【作者】 刘邮洲；

【导师】 王克荣； 陈志谊；

【作者基本信息】 南京农业大学 ， 植物病理学， 2011， 博士

【摘要】 由尖孢镰刀菌番茄专化型[Fusarium oxysporum f.sp.licopersici (sacc) snyderet Hansen]引起的番茄枯萎病和贝伦格葡萄座腔菌梨生专化型Botryosphaeria berengeriana de. Not. f.sp. piricola (Nose)引起的梨轮纹病全国普遍发生。生物防治植物病害能降低农产品表面化学杀菌剂的残留量,保护生态环境和人类健康。我们采集不同地区样本,以两种病原菌为靶标菌,分离、筛选对枯萎病菌或轮纹病菌具有很强拮抗作用的菌株,进行种属鉴定。初步研究拮抗细菌产生的抑菌物质及控病促生作用机制,克隆生防相关基因-几丁质酶基因。同时还研究了拮抗菌株之间互作、微生物-化学物质(杀菌剂、NaHCO3)协同作用与防效之间的相关性。研究结果表明：1、22株拮抗细菌对番茄枯萎病菌具有较强的室内抑制作用,其中菌株PTS-394、H-70、L-1和SJ-280抑制率最高；22株细菌均能分泌蛋白酶；其中菌株C8-8能分泌几丁质酶；有3株细菌能分泌纤维素酶；3株细菌能分泌嗜铁素。盆栽试验结果表明：拮抗细菌PTS-394对番茄枯萎病的防效最高,为77.40%；菌株H-70、L-1和SJ-280对番茄枯萎病的防效均大于60%。对上述四株拮抗细菌进行16s rRNA种属鉴定,均为枯草芽孢杆菌(Bacillus subtilis).2、利用PCR方法从菌株C8-8中克隆到编码几丁质酶的chiA基因。同源性比对发现该基因序列与粘质沙雷氏菌的chiA有97%以上的相似性。酶切后连接到质粒28a中,并转化大肠杆菌DH5α,扩增培养后提取质粒28a(chiA)并转入到表达宿主大肠杆菌DH3中。DH3ChiA菌株在几丁质培养基上能产生透明的水解圈,具有几丁质酶活性,超声波破碎后SDS-PAGE电泳显示为一条相对分子质量约为60KDa的蛋白质,该蛋白具有较强的室内抑菌活性。3、拮抗细菌C8-8的最适培养温度为30℃-35℃、最适培养pH值为7.0-8.0。培养时间、初始接种量对菌株C8-8的生长有明显的影响,培养27h后菌液OD值最高；最佳初始接种量应是被接种培养液体积的0.5-1/300。不同通气量对菌株生长无明显影响。不同培养条件(碳源、氮源、诱导物、温度、时间和pH值)下菌株C8-8产几丁质酶的活力差别较大。N-乙酰氨基葡萄糖能完全抑制菌株C8-8产酶；酵母粉和蛋白胨能使菌株C8-8酶活力明显提高；胶体几丁质能诱导菌株C8-8产酶。培养温度为31℃,pH值为7.3,培养6天时菌株C8-8的酶活最高,为485μg GlcNAc·h-1。4、接种菌株C8-8能诱导番茄植株产生抗性效应,提高植株体内苯丙氨酸解氨酶(PAL)和过氧化物酶(POD)的酶活性；而且还有利于积累活性氧,进一步激发寄主抗性。采用高效液相色谱HPLC对菌株C8-8代谢产物分析表明：该菌株C8-8在NA培养基中的代谢产物内存在大量的产生GA3,推测可能和促生作用相关,培养2d后产生的GA3为158mg/1,而IAA未测出。在基本培养基中该菌株代谢产物未检测出IAA和GA3。5、拮抗细菌之间存在多种互作关系。当混合的2个菌株之间互作关系是亲和的,其生防效果有所提高,如亲和性菌株组合SJ-280+PTS394对番茄枯萎病的防效为47.1%,明显好于单菌株SJ-280(35.3%)和PTS394(38.7%)的防效。当混合的2个菌株之间互作关系是不亲和的,有的菌株组合生防效果下降,如不亲和性菌株组合C8-8+PTS394的防效为23.5%,明显低于单菌株C8-8(52.8%)和PTS394(38.7%)的防效；有的菌株组合生防效果提高,如不亲和性菌株组合L-1+PTS394的防效为50.0%,明显好于单菌株L-1(29.4%)和PTS394(38.7%)的防效。6、分离样本,获得21株对梨轮纹病菌菌丝生长有较强的拮抗能力的菌株,其中拮抗性能最好的细菌sf 628抑菌圈直径达45mm,抑制率达59.21%。梨果接种试验结果表明：21株拮抗能力较强的细菌中有5株拮抗细菌对梨轮纹病菌引起的病斑扩展抑制率大于50%,其中菌株sf 628抑制率达60.61%。进一步的理化特性和分子鉴定结果表明,菌株sf628为枯草芽孢杆菌枯草亚种。7、常见26种化学药剂对梨轮纹病菌的室内毒力测定结果表明：25%敌力脱能有效抑制梨轮纹病菌菌丝生长,ECso值为0.1522μg/ml。将拮抗细菌sf628与25%敌力脱进行复配,结果表明配比为1：3、1：2、1：1、2：1时增效比均大于1.5,表明有增效作用。其中配比为1：3时对梨轮纹病菌的毒力最强,增效比为2.942。拮抗细菌sf628与25%敌力脱配比为3：1时,增效比为1.071,表明两种单剂复配后有相加作用。8、室内抑菌试验结果表明：生防菌单剂对梨轮纹病菌的防效为75.77-85.41%；与NaHCO3协同作用后防效提高,为83.00-90.23%。以生防菌11为例,梨果试验结果表明：先接种病菌再喷施生防菌时,对梨轮纹病的防效为23.83%；与NaHCO3协同作用后防效提高,为48.16-74.57%。先喷施生防菌再接种病菌时,对梨轮纹病的防效为28.72%；与NaHCO3协同作用后防效明显提高,为51.06-80.91%。更多还原

【Abstract】 Tomato Fusarium Wilt caused by F. oxysporum and pear ring rot caused by B. berengeriana are two serious diseases in many areas of the world. Biological control on plant diseases could reduce the remainder of chemical pesticides and protect natural ecological environment. The aim of this study was to screen and evaluate the antagonistic bacteria against F. oxysporum and B. berengeriana. The results showed that:1、There were 367 strains isolated and purified from root and soil of tomato, pepper and watermelon in Ningxia, Jiangsu and Fujian province. Antagonistic examination against F. oxysporum revealed that 22 strains among all tested strains showed strong and steady antagonism and produced protease. The strain C8-8 could produced chitinase. Several strains among the above 22 strains could produce cellulose and siderophore. On pot experiment, strain PTS-394, H-70, L-1 and SJ-280.of 22 strains could control Fusarium Wilt effectively with the control effect 68.64-77.40%. The molecular identification experiments indicated the strain PTS-394, H-70, L-1 and SJ-280 were B. subtilis.2、An open reading frame that coding chiA gene putatively was cloned from the strain C8-8 genomic DNA by PCR method. Sequence alignment showed that the sequence was 97% identical with the ChiA of enzymogenes of S. marcescens. The PCR fragment was cloned into vector 28a. A protein about 60KDa, was expressed by the host strain DH3ChiA. The purified protein could form a clear hydrolyzed zone on the colloidal chitin plate and inhibit the growth of the mycelia of F. oxysporum. It showed that chiA gene from C8-8 could be utilized as a potential biological control factor.3、The strain C8-8, which could produce high-level chitinase, is an antagonistic bacterium against many pathogenic fungi. The proper cultured temperature, pH value and initial inoculum for growth were 30℃-35℃,7.0-8.0 and 0.5-1/300 of total cultured agar volume, respectively. Ventilation had no influence on the growth of the strain C8-8. Using N-Acetyl-D-glucosamine as carbonic sources could inhibit chitinase activity of strain C8-8 totally, but yeast extract powder and peptone could improve chitinase activity of strain C8-8 greatly. It could reach the highest activity,485μg GlcNAc·h-1 when strain C8-8 was cultured at 31℃, pH 7.3 for 6 days.4、The activity of phenylanine ammonia lyase (PAL) and peroxidase(POD) of tomato treated with C8-8 was increased greatly compared with that of non-treated, which implied that the potential function of resistance disease was induced by inoculating strain C8-8. A large amount of GA3 produced by C8-8 in NA medium were measured by HPLC analysis and its value reached 158mg/l. However none of IAA and GA3 was measured in essence medium, which implied that there are likely a tie between plant hormone produced by antagonistic bacterium C8-8 and promoting growth mechanism.5、Multiple interactions were involved in the combinations of antagonistic bacteria. The compatible combination of SJ-280 with PTS394 resulted in enhanced disease suppression (47.1%) against Fusarium Wilt of solanaceous vegetables as compared to the single strain SJ-280 (35.3%) or PTS394 (38.7%). The incompatible combination of C8-8 with PTS394 led to reduced disease suppression. However, increased disease suppression was also observed in the biocontrol by the incompatible combination of L-1 with PTS394.6、There were 871 strains isolated and purified from the diseased leaves, the diseased pomes, the healthy leaves, the healthy pomes, the diseased tress, the healthy tress and soil from the orchards of Nanjing, Lianyungang and Gaoyou, Jiangsu Province. Antagonistic examination against B. berengeriana revealed that 21 strains of all tested strains showed strong and steady antagonism. Strain sf 628 could inhibit the mycelia growth of B. berengeriana effectively with 45 mm inhibition zone and the inhibition rate 59.21%. The inoculating experiments on pear pomes showed that five strains of 21 tested strains could inhibit effectively necrosis spread of B. berengeriana with the inhibition rate more than 50 %, and the inhibition rate of strain sf 628 reached 60.61%. The physicochemical property and molecular (16S rRNA) identification experiments indicated the strain sf 628 was B. subtilis subsp. subtilis.7、Toxicity test of 26 fungicides on B. berengeriana showed 25% Propiconazole could inhibit effectively the mycelia growth of B. berengeriana with EC50 value 0.1522μg/ml. The mixed experiments against B. berengeriana showed that the synergistic ratios(SR) were greater than 1.5 when the mixed ratios were 1:3,1:2,1:1 and 1:2 between antagonistic bacterium sf 628 and Propiconazole and the SR was 1.071 under the mixed ratio was 3:1. The synergism under the ratio of 1:3 between antagonistic bacterium and Propiconazole was the most significant with synergistic ratio 2.942. 8、We also evaluate influence of sodium bicarbonate (SBC) on effectiveness of biocontrol agents (BA) to control ring rot on postharvest pear fruit. The results indicated that the inhibition rate against B. berengeriana was 42.30%,75.77%-85.41%, and 83.00% 90.23% in vitro when the plates were treated with 2% SBC (w/v), BA (strain 11,2,28 and sf628), and the combination, respectively. On fruit, the control effect was 23.83%,12.79%, 71.09%,48.16%,74.57%, and 57.90% when fruit were first inoculated with B. berengeriana (curative activity), then treated with BA 11 30mL, SBC 30mL, BA 11 15mL +SBC 15mL, SBC 15mL+BA 1115mL, BA 11 30mL+SBC 30mL, and SBC 30mL+BA 11 30mL, respectively. When fruit were treated with the same above, then inoculated with the pathogen (preventive activity), the control effect was 28.72%,17.31%,78.25%,51.06%, 80.91%, and 61.50%.更多还原