禾生指葡孢霉的遗传多样性及对苗期青稞的致病性

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

【背景】在青藏高原及周边高海拔地区,青稞是当地唯一的小禾谷类粮食作物,也是主要饲草来源。穗腐病是近年来仅在青藏高原青稞种植区发现的一种新真菌病害,病原菌为禾生指葡孢霉(Dactylobotrys graminicola),它能侵染多种麦类作物及禾本科杂草,给青稞安全生产带来严重的挑战。【目的】研究禾生指葡孢霉遗传多样性、系统进化以及对寄主种子萌发的影响,揭示该病原菌的流行传播、与寄主植物互作关系以及初侵染菌源,为病害防治提供参考。【方法】从病害流行区采集病株标本,并从典型病穗组织分离禾生指葡孢霉菌株27个,依据无性世代形态特征加以鉴定,分离获得菌株的单孢培养物,提取基因组DNA。利用SRAP分子标记分析禾生指葡孢霉的全基因组多态性,获得菌株特异性分子标记;利用LSU和Rpb2保守遗传位点比较禾生指葡孢霉与其近缘种属的进化关系;将禾生指葡孢霉分生孢子悬浮液与未萌发或发芽后的青稞种子共培养,分析其对青稞种子萌发及幼苗的影响。【结果】选用14对SRAP引物组合进行穗腐菌遗传多样性分析,平均一对引物扩增产生90个多态性条带;平均每个穗腐病菌株1.4个特异分子性标记,分离自小麦的菌株Z 13008特异性标记最多(7个),而对照菌株禾谷镰孢(Fusarium graminearum)具有20个特异性标记。禾生指葡孢霉的地域分布与其遗传多样性缺乏明显相关性。在鉴定的菌株中,仅有分离自小麦(Z13008)、黑麦(Z 13024)和2个分离自青稞的菌株(Z 13013和Z 13006)的遗传多样性与其他菌株差异较大,说明病原菌群体的遗传变异水平较低。利用20个分子标记编制DNA指纹二叉式分类检索表,可鉴定21个禾指葡孢霉菌株。系统进化分析揭示禾生指葡孢霉与丛赤壳属真菌遗传距离较近。青稞种子萌发前接种处理对种子萌发、幼苗生长均无明显的抑制作用,在萌发后共培养处理能明显抑制幼苗根系生长,导致根系变为浅褐色,但对幼苗植株生长无明显影响。【结论】与近缘种禾谷镰孢相比,禾生指葡孢霉群体内基因组DNA多态性较低,而且其遗传变异与寄主种类密切相关。禾生指葡孢霉致病性较弱,对青稞种子萌发和幼苗生长无明显的抑制作用。

【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.