【作者】 霍振华；

【导师】 沈其荣；

【作者基本信息】 南京农业大学 ， 植物营养， 2010， 博士

【摘要】 多粘类芽孢杆菌(Paenibcillus polymyxa) SQR-21是本实验室从黄瓜枯萎病发病严重的田块中,采集健康黄瓜植株的根际土壤中分离出来的一株产芽孢的革兰氏阳性细菌。P.polymyxa SQR-21能分泌多种胞外抗菌物质,对多种土传病原菌有抑制活性,能显著提高植物的抗病性,同时能促进作物生长,提高作物的产量,是一种较好的生防促生菌。利用SQR-21能形成抗逆性强的芽孢的特性,将SQR-21接种于能很好保持其活性的腐熟有机物料上经过二次发酵后制成生物有机肥已在得到广泛应用。生物有机肥除了含有较高的有机质外,所含有的有益微生物还表现出对作物生长较好的促进效应。因此,生物有机肥具有提高作物产量、改善作物品质、活化土壤养分、提高土壤生物活性；减少或降低连作作物土传病害等作用。对生产微生物有机肥的企业来说,特殊微生物功能菌种的获取、产品的生产、运输和储存是保证产品质量的几个关键步骤。本实验通过对SQR-21芽孢形成和萌发条件的优化,拟获得高效的产孢条件；研究环境因子对芽孢萌发的影响,根据具体环境因素,通过追加外源物质提高生物有机肥施入土壤后的芽孢萌发效率,芽孢性质受芽孢形成条件的影响,研究产孢温度对芽孢性质的影响,可为获得最优质的芽孢提供依据；通过改变生物有机肥的接种配比、储存温度及含水量,拟获得最佳的有机肥储存条件。1、根据MIDI推荐的Sherlock MIS微生物鉴定方法,对供试菌株进行了磷脂脂肪酸(PLFA)的鉴定分析,共检测到12种PLFA。其中含有15个碳原子的异型饱和脂肪酸的含量最多,达到了60%；不饱和脂肪酸为16:1w7calcohol、16:1 w11c、ISO17:1 w10c、17:1 ANTEISO B/iI 17:1 w10c、17:1 ANTEISO B/iI四种,占PLFA总量的9.8%,同时获得与16SrDNA一致的鉴定结果。通过扫描电镜和透射电镜显微技术,直观地观察了SQR-21芽孢的形态、结构和芽孢形成的主要过程。SQR-21芽孢纵轴长为1.5-1.8μm,横轴长为0.5-0.8μm,芽孢表面皱缩、凹陷,有数条鼓起的脊。超薄切片结果显示,SQR-21芽孢为端生；芽孢核外分别被皮层和芽孢衣包裹。芽孢衣分里、中、外三层,呈波浪状包裹在皮层外侧,有明显的突起,横切面显示大约含有8-11个突起的小棱角；这与已报道的P. polymyxa芽孢的星状横切面略有不同。SQR-21芽孢形成过程与一般芽孢杆菌的相似。在前芽孢和母细胞之间,合成皮层的物质开始积累并最终转化为皮层结构,母细胞膜逐渐被降解,电子致密物质开始合成,并逐渐转化为芽孢衣,完成所有的芽孢结构分化后,孢子囊破裂,释放出成熟的芽孢。2、在实验室摇瓶发酵条件下,最佳碳源为葡萄糖,浓度为1g/L；最佳氮源为牛肉膏,浓度为16g/L; Mn2+、Mg2+、Ca2+和Fe2+最佳浓度分别为0.1 mmol/L、O.5g/L 1 mmol/L和0.5μmol/L;最佳装液量为250ml三角瓶装液100ml；最佳pH为7.0-8.0；最佳培养温度为37℃。在37℃,170r/min振荡培养48小时后,可获得2.22x1010cfu/ml的细菌总数和2.21 x1010cfu/ml的芽孢产量,芽孢产率接近100%。3、芽孢萌发是拮抗微生物在田间发挥生物学效应的重要步骤,因此为了提高生物有机肥的防治效果,进行了芽孢萌发条件研究。本实验以L-天冬酰胺、葡萄糖、果糖和氯化钾(AGFK)组合,以及L-丙氨酸和糖类组合为萌发剂研究P. polymyxa ACCC10252和SQR-21的萌发条件。当AGFK为主要萌发剂,Tris-HCl为萌发缓冲液时,热激活处理和萌发温度对芽孢萌发的影响显得尤为明显。当以PBS为萌发缓冲液时,单一的L-丙氨酸对芽孢萌发的作用很微弱(ACCC10252和SQR-21的OD600的下降百分数分别为17.3%和14.6%),但加入少量的果糖后芽孢能迅速萌发,其中以10mmoll-1 L-丙氨酸和1 mmol l-1果糖组合能获得100%芽孢萌发效率。相反,当萌发缓冲液换成Tris-HCl时,萌发效果并不明显,但10 mmol l-1氯化钠的加入分别能将ACCC 10252和SQR-21的芽孢萌发效率提升19.6%和24.3%。另外,10 mmol l-1 L-丙氨酸和1 mmol l-1果糖诱导的芽孢萌发对pH的适应范围较广。在施用有机肥的过程中,根据土壤环境的差异,我们可以通过添加适当的外源物质,达到促进芽孢迅速繁殖、顺利定殖的目的。4、研究不同的芽孢形成温度(25℃,30℃和37℃)对芽孢性质的影响,包括：吡啶羧酸(DPA)的含量、耐热性、脂肪酸组成及含量、萌发效率及芽孢蛋白组成。结果表明,37℃条件下芽孢形成速度和耐热性均高于25℃和30℃条件下形成的芽孢,然而萌发效率呈下降趋势。总的来说,芽孢萌发效率与芽孢形成温度成负相关,25℃形成的芽孢萌发效率是30℃形成芽孢的1.27倍。通过检测芽孢膜上脂肪酸的组成与含量发现,随着芽孢形成温度的增加,Anteiso-/Iso-值呈下降趋势,这与温度影响芽孢内层膜的流动性有关,Anteiso-/Iso-越高细胞膜的渗透性越高。芽孢衣蛋白质的组成及种类同样随芽孢形成温度的升高而增多,经蛋白质测序分析,在37℃的芽孢衣蛋白中检测到草酸脱羧酶蛋白、芽孢肽聚糖水解蛋白和外层芽孢衣蛋白三种。因此,在拮抗微生物的发酵过程中,我们不仅要考虑芽孢的产量,还要在此基础上获得优质的芽孢性能,这直接关系到生物有机肥的肥效。5、本实验通过平板计数和原位荧光杂交技术(Fluorescence In Situ Hybridization, FISH)研究生物有机肥(bio-organic fertilizer, BIO)的储存温度,含水量以及接种时SQR-21的存在状态(共27个处理),探讨储存BIO最佳条件,为今后的生物有机肥商品化提供实验依据。实验发现FISH计数结果比平板计数高1-2个数量级,这可能与探针的特异性及芽孢的特性受BIO储存环境影响有关。FISH方法更能便捷、高效、直观地反映复杂环境中微生物状态和数量的变化情况。主要实验结果如下：(1)在20℃储存温度下,接种100%芽孢的处理的芽孢形成率明显高于其它处理。其中S20处理中,第60天时,芽孢获得率为95.6%,总菌数和芽孢数分别占初始值的72.2%和69.0%；S30处理中,第60天时,芽孢获得率为97.3%,总菌数和芽孢数分别占初始值的77.0%和75.0%；而S40处理由于含水量较高,芽孢数明显减少,且萌发后的菌体的存活率不高,第60天时,芽孢获得率为81.6%,总菌数和芽孢数分别只占初始值的49.5%和40.4%。(2)在30℃储存温度下,接种100%芽孢,控制含水量在40%时可保证获得最高的芽孢形成率及细菌总数；接种100%营养体细胞不利于BIO的保存；接种芽孢和营养体的混合菌,控制20%的含水量也能获得相对较高的芽孢形成率和总菌数。(3)在40℃储存温度下,接种100%芽孢的处理中,只有S20处理在第60天时仍保持较高的细菌总数和芽孢数,分别占初始细胞总数的79.4%和79.3%；随着含水量的增加,细胞总数显著减少。总的来说,接种100%的芽孢,保持较低含水量,室温下放置时有利于BIO产品的储存。生产中可以根据实际的环境条件,优化生物有机肥的生产流程和储存条件,以达到节约成本,提高生物有机肥料生物效应的目的。更多还原

【Abstract】 Paenibacillus polymyxa (P. polymyxa) strains, that can produce two types of peptide antibiotics and can form endospores and survive efficiently in soil make them very popular in biocontrol. P. polymyxa SQR-21 is a spore-forming Gram-positive bacterium, which was isolated from plant rhizosphere soil by the Plant-Microbial Interaction Laboratory, Nanjing Agricultural University. It has been identified as a potential biological control agent and classified as a plant growth-promoting rhizobacterium (PGPR). SQR-21 strain has also been introduced into matured composts to make bioorganic fertilizer. These fertilizers have been shown to exhibit strong biological control of Fusarium wilt disease in cucumber and watermelon when applied to Fusarium oxysporum infested soils. In this thesis, we studies the factors influencing spore formation and germination of SQR-21, and the application of SQR-21 spores in amino acid organic fertilizer to produce bioorganic fertilizer (BIO). The main results were as follows:1. The cell membrane of strain SQR-21 contains 12 species of PLFA. The content of anteriso-C15 fatty acid was much higher than other PLFA. The unsaturated fatty acids including 16:1w7c alcohol,16:1 w11c, ISO 17:1 w10c and 17:1 ANTEISO B/i I were about 9.8% of total PLFA. The strain was identified as P. polymyxa by the Sherlock Microbial Identification System (Sherlock MIS). The spore and sporulation process of P. polymyxa SQR-21 were examined by electron microscopy. The dimensions of the oval-shaped spore, formed at terminal portion of cell, were 0.5-0.8μm x 1.5-1.8μm. The spore coat of SQR-21 consisted of three morphologically distinguishable layers, the inner, the middle and the outer spore coat. Eight to eleven ridges were found on a cross section image, which was different from other Bacillus. spp with star-shape. The process of sporulation was essentially the same as that of other Bacillus. spp. During the period of prespore and the mother cell membrane, the substances to form cortex began to accumulate. In the later stage of the sporulation, the electron dense materials were synthesized and became coat structure. At the end, the mature spore released from the mother cell. 2. In order to enhance cell growth and spore formation of P. polymyxa SQR-21, the impact of medium composition and culture conditions were examined. The optimal medium composition was determined as follows:glucose 1g/L; beef extract 16g/L; Mn2+ 0.1 mmol/L; Mg2+0.5g/L; Ca2+1 mmol/L and Fe2+0.5μmol/L. The optimal pH and temperature for growth and sporulation were 7.0-8.0 and 37℃respectively. The bottle filling capacity 100ml/250ml was best. The maximum total cell number (2.22 x1010 cfu/ml) and spore yields (2.21 x 1010 cfu/ml) were obtained at 37℃,170r/min for 48h.3. To optimize the role of Paenibacillus polymyxa SQR-21 in bioorganic fertilizer, a study of spore germination under various conditions was conducted. The abilities to induce spore germination of Paenibacillus polymyxa ACCC10252 and SQR-21 by a mixture of L-asparagine, glucose, fructose and K+(AGFK), and single sugar (glucose, fructose, sucrose and lactose) plus L-alanine were evaluated. Spore germination was measured as a decrease in optical density at 600 nm. The effects of heat activation and germination temperature were important for germination of spores of both strains on AGFK in Tris-HCl. L-alanine alone showed a weak ability to induce spore germination. However, fructose plus L-alanine significantly induced spore germination and the maximum spore germination rate was observed with 10 mmol l-1 L-alanine in the presence of 1 mmol l-1 fructose in PBS. In contrast, fructose plus L-alanine hardly induced spore germination in Tris-HCl. However, addition of 10 mmol l-1 NaCl into Tris-HCl, the percentages of OD600 fall increased by 19.6 and 24.3%for ACCC10252 and SQR-21, respectively. AGFK-induced spore germination needed much more strict germination temperature than fructose plus L-alanine. For both strains, fructose plus L-alanine-induced spore germination was not sensitive to pH.4. Effect of spore-forming temperatures on the spore characteristics including dipicolinic acid (DPA) content, heat resistance, fatty acids composition, germination, and spore coat proteins was studied with Paenibacillus polymyxa SQR-21. Spores were formed rapidly at 37℃and showed higher heat resistance than those formed at 25℃and 30℃. However, the germination rate was negatively correlated to the sporulation temperature. The maximum germination rate of spores formed at 25℃was 1.27 times higher than that formed at 30℃, which may be due to the higher permeability of the spores’inner membrane. The pellets of spores formed at higher temperature showed much darker color than those formed at lower temperature and the number of spore coat proteins were increased with the increase of sporulation temperature.5. The influences of BIO storage temperature, water content and SQR-21 physiologically inoculated status on BIO were studied by plating method and FISH method. The spore and vegetative cell numbers detected by FISH were 10-100 times higher than that by plate counting, suggesting that FISH might be a better method to detect bacterial numbers. When inoculated with 100% spores of SQR-21 into the amino acid fertilizer with 20%-30% water content, the maximum spore forming rate and total cell number were obtained at 20℃. When incubated at 30℃, the optimal conditions for BIO storage were 40% water content with 100% spores. When the BIO was placed at 40℃, lower water content and inoculation with 100% spores favored the BIO storage. In all words, inoculation with 100% spores and maintaining of water moisture at the range of 20%-30% was favourable toward BIO storage. These results were useful in producing high quality of bio-organic fertilizers and keeping them in a good way.更多还原