番茄褐色皱果病毒（tomato brown rugose fruit virus，ToBRFV）是2015年首次报道的一种烟草花叶病毒属病毒，是番茄安全生产的严重威胁。该病毒已经传播到美洲、亚洲和欧洲的十个国家。2019年，ToBRFV在中国山东发生。本论文旨在明确ToBRFV山东分离物（ToBRFV-SD）的症状、寄主范围和分子特性，并建立一种有效的检测方法。田间调查ToBRFV-SD在不同品种的症状表现。将ToBRFV-SD接种辣椒、本氏烟、马铃薯、茄子、中烟102和50个番茄品种，鉴定其寄主范围。分段克隆ToBRFV-SD基因组片段，并测定其序列；利用BioEdit version 7.2.6比对ToBRFV所有分离物的全基因组序列，分析序列一致率；利用MEGA version 10.1.5构建系统发育树。根据ToBRFV、烟草花叶病毒（tobacco mosaic virus，TMV）、番茄花叶病毒（tomato mosaic virus，ToMV）和番茄斑萎病毒（tomato spotted wilt virus，TSWV）等四种番茄重要病毒基因组的保守区域设计特异性引物，建立四重RT-PCR检测体系。ToBRFV-SD在番茄叶片引起不同程度的花叶和疱斑，在花萼和花梗上引起坏死，在番茄果实上引起畸形、黄斑和褐色皱缩坏死斑。ToBRFV-SD可侵染番茄、辣椒和本氏烟，隐症侵染马铃薯、茄子和烟草品种中烟102。测试的50个番茄品种均不抗ToBRFV-SD。ToBRFV-SD和以色列分离物ToBRFV-IL基因组的核苷酸和氨基酸一致率最高。在基于全基因组序列的系统进化树中，所有ToBRFV分离物聚集到一个分枝，与烟草花叶病毒分枝距离较近。随后，我们建立了四重RT-PCR检测体系，能够通过一个RT-PCR反应，同时检测并区分ToBRFV、TMV、ToMV和TSWV。本研究明确了ToBRFV-SD的症状、寄主范围和分子特性，建立了能区分ToBRFV、TMV、ToMV和TSWV四重RT-PCR检测体系，对指导ToBRFV的早期检测和防控有积极作用。

In this study, we investigated the potential nitrification and community structure of soil-based ammonia-oxidizing bacteria (AOB) in apple orchard soil during different growth periods and explored the effects of environmental factors on nitrification activity and AOB community composition in the soil of a Hanfu apple orchard, using a culture-dependent technique and denaturing gradient gel electrophoresis (DGGE). We observed that nitrification activity and AOB abundance were the highest in November, lower in May, and the lowest in July. The results of statistical analysis indicated that total nitrogen (N) content, NH4 +-N content, NO3 --N content, and pH showed significant correlations with AOB abundance and nitrification activity in soil. The Shannon-Winner diversity, as well as species richness and evenness indices (determined by PCR-DGGE banding patterns) in soil samples were the highest in September, but the lowest in July, when compared to additional sampled dates. The DGGE fingerprints of soil-based 16S rRNA genes in November were apparently distinct from those observed in May, July, and September, possessing the lowest species richness indices and the highest dominance indices among all four growth periods. Fourteen DGGE bands were excised for sequencing. The resulting analysis indicated that all AOB communities belonged to the β-Proteobacteria phylum, with the dominant AOB showing high similarity to the Nitrosospira genus. Therefore, soil-based environmental factors, such as pH variation and content of NH4 +-N and NO3 --N, can substantially influence the abundance of AOB communities in soil, and play a critical role in soil-based nitrification kinetics.