黄淮海夏玉米主产区茎腐病主要病原菌及优势种分析

doi:10.3864/j.issn.0578-1752.2019.02.006

摘要/Abstract

摘要：

【目的】明确黄淮海夏玉米主产区玉米茎腐病的主要病原菌组成及优势种,为病原菌致病机制、抗性育种及茎腐病防治提供依据。【方法】于2014—2017年采集黄淮海夏玉米主产区3个省（河北、河南、山东）的玉米茎腐病样本850份。通过形态学、通用引物和特异引物对病组织分离物进行鉴定,并采用上述引物直接检测病组织。整合分析分离物鉴定法和组织分子检测法的结果以确定玉米茎腐病的主要病原菌及优势种;分析主要病原菌在各省间以及同一省份不同年度的检出率,揭示主要病原菌的种群动态变化;分析单个样本中病原菌的检出率,探讨多种病原菌的共存模式。【结果】有667份样本检测出真菌或卵菌,占总样本的78.47%;年度间样本检出率存在差异,2014年的检出率不足50%,而2015—2017年的检出率相近,均高于90%。在所有样本中共检出20属46种真菌或卵菌,其中镰孢菌（Fusarium spp.）的检出率最高,为89.96%,包括禾谷镰孢复合种（F. graminearum species complex）、层出镰孢（F. proliferatum）、拟轮枝镰孢（F. verticillioides）、厚垣镰孢（F. chlamydosporum）、亚粘团镰孢（F. subglutinans）、尖镰孢（F. oxysporum）、黄色镰孢（F. culmorum）、藤仓镰孢（F. fujikuroi）、变红镰孢（F. incarnatum）、木贼镰孢（F. equiseti）和茄镰孢（F. solani）11个种;腐霉菌（Pythium spp.）的检出率为34.18%,包括芒孢腐霉（P. aristosporum）、禾生腐霉（P. graminicola）、棘腐霉（P. acanthicum）、孤雌腐霉（P. amasculinum）和寡雄腐霉（P. oligandrum）5个种。拟轮枝镰孢、禾谷镰孢复合种、芒孢腐霉和层出镰孢为4种主要病原菌,检出率依次为62.07%、46.93%、29.09%和28.04%,其中拟轮枝镰孢为优势种。各省之间,上述4种主要病原菌的检出率存在一定差异。拟轮枝镰孢在河北省的检出率为73.98%,明显高于该菌在河南省和山东省的检出率;禾谷镰孢复合种在3个省的检出率相近;层出镰孢和芒孢腐霉在山东省的检出率分别为35.78%和34.31%,均高于在其他两省的检出率。同一省份不同年度上述4种主要病原菌的检出率呈动态变化,其中任何一种病原菌均有可能上升为优势种。进一步分析表明,单个样本中可以检测出一种或多种病原菌,检测出1种菌的样本占38.38%,检出2种和3种病原菌的样本分别占29.24%和19.04%;2种及以上病原菌共存以镰孢菌和腐霉菌、镰孢菌和镰孢菌模式为主。【结论】黄淮海夏玉米主产区茎腐病主要病原菌为拟轮枝镰孢、禾谷镰孢复合种、芒孢腐霉和层出镰孢,其中拟轮枝镰孢为优势种;拟轮枝镰孢在河北省的检出率最高,层出镰孢和芒孢腐霉在山东省的检出率最高,禾谷镰孢复合种在3个省的检出率相近;单个样本中存在多种病原菌共存的模式。

关键词: 玉米茎腐病, 黄淮海地区, 病原菌检测, 优势种, 镰孢菌, 腐霉菌

Abstract:

【Objective】 The objective of this study is to determine the main pathogens and dominant species of maize stalk rot in the main summer maize producing areas of Huang-Huai-Hai, and to provide a basis for understanding the pathogenic mechanism, resistant breeding, and ultimately maize stalk rot management.【Method】 In 2014-2017, 850 samples of maize stalk rot were collected from three provinces (Hebei, Henan and Shandong) in the main summer maize producing areas of Huang-Huai-Hai. The isolated fungi or oomycetes were morphologically characterized and confirmed by using universal and specific primers, those primers were also used for detecting pathogens in diseased tissues. The combined results of isolate identification and tissue molecular detection were analyzed to determine the main pathogens and the dominant species. The detection rates of main pathogens in different provinces and different years of the same province were analyzed to reveal the population dynamics. The detection rates of pathogens in individual samples were also analyzed to determine the coexistence patterns of multiple pathogens.【Result】 Fungi or oomycetes were detected in 667 samples, accounting for 78.47% of all samples. The detection rates were different among years, less than 50% in 2014 and more than 90% in 2015-2017. Detected fungi or oomycetes were classified to 46 species of 20 genera. Fusarium spp. had the highest detection rate of 89.96%, including F. graminearum species complex, F. proliferatum, F. verticillioides, F. chlamydosporum, F. subglutinans, F. oxysporum, F. culmorum, F. fujikuroi, F. incarnatum, F. equiseti and F. solani. In addition, Pythium spp. had the second highest detection rate of 34.18%, including P. aristosporum, P. graminicola, P. acanthicum, P. amasculinum and P. oligandrum. The detection rates of four main pathogens, F. verticillioides, F. graminearum species complex, P. aristosporum and F. proliferatum, were 62.07%, 46.93%, 29.09% and 28.04%, respectively. It indicated that the dominant species was F. verticillioides. There were some differences in the detection rates of the four main pathogens among provinces. The detection rate of F. verticillioides in Hebei Province was 73.98%, which was significantly higher than that in Henan and Shandong Provinces, and the detection rates of F. graminearum species complex in the three provinces were similar. The detection rates of F. proliferatum and P. aristosporum in Shandong Province were 35.78% and 34.31%, respectively, which were higher than those in other two provinces. The detection rates of the four main pathogens from the same province were dynamic in different years, and any one of them can be the dominant species. Furthermore, several pathogens could be detected in a single sample. While 38.38% samples were colonized by only one pathogen, 29.24% and 19.04% samples were colonized by two and three pathogens. The patterns of two or multiple pathogens in single samples were mainly coexistence of Fusarium spp. and Pythium spp., or coexistence of Fusarium spp. and Fusarium spp..【Conclusion】 The main pathogens of maize stalk rot in the main summer maize producing areas of Huang-Huai-Hai are F. verticillioides, F. graminearum species complex, P. aristosporum and F. proliferatum, and the dominant species is F. verticillioides. The detection rate of F. verticillioides in Hebei Province and that of F. proliferatum and P. aristosporum in Shandong Province are the highest, and the detection rate of F. graminearum species complex in the three provinces is similar. There is a coexistence pattern of two or multiple pathogens in a single sample.

Key words: maize stalk rot, Huang-Huai-Hai region, pathogen detection, dominant species, Fusarium spp., Pythium spp.

刘树森,马红霞,郭宁,石洁,张海剑,孙华,金戈. 黄淮海夏玉米主产区茎腐病主要病原菌及优势种分析[J]. 中国农业科学, 2019, 52(2): 262-272.

LIU ShuSen,MA HongXia,GUO Ning,SHI Jie,ZHANG HaiJian,SUN Hua,JIN Ge. Analysis of Main Pathogens and Dominant Species of Maize Stalk Rot in the Main Summer Maize Producing reas of Huang-Huai-Hai[J]. Scientia Agricultura Sinica, 2019, 52(2): 262-272.

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图/表 6

表1

表2

样本中真菌或卵菌的检出率"

属 Genus	种 Species	组织分子检测 Tissue molecular detection		分离物鉴定法 Isolate identification		种检出率 Detection rate of species (%)	属检出率 Detection rate of genus (%)
属 Genus	种 Species	检出真菌或卵菌的样本数 Number of samples infected with fungi or oomycetes	检出率 Detection rate (%)	检出真菌或卵菌的样本数 Number of samples infected with fungi or oomycetes	检出率Detection rate (%)	种检出率 Detection rate of species (%)	属检出率 Detection rate of genus (%)
镰孢属 Fusarium	禾谷镰孢复合种 F. graminearum species complex	264	39.58	141	21.08	46.93	89.96
	层出镰孢 F. proliferatum	155	23.24	64	9.53	28.04
	拟轮枝镰孢 F. verticillioides	380	56.97	152	22.81	62.07
	厚垣镰孢 F. chlamydosporum	1	0.15	22	3.30	3.45
	亚粘团镰孢 F. subglutinans	7	1.05	0	0	1.05
	尖镰孢 F. oxysporum	32	4.80	1	0.15	4.95
	黄色镰孢 F. culmorum	26	3.90	0	0	3.90
	藤仓镰孢 F. fujikuroi	3	0.45	3	0.45	0.90
	变红镰孢 F. incamatum	0	0	12	1.80	1.80
	木贼镰孢 F. equiseti	6	0.90	5	0.76	1.50
	茄镰孢 F. solani	0	0	1	0.15	0.15
腐霉属 Pythium	芒孢腐霉 P. aristosporum	169	25.34	23	3.40	29.09	34.18
	禾生腐霉 P. graminicola	9	1.35	3	0.45	1.20
	棘腐霉 P. acanthicum	1	0.15	0	0	0.15
	孤雌腐霉P. amasculinum	4	0.60	0	0	0.60
	寡雄腐霉 P. oligandrum	4	0.60	0	0	0.60
链格孢属 Alternaria	互隔交链格孢 A. alternate	9	1.35	12	1.80	1.80	2.10
链格孢属 Alternaria	细极链格孢 A. tenuissima	1	0.15	2	0.30	0.30	2.10
曲霉属 Aspergillus	黄曲霉 A. flavus	5	0.75	7	1.05	1.05	1.20
曲霉属 Aspergillus	米曲霉 A. oryzae	0	0	1	0.15	0.15	1.20
枝顶孢霉属 Acremonium	A. zeae	0	0	1	0.15	0.15	0.15
平脐蠕孢属Bipolaris	玉蜀黍离蠕孢B. maydis	0	0	1	0.15	0.15	0.30
平脐蠕孢属Bipolaris	麦根腐平脐蠕孢B. sorokiniana	1	0.15	1	0.15	0.15	0.30
旋孢腔菌属Cochliobolus	异旋孢腔菌C. heterostrophus	1	0.15	1	0.15	0.15	0.15
枝孢霉属Cladosporium	芽枝状枝孢C. cladosporioides	1	0.15	15	2.25	2.25	2.25
突脐蠕孢属Exserohilum	喙突脐蠕孢E. rostratum	0	0	35	5.25	5.25	5.25
附球孢属Epicoccum	黑附球菌E. nigrum	0	0	1	0.15	0.15	0.15
地霉属Geotrichum	白地霉G. candidum	0	0	2	0.30	0.30	0.30
粘帚霉属Gliocladium	粉红粘帚霉G. roseum	0	0	2	0.30	0.30	0.30
属 Genus	种 Species	组织分子检测 Tissue molecular detection		分离物鉴定法 Isolate identification		种检出率 Detection rate of species (%)	属检出率 Detection rate of genus (%)
属 Genus	种 Species	检出真菌或卵菌的样本数 Number of samples infected with fungi or oomycetes	检出率 Detection rate (%)	检出真菌或卵菌的样本数 Number of samples infected with fungi or oomycetes	检出率Detection rate (%)	种检出率 Detection rate of species (%)	属检出率 Detection rate of genus (%)
漆斑菌属Myrothecium	包围漆斑菌M. cinctum	0	0	2	0.30	0.30	0.30
壳球孢属Macrophomina	菜豆壳球孢M. phaseolina	0	0	1	0.15	0.15	0.15
黑孢霉属 Nigrospora	N. osmanthi	0	0	1	0.15	0.15	4.20
	穗黑孢霉N. oryzae	0	0	25	3.75	3.75
	球黑孢霉N. sphaerica	0	0	2	0.30	0.30
青霉属 Penicillium	草酸青霉P. oxalicum	3	0.45	15	2.25	2.25	3.60
	微紫青霉P. janthinellum	0	0	1	0.15	0.15
	产紫青霉P. purpureogenum	0	0	4	0.60	0.60
	绳状青霉P. funiculosum	0	0	4	0.60	0.60
丝核菌属 Rhizoctonia.	立枯丝核菌R. solani	0	0	3	0.45	0.45	0.45
木霉属 Trichoderma	棘孢木霉T. asperellum	0	0	3	0.45	0.45	5.70
	非洲哈茨木霉T. afroharzianum	0	0	7	1.05	1.05
	哈茨木霉T. harzianum	1	0.15	26	3.90	3.90
	钩状木霉T. hamatum	0	0	1	0.15	0.15
	盖姆斯木霉T. gamsii	0	0	1	0.15	0.15
毛霉属Mucor	未知种Unknown species	0	0	4	0.60	0.60	0.60
盾壳霉属Coniothyrium	未知种Unknown species	0	0	1	0.15	0.15	0.15

表2

表3

表4

表5

表6

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引物Primer	引物序列 Primer sequence (5′-3′)	靶标菌 Target	产物大小 Product size (bp)	退火温度 Annealing temperature (℃)	参考文献 Reference
Fg16NF	ACAGATGACAAGATTCAGGCACA	禾谷镰孢复合种 F. graminearum species complex	280	57	[29]
Fg16NR	TTCTTTGACATCTGTTCAACCCA	禾谷镰孢复合种 F. graminearum species complex	280	57	[29]
PRO1	CTTTCCGCCAAGTTTCTTC	层出镰孢 F. proliferatum	588	56	[30]
PRO2	TGTCAGTAACTCGACGTTGTTG	层出镰孢 F. proliferatum	588	56	[30]
VER1	CTTCCTGCGATGTTTCTCC	拟轮枝镰孢 F. verticillioides	578	57	[30]
VER2	AATTGGCCATTGGTATTATATATCTA	拟轮枝镰孢 F. verticillioides	578	57	[30]
Fc01F	ATGGTGAACTCGTCGTGGC	黄色镰孢 F. culmorum	570	59	[29]
Fc01R	CCCTTCTTACGCCAATCTCG	黄色镰孢 F. culmorum	570	59	[29]
SUB1	CTGTCGCTAACCTCTTTATCCA	亚粘团镰孢 F. subglutinans	631	56	[31]
SUB2	CAGTATGGACGTTGGTATTATATCTAA	亚粘团镰孢 F. subglutinans	631	56	[31]
FOF1	ACATACCACTTGTTGCCTCG	尖镰孢 F. oxysporum	340	58	[32]
FOR1	CGCCAATCAATTTGAGGAACG	尖镰孢 F. oxysporum	340	58	[32]
FEF1	CATACCTATACGTTGCCTCG	木贼镰孢 F. equiseti	389	58	[32]
FER1	TTACCAGTAACGAGGTGTATG	木贼镰孢 F. equiseti	389	58	[32]
DC6	GAGGGACTTTTGGGTAATCA	卵菌 Oomycota	1100	58	[33]
ITS4	TCCTCCGCTTATTGATATGC	卵菌 Oomycota	1100	58	[33]
EF-1	ATGGGTAAGGARGACAAGAC	镰孢菌 Fusarium	700	54	[34]
EF-2	GGARGTACCAGTSATCATGTT	镰孢菌 Fusarium	700	54	[34]
ITS1	TCCGTAGGTGAACCTGCGG	真菌通用引物 Universal primer for fungus	550	57	[35]
ITS4	TCCTCCGCTTATTGATATGC	真菌通用引物 Universal primer for fungus	550	57	[35]
EF-1F	CATCGAGAAGTTCGAGAAGG	真菌通用引物 Universal primer for fungus	300	58	[36]
EF-1R	TACTTGAAGGAACCCTTACC	真菌通用引物 Universal primer for fungus	300	58	[36]

省份 Province	检出真菌或卵菌的样本数 Number of samples infected with fungi or oomycetes	检出率Detection rate (%)
省份 Province		拟轮枝镰孢 F. verticillioides	禾谷镰孢复合种 F. graminearum species complex	层出镰孢 F. proliferatum	芒孢腐霉 P. aristosporum
河北Hebei	123	73.98	45.53	21.95	17.89
河南Henan	340	60.59	48.24	25.59	30.00
山东Shandong	204	57.35	45.59	35.78	34.31

省份 Province	年份 Year	检出真菌或卵菌的样本数 Number of samples infected with fungi or oomycetes	检出率Detection rate (%)
省份 Province	年份 Year		拟轮枝镰孢 F. verticillioides	禾谷镰孢复合种 F. graminearum species complex	层出镰孢 F. proliferatum	芒孢腐霉 P. aristosporum
河北 Hebei	2014	24	41.67	29.17	0	4.17
	2015	9	55.56	44.44	11.11	44.44
	2016	35	68.57	48.57	34.29	22.86
	2017	55	94.55	50.91	25.45	16.36
河南 Henan	2014	52	13.46	40.38	0	59.62
	2015	92	51.09	58.70	3.26	25.00
	2016	104	73.08	54.81	48.08	16.35
	2017	92	82.61	34.78	36.96	33.70
山东 Shandong	2014	52	30.77	9.62	11.54	40.38
	2015	52	44.23	55.77	46.15	28.85
	2016	35	68.57	45.71	45.71	25.71
	2017	65	83.08	66.15	41.54	38.46

年份 Year	检出真菌或卵菌的样本数 Number of samples infected with fungi or oomycetes	真菌或卵菌种数及样本比例 Number of species of fungi or oomycetes and percentage of sample (%)
年份 Year		1	2	3	4	≥5
2014	128	77.34	18.75	3.13	0.78	0
2015	153	29.41	39.87	20.26	7.84	2.61
2016	174	36.21	25.86	24.14	12.07	1.72
2017	212	23.11	30.66	23.58	11.79	10.85
总计Total	667	38.38	29.24	19.04	8.85	4.50

年份 Year	检出真菌或卵菌的样本数 Number of samples infected with fungi or oomycetes	镰孢菌和腐霉菌共存模式及样本比例 Coexistence pattern of F. spp. and P. spp. and percentage of samples (%)
年份 Year		F. spp.	P. spp.	Other	F. spp.+ F. spp	P. spp.+ P. spp.	F. spp.+ P. spp.	F. spp.+ Other	P. spp.+ Other	Other + Other
2014	128	40.63	24.22	12.50	2.34	0	15.63	0.78	3.13	0.78
2015	153	21.57	3.27	4.58	22.22	0	24.84	20.92	1.96	0.65
2016	174	30.46	1.15	4.60	31.61	0	23.56	8.62	0	0
2017	212	19.81	0.94	2.36	30.19	0	29.72	15.57	1.42	0
总计Total	667	26.99	6.00	5.40	23.39	0	24.29	12.14	1.50	0.30