OverviewToshihiko Eki, Professor of Applied Chemistry and Life Science (and Research Center for Agrotechnology and Biotechnology), Toyohashi University of Technology developed a high-efficiency method to analyze soil nematodes using 18S ribosomal RNA gene areas as DNA barcodes. This method was able to identify characteristics of nematode communities in fields, gardens, and copses. The target will be extended to include all soil-dwelling organisms found in agricultural soils. This will allow for investigations into the soil's environment as well as bio-diversity. This will contribute to advanced agriculture.More DetailsThe International Year of Soils 2015 was established by the UN. There have been numerous efforts to increase awareness about the importance of soil and conservation. The soil ecosystem is made up of many organisms including bacteria, fungi and protists. A representative soil animal is the nematodes. They are only a few millimeters in length and look a lot like worms. They are important in the cycle of soil materials. Although soil nematodes are bacteria-feeders, they can also feed on plants parasitism or fungi. Plant parasitic nematodes are particularly destructive to crops. From an agricultural perspective, it is important to identify and classify nematodes. There are more than 30,000 species of nematodes. Because nematodes are similar, it is hard to identify nematodes apart from experts.They focused their research on DNA barcoding, which allows species to be identified based on the unique sequences of nucleotides within a barcode gene. Next-generation sequencers are capable of decoding large numbers of nucleotide combinations. This was used to analyze nematode communities in different soil environments. Four DNA barcodes were initially set up for the 18S Ribosomal RNA genes that are shared by eukaryotes. The soil nematodes used in analysis were taken from an uncultivated field, a copse and a home-garden that grew zucchini. The PCR was used for amplifying the four gene fragments of the nematodes' DNA and determining the nucleotide sequences. The nematode-derived variant sequences (SVs), which represent independent nematode species, were also identified. After taxonomical classification, analysis and comparison of the SVs, we discovered that plant parasitizing and bacteria feeder nematodes were both abundant in copse soil, while those from the garden were plentiful in the soil. In addition to the bacteria feeders, predatory and omnivorous larvae were also found in the uncultivated fields.The DNA barcoding technique using a next generation sequencer is used widely for analysis of intestinal microbiota. However, analyses of eukaryotes like nematodes remain in the research stage. This research shows its utility in taxonomic profiling soil nematodes.Development BackgroundToshihiko Eki, Research Team Leader, stated that "Through genetics, I have been working for about 20 years with nematodes (mainly C.elegans). This theme was inspired by research we could do related to agriculture as a member of the university's Research Center for Agrotechnology and Biotechnology. We isolated nematodes in the university's soybean fields and unmanaged flowerbeds and analysed the DNA barcodes for each one. The soybean field was home to many bacteria feeders. This was then used as a comparison with the unmanaged flowerbed where both weed-parasitizing and predator nematodes were plentiful. This was the beginning of our research (Morise et., PLoS ONE 2012). The first generation of the one-by-one DNA sequencer was used. We were able to identify the characteristics of the nematode communities in the three ecologically distinct soil environments.Future OutlookThe research team is currently developing the third-generation DNA-barcoding method. This involves purifying DNA from the soil and analysing the organisms in the entire soil, rather than isolating or analyzing specific soil-dwelling organisms. They are currently studying the soil biota from cabbage fields, among other things. They want to analyze the soil biota of cabbage fields, as well as clarify the effects of crop growth on soil-dwelling organisms. This research will allow crops to be grown and managed logically using biological data from agricultural soils. It can also contribute to smart agriculture in Japan such as the Higashi-Mikawa region.###This research was made possible by the Takahashi Industrial and Economic Research Foundation.Refer toHarutaro Kenmotsu and Masahiro Ishikawa. Tomokazu Nitta. Yuu Hirose. Toshihiko Ei (2021). High-throughput amplified sequencing of four 18S gene regions revealed distinct community structures for soil nematodes at three ecologically diverse sites.PLoS ONE 16(4): e0249571.
