小麦内生细菌的种群多样性调查及其在小麦条锈病生物防治中的利用研究

发布时间：2018-06-09 00:19

  本文选题：小麦条锈病 + 内生细菌　； 参考：《广西大学》2016年博士论文

【摘要】：小麦条锈病是由Puccinia striiformis west.f.sp.tritici Eriks et Henn引起的真菌性病害,也是世界上小麦最严重的病害之一。由于其发生区域广,流行频率高,危害损失重等特点已成为影响小麦稳产高产的主要因素。目前对小麦条锈病的防治主要采用抗性品种、化学药剂和农业综合防治措施。但由于小麦条锈病菌生理小种变异快及施用农药造成药剂污染等原因,生产上迫切需要一种可持续、环保的方法来进行小麦条锈病的防治。内生细菌因其特有的优点及多样的生物学特性,受到众多植物病理学家的关注。但是用小麦内生细菌防治小麦条锈病还鲜有报道。作者对小麦内生细菌的种群多样性进行了调查,从分离的内生细菌中筛选出对小麦具有显著促生、防病和增产效果的菌株,并对其防病促生和增产机理进行了研究,主要结果如下：1、本研究对采自不同地区、不同时期、不同器官的健康小麦样品进行内生细菌的分离并对其种群多样性进行分析。结果表明：从小麦体内共分离出内生细菌313株,其中从南阳市卧龙区样品中分离出的内生细菌数为101株,占总细菌数的32.27%；不同小麦器官的内生细菌数量存在差异,在整个生长期,根部内生细菌的数量最多达11.5×105cfu/g,茎、叶片和籽粒中内生细菌数量分别为688×105cfu/g、4.77×105cfu/g 和 3.47× 105 cfu/g;不同时期内生细菌分离也存在差异,抽穗期内生细菌数量为10.48×105cfu/g,分蘖期、拔节期和蜡熟期内生细菌数量分别为2.73×105cfu/g、6.65×105cfu/g和9.3× 105cfu/g。经过16S rDNA序列比对分析,内生细菌群落共包含23 Operational taxonomic units (OTU),归属于9个属。芽孢杆菌属(Bacillus)为优势菌群,占细菌总数的76.37%,假单胞属次之,占细菌总数的10.23%。蜡样芽孢杆菌(B. cereus)和枯草芽孢杆菌(B.subtilis)在小麦整个生长过程中具有较高的分离频率,分别为9.7%和4.37%。Stenotrophomonas maltophilia作为小麦内生菌为首次报道。2、以127株小麦内生细菌为研究对象,通过室内和室外试验就内生细菌对小麦幼苗促生作用进行研究,采用聚类分析、判别分析和相关分析等综合统计分析法,对实验结果进行分析。结果表明：6株内生细菌(SB127, LD161、RA135、JD204、 RC79、RB132)对小麦的生长指标(叶长、茎叶干重、根干重)、解无机磷与有机磷能力、产生长素量等8个观察指标都较高,且生长指标与解磷能力及产生长素量有显著的正相关性,内生细菌的解磷能力及产生长素量对小麦生长有显著的影响。不同的内生细菌促进小麦生长的作用不同。选择生长素分泌量高、解磷能力强的内生细菌菌株,对促进小麦生长,防治小麦病害有积极意义。3、根据形态学、生理生化特征和16S rDNA序列同源性对6株内生细菌(RC79、SB127、 RB132、 RA135、 LD16、 JD204)进行鉴定,并对2株(LD161、JD204)还用rpoD基因进行了鉴定,初步将其归为蜡样芽孢杆菌(Bacillus cereus)、巨大芽孢杆菌(Bacillus megaterium)、枯草芽孢杆菌(Bacillus subtilis)、荧光假单胞菌(Pseudomonas fluorescens)和恶臭假单胞菌(Pseudomonas putida)。 6株内生细菌的最适生长温度均为28℃～30℃,最适生长pH值均为6～8；菌株RC79、SB127、RB132 与 RA135均能有效利用葡萄糖碳源,菌株LD161与JD204可利用除木糖醇外的供试碳源,6株菌株几乎能利用所有的供试8种氮源。除RB132外所有菌株均检测到精氨酸脱羧酶与赖氨酸脱羧酶活性。定殖实验表明：6株促生菌株均可稳定在小麦体内定殖,并且菌株SB127、RB132和JD204在小麦体内可以向地上部转移。4、内生细菌对14个不同小麦品种的大田防效和增产试验结果表明：用6株内生细菌RC79M、SB127M、RB132M、RA135M、 LD161M和JD204M分别处理14个不同小麦品种后,小麦平均发病率为14.62%～19.9%,约为对照区平均发病率的68.9%～83.4%；内生菌处理后病情指数平均为4.98～7.13,约为对照区平均病情指数的64.7%～92.3%；与各自对照相比,对小麦条锈病正的平均防治效果达到29.6%～45.1%,产量平均增加了4.56%～10.59%。内生细菌JD204M具有高效的田间病害控制和促进小麦增产能力。5、利用内生细菌JD204M诱导小麦植株系统抗性试验结果表明：用内生细菌诱导处理后,在整个生育期抗病品种(新原958)和感病品种(矮抗吨产王)小麦叶片的苯丙氨酸解氨酶(PAL)、多酚氧化酶(PPO)、过氧化物酶(POD)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)活性均比对照有所增强,但两个品种之间的酶活的动态变化有所不同；丙二醛(MDA)、超氧阴离子和H2O2含量比对照降低,同时伴随着脯氨酸、类黄酮含量的增加,可能与内生细菌诱导小麦植株产生的抗病性有关。6、研究了内生突变细菌JD204M对14个不同小麦品种分蘖数、株高、根系活力以及叶片光合特性的影响。结果表明：用内生细菌处理后可提高小麦幼苗的分蘖数,郑麦9023品种分蘖数增加最多,比对照提高了34.14%；有12个品种的株高和根系活力分别比对照平均增加9.21%和13.33%；10个品种小麦叶片叶绿素含量和净光合速率(Pn)分别比对照平均增加7.64%和16.59%；11个品种的蒸腾速率(Tr)和气孔导度(Gs)分别比对照平均增加10.74%和20.32%,9个品种的胞间C02浓度(Ci)平均减少13.18%。12个品种的光化学效率(ΦPS Ⅱ)比对照均有所提高,矮抗吨产王增幅最大,为24.20%；11个品种的Fv/FO和Fv/Fm值与对照相比分别增加了19.45%和10.36%。
[Abstract]:Wheat stripe rust is a fungal disease caused by Puccinia striiformis west.f.sp.tritici Eriks et Henn. It is also one of the most serious diseases in Wheat in the world. It has become the main factor affecting the stable yield and high yield of wheat because of its widespread occurrence, high epidemic frequency and heavy damage to the wheat. Resistant varieties, chemical agents and integrated agricultural control measures. However, due to the rapid variation in physiological races and pesticide pollution caused by the application of pesticides, a sustainable and environmentally friendly method for the prevention and control of wheat stripe rust is urgently needed in production. Endophytic bacteria have their unique advantages and diverse biological characteristics. The control of wheat stripe rust with wheat endophytic bacteria is rarely reported. The population diversity of wheat endophytic bacteria was investigated. From the isolated endophytic bacteria, the strains with significant growth promoting, disease prevention and increasing yield were screened from the isolated endophytic bacteria. The main results are as follows: 1, this study carried out the isolation of endophytic bacteria from the healthy wheat samples from different regions, different periods and different organs. The results showed that 313 strains of endophytic bacteria were isolated from the wheat and the endophytic bacteria isolated from the Wolong region of Nanyang were isolated. The number of 101 strains, accounting for 32.27% of the total number of bacteria, the number of endophytic bacteria in different wheat organs is different, the number of endophytic bacteria is up to 11.5 x 105cfu/g in the whole growth period, the number of endophytic bacteria in the stem, leaves and grains is 688 * 105cfu/g, 4.77 x 105cfu/g and 3.47 x 105 cfu/g, and the endophyte isolation in different periods is also deposited. The number of endophytic bacteria in the heading stage was 10.48 * 105cfu/g, the tillering stage, the jointing stage and the waxy mature period were 2.73 x 105cfu/g, 6.65 x 105cfu/g and 9.3 x 105cfu/g. were analyzed by 16S rDNA sequence alignment, and the endophytic bacteria community included 23 Operational taxonomic units (OTU), belonging to 9 genera. For the dominant bacteria group, 76.37% of the total number of bacteria, Pseudomonas sp., 10.23%. Bacillus cereus (B. cereus) and Bacillus subtilis (B.subtilis) have high separation frequency in the whole growth process of wheat, and 9.7% and 4.37%.Stenotrophomonas maltophilia respectively for wheat endophyte as the first report.2, to 1. 27 endophytic bacteria were used to study the effect of endophytic bacteria on the growth of wheat seedlings through indoor and outdoor tests. The results were analyzed by cluster analysis, discriminant analysis and correlation analysis. The results showed that 6 endophytic bacteria (SB127, LD161, RA135, JD204, RC79, RB132) were used to wheat The growth indexes (leaf length, stem and leaf dry weight, root dry weight), the ability to hydrolyse inorganic phosphorus and organophosphorus and the production of long prime are all higher, and there is a significant positive correlation between the growth indexes and the ability of phosphorus removal and the production of long prime. The ability of phosphorus removal and production of endophytic bacteria has a significant effect on the growth of wheat. Different endophytic bacteria promote the growth of endophytic bacteria. 8 6 endophytic bacteria (RC79, SB127, RB132, RA135, LD16, JD204) were identified for 6 endophytic bacteria (RC79, SB127, RB132, RA135, LD16, and JD204), and 2 strains (LD1). 61, JD204) was also identified with rpoD gene, which was initially classified as Bacillus cereus (Bacillus cereus), Bacillus gigantobacilli (Bacillus megaterium), Bacillus subtilis (Bacillus subtilis), Pseudomonas fluorescens (Pseudomonas fluorescens) and Pseudomonas pustinosa (Pseudomonas putida). The optimum growth temperature of 6 endophytic bacteria The optimum growth pH value is 6~8 at 28 to 30 C, and the strain RC79, SB127, RB132 and RA135 can use the glucose carbon source effectively. The strain LD161 and JD204 can be used as the test carbon source except for the xylitol. The 6 strains can almost use all 8 nitrogen sources. All strains except RB132 have detected the arginine decarboxylase and lysine decarboxylase. The colonization test showed that the 6 strains could be colonized steadily in the wheat, and the strain SB127, RB132 and JD204 could transfer.4 to the ground in the wheat. The results of the endophytic bacteria against the 14 different wheat varieties showed that 6 endophytic bacteria RC79M, SB127M, RB132M, RA135M, LD161M and JD204M were used. After treating 14 different wheat varieties, the average incidence of wheat was 14.62% ~ 19.9%, which was about 68.9% ~ 83.4% of the average incidence rate in the control area, and the average disease index of endophyte was 4.98 to 7.13 after treatment, which was about 64.7% to 92.3% of the average disease index in the control area. Compared with the control, the average control effect on wheat stripe rust was 29. .6% ~ 45.1%, the yield increased averagely by 4.56% ~ 10.59%. endophytic bacteria JD204M with high efficiency of field disease control and increasing wheat yield.5. The results of wheat plant resistance test induced by endophytic bacteria JD204M showed that after the induction of endophytic bacteria, the disease resistant varieties (new original 958) and susceptible varieties (dwarf resistance) were treated with endophytic bacteria. The activity of phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), peroxidase (POD), catalase (CAT) and superoxide dismutase (SOD) in wheat leaves were enhanced, but the dynamic changes of the enzyme activities between the two varieties were different, and the content of malondialdehyde (MDA), superoxide anion and H2O2 was lower than that of the control. With the increase of proline and flavonoid content,.6 may be related to the resistance of endophytic bacteria to wheat plants. The effects of endogenous mutant bacterial JD204M on the number of tillers, plant height, root activity and leaf photosynthetic characteristics of 14 different wheat varieties were studied. The results showed that the endophytic bacteria could improve the wheat seedling score. In the number of tillers, the number of tillers increased most in Zhengmai 9023, and increased by 34.14% compared with the control. The plant height and root activity of 12 varieties increased by 9.21% and 13.33%, respectively. The chlorophyll content and net photosynthetic rate (Pn) of the 10 wheat cultivars increased by 7.64% and 16.59%, respectively, and the transpiration rate (Tr) and stomata of 11 varieties. The conductance (Gs) increased by 10.74% and 20.32%, respectively. The intercellular C02 concentration (Ci) of the 9 varieties decreased on average (Ci) of 13.18%.12 varieties, and the growth of the dwarf resistance tons king was the maximum, 24.20%. The Fv/FO and Fv/Fm values of 11 varieties increased by 19.45% and 10.36%., respectively, compared with the control.
【学位授予单位】：广西大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S435.121.42;S476
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本文编号：1997901