respectively. The larvae cuticle, mainly composed of chitin and proteins, was degraded by cuticle-degrading enzymes, chitinase, and protease in both the bacteria culture and crude enzyme treatments, causing swelling and disintegration of the cuticle. Field application of the bacteria culture was achieved by vehicle-mounted sprayer. Bacterial treatment caused morphological damage on the larvae cuticles and subsequent mortality. Foliar application of the bacteria culture reduced tree defoliation by H. cunea and enhanced growth compared to the control. Especially, L. antibioticus HS124 produced auxins, and increased growth of poplar trees.

Bacillus velezensis CE 100 Inhibits Root Rot Diseases (Phytophthora spp.) and Promotes Growth of Japanese Cypress (Chamaecyparis obtusa Endlicher) Seedlings.

Root rot diseases, caused by phytopathogenic oomycetes, Phytophthora spp. cause devastating losses involving forest seedlings, such as Japanese cypress (Chamaecyparis obtusa Endlicher) in Korea. Plant growth-promoting rhizobacteria (PGPR) are a promising strategy to control root rot diseases and promote growth in seedlings. In this study, the potential of Bacillus velezensis CE 100 in controlling Phytophthora root rot diseases and promoting the growth of C. obtusa seedlings was investigated. B. velezensis CE 100 produced ß-1,3-glucanase and protease enzymes, which degrade the ß-glucan and protein components of phytopathogenic oomycetes cell-wall, causing mycelial growth inhibition of P. boehmeriae, P. cinnamomi, P. drechsleri and P. erythoroseptica by 54.6%, 62.6%, 74.3%, and 73.7%, respectively. The inhibited phytopathogens showed abnormal growth characterized by swelling and deformation of hyphae. B. velezensis CE 100 increased the survival rate of C. obtusa seedlings 2.0-fold and 1.7-fold compared to control, and fertilizer treatment, respectively. Moreover, B. velezensis CE 100 produced indole-3-acetic acid (IAA) up to 183.7 mg/L, resulting in a significant increase in the growth of C. obtusa seedlings compared to control, or chemical fertilizer treatment, respectively. Therefore, this study demonstrates that B. velezensis CE 100 could simultaneously control Phytophthora root rot diseases and enhance growth of C. obtusa seedlings.

Antifungal Activity of Cyclic Tetrapeptide from Bacillus velezensis CE 100 against Plant Pathogen Colletotrichum gloeosporioides.

The aim of this study was to investigate the antifungal activity of a cyclic tetrapeptide from Bacillus velezensis CE 100 against anthracnose-causing fungal pathogen Colletotrichum gloeosporioides. Antifungal compound produced by B. velezensis CE 100 was isolated and purified from ethyl acetate extract of B. velezensis CE 100 culture broth using octadecylsilane column chromatography. The purified compound was identified as cyclo-(prolyl-valyl-alanyl-isoleucyl) based on mass spectrometer and nuclear magnetic resonance analyses. This is the first report of the isolation of a cyclic tetrapeptide from B. velezensis CE 100 culture filtrate. Cyclic tetrapeptide displayed strong antifungal activity at concentration of 1000 µg/mL against C. gloeosporioides mycelial growth and spore germination. Our results demonstrate that the antifungal cyclic tetrapeptide from B. velezensis CE 100 has potential in bioprotection against anthracnose disease of plants caused by C. gloeosporioides.

Metagenomic analysis reveals enhanced biodiversity and composting efficiency of lignocellulosic waste by thermoacidophilic effective microorganism (tEM).

Composting is a microbiological process that converts organic waste into organic soil amendment. We reveal enhanced biodiversity and microbial population with subsequent enhancement of composting efficiency of lignocellulosic waste using thermoacidophilic effective microorganisms (tEM). Composting with tEM + shading (tEMA) or tEM without shading (tEMB) increased the average microbial population by 12.0% or 6.7%, respectively compared to non-tEM composting without shading/control (C). The biodiversity in tEMA or tEMB treated groups was increased by 34.7% or 43.7%, respectively, compared to C. The highest increase in population (31.7% and 9.4%) and diversity (91.2% and 91.6%) were observed in tEMA and tEMB at 30 d, respectively. Regarding microbial structure, the most dominant phylum shifted from Proteobacteria to Bacteroidetes during composting. From 60 to 120 d, tEM notably improved the average abundance of Firmicutes (mainly Bacillus) by 166.7% and 75.8% in tEMA and tEMB groups, respectively. The overall gradation rate of large compost granules (<2 mm) increased by 36.4% and 24.7%, following tEMA and tEMB treatment, respectively. The average rate of increase in bulk density was 42.6% or 33.3% by tEMA or tEMB, respectively, compared to C. We reveal the major differences in microbial structure, including a higher abundance of beneficial microbes like Bacillus in tEM treated composts. The study revealed that tEM could improve biodiversity and population of microbes, especially during thermophilic phase (above 45 °C), with a subsequent increase in composting rate, mineralization, and product quality. The results of this study are particularly invaluable in the areas of environmental conservation and organic agriculture.

Characterization of bacterial endophytes from Myanmar medicinal plants for antimicrobial activity against human and plant pathogens

This research aimed to investigate the antagonistic activity of the bacterial endophytes from Myanmar medicinal plants. Thirty-one bacterial isolates were isolated from Myanmar medicinal plants: Tinospora cordifolia (Wild.) Miers., Catharanthus roseus G. Don., Tectona hamiltoniana Wall. and Boscia variabilis Collett & Hemsl. (Capparaceae). Dual culture and agar well diffusion methods were used for antimicrobial assay. One endophyte from Catharanthus roseus and two bacterial isolates from Boscia variabilis Collett & Hemsl. (Capparaceae) had not only the antibacterial activity towards the human pathogenic bacteria but also the antifungal activity against the plant pathogenic fungi. From 16S rRNA sequencing, one strain from Catharanthus roseus G. Don. was Bacillus amyloliquefaciens DSM7 and two antagonistic strains from Boscia variabilis Collett & Hemsl. (Capparaceae) were Bacillus subtilis subsp. subtilis str. 168 and Bacillus amyloliquefaciens DSM7, respectively. The best medium for the maximum production of the bioactive compounds was Bacillus medium supplemented with the 4% of starch and 0.3% of peptone for B. amyloliquefaciens DSM7 and B. subtilis subsp. subtilis str. 168 showed the maximum antimicrobial compounds production when it was incubated in the medium amended with 3% of starch and 2% of peptone. The optimum conditions for the the maximum production of the antimicrobial compound were the medium pH of 6 at 35 ºC after two days of incubation for B. amyloliquefaciens DSM 7 and B. subtitlis subsp. subtilis str. 168 secreted the maximum concentration of the bioactive compounds at pH 7.5 and 35˚C on second day incubation period. In conclusion, the isolated endophytic bacteria showed the strong antimicrobial activity towards the pathogenic microbes and they could be used in medicine and agriculture as well.