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, RKN) 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 sweetpotato 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), while 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.

Occurrence and distribution of Meloidogyne spp. in fields rotated with sweetpotato and host range of a North Carolina population of Meloidogyne enterolobii.

Root-knot nematodes (RKN, Meloidogyne spp.) are some of the most economically important and common plant parasitic nematodes in North Carolina (NC) cropping systems. Soil samples collected from fields planted with crops rotated with sweetpotato [Ipomoea batatas (L.) Lam.] in 39 NC counties in 2015-2018 were processed at the NC Nematode Assay Laboratory. The occurrence of second-stage juvenile (J2) RKN populations was examined based on collection year, month, county, and previous planted crop. The highest number of RKN positive samples originated from Cumberland (53%), Sampson (48%), and Johnston (48%) counties. The highest average RKN population density was detected in Sampson (147 J2/500 cm3 soil) and Nash (135 J2/500 cm3 soil) counties, while Wayne (7 J2/500 cm3 soil) and Greene (11 J2/500 cm3 soil) counties had the lowest average RKN population density. Meloidogyne enterolobii is a new invasive species that is impacting sweetpotato growers of NC. The host status of a NC population of M. enterolobii, the guava-root knot nematode, was determined by examining eggs per gram of fresh root (ER) and the final nematode egg population divided by the initial population egg count (reproductive factor, RF) in greenhouse experiments. This included eighteen vegetable, field, cover crops and weed species. The tomato 'Rutgers' was used as a susceptible control. Cabbage 'Stonehead', pepper 'Red bull', and watermelon 'Charleston gray' and 'Fascination' were hosts and had similar mean ER values to the positive control, ranging from 64 to 18,717. Among field crops, cotton, soybean 'P5018RX', and tobacco were hosts with ER values that ranged from 185 to 706. Members of the Poaceae family such as sweet corn (Zea mays) and sudangrass (Sorghum x drummondii) were non-hosts to M. enterolobii and the mean ER values ranged from 1.85 to 7. The peanut 'Tifguard' and winter wheat (Triticum aestivum) also had lower ER values than the vegetable hosts. Growers should consider planting less susceptible or non-hosts such as peanut, sudangrass, sweet corn, and winter wheat in 2-3 year crop rotations to lower populations of this invasive nematode.

Evaluation of Weed Species for Host Status to the Root-Knot Nematodes Meloidogyne enterolobii and M. incognita Race 4.

Weeds that compete with valuable crops can also host plant-parasitic nematodes, acting as a source of nematode inoculum in a field and further damaging crops. The host status of 10 weed species commonly found in North Carolina, USA, was determined for the root-knot nematodes Meloidogyne enterolobii and M. incognita race 4 in the greenhouse. Each weed species was challenged with 5,000 eggs/plant of either M. enterolobii or M. incognita race 4, with five replicate plants per treatment in two separate greenhouse trials. Root galling severity and total number of nematode eggs per root system were recorded 60 days after inoculation. Reproduction factor (Rf = final nematode population/initial nematode population) was calculated to determine the host status of each weed species to M. enterolobii and M. incognita race 4. Four weed species (Datura stramonium, Digitaria sanguinalis, Senna obtusifolia, and Cyperus esculentus) were poor hosts (Rf < 1) to both nematode species, and roots of these weed plants did not display galling. Four weed species (Ipomoea hederacea, Amaranthus palmeri, Portulaca pilosa, and Ipomoea lacunosa) were hosts (Rf > 1) to both nematode species, and all had observable root gall formation. Sida rhombifolia and Cyperus rotundus were poor hosts to M. enterolobii but susceptible hosts to M. incognita. This study documents a differential host status of some common weeds to M. enterolobii and M. incognita race 4, and these results highlight the necessity of managing root-knot nematodes through controlling weeds in order to protect valuable crops.

Evaluation of Soybean Genotypes (Glycine max and G. soja) for Resistance to the Root-Knot Nematode, Meloidogyne enterolobii.

Potential resistance to the root-knot nematode (RKN) Meloidogyne enterolobii in 72 Glycine soja and 44 G. max soybean genotypes was evaluated in greenhouse experiments. Approximately 2,500 eggs of M. enterolobii were inoculated on each soybean genotype grown in a steam sterilized 1:1 sand to soil mixture. Sixty days postinoculation, plants were destructively harvested to determine the host status. The host status of each soybean genotype was determined by assessing root galling severity and calculating the final eggs per root system divided by the initial inoculum, or the reproduction factor (Rf). Five G. soja soybean genotypes were identified as resistant (Rf < 1) to M. enterolobii: '407202', '407239', '424083', '507618', and '639621'. None of the tested G. max soybean genotypes were identified as resistant to M. enterolobii. Some of the G. max genotypes determined to be susceptible to M. enterolobii include 'Hagood', 'Avery', 'Rhodes', 'Santee', and 'Bryan'. The genotype 'Bryan' had the lowest Rf values among the group at 5.06 and 6.67 in two independent trials, respectively, which represents a five- to sixfold increase in reproduction of M. enterolobii. Plant genotypes resistant to RKNs are effective in managing the disease and preserving yield, cost-efficient, and environmentally sustainable, and host resistance is often regarded as the most robust management tactic for controlling plant-parasitic nematodes. Resistance to RKNs in soybean genotypes has been identified for other Meloidogyne species, yet there is currently limited data regarding soybean host status to the highly aggressive nematode M. enterolobii. This study adds to the knowledge of potential native resistance to M. enterolobii in wild and cultivated soybean.

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.

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.

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.

Spatiotemporal Attributes and Crop Loss Associated with Tan Spot Epidemics in Baby Lima Bean in New York.

Tan spot, caused by the pycnidial fungi Didymella americana and Boeremia exigua var. exigua, is a foliar disease affecting processing baby lima bean production in New York. Tan spot epidemics are prevalent, occur annually, and may result in substantial defoliation. The disease is controlled by the prophylactic application of fungicides to maximize green leaf area. Information on yield losses due to tan spot on baby lima bean yield and the benefits of fungicide applications is needed to justify investments in disease management. Four small-plot, replicated trials were conducted over 2 years in commercial baby lima bean fields to evaluate the efficacy of fungicides for tan spot control at Piffard and Leicester, NY. Applications of pyraclostrobin or boscalid significantly reduced tan spot incidence and severity compared with nontreated plots, and increased the number of leaves per stem. In 2016, the increase in green leaf area associated with fungicide application was also documented in canopy reflectance values at 830 nm. Despite the decrease in tan spot incidence and corresponding increase in crop health obtained from fungicides, this effect did not translate into significant increases in pod yield. This finding suggested that the relationship between green leaf area and yield is highly variable in baby lima bean. The spatial and spatiotemporal patterns of naturally occurring tan spot epidemics were also characterized in baby lima bean fields across western New York using disease incidence data collected in transects and grids. The spatial pattern of data collected in transects was analyzed using median runs analysis. Disease incidence data collected in two-dimensional grids were analyzed to quantify spatial pattern using spatial analysis by distance indices (SADIE). The association function of SADIE was used to quantify the spatiotemporal patterns of tan spot epidemics after crop emergence and at harvest. These findings suggested that tan spot is likely to initiate at relatively frequent, randomly positioned foci, and that subsequent, limited spread results in significant local aggregation. Hypotheses for inoculum sources and recommendations for tan spot control in baby lima bean fields in New York are discussed.

Spatiotemporal spread of grapevine red blotch-associated virus in a California vineyard.

Grapevine red blotch-associated virus (GRBaV), the causative agent of red blotch disease, is a member of the genus Grablovirus, in the family Geminiviridae and the first known geminivirus of Vitis spp. Limited information is available on the epidemiology of red blotch disease. A 2-hectare Vitis vinifera cv. 'Cabernet franc' vineyard in Napa County, California, USA was selected for monitoring GRBaV spread over a three-year period (2014-2016) based on an initially low disease incidence and an aggregation of symptomatic vines at the edge of the vineyard proximal to a wooded riparian area. The incidence of diseased plants increased by 1-2% annually. Spatial analysis of diseased plants in each year using ordinary runs analysis within rows and Spatial Analysis by Distance IndicEs (SADIE) demonstrated aggregation. Spatiotemporal analysis between consecutive years within the association function of SADIE revealed a strong overall association among all three years (X=0.874-0.945). Analysis of epidemic spread fitting a stochastic spatiotemporal model using the Monte Carlo Markov Chain method identified strong evidence for localized (within vineyard) spread. A spatial pattern consisting of a combination of strongly aggregated and randomly isolated symptomatic vines within 8-years post-planting suggested unique epidemic attributes compared to those of other grapevine viruses vectored by mealybugs and soft scales or by dagger nematodes for which typical within-row spread and small-scale autocorrelation are well documented. These findings are consistent with the existence of a new type of vector for a grapevine virus.