Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (1): 213-224.doi: 10.3864/j.issn.0578-1752.2020.01.020

• RESEARCH NOTES • Previous Articles    

Genetic Diversity of Dactylobotrys graminicola and Its Pathogenicity to Hordeum vulgare var. nudum Seedlings

Lin CHEN1,RuiMing LIN1(),FengTao WANG1,YunXing PANG1,Xue LI1,AiPing ZHAO1,YanXia ZHANG2,JinLing ZHANG1,3,WenXing LI4,SuQin HE5,Jing FENG1,Yun LI1,6,CaiYi WEN6,ShiChang XU1   

  1. 1 State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193
    2 Institute of Agricultural Sciences, Haibei Tibetan Autonomous Prefecture of Qinghai, Haibei 810299, Qinghai
    3 School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang 621010, Sichuan
    4 Shigatse Institute of Agricultural Sciences of Tibet, Shigatse 857000, Tibet
    5 Institute of Plant Protection, Gansu Academy of Agricultural Sciences, Lanzhou 730070
    6 College of Plant Protection, Henan Agricultural University, Zhengzhou 450001
  • Received:2019-06-18 Accepted:2019-08-16 Online:2020-01-01 Published:2020-01-19
  • Contact: RuiMing LIN E-mail:linruiming@caas.cn

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Abstract:

【Background】In the high-latitude agricultural areas of Qinghai-Tibet Plateau and its peripheral regions, hulless or naked barley (Hordeum vulgare var. nudum) is the only small grain cereal crop in the local area, and it is also the major forage. Dactylobotrys spike blight, caused by Dactylobotrys graminicola, is a new fungal disease only found in the hulless barley growing areas in Qinghai-Tibet Plateau in recent years, which can attack several triticeae crops and cereal grasses and make a great challenge to security of hulless barley production.【Objective】The objective of this study is to research the genetic diversity, systemic evolution of the fungal pathogen of D. graminicola and its effects on the host seed germination, reveal the pathogen epidemics and spread ways, host-pathogen interrelationship and the primary inoculum, and to provide valuable information for disease control.【Method】The diseased plant samples were collected from the disease epidemic areas, and 27 strains were isolated from the typical infected host spike tissues. After identification based on its biological traits of anamorph, the single-conidium strains were cultivated and used for genomic DNA extraction. The whole genomic diversity was evaluated with SRAP markers and strain-specific markers were developed. The evolution relationship between D. graminicola and its related genera or species was analyzed using the two conserved loci of LSU and Rpb2. The conidium suspension was co-cultivated with the germinated or ungerminated hulless barely seeds, and its effect on barley seed germination and seedling growth of hulless barely was analyzed.【Result】Fourteen SRAP primer combinations were selected to detect genetic diversity of D. graminicola strains, and on average, 90 polymorphic DNA bands were produced per primer combination. For D. graminicola strains, only 1.4 strain-specific markers were developed averagely. The strain of Z 13008 derived from common wheat possessed the most 7 SRAP markers. For Fusarium graminearum used as a control strain, 20 specific markers were identified. It was confirmed that there is no close relationship between strain origins of D. graminicola and their genetic diversity. Among the identified strains, only those like Z 13008 from common wheat, Z 13024 from rye and another two (Z 13013 and Z 13006) from hulless barley showed higher differences in genetic diversity compared with the others, indicating that the genetic variation level of D. graminicola population is low. Besides, it is feasible to characterize the 21 D. graminicola strains with a DNA dichotomous fingerprinting key constructed with 20 SRAP markers. D. graminicola is genetically closely related to the fungal species of genus Nectria through phylogenetic analysis. When inoculating seeds with conidial suspensions before germination, the pathogen had no significant suppression on seed germination and growth of seedlings and their roots. While it could infect and restrain seedling root growth when co-cultivating the germinated seeds with conidial suspensions, and resulted in the infected young roots changing into light brown color, but had no significant effect on seeding growth.【Conclusion】D. graminicola showed lower genetic diversity compared with its relative species of F. graminearum, and its genetic variation is closely related to its host plant species. The pathogenicity of the pathogen is weak, and it has no obvious inhibiting effect on the germination and seedling growth of hulless barley.

Key words: Hordeum vulgare, Hordeum vulgare var. nudum, Dactylobotrys graminicola, Dactylobotrys spike blight, SRAP, genetic diversity, fingerprinting key

Table 1

Geographic origins, host species and diseased plant tissues of D. graminicola strains"

菌株Strain 来源Origin 寄主种类/组织Host species/tissue 采集日期Collection date
Z 13001 甘肃合作市 Hezuo, Gansu 黑麦/穗 Rye/spike 2013-08
Z 13002 甘肃合作市 Hezuo, Gansu 野燕麦/穗 Wild oat/spike 2013-08
Z 13003 甘肃合作市 Hezuo, Gansu 青稞/穗 Naked barley/spike 2013-08
Z 13004 甘肃卓尼县 Zhuoni, Gansu 青稞/穗 Naked barley/spike 2013-08
Z 13005 甘肃临潭县 Lintan, Gansu 青稞/穗 Naked barley/spike 2013-08
Z 13006 甘肃临潭县 Lintan, Gansu 青稞/穗 Naked barley/spike 2013-08
Z 13007 甘肃临潭县 Lintan, Gansu 黑麦/穗 Rye/spike 2013-08
Z 13008 甘肃临潭县 Lintan, Gansu 小麦/穗 Common wheat/spike 2013-08
Z 13009 甘肃临潭县 Lintan, Gansu 青稞/穗 Naked barley/spike 2013-08
Z 13010 青海湟中县 Huangzhong, Qinghai 青稞/穗 Naked barley/spike 2013-07
Z 13011 青海湟中县 Huangzhong, Qinghai 野燕麦/穗 Wild oat/spike 2013-07
Z 13012 青海互助县 Huzhu, Qinghai 野燕麦/穗 Wild oat/spike 2013-07
Z 13013 青海互助县 Huzhu, Qinghai 青稞/穗 Naked barley/spike 2013-07
Z 13014 青海海晏县 Haiyan, Qinghai 青稞/穗 Naked barley/spike 2013-07
Z 13015 青海海晏县 Haiyan, Qinghai 青稞/穗 Naked barley/spike 2013-07
Z 13016 青海海晏县 Haiyan, Qinghai 青稞/穗 Naked barley/spike 2013-07
Z 13017 青海门源县 Menyuan, Qinghai 野燕麦/穗 Wild oat/spike 2013-07
Z 13018 青海门源县 Menyuan, Qinghai 青稞/穗 Naked barley/spike 2013-07
Z 13019 青海门源县 Menyuan, Qinghai 野燕麦/穗 Wild oat/spike 2013-07
Z 13020 青海门源县 Menyuan, Qinghai 青稞/穗 Naked barley/spike 2013-07
Z 13021 青海门源县 Menyuan, Qinghai 野燕麦/穗 Wild oat/spike 2013-07
Z 13022 青海门源县 Menyuan, Qinghai 野燕麦/穗 Wild oat/spike 2013-07
Z 13023 青海门源县 Menyuan, Qinghai 青稞/穗 Naked barley/spike 2013-07
Z 13024 青海湟源县 Huangyuan, Qinghai 黑麦/穗 Rye/spike 2013-07
Z 13025 青海湟源县 Huangyuan, Qinghai 野燕麦/穗 Wild oat/spike 2013-07
Z 13026 青海湟源县 Huangyuan, Qinghai 青稞/穗 Naked barley/spike 2013-07
Z 13027 青海海晏县 Haiyan, Qinghai 青稞/籽粒 Naked barley/seed 2013-07

Table 2

Primer sequences of SRAP analysis"

正向引物序列 Forward primer sequence 反向引物序列 Reverse primer sequence
Me1 TGAGTCCAAACCGGATA Em1 GACTGCGTACGAATTAAT
Me2 TGAGTCCAAACCGGAGC Em2 GACTGCGTACGAATTTGC
Me3 TGAGTCCAAACCGGAAT Em4 GACTGCGTACGAATTTGA
Me4 TGAGTCCAAACCGGACC Em5 GACTGCGTACGAATTAAC
Me5 TGAGTCCAAACCGGAAG Em6 GACTGCGTACGAATTGCA
Me6 TGAGTCCAAACCGGTAA Em9 GACTGCGTACGAATTCGA
Me7 TGAGTCCAAACCGGTCC Em10 GACTGCGTACGAATTCAG
Me8 TGAGTCCAAACCGGTGC Em11 GACTGCGTACGAATTCCA

Fig. 1

Effects of D. graminicola infection on seed germination and seedling growth of H. vulgare var. nudum"

Table 3

Total polymorphic bands amplified by 14 SRAP primer combinations for genetic diversity analysis on D. graminicola strains"

引物组合
Primer combination		 多态性条带
Polymorphic band		 引物组合
Primer combination		 多态性条带
Polymorphic
band
Me1-Em2 59 Me6-Em5 210
Me3-Em5 102 Me6-Em10 177
Me3-Em11 22 Me7-Em1 67
Me4-Em2 108 Me7-Em4 97
Me4-Em10 97 Me7-Em6 52
Me4-Em11 22 Me7-Em9 47
Me5-Em2 88 Me8-Em5 107

Table 4

Strain-specific molecular markers in D. graminicola generated by SRAP method"

菌株
Strain		 标记数目
Number of markers		 菌株特异性标记
Specific marker details a
F. graminearumb 20 Me6-Em5, 230; Me7-Em1, 70; Me7-Em1, 500; Me7-Em4, 120; Me7-Em4, 550; Me7-Em6, 200; Me7-Em6, 310; Me7-Em6, 400; Me8-Em5, 580; Me8-Em5, 120; Me8-Em5, 250; Me4-Em10, 370; Me4-Em10, 330; Me4-Em2, 330; Me4-Em2, 360; Me3-Em5, 500; Me3-Em5, 260; Me3-Em5, 180; Me3-Em5, 160; Me1-Em2, 430
Z 13003-1 4 Me1-Em2, 430; Me4-Em2, 180; Me4-Em11, 270; Me3-Em2, 250
Z 13003 3 Me3-Em2, 250; Me3-Em5, 600; Me3-Em5, 400
Z 13002 5 Me4-Em2, 330; Me3-Em5, 210; Me3-Em2, 370; Me6-Em5, 570; Me6-Em10, 380
Z 13008 7 Me4-Em2, 180; Me4-Em11, 270; Me4-Em11, 210; Me5-Em2, 530; Me3-Em5, 600; Me6-Em10, 100; Me7-Em4, 280
Z 13006 2 Me6-Em5, 320; Me6-Em10, 230
Z 13009 1 Me7-Em4, 230
Z 13012 1 Me3-Em2, 270
Z 13020 1 Me4-Em11, 270
Z 13021 1 Me6-Em10, 100
Z 13024 5 Me7-Em1, 310; Me7-Em1, 120; Me7-Em1, 380; Me7-Em1, 350; Me7-Em1, 600

Fig. 2

The dendrogram of 21 D. graminicola strains and one F. graminearum control, constructed by UPGMA based on the total genetic differences identified with SRAP method"

Table 5

A dichotomous fingerprinting key for identification of D. graminicola strains and a control strain of F. graminearum using SRAP markers"

SRAP标记 SRAP markera P/Ab 菌株数Strain numberc
1. Me6-Em5, 290 P 4, 6, 8, 12, 15, 16, 19, 20, 21
1.1 Me6-Em10, 450 P 4, 12, 15, 16, 19, 21
1.1.1 Me7-Em4, 270 P 19
1.1.2 Me7-Em4, 270 A 4, 12, 15, 16, 21
1.1.2.1 Me7-Em4, 190 P 4, 12, 16
1.1.2.1.1 Me3-Em2, 450 P 4
1.1.2.1.2 Me3-Em2, 450 A 12, 16
1.1.2.1.2.1 Me4-Em2, 190 P 12
1.1.2.1.2.2 Me4-Em2, 190 A 16
1.2 Me6-Em10, 450 A 6, 8, 20
1.2.1 Me6-Em5, 260 P 6, 20
1.2.1.1 Me6-Em10, 230 P 6
1.2.1.2 Me6-Em10, 230 A 20
1.2.2 Me6-Em5, 260 A 8
2. Me6-Em5, 290 A 1, 2, 3, 5,7 ,9, 10, 11, 13, 14, 17, 18, 22
2.1 Me7-Em4, 130 P 2, 3, 7, 9, 17, 18, 22
2.1.1 Me6-Em10, 330 P 2, 9, 17
2.1.1.1 Me3-Em2, 650 P 2
2.1.1.2 Me3-Em2, 650 A 9, 17
2.1.1.2.1 Me6-Em10, 170 P 9
2.1.1.2.2 Me6-Em10, 170 A 17
2.1.2 Me6-Em10, 330 A 3, 7, 18, 22
2.1.2.1 Me6-Em10, 120 P 7, 21
2.1.2.1.1 Me7-Em4, 130 P 7
2.1.2.1.2 Me7-Em4, 130 A 21
2.1.2.2 Me6-Em10, 120 A 3, 18
2.1.2.2.1 Me3-Em5, 500 P 3
2.1.2.2.2 Me3-Em5, 500 A 18
2.2 Me7-Em4, 130 A 1, 5, 10, 11, 13, 14
2.2.1 Me1-Em2, 480 P 1, 5
2.2.1.1 Me3-Em2, 500 P 1
2.2.1.2 Me3-Em2, 500 A 5
2.2.2 Me1-Em2, 480 A 10, 11, 13, 14
2.2.2.1 Me3-Em2, 450 P 10, 11
2.2.2.1.1 Me7-Em6, 450 P 10
2.2.2.1.2 Me7-Em6, 450 A 11
2.2.2.2 Me3-Em2, 450 A 13, 14
2.2.2.2.1 Me7-Em9, 450 P 13
2.2.2.2.2 Me7-Em9, 450 A 14

Fig. 3

Parsimonious phylogenetic tree of D. graminicola and related fungal species based on genomic sequences of two loci (LSU + Rpb2)"

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