Paula Agudelo

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.

Differential Expression of a β-1,4-Endoglucanase Induced by Diet Change in the Foliar Nematode Aphelenchoides fragariae

We identified and characterized a β-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 β-1,4-endoglucanase and plant-parasitism-related gene described in the genus Aphelenchoides.

First report of a mermithid nematode infecting the invasive Megacopta cribraria (Hemiptera: Plataspidae) in the United States.

Megacopta cribraria (Hemiptera: Plataspidae) has become a pest of soybean, Glycine max (L.), in the United States. While several natural enemies of M. cribraria have been reported, our study is the first to report nematodes beneath the pleural membranes in the abdominal cavities of adults. Morphological and molecular analyses suggest this nematode belongs to the family Mermithidae. This first report of a nematode infection in M. cribraria adds to the current inventory of enemies attacking this insect. Our observations provide a basis for future research to examine the impact of nematodes on M. cribraria mortality and to investigate their capacity to reduce populations.

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.

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.

Validation of a Real-Time Polymerase Chain Reaction Assay for the Identification of Meloidogyne arenaria

Meloidogyne arenaria is an economically important parasite of many crops worldwide. Identification and detection of this species in soil samples is necessary for the design of crop rotation systems, selection of resistant cultivars, and potential use of biological control options. The objective of this study was to develop and validate a real-time polymerase chain reaction (PCR) assay, using species-specific primers and SYBR Green I Dye, for identification of M. arenaria. The specificity of the assay was confirmed by testing for amplification of DNA from other Meloidogyne spp. and from M. arenaria populations of different geographic origins. Field soil samples containing a mixture of M. arenaria and M. incognita were used to compare identification by the real-time PCR assay with identification by esterase phenotype analysis of mature females and by morphometrics of juveniles. The real-time PCR assay provided an accurate and sensitive means for the identification of single juveniles from soil samples.

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.

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.

High genetic diversity and geographic subdivision of three lance nematode species (Hoplolaimus spp.) in the United States.

Lance nematodes (Hoplolaimus spp.) feed on the roots of a wide range of plants, some of which are agronomic crops. Morphometric values of amphimictic lance nematode species overlap considerably, and useful morphological characters for their discrimination require high magnification and significant diagnostic time. Given their morphological similarity, these Hoplolaimus species provide an interesting model to investigate hidden diversity in crop agroecosystems. In this scenario, H. galeatus may have been over-reported and the related species that are morphologically similar could be more widespread in the United States that has been recognized thus far. The main objectives of this study were to delimit Hoplolaimus galeatus and morphologically similar species using morphology, phylogeny, and a barcoding approach, and to estimate the genetic diversity and population structure of the species found. Molecular analyses were performed using sequences of the cytochrome c oxidase subunit 1 (Cox1) and the internal transcribed spacer (ITS1) on 23 populations. Four morphospecies were identified: H. galeatus, H. magnistylus, H. concaudajuvencus, and H. stephanus, along with a currently undescribed species. Pronounced genetic structure correlated with geographic origin was found for all species, except for H. galeatus. Hoplolaimus galeatus also exhibited low genetic diversity and the shortest genetic distances among populations. In contrast, H. stephanus, the species with the fewest reports from agricultural soils, was the most common and diverse species found. Results of this project may lead to better delimitation of lance nematode species in the United States by contributing to the understanding the diversity within this group.

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.