小麦根腐菌拮抗内生菌的筛选及其抑菌活性评估

论文总字数：18273字

摘 要

小麦根腐病(Wheat root rot)是小麦生产中严重的真菌性病害之一，病原物主要为麦根腐离蠕孢菌（Bipolaris sorokinian）。根腐病分布极广，小麦种植国家均有发生，因此，筛选有效防治方法对于控制该病害尤为重要。本文从小麦的根、茎、叶中分离内生菌，并测试其对小麦根腐病菌的抑菌活性，旨在筛选出对小麦根腐病菌有抑菌活性的内生菌，实验取得如下结果：

（1）采用组织分离法从油菜的根、茎、叶部位一共分离得到35株菌株，通过平板培养和筛选得到两株对小麦根腐病菌有明显抑制作用的菌株Z0和ZJ。其中，从油菜根部得到的Z0菌株抑菌效果最好。生物学特征观察、生理生化实验、革兰氏染色及16S rDNA结果表明，拮抗菌Z0为短小杆菌（Curtobacterium spp.）,革兰氏因阳性，菌落乳白色、圆形、不透明、略微凸起。

（2）通过平板对峙法，测量拮抗菌发酵液、菌丝混合液以及上清液不同条件下培养的病原菌抑菌带大小可见，前两者对病原菌有明显的抑制作用，其中效果最好的是原液，抑菌率可达34.79%；在液培条件下，通过称量菌丝干重发现发酵液对病原菌同样有较为明显的抑制作用，防治效果甚至优于固体培养。显微观察结果显示受抑的根腐菌丝畸形，有原生质外泄，甚至菌丝死亡。

关键词：小麦根腐病，筛选，拮抗菌株，生物防治

Abstract: Wheat root rot (WRR), caused by Bipolaris sorokinian, results in a serious and prevalent disease problem for wheat production in countries growing wheat. Therefore, selecting effective biocontrol resources is particularly important for the control the disease. In this study, endophytes from the roots, stems and leaves of the wheat, which had antibacterial activities against B. sorokinian, were screened and identified. The main results are as follows:

(1) Above all, 35 strains were isolated from the roots, stems and leaves, and two strains (Z0 and ZJ) that had inhibitory effects on Bipolaris sorokinian were obtained through plate screening. More than that, the strain Z0 obtained from the root of rapeseed had the better antibacterial effect. The identification results show that strain Z0 was Gram-positive bacteria, and the colonies were milky white, round, opaque and slightly raised, together with the 16S rDNA gene sequence of strainZ0 was highly similar to that of Curtobacterium flaccumfaciens, we confirmed that strain Z0 was Curtobacterium spp.

(2) The antibacterial activity of the antagonistic bacteria fermentation broth, mycelia mixed solution and supernatant was measured by the plate counter-twisting method. The inhibitory bands of the pathogenic bacteria were observed under different conditions. The former two had obvious inhibitory effects on the pathogenic bacteria. Among them, Fermentation stock had the best effect and the antibacterial rate up to 34.79%. Under the condition of liquid culture, the weight of the mycelium was weighed to find out that the fermentation broth also had obvious inhibitory effect on the pathogenic bacteria, and the control effect was even better than that of the solid culture. In addition, the root rot mycelium was suppressed, protoplasm leaked, and even the mycelium died under the microscopic observations.

Keywords: Wheat root rot , filter , Antagonistic strain, Biological control

目录

1 引言 6

1.1 小麦根腐病的研究进展 6

1.1.1 小麦根腐病的发病原因 6

1.1.2 小麦根腐病的发病症状 7

1.1.3 小麦根腐病的侵染循环 7

1.2 小麦根腐病的防治现状 8

1.2.1 小麦根腐病的化学防治 8

1.2.2 小麦根腐病的农业防治 9

1.2.3 小麦根腐病的生物防治 9

1.3 本研究的目的及意义 11

2 材料和方法 11

2.1 材料 11

2.1.1 样品采集 11

2.1.2 供试病原菌 11

2.1.3 供试试剂 11

2.1.4 供试培养基 12

2.2 方法 12

2.2.1 小麦根腐病拮抗菌的筛选 12

2.2.2 拮抗菌Z0的鉴定 12

2.2.3 拮抗菌Z0对病原菌抑菌作用测定 13

2.2.4 拮抗菌Z0对根腐病原菌菌丝形态的影响 14

3 结果与讨论 14

3.1 拮抗菌的分离结果 14

3.2 拮抗菌的筛选结果 14

3.3 拮抗菌Z0对根腐菌在固体培养条件下的抑菌率测定 15

3.4 拮抗菌Z0对根腐菌在液体培养条件下的抑菌率测定 16

3.5 拮抗菌Z0对根腐菌菌丝的形态影响 16

3.6 拮抗菌Z0的鉴定 18

3.6.1 革兰氏染色鉴定及平板观察结果 18

3.6.2 生理生化鉴定结果 18

3.6.3 16SrDNA基因测序结果 19

结论 21

参考文献 22

致谢 24

引言

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