国槐枝枯病病原菌鉴定及其生物学特性

doi:10.3969/j.issn.1004-1524.20230859

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (3): 579-588.DOI: 10.3969/j.issn.1004-1524.20230859

国槐枝枯病病原菌鉴定及其生物学特性

罗芷涵¹(), 刘朋飞¹, 于军², 齐鹤³, 陈小光⁴, 楼兵干¹^,^*()

1.浙江大学生物技术研究所,农业农村部作物病虫分子生物学重点实验室,浙江杭州 310058
2.塔里木大学植物科学学院,新疆阿拉尔 843399
3.巴音郭楞蒙古自治州林业和草原局,新疆库尔勒 841000
4.库尔勒市园林局,新疆库尔勒 841000

收稿日期:2023-07-13 出版日期:2024-03-25 发布日期:2024-04-09
作者简介:罗芷涵(1999—),女,福建南平人,硕士研究生,研究方向为植物真菌病害。E-mail:22116086@zju.edu.cn
通讯作者: *楼兵干,E-mail: bglou@zju.edu.cn
基金资助:
国家重点研发计划(2021YFD1400200)

Identification and biological characteristics of pathogen causing branch dieback on Styphnolobium japonicum (L.) Schott

LUO Zhihan¹(), LIU Pengfei¹, YU Jun², QI He³, CHEN Xiaoguang⁴, LOU Binggan¹^,^*()

1. Ministry of Agriculture and Rural Affairs Key Lab of Molecular Biology of Crop Pathogens and Insects, Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China
2. College of Plant Science, Tarim University, Alar 843399,Xinjiang, China
3. Forestry and Grassland Administration of Bayingol Mongolian Autonomous Prefecture, Korla 841000, Xinjiang, China
4. Landscaping Affair Center of Korla,Korla 841000, Xinjiang, China

Received:2023-07-13 Online:2024-03-25 Published:2024-04-09

摘要/Abstract

摘要：

近年来,枝枯病在我国南疆的发病率高达50%~70%。为明确国槐枝枯病的致病菌及其生物学特性,采用组织分离法对南疆多地的发病国槐枝条进行病原菌分离,并进行致病性测定、形态学和分子生物学鉴定。结果表明,引起国槐枝枯病的病原菌为新暗色柱节孢(Neoscytalidium dimidiatum)。PDA培养基(pH值5.0)、34 ℃条件最适宜该菌的菌丝生长;PDA培养基(pH值7.0~9.0)、34 ℃条件最适宜该菌的分生孢子的形成。较低的相对湿度更有利于该菌的分生孢子萌发和生长。以牛肉膏为氮源、甘露糖为碳源最有利于该菌的菌丝生长,以蛋白胨为氮源最适宜该菌分生孢子的产生;而以木糖、山梨糖为碳源,硝态氮、尿素为氮源时,菌丝生长及产孢速率均有所下降。

关键词: 国槐枝枯病, 病原鉴定, 新暗色柱节孢(Neoscytalidium dimidiatum), 生物学特性

Abstract:

Branch dieback of Styphnolobium japonicum (L.) Schott is a serious disease that has occurred in the southern Xinjiang in recent years. To identify the pathogen of branch dieback in S. japonicum and its biological characteristics, we collected the samples with typical symptoms and the pathogen were isolated and purified. Based on the pathogenicity tests, morphological and molecular identification, we confirmed that the pathogen is Neoscytalidium dimidiatum. The results of biological tests showed that the optimal conditions for mycelial growth of N. dimidiatum were PDA medium (pH 5.0) at 34 ℃. The spore production was optimal on PDA medium (pH 7.0-9.0) at 34 ℃. The best C&N sources for mycelial growth were mannose and beef extract, respectively, while peptone was best for sporulation. In contrast, mycelial growth and spore production rate decreased when xylose and sorbose were used as carbon sources and nitrate nitrogen and urea as nitrogen sources.

Key words: branch dieback of Styphnolobium japonicum (L.) Schott, pathogen identification, Neoscytalidium dimidiatum, biological characteristic

中图分类号:

S763.7

罗芷涵, 刘朋飞, 于军, 齐鹤, 陈小光, 楼兵干. 国槐枝枯病病原菌鉴定及其生物学特性[J]. 浙江农业学报, 2024, 36(3): 579-588.

LUO Zhihan, LIU Pengfei, YU Jun, QI He, CHEN Xiaoguang, LOU Binggan. Identification and biological characteristics of pathogen causing branch dieback on Styphnolobium japonicum (L.) Schott[J]. Acta Agriculturae Zhejiangensis, 2024, 36(3): 579-588.

图/表 12

参考文献 21

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基因 Gene	引物名称 Primer	核苷酸序列 Nucleotide sequence
rDNA-ITS	ITS1	5'-TCCGTAGGTGAACCTGCGG-3'
	ITS4	5'-TCCTCCGCTTATTGATATGC-3'
EF1-α	EF1-728F	5'-CATCGAGAAGTTCGAGAAGG-3'
	EF1-986R	5'-TACTTGAAGGAACCCTTACC-3'
LSU	LROR	5'-ACCCGCTGAACTTAAGC-3'
	LR5	5'-TCCTGAGGGAAACTTCG-3'

基因 Gene	引物名称 Primer	核苷酸序列 Nucleotide sequence
rDNA-ITS	ITS1	5'-TCCGTAGGTGAACCTGCGG-3'
	ITS4	5'-TCCTCCGCTTATTGATATGC-3'
EF1-α	EF1-728F	5'-CATCGAGAAGTTCGAGAAGG-3'
	EF1-986R	5'-TACTTGAAGGAACCCTTACC-3'
LSU	LROR	5'-ACCCGCTGAACTTAAGC-3'
	LR5	5'-TCCTGAGGGAAACTTCG-3'

菌株名称 Strain	序列登录号GenBank accession numbers
菌株名称 Strain	rDNA-ITS	EF1-α	LSU
GH01	OP379832	OQ136617	OP389048
GH02	OQ096643	OQ586044	OQ127403
GH03	OQ096644	OQ586045	OQ127404
GH04	OQ096645	OQ586046	OQ127405
GH05	OQ096646	OQ586047	OQ127406
GH06	OQ096647	OQ586048	OQ127407
GH07	OQ096648	OQ586049	OQ127408
GH08	OQ096649	OQ586050	OQ127409
GH09	OQ096650	OQ586051	OQ127410
GH10	OQ096651	OQ586052	OQ127411
GH11	OQ096652	OQ586053	OQ127412
GH12	OQ096653	OQ586054	OQ127413
GH13	OQ096654	OQ586055	OQ127414
GH14	OQ096655	OQ586056	OQ127415
GH15	OQ096656	OQ586057	OQ127416
GH16	OQ096657	OQ586058	OQ127417
7b	OL376284	OL455961	OL376301
BMP13	MN067769	MN078938	MK968808
CBS 115206	KF766226	GU349030	DQ377882
MFLUCC 17-0359	MK480514	MK495819	MK478925
Mlty_Ma02	MT195553	MT362603	MT195555
NeNo1	KX370828	MF662595	MH899579
PTD-MA	MW605153	MW605154	MW605155
URF_Pt01	MT010216	MT362600	MT010293
CBS456.78	KF766192	EF622063	KF766362
CMW 26167	EU144055	EU144070	KF766333

菌株名称 Strain	序列登录号GenBank accession numbers
菌株名称 Strain	rDNA-ITS	EF1-α	LSU
GH01	OP379832	OQ136617	OP389048
GH02	OQ096643	OQ586044	OQ127403
GH03	OQ096644	OQ586045	OQ127404
GH04	OQ096645	OQ586046	OQ127405
GH05	OQ096646	OQ586047	OQ127406
GH06	OQ096647	OQ586048	OQ127407
GH07	OQ096648	OQ586049	OQ127408
GH08	OQ096649	OQ586050	OQ127409
GH09	OQ096650	OQ586051	OQ127410
GH10	OQ096651	OQ586052	OQ127411
GH11	OQ096652	OQ586053	OQ127412
GH12	OQ096653	OQ586054	OQ127413
GH13	OQ096654	OQ586055	OQ127414
GH14	OQ096655	OQ586056	OQ127415
GH15	OQ096656	OQ586057	OQ127416
GH16	OQ096657	OQ586058	OQ127417
7b	OL376284	OL455961	OL376301
BMP13	MN067769	MN078938	MK968808
CBS 115206	KF766226	GU349030	DQ377882
MFLUCC 17-0359	MK480514	MK495819	MK478925
Mlty_Ma02	MT195553	MT362603	MT195555
NeNo1	KX370828	MF662595	MH899579
PTD-MA	MW605153	MW605154	MW605155
URF_Pt01	MT010216	MT362600	MT010293
CBS456.78	KF766192	EF622063	KF766362
CMW 26167	EU144055	EU144070	KF766333

培养基 Medium	菌落直径 Colony diameter/cm	产孢数量 Spore yield/10⁵
马铃薯葡萄糖琼脂培养基	3.69±0.09 a	166.40±34.26 a
Potato dextrose agar, PDA
固体查氏培养基Czapek	3.74±0.11 a	21.20±3.92 c
甘薯葡萄糖琼脂培养基	3.70±0.16 a	17.80±3.31 c
Sweet potato dextrose agar, SPDA
玉米粒琼脂培养基	3.32±0.18 b	68.60±16.70 b
Corn meal agar, CMA
基本培养基	2.98±0.19 c	27.00±7.82 c
Complete medium, CM
燕麦片琼脂培养基	2.62±0.24 d	85.60±18.78 b
Oat meal agar, OA
改良营养琼脂培养基	2.29±0.14 e	16.20±3.92 c
Nutrient agar, NA
水琼脂培养基	1.94±0.13 f	2.00±0.67 d
Water agar, WA

国槐枝枯病病原菌鉴定及其生物学特性

Identification and biological characteristics of pathogen causing branch dieback on Styphnolobium japonicum (L.) Schott

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参考文献 21

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Metrics

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碳源 C sources	菌落直径 Colony diameter/cm	干重 Dry weight/mg	产孢数量 Spore yield/10⁵
甘露糖Mannose	5.95±0.16 a	218.50±5.20 a	24.01±1.62 d
果糖Fructose	4.82±0.26 b	219.70±9.00 a	37.26±0.89 b
半乳糖Galactose	4.47±0.41 c	204.30±1.50 c	11.21±0.30 gh
蔗糖Sucrose	4.38±0.16 c	205.00±0.50 c	13.37±1.24 fg
葡萄糖Glucose	4.35±0.26 c	207.20±3.00 ab	14.57±1.02 f
乳糖Lactose	3.97±0.17 d	198.40±1.90 cd	30.88±0.83 c
麦芽糖Maltose	3.95±0.46 d	216.80±7.20 ab	9.52±0.41 h
可溶性淀粉Soluble starch	3.77±0.09 d	198.00±1.50 cd	14.10±0.83 f
阿拉伯糖Arabinose	3.44±0.24 e	190.50±2.70 de	16.83±1.55 e
CK	3.11±0.12 f	178.00±3.50 de	125.97±1.39 a
甘油Glycerol	3.11±0.28 f	207.20±3.00 de	10.48±0.41 h
木糖Xylose	3.07±0.30 f	191.10±6.20 de	10.48±0.41 h
山梨糖Sorbose	1.82±0.29 g	185.30±2.00 e	24.77±1.28 d

氮源 N sources	菌落直径 Colony diameter/cm	干重 Dry weight/mg	产孢数量 Spore yield/10⁵
牛肉膏Beef Extract	4.63±0.12 a	252.60±6.00 a	53.61±0.44 c
CK	4.22±0.20 b	180.60±9.00 e	20.05±0.87 e
蛋白胨Peptone	4.11±0.14 bc	243.10±4.90 ab	138.10±0.70 a
氯化铵NH₄Cl	4.05±0.10 cd	212.10±2.00 c	8.85±0.65 i
硫酸铵(NH₄)₂SO₄	3.69±0.09 cd	230.00±7.40 b	15.30±0.50 f
苯丙氨酸Phenylalanine	3.87±0.34 de	202.00±4.90 cd	137.43±1.23 a
硝酸铵NH₄NO₃	3.78±0.13 e	193.00±6.00 de	13.24±0.88 g
硝酸钠NaNO₃	3.76±0.17 ef	191.70±6.30 de	13.24±0.88 g
草酸铵(NH₄)₂C₂O₄	3.61±0.18 g	191.80±6.60 de	84.87±0.66 b
甘氨酸Glycine	3.60±0.20 g	178.50±6.00 e	10.25±0.89 hi
精氨酸Arginine	3.39±0.13 h	236.10±4.00 b	42.08±0.82 d
硝酸钾KNO₃	3.29±0.22 h	185.90±4.10 e	11.64±0.46 h
尿素Urea	1.90±0.12 i	149.40±7.30 f	8.65±0.46 i

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