醋糟基质对土传病害的抑制效果及其拮抗微生物的研究

【作者】 林英；

【导师】 李萍萍；

【作者基本信息】 江苏大学 ， 农业生物环境与能源工程， 2014， 博士

【摘要】 无土基质栽培由于其能够避免土传病害等连作障碍问题而在设施园艺中快速发展。但是目前对基质栽培避免连作障碍主要是靠更换新的基质,成本代价高,因此,研发出具有抑制土传病害作用的新型基质成为了设施园艺发展的重要课题。醋糟基质是将制醋行业的废料通过堆置发酵后加工而成,已有的研究结果表明其连作黄瓜3年都没有出现土传病害。为探究醋糟基质是否具有抑制土传病害作用,对哪些病害具有抑制作用,以及抑病的机制,本论文在温室盆栽实验条件下,对醋糟基质单独使用及其与草炭、蛭石按不同比例混配使用对黄瓜枯萎病和立枯病的抑制效果进行了评估,对醋糟基质中的拮抗微生物进行了深入的研究,初步探明了醋糟基质可以避免黄瓜枯萎病等连作病害的原因,并筛选出23株拮抗微生物,为高效利用醋糟基质并最终研制出具有土传病害抑制作用的新型基质提供了科学依据。主要研究结果如下：本研究得到如下主要结论：1.在温室盆栽条件下,进行了纯醋糟基质及其醋糟与草炭、蛭石不同配比(体积比)的基质对黄瓜枯萎病抑制效果的试验。结果表明：①与纯草炭对比,醋糟基质单独使用或者与草炭、蛭石混配使用对黄瓜枯萎病都有一定的抑制效果。不同混配基质对黄瓜枯萎病的抑制效果按从高到低依次为：醋糟：草炭：蛭石(3：1：1)>纯醋糟>醋糟：草炭：蛭石(6：1：1)>醋糟：草炭：蛭石(1：1：1)>醋糟：草炭(1：1)；②将黄瓜枯萎病的病情指数与基质的理化性状和微生物数量进行Pearson相关性分析发现：基质的容重,C/N与黄瓜枯萎病的病情指数呈显著的正相关,相关系数r值分别为0.4788和0.6994；基质的总孔隙度、通气孔隙度、pH值、EC值与黄瓜枯萎病的病情指数呈显著的负相关,相关系数r值分别为-0.6463，-0.6463,-0.5708和-0.7766；种植前基质中芽孢杆菌的数量,种植后基质中细菌的数量、芽孢杆菌的数量都与黄瓜枯萎病的病情指数呈显著的负相关,相关系数r值分别为-0.5783,-0.5795和-0.3862；③采用主成分分析,以病情指数、苗高、茎粗、地上部分干重、地下部分干重、雌花数为评价对象,对不同混配基质下黄瓜的生长状况进行综合评价发现,在接种黄瓜枯萎病病原菌的情况下,不同混配基质下黄瓜的综合得分由高到低依次为醋糟：草炭：蛭石(3：1：1)>纯醋糟>醋糟：草炭：蛭石(6：1：1)>醋糟：草炭：蛭石(1：1：1)>醋糟：草炭(1：1)>纯草炭。2.在温室盆栽条件下,将醋糟基质与草炭、蛭石按照不同的体积配比,研究不同混配基质对黄瓜立枯病的抑制效果。研究表明：①与纯草炭基质相比,醋糟基质单独使用或者是与草炭或蛭石混配使用对黄瓜由立枯丝核菌引发的立枯病都没有明显的抑制效果；②将黄瓜立枯病的病情指数与基质的理化性质和微生物数量进行Pearson相关性分析发现,黄瓜立枯病的病情指数与基质的物理性状、化学性状、养分性状和微生物数量等各项指标都没有显著的相关性。3.在室内平板实验条件下,将经过过滤灭菌处理、高压灭菌处理和没有经过任何灭菌处理的醋糟浸提液分别添加到PDA培养基中,研究醋糟浸提液对黄瓜枯萎病和立枯丝核病原菌菌丝生长的抑制效果。结果表明：与对照组相比,经过过滤灭菌和高压灭菌处理过的醋糟浸提液对2种病原菌菌丝的生长都没有明显的抑制作用,而没有经过任何灭菌处理的醋糟浸提液对黄瓜枯萎病和立枯丝核菌菌丝生长的抑制率分别为65.16%和40.67%。4.在传统的稀释平板法上略做改进,对醋糟基质中的拮抗细菌进行了分离,根据16S rDNA序列分析对分离的拮抗细菌进行了鉴定。利用平板对峙法对分离的拮抗细菌的广谱抑菌效果进行了验证,采用平板实验通过水解圈的有无对拮抗细菌的水解酶进行了检测；采用无机磷Ca3(PO4)2固体溶磷圈法、CAS平板检测法、JNFb半固体培养基法、Salkowski比色法对拮抗细菌促生长相关的生物学特征如解磷、产铁载体、固氮、产IAA的能力分别进行了检测；并从中选取了5株在室内平板实验条件下对病原菌抑制效果较为突出的拮抗菌在室内盆栽实验条件下观察其抗病促生效果。通过研究发现,从醋糟基质一共分离到了18株芽孢杆菌和1株类芽孢杆菌。分离的这19株拮抗细菌对8种植物病原真菌都具有较好的广谱性抑制效果,类芽孢杆菌还对革兰氏阳性细菌和革兰氏阴性细菌都具有很好的抑制效果。分离的19株拮抗细菌全部都能产生纤维素酶,有11株拮抗菌能产生三种水解酶(纤维素酶、蛋白酶、几丁质酶),13株拮抗细菌(68%)能分泌几丁质酶,18株拮抗细菌(890%)能分泌蛋白酶。分离的每种拮抗细菌至少都能产生2种水解酶。分离的19株拮抗菌中有18株菌(95%)能产生铁载体,没有1株菌具有解磷功能,极少数菌具有固氮和产IAA功能,所有的拮抗细菌都表现出了至少一种与植物促生长相关的生物学特征。温室盆栽实验的结果表明,选取的5株拮抗细菌中有4株对黄瓜枯萎病和黄瓜立枯病都具有很好的抑制效果,有4株拮抗菌对黄瓜小苗的生长表现出了明显的促进效应。5.采用传统的稀释平板法,对醋糟中的拮抗真菌进行了分离,采用18S rDNA序列分析对分离的拮抗真菌进行了鉴定；采用赛璐酚膜法和双格实验法对分离的拮抗真菌产非挥发性和挥发性代谢产物的抑菌活性进行了检测；采用与上节拮抗细菌相同的方法进行了水解酶活性、解磷、产铁载体、产IAA能力等指标检测。结果表明：从醋糟基质中一共分离到了4株拮抗真菌,且这4株菌都为木霉菌。分离的4株木霉菌全部都能产生非挥发性的代谢产物抑制立枯丝核菌的生长,其中1株木霉菌还能产生挥发性的代谢产物抑制立枯丝核菌的生长。分离的4株木霉菌都能分泌几丁质酶、蛋白酶,3株能分泌铁载体,3株能产生IAA,1株具有解磷功能。更多还原

【Abstract】 Soilless cultivation develops rapidly in horticulture because it can alleviate continuous cropping obstacles such as soil-borne diseases. The common approach to eradicate obstacles currently is to replace the substrate. So look for new substrate, which can suppress soil-borne diseases and overcome continuous cropping obstacles has been become the important issue of horticulture. Vinegar residue substrate is a new horticultural substrate made from waste residue produced by the vinegar industry. And the results of current research showed that the continuous cropping obstacles did not appear when continuous planting tomatoes or cucumbers for3years. In order to study whether the vinegar residue substrate has suppressive effects on soil-borne diseases, what is the mechanism? This paper studied the suppressive effects of mixed substrates of vinegar residue mixed with peat and vermiculite in different proportions to wilt caused by Fusarium oxysporum and damping off caused by Rhizoctonia solani of cucumber in pot experiment under greenhouse, especially deeply researched the antagonistic microorganisms in vinegar residue substrate. These rearches primary proved the reasons that the vinegar residue substrate can avoid continuous cropping diseases such as wilt, and isolated23antagonistic microorganisms, which provide scientific basis for using of vinegar residue substrate efficient and developing the new substrate with suppressive effects to soil-borne diseases eventually. The main conclusions of this study areas follows:1. Mixed the vinegar residue substrate with peat and vermiculite in different proportions, their suppressiveness to Fusarium oxysporum wilt were studied in pot experiment under greenhouse. These results showed that:①comparison with pure peat, vinegar residue substrate used alone or mixed with peat or vermiculite all had a certain inhibitory effect on cucumber wilt. The ability to suppress diseases of different mixed vinegar residue substrate followed the order:vinegar residue substrate:peat: vermiculite (3:1:1)> pure vinegar residue substrate> vinegar residue substrate peat:vermiculite (6:1:1)> vinegar residue substrate:peat:vermiculite (1:1:1)> vinegar residue substrate:peat (1:1)(ratios are by volume);②The results of Pearson correlation analysis of cucumber wilt disease index between the physicochemical property and the number of microorganisms found that there was significant positive correlation between wilt disease index and C/N (r=0.4788)、bulk density(r=0.6994) respectively; there was a significant negative correlation between cucumber wilt disease index and total porosity (r=-0.6463)、aeration porosity (r=-0.6463_、pH(r=-0.5708) and EC (r=-0.7766) of substrate; there was a significant negative correlation between cucumber wilt disease index and the number of Bacillus spp.(r=-0.5783) in substrate before planting、the number of bacteriar (=-0.5795) and Bacillus spp.(r=-0.386) after planting;③Using the disease index、height、stem diameter、shoot dry weight、root dry weight as an evaluation object to make a principal component analysis of growth of seedlings, the results of comprehensive evaluation found that under inoculation with the pathogen conditions, The synthesis score of seedlings in different mixed vinegar residue substrate followed the order:vinegar residue substrate:peat:vermiculite (3:1:1)> pure vinegar residue substrate> vinegar residue substrate:peat:vermiculite (6:1:1)> vinegar residue substrate:peat:vermiculite (1:1:1)> vinegar residue substrate:peat (1:1)(ratios are by volume).2. Mixed the vinegar residue substrate with peat and vermiculite in different proportions, their suppressiveness to Rhizoctonia solani damping-off were studied in pot experiment under greenhouse. These results showed that:①comparison with pure peat, vinegar residue substrate used alone or mixed with peat or vermiculite all did not have inhibitory effect on cucumber damping-off;②The results of Pearson correlation analysis of disease index values between the physicochemical property(bulk density, total porosity, aeration porosity, water-holding porosity, aeration porosity/water-holding porosity, pH, EC, total carbon, total nitrogen, total phosphorus, total potassium, available nitrogen, available phosphorus, available potassium and C/N) and the number of microorganisms (fungi, bacteria, Bacillus spp.) found that there was no significant correlation.3. In the plate experimental, each of the non-sterilized, filter-sterilized and heat-sterilized extracts collected from vinegar residue substrate were added to the PDA medium to study their inhibitory effect on mycelial growth of Fusarium oxysporum f.sp. Cucumerinum and Rhizoctonia solani. These results showed that filter-sterilized and heat-sterilized extracts collected from vinegar residue substrate all had no inhibitory effect on mycelial growth of two pathogenic fungi tested, non-sterilized extracts had significant inhibitory effect compared with the control, the inhibitory ratio to Fusarium oxysporum f. sp. Cucumerinum and Rhizoctonia solani were65.16%and40.67%repectilvy.4. Using an improved dilution plate method isolated antagonistic bacteria from vinegar residue substrate; using16S rDNA gene sequencing to identify them; using a dual plate assay to test their broad antifungal inhibitory activity; using plate medium to test their hydrolytic enzymes by cleared zone; some growth-promoting features also were tested, such as mineral phosphate solubilisation activity was determined on dicalcium phosphate plates; the nitrogen fixation ability of the isolates was tested according to the observation of a pellicle formed near the surface of the NFb semi-solid N-free media; the production of indole-3-acetic acid (IAA) by Salkowski colorimetric technique; Five pre-screened prominent antagonistic bacteria in vitro studies were further evaluated individually for their antagonistic potential against disease and plant growth promotion potential under greenhouse conditions. These results showed that: Using the improved method rapidly isolated18Bacillus spp. and1Paenibacillus sp.; All isolated19antagonistic bacteria exhibited broad-spectrum antagonism towards the tested8fungi; Paenibacillus sp. showed antifungal activity against Gram-negative and Gram-positive bacteria; All of the selected19strains exhibited cellulase activity,11strains secreted three hydro lytic enzymes(cellulose, chitinase, protease),13strains (68%) chitinase activity,18strains (95%) protease activity respectively, all of the strains exhibited at least two enzyme activities;18strains (95%) were siderophore producer, no strains phosphatase activity, small strains showed nitrogen-fixing and producing IAA ability, all of strains showed one of the growth-promoting features, four out of five selected strains were found both to be effective in controlling wilt and damping-off disease and four strains showed strong growth-promoting activities for cucumber seedlings under greenhouse conditions.5. Using the traditional dilution plate method, isolated antagonistic fungi from vinegar residue substrate; using18S rDNA gene sequencing to identify them; using cellophane membranes and divided plate method to test their volatile and non-volatile inhibitors; testing phosphate solubilisation activity by Ca3(PO4)2dicalcium phosphate plates; testing the production of indole-3-acetic acid (IAA) by Salkowski colorimetric technique. These results showed that:four antagonistic fungi were isolated from vinegar residue substrate and the four fungus all belonged to the genus Trichoderma; All the four fungus can produce volatile and non-volatile metabolites that may inhibit the growth of R. solani; All the four Trichoderma have cellulase and chitinase activity, Three out of four can secrete siderophore; three out of four were IAA producer, one out of four strains had phosphatase activity.更多还原