Molecular characterization of root-knot nematodes (Meloidogyne spp.) from Arkansas, USA

Root-knot nematodes (Meloidogyne spp.) are the most common major pathogens of many crops throughout the world, impacting both the quantity and quality of marketable yields. In this study, a total of 244 root-knot nematode populations from various hosts from 39 counties in Arkansas were tested to determine the species diversity. Molecular characterization was performed on these populations by DNA sequencing of the ribosomal DNA 18S-ITS-5.8S, 28S D2/D3 and a mitochondrial DNA fragment flanking cytochrome oxidase gene subunit II - the intergenic spacer. Five species were identified, including M. incognita (Kofoid & White, 1919) Chitwood, 1949 from soybean, cotton, corn and various vegetables (232 samples); M. hapla Chitwood, 1949 from rose (1 sample); M. haplanaria Eisenback, Bernard, Starr, Lee & Tomaszewski, 2003 from okra, tomato, peanut, Indian hawthorn, ash, willow and elm trees (7 samples); M. marylandi Jepson & Golden in Jepson, 1987 from grasses (3 samples); and M. partityla Kleynhans, 1986 from pecan (1 sample) through a combined analysis of DNA sequencing and PCR by species-specific primers. Meloidogyne incognita is the most abundant species that was identified in 95% samples and was the only species in field crops including soybean and cotton, except for one population of M. haplanaria from soybean in Logan County (TK201). Species-specific primers were used to verify M. incognita through PCR by species-specific primers. Unlike historical data, M. arenaria, M. javanica and M. graminis were not detected from any of the samples collected during this study. This result is essential for effective and sustainable management strategies against root-knot nematodes in Arkansas.

Although attempts were made to perform DNA sequencing on all three genes for each sample, not all PCR or DNA sequencing was successful. However, at least one gene was sequenced from all RKN populations except for one population (TK42). One hundred forty-two samples (   A phylogenetic tree based on the mitochondrial DNA CoxII-IGS sequences is presented in Fig. 12 rooted with M. partityla (MK102796) based on the multiple sequence alignment whose sequence is most distinct from the other sequences. No outgroup species was included in the analysis because of the large sequence divergency. This tree placed Arkansas RKN in five distinct groups. Brinkley, Arkansas. This sample was found below an oak tree in a mixture of grasses and dicot weeds. Several attempts to find females failed and no DNA study was ever performed. There were some RKN samples forwarded to the second author by the Arkansas Nematode Assay Service and by the Arkansas Plant Health Clinic that contained soil with little or no roots, thus only the second-stage juveniles were available. These second-stage juveniles were reared in a greenhouse using tomato and bermudagrass as possible hosts. While some success in producing www.nature.com/scientificreports www.nature.com/scientificreports/ a population of RKN resulted, most testing resulted in failure. This failure was disappointing in that two samples identified with the second-stage juveniles appeared to be M. arenaria 17 . Another failure was not establishing a RKN population when finding males along with the second-stage juveniles in grass samples in experimental plots from the main University Experiment Station in Fayetteville.
Meloidogyne incognita (Southern RKN) is the most abundant species and was identified in 95% samples. It was the only species found in field crops including soybean and cotton, except for one population of M. haplanaria from soybean in Logan County (TK201). This species has worldwide distribution and numerous hosts and is the most damaging species throughout the tropics and warmer regions of the world. Meloidogyne incognita is predominantly found in warmer climates, at latitudes between 35°S and 35°N 26 . This study revealed M. incognita is the most common and widespread species in field crops in Arkansas.
Meloidogyne hapla (Northern RKN) is widely distributed, particularly in temperate regions and the cooler, higher altitude areas of the tropics. Taylor & Buhrer 27 reported that in the USA, M. hapla was most common north of 39°N. It is polyphagous and affects over 550 crops and weeds 28 including many agricultural and horticultural plants (vegetables, fruits, ornamentals), but few grasses or cereals 28 . From the current and previous study 17 , this species was found from knockout rose, oak, elm and poke weed (Phytolacca americana) from three northern counties including Craighead, Logan, and Washington (Fig. 5), but not from any field crops.
Meloidogyne haplanaria (Texas peanut RKN) was originally found attacking peanut in Texas 29 and was also reported from Arkansas 17 and Mi-resistant tomato in Florida 30 . Host range studies revealed that it can parasitize several legumes and crucifer crops 29 and infect M. arenaria-susceptible cultivars of peanut, garden pea and radish 31 . Although watermelon, cotton, corn, tobacco and wheat are nonhosts for M. haplanaria, peper, eggplant, soybean and common bean are moderate hosts for this nematode 29,31 . In our study, this species was found on ash, tomato, peanut, willow, elm, Indian hawthorn and soybean from six counties including Baxter, Faulkner, Logan, Saline, Van Buren and Washington (Fig. 6). It's worthy to note that only one soybean field (TK201) had M. haplanaria.  40 . From current and previous study 17 , this species was found from grasses from six counties including Craighead, Drew, Hempstead, Logan, Perry and Washington (Fig. 7).  10 and in 1982 by Robbins 6 was believed to be M. marylandi which was described much later in 1987 26 . Before M. marylandi was described in 1987, no DNA analysis was available and species found from grass in Arkansas was assigned as M. graminis. Thus, no M. graminis is really confirmed in Arkansas.
Meloidogyne partityla (pecan RKN) is a plant pathogenic nematode infecting pecan. It was first described in pecan trees in South Africa by Kleynhans (1986) 44 52 . In addition to pecans, they also infect the California black walnut (Juglans hindsii), English walnut (J. regia), shagbark hickory (Carya ovate), post oak (Quercus stellate), water oak (Quercus nigra) and laurel oak (Q. laurifolia). The health of infested trees continues to decline every year 50 . In this study, only one sample from pecan in Logan County was identified as M. partityla (Fig. 8).
In this study, DNA sequencing and PCR by species-specific primers were employed successfully to characterize and identify RKN from a wide range of plants from 39 counties in Arkansas. The results revealed the presence of five RKN species with M. incognita being the most predominant. Their hosts, distribution, DNA sequences of three genes and phylogenetic relationships were investigated. This study provides basic information for future management of these economically important species in Arkansas.

Methods nematode sample collection. A total of 244 RKN populations from various hosts from 39 counties in
Arkansas were sampled in this study from 2014 to 2018 (Table 1) (Fig. 14). These samples were collected during the growing season. No specific permissions were required in sampling for plant-parasitic nematodes and no endangered or protected species were involved. Two hundred and six RKN samples (TK1-TK206) were initially www.nature.com/scientificreports www.nature.com/scientificreports/ collected from soil samples that were taken by Arkansas Cooperative Extension Service agents as a part of a statewide nematode survey sponsored in part by the Arkansas Soybean Promotion Board. Samples were collected during the period from September 1 -November 1 in 2014-2016 and were from fields that were either in soybean in the year they were sampled, or they were cropped to corn, grain sorghum, or cotton as a rotation crop with soybean. Samples were stored and transported to the Arkansas Nematode Diagnostic Laboratory in Hope, Arkansas in plastic bags inside insulated coolers. Samples were stored no longer than two weeks prior to assay. When RKN was extracted through routine elutriation 62 and sugar flotation 63 of a sub-sample, the remaining soil was placed into a 15-cm-diameter clay pot filled with 50:50 mixture of fine builders' sand and sandy loam topsoil. A single www.nature.com/scientificreports www.nature.com/scientificreports/ tomato seedling (Solanum lycopersicon L var. lycopersicum, cv. 'Rutgers') at the age of three to four week old from gemination was grown in the soil in a greenhouse. Tomato plants were then removed from the soil and the root systems were washed to remove excess soil at harvest. Root galls on tomato were collected after 60-70 days of inoculation and shipped to Nematode Lab at Agronomic Division in North Carolina Department of Agriculture. Thirty-eight other populations were collected by the second author. Galls or dissected females were shipped to NCDA without rearing nematodes on tomato.
DNA extraction. RKN females were dissected in water in a 9-cm petri dish under Zeiss Stemi 2000-C microscope (Gottingen, Germany). A single female was pipetted into 10-µl 1X TE buffer (10 mM Tris-Cl, 1 mM EDTA; pH 9.0) on a glass microscope slide (7.5 cm × 2.5 cm). The nematodes were then macerated with a pipette tip into pieces, collected in 50-µl 1X TE buffer and stored at −20 °C. Three DNA replicates per sample were prepared for www.nature.com/scientificreports www.nature.com/scientificreports/ any samples with females. If only the second-stage juveniles were available, 1-10 juveniles were macerated with a pipette tip into pieces and put in one tube as DNA template in 50-µl 1X TE buffer. DNA amplification, cleaning and sequencing. The primers used for ribosomal and mitochondrial DNA PCR and DNA sequencing are shown in Table 2 as previously described 36 . These primers were synthesized by Integrated DNA Technologies, Inc. (Coralville, Iowa, USA). The 25-µl PCR was performed using 12.5-µl 2X Apex Taq red master mix DNA polymerase (Genesee Scientific Corporation, San Diego, CA, USA), 9.5-µl water, 1-µl each of 10-µM forward and reverse primers, and 1 µl of DNA template according to the manufacturer's protocol in a Veriti ® thermocycler (Life Technologies, Carlsbad, CA, USA). The thermal cycler program for PCR was as follows: denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 45 s, and extension at 72 °C for 1 min. A final extension was performed at 72 °C for 10 min. PCR products were cleaned using ExoSap-IT (Affymetrix, Inc., Santa Clara, CA, USA) according to the manufacturer's protocol. DNA sequencing was performed using PCR primers for direct sequencing by dideoxynucleotide chain termination using an ABI PRISM BigDye terminator cycle sequencing ready reaction kit (Life Technologies, Carlsbad, CA, USA) in an Applied Biosystems 3730 XL DNA Analyzer (Life Technologies) by the Genomic Sciences Laboratory (North Carolina State University, Raleigh, NC, USA). The molecular sequences were compared with other nematode species available at the GenBank sequence database using the BLASTn homology search program. phylogenetic analyses. DNA sequences were edited with ChromasPro1.5 2003-2009 (Technelysium Pty Ltd, Helensvale, Australia) and were aligned by Mega7.0.14 64 using default settings. The model of base substitution in the DNA sequence data was evaluated using MODELTEST version 3.06 65 . The Akaike-supported model 66 , the proportion of invariable sites, and the gamma distribution shape parameters and substitution rates were used in phylogenetic analyses using DNA sequence data. Bayesian analysis was performed to confirm the tree topology for each gene separately using MrBayes 3.1.0 67 , running the chain for 1,000,000 generations and setting the 'burnin' at 2,500. Markov Chain Monte Carlo (MCMC) methods were used within a Bayesian framework to estimate the posterior probabilities (pp) of the phylogenetic trees 68 using the 50% majority-rule. The λ 2 test for homogeneity of base frequencies and phylogenetic trees was performed using PAUP* version 4.0 (Sinauer Associates, Inc. Publishers, Sunderland, MA, USA).