Antimicrobial activity of Paenibacillus peoriae strain NRRL BD-62 against a broad spectrum of phytopathogenic bacteria and fungi

AIMS: To investigate the potential antagonistic activity of Paenibacillus peoriae strain NRRL BD-62 against phytopathogenic micro-organisms and to determine the physiological and biochemical characteristics of the antimicrobial compound produced by this strain. METHODS AND RESULTS: Strain NRRL BD-62 showed a broad inhibition spectrum with activity against various phytopathogenic bacteria and fungi. Physico-chemical characterization of the antimicrobial activity showed that it was stable during heat treatment and was retained even after autoclave at 121 degrees C for 10 min. The compound was also stable after the treatment with organic solvents, hydrolytic enzymes and its activity was preserved at a wide range of pH. The partial purification carried out by Sephadex G25 gel filtration showed two profiles of inhibition against the indicator strains tested, suggesting at least two different substances with distinct molecular weight. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the production of antimicrobial substances in P. peoriae. Besides the antimicrobial inhibition capability, the strain NRRL BD-62 is also able to effectively fix molecular nitrogen, and produce chitinases and proteases as well, suggesting that further studies should be addressed to use P. peoriae strain NRRL BD-62 as a plant growth promoter and/or as a biocontrol agent in field experiments.     

Influence of growth conditions on the production of extracellular proteolytic enzymes in Paenibacillus peoriae NRRL BD-62 and Paenibacillus polymyxa SCE2

AIMS: To analyse the extracellular protease profile of two Paenibacillus species, Paenibacillus peoriae and Paenibacillus polymyxa, as well as how different growth media influenced its expression. METHODS AND RESULTS: Both bacteria were cultured in five media [Luria-Bertani broth, glucose broth, thiamine/biotin/nitrogen broth (TBN), trypticase soy broth and a defined medium] for 48 h at 32 degrees C. Our results showed a heterogeneous protease secretion pattern whose expression was dependent on medium composition. However, TBN induced the most quantitative and qualitative protease production on both Paenibacillus. The proteases were detected in neutral-alkaline pH range, being totally inhibited by 1,10-phenanthroline, a zinc-metalloprotease inhibitor. We also analysed the protease expression during the growth and, at least to P. peoriae, the most elevated protease activity was measured at 96 h, in which the highest number of spores and a low concentration of viable cells were observed. CONCLUSIONS: The results presented add P. peoriae and P. polymyxa to the list of neutral-alkaline extracellular protease producers. SIGNIFICANCE AND IMPACT OF THE STUDY: Paenibacillus species are ubiquitous in nature, are capable to form resistant spores and to produce several hydrolytic enzymes, including proteases. However, only few data concerning the production of these enzymes are available. Proteases produced by Paenibacillus strains may represent new sources for biotechnological use.     

Antifungal activity and biocontrol potential of Paenibacillus polymyxa HT16 against white rot pathogen (Coniella diplodiella Speq.) in table grapes

Paenibacillus polymyxa HT16 was evaluated for its ability to reduce white rot disease caused by Coniella diplodiella. HT16 showed significant inhibitory effects on C. diplodiella in vitro, reducing white rot disease in ‘Kyoho’ and ‘Thompson Seedless’ table grape varieties by more than 40% after inoculation. Liquid cultures were more effective than washed cell suspensions or cell-free supernatants, and a higher content of, and longer incubation time with, strain HT16 were more effective in reducing disease incidence. The spore germination rate and mycelial morphology of C. diplodiella were adversely affected by HT16. These results suggested that HT16 may be a promising biocontrol agent against pre-harvest and post-harvest decay in table grapes.     

多粘类芽胞杆菌利用生物柴油副产物粗甘油生产乙偶姻

研究多粘类芽胞杆菌利用廉价底物生物柴油副产物粗甘油批次发酵生产乙偶姻,考察不同pH条件、不同转速下乙偶姻、2,3-丁二醇和乙酸3种主要产物的产量,根据发酵结果设计一种三阶段溶氧调节的方法,结果表明:经76h批次发酵,乙偶姻产量为14.62 g/L,副产物2,3-丁二醇和乙酸分别为1.24和2.93 g/L;副产物量低,且易于分离,可以有效减少后期分离提取产物的成本。     

Inhibitory activity of Paenibacillus polymyxa SCE2 against human pathogenic micro-organisms

Paenibacillus polymyxa strain SCE2 was shown to inhibit the growth of different potential human pathogenic bacterial strains and fungi in vitro. To determine the genetic characterization of this antimicrobial substance, strain SCE2 was transformed with plasmid pTV32(Ts), a delivery vector for Tn917-lac. After transposition, four mutants were shown to have lost their capability to inhibit Micrococcus sp. and Staphylococcus aureus RN450, but they continued to inhibit the growth of Corynebacterium fimi NCTC7547 and Escherichia coli HB101. Hybridization experiments using the DNA of the four mutants digested with different endonucleases and pTV32(Ts) as a probe showed that the place of insertion of Tn917-lac in the chromosome was the same in mutants 4 and 36 and in mutants 31 and 59, but different between these pairs. It is thought possible that more than one antimicrobial substance is being produced by strain SCE2.     

Colonization of peanut roots by biofilm-forming Paenibacillus polymyxa initiates biocontrol against crown rot disease

Aim: To investigate the role of biofilm‐forming Paenibacillus polymyxa strains in controlling crown root rot disease. Methods and Results: Two plant growth‐promoting P. polymyxa strains were isolated from the peanut rhizosphere, from Aspergillus niger‐suppressive soils. The strains were tested, under greenhouse and field conditions for inhibition of the crown root rot pathogen of the peanut, as well as for biofilm formation in the peanut rhizosphere. The strains’ colonization and biofilm formation were further studied on roots of the model plant Arabidopsis thaliana and with solid surface assays. Their crown root rot inhibition performance was studied in field and pot experiments. The strains’ ability to form biofilms in gnotobiotic and soil systems was studied employing scanning electron microscope. Conclusion: Both strains were able to suppress the pathogen but the superior biofilm former offers significantly better protection against crown rot. Significance and Impact of the Study: The study highlights the importance of efficient rhizosphere colonization and biofilm formation in biocontrol.     

多粘类芽孢杆菌HY96-2产脂肽类抗真菌物质的研究

对一株已经商业化的可防治植物枯萎病的生防菌-多粘类芽孢杆菌HY96-2发酵液中抗真菌活性物质进行了分离纯化,采用酸化、正丁醇萃取、乙酸乙酯沉淀、硅胶柱层析、Sephadex LH-20柱层析、高效液相色谱(HPLC)等方法分离得到了一个抗真菌活性化合物6B,经NMR、MS、MS/MS等光谱学方法鉴定其为Fusaricidin A。琼脂扩散法抑菌试验结果表明,6B对西瓜枯萎病菌、水稻纹枯病菌、灰霉病菌等15种植物病原真菌的最小抑菌浓度为12.5～50μg/mL。盆栽实验结果表明,6B对黄瓜灰霉病具有明显的防治效果,当6B的浓度达到250μg/mL时,防治效果高达95%。     

一株抗真菌内生多粘芽孢杆菌的分离鉴定及对水稻恶苗病菌的抑制作用

目的：从番茄叶片中筛选具广谱抑真菌活性的拮抗内生细菌,研究其对水稻恶苗病菌的抑制作用。方法：采用对峙培养法筛选拮抗内生细菌,根据菌株形态、生理生化特性结合16S rRNA基因序列分析鉴定菌株;采用硫酸铵沉淀法提取抗菌粗蛋白,研究其对水稻恶苗病菌菌丝生长和孢子萌发的影响。结果：从番茄叶片中筛选到一株抗真菌内生多粘芽孢杆菌（Paenibacillus polymyxa）SD-6,该菌株具有广谱抑菌活性,对供试的13种植物病原真菌均具较强的抑制作用;该菌株产生的抗菌粗蛋白能够显著抑制水稻恶苗病菌菌丝生长和孢子萌发,并能导致萌发孢子畸形和破裂。结论：从番茄叶片中分离到一株能产生抗真菌蛋白并具有广谱高效抑真菌作用的内生多粘芽孢杆菌,该菌株及其抗菌蛋白具有防治水稻恶苗病的潜力。     

影响多粘类芽胞杆菌发酵液抑菌活性因素的研究

目的以点青霉菌作为指示菌,研究影响植物内生多粘芽胞杆菌发酵液抑菌活性的部分因素,为鉴定发酵液的抑菌物质提供基础研究。方法通过对多粘类芽胞杆菌发酵液进行不同处理（改变pH、加热、乙醇处理和蛋白酶酶解）,采用牛津杯法观察处理后发酵液对点青霉菌抑菌活性的变化。结果多粘类芽胞杆菌发酵液的抑菌效果在酸性条件下稳定,抑菌效果明显;而在中性和碱性范围内不稳定,抑菌效果不明显;多粘类芽胞杆菌发酵液中的有些抑菌物质具备良好的热稳定性;80％乙醇处理的发酵上清液有抑菌作用;经蛋白酶酶解后发酵液的抑菌活性变化不大。结论多粘类芽胞杆菌产生的乙醇沉淀物具有抑菌作用;发酵液中可能含有类细菌素的抑菌物质。     

Effects of milling and surfactants on suspensibility and spore viability in Paenibacillus polymyxa powder formulations

Two milling methods and five surfactants were tested for their effects on the suspensibility of a wettable powder formulation of Paenibacillus polymyxa HY96-2 (P. polymyxa original powder or PPOP). Both hammer milling and jet milling significantly improved suspensibility of PPOP (from 8.1 to 26.4% and 58.3%, respectively), and did not decrease spore viability. Five surfactants (EFW^®, D-425^®, sodium dodecyl benzene sulfonate, tea saponin and sodium lignosulphonate) were each mixed with jet milled powder (JMP). The best results were obtained with sodium lignosulphonate and D425. These gave equal improvement in suspensibility. Sodium lignosulphonate had the best biocompatibility; spore viability values of JMP mixed with this at 1, 3, 5, or 10% (w/w) were all over 95.0%. Addition of sodium lignosulphonate to hammer milled powder (HMP) or JMP at 5% (w/w) increased suspensibility from 26.4 to 38.6% and from 58.3 to 81.1%, respectively. Our results suggest that jet milling and sodium lignosulphonate can synergistically improve the suspensibility of P. polymyxa wettable powder without adverse effects on spore viability. These findings will accelerate the commercialization of P. polymyxa wettable powder and provide a basis for the development of other biopesticides.     

Biological Control of Botrytis cinerea Pers. ex Fr. in Strawberry by Paenibacillus polymyxa (Isolate 18191)

Isolate 18191, obtained from mature strawberry fruit and determined as Paenibacillus polymyxa has shown an antagonistic potential against Botrytis cinerea , the causal agent of grey mould in strawberries. Germ tube growth of conidia of B. cinerea was strongly inhibited by the culture suspension of the antagonist in aqueous strawberry fruit pulp suspension (1%) but germination rate of conidia was not affected. The application of the culture suspension and the washed cells on detached strawberry leaf discs reduced conidiophore density of B. cinerea by 67 and 84%, respectively. The treatment of detached leaf discs with culture suspensions of different cell densities (1 × 10⁶, 1 × 10⁷, 1 × 10⁸) showed that the lowest density already reduced incidence of B. cinerea by 68% after 8 days incubation period. Investigating the influence of the temperature on the effectiveness of P. polymyxa it was observed that the antagonist was highly effective already at 10°C and reduced incidence and conidiophore density of B. cinerea by 53 and 58%, respectively. In 3-year field trials the effectiveness of P. polymyxa was in a range of 24–36% as compared to the water control.     

Optimization of dilution rate, pH and oxygen supply on optical purity of 2, 3-butanediol produced by Paenibacillus polymyxa in chemostat culture

The optimum dilution rate for 2, 3-butanediol (BDL) production by Paenibacillus polymyxa was 0.2 h1 and the optical purity of BDL remained above 98 % at all dilution rates. With decreasing culture pH, ethanol and BDL production increased, whereas the optical purity of BDL decreased to 94 % at pH 5.7. In the chemostat culture at pH 6.3 and a 0.1 h1 dilution rate, the optimum air supply for BDL production was 200 ml min–1 in which the O2 uptake rate was 6.7 mmol l–1 h–1. Under this condition, the optical purity of BDL decreased to 93 %. © Rapid Science Ltd. 1998     

Detection and quantification of Paenibacillus polymyxa in the rhizosphere of wild barley (Hordeum spontaneum) with real-time PCR

Aim:  To detect and quantify the plant drought tolerance enhancing bacterium Paenibacillus polymyxa in a collection of 160 Hordeum spontaneum rhizosphere samples at the 'Evolution Canyon' ('EC'), Israel.
Methods and Results:  PCR primers and a FAM-TAMRA probe (6-carboxyfluorescein, 6-carboxy-tetramethyl-rhodamine) targeting 16S rRNA genes were designed and used to detect and quantify the target strain. Two commercial kits, Bio101 Fast Spin and Mo Bio Ultra Clean Soil DNA, were tested for DNA isolation from the rhizosphere and surrounding soil. Population densities of P. polymyxa were studied in the rhizosphere of wild barley and surrounding soil from the contrasting climatic slopes at the 'EC' using the real-time PCR and culture based methods.
Conclusion:  Paenibacillus polymyxa is one of the best established species in wild barley rhizosphere at the 'EC' slopes. With the real-time PCR assay we are able to detect 1 pg of DNA per PCR corresponding to 100 cells per ml. The results at the 'EC' correlate well to bacterial estimations by culture based methods.
Significance and Impact of the Study:  Significantly higher P. polymyxa cell number was detected in the rhizosphere of arid 'African' microclimate indicating possible role of adaptive co-evolution with plants.     

Inhibitory Effect of Paenibacillus polymyxa GBR-462 on Phytophthora capsici Causing Phytophthora Blight in Chili Pepper

In the present work 25 strains of Paenibacillus polymyxa isolated from rotted ginseng roots were screened for their antimicrobial activity against Phytophthora capsici in vitro . Based on antimicrobial activity, 15 strains categorized as strongly antimicrobial, among them GBR-462 was found as the most active, and five strains each as weekly antimicrobial and no antimicrobial. Antimicrobial activity was influenced by the initial inoculum density, as strains of P. polymyxa with a strong antimicrobial activity (including P. polymyxa GBR-462) showed the antimicrobial activity against P. capsici and could form biofilm only when they were applied at the higher initial inoculums, 10⁸ cfu/ml. No inhibitory effect was noted on the mycelial growth and zoospore germination of the pathogen when applied at the lower inoculum density of 10⁶ cfu/ml of P. polymyxa GBR-462. However, sporangium formation and zoospore release was significantly inhibited at the lower inoculum density. Also light and electron microscopy revealed the structures of sporangia aberrant with no or few healthy nuclei, indicating sporangium and zoospore formation inhibited at the lower inoculum density. Application of P. polymyxa GBR-462 into potted soil suppressed disease progression as well as disease severity; disease severity was reduced by 30% as compared to untreated pots, suggesting P. polymyxa GBR-462 could be a potential biocontrol agent against Phytopthora capsici .     

Effects of initial inoculation density of Paenibacillus polymyxa on colony formation and starch‐hydrolytic activity in relation to root rot in ginseng

Aims: To examine the relationships between population growth and biological characters of the plant‐growth‐promoting rhizobacterium Paenibacillus polymyxa GBR‐1. Methods and Results: Population growth, colony formation, starch‐hydrolytic activity, and ginseng root rot caused by P. polymyxa GBR‐1 isolated from a rotten ginseng root were examined in vitro and in vivo at high [1 × 10⁸ colony‐forming units (CFU) ml^?1] and low (1 × 10⁶ CFU ml^?1) initial inoculum densities. Paenibacillus polymyxa GBR‐1 showed strong starch‐hydrolytic activity on modified starch agar with relatively low starch content, but only at certain incubation temperatures (18 and 23°C); the high‐density inoculum produced bacterial colonies about nine times thicker than those formed from the lower inoculum density. Light, scanning electron, and transmission electron microscopy showed that the thick colonies from the high‐density inoculum were filled with extracellular polymeric substances (EPS), in which a relatively small number of ovoid‐rod‐shaped bacterial cells (mostly endospore‐bearing cells) were distributed. In contrast, the thin colonies from the low‐density inoculum were composed of massive vegetative cells with a rectangular rod shape and minimum EPS. Fluorescent in situ hybridization (FISH) revealed that the β‐amylase gene was expressed only in bacterial cells from the thick colonies formed from the high‐density inoculum, but not in those from the low‐density inoculum. The culture filtrate from the thick colonies produced a hydrolytic clear zone on modified starch agar, degraded starch granules in various manners, and produced rot symptoms on ginseng root tissues. Conclusions: The biological properties of colony formation, starch hydrolysis, and ginseng tissue rotting by P. polymyxa GBR‐1 are interrelated; they are influenced by the initial bacterial population density but not by the in situ and the final population densities. Significance and Impact of the Study: Knowledge of disease‐inducing characters of P. polymyxa GBR‐1 can be used in the development of biocontrol strategies.     

Artificial consortium that produces riboflavin regulates distribution of acetoin and 2,3‐butanediol by Paenibacillus polymyxa CJX518

The introduction of an NADH/NAD⁺ regeneration system can regulate the distribution between acetoin and 2,3‐butanediol. NADH regeneration can also enhance butanol production in coculture fermentation. In this work, a novel artificial consortium of Paenibacillus polymyxa CJX518 and recombinant Escherichia coli LS02T that produces riboflavin (VB₂) was used to regulate the NADH/NAD⁺ ratio and, consequently, the distribution of acetoin and 2,3‐butanediol by P. polymyxa. Compared with a pure culture of P. polymyxa, the level of acetoin was increased 76.7% in the P. polymyxa and recombinant E. coli coculture. Meanwhile, the maximum production and yield of acetoin in an artificial consortium with fed‐batch fermentation were 57.2 g/L and 0.4 g/g glucose, respectively. Additionally, the VB₂ production of recombinant E. coli could maintain a relatively low NADH/NAD⁺ ratio by changing NADH dehydrogenase activity. It was also found that 2,3‐butanediol dehydrogenase activity was enhanced and improved acetoin production by the addition of exogenous VB₂ or by being in the artificial consortium that produces VB₂. These results illustrate that the coculture of P. polymyxa and recombinant E. coli has enormous potential to improve acetoin production. It was also a novel strategy to regulate the NADH/NAD⁺ ratio to improve the acetoin production of P. polymyxa.