Pine wilt disease is caused by the pine wood nematode (Bursaphelenchus xylophilus) and leads to wilting and death of pines. It is one of the most damaging diseases of pines worldwide. Therefore, accurate and rapid detection methods are of great importance for the control of B. xylophilus. Traditional detection methods have some problems, such as being time-consuming and requiring expensive instruments. In this study, the loop-mediated isothermal amplification (LAMP) and clustered regularly interspaced short palindromic repeats (CRISPR) were used to establish a set of intelligent detection and analysis system for B. xylophilus, called LAMP-CRISPR/Cas12a analysis, which integrated field sampling, rapid detection and intelligent control analysis. The process can be completed within 1 hour, from sample pretreatment and detection to data analysis. Compared with the single LAMP method, the LAMP-CRISPR/Cas12a assay uses species-specific fluorescence cleavage to detect target amplicons. This process confirms the amplicon identity, thereby avoiding false-positive results from non-specific amplicons, and the large amounts of irrelevant background DNA do not interfere with the reaction. The LAMP-CRISPR/Cas12a assay was applied to 46 pine wood samples and the samples carrying B. xylophilus nematodes were successfully identified. To meet the needs of different environments, we designed three methods to interpret the data: 1) naked eye interpretation; 2) lateral flow biosensor assay; and 3) integrated molecular analysis system to standardize and intellectualize the detection process. Application of the B. xylophilus detection and analysis system will reduce the professional and technical requirements for the operating environment and operators and help to ensure the accuracy of the detection results, which is important in grass-root B. xylophilus detection institutions.
The pine wood nematode (PWN), Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle., is one of the most dangerous invasive species in the world, causing devastating pine wilt disease (PWD) in pine trees from many countries. The PWN is now established in 18 provinces in China from the south to north, and it has expanded to some areas of Liaoning Province with temperatures that are beyond the ideal range. It has been reported that Pinus koraiensis Siebold & Zucc., one of the representative pine trees of Liaoning Province, has been infected by PWNs. To investigate the pathogenicity of the PWN in P. koraiensis, the reproductive ability of PWNs on fungal culture was compared among three isolates: QH-1, NM-1, and CM-1 (QH-1 from Liaoning Province, NM-1 from Nanjing Province, and CM-1 from Chongqing Municipality). Four-year-old P. koraiensis seedlings were inoculated with QH-1, NM-1, and CM-1 at a rate of 2000 per seedling. Pathogenicity, external symptoms, and nematode migration were all monitored on a daily basis over the next few days. The results from the experiment showed that all three PWN isolates caused wilt in P. koraiensis seedlings, with QH-1 being more virulent than NM-1 and CM-1. In addition, QH-1 exhibited greater reproductive and migration abilities in the seedlings than NM-1 and CM-1. These results indicate that the virulence of the B. xylophilus isolates QH-1, NM-1, and CM-1 can differ in terms of seedling mortality, migration ability, and reproductive ability (in trees).
Pine wilt disease (PWD) has caused extensive mortality in pine forests worldwide. The longicorn beetle Monochamus saltuarius, as the vector of the invasive species Bursaphelenchus xylophilus, plays an important role in the infection cycle. Although the gut microbiota and its contribution to health and disease have been extensively documented, it is unclear whether B. xylophilus affects the longicorn gut microbiota because of a lack of understanding of potential temporal changes in the microbial composition of the vector beetles. In this study, we collected beetles at the emergence and mating stages, and divided them into two groups according to whether they carried nematodes. Based on 16S rDNA sequence analysis, 174 bacterial species were identified that belonged to 112 genera, 53 families, and 8 phyla. Bursaphelenchus xylophilus increased the microflora abundance and diversity of the infected M. saltuarius. In addition, Firmicutes and Bacteroidetes were more abundant in infected M. saltuarius at the same developmental stage. Some of the bacteria in these two phyla were the key species in the co-occurrence network of intestinal flora and represented a unique module in the co-occurrence network of infected M. saltuarius. We found some high abundance colonies in the intestinal tract of infected M. saltuarius during the emergence period that were mostly related to metabolism. Compared with the emergence period, there were more similar microorganisms in the intestinal tract of M. saltuarius during the mating period. With the change in growth environment and continuous feeding, the intestinal microorganisms gradually stabilized and became single species.
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