An unprecedented number of viruses have been discovered by leveraging advances in high-throughput sequencing. Infectious clone technology is a universal approach that facilitates the study of biology and role in disease of viruses. In recent years homology-based cloning methods such as Gibson assembly have been used to generate virus infectious clones. We detail herein the preparation of home-made cloning materials for Gibson assembly. The home-made materials were used in one-step generation of the infectious cDNA clone of a plant RNA virus into a T-DNA binary vector. The clone was verified by a single Illumina reaction and a de novo read assembly approach that required no primer walking, custom primers or reference sequences. Clone infectivity was finally confirmed by -mediated delivery to host plants. We anticipate that the convenient home-made materials, one-step cloning and Illumina verification strategies described herein will accelerate characterization of viruses and their role in disease development.

is a devastating pathogen that infects a wide range of plants and causes Phytophthora rot disease, which has resulted in great economic losses in crop production. Therefore, the rapid and practicable detection of is important for disease monitoring and forecasting. In this study, we developed a lateral flow recombinase polymerase amplification (LF-RPA) assay for the sensitive visual detection of . Specific primers for were designed based on the ras-related protein gene ; all 10 isolates yielded positive detection results, whereas no cross-reaction occurred in related oomycete or fungal species. The detection limit for the LF-RPA assay was 100 fg of genomic DNA under optimized conditions. Combined with a simplified alkaline lysis method for plant DNA extraction, the LF-RPA assay successfully detected in naturally diseased strawberry samples without specialized equipment within 40 min. Thus, the LF-RPA assay developed in this study is a rapid, simple, and accurate method for the detection of , with the potential for further application in resource-limited laboratories.

Apple rootstock genotypes confer different levels of tolerance to apple replant disease (ARD) and vigour to a newly replanted apple tree. A hybrid management system of rotating the rootstock genotype planted between successive generations and inter-row planting in the alleyways of orchards may minimise the severity of ARD symptoms. High-throughput sequencing of the fungal ITS and bacterial 16S rDNA regions was used to investigate the diversity, and differential taxa present in soils displaying symptoms of ARD. Candidate pathogens and beneficial microorganisms were correlated with the above-ground establishment of each rootstock genotype in a UK cider orchard. Our results suggest that the same rootstock or rootstock with closely related parentage to the previous rootstock had more severe ARD symptoms. Planting in the alleyway appeared an effective strategy to minimise the severity of symptoms irrespective of rootstock genotype. The planting location effect had a higher contribution to the variation in the rhizosphere microbiome than that of the rootstock genotype. No predicted causal agents for ARD could be identified to a taxonomic level to predict their function but two species associated with mycorrhizae, and were identified as inversely correlated with ARD severity and could be candidate beneficial species for apple, warranting further investigation and research. Our findings suggest that planting in the alleyways and planting rootstocks genetically dissimilar to the previously planted rootstock can be beneficial for tree establishment. We have also identified species inversely associated with ARD severity, making candidates for future research to test the antagonistic effect of the species against ARD pathogens in apple roots.

Maize chlorotic mottle virus (MCMV) is one of the important quarantine pathogens in China. It often co-infects with one or two viruses in the family and causes maize lethal necrosis disease. Therefore, an accurate and sensitive method for the detection of MCMV is urgently needed. Combined with reverse transcription and recombinase-aided amplification, we developed a CRISPRCas12a-based visual nucleic acid detection system targeting the MCMV coat protein gene. The whole process can be completed within 45 min with high sensitivity. This system could detect cDNAs diluted up to 10 when 2000 ng of total RNA was used for reverse transcription. The Cas12a/crRNA complex designed for MCMV detection could recognize and cleave the targeted double-stranded DNA, and ultimately cleave the single-stranded DNA probes and produce fluorescent signals. The green fluorescence produced under blue light (440-460 nm) in this procedure could be observed by the naked eye. Since this novel method is specific, rapid, sensitive and does not require special instruments and technical expertise, it should be suitable for on-site visual detection of MCMV in seeds, plants of maize and potentially in its insect vectors.

In the last two decades, fire blight has progressively spread eastward from Europe and the Mediterranean area to several pome-fruit producing regions of Asia. Its causative agent, the bacterial pathogen , was detected in several new countries, including Georgia, Kyrgyzstan, and Kazakhstan. In the latter two states, the disease creates a threat not only to the commercial apple and pear production, but also to the wild and species that constitute the basis of the local forest ecosystems. In this study, we investigated the genetic diversity of the pathogen in Central Asia and the Caucasus region utilizing CRISPR Repeat Regions (CRRs) genotyping and genome sequencing, with the aim to understand its dissemination patterns across the continent. Genome sequence analysis revealed that all strains from these two regions exclusively derived from the archetypal CRR1 genotype A. Our analysis revealed three main clades in Central Asia, with distinct yet partial overlapping geographical distributions. Genomic relationships among isolates indicate that Central Asian strains are genetically closest to those from the Persian region and the Middle East, while the Georgian population is genetically more distant and can align with strains from the Volga District in southern Russia and the Eastern Mediterranean area. Notably, this study also includes strains from the first confirmed occurrences of fire blight in Uzbekistan, Tajikistan, and China. Our findings highlight the importance of phylogenetic analysis and genome sequencing in understanding the phytopathogen epidemics and protecting key agricultural species and the genetic resources of their wild counterparts in the forest.