Observations on the Population Genetic Structure of the Leaf Galling Nematode, Ditylenchus gallaeformans.

Ditylenchus gallaeformans is a plant parasitic nematode that induces galls on aboveground parts of Melastomataceae plants. It differs from most gall-inducing nematodes in that it is not an endoparasite and has been considered as a possible biological control agent against invasive species of Miconia. Little is known about D. gallaeformans biology, genetic differences among populations, and host preferences. This study examined the genetic differences among D. gallaeformans populations from different locations and host species and the phylogenetic relationships among them. Nematodes were collected from galls in plants from Costa Rica, Dominica, and Trinidad. The Cytochrome c oxidase 1 (cox1) region was sequenced from a total of 33 individual nematodes isolated from 33 different plant individuals, representing 21 species of Melastomataceae. Phylogenetic reconstructions, haplotype networks, and analysis of molecular variance showed that the species is monophyletic and has three major clades, which were mostly consistent with geographic location but not with host species. The first clade was composed by two subclades, one with individuals from Costa Rica and one with individuals from Dominica. The second and third clades comprised nematodes only from Trinidad. Overall, there is no evidence of host-species specialization in D. gallaeformans. Biocontrol efforts using the nematode against invasive Miconia could focus on geographical location matching but likely will not need to match host species.

Classification Methods and Identification of Reniform Nematode Resistance in Known Soybean Cyst Nematode-Resistant Soybean Genotypes.

Plant parasitic nematodes are a major yield-limiting factor of soybean in the United States and Canada. It has been indicated that soybean cyst nematode (SCN; Heterodera glycines Ichinohe) and reniform nematode (RN; Rotylenchulus reniformis Linford and Oliveira) resistance could be genetically related. For many years, fragmentary data have shown this relationship. This report evaluates RN reproduction on 418 plant introductions (PIs) selected from the U.S. Department of Agriculture Soybean Germplasm Collection with reported SCN resistance. The germplasm was divided into two tests of 214 PIs reported as resistant and 204 PIs reported as moderately resistant to SCN. The defining and reporting of RN resistance changed several times in the last 30 years, causing inconsistencies in RN resistance classification among multiple experiments. Comparison of four RN resistance classification methods was performed: (i) ≤10% as compared with the susceptible check, (ii) using normalized reproduction index (RI) values, and using (iii) transformed data log10(x), and (iv) transformed data log10(x + 1) in an optimal univariate k-means clustering analysis. The method of transformed data log10(x) was selected as the most accurate for classification of RN resistance. Among 418 PIs with reported SCN resistance, the log10(x) method grouped 59 PIs (15%) as resistant and 130 PIs (31%) as moderately resistant to RN. Genotyping of a subset of the most resistant PIs to both nematode species revealed their strong correlation with rhg1-a allele. This research identified genotypes with resistance to two nematode species and potential new sources of RN resistance that could be valuable to breeders in developing resistant cultivars.

First Genomic Resource of the Columbia Lance Nematode Hoplolaimus columbus.

The Columbia lance nematode Hoplolaimus columbus has been reported frequently from North America due to its negative impact on agricultural production. In this study, for the first time, we sequenced the whole genome of a female specimen by using whole-genome amplification and Illumina MiSeq. Data were de novo assembled to form scaffolds of 205.75 Mbp consisting of 118,374 contigs. The largest scaffold was 636,881 bp. Benchmarking Universal Single-Copy Orthologs completeness was 66.6% (eukaryotic dataset), and >8,000 unique genes were predicted by GeneMark-ES. In total, 61,855 protein sequences were predicted by AUGUSTUS, and 10,085 of them were annotated by PANNZER2 with at least one function. These data will provide valuable resources for studies focusing on pathogenicity and phylogenomics of plant-parasitic nematodes.

First report of Meloidogyne javanica pathogenic on hybrid lavender (Lavandula ×intermedia) in the United States.

Root-knot nematodes (RKNs), Meloidogyne spp., are some of the most economically important pathogens of cultivated plants. Meloidogyne javanica is one of the most destructive RKN species and is well known for its broad host range and the severe damage it causes to plant roots (Perry et al. 2009). In Feb 2018, four mature dead and dying hybrid lavender plants (Lavandula ×intermedia 'Phenomenal') were collected in Edgefield County, South Carolina, and suspected of having Phytophthora root and crown rot (Dlugos and Jeffers 2018). Greenhouse-grown plants had been transplanted in Dec 2016 and Jan 2017 into a sandy loam soil on a site that had been fallow or in pasture for over 30 years. Some plants began to turn gray and die in summer 2017, and approximately 40% of 1230 plants were symptomatic or dead by Feb 2018. Phytophthora spp. were not isolated from the collected plants but were isolated from plants collected on subsequent visits. Instead, all four plants had small, smooth galls on the roots. Lavender roots were examined microscopically (30-70×), and egg masses of RKNs were observed on the galls. Mature, sedentary RKN females were handpicked from galled roots, and perineal patterns of 10 specimens were examined and identified as M. javanica. Juveniles and eggs were extracted from lavender roots by the method of Coolen and D'herde (1972). To confirm species identification, DNA was extracted from 10 individual juveniles, and a PCR assay was conducted using species-specific primers for M. javanica, Fjav/Rjav (Zijlstra et al. 2000). A single amplicon was produced with the expected size of approximately 720 bp, which confirmed identity as M. javanica. To determine pathogenicity, M. javanica from lavender roots were inoculated onto susceptible tomato plants for multiplication, and severe gall symptoms occurred on tomato roots 60 days later. Nematodes were extracted from tomato roots and inoculated onto healthy, rooted cuttings of 'Phenomenal' lavender plants growing in pots of soilless medium in a greenhouse. Plants were inoculated with 0, 1000, 2000, 5000, or 10000 eggs and juveniles of M. javanica. Five single-plant replicates were used for each treatment, and plants were randomized on a greenhouse bench. Plants were assessed 60 days after inoculation, and nematodes were extracted from roots and counted. The reproduction factor was 0, 43.8, 40.9, 9.1, 7.7, and 2.6 for initial nematode populations 0, 1000, 2000, 5000, and 10000, respectively, which confirmed pathogenicity (Hussey and Janssen 2002). Meloidogyne javanica also was recovered in Mar 2018 from galled roots on a 'Munstead' (L. angustifolia) lavender plant from Kentucky (provided by the Univ. of Kentucky Plant Disease Diagnostic Laboratories), and an unidentified species of Meloidogyne was isolated in Aug 2020 from a 'Phenomenal' plant grown in Florida. COI mtDNA sequences from the SC (MZ542457) and KY (MZ542458) populations were submitted to Genbank. M. javanica previously was found associated with field-grown lavender (hybrid and L. angustifolia) in Brazil, but pathogenicity was not studied (Pauletti and Echeverrigaray 2002). To our knowledge, this is the first report of M. javanica pathogenic to L. ×intermedia in the USA, and the first time RKNs have been proven to be pathogenic to Lavandula spp. following Koch's Postulates. Further studies are needed to investigate the geographic distribution of M. javanica on lavender and the potential threat this nematode poses to lavender production in the USA.

Identification of Sweet Potato Germplasm Resistant to Pathotypically Distinct Isolates of Meloidogyne enterolobii from the Carolinas.

Meloidogyne enterolobii (syn. mayaguensis) is an emergent species of root-knot nematode that has become a serious threat to sweet potato (Ipomoea batatas) production in the southeastern United States. The most popular sweet potato cultivars grown in this region are highly susceptible to M. enterolobii. As a result, this pest has spread across most of the sweet potato growing counties in the Carolinas, threatening the industry as well as other crops in the region. The development and release of new sweet potato cultivars with resistance to M. enterolobii would help to manage and slow the spread of this pest. To support sweet potato resistance breeding efforts, 93 accessions selected from the U.S. Department of Agriculture germplasm collection and breeding programs in the United States were screened to identify 19 lines with strong resistance to M. enterolobii. The resistance in these accessions was tested against two M. enterolobii isolates that were collected from sweet potato production fields in the Carolinas. These isolates were found to have distinct pathotypes, with galling and nematode reproduction differences observed on cotton as well as sweet potato. This study is the first report of intraspecific pathotypic variation in M. enterolobii, and it identifies sweet potato germplasm with resistance against both pathogenic variants of this nematode.

First report of Seville root-knot nematode, Meloidogyne hispanica (Nematoda: Meloidogynidae) in the USA and North America.

A high number of second stage juveniles of the root-knot nematode were recovered from soil samples collected from a corn field, located in Pickens County, South Carolina, USA in 2019. Extracted nematodes were examined morphologically and molecularly for species identification which indicated that the specimens of root knot juveniles were Meloidogyne hispanica. The morphological examination and morphometric details from second-stage juveniles were consistent with the original description and redescriptions of this species. The ITS rRNA, D2-D3 expansion segments of 28S rRNA, intergenic COII-16S region, nad5 and COI gene sequences were obtained from the South Carolina population of M. hispanica. Phylogenetic analysis of the intergenic COII-16S region of mtDNA gene sequence alignment using statistical parsimony showed that the South Carolina population clustered with Meloidogyne hispanica from Portugal and Australia. To our best knowledge, this finding represents the first report of Meloidogyne hispanica in the USA and North America.

Quantitative Trait Loci Associated with Rotylenchulus reniformis Host Suitability in Soybean.

Reniform nematode (Rotylenchulus reniformis) is a yield-limiting pathogen of soybean (Glycine max) in the southeastern region of the United States. A population of 250 recombinant inbred lines (RIL) (F2:8) developed from a cross between reniform nematode resistant soybean cultivar Forrest and susceptible cultivar Williams 82 was utilized to identify regions associated with host suitability. A genetic linkage map was constructed using single-nucleotide polymorphism markers generated by genotyping-by-sequencing. The phenotype was measured in the RIL population and resistance was characterized using normalized and transformed nematode reproduction indices in an optimal univariate cluster analysis. Quantitative trait loci (QTL) analysis using normalized phenotype scores identified two QTLs on each arm of chromosome 18 (rrn-1 and rrn-2). The same QTL analysis performed with log10(x) transformed phenotype data also identified two QTLs: one on chromosome 18 overlapping the same region in the other analysis (rrn-1), and one on chromosome 11 (rrn-3). While rrn-1 and rrn-3 have been reported associated with reduced reproduction of reniform nematode, this is the first report of the rrn-2 region associated with host suitability to reniform nematode. The resistant parent allele at rrn-2 showed an inverse relationship with the resistance phenotype, correlating with an increase in nematode reproduction or host suitability. Several candidate genes within these regions corresponded with host plant defense systems. Interestingly, a characteristic pathogen resistance gene with a leucine-rich repeat was discovered within rrn-2. These genetic markers can be used by soybean breeders in marker-assisted selection to develop lines with resistance to reniform nematode.

Detoxification-related gene expression accompanies anhydrobiosis in the foliar nematode (Aphelenchoides fragariae).

The foliar nematode (Aphelenchoides fragariae) is a quarantined pest that infects a broad range of herbaceous and woody plants. Previous work has demonstrated its remarkable ability to survive rapid and extreme desiccation, although the specific molecular mechanisms underlying its anhydrobiotic response have not been characterized. The authors used RNA sequencing and de novo transcriptome assembly to compare patterns of gene expression between hydrated and 24-hr desiccated nematodes. In total, 2,083 and 953 genes were significantly up- and downregulated, respectively, in desiccated nematodes. Of the 100 annotated genes with the largest positive fold-changes, more than one third encoded putative detoxification-related proteins. Genes encoding enzymes of Phase I and Phase II detoxification systems were among the most strongly upregulated in the transcriptome, including 35 cytochrome p450s, 23 short chain dehydrogenase/reductases, 5 glutathione-S-transferases, and 22 UDP-glucuronosyltransferases. Genes encoding heat shock proteins, unfolded protein response enzymes, and intrinsically disordered proteins were also upregulated. Anhydrobiosis in A. fragariae appears to involve both strategies to minimize protein misfolding and aggregation, and wholesale induction of the cellular detoxification machinery. These processes may be controlled in part through the activity of forkhead transcription factors similar to Caenorhabditis elegans' daf-16, a number of which were differentially expressed under desiccation.The foliar nematode (Aphelenchoides fragariae) is a quarantined pest that infects a broad range of herbaceous and woody plants. Previous work has demonstrated its remarkable ability to survive rapid and extreme desiccation, although the specific molecular mechanisms underlying its anhydrobiotic response have not been characterized. The authors used RNA sequencing and de novo transcriptome assembly to compare patterns of gene expression between hydrated and 24-hr desiccated nematodes. In total, 2,083 and 953 genes were significantly up- and downregulated, respectively, in desiccated nematodes. Of the 100 annotated genes with the largest positive fold-changes, more than one third encoded putative detoxification-related proteins. Genes encoding enzymes of Phase I and Phase II detoxification systems were among the most strongly upregulated in the transcriptome, including 35 cytochrome p450s, 23 short chain dehydrogenase/reductases, 5 glutathione-S-transferases, and 22 UDP-glucuronosyltransferases. Genes encoding heat shock proteins, unfolded protein response enzymes, and intrinsically disordered proteins were also upregulated. Anhydrobiosis in A. fragariae appears to involve both strategies to minimize protein misfolding and aggregation, and wholesale induction of the cellular detoxification machinery. These processes may be controlled in part through the activity of forkhead transcription factors similar to Caenorhabditis elegans' daf-16, a number of which were differentially expressed under desiccation.

Cucurbit Rootstocks Resistant to Fusarium oxysporum f. sp. niveum Remain Resistant When Coinfected by Meloidogyne incognita in the Field.

Interspecific hybrid squash (Cucurbita maxima × Cucurbita moschata) rootstocks used to graft watermelon (Citrullus lanatus var. lanatus) are resistant to Fusarium oxysporum f. sp. niveum, the fungus that causes Fusarium wilt of watermelon, but they are susceptible to Meloidogyne incognita, the southern root knot nematode. A new citron (Citrullus amarus) rootstock cultivar Carolina Strongback is resistant to F. oxysporum f. sp. niveum and M. incognita. The objective of this study was to determine if an interaction between M. incognita and F. oxysporum f. sp. niveum race 2 occurred on grafted or nongrafted triploid watermelon susceptible to F. oxysporum f. sp. niveum race 2. In 2016 and 2018, plants of nongrafted cultivar Fascination and Fascination grafted onto Carolina Strongback and interspecific hybrid squash cultivar Carnivor were inoculated or not inoculated with M. incognita before transplanting into field plots infested or not infested with F. oxysporum f. sp. niveum race 2. Incidence of Fusarium wilt and area under the disease progress curve did not differ when hosts were inoculated with F. oxysporum f. sp. niveum alone or F. oxysporum f. sp. niveum and M. incognita together. Fusarium wilt was greater on nongrafted watermelon (78% mean incidence) than on both grafted rootstocks and lower on Carnivor (1% incidence) than on Carolina Strongback (12% incidence; P ≤ 0.01). Plants not inoculated with F. oxysporum f. sp. niveum did not wilt. At the end of the season, Carnivor had a greater percentage of the root system galled than the other two hosts, whereas galling did not differ on Fascination and Carolina Strongback. F. oxysporum f. sp. niveum reduced marketable weight of nongrafted Fascination with and without coinoculation with M. incognita. M. incognita reduced marketable weight of Fascination grafted onto Carnivor compared with noninoculated, nongrafted Fascination. In conclusion, cucurbit rootstocks that are susceptible and resistant to M. incognita retain resistance to F. oxysporum f. sp. niveum when they are coinfected with M. incognita.

Multiple Nodulation Genes Are Up-Regulated During Establishment of Reniform Nematode Feeding Sites in Soybean.

The semi-endoparastic reniform nematode (Rotylenchulus reniformis) infects over 300 plant species. Females penetrate host roots and induce formation of complex, multinucleate feeding sites called syncytia. While anatomical changes associated with reniform nematode infection are well documented, little is known about their molecular basis. We grew soybean (Glycine max) in a split-root growth system, inoculated half of each root system with R. reniformis, and quantified gene expression in infected and control root tissue at four dates after inoculation. Over 6,000 genes were differentially expressed between inoculated and control roots on at least one date (false discovery rate [FDR] = 0.01, |log2FC| ≥ 1), and 507 gene sets were significantly enriched or depleted in inoculated roots (FDR = 0.05). Numerous genes up-regulated during syncytium formation had previously been associated with rhizobia nodulation. These included the nodule-initiating transcription factors CYCLOPS, NSP1, NSP2, and NIN, as well as multiple nodulins associated with the plant-derived peribacteroid membrane. Nodulation-related NIP aquaporins and SWEET sugar transporters were induced, as were plant CLAVATA3/ESR-related (CLE) signaling proteins and cell cycle regulators such as CCS52A and E2F. Nodulins and nodule-associated genes may have ancestral functions in normal root development and mycorrhization that have been co-opted by both parasitic nematodes and rhizobial bacteria to promote feeding site and nodule formation.

Molecular Characterization and Identification of Stubby Root Nematode Species From Multiple States in the United States.

Stubby root nematodes (SRN) are important plant parasites infecting many crops and widely distributed in many regions of the United States. SRN transmit Tobacco rattle virus, which causes potato corky ringspot disease, thereby having a significant economic impact on the potato industry. In 2015 to 2017, 184 soil samples and 16 nematode suspensions from North Dakota, Minnesota, Idaho, Oregon, Washington, South Carolina, North Carolina, and Florida were assayed for the presence of SRN. SRN were found in 106 soil samples with population densities of 10 to 320 SRN per 200 g of soil and in eight of the nematode suspensions. Sequencing of ribosomal DNA (rDNA) or species-specific polymerase chain reaction assays revealed the presence of four SRN species, including Paratrichodorus allius, P. minor, P. porosus, and Trichodorus obtusus. Accordingly, their rDNA sequences were characterized by analyzing D2-D3 of 28S rDNA, 18S rDNA, and internal transcribed spacer (ITS) rDNA obtained in this study and retrieved from GenBank. Both intra- and interspecies variations were higher in ITS rDNA than 18S rDNA and D2-D3 of 28S rDNA. Based on phylogenetic analysis, the four SRN species formed a monophyletic group, with P. allius more closely related to P. porosus than P. minor and T. obtusus. Indel variation of ITS2 rDNA was present in P. allius populations from the same geographic regions. This study documented the occurrence of SRN species across multiple states. The intra- and interspecies genetic diversity of rDNA in this study will provide more information for understanding the evolutionary relationships of SRN and will be valuable for future studies of SRN species identification and management.

Distribution of Hoplolaimus Species in Soybean Fields in South Carolina and North Carolina.

Hoplolaimus columbus is an important nematode pest of soybean in South Carolina and North Carolina. Tolerant cultivars are available for the management of this plant-parasitic nematode; however, variation in the response of soybean cultivars to H. columbus populations has been observed. This variation may be due to the presence of different species or high genetic diversity of H. columbus populations. The objective of this study was to identify the Hoplolaimus spp. present in fields representing the main soybean-growing regions in South Carolina and North Carolina and to examine the genetic variability of these populations. In South Carolina, the only species found associated with soybean was H. columbus but, in North Carolina, H. stephanus was the dominant species. The two species were never found together. Genetic variability analyses of a mitochondrial and a nuclear marker showed that only one haplotype was shared by the H. columbus populations. H. stephanus showed higher genetic variability, with private haplotypes per sampling location. Knowledge of the distribution and genetic variability of these two Hoplolaimus spp. is valuable to growers to determine potentially damaging infestations of these plant-parasitic nematodes in soybean fields.

Agamermis (Nematoda: Mermithidae) Infection in South Carolina Agricultural Pests.

Native and invasive stink bugs (Hemiptera: Pentatomidae) and the closely related invasive Megacopta cribraria (Hemiptera: Plataspidae) are agricultural pests in the southeastern United States. Natural enemies, from various phyla, parasitize these pests and contribute to population regulation. We specifically investigated Nematoda infections in pentatomid and plataspid pests in one soybean field in South Carolina in 2015. Nematodes were identified through molecular and morphological methods and assigned to family Mermithidae, genus Agamermis. This study reports mermithid nematode infection in immature M. cribraria for the first time and provides the first mermithid host record for the stink bugs Chinavia hilaris, Euschistus servus, and another Euschistus species, and a grasshopper (Orthoptera: Acrididae) in South Carolina. The same Agamermis species infected all hosts. The broad host range and prevalence suggests that Agamermis may be an important contributor to natural mortality of pentatomid and plataspid pests. Previous mermithid host records for the Pentatomidae and Plataspidae worldwide are summarized. Further work is needed to assess the impact of infection on populations over a broader range of agricultural fields and geographic localities.

Spatial distribution of reniform nematode in cotton as influenced by soil texture and crop rotations.

Reniform nematode (RN) is an important pest in cotton production. Knowledge of the distribution patterns of RN is essential for selecting sampling strategies and for site-specific management. A 3-year study was conducted in two fields in South Carolina with the purpose of characterizing the distribution of RN using a fine-scale sampling scheme in plots representing different soil textures (field 1), and using a large-scale arbitrary sampling scheme (field 2). Horizontal distribution data showed an aggregated pattern of RN densities at planting and after harvest in both fields each year, with patches ranging from 8 to 12 m. However, a significant neighborhood structure was only detected when suitable hosts (cotton and soybean) were planted. Correlations between RN densities and percent sand and silt were detected, showing nematode densities peaked when sand content was around 60% and declined when sand content increased above 60 to 65%. When fewer samples were taken in the field with more uniform sand content, percentage of sand was a less reliable predictor of RN densities. Vertical sampling showed the highest numbers of RN were found at 15 to 30 cm deep after cotton, but were deeper after a nonhost crop. Understanding distribution patterns of RN within a field may improve the effectiveness of management practices.

Differential expression of a ß-1,4-endoglucanase induced by diet change in the foliar nematode Aphelenchoides fragariae.

We identified and characterized a ß-1,4-endoglucanase, Afr-ENG-1, in the foliar nematode Aphelenchoides fragariae that is differentially expressed when the nematode feeds on fungi or plants. When individuals from hosta plants were transferred to a fungus culture, expression of the enzyme decreased 1,812-fold after five generations on the fungus diet. Afr-eng-1 was readily detected in the genome of 75% of nematodes from the plant population but only in 38% of the diet-changed population. The gene cannot be detected in nematodes maintained on fungus for over 100 generations. Diet was also associated with changes in nematode body size and in the severity of symptoms caused on hosta leaves. Plant-diet nematodes caused larger lesions and were longer and thinner than fungus-diet nematodes. Nematodes moved from a plant diet to a fungus diet for five generations had the same body size as the nematodes that had fed on the fungus for 100 generations. Full-length sequences of Afr-eng-1 were obtained and found to encode a glycosyl hydrolase family 5 protein. This is the first ß-1,4-endoglucanase and plant-parasitism-related gene described in the genus Aphelenchoides.

A Protocol for Assessing Resistance to Aphelenchoides fragariae in Hosta Cultivars.

The use of resistant and tolerant cultivars is an important component of an integrated management plan for foliar nematodes on hosta. In order to identify tolerance and resistance in commercial hosta cultivars, reliable and efficient screening methods are required. To optimize the screening protocol, a series of greenhouse experiments was conducted using six hosta cultivars and two types of nematode inoculum. The pathogenicity and reproduction of Aphelenchoides fragariae maintained on fungal cultures versus maintenance on hosta were evaluated with two inoculation methods (with injury and without injury). Both sources of inoculum were pathogenic on all six cultivars tested but the plant inoculum caused two to eight times larger lesions than the fungus inoculum. Both inocula caused larger lesions and resulted in higher reproduction rates on injured leaves than on noninjured leaves. Water soaking was more efficient than traditional Baermann funnel extraction methods. Correlations between foliage symptom severity and nematode reproduction were low or nonexistence. A numerical scale for faster assessment of disease severity was developed, and recommendations for a reliable protocol for assessment of resistance and tolerance are discussed.

Effect of Crop Rotations on Rotylenchulus reniformis Population Structure.

Rotylenchulus reniformis is a highly variable nematode species and an economically important pest in many cotton fields across the south-eastern United States. Rotation with resistant or poor host crops is a method for management of reniform nematode. We studied the effect of six planting schemes covering four 120-day planting cycles on the predominant genotype of R. reniformis. Rotations used were: (i) cotton to corn; (ii) susceptible soybean to corn; (iii) resistant soybean to cotton; (iv) corn to cotton; (v) continuous susceptible soybean; (vi) continuous cotton. After each 120-day cycle, amplified fragment length polymorphisms (AFLPs) produced from four primer pairs were used to determine the effect of crop rotation on the predominant genotype of reniform nematode. A total of 279 polymorphic bands were scored using four primer combinations. Distinct changes in genotype composition were observed following rotations with resistant soybean or corn. Rotations involving soybean (susceptible and resistant) had the greatest effect on population structure. The characterization of field population variability of reniform nematode and of population responses to host plants used in rotations can help extend the durability of resistant varieties and can help identify effective rotation schemes.

Genetic Variability of Rotylenchulus reniformis.

Rotylenchulus reniformis, reniform nematode, is a polyphagous pest commonly found parasitizing cotton in the southeastern United States. We developed and optimized 10 polymorphic microsatellite loci found in reniform nematode and tested them on 160 individual reniform nematodes to determine informative genetic variation of isolates from the southeastern United States, Colombia, Japan, and from the species Rotylenchulus parvus. No significant gametic disequilibrium was detected between any pair of loci, and most loci were not in expected Hardy-Weinberg proportions. A positive FIS coefficient was observed at all 10 loci, suggesting a high level of inbreeding at these loci. Most isolate locations exhibited significant genotypic differentiation and moderate to very high genetic differentiation based on FST analysis. The most consistently differentiated isolates were found reproducing parthenogenetically in Japan. These isolates were also found to represent the most basal locality in this study based on unweighted pair group method with arithmetic mean (UPGMA) clustering analysis and were distinct from other localities based on STRUCTURE V 2.3 analysis. These results support previous reports suggesting that the parthenogenetically reproducing isolates from Japan are another species. Taken together, our results can serve as the foundation for more extensive characterization of population structure and genetic variation among isolates of R. reniformis variants to help discern the impact of alternative processes on genetic connectivity and differentiation in the genetically undercharacterized reniform nematode.

Conventional PCR Detection and Real-Time PCR Quantification of Reniform Nematodes.

Reniform nematode (Rotylenchulus reniformis) is a relatively recent introduction into the continental United States that can cause major yield losses on a variety of important crops including cotton and soybeans. DNA sequences from the internal transcribed spacer (ITS) region of this nematode were used to design primers for conventional and real-time PCR, as well as a TaqMan probe. These primers amplified DNA of reniform nematode isolates from a wide geographic range but did not detect genetically related species or other pathogenic nematodes found in production fields including Meloidogyne incognita and Heterodera glycines. Both SYBR green and TaqMan assays reliably quantified as little as 100 fg of reniform nematode DNA, and could be used to quantify as few as five reniform nematodes. An inexpensive and rapid DNA extraction protocol for high throughput diagnostic assays is described.

Induction of Glutaredoxin Expression in Response to Desiccation Stress in the Foliar Nematode Aphelenchoides fragariae.

Desiccation tolerance plays an important role in the overwinter survival of the foliar nematode Aphelenchoides fragariae. Survival rates of A. fragariae were compared with those of the anhydrobiotic soil-dwelling nematode Aphelenchus avenae after desiccation (90% RH), cold (4°C) and osmotic (500 mM sucrose) stress treatments. A. fragariae formed aggregates during desiccation and showed higher survival rates than A. avenae under desiccation and osmotic stress. Analysis of transcripts with Illumina RNA-seq indicated that glutaredoxin and other antioxidant-related genes were up-regulated under desiccation stress. Quantitative RT-PCR demonstrated 2.8 fold and 1.3 fold up-regulation of a glutaredoxin gene under desiccated and osmotic stress, respectively, suggesting the participation of antioxidant mechanisms in desiccation tolerance of A. fragariae.