【作者】 李明；

【导师】 张荣意；

【作者基本信息】 海南大学 ， 植物病理学， 2011， 硕士

【摘要】 1香蕉冠腐病菌致病机制研究应用分光光度法对活体内外半裸镰刀菌产生的细胞壁降解酶进行活性分析,初步明确了果胶酶类和纤维素半纤维素酶类在该菌侵染香蕉果实过程中起关键作用。1.1活体外半裸镰刀菌产生的细胞壁降解酶活性的变化随着培养天数的增加F.s分泌的PME和PGTE的活性在第3d呈现急剧上升趋势,前者总是先于后者到达峰值,Cx和β-G的活性都于第二天开始显著增强,Cx也是先于β-G到达峰值。前期各种酶活性上升的很快,接着所有酶在达到培养期间的最大值后,都陆续开始下降。1.2活体内细胞壁降解酶活性的变化蕉梳切口接种F.s后,接种组织内没有检测到PG, PMG活性,而PME、PGTE、PMTE活性迅速增高,PME活性的最大值要先于PGTE和PMTE到达,说明PME在时间上要先于果胶裂解酶产生。果指切口受病原菌侵染后,Cx和β-葡萄糖苷酶活性迅速增加,同时由于Cx两次活性高峰出现的都比β-G出现的早,表明其对寄主细胞壁降解先于β-葡萄糖苷酶。而半纤维素酶(β-D-Xyl)尽管在相对数值上要小于纤维素酶,但在病菌侵染寄主组织产生半纤维素酶的时序性上却要先于纤维素酶。1.3 B68发酵滤液对F.s细胞壁降解酶活体外合成的影响当发酵滤液浓度为50%时,完全抑制了半裸镰刀菌的各种果胶酶的合成,但没有完全抑制Cx和β-葡萄糖苷酶,β-D木聚糖苷酶合成。除PG和PMG之外的六种酶在几种浓度处理下的酶活性均与对照达极显著差异,而PG和PMG也在除5%浓度处理外的其它四种浓度处理下与对照达极显著差异。2.拮抗菌处理后香蕉果实抗病机制研究选择抗氧化物酶类APX, SOD, POD, CAT和防御酶类PPO, PAL,β-1-3葡聚糖酶,几丁质外切酶,几丁质内切酶作为抗病性反应的指标,对接种半裸镰刀菌(F.s)和拮抗菌(B68)后香蕉果实内这些酶活性水平变化进行比较,结果表明,抗氧化物酶类：拮抗菌(B68)+半裸镰刀菌(F.s),拮抗菌(B68),半裸镰刀菌(F.s)三个处理的POD活性最先于第三天达到最大值,B68和F.s处理的SOD活性于第四天达到最大值,三个处理的CAT和APX活性分别于第5d和第6d达到最大值。B68+F. s和F.s处理的PAL活性于第3d达到最大值,而三个处理的PPO活性于第5d和三个处理的β-1-3葡聚糖酶,几丁质外切酶,几丁质内切酶活性于第6d都分别达到极大值。这些酶活性高峰出现的早迟可能与它们在植物体内所起作用的时间有关。比较不同接种处理诱导产生各种酶活性的强弱可发现在大多少时间内B68+F. s处理的酶活性要大于其他处理的酶活性。拮抗菌十病原菌处理的木质素和HRGP含量都于第四天达到最大值,F.s和B68处理的HRGP含量于第5d到达最大值,而F.s和B68处理的木质素含量都于第7d达到极大值,在整个试验期间B68+F. s处理的木质素和HRGP含量均高于另外两个处理的木质素含量和HRGP含量。β-1,3-葡聚糖酶,几丁质酶,β-1,3-葡聚糖+几丁质3种粗酶液在体外对半裸镰刀菌孢子萌发的抑制程度表现不同,混合酶的抑制作用最强要显著大于单一酶液的抑制作用,当粗酶液浓度为50%时,抑制率达到90.43%,孢子萌发抑制率(Y)和混合酶液浓度(X), Y=l.5093X+14. 686, R2=99.29%说明随着混合酶液浓度提高,孢子萌发抑制率按线性规律下降。3.B68在香蕉果实表-面的定殖及其部分增效因子增效作用机制研究生防菌B68在香蕉果表的定殖与引进浓度、接种灰霉病菌时间和环境因子有关,拮抗细菌在香蕉果表的定殖能力与最初引进的细菌浓度呈正相关,即引进的浓度越高,定殖时间越长,定殖的菌量也越大。与对照相比,接种半裸镰刀菌对B68的定殖有一定的影响。但是,先接种B68后接种半裸镰刀菌(间隔ld)要比B68与半裸镰刀菌同时接种以及先接种半裸镰刀菌后接种B68影响小。这表明半裸镰刀菌与1368间存在营养、空间的竞争,这也显示生防菌B68在冠腐病害发生前使用要比发生后使用效果好。试验得出,最有利于细菌定殖的条件是温度25℃-30℃,相对湿度95%-100%。初步研究了分散剂和湿润剂类增效因子对枯草芽孢杆菌B68的促生长作用、增效因子引起B68在香蕉果实上的定殖差异。结果发现：NNO、木质素磺酸钠、CMC、茶皂素、皂角粉、SDS6种增效因子可不同程度地促进芽抱杆菌生长：分散剂MF、湿润剂茶皂素在香蕉果实贮藏期均可促进生防细菌B68在果面的定殖。更多还原

【Abstract】 1.Study on the Pathogenesis of Fusarium semitectumActivities of the cell wall degrading enzymes(CWDEs)produced by Fusarium semitectum both in vitro and in vivo were studied.The results primarily demonstrated that pectinase enzymes and cellulose and hemicellulose play a key role in the process of infecting banana fruit by F.s. 1.1 Changes in activities of CWDE produced by F.s. in vitro PME and PGTE activities showed a sharp rise trend in the third day produced by F.s with the increase of culturing days,the former always precede the latter to reach the peak,, Cx, andβ-G activity both significantly enhanced in the next day,Cx always precedeβ-G to reach the peak. Enzyme activities increased quickly in the early days,and then all the enzymes began to come down when they had reached the maximum during the incubation period,1.2 Changes in activities of CWDE produced by F.s. in vivoinoculated tissue hadn’t been detected PG, PMG activity when banana comb incision was inoculated by F.s, while PME, PGTE, PMTE activity increased rapidly,while the maximum of PME arrived prior to PGTE, PMTE. Cx, andβ-glucosidase activity in the incision spot increased rapidly after being infected by F.s,it bad been demonstrated that Cx degrade the cell wall beforeβ-glucosidase owing to twice peak of Cx activity prior toβ-G activity.Alougth the semi-cellulose (β-D-Xyl) was lower than the cellulose in the relative value,the former emerged before the latter on the timing.1.3 The effect of ferment filtrate of B68 on the synthesis of CWDE produced by F.s in vitroWhen the concentration of fermentation filtrate come to 50%, the synthesis of pectinase had been completely inhibited, but did not completely inhibit the Cx andβ-glucosidase.β-D xylan glycosidase synthesis.the six enzymes activities in addition to PG,PMG differed significantly from the CK in several kinds of treatment concentration, while the PG,JPMG activities differed significantly from the CK in four kinds of treatment concentration except 5% concentration treatment.2.Study on the disease resistance mechanism after being treated with antagonist and pathogenSelecting antioxidant enzymes APX, SOD, POD, CAT and defense enzymes PPO, PAL.β-1-3 glucanase, chitinase exonuclease, chitinase enzyme reaction as a disease resistance Indicators,the change in the level of enzyme activities in the banana fruit after been inoculated by the F.s and B68 had been compared.The results showed that antioxidant enzymes:POD activity under the treatment of antagonists (B68)+Fusarium semitectum (Fs). antagonists (B68), Fusarium semitectum (Fs) firstly reached the maximum on the third day,SOD activity under the treatment of antagonists (B68), Fusarium semitectum (Fs) reached the maximum on the fourth day,CAT and APX activities with the four treatments reached the maximum on the fifth and sixth day respectively. PAL activity under the treatment of B68+F.s and F.s reached the maximum on the third day,PPO activity with three treatment andβ-1-3 glucanase, exon-chitinase,endo-chitinase activities with three treatment reached the maximum on the fifth and sixth day respectively.The peak of enzyme activity may occur relevant to their functioning time in banana.varieties of enzyme activities.Comparison of he strength of varieties of enzyme activities induced by different inoculation can be found that many of the enzyme activities dealt with the B68+Fs was larger than the enzyme activities of other treatments.in large amount of time.The content of lignin and HRGP after being inoculated with B68+F.s reached the maximum value on the fourth day.the content of HRGP inoculated with F.s and B68 peak at fifth day,while the content of lignin inoculated with F.s and B68 both peak at seventh day.the content of lignin and HRGP inoculated with B68+F.s were higher than those inoculated with F.s and B68β-1,3-glucanase, chitinase,β-1,3-glucan+chitin presented different inhibition to spore germination. The mixture ofβ-1,3-glucanase and chitinase presented obvious inhibition to spore germination. Inhibition of the mixture was significantly higher than that of singleβ-1,3-glucanase.when the crude enzyme concentration reached 50%, the inhibition rate reached 90.43 percent, spore germination rate (Y) and mixed enzyme concentration (X). Y= 1.5093X+14.686, R2= 99.29%, spore germination rate decreased by a linear law with increase of mixture of enzyme concentration.3.Bs68 on the colonization of banana fruit surface and study on the machanism upon part of synergistic factorThe colonizing ability of B68 was related to temperature,humidity,introduced bacterial concentration and inoculation pathogen.The colonizing capacity of antagonistic bacterium on fruit surface had positive relationship to the introduced bacterial concentration. the higher concentration of the introduction, the longer colonization is, the greater amount of bacteria colonization is.Inoculation F.s had some influences on the colonization of antagonistic bacterium.The bacterial amount of inoculating pathogens after introducing bacteria was more than that of inoculating pathogens and bacteria together and introducing bacteria after pathogens.This indicated that there existed nutrition, space competition,which also showed the use of B68 prior to occurrence of banana crown rot was superior to the use of B68 later on occurrence of banana crown rot.It had been concluded that the most favorable conditions for bacterial colonization was 25℃-30℃,the relative humidity was 95%-100%.It had been preliminarily studied the effect of dispersant and wetting agent class as synergistic factor on the promoting B68 growth,the effect of synergistic factor on the B68 colonization differences.The results showed that:NNO, Lignin sulfonate, CMC, tea saponin, saponins powder, SDS 6 species of factors may contribute to the growth of Bacillus spores by degree.dispersing agent MF, tea saponin wetting agent could promote colonization of bacterial strain B68 on the surface of storage life of banana fruit.更多还原