NEMATODE MANAGEMENT IN ANNUAL CROPS WITH EMPHASIS ON HOST PLANT RESISTANCE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1011608
• Project Start Date: Nov 22, 2016
• Project End Date: Sep 30, 2021
• Program Code: [(N/A)]- (N/A)

Project Director
Roberts, P.

Recipient Organization
UNIVERSITY OF CALIFORNIA, RIVERSIDE
(N/A)
RIVERSIDE,CA 92521

Performing Department
Nematology, Riverside

Non Technical Summary
Nematodes attacking crop plants cause more than $1 billion in crop losses worldwide each year. Current control of nematodes is based mostly on chemical nematicides which have significant human health and environmental risks associated with their use. This project will focus on the most important group of nematode pests attacking California field and vegetable crops, the root-knot nematodes. The project will create and extend new knowledge on nematode pest biology and genetics, emphasizing nematode relationships with host plant resistance. There are valuable natural nematode resistance trait resources in the germplasm of the target crops, which will be minedfor use in breeding resistant varieties for growers. The knowledge will be used to develop new nematode resistant crop varieties, develop molecular markers for use in marker-assisted selection in crop breeding programs, and determine the best strategies for implementing resistant crops in annual cropping systems using field-based experimentation. Cutting edge genomics technologies will be applied to study nematode and plant genetics and for new variety breeding. These include lab, greenhouse and field-based screening for plant resistance traits, and the use of molecular sequence data for development and application of markers for resistance traits. This will expedite the breeding selection process to advance the best performing breeding lines carrying resistance.The project will focus on carrots, beans, cowpeas and cotton, which are grown in rotation with many other field and vegetable crops. This work will improve nematode management programs and thereby help enhance the productivity and profitability of California farmers. As such, this project supports and broadens the approaches and tools available for management of the most important endemic nematode pests of California crops. New knowledge and tools outputs will be shared with public and private sector plant breeders, seed companies, and grower and processor groups for the targeted crops, via peer and popular publications, field days and conference presentations.The project is important to agriculture as a whole and to Californiaand similar production areas.Plant parasitic nematodes cause significant losses in production and quality of field and vegetable crops worldwide. These losses occur in both subsistence and highly mechanized agricultural systems. Effective management practices for nematodes can increase productivity of land and growers' income, improve quality of food and fiber and give more uniform emergence, growth and maturity of crop plants. Over the last fifty years, nematode management has been based primarily on the use of soil-applied nematicidal agents that either kill nematodes or inhibit their parasitic ability. However, a major shift away from the use of nematicides is occurring because of problems associated with human health and environment risks, and the prohibitive cost structure of developing new nematicides and of using currently available nematicides in many low cash value production systems.Through better understanding of the ecology, biology and pathology of known or suspected nematode pathogens of plants, non-chemical management strategies may be made more effective and, where suitable, might be integrated with existing or modified nematicide use in a broad based management program. Currently the best alternative controls in field and vegetable crops are based on host plant resistance that protects both the resistant crop and the following susceptible crops in annual rotations. Natural host plant resistance is a major resource in agriculture for developing nematode management tools and programs. The potential for resistance has been achieved in some crops and situations but not in many others, reflecting that only a small portion of natural host plant resistance has been utilized.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	1719	1080	30%
212	1499	1120	40%
212	2499	1120	30%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1499 - Vegetables, general/other; 1719 - Cotton, other; 2499 - Plant research, general;

Field Of Science
1120 - Nematology; 1080 - Genetics;

Keywords

carrot, cowpea, lima bean, common bean, cotton

nematode resistance breeding

integrated nematode pest management

crop genetics

Goals / Objectives
Thegoalof this project is to identify, analyze and implement host plant resistance traits wherever feasible. The justification for this approach is based on several considerations: 1) resistance has a proven track record of effectiveness in managing nematodes in agriculture, 2) resistance genes in crops provide an environmentally benign pest management protocol, 3) the cost of resistance use is compatible with both subsistence level as well as high input agricultural systems, and 4) a huge untapped resource of natural host plant resistance genes is available for possible development. The most effective developments in this area will be based on enhanced and new knowledge of the identity, nature, mode of operation and effects in cropping systems of resistance genes. Coupled with understanding resistance in the plant is the need to understand the relationship of resistance to the matching parasitic ability in the nematode. That gene-for gene matching of plant resistance genes and nematode virulence genes occurs has been established, and the genetic basis for the virulence condition in the nematode requires much further research. New knowledge in this area should lead to more effective selection and deployment of resistance genes for crop protection, and also to the promotion of resistance gene durability. An additional aspect of resistance use is the examination of integrative procedures for nematode management programs, whereby resistance is combined with other nematode and interacting plant pathogen management tactics.Specific Objectives:1. Examine nematode biology in nematode interactions with soil and with plant hosts, specifically including the abiotic factors temperature and moisture, and biotic factors of interacting organisms (fungi, bacteria, other nematodes) and plant host status.2. Develop, refine and implement management strategies for root-knot and comparative nematodes on field and vegetable crops, emphasizing non-host rotations, resistant and tolerant cultivars and nematicides, singly or integrated where appropriate.3. Identify, characterize, introgress and implement host plant resistance traits, corresponding to genetic variation for parasitism in nematode populations.

Project Methods
Objective 1. In both field station (UCR Agricultural operations, South Coast REC, Kearney REC), and grower field sites, established root-knot nematode infestations will be assessed for population density and uniformity. Soil samples and root samples will be processed for total nematode population densities using standard protocols to determine trial sites for field phenotyping for resistance and infection responses. Laboratory, greenhouse and field tests will be used to establish the resistance response in target crop germplasm and genetic populations based on levels of root galling and nematode reproduction in roots, according to standard techniques. Population studies under laboratory and field conditions will be used in conjunction with chemical and other control procedures to determine damage functions and tolerance thresholds of selected nematodes with respect to crop plant genotype differences. Population dynamics and pathogenic capabilities of commonly occurring mixed populations of different nematode species and interacting organisms will be evaluated under greenhouse and field conditions. The relationships between root-knot nematodes and Fusarium wilt fungi on cowpea and cotton plants will be studied for genotype x environment interactions in multifactorial experiments. This will be done by seasonal monitoring and measurement of disease progression and plant phenology, using plant genotypes with specific resistance and susceptibility to nematode and wilt populations, singly and in various combinations using published procedures.Objective 2. Field experiments will be used to determine the feasibility of non-host and resistant cultivar based crop rotations for root-knot nematode management. The frequency, sequence and combinations of resistance traits in different crops will be studied in small field plot and microplot experiments. Multiple year experiments will be run in which changes in nematode population densities in soil will be assayed using standard sampling and extraction techniques at the beginning and end of each crop cycle. Seasonal multiplication rates will be calculated from the soil density assays. Root infection based on root-galling indices and nematode egg numbers per unit weight of root and plant growth or yield based on fresh and dry weights of fruit or shoots will be measured. Experiments will be designed as randomized complete block, split-block, or split-plot arrangements with four to six replications. Plant genotype treatments will be compared with and without nematode inoculum, and for each crop with a resistant cultivar, a susceptible cultivar check treatment will be compared. Crops of major focus with resistance to root-knot nematode (Meloidogyne spp.) will include Lima bean (Phaseolus lunatus) common bean (P. vulgaris), cotton (Gossypium hirsutum and G. barbadense), carrot (Daucus carota), and cowpea (Vigna unguiculata). Data will be analyzed using standard ANOVA tests, orthogonal contrasts, and simple and multiple regression.Objective 3. In cooperation with plant breeders, current techniques will be used to assess the level of nematode resistance or susceptibility to species and biotypes of root-knot nematodes, including growth pouch, greenhouse-pot, and field-plot screens). Of particular interest are the Meloidogyne resistance gene systems in cotton, carrot, cowpea, Lima bean and common bean. Characterization of genetic control of resistance traits will be made using classical Mendelian approaches in conjunction with molecular marker development, genotyping, and application. Crossing and selfing will be used to generate segregating populations, including recombinant inbred and near-isogenic lines, for use in marker development and resistance gene mapping. Emphasis will be placed on SSR (microsatellite) and SNP analyses for generating molecular markers and for mapping resistance traits. Methods will be based on those employed previously, for example as described by Boiteux et al. (2004), and Wang et al. (2008), and Muchero et al. 2009a, 2011). This will enable tests on resistance expression using plants of known genetic constitution. Genetic analyses will provide plant populations and genetic mapping information to develop marker-assisted breeding protocols and for map-based gene-cloning. We will focus on using SNP markers derived from ESTs or genotyping -by-sequencing. SNP genotyping in cowpea and cotton will be conducted using the newly developed Illumina iSelect SNP arrays for each crop plant, coupled with QTL analysis using MapQTL 5.0 software. Synteny relationships with the annotated soybean and common bean, cotton and carrotsequences and with other sequenced plant genomes will be used for candidate gene identification, as we have done for Macrophomina resistance (Muchero et al., 2011). BAC libraries in cotton and cowpea will be utilized for physical mapping and isolation of target resistance genes, and for increasing marker density in the QTL regions of interest. Molecular and genomic characterization of resistance genes will guide decisions on the priority and combinations of resistance gene introgression for crop improvement, including preferred genes for cloning.A living collection of Meloidogyne spp. will continue to be maintained by greenhouse culture and in cryopreservation. The collection of isolates with specific (a)virulence characteristics matching host resistance genes represent a diverse collection of M. arenaria, M. javanica, M. hapla and M. incognita populations. The analysis of the (a)virulence traits and specificities in the nematode isolates will be determined in isofemale lineages of M. incognita as used previously, and in controlled matings and molecular fingerprinting in populations of M. hapla. Absolute and relative fitness assessments will be made by measuring life-stage development and fecundity in selected and unselected nematode lines raised for multiple generations on plant genotypes with or without known resistance genes.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Primary target audiences were commercial producers (growers) of cotton, grain legumes, and fresh market carrots. Secondary target audiences included public and private cotton, grain legume, and carrot breeders and seed producers. Through these secondary target audiences, the new knowledge and availability of nematode resistance genes, associated molecular markers, and advanced breeding lines or varieties carrying resistance are being applied for modern breeding of improved crop varieties. Changes/Problems:No major changes or problems were encountered. What opportunities for training and professional development has the project provided?Four undergraduate students have worked on various aspects of the project research in the reporting period, plus two graduate students and two postdoctoral fellows. The project provides an excellent training ground for general research experience and more specifically for interest in plant nematology and pathology, and plant genetics, genomics and breeding. Also, with an international focus on the cowpea genetics and breeding part of the project, these students are gaining an international educational training and perspective, with collaborating students and scientists from Africa having visited UCR during the report period. How have the results been disseminated to communities of interest?Results were disseminated through three field days at field experimentation sites, annual reports to the agricultural industry and funding agencies, through peer-reviewed journal and commodity report publications, and presentations at three grower-industry meetings and three professional society meetings. What do you plan to do during the next reporting period to accomplish the goals?Continuation of experiments conducted in the last reporting period are planned in the next reporting period. The best improved breeding lines will be screened with multiple nematode isolates, and advanced to the next generation. Application of new genomic marker technologies will be made for genetic analysis of resistance traits in the three target crop species. Repeat experiments are needed to validate and confirm results from the current and previous years.

Impacts
What was accomplished under these goals? Host plant resistance to root-knot nematodes (Meloidogyne spp.) and interacting organisms was studied as a major integrative tactic for nematode management programs in annual field and vegetable cropping systems. Resistance traits in carrot, cotton and cowpea (blackeye beans) were investigated at the molecular and genome organization levels, in conjunction with trait determinants of agronomic characters (e.g. cowpea seed size and color; carrot root shape, color and nutritional quality; cotton fiber quality) and resistance to other biotic stresses including fungal pathogens and insects. QTL for resistance to root-knot nematodes and to the interacting charcoal rot or ashy stem blight disease were mapped in cowpea populations using field and greenhouse phenotyping screens. Previously we mapped the Rk locus in cowpea for resistance to M. incognita and some M. javanica populations on cowpea linkage group VuLG11. Additional analysis confirmed the position of the Rk locus, and also identified a second locus for gene Rk2 on cowpea VuLG9 associated with resistance to M. javanica reproduction. Flanking SNP markers for the locus were identified for use in marker-assisted breeding for nematode resistance. Transcriptome analysis on resistant and susceptible near-isogenic resistant and susceptible lines three and nine days after inoculation revealed six differentially expressed genes within the two resistance gene QTL intervals, which belong to the TIR-NBS-LRR family of resistance genes of which three were upregulated at one or more time-points. Quantitative RT-PCR validated gene expression to be positively correlated with RNA-seq expression pattern for these candidate R genes. Functional analysis of these cowpea R genes is almost complete, using Agrobacterium rhizogenes-mediated root transformation of susceptible cowpea with sequence inserts of each candidate R gene, singly and in combinations. Two infested field nurseries at the UC Kearney Research Station in Parlier, CA were used for selection of nematode resistance among more than 1100 fresh market carrot lines developed for high nutritional content and resistance to M. javanica and M. incognita. In 2019 more than 70 percent of the morst advanced breeding lines showed strong dual resistance in these trials. The high percentage of very resistant entries indicates selection for resistance in the 2015 to 2018 field nursery trials under intense nematode pressure was very effective. Roots from the most resistant lines and with preferred root shape and color were sent to USDA Wisconsin for selfing or crossing. A carrot resistance panel representing the 11 best diverse sources and combinations of resistance genes was greenhouse screened for infection response to additional isolates of M. hapla. Indices of virulence and resistance were developed based on nematode root-galling symptoms. Lines resistant to all nematode isolates were identified, in particular, a selection from carrot line Homs with very strong resistance, from which a heterozygous plant was selfed to form a segregating F2 population for genetic analysis of the resistance. This population was greenhouse-phenotyped with M. hapla and genotyped by GBS (genotyping-by sequencing); the data were used for genetic mapping of the resistance, from which one major effect QTL was located in the carrot genome. A sub-set of the resistance panel was tested with M. javanica in temperature tanks to determine sensitivity of resistance to high temperature. Sources of resistance were identified that were effective against M. javanica at 31 C and 34 C constant soil temperatures.

Publications

• Type: Other Status: Published Year Published: 2019 Citation: Roberts PA, Huynh BL, Clark NE, Matthews WC, Frate CA. 2019. Blackeye Varietal Improvement. p. 1-9. In University of California Dry Bean Research 2018 Progress Report, California Dry Bean Advisory Board, Dinuba, CA.
• Type: Other Status: Published Year Published: 2019 Citation: Roberts PA, Matthews WC, Simon PS. 2019. Identification of gene sources for resistance to root-knot nematodes. pp. 40-61. In 2018 Annual Report, California Fresh Carrot Advisory Board, Dinuba, CA.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Batieno TBJ, Souleymane O, Tignegre J, Huynh B-L, Kusi F, Poda SL, Close TJ, Roberts P, Danquah E, Ofori K, Ouedraogo TJ (2018) Single nucleotide polymorphism (SNP)-based genetic diversity in a set of Burkina Faso cowpea germplasm. African Journal of Agricultural Research 13:978-987
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Lonardi S, Mu�oz-Amatria�n M, Liang Q, Shu S, Wanamaker SI, Lo S, Tanskanen J, Schulman AH, Zhu T, Luo M-C, Alhakami H, Ounit R, Hasan AM, Verdier J, Roberts PA, Santos JRP, Ndeve A, Dole~el J, Vr�na J, Hokin SA, Farmer AD, Cannon SB, Close TJ (2019) The genome of cowpea (Vigna unguiculata [L.] Walp.) The Plant Journal 98:767782. DOI: 10.1111/tpj.14349.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Huynh BL, Ehlers JD, Close TJ, Roberts PA (2019) Registration of a cowpea [Vigna unguiculata (L.) Walp.] multiparent advanced generation intercross (MAGIC) population. Journal of Plant Registrations 13: 281-286.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Ndeve AD, Santos JRP, Matthews WC, Huynh B-L, Guo Y-N, Lo S, Mu�oz-Amatria�n M, Roberts PA (2019) A novel root-knot nematode resistance QTL on chromosome Vu01 in cowpea. G3 (Genes, Genomes, Genetics) 9:1199-1209.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Primary target audiences were commercial producers (growers) of cotton, grain legumes, and fresh market carrots. Secondary target audiences included public and private cotton, grain legume, and carrot breeders and seed producers. Through these secondary target audiences, the new knowledge and availability of nematode resistance genes, associated molecular markers, and advanced breeding lines or varieties carrying resistance are being applied for modern breeding of improved crop varieties. Changes/Problems:No major changes or probelms were encountered. What opportunities for training and professional development has the project provided?Four undergraduate students have worked on various aspects of the project research in the reporting period, plus two graduate students andtwo postdoctoral fellows. The project provides an excellent training ground for general research experience and more specifically for interest in plant nematology and pathology, and plant genetics, genomics and breeding. Also, with an international focus on the cowpea genetics and breeding part of the project, these students are gainng an international educational training and perspective, with collaborating students and scientists having visited UCR during the report period. How have the results been disseminated to communities of interest?Results were disseminated through three field days at field experimentation sites, annual reports to the agricultural industry and funding agencies, through peer-reviewed journal and and commodity report publications, and presentations at three grower-industry meetings and three professional society meetings. What do you plan to do during the next reporting period to accomplish the goals?Continuation of experiments conducted in the last reporting period are planned in the next reporting period. The best improved breeding lines will be screened with multiple nematode isolates, and advanced to the next generation. Application of new genomic marker technologies will be made for genetic analysis of resistance traits in the target crop species. Repeat experiments are needed to validate and confirm results from the current and previous years.

Impacts
What was accomplished under these goals? Host plant resistance to root-knot nematodes (Meloidogyne spp.) and interacting organisms was studied as a major integrative tactic for nematode management programs in annual field and vegetable cropping systems. Resistance traits in carrot, cotton and cowpea (blackeye beans) were investigated at the molecular and genome organization levels, in conjunction with trait determinants of agronomic characters (e.g. cowpea seed size and color; carrot root shape, color and nutritional quality; cotton fiber quality) and resistance to other biotic stresses including fungal pathogens and insects. QTL for resistance to root-knot nematodes and to the interacting charcoal rot or ashy stem blight disease were mapped in cowpea populations using field and greenhouse phenotyping screens. Previously we mapped the Rk locus in cowpea for resistance to M. incognita and some M. javanica populations on cowpea linkage group VuLG11. Additional analysis confirmed the position of the Rk locus, and also identified a second locus for gene Rk2 on cowpea VuLG9 associated with resistance to M. javanica reproduction. Flanking SNP markers for the locus were identified for use in marker-assisted breeding for nematode resistance. Transcriptome analysis on resistant and susceptible near-isogenic resistant and susceptible lines three and nine days after inoculation revealed 13 important differentially expressed genes within the two resistance gene QTL intervals. Six of these genes belong to the TIR-NBS-LRR family of resistance genes and three were upregulated at one or more time-points. Quantitative RT-PCR validated gene expression to be positively correlated with RNA-seq expression pattern for eight genes. Functional analysis of these cowpea R genes is being pursued using Agrobacterium rhizogenes mediated root transformation of susceptible cowpea with each candidate R gene. Two infested field nurseries at the UC Kearney Research Station in Parlier, CA were used for selection of nematode resistance among more than 1600 fresh market carrot lines developed for high nutritional content and resistance to M. javanica and M. incognita. In 2018 more than 75 percent of the more advanced breeding lines showed strong dual resistance in these trials. The high percentage of very resistant entries indicates selection for resistance in the 2015to 2017 field nursery trials under intense nematode pressure was very effective. Roots from the most resistant lines and with preferred root shape and color were sent to USDA Wisconsin for selfing or crossing. A carrot resistance panel representing the 11 best diverse sources and combinations of resistance genes was greenhouse re-screened for infection response to 11 isolates of M. hapla. Indices of virulence and resistance were developed based on nematode root-galling symptoms. Lines resistant to all nematode isolates were identified, in particular, a selection from carrot line Homs with very strong resistance, from which a heterozygous plant was selfed to form a segregating F2 population for genetic analysis of the resistance. Field and grreenhouse screening of a set of 694 carrot germplasm entries obatined from the USDA germplasm center in Ames, Iowa, revelqed a subset of about 20 entries with resisanace to M. incognita, some of which were also resistant to M. javanica and M. hapla. These were sent to USDA-ARS, Madison Wisconsin for developing new carrot breeding populations.

Publications

• Type: Other Status: Published Year Published: 2018 Citation: Roberts PA, Huynh BL, Matthews WC, Frate CA. 2018. Blackeye Varietal Improvement. p. 1-9. In University of California Dry Bean Research 2017 Progress Report, California Dry Bean Advisory Board, Dinuba, CA.
• Type: Other Status: Published Year Published: 2018 Citation: Roberts PA, Matthews WC, Simon PS. 2018. Identification of gene sources for resistance to root-knot nematodes. pp. 41-64. In 2017 Annual Report, California Fresh Carrot Advisory Board, Dinuba, CA.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Ndeve, A. D., W. C. Matthews, J. R. P. Santos, B.-L. Huynh and P. A. Roberts. 2018. Broad-based root-knot nematode resistance identified in cowpea gene-pool two. Journal of Nematology 50:545-558. Doi: 10.21307/jofnem-2018-046
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Santos JRP, Ndeve A, Huynh BL, Matthews WC, Roberts PA. 2018. Transcriptome analysis of cowpea near-isogenic lines reveals candidate genes for root-knot nematode resistance. PLoS ONE 13 (1): e0189185.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Wang C, Ulloa M, Duong TT, Roberts PA. 2018. QTL analysis of transgressive nematode resistance in tetraploid cotton reveals complex interactions on chromosome 11 regions. Frontiers in Plant Science 8: 1979 p.1-12. doi: 10.3389/fpls.2017.01979
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Huynh B-L, Ehlers JD, Huang BE, Mu�oz-Amatria�n M, Lonardi S, Santos J, Ndeve A, Batieno BJ, Boukar O, Cisse N, Drabo I, Fatokun C, Kusi F, Agyare RY, Guo Y-N, Herniter I, Lo S, Wanamaker SI, Xu S, Close TJ, Roberts PA. 2018. A multiparent advanced generation inter?cross (MAGIC) population for genetic analysis and improvement of cowpea (Vigna unguiculata L. Walp.). The Plant Journal 93, 11291142.

Progress 11/22/16 to 09/30/17

Outputs
Target Audience:Primary target audiences were commercial producers (growers) of cotton, grain legumes, and fresh market carrots. Secondary target audiences included public and private cotton, grain legume, and carrot breeders and seed producers. Through these secondary target audiences, the new knowledge and availability of nematode resistance genes, associated molecular markers, and advanced breeding lines or varieties carrying resistance are being applied for modern breeding of improved crop varieties. Changes/Problems:No changes or problems. What opportunities for training and professional development has the project provided?Four undergraduate students have worked on various aspects of the project research in the reporting period, plus two graduate students and one postdoctoral fellow. The project provides an excellent training ground for general research experience and more specifically for interest in plant nematology and pathology, and plant genetics, genomics and breeding. ? How have the results been disseminated to communities of interest?Results were disseminated through three field days at field experimentation sites, annual reports to the agricultural industry and funding agencies, through peer-reviewed journal and book chapter publications, and presentations at three grower-industry meetings and three professional society meetings. What do you plan to do during the next reporting period to accomplish the goals?Continuation of experiments conducted in the last reporting period are planned in the next reporting period. The best improved breeding lines will be screened with multiple nematode isolates, and advanced to the next generation. Application of new genomic marker technologies will be made for genetic analysis of resistance traits in the target crop species. Repeat experiments are needed to validate and confirm results from the current year.

Impacts
What was accomplished under these goals? Host plant resistance to root-knot nematodes (Meloidogyne spp.) and interacting organisms was studied as a major integrative tactic for nematode management programs in annual field and vegetable cropping systems. Resistance traits in carrot, cotton and cowpea (blackeye beans) were investigated at the molecular and genome organization levels, in conjunction with trait determinants of agronomic characters (e.g. cowpea seed size and color; carrot root shape, color and nutritional quality; cotton fiber quality) and resistance to other biotic stresses including fungal pathogens and insects. QTL for resistance to root-knot nematodes and to the interacting Fusarium vascular wilt disease were mapped in cowpea populations using field and greenhouse phenotyping screens. Previously we mapped the Rk locus in cowpea for resistance to M. incognita and some M. javanica populations on cowpea linkage group VuLG11. Additional analysis confirmed the position of the Rk locus, and also identified a second locus for gene Rk2 on cowpea VuLG9 associated with resistance to M. javanica reproduction. Flanking SNP markers for the locus were identified for use in marker-assisted breeding for nematode resistance. Transcriptome analysis on resistant and susceptible near-isogenic resistant and susceptible lines three and nine days after inoculation revealed up-regulation of 109 and 98 genes and down-regulation of 110 and 89 genes, respectively, out of 19,922 unique genes mapped to the common bean reference genome. Among the differentially expressed genes, 13 genes were found within the two resistance gene QTL intervals. Six of these genes belong to the TIR-NBS-LRR family of resistance genes and three were upregulated at one or more time-points. Quantitative RT-PCR validated gene expression to be positively correlated with RNA-seq expression pattern for eight genes. Future functional analysis of these cowpea genes will enhance our understanding of Rk-mediated resistance and identify the specific gene responsible for the resistance. Two field trials were conducted on root-knot nematode sites infested with M. incognita and M. javanica at the Kearney Research Station, Parlier, to assay for grain yield among lines carrying different combinations of resistance genes. Two infested field nurseries at the UC South Coast Research Station in Irvine, CA were used for selection of nematode resistance among more than 1400 fresh market carrot lines developed for high nutritional content and resistance to M. javanica and M. incognita. In 2017 more than 75 percent (262 out of 367) of the more advanced breeding lines showed strong dual resistance in these trials. The high percentage of very resistant entries indicates selection for resistance in both the 2015 and 2016 field nursery trials under intense nematode pressure was very effective. Roots from the most resistant lines and with preferred root shape and color were sent to USDA Wisconsin for selfing or crossing. A carrot resistance panel representing the 11 best diverse sources and combinations of resistance genes was greenhouse re-screened for infection response to 11 isolates of M. hapla. Indices of virulence and resistance were developed based on nematode root-galling symptoms. Lines resistant to all nematode isolates were identified, in particular, a selection from carrot line Homs with very strong resistance, from which a heterozygous plant was selfed to form a segregating F2 population for genetic analysis of the resistance. Molecular analysis of M. incognita and Fusarium wilt races 1 and 4 resistance in Upland (Gossypium hirsutum) and Pima (G. barbadense) cotton was continued for marker development and mapping. The project employed a recombinant inbred line (RIL) population developed from a marker-validated chromosome 17 substitution line (carrying a G. barbadense substitution of chromosome 17 in G. hirsutum with influence on Fusarium race 4 resistance) which pinpointed a Fusarium race 4 resistance locus. An interspecific RIL population (Pima S7 x Acala Nemx) was also used for genetic mapping of resistance traits. Results confirmed the locations of resistance trait determinant QTL which had been identified previously by QTL mapping using SSR markers. Additional markers in the nematode and wilt resistance regions on chromosomes 11 and 21 were developed using the G. arboreum A2 whole genome sequence. These were compared to sequence of BAC clones from the two chromosomes and were remapped in populations segregating for nematode resistance. Results showed that only chromosome 11 contributed resistance to the nematode and Fusarium wilt pathogens, whereas its homoeologous chromosome 21 had no significant contribution to resistance. We completed analysis of resistance to M. incognita and Fusarium wilt races 1 and 4 in RILs generated from the Pima S-7 x Acala NemX cross. Transgressive segregation for nematode resistance was observed, confirming both parents contributed genes enhancing resistance expression. Under greenhouse conditions, Pima S-7 showed resistance to wilt race 1 and susceptibility to wilt race 4, but the reverse was found for Acala NemX (susceptible to race 1 and resistant to race 4). We extracted high quality DNA from leaf tissue of the RILs and the parents, which was used for SNP genotyping the population using the Illumina International Cotton SNP Consortium 63K Array. A total of 117 RI lines plus parents and controls were SNP genotyped. Integration of the SSR markers previously mapped to resistance regions with the new SNP markers is underway, as a fine-mapping approach to the genome regions with the resistance trait determinants. The SNP plus SSR integration will provide a new genetic map for the interspecific Pima S-7 x Acala NemX RIL population.

Publications

• Type: Book Chapters Status: Awaiting Publication Year Published: 2018 Citation: Sikora, R.A., Roberts, P.A. 2018. Management practices: an overview of integrated nematode management technologies. In Plant Parasitic Nematodes in Subtropical and Tropical Agriculture, 3rd edition. Editors: R. Sikora, J. Halleman, D. Coyne, P. Timper, eds. CAB International, Wallingford, UK. Pp. 1-62. (in press)
• Type: Book Chapters Status: Published Year Published: 2017 Citation: Roberts, P.A. 2017. Diseases caused by Nematodes. In: Compendium of Lettuce Diseases and Pests. 2nd edition. Editors: K.V. Subbarao, R.M. Davis, R.L. Gilbertson, R.N. Raid. American Phytopathological Society. APS Press, St Paul. p. 1-5.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: 1. Frigulti T, M Ulloa, RB Hutmacher, M Keeley, SD Wright, J Burke, RL Nichols, PA Roberts. 2017. Exploring Pima and Upland cross-combinations to identify Fusarium oxysporum f. sp. vasinfectum Race 4 resistant cottons by combining ability of superior cultivars. Proceedings Beltwide Cotton Conference, Dallas, TX, January 2017.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Leal-Bertioli, S.C.M., Moretzsohn, M.C., Roberts, P.A., Ball�n-Taborda, C., Borba, T.C.O., Valdisser, P.A., Vianello, R.P., Ara�jo, A.C.G., Guimar�es, P.M., Bertioli, D.J. 2016. Genetic mapping of resistance to Meloidogyne arenaria in Arachis stenosperma: a new source of nematode resistance for peanut. G3 Genes, Genomes, genetics Vol 6:377 p.p.1-14. doi: 10.1534/g3.115.023044.
• Type: Other Status: Published Year Published: 2017 Citation: Roberts, PA 2017 Identification of gene sources for resistance to root-knot nematodes: 2016 research report California Fresh Carrot Advisory Board p. 1-12 California Fresh Carrot Advisory Board, Dinuba, CA
• Type: Other Status: Published Year Published: 2017 Citation: Roberts, PA, Huynh, BL, Clark NE, Matthews, WC, Frate, CA 2017. Blackeye varietal improvement: University of California Dry Bean Research 2016 California Dry Bean Advisory Board, Dinuba California California Dry Bean Advisory Board p 1-7
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Santos, J.R.P., Ndeve, A., Huynh, B.L., Matthews, W.C., Roberts, P.A. 2018. Transcriptome analysis of cowpea near-isogenic lines reveals candidate genes for root-knot nematode resistance. PLoS ONE 13(1): e0189185. doi: 10.1371/journal.pone.0189185
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Ulloa, M., Wang, C., Saha, S., Hutmacher, R.B., Stelly, D.M., Jenkins, J.N., Burke, J., Roberts, P.A. 2016. Analysis of root-knot nematode and fusarium wilt disease resistance in cotton (Gossypium spp.) using chromosome substitution lines from two alien species. Genetica 144: 167-179. doi: 10.1007/s10709-016-9887-0