First Report of a Root Lesion Nematode (Pratylenchus brachyurus) on Cassava in Taiwan

Abstract

The root lesion nematode, Pratylenchus spp., has a wide host range affecting many economically important crops (Castillo and Vovlas 2007). Cassava (Manihot esculenta Crantz) is an important food crop in several countries, commonly used for the material of bioethanol, animal feed, and starch extraction (Howeler 2014). Soil samples were collected from a mono-cropping cassava farm located in the Houbi district, Tainan city, in Southern Taiwan in October 2021 in a routine soil survey, and there is no obvious above ground symptoms. The cassava is a local cultivar, the sampled tuber did not have lesions, but small brown lesions were present on the roots. Nematodes were extracted using the modified Baermann funnel method (Tsay et al. 2004) for 24 h. Root lesion nematodes were the dominant genera in this sample, containing over 20 individuals per 100 cm3 of soil. Females of the root lesion nematodes were picked, and surface-sterilized with 2000 ppm malachite green for 30 sec and streptomycin for 30 mins. After sterilization, a single female was transferred onto the carrot discs to establish a pure line (Coyne et al. 2014). After 2 months, nematodes were extracted from that pure culture for morphometric, molecular identification, and pathogenicity tests. The female has a moderately slender body, a low and flat lip region, a sclerotized head frame, and a short ventral overlap of the esophagus, monovarial, prodelphic, and post-uterine sac. The tail is conical and the tip is rounded or flattened. The body measures of 20 females were: body length 564.43 μm (511 to 619 μm), stylet length 18.64 μm (18.1 to 19.5 μm), tail length 32.43 μm (27.1 to 38.5 μm), post uterine sac length 12.79 μm (9.41 to 16.9 μm), and V value 85.16% (84.1 to 86.6%). Values of a, b, c, and c’ ratios were 22.32 (18.9 to 26.1), 6.11 (5.36 to 6.73), 17.55 (13.8 to 20.5), and 1.18 (0.92 to 1.5), respectively. All morphometric data were similar to the previous description of P. brachyurus (Castillo and Volvlas 2007). DNA was extracted from three nematodes of the pure cultures using Viagen DirectPCR lysis buffer, and used for PCR amplification of the 18S rRNA fragment and the D2-D3 expansion segment of 28S rRNA using primer sets D2A/D3B and 988F/2646R, respectively (Holterman et al. 2006; Subbotin et al. 2006). The sequence of 18S ribosomal RNA (OP020594) shared 99% similarity with the P. brachyurus sequence deposited in the GenBank database (KY424148), and the sequence of 28S rRNA (OP020593) also shared 99% similarity with several P. brachyurus sequences (e.g. KF712473, MG745329). Bayesian consensus trees, constructed from both 18S and 28S sequences revealed that the nematodes collected in this study are clustered together with P. brachyurus sequences from other countries(Subbotin, et al. 2008). Therefore, the nematodes collected from cassava were identified as P. brachyurus based on morphology, molecular data, and phylogenetic relationship. To determine the pathogenicity, three eight-week-old cassava plants (cv. TMS 60444) were planted in 12-cm-diameter pots filled with 600 cm3 of sterile peat moss: sand (1:1, W: W) and inoculated with 50 nematodes containing different stages. Two plants treated with water were used as the mock control. Seventy-five days after inoculation, nematodes in the soil were recovered using the modified Baermann funnel method for 24 h, and the nematodes inside the root were stained with acid fuchsin. The average reproduction factor (final population/initial population) was 3.93, thus confirming cassava as a host of P. brachyurus. P. brachyurus was previously reported on peanuts and bananas in Taiwan, and wasn’t a dominant species in the field. Finding this nematode on this cassava farm suggests this nematode might have a wider distribution than expected.