Cloning and Characterization of a Novel Alginate Lyase from Paenibacillus sp. LJ-23

As a low molecular weight alginate, alginate oligosaccharides (AOS) exhibit improved water solubility, better bioavailability, and comprehensive health benefits. In addition, their biocompatibility, biodegradability, non-toxicity, non-immunogenicity, and gelling capability make them an excellent biomaterial with a dual curative effect when applied in a drug delivery system. In this paper, a novel alginate lyase, Algpt, was cloned and characterized from a marine bacterium, Paenibacillus sp. LJ-23. The purified enzyme was composed of 387 amino acid residues, and had a molecular weight of 42.8 kDa. The optimal pH of Algpt was 7.0 and the optimal temperature was 45 °C. The analysis of the conserved domain and the prediction of the three-dimensional structure indicated that Algpt was a novel alginate lyase. The dominant degradation products of Algpt on alginate were AOS dimer to octamer, depending on the incubation time, which demonstrated that Algpt degraded alginate in an endolytic manner. In addition, Algpt was a salt-independent and thermo-tolerant alginate lyase. Its high stability and wide adaptability endow Algpt with great application potential for the efficient preparation of AOS with different sizes and AOS-based products.     

固氮类芽孢杆菌的分离鉴定及其促生、抑菌活性的测定

固氮类芽孢杆菌是一类能产生芽孢、具有固氮能力的革兰氏阳性细菌。这类菌除固氮活性和抗逆力强外,有些菌株还具有促进植物生长、对植物病原菌有抑制作用、耐贮藏等特点,被认为是制备微生物肥料的优良菌种之一,但目前经过鉴定的菌种资源十分有限。从全国20个省不同地理环境、不同种类植物根际采集分离筛选获得500个菌株,从中鉴定出60个具有固氮结构基因(nif H)的分离株,通过对其中17个分离株的nif H和16S rRNA基因序列的比对分析,初步确定了它们的系统发育地位,17个分离株均属于固氮类芽孢杆菌,而且这17个菌株全部具有固氮酶活性,最高达到2 109.9±131.1 nmol/mg·h。进一步分析,其中5株具有IAA分泌能力,最高为49.908±3.6μg/m L,11株对4种常见植物病原菌表现出抑制作用。在17个分离株中,共发现了5株兼具固氮、分泌IAA以及抑制植物病原菌的特点,其中IAA分泌能力最低的BJ-18对番茄幼苗仍具有显著的促生作用。分离和鉴定的固氮类芽孢杆菌丰富了固氮微生物菌种资源,发现的兼具固氮、分泌IAA以及抑制植物病原菌的固氮类芽孢杆菌在农业生产中具有潜在的广泛应用前景。     

Biocontrol traits of two Paenibacillus polymyxa strains SQR‐21 and WR‐2 in response to fusaric acid,a phytotoxin produced by Fusarium species

The aim of the present study was to investigate the effects of the phytotoxin fusaric acid (FA) on the biocontrol traits of two biocontrol strains Paenibacillus polymyxa WR‐2 and SQR‐21. The results showed that the growth of both WR‐2 and SQR‐21 decreased with increasing FA concentration, and at 70 and 80 μg mL^?1 FA, respectively, the strains were unable to grow. The biocontrol traits of both strains were negatively affected by FA concentration higher than 2·5 μg mL^?1. However, at 2·5 μg mL^?1 FA, biofilm formation and root colonization were not affected and there was even a positive effect on the production of spores and hydrolytic enzymes (protease and β‐l,3‐glucanase). The production of fusaricidin‐type antifungal compounds was increased with an increase in FA concentration up to 50 and 60 μg mL^?1 for WR‐2 and SQR‐21, respectively. The production of antifungal volatile organic compounds by WR‐2 and SQR‐21 was increased only at 2·5 μg mL^?1 FA. The effect of FA on the overall metabolic activity of WR‐2 and SQR‐21 was also determined. This study will help to understand the response of P. polymyxa strains to FA and will help to improve their biocontrol efficiency.     

Pre-symbiotic and symbiotic interactions between Glomus intraradices and two Paenibacillus species isolated from AM propagules. In vitro and in vivo assays with soybean (AG043RG) as plant host

Two indole-producing Paenibacillus species, known to be associated with propagules of arbuscular mycorrhizal (AM) fungi, were examined for their mycorrhization helper bacteria activity at pre-symbiotic and symbiotic stages of the AM association. The effects were tested under in vitro and in vivo conditions using an axenically propagated strain of the AM fungus Glomus intraradices and Glycine max (soybean) as the plant host. The rates of spore germination and re-growth of intraradical mycelium were not affected by inoculation with Paenibacillus strains in spite of the variation of indole production measured in the bacterial supernatants. However, a significant promotion in pre-symbiotic mycelium development occurred after inoculation of both bacteria under in vitro conditions. The Paenibacillus rhizosphaerae strain TGX5E significantly increased the extraradical mycelium network, the rates of sporulation, and root colonization in the in vitro symbiotic association. These results were also observed in the rhizosphere of soybean plants grown under greenhouse conditions, when P. rhizosphaerae was co-inoculated with G. intraradices. However, soybean dry biomass production was not associated with the increased development and infectivity values of G. intraradices. Paenibacillus favisporus strain TG1R2 caused suppression of the parameters evaluated for G. intraradices during in vitro symbiotic stages, but not under in vivo conditions. The extraradical mycelium network produced and the colonization of soybean roots by G. intraradices were promoted compared to the control treatments. In addition, dual inoculation had a promoting effect on soybean biomass production. In summary, species of Paenibacillus associated with AM fungus structures in the soil, may have a promoting effect on short term pre-symbiotic mycelium development, and little impact on AM propagule germination. These findings could explain the associations found between some bacterial strains and AM fungus propagules.     

多粘类芽孢杆菌抗菌物质和防病机制之研究进展

多粘类芽孢杆菌是一种具有防病和促生作用的生防菌,在植物病害防治方面具有很大的应用潜力。笔者对多粘类芽孢杆菌所产生的抗菌物质及其对病害生防机制的研究进展进行了综述,并对多粘类芽孢杆菌在农业中的应用前景进行了展望。     

多粘类芽胞杆菌胞外多糖在微生物农药剂型中的功能研究

为探明生防菌胞外多糖在微生物农药制剂加工中的应用潜力及价值,考察了多糖粗提物对农药载体的助悬浮作用及对生防菌的热保护和紫外保护作用。结果表明:多粘类芽胞杆菌Paenibacillus polymyxa (HY96-2)胞外多糖对硅藻土、高岭土和碳酸钙3种载体具有明显的助悬浮作用,当载体中多糖粗提物添加量为0.25 g/g时,3种载体的悬浮率可分别提高62.6%、161.8%和284.0%。此外,该胞外多糖对HY96-2芽胞还表现出明显的热保护作用,对HY96-2和海洋芽胞杆菌Bacillus marinus(B-9987)的营养细胞及芽胞表现出明显的紫外保护作用:添加质量体积分数为0.5%的多糖粗提物后,HY96-2芽胞在80 ℃处理20 min后的存活率比对照提高了31.0%,经紫外(UV-B,290~320 nm)照射6 h后,HY96-2营养细胞和芽胞的存活率分别比对照提高了14.1%和22.6% ,B-9987营养细胞和芽胞的存活率分别比对照提高了36.4%和29.5%。该胞外多糖可用作多粘类芽胞杆菌可湿性粉剂加工中的助剂,以替代或减少化学助剂的使用。     

多黏芽孢杆菌HD-1产β-甘露聚糖酶的条件优化

采用DNS比色法,通过单因素试验和正交试验对多黏芽孢杆菌(Bacillus polymyxa)HD-1产β-甘露聚糖酶的培养基主要成分和培养条件进行优化。结果表明,该菌种产β-甘露聚糖酶的最适培养基为魔芋粉0.75%、酵母膏1.00%、磷酸二氢钾0.04%、硫酸镁0.05%,培养基初始p H 9.0;最适培养条件为接种量6%,培养温度31℃,培养时间27 h。在此优化条件下,多黏芽孢杆菌产β-甘露聚糖酶活性可达72.6U/m L。     

Genome Analysis of a Novel Polysaccharide-Degrading Bacterium Paenibacillus algicola and Determination of Alginate Lyases

Carbohydrate-active enzymes (CAZymes) are an important characteristic of bacteria in marine systems. We herein describe the CAZymes of Paenibacillus algicola {"type":"entrez-nucleotide","attrs":{"text":"HB172198","term_id":"239334117"}}HB172198^T, a novel type species isolated from brown algae in Qishui Bay, Hainan, China. The genome of strain {"type":"entrez-nucleotide","attrs":{"text":"HB172198","term_id":"239334117"}}HB172198^T is a 4,475,055 bp circular chromosome with an average GC content of 51.2%. Analysis of the nucleotide sequences of the predicted genes shows that strain {"type":"entrez-nucleotide","attrs":{"text":"HB172198","term_id":"239334117"}}HB172198^T encodes 191 CAZymes. Abundant putative enzymes involved in the degradation of polysaccharides were identified, such as alginate lyase, agarase, carrageenase, xanthanase, xylanase, amylases, cellulase, chitinase, fucosidase and glucanase. Four of the putative polysaccharide lyases from families 7, 15 and 38 were involved in alginate degradation. The alginate lyases of strain {"type":"entrez-nucleotide","attrs":{"text":"HB172198","term_id":"239334117"}}HB172198^T exhibited the maximum activity 152 U/mL at 50 °C and pH 8.0, and were relatively stable at pH 7.0 and temperatures lower than 40 °C. The average degree of polymerization (DP) of the sodium alginate oligosaccharide (AOS) degraded by the partially purified alginate lyases remained around 14.2, and the thin layer chromatography (TCL) analysis indicated that it contained DP2-DP8 oligosaccharides. The complete genome sequence of P. algicola {"type":"entrez-nucleotide","attrs":{"text":"HB172198","term_id":"239334117"}}HB172198^T will enrich our knowledge of the mechanism of polysaccharide lyase production and provide insights into its potential applications in the degradation of polysaccharides such as alginate.     

棉花黄萎病生防内生细菌Jaas cd的鉴定及田间防效

对棉花黄萎病生防内生细菌Jaas cd(原名73a)进行了菌种鉴定和田间应用方式的改进。抑菌谱测定结果表明,该菌株对12种病原真菌均有较强的抑制作用。通过形态观察、生理生化鉴定、Biolog鉴定以及16S rD-NA序列比对,证明该菌株为多黏类芽孢杆菌(Paenibacillus polymyxa)。菌株Jaas cd的16S rDNA序列与多黏类芽孢杆菌(AM062684)的相似性为99.7%,GenBank登录号为AY942618。田间大区示范试验结果表明,用菌株Jaascd的发酵液在棉苗移栽前喷施棉苗和移栽时灌根2种方式处理,都能有效防治棉花黄萎病和提高棉花产量,但移栽前喷施棉苗操作更加简便易行。     

抗菌多肽 APPLM3 乳剂的制备及其对稻瘟病的防效

本试验以来源于多粘类芽孢杆菌（Paenibacillus polymyxa）LM-3菌株的极端嗜热多肽APPLM3为主效成分配得5种微生物源生防乳剂，并用它们对水稻苗瘟进行了田间防效试验。试验结果表明，这5种乳剂的防效都优于LM-3拮抗液直接喷雾，其中处理AB4的防效最佳，达80．73％，其拮抗成分、吐温80、乳化剂OP、溶剂的质量比为50．00：6．50：1．75：41．75。本文报道了常温细菌产生的极端嗜热多肽对稻瘟病菌表现出强的拮抗作用。