枸杞根腐病病原鉴定及拮抗菌筛选

doi:10.3969/j.issn.1004-1524.20231082

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (10): 2283-2297.DOI: 10.3969/j.issn.1004-1524.20231082

枸杞根腐病病原鉴定及拮抗菌筛选

纪嵩岩¹(), 邵长琪¹, 齐文康¹, 何煜晖¹, 张欣¹^,², 王翠平¹^,³^,⁴^,^*()

1.北方民族大学生物科学与工程学院,宁夏银川 750021
2.北方民族大学经济林遗传改良创新团队,宁夏银川 750021
3.林木资源高效生产全国重点实验室,宁夏银川 750004
4.宁夏葡萄与葡萄酒技术创新中心,宁夏银川 750021

收稿日期:2023-09-08 出版日期:2024-10-25 发布日期:2024-10-30
作者简介:纪嵩岩(2002—),男,湖北大冶人,本科生,研究方向为生态学和分子生物学。E-mail:jisy2024@lzu.edu.cn
通讯作者: ^*王翠平, E-mail:wangcuiping@nmu.edu.cn
基金资助:
北方民族大学青年人才培育项目(2023QNPY11);宁夏自然科学基金(2023AAC03314);中国科学院“西部之光”项目(XAB2022YW08)

Identification of Lycium barbarum root rot disease pathogens and biocontrol funguses against root rot disease

JI Songyan¹(), SHAO Changqi¹, QI Wenkang¹, HE Yuhui¹, ZHANG Xin¹^,², WANG Cuiping¹^,³^,⁴^,^*()

1. School of Biological Science and Engineering, North Minzu University, Yinchuan 750021, China
2. Innovation Team for Genetic Improvement of Economic Forests, North Minzu University, Yinchuan 750021, China
3. State Key Laboratory of Efficient Production of Forest Resources, Yinchuan 750004, China
4. Ningxia Grape and Wine Technology Innovation Center, Yinchuan 750021, China

Received:2023-09-08 Online:2024-10-25 Published:2024-10-30

摘要/Abstract

摘要：

枸杞根腐病是发生于枸杞栽植区的一种常见土传病害,由多种致病菌复合侵染引起,目前主要用化学手段进行防控。该研究旨在明确宁夏地区枸杞根腐病的主要致病菌,筛选优良拮抗菌株。以宁夏银川植物园枸杞种植园宁杞1号枸杞患病植株根部和患病植株根际土为材料,用组织分离法分离纯化致病菌及拮抗根际微生物;采用离体回接试验与针刺接种试验研究病原菌对枸杞根腐病的致病作用;采用对峙法测定拮抗菌对分离得到的致病菌的抑制作用;根据菌落形态特征及rDNA-ITS序列对致病菌和拮抗真菌进行鉴定。结果表明,枸杞发病根部组织中分离出的菌株主要为尖孢镰孢菌(Fusarium oxysporum)、茄腐皮镰孢菌(Fusarium solani)、单隔镰孢菌(Fusarium dimerum)、木贼镰孢菌(Fusarium equiseti)、嗜石油腐皮镰孢(Fusarium petroliphilum)和高山被孢霉(Mortierella alpina),分离频率分别为29.5%、16.8%、13.7%、 8.9%、8.9%和7.4%;尖孢镰孢菌(F. oxysporum)、茄腐皮镰孢菌(F. solani)、单隔镰孢菌(F. dimerum)、木贼镰孢菌(F. equiseti)等具有较强致病性。根际土分离到的根际微生物中土曲霉(Aspergillus terreus)和长枝木霉(T. longibrachiatum)对主要致病菌有明显抑制作用,抑制率均在37%以上。

关键词: 枸杞, 根腐病, 致病性, 生防菌, 尖孢镰孢菌, 土曲霉, 长枝木霉

Abstract:

Root rot disease of Lycium barbarum is a common soil-borne disease that occurs in L. barbarum cultivation areas. It is caused by a complex infection of multiple pathogenic fungi and is currently primarily controlled through chemical means. The aim of this study is to explore the feasibility of rapid screening of highly effective, broad-spectrum and practical biocontrol fungi with disease prevention and growth promotion effects, and identify the main pathogenic bacteria of L. barbarum root rot in Ningxia region and screen excellent antagonistic strains. Using the diseased root of Ningqi No.1 L. barbarum and the rhizosphere soil as materials, pathogenic funguses and rhizosphere microorganisms were isolated and purified using tissue separation method; the inhibitory effect of rhizosphere microorganisms on isolated pathogenic funguses was determined using the confrontation method; the antagonistic fungi was identified based on colony culture, morphological characteristics, and rDNA-ITS sequence. The results showed that the main strains isolated from the diseased root tissues of L. barbarum were Fusarium oxysporum, Fusarium solani, Fusarium dimrum, Fusarium equiseti, Fusarium petroliphilum, and Mortierella alpina, with frequencies of 29.5%, 16.8%, 13.7%, 8.9%, 8.9%, and 7.4%, respectively. Aspergillus terreus and Trichoderma longibrachiatum have obvious inhibition effect on the main pathogenic funguses, with the inhibition rate of more than 37%, which can effectively adjust the microbial flora of the root zone of L. barbarum and promote the restoration and balance of soil micro-ecology.

Key words: Lycium barbarum, root rot disease, pathogen, biocontrol funguses, Fusarium oxysporum, Aspergillus terreus, Trichoderma longibrachiatum

中图分类号:

S436.65

纪嵩岩, 邵长琪, 齐文康, 何煜晖, 张欣, 王翠平. 枸杞根腐病病原鉴定及拮抗菌筛选[J]. 浙江农业学报, 2024, 36(10): 2283-2297.

JI Songyan, SHAO Changqi, QI Wenkang, HE Yuhui, ZHANG Xin, WANG Cuiping. Identification of Lycium barbarum root rot disease pathogens and biocontrol funguses against root rot disease[J]. Acta Agriculturae Zhejiangensis, 2024, 36(10): 2283-2297.

图/表 11

图1 枸杞根腐病发病特征 A,根基部发病;B,根中部发病;C,野外发病特征。

Fig.1 The symptoms of root rot of L. barbarum in the field A, Root site disease; B, Root middle segment disease; C, Characteristics of field incidence.

表1 枸杞根腐病病原菌分离率

Table 1 Fungi isolated from the rotten roots of L. barbarum

编号 No.	菌株 Strain	分离数 Number	分离率 Rate/%
Ⅰ	N. rubicola	9	4.7
Ⅱ	A. niger	7	3.7
Ⅲ	F. dimerum	26	13.7
Ⅳ	F. falciforme	6	3.2
Ⅴ	F. solani	32	16.8
Ⅵ	M. alpina	14	7.4
Ⅶ	F. oxysporum	56	29.5
Ⅷ	F. equiseti	17	8.9
Ⅸ	F. petroliphilum	17	8.9
Ⅹ	A. carneus	5	2.6
ⅩⅠ	S. lignicola	1	0.5

图2 致病菌菌落形态及孢子、菌丝、孢子囊 A~E 依次是B9,N7,A1,A4和A7。F~K 依次是A8,B2c,B2d,B2,B4和N4。标尺=20 μm。

Fig.2 Pathogenic funguses colony morphology and photos of spores, hyphae, and sporangia A-E, in turn, are B9, N7, A1, A4 and A7. F-K are A8, B2c, B2d, B2, B4 and N4 in order. Bar=20 μm.

表2 枸杞根离体接种发病情况

Table 2 The pathogenicity of pathogens inoculated on L. barbarum root

致病菌 Pathogenic fungi	发病情况 Incidence	致病性 Pathogenicity
B9(N. rubicola)	3条根被侵染变为棕色,根上产生少量黄色液滴 Three roots were infected and turned brown, with a small amount of yellow droplets produced on the roots	++
N7(A. niger)	12条根全被侵染变为黑色,且大量腐烂成产生黄色液滴 All 12 roots were infected and turned black, and a large number of them decayed into yellow droplets	++++
A1(F. dimerum)	无明显效果No significant effect
A4(F. falciforme)	无明显效果No significant effect
A7(F. solani)	12条根全被侵染变为棕色,且产生少量黄色液滴 All 12 roots were infected and turned brown, with a small amount of yellow droplets produced	+++
A8(M. alpina)	2条根被侵染变为棕色,根上产生少量黄色液滴 Two roots were infected and turned brown, with a small amount of yellow droplets produced on the roots	++
B2c(F. oxysporum)	12条根全被侵染变为棕色,且大量腐烂成产生黄色液滴 All 12 roots were infected and turned brown, with a large amount of decay resulting in the production of yellow droplets	++++
B2d(F. equiseti)	4条根被侵染变为棕色,根周围产生大量黄色液滴,根上长出黑色斑点 Four roots were infected and turned brown, with a large number of yellow droplets produced around the roots and black spots growing on the roots	+++
B2(F. solani)	12条根全被侵染变褐色,且根周围产生大量黄色液滴 All 12 roots were infected and turned brown, and a large number of yellow droplets were produced around the roots	++++
B4(A. carneus)	12条根全被侵染变褐色,少量变为黑色,且部分腐烂成黄色液滴 All 12 roots were infected and turned brown, with a small amount turning black, and some rotting into yellow droplets	++++
对照Control	12 条根全部保持正常浅黄色All 12 roots remain normal and light yellow in color

表2 枸杞根离体接种发病情况

Table 2 The pathogenicity of pathogens inoculated on L. barbarum root

致病菌 Pathogenic fungi	发病情况 Incidence	致病性 Pathogenicity
B9(N. rubicola)	3条根被侵染变为棕色,根上产生少量黄色液滴 Three roots were infected and turned brown, with a small amount of yellow droplets produced on the roots	++
N7(A. niger)	12条根全被侵染变为黑色,且大量腐烂成产生黄色液滴 All 12 roots were infected and turned black, and a large number of them decayed into yellow droplets	++++
A1(F. dimerum)	无明显效果No significant effect
A4(F. falciforme)	无明显效果No significant effect
A7(F. solani)	12条根全被侵染变为棕色,且产生少量黄色液滴 All 12 roots were infected and turned brown, with a small amount of yellow droplets produced	+++
A8(M. alpina)	2条根被侵染变为棕色,根上产生少量黄色液滴 Two roots were infected and turned brown, with a small amount of yellow droplets produced on the roots	++
B2c(F. oxysporum)	12条根全被侵染变为棕色,且大量腐烂成产生黄色液滴 All 12 roots were infected and turned brown, with a large amount of decay resulting in the production of yellow droplets	++++
B2d(F. equiseti)	4条根被侵染变为棕色,根周围产生大量黄色液滴,根上长出黑色斑点 Four roots were infected and turned brown, with a large number of yellow droplets produced around the roots and black spots growing on the roots	+++
B2(F. solani)	12条根全被侵染变褐色,且根周围产生大量黄色液滴 All 12 roots were infected and turned brown, and a large number of yellow droplets were produced around the roots	++++
B4(A. carneus)	12条根全被侵染变褐色,少量变为黑色,且部分腐烂成黄色液滴 All 12 roots were infected and turned brown, with a small amount turning black, and some rotting into yellow droplets	++++
对照Control	12 条根全部保持正常浅黄色All 12 roots remain normal and light yellow in color

图3 离体接种结果 A-D依次是 B9(N. rubicola)、A7(F. solani)、A8(M. alpina)和B2c(F. oxysporum);E-H依次是B2d(F. equiseti)、B2(F. solani)、B4(A. carneus)和 N4(S. lignicola)。

Fig.3 The result of inoculation on roots A-D, in turn, are B9 (N. rubicola), A7 (F. solani), A8 (M. alpina) and B2c (F. oxysporum). E-H are B2d (F. equiseti), B2 (F. solani), B4 (A. carneus) and N4 (S. lignicola) in order.

图4 针刺接种结果 A-D依次是 B9(N. rubicola)、N7(A. niger)、A1(F. dimerum)和A8(M. alpina);E-H依次是B2c(F. oxysporum)、B2d(F. equiseti)、B2(F. solani)和B4(A. carneus)。

Fig.4 The result of inoculation on plants A-D, in turn, are B9 (N. rubicola), N7 (A. niger), A1 (F. dimerum) and A8 (M. alpina). E-H are B2c (F. oxysporum), B2d (F. equiseti), B2 (F. solani) and B4 (A. carneus) in order.

图5 致病菌基于 ITS 序列的系统发育树

Fig.5 Phylogenetic tree of pathogenic fungi strains from L. barbarum root-zone based on rDNA-ITS sequences homology

图6 拮抗菌对枸杞根腐病病原菌生长的抑制作用 A,B9 与PDA-2对峙;B,A1 与PDA-2对峙;C,B2 与PDA-2对峙;D,B2c 与PDA-2对峙;E,B4与PDA-2对峙。

Fig.6 Inhibition of the growth of L. barbarum root rot pathogens by antagonistic fungi A, B9 and PDA-2 confront each other; B, A1 and PDA-2 confront each other; C, B2 and PDA-2 confront each other; D, B2c and PDA-2 confront each other; E, B4 and PDA-2 confront each other.

表3 两种拮抗菌对枸杞根腐病病原菌生长的抑制作用

Table 3 Inhibition of the growth of L. barbarum root rot pathogens by two antagonistic fungi %

菌株 Strain	生长抑制率Percentage of growth inhibition
菌株 Strain	B9	A1	A7	B2c	B4
PDA-2	50.53±1.53	47.53±2.46	37.87±0.92	57.23±1.79	51.67±1.26
B1	45.58±1.53	37.93±1.33	36.12±1.20	42.29±1.79	51.11±1.40

图7 拮抗菌菌落形态及孢子、菌丝、孢子囊 A,B1菌落形态及孢子、菌丝、孢子囊;B,PDA-2菌落形态及孢子、菌丝、孢子囊。标尺=20 μm。

Fig.7 The colony morphology, spores, hyphae, and spore sacs of PDA-2 A, The colony morphology, spores, hyphae, and spore sacs of B1; B, The colony morphology, spores, hyphae, and spore sacs of antagonistic fungi. Bar=20 μm.

图8 拮抗菌基于ITS 序列的系统发育树

Fig.8 Phylogenetic tree of dominant fungi strains from L. barbarum root-zone based on rDNA-ITS sequences homology

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枸杞根腐病病原鉴定及拮抗菌筛选

Identification of Lycium barbarum root rot disease pathogens and biocontrol funguses against root rot disease

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