葡萄生单轴霉菌围可培养微生物多样性及潜在生防菌研究

Diversity of culturable microorganisms in pathosphere of Plasmopara viticola and screening of potential biocontrol strains

摘要:

【目的】揭示葡萄生单轴霉(Plasmoparaviticola)菌围可培养细菌和真菌的多样性特征，筛选对葡萄霜霉病有较强稳定防治效果的生防菌。【方法】连续两年从我国南北方具有代表性的7个葡萄产区采集葡萄霜霉病叶，镊子夹取经保湿培养获得的新鲜霉层并配制孢子囊悬浮液，采用传统分离培养法，结合形态分类、BOX-PCR指纹图谱分析以及分子鉴定结果，对葡萄生单轴霉菌围的可培养细菌和真菌进行聚类分析；采用菌株及其发酵液与病原菌孢子囊悬浮液等体积混合培养测定其对孢子囊的抑制作用，离体叶片接种法检测该菌株及其发酵液对霜霉病的防治效果。【结果】分离获得了90株细菌和110株真菌，分别归属于8个细菌属和14个真菌属，且相同地区不同葡萄品种葡萄生单轴霉菌围的细菌和真菌在同年处于同一分支。假单胞菌属(Pseudomonas)和枝孢属(Cladosporium)稳定存在于各地区不同品种葡萄霜霉病叶上葡萄生单轴霉菌围；在两年间稳定存在的菌株占比多数在80.0%以上且均具有较高的生防作用；其中，广泛分布的6株枝顶孢属(Acremonium)真菌对葡萄霜霉病的防治效果均较好，最高可达100.0%；防治效果较高的11个菌株的无菌发酵液中，黑曲霉(Aspergillusniger) NX2F、苋楔孢黑粉菌(Thecaphora amaranthi) BJ1G和匍枝根霉(Rhizopus stolonifer) BM1L的无菌发酵液防治效果均为100.0%。【结论】葡萄生单轴霉菌围的可培养细菌和真菌群落主要受地区因素影响，有较高的稳定性和生防作用，揭示了枝顶孢属真菌在我国葡萄主要产区葡萄生单轴霉菌围附生的普遍性，为葡萄霜霉病的防治提供了丰富和宝贵的资源。

Abstract:

[Objective] To investigate the diversity of bacteria and fungi in the pathosphere of Plasmopara viticola and screen out the strains with potential biocontrol effects on grape downy mildew. [Methods] The leaves infected by P. viticola were collected from seven representative grape-producing regions in northern and southern China in two consecutive years. The collected leaves were cultured in a humid environment, and the newly growing downy mildew was aseptically picked by forceps to prepare the sporangial suspensions of P. viticola. The strains were isolated by the conventional culture method and identified based on the morphological characteristics, BOX-PCR fingerprints, and molecular sequences. Furthermore, the clustering analysis of different strains was conducted. Sporangial inhibition was tested with equal volumes of strain suspension or fermentation mixed with the sporangial suspension of P. viticola, and the control effects of isolates and their sterile fermentation against grape downy mildew were tested on detached grape leaves. [Results] A total of 90 bacterial strains and 110 fungal strains were isolated, belonging to eight bacterial genera and 14 fungal genera, respectively. The pathosphere of P. viticola in the same province and the same year exhibited similar microbial community composition. Notably, strains of Pseudomonas spp. and Cladosporium spp. exhibited stable populations on grape cultivars collected from different provinces. A majority (over 80.0%) of strains with stable populations in two consecutive years demonstrated significant biocontrol effects against grape downy mildew. Six Acremonium strains with ubiquitous distribution demonstrated the biocontrol effect up to 100.0%. Sterile fermentation of the fungal strains Aspergillus niger NX2F, Thecaphora amaranthi BJ1G, and Rhizopus stolonifer BM1L showed the control effects of 100.0% against grape downy mildew. [Conclusion] The culturable bacterial and fungal communities in the pathosphere of P. viticola were mainly affected by geographical factors in different provinces, and most of the culturable microorganisms presented stable and strong biocontrol effects on grape downy mildew. To the best of our knowledge, it is the first comprehensive report that Acremonium spp. were epibiotic fungi and consistently associated with P. viticola, providing rich and valuable biocontrol resources for grape downy mildew.