First Report of Foliar Nematode (Aphelenchoides pseudobesseyi) on Soybean in Colombia

Soybean (Glycine max L.) is produced in over 70,000 ha in the Altillanura Region, eastern Colombia (Agronet 2023). From 2018 to 2020, foliar symptoms like green stem and foliar retention of soybean, which in Brazil can cause up to 100% soybean yield losses (Meyer et al. 2017), were observed in soybean fields in Colombia. During 2020, samples from symptomatic plants in reproductive stages (R1-R8) were collected from different commercial soybean fields in the Altillanura Region. Over 200 samples were processed, using an incubation method described in Coyne et al. (2014). Nematodes were recovered from photosynthetic leaf tissues and enlarged nodes/buds with population densities ranging from 13 to 132 and 36 to 936 nematodes/10g, respectively. Adult females were morphologically and molecularly characterized as Aphelenchoides pseudobesseyi (Oliveira et al. 2019; Subbotin et al. 2020). Female body length (n = 20) ranged from 653.3 to 806.3 μm (mean = 723 μm ± 52.7), stylet length from 11.0 to 12.3 μm (11.8 μm ± 0.3), body diameter from 14.8 to 17.9 μm (16.3 μm ± 1.1), post-uterine sac length from 38.7 to 51.9 μm (44.6 μm ± 5.1), vulva to anus from 145.5 to 223.2 μm (172.2 μm ± 22.4), and 26% of the vulva-anus distance. Genomic DNA was extracted (QIAGEN DNeasy® Blood & Tissue kit) from a pool of nematodes. The D2A/D3B (Tenente et al. 2004) primers were used to amplify and sequence the D2/D3 expansion region of the 28S rRNA gene. PCR product (~759 bp) was purified, sequenced, deposited in GenBank (OQ930285), and compared to previously deposited sequences (e.g., KX356756, KY510840, KY510839, KY510841, KT692694, KY510842, MH187565) by means of the BLAST algorithm. Similarly, 988F and 18SR-Burs (De Jesus et al. 2016) primers were used to amplify and sequence the near full-length 18S RNA gene (SSU). PCR product was purified, sequenced, deposited in GenBank (OQ954344), and compared to previously deposited sequences (e.g., KT454962, KT943534, KT943535, KY510835, KY510836, KY510837, KY510838, MH187565). Phylogenetic Bayesian analysis (Ronquist et al. 2012) of the of the D2/D3 and 18S regions placed this nematode from Colombia in the A. pseudobesseyi clade (PP = 100). To fulfill a modified Koch's postulates, the A. pseudobesseyi population described above was used in a greenhouse assay. In total, 120 soybean plants (cv. Flor Blanca) were infected with 200 A. pseudobesseyi (females + males)/plant. Briefly, at cotyledon stage (VC), 50 µl aliquot containing 50 A. pseudobesseyi was delivered onto each cotyledon and unifoliolate leaves (200 nematodes/plant). Sterile water was delivered to 80 plants which served as control. Plants were kept in the greenhouse at approximately 25°C and covered with clear plastic bag for 72 h to maintain over 90% relative humidity. After 15, 30, 45, and 60 days, soybean plants (n = 20) were processed, A. pseudobesseyi quantified, and the average reproduction factor (final population/initial population) was 0.1, 2.9, 14.0, and 1.8, respectively. Infected plants showed symptoms of blistering leaves with malformation (midrib vein twist), and A. pseudobesseyi was not observed in control plants. To our knowledge, this is the first report of A. pseudobesseyi parasitizing soybean buds and leaves in Colombia. Soybean is an important commodity for the Altillanura Region, and it is important to monitor the risk posed by this nematode. Furthermore, a better understanding of the nematode-host interaction and epidemiology in Colombia soybean producing regions is needed.