Pseudostellaria heterophylla (Miq.) Pax (P. heterophylla) was a valued traditional Chinese herbal medicine. Previous studies have shown that P. heterophylla TuMV spreads during the vegetative propagation cycle using tuberous roots as carriers. However, the transmission mechanism of TuMV in P. heterophylla and its effects on host growth remain to be elucidated. In this study, virus-free P. heterophylla culture seedlings were infected with control, TuMV-ZR, and TuMV-ZR-EGFP, thereby resulting in the initial infection cycle of IF1 (TIF1, TEIF1) and control NIF1, and used these roots to propagate the subsequent infection cycle IF2 (TIF2, TEIF2) and control NIF2. The transmission of TuMV-ZR seedlings was tracked by EGFP signal, and their yield, quality, and resistance were analyzed simultaneously in the critical growth period of the plants. The results indicated that TuMV-ZR accumulated in the tuberous roots of IF1 plants, subsequently migrated to IF2 during seedling growth, and was re-stored in IF2 tuberous roots, thereby forming a simple virus transmission cycle. Meanwhile, the tuberous roots of IF1 and IF2 P. heterophylla showed lower fresh weight, dry weight, soluble sugar, and saponin levels compared to NIF1 and NIF2, respectively. TuMV caused a significant reduction in chlorophyll synthesis in IF1 and IF2 P. heterophylla, resulting in impairment to their photosynthetic organs and efficiency. The measurement of stress resistance in IF1 and IF2 P. heterophylla revealed that continuous viral infection disrupted antioxidant enzyme activity, increased the content of MDA, enhanced the activity of PAL, and elevated the levels of intracellular osmotic substances in both propagation cycles. The findings indicated that the accumulation of the TuMV-ZR virus during two successive vegetative propagation cycles induced physiological stress, impaired photosynthesis, and caused progressive yield and quality decline with each cycle. This study systematically examined the impact of TuMV-ZR persistence during vegetative propagation on key physiological and biochemical indices in P. heterophylla, providing critical data to clarify vegetative-propagation-mediated germplasm degradation.

Phenolic allelochemicals from root exudates dominate rhizosphere formation, lead to autotoxicity in plants subjected to continuous monoculture (CM) stress and induce the emergence of replant disease. However, the regulatory mechanisms governing the transport of phenolics from plant roots to the rhizosphere remain poorly understood. A potential phenolic efflux transporter from Rehmannia glutinosa, designated RgMATE35, has been preliminarily characterized. The objective of this study was to elucidate the molecular function of RgMATE35 in the secretion of phenolics and to investigate its role in the development of plant replant disease using quantitative real-time PCR (qRT-PCR), genetic transformation, HPLC-Q-TOF-MS and other analytical techniques. A tissue expression pattern analysis of RgMATE35 revealed that it is highly expressed in plant roots. Transient expression analysis confirmed the localization of the protein in plasma membranes. An assessment of the transport activity of RgMATE35 in Xenopus oocytes indicated that it plays a role in facilitating the efflux of labeled ferulic acid ([²H₃]-FA) and trans-p-coumaric acid [²H₆]-pCA. The results of functional studies in R. glutinosa demonstrated that RgMATE35 positively mediates the secretion of FA and pCA from plant roots into the rhizosphere. A molecular and physiological analysis of RgMATE35 transgenic plants subjected to CM stress revealed that the overexpression or repression of RgMATE35 resulted in notable changes in the degree of autotoxic injury in plants. These findings demonstrate that RgMATE35 plays a positive role in the development of replant disease through the secretion of phenolic acids from plant roots. They also provide a fundamental framework for elucidating the molecular regulatory mechanism through which MATEs regulate replant disease through the root secretion of allelochemicals.

Pseudostellaria heterophylla (Miq.) Pax, a highly valued Chinese medicinal plant, is experiencing a notable decline in yield and quality due to viral diseases, particularly caused those by TuMV and BBWV2. Currently, the absence of a quantitative detection method for these viruses in P. heterophylla impedes the accurate diagnosis. The development of an accurate quantitative detection method is thus essential for effectively managing viral diseases in this plant. In this study, singleplex and duplex TaqMan qPCR were developed for the detection of the two viruses, based on two viral cloning vectors. Concurrently, the levels of both viruses were examined in the main produced regions of P. heterophylla. Furthermore, the levels of BBWV2 were examined during its infection of P. heterophylla. The optimal singleplex qPCR employed 0.1 μM of hydrolysis probe and 0.1 μM of primer for TuMV, while 0.2 μM of hydrolysis probe and 0.1 μM of primer were utilised for BBWV2. In contrast, the duplex qPCR employed the use of 0.1 μM of the upstream/downstream primer from each primer pair, with 0.2 μM of the respective hydrolysis probes. The 95% limit of detection (LOD) for singleplex qPCR was 734 copies for TuMV and 20 copies for BBWV2, while the 95% LOD for duplex qPCR was 945 copies for TuMV and 47 copies for BBWV2. Furthermore, the intra- and inter-assay coefficients of variation were found to be less than 1.2% for both singleplex and duplex qPCR. Of the P. heterophylla sampled 60 sites, 96% were found to be infected by one of two viruses. The levels of BBWV2 in N. benthamiana and P. heterophylla demonstrated an initial increase, followed by a subsequent decrease. The TaqMan qPCR methods provide a technical foundation for the monitoring of virus infections in P. heterophylla.

Rehmannia glutinosa Libosch. is a perennial herbaceous plant used as one of the basic herbal medicines in 50 traditional Chinese medicines (Zhang et al. 2016). From 2017 to 2019, outbreaks of leaf ...