Loop-mediated isothermal amplification (LAMP) detection tools have great potential for diagnosing the causal agents of infectious diseases in clinics and in agriculture. In this work, we developed two alternative LAMP protocols for detecting the pathogenic nematode Bursaphelenchus xylophilus, causal agent of pine wilt disease. We first identified a pectate lyase 3 gene as a biomarker for developing a LAMP Detection Kit, as there was no homologue in non-pathogenic nematodes that live in pine timber or bark and show structural similarities to B. xylophilus. The first LAMP protocol used the Genie II equipment and an isothermal master mix containing Geobacillus sp. M 2.0 large fragment DNA polymerase showed approximately 10 times greater sensitivity with a shorter incubation period compared with that of the second LAMP protocol, which utilized a fluorescence metal indicator, calcein and an engineered Bacillus stearothermophilus DNA polymerase I, large fragment (Bst 2.0 DNA polymerase). However, the LAMP reactions with calcein and Bst 2.0 polymerase were the cost-effective method because the reaction could be performed using a simple isothermal block and relatively inexpensive calcein as a fluorescence indicator visible to the naked eye.
This study describes a new technique to investigate how the pine wood nematode (PWN), Bursaphelenchus xylophilus, kills pine epithelial cells. After inoculating PWN into 20-cm-long Pinus thunbergii stem cuttings and incubating for 1, 3 or 7 days, the cuttings were split into 2.5 cm segments. The segments were tangentially cut so that the epithelia of several cortical resin canals were exposed, and these were stained with Evans Blue for the detection of dead epithelial cells. While almost no dead epithelial cells were found in the cortical resin canals of non-PWN-inoculated control cuttings up to day 7 of the experiment, dead epithelial cells were distributed sparsely in the epithelium of cortical resin canals throughout pine cuttings inoculated with PWN 1, 3 and 7 days after inoculation. The sparse and sporadic distribution of dead pine cells in the epithelium suggested that individual PWN attacked one epithelial cell at a time with its stylet and migrated between attacks.
The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a mycophagous and phytophagous pathogen responsible for the current widespread epidemic of the pine wilt disease, which has become a major threat to pine forests throughout the world. Despite the availability of several preventive trunk-injection agents, no therapeutic trunk-injection agent for eradication of PWN currently exists. In the characterization of basic physiological properties of B. xylophilus YB-1 isolates, we established a high-throughput screening (HTS) method that identifies potential hits within approximately 7 h. Using this HTS method, we screened 206 compounds with known activities, mostly antifungal, for antinematodal activities and identified HWY-4213 (1-n-undecyl-2-[2-fluorphenyl] methyl-3,4-dihydro-6,7-dimethoxy-isoquinolinium chloride), a highly water-soluble protoberberine derivative, as a potent nematicidal and antifungal agent. When tested on 4 year-old pinewood seedlings that were infected with YB-1 isolates, HWY-4213 exhibited a potent therapeutic nematicidal activity. Further tests of screening 39 Caenorhabditis elegans mutants deficient in channel proteins and B. xylophilus sensitivity to Ca2+ channel blockers suggested that HWY-4213 targets the calcium channel proteins. Our study marks a technical breakthrough by developing a novel HTS method that leads to the discovery HWY-4213 as a dual-acting antinematodal and antifungal compound.
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