Characterization of a novel antifungal protein produced by Paenibacillus polymyxa isolated from the wheat rhizosphere.

BACKGROUND: Fusarium head blight (FHB) is one of the disasters that seriously harm wheat and other small grain crops. It causes spoilage and mildew of the grain leading to a significant decline in the yield and quality of the grain. This research aimed to isolate antagonistic bacteria to purify antifungal proteins. A strain was isolated from the rhizosphere of healthy wheat in a wheat field affected by a severe FHB epidemic. This isolated strain was tentatively identified as Paenibacillus polymyxa 7F1, which displayed a strong inhibitory effect against several other pathogens. One novel antifungal protein was purified from the P. polymyxa 7F1 and successfully expressed. RESULTS: A crude culture of P. polymyxa 7F1 demonstrated antifungal activity that was stable at a temperature range of 60-90 °C and a pH range of 2.6-9.0. However, the antifungal activity of the P. polymyxa 7F1 was inhibited with proteinase K, trypsin, and neutral protease treatment. A 36 kDa protein with broad-spectrum antifungal activity was purified from the P. polymyxa 7F1. A glycosyl hydrolase domain was identified from this protein through liquid chromatography-mass spectrometry (LC-MS) analysis. A recombinant plasmid pET32a(+)/36kd for prokaryotic expression was constructed, and the renatured p36kd protein demonstrated similar antifungal activity to the 36 kDa protein purified from the P. polymyxa 7F1. CONCLUSION: A novel antifungal protein produced by P. polymyxa 7F1 was purified and expressed. The recombinant protein showed good antifungal activity as the novel purified protein. The novel antifungal protein provides an effective way to control the Fusarium head blight. © 2020 Society of Chemical Industry.

Integrated use of phosphate-solubilizing Bacillus subtilis strain IA6 and zinc-solubilizing Bacillus sp. strain IA16: a promising approach for improving cotton growth.

Mineral nutrition of crop plants is one of the major challenges faced by modern agriculture, particularly in arid and semi-arid regions. In alkaline calcareous soils, the availability of phosphorus and zinc is critically less due to their fixation and precipitation as complexes. Farmers use fertilizers to fulfill crop requirements, but their efficacy is less, which increases production costs. Plant growth-promoting rhizobacteria (PGPR) can improve the availability of crop nutrients through solubilizing the insoluble compounds of phosphorus and zinc in soil. In the present study, a total of 40 rhizobacterial isolates were isolated from cotton rhizosphere and screened for improving cotton growth through the solubilization of phosphorus and zinc. Out of these 40 isolates, seven isolates (IA2, IA3, IA6, IA7, IA8, IA13, and IA14) efficiently solubilized insoluble rock phosphate while seven isolates (IA10, IA16, IA20, IA23, IA24, IA28, and IA30) were more efficient in solubilizing insoluble zinc oxide. In liquid media, strain IA7 (2.75 µg/mL) solubilized the highest amount of phosphate while the highest concentration of soluble zinc was observed in the broth inoculated with strain IA20 (3.94 µg/mL). Seven phosphate-solubilizing and seven zinc-solubilizing strains were evaluated using jar trial to improve the growth of cotton seedlings, and the results were quite promising. All the inoculated treatments showed improvement in growth parameters in comparison with control. Best results were shown by the combined application of IA6 and IA16, followed by the combination of strains IA7 and IA20. Based on the jar trial, the selected isolates were further characterized by plant growth-promoting characters such as siderophores production, HCN production, ammonia production, and exopolysaccharides production. These strains were identified through 16S rRNA sequencing as Bacillus subtilis IA6 (accession # MN005922), Paenibacillus polymyxa IA7 (accession # MN005923), Bacillus sp. IA16 (accession # MN005924), and Bacillus aryabhattai IA20 (accession # MN005925). It is hence concluded that the integrated use of phosphate-solubilizing and zinc-solubilizing strains as potential inoculants can be a promising approach for improving cotton growth under semi-arid conditions.

Electrochemical detection of food-spoiling bacteria using interdigitated platinum microelectrodes.

The fast and non-destructive detection of bacterial attachment on food contact surfaces is important for the prevention of the unwanted formation of biofilms. Biofilms constitute a protected growth mode that allows bacteria to survive even in hostile environments. Therefore, the fast detection of bacterial attachment may be an effective strategy for biofilm control. In this study cyclic voltammetry (CV) was used to detect Bacillus subtilis ssp. subtilis, Paenibacillus polymyxa, Pseudomonas fragi attachment on interdigitated microelectrodes. The differences in current between the uncolonized sterile microelectrodes and the microelectrodes after bacterial attachment were determined. In addition, the surface coverage of microelectrodes was visualized using microscopy techniques. The results showed that the cyclic voltammetry in combination with interdigitated platinum microelectrodes can be used to detect bacterial biofilms.

Paenibacillus polymyxa Associated with the Stingless Bee Melipona scutellaris Produces Antimicrobial Compounds against Entomopathogens.

Social insects are frequently observed in symbiotic association with bacteria that produce antimicrobial natural products as a defense mechanism. There is a lack of studies on the microbiota associated with stingless bees and their antimicrobial compounds. To the best of our knowledge, this study is the first to report the isolation of Paenibacillus polymyxa ALLI-03-01 from the larval food of the stingless bee Melipona scutellaris. The bacterial strain was cultured under different conditions and produced (L)-(-)-3-phenyllactic acid and fusaricidins, which were active against entomopathogenic fungi and Paenibacillus larvae. Our results indicate that such natural products could be related to colony protection, suggesting a defense symbiosis between P. polymyxa ALLI-03-01 and Melipona scutellaris.

An endophytic strain of genus Paenibacillus isolated from the fruits of Noni (Morinda citrifolia L.) has antagonistic activity against a Noni's pathogenic strain of genus Aspergillus.

Endophytes are microbes capable of colonizing the tissues of healthy plants and subsequently establishing a harmonious relationship with their hosts. In this research, the endophytic strain Paenibacillus sp. NEB was isolated from fruits of healthy Noni (Morinda citrifolia L.). Strain NEB was identified as Paenibacillus polymyxa using MALDI-TOF Mass Spectrometry. Pathogenic fungal strain NP-1 was isolated from Noni fruits infected by smut, and was identified as Aspergillus aculeatus by polyphasic taxonomy basing on morphological identification, and ITS-5.8S rDNA and ß-tubulin gene phylogenetic analyses. Through the antagonistic test against the pathogenic strain Aspergillus aculeatus NP-1, the results showed that strain NEB had a good antagonistic activity against smut pathogen of Noni. By sequencing with Illumina HiSeq 2000, the draft genome of Paenibacillus sp. NEB was acquired, and 3 CDSs for glucanases were annotated and potentially correlated to the antagonistic activity of this strain. Using realtime-PCR method with specific primers to amplify the biocontrol gene, ß-1,3-1,4- glucanase gene (gluB), it was found in Paenibacillus polymyxa NEB. This study would provide a theoretical and microbial basis for the rationally developing and using Noni beneficial microbial inoculants against its pathogenic strain in the future.

Bacterial polysaccharides inhibit sucrose-induced hyperglycemia in silkworms.

Diabetes and obesity result from sucrose-induced hyperglycemia. Prevention of hyperglycemia contributes to inhibit the onset of these life-related diseases. Here we show that polysaccharides obtained from soil bacteria inhibit sucrose-induced hyperglycemia in an in vivo silkworm evaluation system. Ethanol precipitates of extracellular polysaccharides were prepared from viscous bacterial colonies. Among 24 samples obtained from different bacterial species, oral administration of 6 samples from Rhizobium altiplani, Cupriavidus sp., Paenibacillus polymyxa, Pantoea eucalypti, Variovorax boronicumulans, and Xanthomonas cynarae suppressed sucrose-induced hyperglycemia in silkworm insect larvae. The R. altiplani fraction treated further with DNase I, RNase A, and proteinase K, followed by phenol extraction also exhibited suppressive activity. Our results suggest that silkworms provide an efficient screening system of bacterial polysaccharides that inhibit sucrose-induced hyperglycemia.

Antibacterial activity and probiotic characterization of autochthonous Paenibacillus polymyxa isolated from Anabas testudineus (Bloch, 1792).

Microbial fish pathogens are prevalent in aquaculture. Control of bacterial fish pathogens is important and bio control of pathogenic bacteria is a novel field of study. The aim of this study was to evaluate the antagonistic activity of bacteria isolated from Anabas testudineus against potent fish pathogens. The cellular components/preparations and filtered cell free culture supernatants were effective against six fish pathogens. Altogether 110 strains were isolated from fish proximal and distal intestine, out of which 10 strains were selected through well diffusion method. From them a strain HGA4C having prominent antimicrobial activity was selected as candidate probiotic strain. The whole-cell product, heat-killed whole-cell product and the filtered broth were exhibited bactericidal activity against the tested pathogens. Among them cell free culture supernatant showed maximum inhibition. In addition, isolated candidate probiotic bacterium was capable of producing extracellular enzymes important for the digestion of food ingredients and was effectively grown in fish mucus obtained from Oreochromis niloticus. The strain tolerated gradient of bile juice secreted by the host and effectively produced biofilm. Analysis of 16S rDNA sequence revealed that isolated strain HGA4C was Paenibacillus polymyxa (MF457398.1). Furthermore intraperitoneal injection of the bacterium did not induce any pathological anomalies or mortalities in Oreochromis niloticus and disclosed the safety of this bacterium as a candidate probiotic in aquaculture.

Characterization of Bacillus megaterium, Bacillus pumilus, and Paenibacillus polymyxa isolated from a Pinot noir wine from Western Washington State.

This report provides the first confirmed evidence of Bacillus-like bacteria present in a wine from Washington State. These bacteria were isolated from a 2013 Pinot noir wine whose aroma was sensorially described as being 'dirty' or 'pond scum.' Based on physiological traits and genetic sequencing, three bacterial isolates were identified as Bacillus megaterium (strain NHO-1), Bacillus pumilus (strain NHO-2), and Paenibacillus polymyxa (strain NHO-3). These bacteria grew in synthetic media of low pH (pH 3.5) while some survived ethanol concentrations up to 15% v/v. However, none tolerated molecular SO2 concentrations ≥0.4 mg/l. Growth of strains NHO-1 and NHO-3 in a Merlot grape juice resulted in increases of titratable and volatile acidities while decreases in titratable acidity were noted for NHO-2.

Isolation of a potential biocontrol agent Paenibacillus polymyxa NSY50 from vinegar waste compost and its induction of host defense responses against Fusarium wilt of cucumber.

Fusarium wilt caused by Fusarium oxysporum f. sp. cucumerinum (FOC) is one of the major destructive soil-borne diseases infecting cucumber. In this study, we screened 60 target strains isolated from vinegar waste compost, from which 10 antagonistic strains were identified to have the disease suppression capacity of bio-control agents. The 16S rDNA gene demonstrated that the biocontrol agents were Paenibacillus polymyxa (P. polymyxa), Bacillus amyloliquefaciens (B. amyloliquefaciens) and Bacillus licheniformis (B. licheniformis). Based on the results of antagonistic activity experiments and pot experiment, an interesting strain of P. polymyxa (named NSY50) was selected for further research. Morphological, physiological and biochemical characteristics indicated that this strain was positive for protease and cellulase and produced indole acetic acid (22.21±1.27µg mL-1) and 1-aminocyclopropane-1-carboxylate deaminase (ACCD). NSY50 can significantly up-regulate the expression level of defense related genes PR1 and PR5 in cucumber roots at the early stages upon challenge with FOC. However, the gene expression levels of a set of defense-related genes, such as the plant nucleotide-binding site (NBS)-leucine-rich repeat (LRR) gene family (e.g., Csa001236, Csa09775, Csa018159), 26kDa phloem protein (Csa001568, Csa003306), glutathione-S-transferase (Csa017734) and phenylalanine ammonia-lyase (Csa002864) were suppressed by pretreatment with NSY50 compared with the single challenge with FOC after nine days of inoculation. Of particular interest was the reduced expression of these genes at disease progression stages, which may be required for F. oxysporum dependent necrotrophic disease development.

Isolation, identification and characterization of Paenibacillus polymyxa CR1 with potentials for biopesticide, biofertilization, biomass degradation and biofuel production.

BACKGROUND: Paenibacillus polymyxa is a plant-growth promoting rhizobacterium that could be exploited as an environmentally friendlier alternative to chemical fertilizers and pesticides. Various strains have been isolated that can benefit agriculture through antimicrobial activity, nitrogen fixation, phosphate solubilization, plant hormone production, or lignocellulose degradation. However, no single strain has yet been identified in which all of these advantageous traits have been confirmed. RESULTS: P. polymyxa CR1 was isolated from degrading corn roots from southern Ontario, Canada. It was shown to possess in vitro antagonistic activities against the common plant pathogens Phytophthora sojae P6497 (oomycete), Rhizoctonia solani 1809 (basidiomycete fungus), Cylindrocarpon destructans 2062 (ascomycete fungus), Pseudomonas syringae DC3000 (bacterium), and Xanthomonas campestris 93-1 (bacterium), as well as Bacillus cereus (bacterium), an agent of food-borne illness. P. polymyxa CR1 enhanced growth of maize, potato, cucumber, Arabidopsis, and tomato plants; utilized atmospheric nitrogen and insoluble phosphorus; produced the phytohormone indole-3-acetic acid (IAA); and degraded and utilized the major components of lignocellulose (lignin, cellulose, and hemicellulose). CONCLUSIONS: P. polymyxa CR1 has multiple beneficial traits that are relevant to sustainable agriculture and the bio-economy. This strain could be developed for field application in order to control pathogens, promote plant growth, and degrade crop residues after harvest.