Citrus yellow mosaic virus (CYMV) is a member of badnaviruses that was first discovered in India and caused serious damage to its citrus industry. At present, CYMV has been listed as a quarantine pest in the United States, Japan, New Zealand, Europe and the Mediterranean region, which has the potential risk of being introduced into China.

The objective of this study is to establish a real-time quantitative PCR (qPCR) detection system for CYMV, screen sensitive citrus varieties to CYMV, elucidate the spatial and temporal distribution of CYMV in host plant, and to provide technical support for monitoring of the virus.

According to the conserved sequences of CYMV coat protein (CP) gene in NCBI, eight pairs of qPCR primers were designed using software Primer Premier 5, and were screened by conventional PCR for primer pairs with good amplification effect and high specificity. The concentration and annealing temperature of the primers were optimized to establish the CYMV qPCR detection system. The specificity of the system was evaluated by detecting different citrus pathogens. Conventional PCR and qPCR were performed in parallel with 1.98×10⁹-1.98 copies/μL of plasmid standards diluted in a gradient as template to evaluate the sensitivity of the established system. The field citrus samples were collected randomly and detected using conventional PCR and qPCR in parallel to evaluate the applicability of the method. Fifteen citrus varieties including Yuhuanyou (Citrus grandis), Chandler pomelo (C. grandis), No.22 karatachi (Poncirus trifoliata), etc., were inoculated with CYMV for symptom observation and molecular detection to screen sensitive indicator plants. Samples from different tissues of Madam Vinous orange (C. sinensis) plants were collected at different times post inoculation, and qPCR assays were performed with C. sinensis elongation factor 1 alpha as an internal reference gene to clarify the spatial and temporal distribution patterns of CYMV in the host plants.

The qPCR detection system for CYMV was established with the optimal primer of CYMV-qF7/R7, the optimal concentration 200 nmol·L^-1 and the optimal annealing temperature 63 ℃. The results of the evaluation experiments showed that the specificity of the established qPCR system is strong, and the detection sensitivity is 1 000 times that of the conventional PCR. The detection results of 660 field citrus samples from different regions showed that the qPCR assay was consistent with the conventional PCR assay, and no CYMV-positive plants were detected except for the positive control. Among the 15 citrus varieties in the inoculation experiment, Yuhuanyou and Chandler pomelo showed strong and typical yellowing mosaic symptoms of CYMV infection at the earliest, suggesting that both could be used as sensitive indicator plants. The detection results of qPCR showed that the titer of CYMV in old leaves was the highest, the titer of virus in old cortex and root was higher, and the titer in tender cortex was lower. The relative content of CYMV in plants was highest from July to September, then decreased month by month, and reached the lowest value in January of the next year. From February of the next year, the relative content of CYMV began to rise, which was consistent with the change trend of the environmental temperature.

A qPCR assay for CYMV detection with high specificity and sensitivity was established, and the spatial and temporal distribution pattern of CYMV in the host plants was determined. In addition, Yuhuanyou and Chandler pomelo can be used as CYMV-sensitive indicator plants.

Citrus Huanglongbing (HLB) is a destructive citrus disease primarily caused by Candidatus Liberibacter asiaticus (CLas). The objective of this research is to investigate the growth pattern of CLas in citrus hairy roots at the initial stage of infection, optimize the rapid evaluation system of Agrobacterium rhizogenes-mediated HLB resistance and apply it to the resistance evaluation of antimicrobial peptides.

A plant expression vector, pGNGM1300, was employed to transform different citrus varieties via the hairy root transformation system, which was mediated by A. rhizogenes K599. Subsequently, varieties with rapid induction and high transformation rate were then selected. Following inoculation with CLas, the presence of CLas in hairy roots was consistently demonstrated through the use of real-time quantitative PCR (qPCR), which allowed for the monitoring of its growth pattern. Further, the optimization of the HLB resistance evaluation system permitted the HLB resistance evaluation of antimicrobial peptides to be conducted via (RT-) qPCR analysis of CLas and related genes, quantification of callose deposition, microstructure observation, and symptom assessment.

Among the 11 tested citrus varieties, citron (Citrus medica) exhibited the fastest induction of hairy roots (15 d), accompanied by the highest induction rate (73.75%) and a substantial transgene positivity rate (53.54%) in hairy roots. The results of regular qPCR monitoring indicated that CLas began to colonize roots on 20-30 days post-inoculation (dpi). At 30-50 dpi, there was a notable increase in the CLas content. The content of the CLas exhibited a slight fluctuation at a range of 60 to 120 dpi, though the discrepancy with 50 dpi was not statistically significant. The results of the antimicrobial peptide resistance evaluation, conducted on MaSAMP (stable antimicrobial peptide), HBD-4 (homo sapiens defensin beta 4), and CB (cecropin B), revealed that at 50-120 dpi, the content of CLas in both MaSAMP- and CB-expressing plants was significantly lower than that of the control. However, except that at 90 dpi, the CLas content was also significantly lower in HBD-4-expressing plants than the control. The callose content in all three plants expressing antimicrobial peptides was significantly lower than that of the control (60 dpi). There was no visible cell wall thickening in the phloem, and no significant deposition of starch grains or callus was observed. Furthermore, none of the plants exhibited signs of root death at 90 dpi.

This study elucidates the growth dynamics of CLas in citrus hairy roots during the early stages of infection and optimizes a rapid scoring system for A. rhizogenes-mediated HLB resistance. In addition, the results show that overexpression of MaSAMP, HBD-4 and CB effectively suppresses CLas proliferation, reduces callose deposition and alleviates HLB symptoms. These findings have the potential to be used in the prevention and control of HLB.