Nematodes are an economically important group of plant pathogens capable of causing billions of dollars in yield losses annually, with many species reported as being parasites of corn. One of the first steps to impact and damage assessment is species identification, because the level of damage caused may vary among genera and species within genera, among other factors. The first step in this research was to identify plant-parasitic nematodes (PPNs) found in corn fields in Ohio to species with morphological and molecular approaches. Morphometrics, morphological characters, and De Man ratios were used along with diagnostic keys for initial species identification. Polymerase chain reaction in combination with Sanger sequencing was used for molecular species identification . Ten PPN species were characterized, including Pratylenchus scribneri, P. thornei, P. crenatus, Helicotylenchus pseudorobustus, Paratrichodorus allius, Paratylenchus neoamblycephalus, Hoplolaimus magnistylus, Tylenchorhynchus claytoni, T. annulatus, and Xiphinema rivesi.
Because some Pratylenchus species are known pathogens of corn, which may be related to their migratory endoparasitic life history, I examined the vertical and temporal variations of population densities in three commercial fields in Wayne and Fulton counties, Ohio during the 2016 and 2017 growing seasons. Soil samples were collected at corn growth stages VE-V1, V3-V4, V6-V7, V9-V10 or soybean growth stages VE-V1, V3-V4, V6-V8, V10-RI from 25 plots in each experiment. From each plot, 15 soil cores were removed to a depth of 70 cm and divided in seven 10-cm sections. Nematodes were extracted from the soil fraction and the root fraction of each section. Pratylenchus crenatus and P. thornei were the predominant species of lesion nematodes found in the fields sampled in Wayne and Fulton counties, respectively. Pratylenchus crenatus population densities generally increased from the 0-10 to the 20-30 cm sampling depths, and then decreased with increasing depth, whereas P. thornei population densities generally increased from the 0-10 to 30-40 cm depth, and then decreased in 2016, and from 0-10 to 10-20 cm and then decreased in 2017. Both species were detected at consistently higher population densities across depths at growth stages V3-V4 in the soil fraction compared to other growth stages tested for corn and soybean. In contrast, in the root fraction, higher counts were observed between growth stages V6 and V10. Soil depth was the most important factor affecting population density. Mean population densities of both species were higher at 15, 25, and 35 cm than at the reference 45 cm depth (the maximum depth at which samples are usually collected for nematode in corn), and lower at 55 and 65 cm. In general, the highest peaks in estimated predicted population densities were recorded at later growth stages. The results suggest that sampling between V6 and V10 to a depth of 40 cm should be recommended for reliable estimation of peak population densities of P. crenatus and P. thornei.
In a parallel study, variations in the population density of the migratory ectoparasitic spiral, pin and stunt nematodes and diversity of PPNs in general with growth stage and sampling depth were investigated in the same four experiments in commercial fields in Wayne and Fulton counties, Ohio, during the 2016 and 2017 growing seasons. Samples were collected and processed as described above for the lesion nematode. Ten different morphological groups of PPNs were identified, but the number of groups and population density of each group varied among experiments, depths, and growth stages. Simpson diversity (D) and Pielou evenness (J’) indices were estimated as measures of diversity. The main effect of sampling depth on D and J’ was statistically significant (P < 0.05) in most cases. D and J’ tended to be highest and comparable among depths in the middle of the sampling range (25 to 45 cm). Helicotylenchus pseudorobustus, P. neoamblycephalus, and T. claytoni were species of spiral, pin and stunt nematodes recovered. The main effect of sampling depth on population density of these nematodes was statistically significant in all experiments, whereas the effects of growth stage and its interaction with depth were not in most cases. Spiral and pin nematodes were found predominantly in the upper 20 cm of the soil at all growth stages, with population density decreasing exponentially with sampling depth. At 3 to 11 cm and 8 to 14 cm, population densities of spiral and pin nematodes were reduced by 50%, respectively. In contrast, stunt nematodes (Tylenchorhynchus spp.) were found predominantly in the upper 40 cm of the soil, and in one of the two experiments in which this nematode was common , population density also decreased exponentially, but at a much slower rate than observed for the spiral or pin nematodes. At the 16 to 30 cm sampling depth, stunt nematode population density was reduced by 50%. Findings from this and the previous study will be useful for developing nematode sampling protocols.
Fourthly, two studies were conducted in 2016, 2017, and 2018 to compare two nematode extraction methods (Study 1) and determine the influence of sample storage time on the recovery of PPNs (Study 2). In Study 1, the two extraction methods compared were (1) the manual decanting, sieving, centrifugal flotation, and Baermann funnel technique (METHOD 1, considered the standard) and (2) the semi-automatic elutriator , centrifugal flotation, and Baermann funnel technique (METHOD 2). In Study 2, the presence and population densities of morphological groups of nematodes were estimated at monthly intervals after sampling and storage at 4oC. Overall, ten different morphological groups were recovered, with lesion, spiral, stunt, dagger, pin, cyst, and tylenchid being more frequent than ring, stubby-root, and lance. In both studies, presence-absence of each morphological group was recorded in each sample as a binary response, and Simpson index (D) and Pielou evenness indices (J’) were estimated as measures of nematode community structure. The Cohen’s kappa coefficients (K) were estimated as measures of agreement between the two extraction methods in terms of presence/absence of key morphological groups. Concordance correlation coefficients (CCC) based on variance components from the fit of linear (for D and J’) and generalized linear (for density, over dispersed count data) mixed models were estimated as measures of agreement between the two methods in terms of population diversity and density. For both K and CCC, a value of 1 signifies perfect agreement, whereas a value of 0 signifies no agreement. K values were positive and significantly different from zero (P < 0.05) in most cases, indicating agreement between the extraction methods. Agreement was substantial (0.61 to 0.80) for the stunt nematode, fair (0.21 to 0.71) to substantial for the spiral nematode, and moderate (0.41 to 0.60) for the pin and cyst nematode. Agreement between the methods in terms of population density (CCC) was fair to high for lesion (0.36 to 0.69) and spiral (0.38 to 0.75) nematodes, fair to moderate for the stunt nematode (0.29 to 0.52), and very low for the dagger nematode (0.09 to 0.13). In all cases, less-than-perfect agreement was due to the nematode presence and population density being underestimated with METHOD 2. Agreement between the methods was moderate for D and J’, with CCC between 0.46 and 0.54. For Study 2, results from the fit of negative binomial regression models to quantify relationships between sample processing time and nematode population density on the link scale showed that slopes were not significantly different from zero (P > 0.05) in most cases. This indicated that mean population density did not change over the ten months of storage for most of the nematodes investigated. However, both D and J’ decreased over the ten-month period in storage, with slopes that were significantly different from zero (P < 0.05) in 2016 and 2017, but not in 2018. Findings from this study suggest that adjustments will need to be made to METHIOD 2 to improve extraction of certain types of nematodes. An alternative would be to use results from this study to devise correction factors to adjust estimates derived from METHOD 2.
Finally, the impact and management of plant-parasitic nematodes (PPNs) were investigated in 22 on-farm trials conducted over a three-year period in 16 commercial corn fields in Fulton and Wayne counties, Ohio. In this study, associations between grain yield and naturally occurring field population density and diversity of PPNs were quantified (Study 1), and the effects of the nematicide seed treatments Avicta, Poncho/Votivo, and Nemastrike on grain yield, plant height, and stand count, and on the vertical distribution of PPN populations at four growth stages were determined (Study 2). In Study 1, 18 experiment fields were established in 2016, 2017, and 2018. Samples were collected between growth stages V3 and V6 to a depth of 50 cm from 25 plots per field. In Study 2, four commercial fields were used to establish experiments in Fulton, and Wayne counties. Soil cores were collected at four growth stages to a depth of 50 cm and then divided into five 10-cm segments from each of which nematodes were extracted and enumerated. Lesion, spiral, stunt, pin nematodes were the most frequently recovered and abundant morphological groups. Regression slopes from simple linear mixed model regression analyses of relationships between grain yield and lesion, spiral, stunt, and total PPN population density, D and J’ were not significantly different from zero in most cases. For the D x grain yield and J' x grain yield relationships, there were more experiments with negative than with positive slopes than observed for relationships between population densities and grain yield. However, results from meta-analyses showed that overall estimated slopes for the population of studies summarized in this investigation were not significantly different from zero for any of the relationships, except for total PPN. Results from the meta-analyses showed that the overall mean slopes for the D x grain yield and J’ x grain yield relationships were negative but not significantly different from zero (P > 0.05). Lesion, spiral, and stunt nematodes were most frequently found in Fulton 2016 and Fulton 2017 , and lesion, spiral, and pin in Wayne 2016 and Wayne 2017 in Study 2. Trends in terms of temporal and spatial variations of PPN population densities were similar between treatments in Fulton 2016 and Fulton 2017 , with few exceptions. Based on results from linear and generalized linear mixed model analyses, the effects of nematicide seed treatments on stand count, plant height, and grain yield were not statistically significant (P > 0.05). Avicta, Poncho/Votivo, and to a lesser extent, Nemastrike, significantly reduced the population densities of some morphological groups of nematodes and affected D and J’ at certain, but not all, sampling depths and growth stages. However, results were not consistent across experiments. Establishing that the existing PPN population densities may not be impacting grain yield and that the tested nematicide seed treatments may not be warranted in the tested fields in Fulton and Wayne County is valuable information for growers and other stakeholders concerned about PPNs.