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First report of a Sheathoid Nematode (Hemicriconemoides litchi) on Longan in Taiwan.

Longan (Dimocarpus longan Lour.) is a subtropical fruit, widely grown in China, Taiwan, and Southeast Asia. Longan has a high commercial value, US$242,200 of dried longan fruits are exported to the United States from Taiwan every year (Wang et al. 2010). A soil sample from a longan orchard located in Changhua County, Taiwan (24.0162657, 120.529-1457) was collected in June 2019. Sheathoid nematodes were the dominant species in this sample and over 40 adult individuals per 100 g soil were found. In December 2019, sheathoid nematodes were collected again from the same tree and processed for identification by morphological and molecular characteristics. Nematodes were extracted using the modified Baermann funnel method (Wu et al. 2010) for 24 h. The morphometric data from fifteen females with variant tail types were taken. All individuals had a closely sheath, lip region set off with two annuli, stylet frequently slightly dorsally curved with rounded knobs, no vulval flaps, tail narrowing to a broadly rounded terminus, or tapering to a truncate end, anus situated 3-4 annuli posterior to the vulva. The body length = 552 μm (483 to 616 μm), body width at mid-body = 34.02 μm (27.75 to 40.03 μm), a = 15.93 (14.73 to 18.87), b = 4.65 (4.04 to 5.06), V% = 91.57 (86.74 to 92.56), stylet length = 66.66 μm (63.51 to 69.6 μm), tail length = 28.48 μm (23.56 to 37.45 μm), ring number 114 to 130, Rs = 14 to 19, Roes = 22 to 28, RV = 9 to 13, Ran = 5 to 9 and RVan = 3 to 4. Since the stylet length were less than 70μm, they are more fitted to be Hemicriconemoides litchi (Van den Berg et al. 2015). DNA samples extracted using VIAGEN® DirectPCR lysis buffer from single females (n = 10) were processed to amplify the 28S D2-D3 expansion segment and the ITS region using primers sets D2A and D3B, TW81 and AB28, respectively (Van den Berg et al. 2014). The D2-D3 region of the nematodes collected in this study (MT-539384) shared 99% similarity with several H. litchi sequences deposited in the GenBank database (e.g. KP192481, KF856540), and the ITS region (MT556011) also shared 99% similarity with several H. litchi sequences (e.g. KP192482, GQ354786). Therefore, based on morphological, molecular data and phylogenetic relationship analysis (Nguyen et al. 2020) the nematodes from the Longan orchard were determined to be H. litchi. To measure the reproductive factor (Rf), 60 sheathoid nematodes (57 females and three males) recovered from Baermann extraction were inoculated onto root systems of a longan tree in a 15-cm-d pot filled with sterilized soil. One hundred and sixty eight days after inoculation, three 100 cm3 subsamples of soil from the pot were processed as mentioned previously, and the average number was 48/100 cm3 of soil. The final population was approximately 768 nematodes per 1600 cm3 of soil with the Rf value of 12.8 confirming Longan as a host, although no symptoms were observed. The DNA from three individuals in the three subsamples with variant tail types were used to obtain D2-D3 and ITS region sequences, and confirmed the species as H. litchi. Hemicriconemoides spp. is associated with root malformation and nutrient deficiencies on agricultural fruit trees (Milne et al. 1971; McSorley et al. 1980); however, aboveground evidence of damage may not become immediately obvious and the importance of sheathoid nematodes is easily overlooked (Chen et al. 2011). This is the first report of H. litchi as a parasite of longan tree in Taiwan.