A Comparison of Three Automated Root-Knot Nematode Egg Counting Approaches Using Machine Learning, Image Analysis, and a Hybrid Model.

Meloidogyne spp. (root-knot nematodes [RKNs]) are a major threat to a wide range of agricultural crops worldwide. Breeding crops for RKN resistance is an effective management strategy, yet assaying large numbers of breeding lines requires laborious bioassays that are time-consuming and require experienced researchers. In these bioassays, quantifying nematode eggs through manual counting is considered the current standard for quantifying establishing resistance in plant genotypes. Counting RKN eggs is highly laborious, and even experienced researchers are subject to fatigue or misclassification, leading to potential errors in phenotyping. Here, we present three automated egg counting models that rely on machine learning and image analysis to quantify RKN eggs extracted from tobacco and sweet potato plants. The first method relied on convolutional neural networks trained using annotated images to identify eggs (M. enterolobii R2 = 0.899, M. incognita R2 = 0.927, M. javanica R2 = 0.886), whereas a second contour-based approach used image analysis to identify eggs from their morphological characteristics and did not rely on neural networks (M. enterolobii R2 = 0.977, M. incognita R2 = 0.990, M. javanica R2 = 0.924). A third hybrid model combined these approaches and was able to detect and count eggs nearly as well as human raters (M. enterolobii R2 = 0.985, M. incognita R2 = 0.992, M. javanica R2 = 0.983). These automated counting protocols have the potential to provide significant time and resource savings annually for breeders and nematologists and may be broadly applicable to other nematode species.

Reaction of Winter Cover Crops to Meloidogyne enterolobii and Glasshouse Bioassay for Evaluating Utility in Managing M. enterolobii in Soybeans.

Meloidogyne enterolobii is an invasive and highly aggressive root-knot nematode pathogen impacting the Southeastern United States. Winter cover cropping may be a cost-effective method for reducing populations of M. enterolobii in between summer cash crops, yet a gap in the knowledge remains about the response of these cover crops to M. enterolobii and their utility in suppressing nematode populations prior to a cash crop. A "two-step" glasshouse bioassay was performed to evaluate eight winter cover crops popular in North Carolina for their direct response to M. enterolobii infection, and to quantify their effect in reducing nematode populations for the following soybean plants. Data on cover crop root galling, soybean root galling, soybean shoot fresh weight, soybean root fresh weight, eggs per gram of soybean root, and a modified reproductive factor were collected. Cereal cover crops did not display root galling, and there was significantly less root galling in those soybean plants following cereal winter cover crops when compared to those following broadleaf winter cover crops. Broadleaf winter cover crops resulted in significantly higher eggs per gram of soybean root and modified reproductive factor in the soybean plants, compared to cereal cover crops and non-inoculated controls. Results from this study suggest that cereal winter cover crops may be poor-hosts to M. enterolobii and may significantly reduce M. enterolobii populations before a soybean crop, compared to broadleaf winter cover crops. This study lays the groundwork for management recommendations and future field trials to assess management of M. enterolobii through winter cover cropping.

Development of a Species-Specific PCR for Detection and Quantification of Meloidogyne hapla in Soil Using the 16D10 Root-Knot Nematode Effector Gene.

The Northern root-knot nematode (Meloidogyne hapla) is an important soilborne pathogen of numerous agricultural crops in temperate regions. Accurate detection and quantification is vital to supporting informed pest management decisions. However, traditional methods of manual nematode extraction and morphology-based identification are time-consuming and require highly specialized training. Molecular methods may expand the diagnostician's toolkit beyond those methods that rely on this disappearing specialized skillset. However, molecular assays targeting the internal transcribed spacer region may lead to inaccurate results because of intraspecific variability. The Meloidogyne spp. effector gene 16D10 was assessed as a target for a SYBR Green I quantitative PCR (qPCR) assay for detection and quantification of M. hapla. M. hapla-specific qPCR primers were developed and evaluated for specificity against five M. hapla isolates and 14 other plant-parasitic nematodes. A standard curve was generated by relating the quantification cycle (Cq) to the log of M. hapla population densities artificially introduced into soil. The influence of soil inhibitors on quantitative amplification was assessed by generating a dilution series from DNA extracted from pure nematode cultures and inoculated soil. Extracts from soil produced significantly higher Cq values than those produced from pure culture extracts. The utility of the qPCR was evaluated using soil samples collected from three naturally infested potato fields, resulting in a significant positive relationship between populations estimated using qPCR and populations derived from manual counting. The qPCR developed in this study provides a useful method for detecting and quantifying M. hapla in soil and demonstrates the utility of effector genes in plant-parasitic nematode diagnostics. The ability to use effector genes as targets for qPCR and other molecular detection and quantification methods may open additional avenues of novel research and support development of improved species-level diagnostics.

Meloidogyne enterolobii, a Major Threat to Tomato Production: Current Status and Future Prospects for Its Management.

The guava root-knot nematode, Meloidogyne enterolobii (Syn. M. mayaguensis), is an emerging pathogen to many crops in the world. This nematode can cause chlorosis, stunting, and reduce yields associated with the induction of many root galls on host plants. Recently, this pathogen has been considered as a global threat for tomato (Solanum lycopersicum L.) production due to the lack of known resistance in commercially accepted varieties and the aggressiveness of M. enterolobii. Both conventional morphological and molecular approaches have been used to identify M. enterolobii, an important first step in an integrated management. To combat root-knot nematodes, integrated disease management strategies such as crop rotation, field sanitation, biocontrol agents, fumigants, and resistant cultivars have been developed and successfully used in the past. However, the resistance in tomato varieties mediated by known Mi-genes does not control M. enterolobii. Here, we review the current knowledge on geographic distribution, host range, population biology, control measures, and proposed future strategies to improve M. enterolobii control in tomato.