水稻细菌性条斑病菌拮抗放线菌的鉴定及其防病和促生功能

doi:10.3864/j.issn.0578-1752.2025.17.006

摘要/Abstract

摘要：

【目的】水稻细菌性条斑病（rice bacterial leaf streak，BLS）为我国检疫性植物病害，由稻黄单胞杆菌稻生致病变种（Xanthomonas oryzae pv. oryzicola，Xoc）侵染引起，严重威胁水稻产量和稻米品质，是我国水稻生产区的主要病害之一。本研究旨在利用优质放线菌资源，为研发防治农作物病害的微生物源产品打下理论基础。【方法】采用梯度稀释涂布法，从不同植物根际土壤样品中分离纯化放线菌菌株，通过测量抑菌圈直径比较不同菌株对Xoc的抑菌能力，选择抑菌效果最好的菌株保藏并进行后续形态学、生理生化特征和多基因比对分析。采用扫描电镜观察、傅里叶变换红外光谱分析、胞内β-半乳糖苷酶的泄露情况测定以及SDS-PAGE凝胶电泳法探究拮抗放线菌对Xoc生理特征的影响，开展盆栽防效试验进行防治水稻细菌性条斑病的实际效益研究。采用特定培养基分析拮抗放线菌的促生特性，并通过浇灌促生试验探究其对水稻幼苗生长发育的影响。【结果】共分离获得80株放线菌，其中Sv-6菌株拮抗Xoc能力最强，抑菌圈直径为（44.87±0.26）mm，经形态特征及系统发育分析，将Sv-6菌株鉴定为弗吉尼亚链霉菌（Streptomyces virginiae）。Xoc经Sv-6菌株培养滤液处理后产生细胞溶胀、皱缩和聚集，同时膜表面物质组成改变、通透性增加，以及蛋白质表达下降。盆栽防效试验结果显示，水稻易感品种甬优15和湘两优900经Sv-6菌株培养液处理后，病斑抑制率达到57.98%—88.25%，且对预防Xoc侵染水稻效果显著。促生特性测定证明Sv-6菌株具有产铁载体、溶无机磷以及产生吲哚乙酸（IAA）的能力。浇灌促生试验验证了Sv-6菌株对水稻幼苗生长具有促进作用，处理后根长的增幅可达48.50%。【结论】弗吉尼亚链霉菌Sv-6菌株对水稻细菌性条斑病具有较好的防治效果，有开发成为绿色生防制剂和微生物菌肥的潜力。

关键词: 水稻细菌性条斑病, 稻黄单胞杆菌稻生致病变种, 弗吉尼亚链霉菌, 生物防治, 促生评价

Abstract:

【Objective】Rice bacterial leaf streak (BLS) is a quarantine-regulated bacterial disease in China caused by Xanthomonas oryzae pv. oryzicola (Xoc), which has severely threatened rice yield and grain quality, becoming one of the main diseases in rice production areas. This study aims to utilize high-quality actinomycete strains to lay a theoretical foundation for developing the products of microbial origin to mitigate crop diseases.【Method】Actinomycete strains were isolated and purified from rhizosphere soil samples of various plants using the serial dilution plating technique. The inhibition ability of different strains against Xoc was compared by measuring the diameter of the inhibition zone. The target strain with the best inhibition effect was selected for preservation and identified through polyphasic characterization integrating morphological traits, physiological-biochemical experiments, and multi-gene alignment analysis. The effects of antagonistic actinomycete on the physiological characteristics of Xoc were investigated by scanning electron microscopy, Fourier-transform infrared spectroscopy (FTIR), intracellular β-galactosidase leakage determination and SDS-PAGE gel electrophoresis. The greenhouse pot control effect test was carried out to study the actual benefit of controlling BLS. The growth-promoting characteristics of antagonistic actinomycete were analyzed by designated medium, and its effects on the growth and development of rice seedlings were investigated by watering growth-promoting experiments.【Result】A total of 80 actinomycete strains were isolated. Among them, strain Sv-6 exhibited the most potent antagonism against Xoc, with inhibition zone diameter of (44.87±0.26) mm. Based on the morphological characteristics and phylogenetic analysis, strain Sv-6 was identified as Streptomyces virginiae. After treatment with the culture filtrate of Sv-6 strain, Xoc cells swelled, shrunk and aggregated. At the same time, the material composition of the membrane surface changed, the permeability increased, and the protein expression decreased. The results of greenhouse pot experiments showed that the lesion inhibition rates of susceptible rice varieties Yongyou 15 and Xiangliangyou 900 were 57.98%-88.25% after being treated with strain Sv-6 culture fluid, which exhibited a good preventive effect on rice infected with Xoc. The growth-promoting characterization confirmed that strain Sv-6 exhibited siderophore production, inorganic phosphate solubilization, and IAA production. The growth-promoting irrigation experiments confirmed that strain Sv-6 enhanced the growth of rice seedlings, and the root length increased by 48.50% after treatment.【Conclusion】S. virginiae Sv-6 exhibits a good control effect on BLS, and has the potential to be developed into green biocontrol agent and microbial fertilizer.

Key words: rice bacterial leaf streak (BLS), Xanthomonas oryzae pv. oryzicola (Xoc), Streptomyces virginiae, biological control, growth-promoting evaluation

胡佳燕, 沈之涵, 温丽慧, 郁嘉豪, 章雨君, 蒋冬花. 水稻细菌性条斑病菌拮抗放线菌的鉴定及其防病和促生功能[J]. 中国农业科学, 2025, 58(17): 3434-3450.

HU JiaYan, SHEN ZhiHan, WEN LiHui, YU JiaHao, ZHANG YuJun, JIANG DongHua. Identification and Evaluation of Biocontrol Actinomycetes Against Xanthomonas oryzae pv. oryzicola for Disease Suppression and Growth Promotion in Rice[J]. Scientia Agricultura Sinica, 2025, 58(17): 3434-3450.

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引物名称Primer name	序列Sequence (5′→3′)
recA-F	ATGGCAGGAACCGACCGCGAGAAGG
recA-R	TCAGCTCTTGGCCGCCGCGGCCTTG
rpoB-F	TTGGCCGCCTCGCGCAATGCCTCGA
rpoB-R	TCAGACCTCTTCGACGCTGCTCGGC
gyrB-F	GTGGCCGATTCCGGCAACCCCAACG
gyrB-R	TCAGATGTCGAGGAAGCGGACGTCC
16S rRNA-27F	AGTTTGATCCTGGCTCAG
16S rRNA-1492R	TACGGTTACCTTGTTACGACTT

鉴别培养基 Differential medium	基内菌丝 Substrate mycelium	气生菌丝 Aerial mycelium	可溶性色素 Water-soluble pigment	菌落生长 Colony growth
察氏Czapek Dox	白色White	淡粉色Light pink	无None	稀疏Sparse
ISP1	淡黄色Faint yellow	白色White	无None	旺盛Lush
ISP2	橘黄色Croci	白色White	无None	旺盛Lush
ISP5	透明Transparente	淡粉色Light pink	无None	稀疏Sparse

碳源种类Type of carbon sources	试验结果Test result	氮源种类Type of nitrogen sources	试验结果Test result
蔗糖Sucrose	+	KNO₃	+
葡萄糖Glucose	+	L-天冬酰胺L-asparagine	+
山梨醇Sorbitol	+	γ-氨基丁酸γ-aminobutyric acid	+
α-乳糖α-lactose	+	L-酪氨酸L-tyrosine	+
甘露醇Mannitol	+	DL-色氨酸DL-tryptophan	+
D（+）-木糖D（+）-xylose	-	(NH₄)₂SO₄	-
D-木糖D-xylose	-	NH₄Cl	-
+：阳性Positive；-：阴性Negative

振动峰分配 Vibrational peak assignment	分类 Classification	振动峰<BOLD>V</BOLD>ibrational peak (cm^-1)
振动峰分配 Vibrational peak assignment	分类 Classification	CK	1/2	1
碳水化合物中的C-O、C-C str、C-O-H、C-O-C伸缩振动峰 C-O, C-C str, C-O-H, C-O-C def of carbohydrates	糖原和核酸 Glycogen and nucleic acids	1064.15	1064.51	1058.73
磷酸二酯>PO₂中P=O的不对称伸缩振动峰 P=O stretching (antisymmetric) of >PO₂	主要是核酸 Mainly nucleic acids	1226.02	1229.78	1227.47
COO-中C=O对称伸缩振动峰 C=O str (sym) of COO-	氨基酸侧链和脂肪酸 Amino acid side chains, fatty acids	1385.60	1388.50	1384.64
酰胺II中蛋白质N-H弯曲振动峰和C-N拉伸振动峰 Amide II (protein N-H bend, C-N stretch)	α螺旋α helices	1525.90	1529.27	1527.83
酰胺I的β-折叠结构β-pleated sheet structures of amide I	β-折叠β-pleated sheet	1635.82	1638.23	1637.75
>CH₂中C-H不对称伸缩振动峰C-H str (asym) of >CH₂	主要是脂类Mainly lipids	2921.15	2919.22	2928.86
羟基O-H伸缩振动峰O-H str of hydroxyl groups	多糖和蛋白质Polysaccharides, proteins	3284.66	3280.81	3288.04