水稻纹枯病拮抗细菌7-5的鉴定及其生防机制初步研究

doi:10.16819/j.1001-7216.2018.7074

摘要/Abstract

摘要：

【目的】旨在获得对水稻纹枯病有生防效果的拮抗细菌,探索生防细菌作用机理。【方法】通过苗期防病试验从本实验室保存的7株拮抗细菌中筛选对水稻纹枯病有较好防病效果的菌株。结合生理生化性状和16S rDNA基因序列同源性分析,对生防细菌7-5进行初步鉴定,并通过PCR扩增、薄层层析等探讨7-5产生抗生素的种类。【结果】供试7株细菌中7-5、4-74和4-78对水稻纹枯病防效分别可达69.0%、70.8%和75.4%。其中4-74和4-78抑菌谱窄,7-5对水稻纹枯病菌、桃褐腐病菌和草莓灰霉病菌都具有较强抑菌能力。菌株7-5 鉴定为绿针假单胞菌;该菌株可产生吩嗪-1-羧酸、嗜铁素、氢氰酸、蛋白酶、藤黄绿脓菌素和2,4-二乙酰基间苯三酚等抗菌物质,不产生硝吡咯菌素、几丁质酶和纤维素酶。【结论】绿针假单胞菌7-5主要通过产生抗生素防治水稻纹枯病。

关键词: 绿针假单胞菌, 水稻纹枯病, 抗生素, 生物防治

Abstract:

【Objective】The objective is to obtain antagonistic bacteria for biocontrol on rice sheath blight and reveal the biological control mechanism.【Method】The control experiment of rice sheath blight by seven antagonistic bacteria stored in our lab was performed using pot cultures at the seedling stage. The strain 7-5 was identified based on a 16S rDNA BLAST and the strain’s physiological and biochemical characterization. The antifungal compounds produced by bacterial strain 7-5 were detected by PCR amplification and thin layer chromatography (TLC). 【Result】The suppressive effects of bacteria 7-5, 4-74 and 4-78 on the rice sheath blight in a greenhouse were as high as 69.0%, 70.8% and 75.4%, respectively. Strain 7-5 could strongly inhibit the growth of Rhizoctoniasolani, Moniliniafructicola and Botrytis cinerea, whereas both strains 4-74 and 4-78 had narrow inhibitory spectra. Strain 7-5 was identified as Pseudomonas chlororaphis and produced many antifungal factors, including phenazine-1-carboxylicacid, 2,4-diacetylphloroglucinol, pyoluteorin, siderophores, hydrogen cyanide and extracellular proteinase, but not including pyrrolnitrin, chitinase and cellulase.【Conclusion】The production of antibiotics plays a key role for P. chlororaphis7-5 to control the rice disease.

Key words: Pseudomonas chlororaphis, rice sheath blight, antibiotics, biological control

中图分类号:

张清霞, 张迎, 何玲玲, 陈夕军, 童蕴慧, 纪兆林. 水稻纹枯病拮抗细菌7-5的鉴定及其生防机制初步研究[J]. 中国水稻科学, 2018, 32(3): 277-284.

Qingxia ZHANG, Ying ZHANG, Lingling HE, Xijun CHEN, , Zhaolin JI. Identification of Strain 7-5, Antagonistic to Rice Sheath Blight, and Preliminary Study of Its Biocontrol Mechanism[J]. Chinese Journal OF Rice Science, 2018, 32(3): 277-284.

图/表 7

表1 本研究所用引物

Table 1 Primers used in this study.

引物 Primer	序列 Sequence (5’-3’)	基因 Amplified gene	片段大小 Amplified segment/bp	文献 Reference
16S-63F	CAGGCCTAACACATGCAAGTC	16SrDNA	1500	[19]
16S-1387R	GGGCGGTGATGTACAAGGC		1500	[19]
phl2a	GAGGACGTCGAAGACCACCA	phlD	745	[20]
phl2b	ACCGCAGCATCGTGTATGAG	phlD	745	[20]
PCA2a	TGCCAAGCCTCGCTCCAAC	phzCD	1400	[20]
PCA3b	CCGCGTTGTTCCTCGTTCAT	phzCD	1400	[20]
pltcf	GAAGCCGAAGGCATGGACCC	pltC	450	本研究This study
pltcr	GCCGCGTCGAAGGTCTTGCA	pltC	450	本研究This study
prnCF	CCACAAGCCCGGCCAGGAGC	prnC	720	[21]
prnCR	GAGAAGAGCGGGTCGATGAAGCC	prnC	720	[21]

图1 拮抗细菌对水稻纹枯病的防治效果柱上不同小写字母表示差异显著。

Fig.1. Control effects of antagonistic bacteria on rice sheath blight. Different small letters above bars mean significant difference at 0.05 level.

图2 拮抗细菌对水稻纹枯病菌的抑菌活性

Fig.2. Inhibition abilities of antagonistic bacteria to R. solani. A,4-74; B, 4-78;C, 7-5, D, W10

图3 拮抗细菌7-5菌株16S rDNA 系统发育树

Fig. 3. Phylogenetic tree based on 16S rDNA sequence homology of antagonistic bacteria strain 7-5.

图4 抗生素基因的PCR检测 M—2000 bp标记;1~3–菌株FD6、7-5、2P24 prn扩增产物;4~6–菌株PAO17-5、FD6产生的pca扩增产物;7~9–菌株FD6、7-5、2P24 产生的plt扩增产物;10~12–菌株FD6、7-5、L5phl扩增产物。

Fig.4. PCR amplification of antibiotics gene from bacterial strain 7-5. M,2000 bp marker;1–3,PRN amplification productsof FD6, 7-5 and 2P24;4–6,PCAamplification products of PAO17-5 and FD6;7–9,PLT amplification productsof FD6, 7-5 and 2P24;10–12,PHL amplification productsof FD6, 7-5 and L5．

图5 7-5产生抗生素的薄层层析鉴定

Fig. 5. Detection of antibiotics produced by strain 7-5 through TLC.

图6 7-5产生代谢分泌物鉴定 A—蛋白酶;B—嗜铁素;C—氢氰酸;左—W10;右—7-5。

Fig. 6. Assay of antifungal compounds produced by strain 7-5. A, Proteinase; B, Siderophore; C, HCN; Left, W10; Right, 7-5.

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