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Author: search.filters.author.Akimitsu, Kazuya

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    Species-specific detection of environmental DNA by endpoint PCR using mismatch base introducing primer design and nested PCR
    (2025-04-01)
    Ishii, Ryosuke
    ;
    Poti, Teeranai
    ;
    Akimitsu, Kazuya
    ;
    Narumi-Kawasaki, Takako
    ;
    Yamada, Yoshihiro
    Environmental DNA (eDNA) has been used effectively for species identification in aquatic macrobiological surveys. To popularize this method, a cost-effective analysis method with high sensitivity is required. In this study, we developed an eDNA detection method that is as sensitive as conventional quantitative real-time PCR (qPCR), which does not require expensive equipment, and with heightened sensitivity that was achieved through mismatch-introducing primer design and nested PCR. The subject of the experiment was Micropterus nigricans (largemouth bass). Standard PCR with mismatch-free primers detected amplicons not only in M. nigricans but also in M. dolomieu and Lateolabrax japonicus. However, the mismatched primers only amplified DNA from M. nigricans and did not show amplicons for the non-target species. Endpoint PCR was less sensitive than qPCR, but it was able to specifically detect the target species in environmental samples. A similar approach was applied to the bitterling fish species, a species to which many other closely related species are known, and Acheilognathus cyanostigma eDNA was specifically detected. This eDNA analysis method is cost-effective because it is comparable in sensitivity to conventional qPCR methods and does not require expensive equipment.
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    Isolates of Colletotrichum truncatum with Resistance to Multiple Fungicides from Soybean in Northern Thailand
    (2023-09-01)
    Poti, Teeranai
    ;
    Thitla, Tanapol
    ;
    Imaiam, Naphatsawan
    ;
    Arunothayanan, Hatthaya
    ;
    Doungsa-Ard, Chanintorn
    In Thailand, four systemic fungicides-carbendazim (Car), azoxystrobin (Azo), difenoconazole (Dif), and penthiopyrad (Pen)-are commonly used to control soybean anthracnose caused by Colletotrichum truncatum; however, the pathogen has developed resistance. From 2019 to 2020, fungicide resistance in C. truncatum from fields in Chiang Rai and Chiang Mai was monitored. In tests of 85 C. truncatum isolates for resistance to multiple fungicides, 15.3% were CarRAzoR, 34.1% were triple resistant (CarRAzoRDifR or CarRAzoRPenR), and 50.6% were CarRAzoRDifRPenR. Surprisingly, all isolates tested had lost their sensitivity to one or more of the fungicides tested. The carbendazim-resistant isolates carried a point mutation in the b-tubulin gene at codon 198 (E198A) or 200 (F200Y), and all azoxystrobin-resistant isolates had a mutation in the cytochrome b gene at codon 143 (G143A) or 129 (F129L). Moreover, a novel mutation at codon 208 (S208Y) in the gene encoding succinate dehydrogenase subunit B was detected in all of the isolates highly resistant to penthiopyrad. No mutation linked with difenoconazole resistance was detected in the genes encoding cytochrome P450 sterol 14a-demethylase. To the best of our knowledge, this is the first report of C. truncatum isolates resistant to multiple fungicides and serves as a warning to take measures to prevent the occurrence and distribution of these multiple-fungicide-resistant populations in soybean fields.