Identification of pathogens causing brown spot disease on cigar tobacco and their pathogenicity to cigar tobacco varieties

doi:10.3969/j.issn.1004-1524.20250293

Acta Agriculturae Zhejiangensis ›› 2026, Vol. 38 ›› Issue (4): 707-719.DOI: 10.3969/j.issn.1004-1524.20250293

• Plant Protection • Previous Articles Next Articles

Identification of pathogens causing brown spot disease on cigar tobacco and their pathogenicity to cigar tobacco varieties

LI Shuo¹(), CHEN Tao², GU Gang³, HU Zhongliang², PAN Xi², YIN Changlin², HUANG Shan², LIANG Haoyi¹, XIAO Shun¹, LIN Zhihui²^,^*(), LIU Guokun¹^,^*()

¹ College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou 350002, China
² Sanming Tobacco Company of Fujian Province, Sanming 365000, Fujian, China
³ Institute of Tobacco Science, Fujian Provincial Tobacco Company, Fuzhou 350003, China

Received:2025-04-11 Online:2026-04-25 Published:2026-05-08
Contact: LIN Zhihui,LIU Guokun

Abstract

Abstract:

Brown spot disease caused by Alternaria spp. is an important disease on flue-cured tobacco, but there are few studies on the pathogen species causing brown spot on cigar tobacco. This study aimed to clarify the pathogen species causing brown spot on cigar tobacco in Sanming region of Fujian Province and their pathogenicity to cigar tobacco varieties. Brown spot samples were collected from the cigar tobacco Haiyan 101 grown in the Sanming region. Pathogens were isolated and purified on PDA medium. Identification was performed by combining morphological and molecular biological methods [multigene (ITS, TEF1, RPB2, GAPDH, Alta1) phylogenetic analysis and His3 single-gene supplementary analysis]. The results showed that the 45 isolates belonged to four species: 9 were Alternaria alternata, 21 were Alternaria tenuissima, 12 were Alternaria longipes, and 3 were Alternaria gaisen. Pot inoculation pathogenicity tests showed that 3 d after inoculating Haiyan 101, all four Alternaria species could produce circular brown lesions on leaves. Among them, A. longipes exhibited the strongest pathogenicity, while A. gaisen showed the weakest pathogenicity. This is the first report of A. gaisen causing tobacco brown spot disease. Field inoculation tests showed that A. tenuissima and A. longipes were highly pathogenic to all five cigar tobacco varieties (Haiyan 101, QX107, QX105, CX80 and QX103). A. alternata showed weak pathogenicity to varieties QX107 and QX105 but strong pathogenicity to the other three varieties. A. gaisen showed weak pathogenicity to all five varieties. The findings of this study provided guidance for the control of brown spot disease on cigar tobacco and the breeding of resistant varieties.

Key words: cigar tobacco, brown spot disease, Alternaria spp., pathogen identification, multi-gene phylogenetic analysis, pathogenicity determination, variety resistance

CLC Number:

S432.44

LI Shuo, CHEN Tao, GU Gang, HU Zhongliang, PAN Xi, YIN Changlin, HUANG Shan, LIANG Haoyi, XIAO Shun, LIN Zhihui, LIU Guokun. Identification of pathogens causing brown spot disease on cigar tobacco and their pathogenicity to cigar tobacco varieties[J]. Acta Agriculturae Zhejiangensis, 2026, 38(4): 707-719.

Figures/Tables 10

References 28

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靶标基因 Target gene	正向引物序列(5'→3') Forward primer sequence (5'→3')	反向引物序列(5'→3') Reverse primer sequence (5'→3')	参考文献 Reference
ITS	TCCGTAGGTGAACCTGCGG	TCCTCCGCTTATTGATATGC	[4]
TEF1	CATCGAGAAGTTCGAGAAGG	TACTTGAAGGAACCCTTACC	[17]
RPB2	GGGGWGAYCAGAAGAAGGC	CCCATRGCTTGTYYRCCCAT	[7]
Alta1	ATGCAGTTCACCACCATCGC	ACGAGGGTGAYGTAGGCGTC	[18]
GAPDH	CAACGGCTTCGGTCGCATTG	GCCAAGCAGTTGGTTGTGC	[19]
His3	ACTAAGCAGACCGCCCGCAGG	GCGGGCGAGCTGGATGTCCTT	[20]

靶标基因 Target gene	正向引物序列(5'→3') Forward primer sequence (5'→3')	反向引物序列(5'→3') Reverse primer sequence (5'→3')	参考文献 Reference
ITS	TCCGTAGGTGAACCTGCGG	TCCTCCGCTTATTGATATGC	[4]
TEF1	CATCGAGAAGTTCGAGAAGG	TACTTGAAGGAACCCTTACC	[17]
RPB2	GGGGWGAYCAGAAGAAGGC	CCCATRGCTTGTYYRCCCAT	[7]
Alta1	ATGCAGTTCACCACCATCGC	ACGAGGGTGAYGTAGGCGTC	[18]
GAPDH	CAACGGCTTCGGTCGCATTG	GCCAAGCAGTTGGTTGTGC	[19]
His3	ACTAAGCAGACCGCCCGCAGG	GCGGGCGAGCTGGATGTCCTT	[20]

种类 Species		菌株号 Strain No.	基因序列登录号GenBank accession No.
种类 Species		菌株号 Strain No.	ITS	TEF1	RPB2	GAPDH	Alta1	His3
标准菌株	Alternaria	CBS 612.72	KP124308	KP125084	KP124777	KP124165	KP123861	—
Standard strains	alternata	CBS 194.86	KP124316	KP125092	KP124784	KP124172	KP123869	—
		CBS 877.95	KP124321	KP125097	KP124789	KP124176	KP123871	—
		CBS 965.95	KP124323	KP125099	KP124791	KP124178	KP123872	—
		CBS 109455	KP124335	KP125111	KP124803	KP124189	KP123883	—
		CBS 112252	KP124340	KP125116	KP124808	KP124194	KP123888	—
		CBS 115200	KP124352	KP125128	KP124820	KP124206	KP123900	—
		CBS 130262	KP124389	KP125167	KP124859	KP124241	KP123937	—
	A. longipes	CBS 539.94	KP124441	KP125220	KP124911	KP124290	KP123987	—
		CBS 540.94	AY278835	KC584667	KC584409	AY278811	AY563304	—
		CBS 917.96	KP124442	KP125221	KP124912	KP124291	KP123988	—
	A. gaisen	CBS 118488	KP124427	KP125206	KP124897	KP124279	KP123977	—
		CPC 25268	KP124428	KP125207	KP124898	KP124280	JQ646393	—
	A. alternantherae	CBS 124392	KC584179	KC584633	KC584374	KC584096	KP123846	—
	A. arborescens	CBS 105.49	KP124396	KP125174	KP124866	KP124248	KP123944	—
		CBS 126.60	KP124397	KP125175	KP124867	KP124249	JQ646390	—
		CBS 109730	KP124399	KP125177	KP124869	KP124251	KP123946	—
	A. gossypina	CBS 100.23	KP124429	KP125208	KP124899	KP124280	KP123977	—
		CBS 107.36	KP124431	KP125210	KP124901	JQ646310	JQ646393	—
	A. tenuissima	SN6-2	LC621622	LC621724	LC621690	LC621656	LC631828	—
		CBS 918.96	AF347032	KC584693	KC584435	AY278809	AY563302	—
三明市雪茄烟上分离的菌株	A. longipes	FSM 01075	PQ151360	PQ151352	PQ163711	PQ163688	PQ151341	—
Strain isolated from Sanming		FSM 01079	PQ151364	PQ151356	PQ163712	PQ163689	PQ151345	—
cigar tobacco	A. tenuissima	FSM 01076	PQ151361	PQ151353	PQ163713	PQ163690	PQ151342	PQ164349
		FSM 01077	PQ151362	PQ151354	PQ163714	PQ163691	PQ151343	PQ164350
		FSM 01078	PQ151363	PQ151355	PQ163715	PQ163692	PQ151344	PQ164351
		FSM 01081	PQ151366	PQ151358	PQ163716	PQ163693	PQ151347	PQ164352
	A. alternata	FSM 01080	PQ151365	PQ151357	PQ163717	PQ163694	PQ151346	PQ164353
	A. gaisen	FSM 01082	PQ151367	PQ151359	PQ163718	PQ163695	PQ151348	—

种类 Species		菌株号 Strain No.	基因序列登录号GenBank accession No.
种类 Species		菌株号 Strain No.	ITS	TEF1	RPB2	GAPDH	Alta1	His3
标准菌株	Alternaria	CBS 612.72	KP124308	KP125084	KP124777	KP124165	KP123861	—
Standard strains	alternata	CBS 194.86	KP124316	KP125092	KP124784	KP124172	KP123869	—
		CBS 877.95	KP124321	KP125097	KP124789	KP124176	KP123871	—
		CBS 965.95	KP124323	KP125099	KP124791	KP124178	KP123872	—
		CBS 109455	KP124335	KP125111	KP124803	KP124189	KP123883	—
		CBS 112252	KP124340	KP125116	KP124808	KP124194	KP123888	—
		CBS 115200	KP124352	KP125128	KP124820	KP124206	KP123900	—
		CBS 130262	KP124389	KP125167	KP124859	KP124241	KP123937	—
	A. longipes	CBS 539.94	KP124441	KP125220	KP124911	KP124290	KP123987	—
		CBS 540.94	AY278835	KC584667	KC584409	AY278811	AY563304	—
		CBS 917.96	KP124442	KP125221	KP124912	KP124291	KP123988	—
	A. gaisen	CBS 118488	KP124427	KP125206	KP124897	KP124279	KP123977	—
		CPC 25268	KP124428	KP125207	KP124898	KP124280	JQ646393	—
	A. alternantherae	CBS 124392	KC584179	KC584633	KC584374	KC584096	KP123846	—
	A. arborescens	CBS 105.49	KP124396	KP125174	KP124866	KP124248	KP123944	—
		CBS 126.60	KP124397	KP125175	KP124867	KP124249	JQ646390	—
		CBS 109730	KP124399	KP125177	KP124869	KP124251	KP123946	—
	A. gossypina	CBS 100.23	KP124429	KP125208	KP124899	KP124280	KP123977	—
		CBS 107.36	KP124431	KP125210	KP124901	JQ646310	JQ646393	—
	A. tenuissima	SN6-2	LC621622	LC621724	LC621690	LC621656	LC631828	—
		CBS 918.96	AF347032	KC584693	KC584435	AY278809	AY563302	—
三明市雪茄烟上分离的菌株	A. longipes	FSM 01075	PQ151360	PQ151352	PQ163711	PQ163688	PQ151341	—
Strain isolated from Sanming		FSM 01079	PQ151364	PQ151356	PQ163712	PQ163689	PQ151345	—
cigar tobacco	A. tenuissima	FSM 01076	PQ151361	PQ151353	PQ163713	PQ163690	PQ151342	PQ164349
		FSM 01077	PQ151362	PQ151354	PQ163714	PQ163691	PQ151343	PQ164350
		FSM 01078	PQ151363	PQ151355	PQ163715	PQ163692	PQ151344	PQ164351
		FSM 01081	PQ151366	PQ151358	PQ163716	PQ163693	PQ151347	PQ164352
	A. alternata	FSM 01080	PQ151365	PQ151357	PQ163717	PQ163694	PQ151346	PQ164353
	A. gaisen	FSM 01082	PQ151367	PQ151359	PQ163718	PQ163695	PQ151348	—

病菌种类 Pathogen species	菌落颜色(中心) Colony color (center)	菌落颜色(边缘) Colony color (margin)	菌落形状与质地 Colony shape and texture	菌落直径/mm Colony diameter/mm	分生孢子长/μm Conidium length/μm	分生孢子宽/μm Conidium width/μm
交链格孢 A. alternata	灰色至褐色 Grayish-brown	白色 White	圆形,毡状 Circular, felt-like	71~73	10~66	7~20
长柄链格孢 A. longipes	褐色 Brown	白色 White	近圆形、边缘不规则,绒毛状 Subcircular, irregular margin, floccose	41~45	12~75	5~18
梨黑斑链格孢 A. gaisen	灰色至褐色 Grayish-brown	灰白相间 Gray-white mottled	圆形,毡状 Circular, felt-like	73~75	12~60	5~20
细极链格孢 A. tenuissima	灰色至褐色 Grayish-brown	白色 White	圆形,毡状 Circular, felt-like	62~66	10~55	5~15

Identification of pathogens causing brown spot disease on cigar tobacco and their pathogenicity to cigar tobacco varieties

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处理 Treatment	第7天病斑直径/mm Lesion diameter on day 7/mm	发病率/% Disease incidence/%
对照Control	0	0
长柄链格孢A. longipes	13.0±1.46 a	100
交链格孢A. alternaria	4.8±0.31 b	100
细极链格孢A.tenuissima	5.0±0.76 b	100
梨黑斑链格孢A. gaisen	1.4±0.53 c	100

菌株 Strain	品种 Cultivar	第7天平均病斑直径/mm Lesion diameter on day 7/mm	发病率/% Disease incidence/%
交链格孢A. alternaria	QX107	0 c	0
	QX105	0.75±0.41 b	25
	CX80	6.88±0.61 a	100
	QX103	5.67±0.33 a	100
	海研101 Haiyan 101	6.00±0.33 a	100
长柄链格孢A. longipes	QX107	7.30±1.04 a	100
	QX105	7.00±0.58 a	100
	CX80	9.25±0.68 a	100
	QX103	9.50±1.38 a	100
	海研101 Haiyan 101	7.25±0.57 a	100
细极链格孢A. tenuissima	QX107	9.10±1.58 a	100
	QX105	7.92±0.85 a	100
	CX80	7.58±0.56 a	100
	QX103	9.00±0.73 a	100
	海研101 Haiyan 101	9.42±1.16 a	100
梨黑斑链格孢A. gaisen	QX107	1.80±0.74 a	75.0
	QX105	0.58±0.40 a	16.7
	CX80	2.50±0.50 a	83.3
	QX103	1.00±0.63 a	33.3
	海研101 Haiyan 101	2.58±0.47 a	83.3

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