First Report of the Reniform Nematode (Rotylenchulus reniformis) from Soybean in Paraguay

Reniform nematodes are sedentary semiendoparasites commonly found in subtropical and tropical regions (Robinson 1997). During the 2014 to 2015 growing season, composite soil samples were collected from soybean fields arbitrarily chosen across eight soybean-producing departments in Paraguay as part of a nationwide survey funded by the Instituto de Biotecnología Agrícola. Nematodes were extracted from 100 cm³ of soil by decanting and sieving followed by sucrose centrifugal flotation. Soil samples from two soybean fields planted to cultivar Coodetec CD 202 in the department of Alto Paraná had reniform nematode densities of 1,400 and 1,800 mixed vermiform life stages per 100 cm³ of soil, respectively. Symptoms were not observed in these fields at sampling (soybean stages R3 to R4). Immature females, males (always present), and juveniles were observed with lateral fields containing four incisures and continuous, conoid labial regions. Immature females were observed with stylets having rounded, posteriorly sloping knobs. Individual immature females were identified as Rotylenchulus reniformis Linford and Oliveira 1940, according to morphological and morphometric characteristics (Robinson 1997). Body length of immature females (n = 20) ranged from 360.9 to 463.9 μm (mean = 417.8 μm), stylet length from 18.2 to 20.4 μm (mean = 19.5 μm), and vulva position from 64.7 to 73.7% (mean = 71.0%). DNA was extracted from 10 immature females. The D2A/D3B (Tenente et al. 2004) and TW81/AB28 (Subbotin et al. 2000) primers were used to amplify and sequence the D2/D3 expansion region of the 28S rRNA gene and the rDNA internal transcribed spacer (ITS) region, respectively. The polymerase chain reaction products were purified and sequenced. The D2/D3 and ITS sequences were deposited in GenBank (accession nos. MG835409 and MG835410, respectively) and compared with previously deposited sequences by means of BLAST. The comparisons revealed a 99% sequence similarity with R. reniformis for the D2/D3 (e.g., KP054126.1, HM131883.1, and GU120091.1) and the ITS (e.g., LC335963.1, KP018562.1, and AY335191.1) sequences. To confirm reproduction on soybean, the reniform nematode population described above was used in a greenhouse assay to fulfill modified Koch’s postulates. Three pots, each with three soybean plants (cv. Williams 82) were infested with 200 R. reniformis (mixed vermiform life stages) per plant. Three additional pots did not receive R. reniformis and served as a control. Pots were kept in the greenhouse at 27°C, and after 8 weeks each pot had reproduction factors (final population/initial population) greater than 1 (12.8 to 20.9). R. reniformis was not observed in control pots, and symptoms were not observed in infested or control plants. Of the 11 reniform nematode species (Van den Berg et al. 2016), R. reniformis is the most economically important with the ability to parasitize more than 300 plant species including soybean (Robinson 1997). Significant soybean yield reduction has been reported in Brazil (neighboring country to Paraguay), where this nematode is considered a serious soybean pathogen (Cardoso et al. 2014). Soybean is an important commodity for Paraguay’s economy, and the risk this nematode poses to soybean production must be monitored. Management recommendations will depend on the nematode distribution and population densities, as well as the availability of soybean resistance to this pathogen. To our knowledge, this is the first report of R. reniformis in Paraguay.