ABSTRACT
Citrus cultivation plays a pivotal role, making a significant contribution to global fruit production and dietary consumption. Accurate identification of viral pathogens is imperative for the effective management of plant viral disease in citrus crops. High-throughput sequencing serves as an alternative approach, enabling comprehensive pathogen identification on a large scale without requiring pre-existing information. In this study, we employed HTS to investigate viral pathogens infecting citrus in three different regions of South Korea: Jejudo (Jeju), Wando-gun (Wando), and Dangjin-si (Dangjin). The results unveiled diverse viruses and viroids that exhibited regional variations. Notably, alongside the identification of well-known citrus viruses such as satsuma dwarf virus, citrus tatter leaf virus, and citrus leaf blotch virus (CLBV), this study also uncovered several viruses and viroids previously unreported in Korean citrus. Phylogenetic analysis revealed that majority of identified viruses exhibited the closest affilations with isolates from China or Japan. However, CLBV and citrus viroid-I-LSS displayed diverse phylogenetic positions, reflecting their regional origins. This study advances our understanding of citrus virome diversity and regional dynamics through HTS, emphasizing its potential in unraveling intricate viral pathogens in agriculture. Consequently, it significantly contributes to disease management strategies, ensuring the resilience of the citrus industry.
ABSTRACT
Phalaenopsis orchids are one of the most popular ornamental plants. More than thirty orchid viruses have been reported, and virus-infected Phalaenopsis orchids significantly lose their commercial value. Therefore, the development of improved viral disease detection methods could be useful for quality control in orchid cultivation. In this study, we first utilized the MinION, a portable sequencing device based on Oxford Nanopore Technologies (ONT) to rapidly detect plant viruses in Phalaenopsis orchids. Nanopore sequencing revealed the presence of three plant viruses in Phalaenopsis orchids: odontoglossum ringspot virus, cymbidium mosaic virus, and nerine latent virus (NeLV). Furthermore, for the first time, we detected NeLV infection in Phalaenopsis orchids using nanopore sequencing and developed the reverse transcription-recombinase polymerase amplification (RT-RPA)-CRISPR/Cas12a method for rapid, instrument-flexible, and accurate diagnosis. The developed RT-RPA-CRISPR/Cas12a technique can confirm NeLV infection in less than 20 min and exhibits no cross-reactivity with other viruses. To determine the sensitivity of RT-RPA-CRISPR/Cas12a for NeLV, we compared it with RT-PCR using serially diluted transcripts and found a detection limit of 10 zg/µL, which is approximately 1000-fold more sensitive. Taken together, the ONT platform offers an efficient strategy for monitoring plant viral pathogens, and the RT-RPA-CRISPR/Cas12a method has great potential as a useful tool for the rapid and sensitive diagnosis of NeLV.
Subject(s)
Amaryllidaceae , Latent Infection , Nanopore Sequencing , Orchidaceae , CRISPR-Cas Systems , Cross Reactions , RecombinasesABSTRACT
Citrus tristeza virus (CTV) is a highly destructive viral pathogen posing a significant threat to citrus crops worldwide. The disease management and crop protection strategies necessitate the development of rapid and accurate detection methods. In this study, we employed Oxford Nanopore sequencing (ONT) to detect CTV in Citrus unshiu samples. Subsequently, we developed a specific and sensitive detection assay combining CRISPR/Cas12a with reverse transcription-recombinase polymerase amplification. The CRISPR-Cas12a assay exhibited exceptional specificity for CTV, surpassing conventional RT-PCR by at least 10-fold in sensitivity. Remarkably, the developed assay detected CTV in field samples, with zero false negatives. This diagnostic approach is user-friendly, cost-effective, and offers tremendous potential for rapid on-site detection of CTV. Therefore, the CRISPR-Cas12a assay plays a significant role in managing and preserving citrus trees that are free from viruses in the industry.
