First report of the root-knot nematode Meloidogyne enterolobii on sweet potato in Georgia, United States.

Five species of root-knot nematodes (RKN), Meloidogyne spp. (M. haplanaria, M. incognita, M. floridensis, M. javanica, and M. arenaria) were detected in 67% of vegetable-growing regions in Georgia, United States (Marquez et al. 2021a, b; Marquez and Hajihassani 2022a, b). In October 2021, two sweet potato (Ipomoea batatas) samples (FF1 and FF2) collected at harvest from a field located in Tattnall County, Reidsville, GA were received for assessment. Symptoms of RKN damage on storage roots included a veiny appearance, surface cracking, and bumpy yellow to brown colored specks (Fig. 1). The population density of RKN second-stage juveniles (J2) was 148 and 180 J2/100 cm3 of soil for FF1 and FF2 samples, respectively. Genomic DNA was isolated by smashing females (n=10) individually in 20 µL of PCR-grade water, followed by freezing at -20°C overnight and thawing at 95°C for 1 min. Each DNA sample was first analyzed with a duplex PCR method using RKN species-specific primer sets for the most common nematode species: Mi2F4/Mi1R1 (M. incognita) and Far/Rar (M. arenaria) and SEC-1F/SEC-1R (M. incognita) and Fjav/Rjav (M. javanica) (Zijlstra et al. 2000; Hajihassani et al. 2022). Since this method failed to identify the RKN species, DNA samples were amplified with Me-F and Me-R primers, specific for diagnosing M. enterolobii (Long et al. 2006). Species-specific PCR produced a 240 bp DNA fragment for FF1 and FF2 samples (Fig. 2), corresponding to M. enterolobii. RKN species identification was confirmed by DNA sequencing using C2F3/1108 and TRNAH/MRH106 primers (Powers et al. 2018; Stanton et al. 1997). Products of C2F3/1108 (GenBank accessions ON320401 and ON320405) were 100% identical with 100% query coverage to a North Carolina M. enterolobii isolate (MN809527), while TRNAH/MHR106 sequences (ON320402 and ON320406) was 99.4-99.8% identical with 89-91% query coverage to a China isolate (MF467278). Measurements [Mean (range)] of body length of M. enterolobii J2 (BL): 454.0 (411.3-485.1) µm; maximum body width (BW): 15.1 (13.8-17.0) µm; stylet length: 14.3 (12.7-15.2) µm; total BL/greatest BW: 30.1 (28.6-31.4) µm; and BL/head end to posterior end of metacorpus: 7.3 (6.5-8.2) µm. Morphological measurements of J2 were comparable to the original description of M. enterolobii Yang and Eisenback. The host suitability of sweet potato varieties [Covington (susceptible to M. enterolobii), Beauregard (susceptible to intermediate), and Regal (resistant)] to the M. enterolobii isolate was assessed (Rutter et al. 2021). Nematode eggs were extracted from skin/bumps of samples FF1 and FF2 by blending (15 sec) and shaking in a 0.5% NaOCl solution (5 min), followed by washing and centrifugation in a standard sucrose solution. Sweet potato slips were transplanted in 10.8-cm-diam. pots filled with sand and steamed field soil (1:1 v/v), and two days after planting, were inoculated with 10,000 eggs of M. enterolobii (six replicates per plant variety). Plants were maintained in a greenhouse at 25-28°C for 85 days in a completely randomized design. Root galling index (scale of 0 to 5) of 4.4, 4.2, and 0.8 (Fig. 3) and reproduction factor (final egg numbers/initial egg number) of 8.2, 7.5, and 0.01 were obtained for Covington, Beauregard, and Regal, respectively confirming that Covington and Beauregard are susceptible to M. enterolobii while Regal is resistant. Meloidogyne enterolobii has not been reported in GA and this is the first report of the nematode on sweet potato in the state. This RKN species is an emerging pest of economic importance in many crops in the Southern United States (Brito et al. 2004; Rutter et al. 2018; Ye et al. 2013). Development of effective short- and long-term control procedures is urgently needed for managing M. enterolobii.