导　　师： 夏延斌

学科专业： 083201

授予学位： 硕士

作　　者： ;

机构地区： 湖南农业大学

摘　　要： 果蔬的采后病害是影响其保鲜和贮藏的主要因素之一,目前使用防腐剂或保鲜剂来减少病害仍是主要措施,但剂量不适易造成过多残留,危害人们的健康；连续使用易诱发病原菌的抗药性。而健康的植物体内存在大量的内生菌,其中有一些菌对病原菌具有明显的拮抗作用,可作为控制果蔬采后病害的一种新资源开发,具有良好的应用前景。 本研究以健康辣椒植株为材料,研究了从中分离内生菌方法、并以辣椒疫霉病菌/(Phytophthora capsici/)、辣椒黑霉病菌/(Stemphylium botryosum/)、辣椒炭疽病菌/(C. gloeosporioides/)为病原菌来筛选拮抗菌,然后优化培养基和发酵条件,将培养的拮抗菌种对辣椒进行保鲜,主要结论如下： 1、以辣椒疫霉病菌、辣椒黑霉病菌、辣椒炭疽病菌为病原菌,采用稀释分离法从健康辣椒植株的茎、叶、果肉中选择性分离有拮抗作用的内生菌菌株。通过初筛和复筛,分离出一株具有明显拮抗作用的菌株X,研究得出该菌株对三种病原菌的抑制率在34.80/%-48.0/%之间。经过形态学观察,该菌菌落较小、呈浅白色、不透明、中间突起、半湿润,边缘整齐；该菌是革兰氏阳性的芽孢杆菌、个体较小、呈杆状、成对,有时成链排列。经UltrafleXtreme MALDI-TOF-TOF生物质谱仪分析,得出该菌株与芽孢杆菌属/(Bacillus/)的同源性为99/%,故确定该菌株为解淀粉芽孢杆菌/(B. amyloliquefaciens/),命名为X。 2、通过单因素试验得到拮抗内生菌X的最适温度为28℃；最适pH为7.0；接种量从0.6/%增加到1/%时,其抑制率从37.67/%也随之增加到65.78/%；该菌在不同的培养条件下,抑制率发生变化,在培养基中添加不同的碳源时,葡萄糖抑制率为70.11/%,蔗糖、可溶性淀粉、麦芽糖和乳糖的抑制率分别为65.34/%、60.32/%、44.12/%、35.68/%；氮源中酵母膏、牛肉膏、蛋白胨、NH4ClO和KN03其抑菌作用依次降低,抑菌率分别为72.64/%、60.13/%、58.97/%、42.55/%、32.78/%；不同金属离抑菌效果不一样,NaCl、CaCl2、KH2PO4、FeSO4和MnSO4,抑制率分别为为65.11/%、57.28/%、53.23/%、45.46/%和40.25/%。以单因素试验结果为依据,进行多因素试验,通过4因素5水平的响应面实验,得到最适合菌株x的生长和最佳抑菌效果条件,最佳培养基组成为接种量0.80/%,酵母膏0.5/%,葡萄糖2.5/%,NaCl0.13/%, pH7.0。培养条件为温度28℃、转速180r//min。在此条件下菌株X对病原菌的抑制率达到最大为73.02/%,明显高于未经优化培养条件下测得的抑制率48.0/%。 3、拮抗内生菌X对新鲜辣椒的采后保鲜及抑菌效果研究。将采收的辣椒用拮抗菌发酵液浸泡处理,在室温20℃28℃条件下贮藏期间的主要病害是辣椒黑霉病菌和辣椒疫霉病菌,在室温25℃条件下,用1/%、2/%、3/%浓度的拮抗内生菌发酵滤液浸泡辣椒,辣椒疫霉病的发病率分别为45/%、38/%、56/%,无菌水对照组的发病率为79/%；辣椒黑霉病的发病率21/%、18/%和14/%,其中对照组为47/%。在5℃贮藏发生的主要病害是辣椒疫霉病,1/%、2/%、3/%浓度的拮抗内生菌发酵滤液浸泡辣椒,辣椒疫霉病的发病率分别为10/%、8/%、7/%,无菌水对照组的发病率为27/%,辣椒黑霉病的发病率0/%、0/%和0/%,其中对照组为5/%,说明在5℃条件下,辣椒黑霉病基本得到控制。将致病菌接种于辣椒上的人工圆形伤口,内生菌发酵滤液对其辣椒病害的抑制效果为：浓度为3/%时,其抑菌圈直径最小为36mm,1/%时为45mm,2/%时为57mm；而无菌水对照组的抑菌圈直径为68mm。另外发现在试验期内浸泡组辣椒的失重率随贮藏时间的延长均呈上升趋势,但上升趋势不同；可溶性固形物的含量随着时间的延长而下降,从4.06/%降到1.31/%：而有机酸的含量也呈下降趋势,3.15mmol//100g降为0.96mmol//100g,Vc含量随贮藏时间的延长均呈下降趋势,但下降趋势不同。 Postharvest disease of fruits and vegetables is one of the main factors which affect the preservation and storage, the use of preservatives or antistaling agents is still the main measures to reduce the disease at the recent, but the residual pollution is caused when the dosage is no well, continuous use is easy to induce drug resistance of pathogenic bacteria, environmental pollution is caused, damage people's health and so on. There are large amounts of endophytes can be used as bio-control bacteria will be a new repository in healthy plants, and they would have a good application prospect in terms of researching for new controlling postharvest diseases of fruits and vegetables. This paper is aim to select a endogenous strain of antagonism X which isolated from healthy plants, the strain has obvious antibacterial effect to Phytophthora capsici, Pepper green mildew, and C.gloeosporioides. And mainly introduce the strain isolation, screening and identification, the culture medium and fermentation conditions is optimized is to improve the ability of its bacteriostatic effect, application effect of postharvest fresh-keeping of pepper is evaluated and biological control is preliminary researched of postharvest diseases of pepper. The main conclusions are as follows: 1. Phytophthora capsici, Pepper green mildew, and C.gloeosporioides as pathogens, antagonism effect of endogenous bacteria strains are selectivity separated from a health chili plant stem, leaf, fruit pulp with the method of dilute separation. A strain X of obvious antagonism effect is isolated with the method of screening, and concludes the inhibition rate between34.80/%,48.0/%.With the morphological observation, the bacteria colony is lesser, the pale white, not transparent, intermediate processes, semi-humid, edge neat; The bacteria is gram positive bacillus, individual small, rods, in pairs, sometimes into a chain. With the analysis of UltrafleXtreme MALDI-TOF-TOF sequences, find that the homology between gene sequence and the strain of Bacillus genus /(Bacillus/) is99/%,therefore,the strain is determined B.amyloliquefaciens, and named X. 2. The optimum temperature of the endogenous bacteria X is28℃with the method of single factor experiment; When pH7.0and the inoculation quantity increases from0.6/%to1.0/%,the inhibition rate also increases from37.67/%to65.78/%; Different carbon source is added in the culture medium, and the glucose is added in the nutrient solution, the best inhibition of pathogenic bacteria is70.11/%; Followed by soluble starch, sucrose, maltose and lactose,65.34/%,60.32/%,44.12/%and35.68/%respectively, the minimal inhibitory effect of lactose among them. When different nitrogen source, as yeast extract, beef extract peptone, NH4ClO and KNO3is added, the inhibitory action of antagonistic bacteria reduce in proper sequence, the rate of inhibition of pathogenic bacteria is72.64/%when yeast extract is added. Followed by beef cream, peptone,NH4ClO and KNO3,the inhibition rate of pathogenic bacteria is60.13/%,58.97/%,42.55/%,58.97/%respectively, The minimum inhibitory effect is KNO3among them; with different metal ions are added in the nutrient solution, the best inhibition rate is65.11/%when NaCl is added, Followed by CaCl2, KH2PO4, FeSO4and MnSO4, inhibition rate of pathogenic bacteria is57.28/%、53.23/%、45.46/%、40.25/%respectively, of which the synergy of MnSO4is minimal. The multifactor experiment was carried out based on single factor test results, through the response surface experiment of four factors, five levels, the most suitable to the growth of strains X and best bacteriostatic effect is obtained, the inoculum quantity0.80/%, yeast extract0.5/%,glucose2.5/%,NaCl0.13/%, the maximum inhibition rate of pathogenic bacteria is73.02/%on the condition of the strains of pathogenic bacteria X, higher than the inhibition rate48.0/%without optimization of cultivation conditions. The best medium of ntagonism of endophytes X is the inoculum quantity0.80/%, yeast extract0.5/%,glucose2.5/%, NaCl0.13/%,pH7.0. the temperature of Culture conditions is28℃, and rotational speed180r//min. 3. Antagonistic endophytic bacteria X has a certain impact on postharvest fresh-keeping and bacteriostasis effect of fresh chili and the storage time and temperature factors. Either in room temperature25℃or in cold storage conditions5℃, the fermentation filtrate of antagonistic bacteria all has bacteriostatic effect to pathogenic bacteria. This paper shows that the main disease of the chili are soaked during the storage at room temperature is Phytophthora capsici, Pepper green mildew, but the disease is Phytophthora capsici in cold storage conditions5℃. Under the condition of room temperature25℃,the concentrations of1/%,2/%and3/%antagonism of endophytic fungus fermentation filtrate cause the Phytophthora capsici of soaked chili,the disease incidence is45/%,38/%and56/%respectively,but under the condition of the control group of sterile water, the disease incidence is79/%,and the best concentration is3/%;the disease incidence of Pepper green mildew is21/%,18/%and14/%on the decline, and the control group was47/%.Under the condition of room temperature5℃,the concentrations of1/%,2/%and3/%antagonism of endophytic fungus fermentation filtrate cause the Phytophthora capsici of soaked chili, the disease incidence is10/%,8/%and7/%respectively, but under the condition of the control group of sterile water, the disease incidence is27/%,and the best concentration is1/%;the disease incidence of Pepper green mildew is0/%respectively, it shows that Pepper green mildew is controlled in the condition of room temperature5℃.After the artificial chili wound, the fermentation filtrate of antagonistic bacteria has bacteriostatic effect to pathogenic bacteria. It shows when the concentration was3/%, it has the smallest diameter of the antibacterial circle,36mm. When its concentration is1/%, the diameter of the antibacterial circle is45mm; when its concentration is2/%, the diameter of the antibacterial circle is57mm; but the control group of sterile water, the diameter of the antibacterial circle is68m. The weightlessness rate of those chili with the extension of storage time are on the rise, but rising trend; In the test;the content of soluble solids in chili falls along with the time, from4.06/%to1.31/%; and the content of the organic acid is a downward trend, from3.15mmol//100g to0.96mmol//100g;Vc content in the extension of storage time are declining, but the downward trend is different.

关 键 词： 辣椒 拮抗内生菌 筛选 鉴定 效果评价

分 类 号： [TS255.5]

领　　域： [轻工技术与工程] [轻工技术与工程]