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Plasmodiophora brassicae (P. brassicae) is a soil-born pathogen worldwide and can infect most cruciferous plants, which causes great yield decline and economic losses. It is not well known how microbial diversity and community composition change during P. brassicae infecting plant roots. Here, we employed a resistant and a susceptible pakchoi cultivar with and without inoculation with P. brassicae to analyze bacterial and fungal diversity using 16S rRNA V3-V4 and ITS_V1 regions, respectively. 16S rRNA V3-V4 and ITS_V1 regions were amplified and sequenced separately. Results revealed that both fungal and bacterial diversity increased, and composition was changed in the rhizosphere soil of the susceptible pakchoi compared with the resistant cultivar. In the four groups of R_mock, S_mock, R_10d, and S_10d, the most relatively abundant bacterium and fungus was Proteobacteria, accounting for 61.92%, 58.17%, 48.64%, and 50.00%, respectively, and Ascomycota, accounting for 75.11%, 63.69%, 72.10%, and 90.31%, respectively. A total of 9488 and 11,914 bacteria were observed uniquely in the rhizosphere soil of resistant and susceptible pakchoi, respectively, while only 80 and 103 fungi were observed uniquely in the correlated soil. LefSe analysis showed that 107 and 49 differentially abundant taxa were observed in bacteria and fungi. Overall, we concluded that different pakchoi cultivars affect microbial diversity and community composition, and microorganisms prefer to gather around the rhizosphere of susceptible pakchoi. These findings provide a new insight into plant–microorganism interactions. [ABSTRACT FROM AUTHOR]

Plasmodiophora brassicae (Wor.) is an obligate plant pathogen affecting Brassicae worldwide. To date, there is very little information available on the biology and molecular basis of P. brassicae primary and secondary zoospore infections. To examine their roles, we used microscope to systematically investigate the infection differences of P. brassicae between samples inoculated separately with resting spores and secondary zoospores. The obvious development of P. brassicae asynchrony that is characterized by secondary plasmodium, resting sporangial plasmodium, and resting spores was observed at 12 days in Brassica rapa inoculated with resting spores but not when inoculated with secondary zoospores at the same time. Inoculation with resting spores resulted in much more development of zoosporangia clusters than inoculation with secondary zoospores in non-host Spinacia oleracea. The results indicated that primary zoospore infection played an important role in the subsequent development. To improve our understanding of the infection mechanisms, RNA-seq analysis was performed. Among 18 effectors identified in P. brassicae, 13 effectors were induced in B. rapa seedlings inoculated with resting spores, which suggested that the pathogen and host first contacted, and more effectors were needed. Corresponding to those in B. rapa, the expression levels of most genes involved in the calcium-mediated signaling pathway and PTI pathway were higher in plants inoculated with resting spores than in those inoculated with secondary zoospores. The ETI pathway was suppressed after inoculation with secondary zoospores. The genes induced after inoculation with resting spores were suppressed in B. rapa seedlings inoculated with secondary zoospores, which might be important to allow a fully compatible interaction and contribute to a susceptible reaction in the host at the subsequent infection stage. The primary zoospores undertook an more important interaction with plants. [ABSTRACT FROM AUTHOR]

Clubroot disease caused by the soil-borne Plasmodiophora brassicae is devastating to Brassicaceae crops and spreading rapidly in China in recent years, resulting in great yield losses annually. Virulence of P. brassicae populations specializes and is in dynamic change in the fields. Information on the pathotypes and their distributions is crucial to control the clubroot disease. Presently, the pathotypes of P. brassicae prevalent in China, however, are not well determined. In this study, we used 16 Brassica hosts, including the European Clubroot Differential (ECD) and Williams sets, to designate the pathotypes of 33 P. brassicae populations from 13 provinces. The 33 P. brassicae populations could be divided into 26 pathotypes by the ECD set or seven pathotypes by the Williams set, revealing ECD16/15/31 and ECD16/31/31 or P4 and P2 as the predominant pathotypes. We found that the Brassica rapa differentials ECD01 to ECD04 showed stable and high levels of resistance to most pathotypes of P. brassicae in China, thereby providing valuable resources for clubroot-resistance breeding of Brassicaceae crops. The ECD set exhibited much higher discernibility and further divided the isolates that belonged to the P4 pathotype into 10 ECD pathotypes. Isolates of ECD16/23/31 and ECD16/15/31 were strongly virulent on Huashuang 5R, the first and widely used clubroot-resistant cultivar of oilseed rape in China. As we learn, 26 pathotypes are the most diverse populations of P. brassicae characterized until now in China. Our study provides new insights into virulence specialization of P. brassicae and their geographical distributions, contributing to exploitation of clubroot-resistant resources and the field layout of the present resistant Brassica crops in China., Competing Interests: The author(s) declare no conflict of interest.

Clubroot caused by the obligate parasite Plasmodiophora brassicae is a devastating disease affecting the canola industry worldwide. The socio-economic impact of clubroot can be significant, particularly in regions where Brassica crops are a major agricultural commodity. The disease can cause significant crop losses, leading to reduced yield and income for farmers. Extensive studies have been conducted to understand the biology and genetics of the pathogens and develop more effective management strategies. However, the basic procedures used for pathogen storage and virulence analysis have not been assembled or discussed in detail. As a result, there are discrepancies among the different protocols used today. The aim of this article is to provide a comprehensive and easily accessible resource for researchers who are interested in replicating or building upon the methods used in the study of the clubroot pathogen. Here, we discuss in detail the methods used for P. brassicae spore isolation, inoculation, quantification, propagation, and molecular techniques such as DNA extraction and PCR. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Extraction of Plasmodiophora brassicae resting spores and propagation Support Protocol 1: Evans blue staining to identify resting spore viability Support Protocol 2: Storage of Plasmodiophora brassicae Basic Protocol 2: Generation of single spore isolates from P. brassicae field isolates Basic Protocol 3: Phenotyping of Plasmodiophora brassicae isolates Basic Protocol 4: Genomic DNA extraction from Plasmodiophora brassicae resting spores Basic Protocol 5: Molecular detection of Plasmodiophora brassicae., (© 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.)