摘要

目的：近年来，在新疆库尔勒地区梨园梨树的嫩梢上观察到黑褐色枯枝，根据发病特点，暂 时将此病害命名为“梨枝枯病”，因此，本课题开展了病原菌的分离和鉴定工作。另外，本研究从健康梨树的根围土壤中筛选出对解淀粉欧文氏菌具有拮抗作用的细菌，并进行了离体杜梨叶、树枝和库尔勒香梨的室内防效测定，研究结果为“梨枝枯病”的生物防治奠定基础。

方法：采用组织分离法从染病的梨树枝条中分离病原菌；采用平板对峙法筛选土壤中的拮抗菌，选取拮抗效果最佳的拮抗菌进行离体梨树组织的室内防效测定；采用薄层层析法（TLC）和高分辨质谱法（HRMS）鉴定拮抗菌甲醇提取物中的活性物质；通过侵染拟南芥种子探究拮抗菌对种子萌发及幼苗生长的促生作用。

结果：

1.本研究从库尔勒香梨染病枝条上分离到一株细菌C1，经形态学、生理生化和分子生物学鉴定为解淀粉欧文氏菌，命名为Erwinia amylovora C1。将分离得到的E. amylovora C1和标准菌株Erwinia amylovora 0017回接到杜梨树枝、叶片和库尔勒香梨的果实，结果表明，接种E. amylovora C1的健康梨树组织产生了与标准菌株在田间“梨枝枯病”相似的症状，进一步证明了C1为解淀粉欧文氏菌。

2.从梨树的根围土壤中分离得到9株拮抗菌，经16S rDNA鉴定，拮抗菌分别为萎缩芽孢杆菌（Bacillus atrophaeus）、巨大芽孢杆菌（Priestia megaterium）和粘质沙雷菌（Serratia marcescens）。平板对峙试验中，巨大芽孢杆菌（Priestia megaterium）KD7的拮抗效果最佳。对P. megaterium KD7进行离体梨树组织的保护性试验和治疗性试验，结果显示，P. megaterium KD7能有效防治“梨枝枯病”对植物的侵染，保护性试验防治效果优于治疗性试验。

3.研究表明，P. megaterium KD7的无细胞上清液和甲醇提取液对E. amylovora C1有较高的抑菌活性。随后采用薄层层析法（TLC）检测甲醇提取物的抗菌成分，其Rf值为0.71，表明甲醇提取物中存在氨基酸类物质。进一步采用高分辨质谱法（HRMS）检测甲醇提取物中的拮抗物质，鉴定得到三种脂肽类抗生素，即C13-surfactin [M+H]⁺、C15-surfactin [M+H]⁺和C14-iturin A [M+H]⁺。

4.拮抗菌的促生试验表明，P. megaterium KD7可降解大分子有机物（淀粉和蛋白）促进拟南芥种子萌发和幼苗的生长。

结论：在新疆库尔勒地区梨园染病的梨树枝条上分离出1株“梨枝枯病”的病原菌。从梨园根围土壤中筛选得到9株拮抗菌，其中P. megaterium KD7的拮抗效果最佳，可以有效的保护梨树组织免受“梨枝枯病”的侵害。P. megaterium KD7可能通过向胞外分泌表面活性素和伊枯草菌素A抑制E. amylovora C1的活性。P. megaterium KD7具有蛋白酶和淀粉酶活性，对植物种子萌发和幼苗生长具有一定促生效应。

Abstract

Objective: In recent years, black-brown dead branches were observed on the shoots of pear trees in the orchard in Korla, Xinjiang, which was similar to the symptoms of pear branch blight. The pathogenic bacteria from infected from Korla fragrant pear was isolated and identified in this study. Then, bacteria with antagonistic activity against E. amylovora were screened from the rhizosphere soil of healthy pear trees, and the indoor control effect of antagonism was determined using isolated leaves and twigs of Pyrus betulifolia Bunge and fuits from Korla fragrant pear. These results help search for potential and effective biocontrol agents to control pear branch blight.

Methods: In this study, the pathogenic bacteria were isolated from infected Korla fragrant pear twigs by tissue isolation method. The gradient dilution method was used to screen the antagonistic bacteria in the soil, and the most effective antagonistic bacteria were selected for the indoor control effect test using pear tissues in vitro. Then thin layer chromatography (TLC) and high resolution mass spectrum (HRMS) mass spectrometry were used to analyze the active components in the methanol extract of antagonistic bacteria P. megaterium KD7. Finally, the germination rate and seedling growth of Arabidopsis thaliana seeds treated with antagonistic bacteria were observed to evaluate the promoting effect.

Result:

1. In this study, a bacterial strain C1 was isolated from the infected branches of Korla fragrant pear, which was identified as Erwinia amylovora C1 by morphology, physiology, biochemistry and molecular biology method. Strain C1 and the standard Erwinia amylovora 0017 were return inoculated with pear branches and leaves of Pyrus betulifolia Bunge and fruits of Korla fragrant pear. The results showed that the healthy pear tree tissue produced symptoms similar to that of the standard strain in the field of pear branch blight, which further confirmed that C1 was E. amylovora.

2. Nine antagonistic bacteria were isolated from the rhizosphere soil of Korla fragrant pear trees. According to 16S rDNA identification, the antagonistic bacteria belonged to Bacillus, Priestia and Serratia respectively. Among them, Priestia megaterium KD7 has the best antagonistic effect on E. amylovora. Next, the P. megaterium KD7 was used to carry out protective and therapeutic tests on pear tree tissue in vitro. The result showed that P. megaterium KD7 can effectively control the infection of pear branch blight on pear leaves, branches and fruits, and the protective test was better than the therapeutic test.

3. The cell-free supernatant and methanol extract from P. megaterium KD7 showed antibacterial effect against E. amylovora C1. the antibacterial substances of the methanol extract were detected by TLC method, and the Rf was 0.71, indicating that amino acids were existed in the methanol extract. Furthermore, the antibacterial components from methanol extract were identified by HRMS method and three lipopeptides were identified, which were C13-surfactin [M+H]⁺、C15-surfactin [M+H]⁺ and C14-iturin A [M+H]⁺。

4. P. megaterium KD7 degrades macromolecular organic substances (starch and protein) to promote the germination of Arabidopsis thaliana seeds and the growth of seedlings.

Conclusion: A pathogen of pear branch blight was isolated from the pear orchard in Korla, Xinjiang. Nine strains of antagonistic bacteria were screened from the soil, among which the antagonistic effect of P. megaterium KD7 was the best, which can effectively protect the pear tissue from the infection of pear branch blight. P. megaterium KD7 secretes surfactin and iturin A into the extracellular space to inhibit the activity of E. amylovora C1. P. megaterium KD7 also has protease and amylase activities to promote the decomposition of macromolecular substances in soil to promote plant growth.

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