The Origin of the Problem: Characterization of Paraguayan Septoria steviae, Causal Agent of Septoria Leaf Spot of Stevia, Based on Multilocus Sequence Analysis.

Septoria leaf spot is a significant disease affecting cultivated stevia, potentially reducing yields by > 50%. The disease is caused by Septoria steviae, first identified in 1978 in Japan as a new pathogen of stevia. Understanding the origin of S. steviae could clarify how it spread to new production areas. To investigate this, 12 isolates of Septoria sp. were obtained from stevia's native range in the Amambay forests and field plantings in Paraguay from 2018 to 2020. These isolates underwent colony morphology and molecular characterization of Actin, ß-Tubulin, Calmodulin, ITS, LSU, RPB2, and TEF1α loci. GenBank sequences from S. steviae isolates collected in France, Japan, and the United States were included. Multilocus sequence phylogenetic analysis generated a maximum likelihood (ML) tree. The morphological characteristics of Paraguayan isolates were similar to those of previously reported S. steviae type cultures from Japan. The ML analysis showed that Paraguayan isolates formed a monophyletic group with S. steviae isolates from France, Japan, and the United States. During blotter tests, pycnidia and cirri of S. steviae were observed on multiple stevia seed surfaces from different sources. Further characterization confirmed viable pathogenic conidia of S. steviae. This observation suggests that S. steviae could be associated with stevia seed, possibly spreading from the center of origin to other countries. This research is the first to genetically characterize S. steviae from Paraguay and propose its potential spread mechanism from the center of origin to the rest of the world.

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 (RKNs, 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 to 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 of soil) and Nash (135 J2/500 cm3 of soil) counties, while Wayne (7 J2/500 cm3 of soil) and Greene (11 J2/500 cm3 of 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 an NC population of M. enterolobii, the guava RKN, 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 18 vegetable, field, and 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 × drummondii) were nonhosts 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 hosts or nonhosts such as peanut, sudangrass, sweet corn, and winter wheat in 2- to 3-year crop rotations to lower populations of this invasive nematode.

Validation of Standard Area Diagrams to Estimate the Severity of Septoria Leaf Spot on Stevia in Paraguay, Mexico, and the United States.

Septoria leaf spot (SLS) affects stevia leaves, reducing their quality. Estimates of SLS severity on different genotypes are made to identify resistance and as a basis to compare management approaches. The use of standard area diagrams (SADs) can improve the accuracy and reliability of severity estimates. In this study, we developed new SADs with six illustrations (0.5, 1, 10, 25, 40, and 75% severity). The SADs were validated by raters with and without experience in estimating SLS. Raters evaluated 40 leaf photos with SLS severities ranging from 0 to 100% without and with the SADs. Agreement (ρc), bias (Cb), precision (r), and intracluster correlation (ρ) coefficients were significantly closer to "true" severity values when the SADs was used by inexperienced (ρc = 0.89; Cb = 0.97; r = 0.90, ρ = 0.81) and experienced (ρc = 0.94; Cb = 0.99; r = 0.95, ρ = 0.91) raters. The SADs were tested under field conditions in Paraguay, Mexico, and the United States, with inexperienced raters assigned to two groups, one SADs trained and the other not trained, that estimated SLS severity three times: first, all raters without SADs and no time limit for the estimates; second, only the SADs-trained group used SADs and no time limit; and third, only the SADs-trained group used SADs, with a time limit of 10 s imposed per specimen assessment. Agreement and reliability of SLS severity estimates significantly improved when raters used the SADs without a time limit. The use of the new SADs improved the accuracy, precision, and reliability of SLS severity estimates, enhancing the uniformity in assessment across different stevia programs.

Meta-Analysis of the Field Efficacy of Seed- and Soil-Applied Nematicides on Meloidogyne incognita and Rotylenchulus reniformis Across the U.S. Cotton Belt.

Meta-analysis was used to compare yield protection and nematode suppression provided by two seed-applied and two soil-applied nematicides against Meloidogyne incognita and Rotylenchulus reniformis on cotton across 3 years and several trial locations in the U.S. Cotton Belt. Nematicides consisted of thiodicarb- and fluopyram-treated seed, aldicarb and fluopyram applied in furrow, and combinations of the seed treatments and soil-applied fluopyram. The nematicides had no effect on nematode reproduction or root infection but had a significant impact on seed cotton yield response ([Formula: see text]), with an average increase of 176 and 197 kg/ha relative to the nontreated control in M. incognita and R. reniformis infested fields, respectively. However, because of significant variation in yield protection and nematode suppression by nematicides, five or six moderator variables (cultivar resistance [M. incognita only], nematode infestation level, nematicide treatment, application method, trial location, and growing season) were used depending on nematode species. In M. incognita-infested fields, greater yield protection was observed with nematicides applied in furrow and with seed-applied + in-furrow than with solo seed-applied nematicide applications. Most notable of these in-furrow nematicides were aldicarb and fluopyram (>131 g/ha) with or without a seed-applied nematicide compared with thiodicarb. In R. reniformis-infested fields, moderator variables provided no further explanation of the variation in yield response produced by nematicides. Furthermore, moderator variables provided little explanation of the variation in nematode suppression by nematicides in M. incognita- and R. reniformis-infested fields. The limited explanation by the moderator variables on the field efficacy of nematicides in M. incognita- and R. reniformis-infested fields demonstrates the difficulty of managing these pathogens with nonfumigant nematicides across the U.S. Cotton Belt.

Effect of Fungicide Mobility and Application Timing on the Management of Grape Powdery Mildew.

Grape powdery mildew (GPM) fungicide programs consist of 5 to 15 applications, depending on region or market, in an attempt to achieve the high fruit quality standards demanded by the market. Understanding how fungicides redistribute and targeting redistributing fungicide to critical crop phenological stages could improve fungicide protection of grape clusters. This study evaluated fungicide redistribution in grapevines from major fungicide groups labeled for GPM control. Translaminar and xylem redistribution was examined by placing fungicide-impregnated filter disks on the adaxial or abaxial leaf surface of detached leaves for 10 min and then incubating for 48 h before inoculating the abaxial surface with conidia. Vapor redistribution used Teflon disks sprayed with fungicides and placed on the abaxial leaf surface of detached leaves 48 h before inoculation. Disease development was rated 10 days later. Translaminar movement through calyptra was tested using flowering potted vines. All fungicides tested redistributed through at least one mechanism. Fungicide timing at critical phenological stages (early, mid, and late bloom) was assessed in small plots of cultivar Pinot noir vines. The application of trifloxystrobin, quinoxyfen, or fluopyram at different bloom stages showed that applications initiated at end of bloom resulted in the lowest berry infection probabilities of 0.073, 0.097, and 0.020, respectively. The results of this study suggest that integrating two carefully timed applications of redistributing fungicides initiated at end of bloom into a fungicide program may be an effective strategy for wine grape growers in western Oregon to produce fruit with low GPM infection.

Development of a quantitative loop-mediated isothermal amplification assay for the field detection of Erysiphe necator.

Plant pathogen detection systems have been useful tools to monitor inoculum presence and initiate management schedules. More recently, a loop-mediated isothermal amplification (LAMP) assay was successfully designed for field use in the grape powdery mildew pathosystem; however, false negatives or false positives were prevalent in grower-conducted assays due to the difficulty in perceiving the magnesium pyrophosphate precipitate at low DNA concentrations. A quantitative LAMP (qLAMP) assay using a fluorescence resonance energy transfer-based probe was assessed by grape growers in the Willamette Valley of Oregon. Custom impaction spore samplers were placed at a research vineyard and six commercial vineyard locations, and were tested bi-weekly by the lab and by growers. Grower-conducted qLAMP assays used a beta-version of the Smart-DART handheld LAMP reaction devices (Diagenetix, Inc., Honolulu, HI, USA), connected to Android 4.4 enabled, Bluetooth-capable Nexus 7 tablets for output. Quantification by a quantitative PCR assay was assumed correct to compare the lab and grower qLAMP assay quantification. Growers were able to conduct and interpret qLAMP results; however, the Erysiphe necator inoculum quantification was unreliable using the beta-Smart-DART devices. The qLAMP assay developed was sensitive to one spore in early testing of the assay, but decreased to >20 spores by the end of the trial. The qLAMP assay is not likely a suitable management tool for grape powdery mildew due to losses in sensitivity and decreasing costs and portability for other, more reliable molecular tools.