Brief Summary of Minutes of Annual Meeting

Project No. and Title – S-1066: Development of sustainable crop production practices for integrated management of plant=pathogenic nematodes

Period Covered:  10/01/2016 to 09/30/17

Participants:

Lawrence, Kathy (AL); Robbins, Robert (AK); Grabau, Zane (FL); Dickson, Donald (FL); Hajihassani, Abolfazl (GA); Overstreet, Charles (LA); Davis, Rick (NC); Agudelo, Paula (SC); Lacewell, Ron (TX); Eisenback, Jon (VA); Johnson, Chuck (VA).

Guest: 

Groover, Will, (AL); Klepadlo, Mariola, (MO); Ye, Weimin (NC); Thiessen, Lindsey (NC); Ruark, Casey (NC); Rutter, Will (ARS-USDA, SC); Li, Wei & Wilkes, Juliet (SC).

12 November – Arrival and informal dinner.

13 November – Meeting held at the North Carolina Department of Agricultural Facility.

 Welcoming comments and meeting called to order by Rick Davis, chairperson and host. 

1. W. Dickson appointed secretary.

Introductions of members and guests – S-1066 is comprised of 23 members representing 17 states. 

Administrator Ron Lacewell updated group on budgeting items from US Congress, and discussed information regarding future funding for grants especially related to sustainable agricultural and multidisciplinary projects.

Reports presented by committee members from SC, VA, NC, and AL.

            Tour of the NC Department of Agricultural Nematode Assay Laboratory by Weimin Ye.

14 November – Reports continued to be presented by committee members from MO, FL, LA, and AR.

Business Meeting.  

            Approval of 2016 Minutes.  Florida selected to host the 2018 S-1066 annual meeting. D. W. Dickson appointed chairperson and local arrangement host for 2018.   In state location and date to be determined.

Meeting adjourned at noon.

Objective 1:   Advance the tools for identification of nematode species and characterization of intraspecific variability.

Alabama (K. Lawrence). Species identification of Meloidogyne spp. (root-knot nematode, RKN) is an important tool to offer growers in the state of Alabama because it is beneficial for planning and implementing a crop rotation to reduce the impact of these yield-limiting nematodes. The goal of this project was to evaluate multiple species identification techniques and determine the best combination of methods for implementing a practical and efficient assay for RKN species identification. To do this, three different techniques were evaluated for their ability to quickly and accurately identify RKN species. The techniques used in this study were morphological measurements, differential-host test, and molecular analysis. Each of these techniques was used on multiple RKN populations, starting with a known M. incognita race 3 population. This greenhouse population was previously identified via the differential-host test. Initial results showed a confirmation of species with the differential-host test and PCR amplification, but morphological measurements of juveniles did not distinguish our test population from M. arenaria and M. javanica. Soil and root samples were then collected from throughout Alabama for RKN species identification. Overall, 75 samples from 14 counties in Alabama were collected from grower fields for species analysis. Crops sampled during collection included cotton, soybean, corn, peanut, sweet potato, squash, pepper, kiwi, turmeric, and turf. Both molecular analysis (PCR) and the differential-host test were used for species identification. Primers used for PCR include those that identify commonly found RKN species: M. incognita, M. arenaria, M. javanica, M. hapla, M. fallax, M. chitwoodi, and M. enterolobii. Of these samples, 73 were identified as M. incognita (97%), and two were identified as M. arenaria (3%). These species were identified through the differential-host test and PCR using primer sets IncK-14F/IncK-14R (M. incognita) and Far/Rar (M. arenaria). Overall, M. incognita is the most prevalent species of root-knot nematode that has been found on cropping systems in Alabama during this project.

Arkansas (R. Robbins).  In working with soybean breeders from Missouri I tested 214 soybean Plant Introductions reported to have a high level of Soybean Cyst Nematode (SCN) resistance and 204 with reported moderate SCN for resistance to the reniform nematode (Rotylenchulus reniformis). These tests for reproduction were conducted in the greenhouse using our standard Arkansas reniform culture. In two tests, at different times, of those with a high level of resistance to SCN I found 44 PI’s of both tests (times) to have resistance to reniform nematodes and an additional 8 in the second test when compared to the resistant check “Hartwig.”  For 204 PI’s with reported moderate resistance I found 5 PI’s with reniform reproduction not different than “Hartwig.” Cooperators in Missouri are working to find correlation with my PI’s reproduction data with phenotypic data. Correlation of reproduction and phenotypes could be useful in identifying soybean lines with resistance to both SCN and reniform nematodes.  I tested 12 species of Oaks as hosts of the Pecan Root-Knot nematode (Meloidogyne partityla). Of the 12 two (Cork and Pin oak) produced galls and egg masses, while Holly, Tabor, and burr Oaks produced galls only. English Walnut (Juglans regia) also produced galls and egg masses.

South Carolina (P. Agudelo).  We continued to collect and study intra- and interspecific variability of lance nematodes.  We described a new Hoplolaimus species from the Smoky Mountains.  We sequenced the mitochondrial genome of for two lance nematode species to provide references for comparative genomics, speciation, and phylogeography studies. 

Virgina. (C. Johnson and J. Eisenback).  A new species of root-knot nematodes is currently being described parasitizing yellow and purple nut-sedge in New Mexico. The female is very small and the neck is offset from the tail terminus which protrudes from the posterior end. A complete description using light and scanning electron microscopes and molecular characteristics is currently underway. A re-description of Meloidogyne kikuyensis has shown that this nematode is a putative primitive species.  It has just 7 large chromosomes, unlike that majority of species in the genus that have numerous small chromosomes. Also, molecular characters support the idea that it is a basal species within the genus.  The plant-host parasite has shown that the nodule-like galls are of unique origin and are very similar to that of nodules produced by nitrogen fixing bacteria.

Objective 2:  Elucidate molecular and physiological mechanisms of plant-nematode interactions to improve host resistance.

Mississippi (G. Lawrence and V. Klink).  A harpin elicitor induces the expression of a coiled-coil nucleotide binding leucine rich repeat (CC-NB-LRR) defense signaling gene and others functioning during defense to parasitic nematodes. The bacterial effector harpin induces the transcription of the Arabidopsis thaliana (thale cress) NON-RACE SPECIFIC DISEASE RESISTANCE 1/HARPIN INDUCED1 (NDR1/HIN1) coiled-coil nucleotide binding leucine rich repeat (CC-NB-LRR) defense signaling gene. In Glycine max (soybean), Gm-NDR1-1 transcripts have been detected within root cells undergoing a natural resistant reaction to parasitism by the syncytium-forming nematode Heterodera glycines, (soybean cyst nematode [SCN]) functioning in the defense response. Expressing Gm-NDR1-1 in Gossypium hirsutum (cotton) leads to resistance to Meloidogyne incognita (root knot nematode [RKN]) parasitism. In experiments presented here, the heterologous expression of Gm-NDR1-1 in G. hirsutum impairs Rotylenchulus reniformis (reniform nematode) parasitism. These results are consistent with the hypothesis that Gm-NDR1-1 expression functions broadly in generating a defense response. To examine a possible relationship with harpin, G. max plants topically treated with harpin result in induction of the transcription of Gm-NDR1-1. The result indicates the topical treatment of plants with harpin, itself, may lead to impaired nematode parasitism. Topical harpin treatments are shown to impair G. max parasitism by H. glycines, M. incognita and R. reniformis and G. hirsutum parasitism by M. incognita and R. reniformis. How harpin could function in defense has been examined in experiments showing it also induces transcription of G. max homologs of the proven defense genes ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1), TGA2, galactinol synthase, reticuline oxidase, xyloglucan endotransglycosylase/hydrolase, alpha soluble N-ethylmaleimide-sensitive fusion protein (-SNAP) and serine hydroxymethyltransferase (SHMT). In contrast, other defense genes are not directly transcriptionally activated by harpin. The results indicate harpin induces pathogen associated molecular pattern (PAMP) triggered immunity (PTI) and effector-triggered immunity (ETI) defense processes in the root, activating defense to parasitic nematodes. RNA has been isolated from Glycine max (soybean) root cells undergoing the process of defense to Heterodera glycines (soybean cyst nematode). The RNA has been used in gene expression analyses. The procedure has led to the identification of candidate resistance genes. A gene testing platform has been developed to functionally test these genes. The procedure has examined hundreds of genes with some functioning effectively in defense. The analysis has demonstrated the importance of various cellular processes to defense and has identified genes that previously had no known role in defense.  A functional developmental genomics screen is identifying genes functioning within cells that function in plant to a root pathogen.  RNA has been isolated from Glycine max (soybean) root cells undergoing the process of defense to a root pathogen. The RNA has been used in gene expression analyses, leading to the identification of candidate resistance genes. A gene testing platform has been developed to functionally test these genes with the aim of determining if the genes function during the process of defense. The procedure has examined hundreds of genes with some functioning effectively in defense. The analysis has demonstrated the importance of various cellular processes to defense and has identified genes that previously had no known role in defense.

Missouri (H. Nguyen and M. Klepadlo).  Discovery of new resistance sources.  Since 2008, 584 soybean plant introductions (PIs) with maturity group (MG) 000-II were screened against SCN race 2 and 3, and 636 PIs with MG III-V were screened against SCN race 1, 2, 3, 4, 5 and 14. A subset of 76 PIs were selected and classified under Peking-type, PI 88788-type and potential new resistance subgroups, and proceeded with screening against SRKN and RN. Among 76 PIs, 56 and 12 of them were resistant to two and three nematode species. 

Over 1,000 soybean germplasm, including exotic plant introductions (PIs), breeding lines, and varieties, were sequenced using the next-generation sequencing (NGS) technology and will provide a fundamental tool in genome mining. In addition, a new reference southern soybean genome ‘Lee’ will be available to public in the end of 2018. Genome-wide haplotype clustering and structural variation analysis are routinely used for identification of nematode resistance genes and corresponding molecular markers for diagnostic use. Genetic analysis of new sources. Two major QTL responsible for resistance to different SCN races were consistently mapped at the same genomic locations on Chrs. 10 (LG O) and 18 (LG G), as previously reported in PI 567516C and PI 567305.These PIs are also highly resistant to other nematode species: SRKN and RN. Genetic analyses were conducted in a recombinant inbred line (RIL) populations to identify and map genomic regions for multi-nematode resistance. Whole-genome sequencing (WGS) data and haplotype analysis indicated that these two PIs shared similar genome component in both QTL regions. PI 438489B was reported to be highly resistant to multi-SCN races, SRKN and RN. Genetic analysis confirmed two major loci, rhg1 (Peking-type) and Rhg4 for resistance to SCN and three QTL for resistance to RKN on Chrs. 8, 10, and 13. Identification of RN resistance was done in collaboration with Dr. Robbins. Two linkage maps were used using Universal Soybean Linkage Panel and Whole Genome Sequencing technology. Three QTL were detected on Chr. 18, 11 and 3. Candidate genes are going through extensive haplotype and phylogenetic analyses and final candidate genes will be tested with CRISPR-Cas9 system to confirm their functions.  Fine-mapping of novel QTL. Two novel SCN QTL, Chr. 10 (LG O, locus O) and Chr. 18 (LG G, Locus 2G), detected in PI 567516C are the target for fine-mapping and cloning. Backcrossing populations were developed to fine-map these QTL regions. For locus O, more than 1,000 BC4F2 plants were genotyped. Seven BC4F2:4 NILs were developed in the target region and are undergoing SCN phenotyping. The QTL on Chr. 18 was genetically distant from the known rhg1 locus and tentatively designated as the 2G QTL. For locus 2G, 12 BC4F2:4 NILs were developed and screened with multiple SCN races. Fine-mapping of these loci will continue in 2018.

North Carolina (E. Davis).  Five viruses [ScNV, ScPV, ScRV, ScTV, and SbCNV-5] previously found to infect SCN greenhouse populations in Illinois were also detected by RT-PCR within SCN from 43 greenhouse cultures and 25 field populations from North Carolina (NC) and Missouri (MO). Viral titers within SCN greenhouse cultures were similar throughout juvenile development, and the presence of viral anti-genomic RNAs within egg, second-stage juvenile (J2), and pooled J3 and J4 stages suggests active viral replication within the nematode. Viruses were found at similar or lower levels within field populations of SCN compared to greenhouse cultures of NC populations. Five greenhouse cultures [LY1, LY2, MM2, TN7, and TN22] harbored all five known viruses whereas in most populations a mixture of fewer viruses was detected. In contrast, three greenhouse cultures [MM21, MM23, MM24] of similar descent to one another did not possess any detectable viruses and primarily differed in location of the cultures (NC versus MO). Viruses ScNV, ScPV, and ScTV were also detected in Heterodera trifolii (clover cyst) and viruses ScPV and ScRV were detected in a greenhouse population of Heterodera schachtii (beet cyst), but none of the five SCN viruses were detected in other cyst, root-knot, or reniform nematode populations tested. The viruses were not detected within soybean host plant tissue.  Constitutive expression of the Hs25A01 cDNA under the control of the 35S promoter in Arabidopsis plants caused a small increase in root length accompanied by a 35% increase in susceptibility to H. schachtii. A plant-expressed RNAi construct targeting Hs25A01 transcripts in invading nematodes significantly reduced host susceptibility to H. schachtii.  These data document that Hs25A01 has physiological functions in planta and is conducive to cyst nematode parasitism.  To broaden SCN resistance breeding resources and to mitigate nematode damage, we used Glycine soja, a wild soybean progenitor that shows much higher genetic diversity than cultivated soybean, to identify resistant accessions and to dissect the genetic basis of resistance to HG 2.5.7. A total of 235 G. soja accessions were evaluated; 43 were found to be resistant to SCN HG 2.5.7 (female index < 30) and could be considered exotic and novel SCN-resistant resources. We further conducted a genome-wide association study (GWAS) of HG 2.5.7 resistance with an association panel containing 235 wild soybean accessions using 41,087 single nucleotide polymorphisms (SNPs). A total of 10 SNPs distributed on chromosome 18 and chromosome 19 were found to be significantly associated with SCN HG 2.5.7 resistance.

South Carolina (P. Agudelo).  We have focused on reniform nematode infection in cotton and soybean roots.  To document plant responses, we set up a split-root growth system to collect tissues from infected and uninfected portions of the same root system.  A 12-day time course of histology and gene expression of infected roots were generated.  Histological observations recorded the developmental process of the permanent feeding structure, and we investigated the effect of reniform nematode parasitism on lateral root formation.  Nematode infection resulted in significantly higher branching complexity in cotton roots and alters hormone-associated gene expression.  Monoclonal antibodies were used to investigate potential modifications of cell wall components in infected cotton roots.

Tennessee (Tarek Hewezi, Feng Chen and Reza Hajimorad).  Recent studies report on key regulatory role of microRNA (miRNA) genes in regulating plant responses to cyst nematode infection. We generated whole-genome DNA methylation map at single-base resolution during the compatible interaction between soybean (Williams 82) and the soybean cyst nematode (SCN; Heterodera glycines). We investigated DNA methylation changes occurring in the promoter (2 kb upstream of the primary miRNA sequence) of all known soybean miRNA genes. We identified 28 miRNAs that are significantly (q value < 0.01) differentially methylated (methylation difference ≥ 25%). Differential DNA methylation was found mainly in the CG and CHG sequence contexts. Also we found that DNA hypomethylation (loss of methylation) in miRNA promoters occurs to a much higher level than hypermethylation (gain of methylation). We predicted target genes of these differentially methylated miRNAs using computational tools. Many of the predicted targets are among the confirmed miRNA targets in publically available degradome datasets. Interestingly, several of these target genes have been previously identified as syncytium differentially expressed genes, pointing into a role of DNA methylation, as a key epigenetic mark, in controlling the regulatory function of miRNAs during soybean response to SCN infection. We further investigated the function of one of these miRNAs using soybean transgenic hairy root system.  The primary transcript sequence of this miRNA was cloned in a binary vector and overexpressed in soybean cultivar Williams 82. The transgenic hairy roots were selected using GFP reporter. Transgenic hairy roots expressing only GFP were used as negative control. qPCR analysis confirmed the increased expression of the mature miRNA. The transgenic hairy root plants were arranged in three replicates each with 5 plants to determine plant susceptibility to SCN (race 3) in a greenhouse experiment. SCN infection assay showed a significant (P value < 0.001) increase (~200%) in nematode susceptibility of the hairy roots overexpressing the miRNA gene relative to the control plants expressing GFP only. Together, these data indicate that DNA methylation contribute to the regulatory function of miRNA genes during soybean × SCN interaction.  A number of putative soybean defense genes against SCN, including a salicylate acid methyltransferase gene (SAMT), a jasmonic acid methyltransferase gene (JAMT) and several terpene synthase genes (TPS), have been identified. The role of SAMT and a TPS gene in SCN resistance have been evaluated and validated using molecular biology, biochemistry and transgenic approaches. Some TPS genes of other biological sources have been characterized, which may be used as molecular tools for improving SCN-resistance of soybean.

Virgina. (C. Johnson and J. Eisenback).  Next generation sequencing datasets of Pratylenchus penetrans were combined spatially and temporally to resolve candidate genes selected for the discovery of a panel of effector genes for this species. The spatial expression of the transcripts of 22 candidate effector genes within the esophageal glands were revealed by in situ hybridization. They were chosen from more than 100 genes identified from the nematode. These comprised homologues of known effectors of other plant-parasitic nematodes with diverse putative functions, as well as eight novel pioneer effectors specific to this nematode. They were combined in situ for localization of effectors with available genomic data to identify a non-coding motif that are enriched promoter regions of a subset of P. penetrans effectors. Using RT-qPCR analyses, a select subset of candidate effectors was shown to be actively expressed during the early steps of plant infection These results provide the most comprehensive panel of effector genes found for P. penetrans. Considering the damage caused by this nematode, valuable data to elucidate the basis of pathogenicity offers useful tactic to provide potential specific target effector genes to control this important pathogen.

Objective 3:  Integrate nematode management agents (NMAs) and cultural tactics with the use of resistant cultivars to develop sustainable crop production systems.

Alabama (K. Lawrence).  An increased attention has been placed on biological control of plant-parasitic nematodes using various fungi and bacteria. In our study we evaluated the potential of 662 plant growth-promoting rhizobacteria (PGPR) strains for mortality to Meloidogyne incognita J2 in vitro.  Results indicated that the mortality of M. incognita J2 by the PGPR strains ranged from 0 to 100% with an average of 39%. Among the PGPR strains examined, 212 of 662 strains (or 33%) caused significantly greater mortality percent of M. incognita J2 than the untreated control. Bacillus was the bacterial genus most often inducing mortality when compared with the other genera. In subsequent greenhouse trials trials, B. velezensis strain Bve2 reduced M. incognita eggs per gram of cotton root in similarly to the commercial standards Abamectin and Clothianidin plus B. firmus I-1582. Bacillus mojavensis strain Bmo3, B. velezensis strain Bve2, B. subtilis subsp. subtilis strain Bsssu3, and the Mixture 2 (Abamectin + Bve2 + B. altitudinis strain Bal13) suppressed M. incognita eggs per gram of root in the microplot trials. Bacillus velezensis strains Bve2 and Bve12 also increased seed-cotton yield in the microplot and field trials. Overall, results indicate that B. velezensis strains Bve2 and Bve12, B. mojavensis strain Bmo3, and Mixture 2 (Abamectin + Bve2 + B. altitudinis strain Bal13) have potential to reduce M. incognita population density and to enhance growth of cotton when applied as in-furrow sprays at planting. Common turmeric (Curcuma longa L.), a spice crop native to India, used for the yellow color in products ranging from foods to pharmaceuticals. The medicinal plant is in early stages of evaluation as niche crop for Alabama and afresh market demand of turmeric is rising in local farmers markets. IN 2015, turmeric plants grown on the campus of Auburn University, exhibited small galls on root systems. Symptoms appeared throughout all eight selections of Curcuma longa grown at Auburn University. Plants in early summer exhibited symptoms including chlorosis, stunting, and marginal leaf necrosis. Symptomatic plants were collected and root systems exhibited numerous galls, typical of Meloidogyne infection. Nematode eggs were extracted from root systems and enumerated. Eggs were hatched to the second juvenile stage (J2) for species identification. Individual juveniles (1-10 per sample) were picked out of the population.  Each juvenile was then smashed into several pieces by a 100 µL pipette tip via the smashing method and immediately used for PCR (Harris and Powers, 1993).  The J2 DNA was amplified via PCR using primers IncK-14F and IncK-14R that are specific for amplification of M. incognita (Randig et al., 2002).  Primers specific for M. arenaria (Far/Rar), M. javanica (Fjav/Rjav), M. hapla (JMV1/JMV hapla), and M. enterolobi (Me-F/Me-R) were also used, but failed to amplify any of the unknown nematode DNA (Randig et. al. 2002; Zijlstra et. al. 2000).  PCR was run on unknown samples as well as a positive control sample of M. incognita DNA obtained from the greenhouse stock cultures that have previously been identified as M. incognita by this research group (Groover and Lawrence, 2016).  Approximately 45-50 J2’s were tested with each primer set, and the IncK-14F/IncK-14R primer set amplified about 30 as M. incognita, giving an amplification rate around 65%.  The amplified PCR product was then run on a 1% agarose gel and a 400 base pair fragment was observed under a UV light, confirming the population to be M. incognita (Randig et. al. 2002).  M. incognita-inoculated turmeric selections exhibited reduced average plant height, shoot fresh weight, and root fresh weight with the measurements being 26% to 50%of those of the control. Final nematode population densities on ranged from 19 to 4703 eggs per gram of root of the turmeric selections. Reproductive factor (RF), defined as the final nematode population density divided by the initial inoculum density, was calculated to be as low as 0.6 up to 4.1. Most turmeric selections were susceptible to M. incognita, however selection CL2, was somewhat resistant to the nematode as its RF value was less than 1. To our knowledge, this is the first report of M. incognita infecting Curcuma longa in the United States. Because M. incognita has been recorded in 46 out of Alabama’s 67 counties, potential growers of turmeric should consider nematode management and variety selection as an important step to successful turmeric production in the state of Alabama.

Arkansas (T. Faske).  During the 2017 cropping season my program evaluated 48 soybean cultivars for susceptibility to the southern root-knot nematode, which is the most important plant-parasitic nematode that affects soybean production in the mid-South, including Arkansas.  This provides some information on cultivar selection in fields with a high population density of root-knot nematodes.  My program also evaluated several of the new seed-applied nematicides such as AVEO, Nemastrike, BioST, and ILeVO, and in-furrow applied nematicides like AgLogic, and Salibro in soybean.  Similarly in cotton we evaluated seed-applied nematicides like Nemastrike, COPeO, and BioST and in-furrow applied nematicides like Velum Total and AgLogic. Summary of these trials will be reported as plant disease management reports or used to at winter extension meetings and in-service trainings.

Arkansas (R. Robbins).  I tested 66 soybean breeder’s lines for reniform nematode resistance; 10 lines from Arkansas, 10 form Clemson, 16 form Georgia, and 20 from Missouri. Of these 66 lines two each from Clemson and Georgia, ten of Missouri, and none from Arkansas did not reproduce more than the resistant check “Hartwig” and may be useful in breeding for reniform nematode resistance in commercial lines.

Florida (D. Dickson).  Tifguard, which was released as a peanut cultivar resistant to root-knot nematode and tomato spotted wilt virus in 2007, was found to be heavily infected by Meloidogyne arenaria in several production fields in Florida in 2012. The goal of this project was to determine why the cultivar that was reported to be highly resistant succumbed to root-knot nematode infection. The objectives were to assess the resistance of three different sources of Tifguard seeds; to determine the vertical population densities and seasonal population changes of M. arenaria on resistant and susceptible peanut; to determine the effects of high soil temperature on the resistance in Tifguard, and to evaluate the yield of Tifguard, isogenic Tifguard and Georgia-06G treated vs. nontreated with 1,3-dichloropropene. In three M. arenaria infested field sites a comparison of Tifguard seed obtained from three sources showed that 3, 30, and 40% of plants that were infected by the nematode were negative for the nematode resistance gene. Comparison of vertical population densities of M. arenaria on Georgia-06G in two different soil types showed that greater numbers occurred in the upper 60 cm of soil during the growing season in a Candler sand, whereas in a Norfolk loamy sand greater densities were found only in the top 45 cm. The seasonal distribution of J2 in the soil followed similar trends in the two locations, with a peak occurring during late summer and early fall at harvest. Number of J2 dropped following harvest and reached a density less than 10 J2/200 cm³ of soil in February. The population densities of M. arenaria on Georgia-06G at all depths were much greater in Norfolk loamy sand than that in the Candler sand. Tifguard reduced the nematode population to less than 20 and 70 in the Candler sand and the Norfolk loamy sand, respectively over the experimental periods. Comparison of nematode numbers from different developmental stages at different temperatures demonstrated that the high soil temperature increased nematode infection rate and accelerated nematode development in Georgia-06G. No further development of J2 occurred in Tifguard roots at 28 or 31 ℃, however at 34 ℃ a few J3-J4, females, egg laying females, and males of M. arenaria were observed.

Florida (Z. Grabau).  Investigated use of conventional and alternative crop rotations for reniform nematode management.  Investigated integration of nematicide application with crop rotation for nematode management.  Investigated impacts on agricultural management practices on soil ecology based on free-living nematodes 

Louisiana (C. Overstreet and E. McGawley).  Experiments were conducted at the Northeast Research Station to evaluate the effectiveness of site-specific application of nematicides on soybean in a field that had variable soil texture and was infested with both Southern root-knot and reniform nematodes. The field was divided into soil zones based on apparent electrical conductivity (EC_a). Zones 1, 2, 3, 4, and 5 had ranges of EC_a-deep values of 20.2-33.7, 33.7-49.4, 49.4-67.0, 67.0-84.6, and 84.6-118.0 mS/m, respectively. Treatments included the nematicide Telone at 3 gallons/acre, Avicta Complete Bean as a seed treatment, the combination of the two nematicides together, and an untreated control. Each treatment was replicated 40 times to ensure occurrence in all the soil zones.  The fumigant significantly reduced population levels of reniform nematode in zone 1 and numerically in zones 2 and 3 at planting. Root-knot populations were very low in all plots at the time of planting. The Avicta Complete Bean did not significantly impact populations of the nematode or yield and data was combined between Telone treated or not treated. Reniform populations at harvest were 38,462 and 26,697 per 500 cc of soil for the untreated in zones 1 and 2, respectively which was significantly less than the Telone treatments of 11,590 and 14,053. Southern root-knot populations in the untreated were significantly higher in zones 2, 3, 4, and 5 than the Telone treatment. Soybean yield was significantly higher in zones 1 and 3 with the application of Telone and numerically in all the others. However, the differences in yield were insignificant (2 bushels per acre) in zones 4 and 5. This test indicated that management zones could be established in soybean that reflected more of the soil influence on nematicide response than simply populations of the nematode. A second experiment was conducted that was similar to the first one that used two different varieties that were treated or not treated with the fumigant Telone at 3 gallons/acre preplant. Asgrow 54X6 variety is susceptible to both Southern root-knot and reniform nematode and Armor 53D04 has some resistance against Southern root-knot and none against reniform nematode. This site was divided into three zones based on EC_a-deep values that ranged from 18.2-42.8, 42.8-71.8, and 71.8-118.0 mS/m, respectively for zones 1, 2, and 3. Populations of reniform nematode were significantly reduced in 53D04 untreated from treated averaging 5111 and 690 vermiform life stages of reniform nematode per 500 cc of soil, respectively. Telone numerically reduced populations of reniform nematode of both varieties in all zones. Populations of reniform nematode were significantly higher on the untreated on AG54X6 in zone 2 and 53D04 had higher population in the untreated in zones 1 and 2. No differences were observed with treated or untreated with either variety in zone 3. Root-knot nematode populations were significantly higher compared to the treated with AG54X6 in zones 1 and 2. Populations of root-knot remained low across soil zones and treatments with 53D04. Soybean yield of AG54X6 was not impacted by fumigation across soil zones. The variety 52D04 did show a significant response to the fumigant in zone 1 of 4.2 bushels per acre but not in the other two zones. This study indicates that variety selection may also be important in development of management zones for nematodes in soybean.  Grain sorghum is considered to be an acceptable rotation crop to manage Southern root-knot nematode in many states. Experiments were established to determine if grain sorghum varieties would be impacted by Southern root-knot nematode and the impact on population development of the nematode for succeeding crops. In test one, three sorghum varieties (83P17, NK6638, and REV9782) were selected that had various levels of resistance against Southern root-knot nematode and determined to be very susceptible, moderately susceptible, and moderately resistant, respectively. Each of these varieties was treated with or without Telone II at 3 gallons per acre preplant. Although populations of Southern root-knot nematode were low at the time of planting, Telone significantly reduced the nematode in NK6638. Final populations of Southern root-knot nematode were much higher after harvest and averaged 512, 352, and 256 juveniles per 500 cc of soil for 83P17, NK6638, and REV9782, respectively. The fumigant was very effective and resulted in no root-knot juveniles in the soil in any of the varieties. The fumigant did not significantly improve yield in any of the varieties. Sorghum yields were low and averaged 80, 88, and 76 bushels per acre for 83P17, NK6638, and REV9782, respectively. A second sorghum trial evaluated five varieties that were very susceptible or moderately susceptible to Southern root-knot nematode for the influence of the fumigant Telone and impact of population development at harvest. The fumigant either significantly reduced or numerically decreased populations on all varieties. Two varieties had very high populations of root-knot nematode at harvest in the untreated averaging 2560 and 1568 juveniles per 500 cc of soil for 83P99 (very susceptible) and DkS51-01 (moderately susceptible). These population levels would be considered to be very damaging to susceptible crops that would be planted in rotation with sorghum. Yields were similar between treated and untreated for each variety. Yields were also high for 83P99 which would make it a variety more likely to be planted by producers and more likely to cause problems to the next crop. Differences in population development and pathogenicity in isolates of reniform nematode have been reported from different states. Experiments were conducted to evaluate soybean responses to indigenous isolates of the reniform nematode (Rotylenchulus reniformis) in Louisiana. Microplot and greenhouse experiments were conducted to evaluate the comparative reproduction and pathogenicity of single egg-mass populations of R. reniformis isolated from West Carroll (WC), Rapides, Tensas and Morehouse (MOR) parishes of Louisiana. Data from full-season microplot trials displayed significant differences in reproduction and pathogenicity of the nematode with the commercial soybean cultivars REV 56R63, Pioneer P54T94R, and Dyna-Gro 39RY57. Significantly low population density was observed in the isolate from the MOR parish compared to that of the least reproducing WC isolate. The MOR isolate was also the most pathogenic and resulted in significantly less soybean plant and pod weights compared to the control. In 60 day greenhouse trials, susceptible cultivar Progeny P4930LL and the resistant germplasm lines PI 90763 and PI 548316 were added together with the same cultivars used in the microplot trials.  Similar to the microplot trials, the MOR isolate had the least level of reproduction compared to that of WC and presented the greatest level of pathogenicity. In both microplot and greenhouse trials, the soybean cultivar REV 56R63 had a significant reduction in reniform numbers compared to cultivars Pioneer P54T94R and Dyna-Gro 39RY57. A similar study was conducted to evaluate indigenous populations of reniform nematode on cotton. Comparative reproduction and pathogenicity of reniform nematode populations derived from single-egg mass and collected form West Carroll (WC), Rapides (RAP), Morehouse (MOR), and Tensas (TEN) parishes were evaluated on cotton in full-season (150 days) microplot, and 60-day greenhouse trials. Data from microplot trials showed significant differences among isolates of reniform nematode in both reproduction and pathogenicity on upland cotton cultivars Phytogen 499 WRF, Deltapine 1133 B2RF, and Phytogen 333 WRF. Across all cotton cultivars, MOR and RAP isolate had the greatest and the least reproduction value of 331.8 and 230.2, respectively. Reduction in plant dry weight, number of bolls, seed cotton weight, and lint weight was the greatest and the least for MOR and RAP isolate, respectively. The reproduction and pathogenicity of WC and TEN isolate was intermediate. In the greenhouse experiment, reproduction of MOR and RAP isolate across all cotton genotypes (three cultivars used in microplot experiment, one susceptible cultivar Stoneville 4947, and two germplasm lines MT2468 Ren3, and M713 Ren5) was the greatest (reproduction value 10.7) and the least (reproduction value 7.9), respectively. Although reproductions of reniform nematode were lower in the germplasm lines than the cultivars, the germplasm lines sustained greater plant weight loss. The variability in reproduction and pathogenicity among endemic populations of reniform nematode in both the microplot and greenhouse experiments adds further support to the hypothesis that virulence phenotypes of R. reniformis exist. Experiments were established to develop a short during test to detect variability among isolates of cotton and soybean using root-associated life stages. Isolates of the nematode from eight cotton-producing parishes focused solely on reproduction of the root-associated infective and swollen female life stages with and without attached egg masses on the cotton genotypes MT2468 Ren 3, M713 Ren 5, and Stoneville 4946 and the soybean genotypes PI 548316, PI 90763, and Progeny 4930LL.  Data from greenhouse-based, 30-d-duration tests showed significant differences in life stage totals per root system among the eight isolates. Data from subsequent greenhouse studies with isolates of the nematode from West Carroll (WC), Morehouse (MOR), Rapides (RAP), and East Carroll (EC) parishes showed that on cotton there were significantly greater numbers of females with egg masses and total life stages on roots for the isolate from WC than for the other 3 isolates. Subsequent laboratory tests with durations of 14-21 days employed the same isolates previously described. Soybean and cotton plants were grown either in steam-sterilized soil or in soilless Cyg germination pouches. Overall, genotypes of cotton were better able to distinguish populations of the nematode on roots than were the genotypes of soybean. After 14 days for both cotton and soybean, the greatest numbers of infective and swollen females and root totals were observed with the WC isolate of the nematode. After 21 days, numbers of swollen females with egg masses and root totals for cotton were significantly greater for the WC isolate than for the other isolates. Germination pouches showed that, on tomato, the WC and RAP isolates had greater numbers of swollen females and total root stages than the other two isolates. Total egg mass contents, the sum of the numbers of eggs and hatched juveniles, were greatest for the WC isolate of the nematode and averaged 30 per egg mass. White tip disease of rice caused by Aphelenchoides besseyi has been considered a minor pest of rice during the past 50 years in the United States. Recently this nematode has been found in a number of quarantine samples in Louisiana and Arkansas. Objectives of this research were to determine incidence of this nematode in commercial seed sold to producers in Louisiana and to determine the host status of major cultivars currently produced in the state. During 2015-2016, a total of 286 seed samples representing 3 medium grain, 18 long grain, and 4 long grain hybrid cultivars were examined for A. besseyi. The nematode was detected in 12% of the samples and the highest incidence occurred on long grain hybrids with 30% of the 63 samples infested. Nineteen-week-duration greenhouse studies were conducted to evaluate reproduction of the nematode and pathogenicity to three medium, three long grains, and three long grain hybrid rice cultivars currently popular in Louisiana. Reproductive values of 11.9 and 2.9 were obtained for medium grain cultivar Jupiter and long hybrid XL 753, respectively. Grain weights of Jupiter, CL 111 and XL 753 plants inoculated with A. besseyi were significantly reduced below those of non-inoculated controls. There were significant reductions in plant height for all cultivars, except the long grain cultivar CL 152. Weights of Jupiter, CL 111, CL 152, XL 745 and XL 753 plants were reduced significantly when inoculated with A. besseyi. Germination and seedling growth studies conducted in the laboratory and greenhouse indicated that A. besseyi had a negative effect of 27% on percentage of seeds germinating of medium grain Jupiter. However, the nematode had a significant negative impact of 0.64 in average on the rate of germination for all cultivars except the medium grain Caffey.

Minnesota (S. Chen).  In 2017, a total of 93 private and public soybean cultivars were assayed for their resistance to SCN HG Type 7 (race 3) in the greenhouse. A number of soybean germplasm lines, most of which were in MG 000-II, were retested for their resistance to SCN race 1 and/or race 14.  Soybean breeding lines with PI 567615C source of resistance were evaluated for their resistance to SCN populations in the greenhouse, and a few of them were tested for yield in fields.  A total of 119 pennycress germplasm lines in the UMN breeding program were evaluated for their resistance to SCN.  None of the pennycress lines are highly resistant to the nematode. Biological seed treatments for Soybean Cyst Nematode (Heterodera glycines) management. One strategy for H. glycines management is with biological control. A test was conducted in the greenhouse at Mississippi State University to determine the efficacy of selected biological products. Treatments included seeds treated with ALB EXP Bacteria 1, 2, & 3, Burkholderia sp. alone and in combination with Bacterial metabolite, Saponin, and Harpin, a standard Abamectin and an untreated control. All seeds were treated by Albaugh, LLC. The study included effects on plant growth and nematode life stage development. Seeds were placed in 2.54 cm depressions in a steam sterilized sand: soil mix in 10 cm dia clay pots. H. glycines, 2500 eggs, was added on top of the seed for each treatment. The test was arranged in a RCB with 5 replications and ran for 60 days. At harvest, no negative effects were recorded from any treatment on soybean growth.  Seed treatments significantly reduced eggs and cysts of H. glycines compared with the untreated control. Seed treatments were similar in efficacy to the standard, Abamectin. H. glycines J2 numbers were significantly lower in the seed treatments compared with the control except in treatments ALB EXP Bacteria 1 and 2.  When two systemic acquired resistant products were added to Burkholderia sp., both cyst and egg numbers were lower compared to Burkholderia alone. Future research will focus on stacking different modes of action to enhance nematicidal activity.

Mississippi (G. Lawrence and V. Klink).  Agricultural chemical companies and developmental products currently designed for nematode control in row and vegetable crops. Efficacy studies have been conducted in 2017 with the products listed in Table 1 to determine their effect on nematode infestations of field crops. Many are still in their early developmental stages therefore only numbers or codes are available for some of the listed products.

Table 1. Experimental and Existing Nematicide Products examined in Mississippi by Company, Product and Application Method.

Missouri (H. Nguyen and M. Klepadlo).  Development of markers and genotyping assays.  For diagnostic purposes we use rhg1-2 and rhg1-5 SNP markers for detection Peking-type vs. PI 88788-type of rhg1, Rhg4-3 and Rhg4-5 for Rhg4, and O-8 and B1-7 for detection of SCN QTL on Chr. 10 and 11, respectively. We are advancing in development of SNP markers and KASP assay for detection RN resistance QTL on Chrs. 11 and 18.  Breeding and germplasm development.  Breeding and germplasm development is done in collaboration with soybean breeder in Southern MO breeding station at Fisher Delta Center in Portageville MO. We use genotyping methods to confirm source of known resistance in experimental breeding lines before commercial release. Nguyen lab developed experimental lines with pyramided genes in various combinations to test impact of each gene to resistance to different SCN races. Moreover, we extensively work on introducing novel SCN resistance QTL from PI 567516C into high yielding backgrounds.

Tennessee (Tarek Hewezi, Feng Chen and Reza Hajimorad).  We have hypothesized that nematodes, similar to other organisms, are hosts to viruses. The pathogenic viruses of nematodes can be used directly as bionematicide while those non-pathogenic can be modified genetically for application as bionematicide. As reported in “Annual Progress Report 2016”, for the sake of evaluating any potential pathogenic virus of SCN, a virus-free nematode population is needed. However, according to the literature, all SCN laboratory races or naturally occurring field populations harbor at least one or more persistent viruses. In search of a virus-free nematode population serving as an experimental nematode, we have focused on sugar beet cyst nematode (BCN) and have examined, via Illumina sequencing combined with bioinformatics, its transcriptomes derived from eggs and J2s for absence of viruses. It should be noted that BCN (Heterodera schachtii) is a close relative of, and can intermate with, SCN (Heterodera glycines). The two species of nematodes differ primarily in their preferred hosts. BCN feeds preferentially on cruciferous plants, while SCN feeds primarily on soybean. Following transcriptome sequencing, they were trimmed and the non-nematode-like sequence reads were assembled into a total of 35,232 and 26,196 contigs for eggs and J2, respectively. These contigs were searched for the presence of known SCN viruses. The known viruses of SCN are SCN nyavirus (ScNV) (family: Nyamiviridae), SCN rhabdovirus (ScRV) (Family: Rhabdoviridae), SCN phlebovirus (ScPV) (Family: Bunyaviridae), SCN tenuivirus (ScTV) (Family: Bunyaviridae) and SCNV 5 pestivirus (SbCNV-5) (Family: Flaviviridae). The genomes of all these viruses, except that of SbCNV-5, consist of single-stranded negative-sense RNA. SbCNV-5 is currently the only known virus from SCN with single-stranded positive-sense RNA genome. The contigs derived from BCN transcriptome data for both eggs and J2s were screened against respective sequences of the above viruses available in GenBank using TBLASTX. Out of 35,232 contigs that assembled from SBCN eggs transcriptome sequence data, a total of 250 contigs were significantly (e-value <0.01) similar to the partial genomic sequences of the L segment of the ScTV with the longest being 6430 nucleotide long (e-value 9.40e-79). Out of 25,196 contigs assembled from the transcriptome sequence data derived from J2s, a total of 218 of contigs were significantly (e-value <0.01) similar to the partial genomic sequence of the ScTV with the longest being 6418 nucleotide long (e-value 8.24e-79). This virus was provisionally named Sugar beet cyst nematode virus 1 (SBCNV-1). The result of a BLASTP search against the GenBank database using the deduced amino acid sequence of SBCNV-2 showed 28% identity with the L-segment of Uukuniemi virus encoding RNA-dependent-RNA polymerase (RdRp) (e-value =7e-145). Hence, SBCNV-1 likely belongs to the genus Phlebovirus in the family Bunyaviridae. Based on pair-wise comparison of the deduced amino acids of its putative RdRp, it showed 27% identity (e-value = 1e-117) with ScTV and 25% with ScPV (e-value 1e-112). As far as any other known SCN viruses is concerned, none of the virus-like contigs in our study showed high similarity (e-value ranging e-08 to e-05) to their respective genomic sequences. Thus, our BCN culture likely lacked transcriptome sequences corresponding to all of the known SCN viruses. However, perhaps transcriptome data from a much diverse laboratory cultures along with naturally occurring populations of BCN are needed to fully identify its RNA viromes.  Interestingly, we have also identified in the sequence pools derived from both eggs and J2s a novel positive-sense RNA virus that provisionally named beet cyst nematode virus-2 (BCNV-2). This novel RNA virus was present in BCN populations from Iowa and Missouri as confirmed by RT-PCR reactions.  We have almost completed its full-length genomic sequence that is ~9503 nucleotides long. Determination of its precise 5' end sequences by RACE as well as its phylogenetic relationships with related viruses affecting other organisms is underway.

Texas (T. Wheeler).  Combination of crop rotationiIrrigation rate/Variety:  With declining irrigation pumping capacity in the Southern High Plains, many producers are choosing to plant wheat in the fall after cotton harvest, and then fallow the land the following year after the wheat is harvested.  The effects of this rotation were compared with continuous cotton, when root-knot nematode resistant cultivars and three irrigation rates were also incorporated into the management program.  The varieties included in this 3-year study were: DP 1454NRB2RF (2 gene resistance, possibly on chromosome 11 and 7); FM 2011GT (no known genes for resistance, possibly some tolerance); NG 1511B2RF (root-knot susceptible variety with good yield potential in this region); PHY 417WRF (2-genes for resistance on chromosome 11 and 14); ST 4946GLB2 (1-gene for resistance).  The root galling early in the season was higher in continuous cotton than the wheat/cotton rotation.  The highly resistant PHY 417WRF had fewer root galls than any other variety (Table 1).  FM 2011GT had the highest number of galls.  There were no differences in the number of galls between the varieties in the wheat/cotton rotation (Table 1).  Root-knot nematode density was lower for PHY 417WRF in both cropping systems compared to all other varieties.  There were no differences in nematode density for the continuous cotton system, but in the wheat/cotton rotation, DP 1454NRB2RF and ST 4946GLB2 had lower root-knot nematode densities than FM 2011GT (Table 1).

Table 1. Influence of cotton/winter wheat/summer fallow (WC) cropping system compared to continuous cotton (CC) with a wheat or rye cover crop, on root-knot nematodes, 2014 to 2016.

¹Means followed by a different letter indicate that the varieties were significantly different at P=0.05.  The root-knot nematode densities were LOG10(x+1) transformed before analyzing.

Cotton yields in these two cropping systems and three irrigation rates, were analyzed for variety and variety x irrigation rate effects for 2014 - 2016.  There was no variety x irrigation rate interaction, so the main effects of variety are presented. The wheat/cotton rotation yielded more (1,003 lbs of lint/acre) than the continuous cotton system (713 lbs of lint/acre, P < 0.001).  In the continuous cotton system, ST 4946GLB2 had higher yields than all varieties except for PHY 417WRF (Table 2).  In the wheat/cotton rotation, ST 4946GLB2 and NG 1511B2RF had higher yields than DP 1454NRB2RF and PHY 417WRF.  There did not appear to be any advantage to nematode resistant genes in the wheat/cotton cropping system, but there did appear to be one in the continuous cotton system.  The root-knot nematode susceptible varieties yielded as well or better than the root-knot nematode resistant varieties.  DP 1454NRB2RF did not yield well in either system, but this is a long season variety which is a poor fit for the southern High Plains of Texas. There were probably other benefits besides a reduction in root-knot nematode density to the wheat/cotton cropping system, particularly with storage of moisture in the soil profile. There was a 290 lb/acre lint increase on average in the wheat/cotton system, and even highly resistant PHY 417WRF had a 188 lb/acre lint increase.  Root-knot nematode was almost eliminated in the PHY 417WRF plots in the wheat/cotton system. This rotation system was not as successful when examined 20 years ago, when pumping capacities were higher. However, with declining water (the irrigation rates in this study averaged 3.1, 4.6, and 6.2 inches/growing season), the advantages of taking part of the land out of cotton production each year and planting a winter crop like wheat and then fallowing the land can be profitable.  Leaving the land bare (only fallow), will allow more runoff of rain, and probably promote more weed issues.  Planting dryland cotton on half of the circle to conserve water will not result in storage of moisture in the soil profile. This wheat/fallow/cotton system appears to offer advantages to this region, especially when combined with several years of root-knot nematode resistant varieties. The cropping system/irrigation rate studied started a new cycle of varieties in 2017, that included four root-knot nematode susceptible varieties and ST 4946GLB2.

Table 2. Effect of cotton/winter wheat/summer fallow (WC) cropping system compared to continuous cotton (CC) with a wheat or rye cover crop, on cotton lint yield, 2014 to 2016.

¹Means followed by a different letter indicate that the varieties were significantly different at P=0.05. 

Field testing varieties for nematode resistance:  Small plot variety/advanced commercial line trials were conducted in several commercial cotton fields.  Plots were 2 to 4-rows wide, 36 feet long, on 40-inch centers.  All trials were irrigated by the producers.  Varieties with either 2-gene resistance (DP 1558NRB2RF, PHY 417WRF) or 1-gene resistance (ST 4946GLB2) were included in the trials.  Several susceptible varieties were also included. There appears to be more interest by producers in planting susceptible varieties in root-knot nematode fields, than using varieties with some resistance/tolerance to root-knot nematodes. Of the new experimental lines tested in 2017, Monsanto 16R246NRB2XF and 17R942NRB3XF appear to reduce root-knot nematode densities (Table 3). Phytogen experimentals with good root-knot nematode resistance include PX2AX4W3FE, PX3A82W3FE, and PX4A52W3FE. 

Table 3. Root-knot nematode (RK) densities on cultivars for trials in 2017.