Transcriptome Analysis of Resistant Cotton Germplasm Responding to Reniform Nematodes.

Reniform nematode (Rotylenchulus reniformis) is an important microparasite for Upland cotton (Gossypium hirsutum L.) production. Growing resistant cultivars is the most economical management method, but only a few G. barbadense genotypes and some diploid Gossypium species confer high levels of resistance. This study conducted a transcriptome analysis of resistant genotypes to identify genes involved in host plant defense. Seedlings of G. arboreum accessions PI 529728 (A2-100) and PI 615699 (A2-190), and G. barbadense genotypes PI 608139 (GB 713) and PI 163608 (TX 110), were inoculated with the reniform nematode population MSRR04 and root samples were collected on the fifth (D5) and ninth (D9) day after inoculation. Differentially expressed genes (DEGs) were identified by comparing root transcriptomes from inoculated plants with those from non-inoculated plants. Accessions A2-100 and A2-190 showed 52 and 29 DEGs on D5, respectively, with 14 DEGs in common, and 18 DEGs for A2-100 and 11 DEGs for A2-190 on chromosome 5. On D9, four DEGs were found in A2-100 and two DEGs in A2-190. For GB 713, 52 and 43 DEGs were found, and for TX 110, 29 and 117 DEGs were observed on D5 and D9, respectively. Six DEGs were common at the two sampling times for these genotypes. Some DEGs were identified as Meloidogyne-induced cotton (MIC) 3 and 4, resistance gene analogs, or receptor-like proteins. Other DEGs have potential roles in plant defense, such as peroxidases, programmed cell death, pathogenesis related proteins, and systemic acquired resistance. Further research on these DEGs will aid in understanding the mechanisms of resistance to explore new applications for the development of resistant cultivars.

Influence of Curly Leaf Trait on Cottonseed Micro-Nutrient Status in Cotton (Gossypium hirsutum L.) Lines.

Cottonseed is a source of nutrients, including protein, oil, and macro- and micro-nutrients. Micro-nutrients such as boron (B), copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn) are essential for plant and human health. Deficiencies of these micro-nutrients in soil lead to poor crop production and poor seed quality. Micro-nutrient deficiencies in the human diet lead to malnutrition and serious health issues. Therefore, identifying new cotton lines containing high nutritional qualities such as micro-nutrients, and understanding plant traits influencing micro-nutrients are essential. The objective of this research was to investigate the effects of leaf shape (curly leaf: CRL) on cottonseed B, Cu, Fe, Mn, Ni (nickel), and Zn in two near-isogenic cotton lines differing in leaf shape (DP 5690 wild-type with normal leaves and DP 5690 CRL). We also used Uzbek CRL, the source of the curly leaf trait, for comparison. A field experiment was conducted in 2014 and 2015 in Stoneville, MS, USA. The experiment was a randomized complete block design with three replicates. The results showed that, in 2014, both DP 5690 wild-type and Uzbek CRL had higher seed B, Cu, Fe, and Ni than in DP 5690 CRL. The accumulation of Mn and Zn in seeds of DP 5690 CRL was higher than in DP 5690 wild-type and Uzbek CRL. However, in 2015, the concentrations of B, Cu, Fe, and Ni, including Mn and Zn, were higher in both DP 5690 wild-type and Uzbek CRL than in DP 5690 CRL. Positive and negative correlations existed in 2014; however, only positive correlations existed between all nutrients in 2015. This research demonstrated that leaf shape can alter cottonseed micro-nutrients status. As Uzbek CRL behaved similar to wild-type, both leaf shape and other factors contributed to the alteration in seed micronutrients, affecting seed nutritional qualities. Therefore, leaf-shape partially contributed to the changes in micro-nutrients in cottonseed. The negative and positive correlations in 2014, and only positive correlations in 2015, were likely due to the heat difference between 2014 and 2015 as 2015 was warmer than 2014. Significant levels of seed micro-nutrients were shown between these lines, providing opportunities for breeders to select for high seed micro-nutrients in cotton. Additionally, the current research provides researchers with physiological information on the impact of leaf shape on seed nutritional quality. The leaf shape trait can also be used as a tool to study leaf development, physiological, biochemical, and morphological processes.

Cottonseed Protein, Oil, and Minerals in Cotton (Gossypium hirsutum L.) Lines Differing in Curly Leaf Morphology.

Cottonseed is an important source of protein, oil, and minerals for human health and livestock feed. Therefore, understanding the physiological and genetic traits influencing the nutrient content is critical. To our knowledge, there is no information available on the effects of leaf shape-curly leaf (CRL)-on cottonseed protein, oil, and minerals. Therefore, the objective of the current research was to investigate the effect of the curly leaf trait on cottonseed protein, oil, and minerals in cotton lines differing in leaf shape. Our hypothesis was that since leaf shape is known to be associated with nutrient uptake, assimilation, and photosynthesis process, leaf shape can influence seed protein, oil, and minerals. A two-year field experiment using two curly leaf lines (Uzbek CRL and DP 5690 CRL) and one normal leaf (DP 5690 wild type) line was conducted in 2014 and 2015 in Stoneville, MS, USA. The experiment was a randomized complete block design with three replicates. The results showed that both Uzbek CRL and DP 5690 wild type lines had higher seed oil, and nutrients N, P, K, and Mg than DP 5690 CRL. Calcium was higher in DP 5690 CRL for two years and protein was only higher than the parents in 2015. Consistent significant positive and negative correlations between some nutrients were observed, which may be due to environmental conditions, especially heat. This indicates that curly leaf trait may partially regulate the accumulation of these nutrients in seeds. The results demonstrated that leaf shape trait-curly leaf-can affect cottonseed nutritional qualities. This research is important to breeders for cotton selection for high seed oil or protein, and to other researchers to further understand the genetic impact of leaf shapes on seed nutritional quality. It is also important for scientists to use leaf shape as a tool for physiological, biochemical, and morphological research related to leaf development.

Single Nucleotide Polymorphism Analysis Using KASP Assay Reveals Genetic Variability in Rotylenchulus reniformis.

This study employed single nucleotide polymorphisms (SNPs) to determine the genetic variability present in 26 isolates of Rotylenchulus reniformis from Louisiana, Mississippi, Arkansas, South Carolina, Georgia, Hawaii, and Alabama. Genomic DNA from reniform nematode was extracted and increased quantitatively using the process of whole genome amplification. More than 162 putative SNPs were identified, 31 of which were tested using a KASP kompetitive allele-specific PCR genotyping assay. Of the SNPs tested, 13, 17, and 19 SNPs revealed genetic variability within reniform nematode isolates from Louisiana, Mississippi, and Arkansas, respectively. Seven SNPs elucidated genetic differences among isolates of reniform nematode from Louisiana, Mississippi, and Arkansas. Eight SNPs determined genetic variability among individual isolates from South Carolina, Georgia, Hawaii, and Alabama. This study is the first to report genetic variability in geographic isolates of reniform nematode employing a SNP assay. This study also demonstrated that SNP markers can be used to evaluate isolates of R. reniformis and could be useful to assess their genetic diversity, origin, and distribution. Such information would be extremely useful in resistance breeding programs.

Screening Cotton Genotypes for Reniform Nematode Resistance.

A rapid non-destructive reniform nematode (Rotylenchulus reniformis) screening protocol is needed for the development of resistant cotton (Gossypium hirsutum) varieties to improve nematode management. Most protocols involve extracting vermiform nematodes or eggs from the cotton root system or potting soil to determine population density or reproduction rate. These approaches are generally time-consuming with a small number of genotypes evaluated. An alternative approach is described here in which the root system is visually examined for nematode infection. The protocol involves inoculating cotton seedling 7 days after planting with vermiform nematodes and determining the number of females attached to the root system 28 days after inoculation. Data are expressed as the number of females per gram of fresh root weight to adjust for variation in root growth. The protocol provides an excellent method for evaluating host-plant resistance associated with the ability of the nematode to establish an infection site; however, resistance that hinders nematode reproduction is not assessed. As with other screening protocols, variation is commonly observed in nematode infection among individual genotypes within and between experiments. Data are presented to illustrate the range of variation observed using the protocol. To adjust for this variation, control genotypes are included in experiments. Nonetheless, the protocol provides a simple and rapid method to evaluate host-plant resistance. The protocol has been successfully used to identify resistant accessions from the G. arboreum germplasm collection and evaluate segregating populations of more than 300 individuals to determine the genetics of resistance. A vegetative propagation method for recovering plants for resistance breeding was also developed. After removal of the root system for nematode evaluation, the vegetative shoot is replanted to allow the development of a new root system. More than 95% of the shoots typically develop a new root system with plants reaching maturity.

Genome-wide association study of Gossypium arboreum resistance to reniform nematode.

BACKGROUND: Reniform nematode (Rotylenchulus reniformis) has emerged as one of the most destructive root pathogens of upland cotton (Gossypium hirsutum) in the United States. Management of R. reniformis has been hindered by the lack of resistant G. hirsutum cultivars; however, resistance has been frequently identified in germplasm accessions from the G. arboreum collection. To determine the genetic basis of reniform nematode resistance, a genome-wide association study (GWAS) was performed using 246 G. arboreum germplasm accessions that were genotyped with 7220 single nucleotide polymorphic (SNP) sequence markers generated from genotyping-by-sequencing. RESULTS: Fifteen SNPs representing 12 genomic loci distributed over eight chromosomes showed association with reniform nematode resistance. For 14 SNPs, major alleles were shown to be associated with resistance. From the 15 significantly associated SNPs, 146 genes containing or physically close to these loci were identified as putative reniform nematode resistance candidate genes. These genes are involved in a broad range of biological pathways, including plant innate immunity, transcriptional regulation, and redox reaction that may have a role in the expression of resistance. Eighteen of these genes corresponded to differentially expressed genes identified from G. hirsutum in response to reniform nematode infection. CONCLUSIONS: The identification of multiple genomic loci associated with reniform nematode resistance would indicate that the G. arboreum collection is a significant resource of novel resistance genes. The significantly associated markers identified from this GWAS can be used for the development of molecular tools for breeding improved reniform nematode resistant upland cotton with resistance introgressed from G. arboreum. Additionally, a greater understanding of the molecular mechanisms of reniform nematode resistance can be determined through genetic structure and functional analyses of candidate genes, which will aid in the pyramiding of multiple resistance genes.

The Elusive Search for Reniform Nematode Resistance in Cotton.

The reniform nematode (Rotylenchulus reniformis Linford and Oliveira) has emerged as the most important plant-parasitic nematode of cotton in the United States cotton belt. Success in the development of reniform nematode-resistant upland cotton cultivars (Gossypium hirsutum L.) has not been realized despite over three decades of breeding efforts. Research approaches ranging from conventional breeding to triple species hybrids to marker-assisted selection have been employed to introgress reniform nematode resistance from other species of cotton into upland cultivars. Reniform nematode-resistant breeding lines derived from G. longicalyx were developed in 2007. However, these breeding lines displayed stunting symptoms and a hypersensitive response to reniform nematode infection. Subsequent breeding efforts focused on G. barbadense, G. aridum, G. armoreanum, and other species that have a high level of resistance to reniform nematode. Marker-assisted selection has greatly improved screening of reniform nematode-resistant lines. The use of advanced molecular techniques such as CRISPER-Cas9 systems and alternative ways such as delivery of suitable "cry" proteins and specific double-stranded RNA to nematodes will assist in developing resistant cultivars of cotton. In spite of the efforts of cotton breeders and nematologists, successes are limited only to the development of reniform nematode-resistant breeding lines. In this article, we provide an overview of the approaches employed to develop reniform nematode-resistant upland cotton cultivars in the past, progress to date, major obstacles, and some promising future research activity.

Gossypium arboreum Accessions Resistant to Rotylenchulus reniformis.

In the southeastern United States, reniform nematode (Rotylenchulus reniformis) is a serious pest of upland cotton (Gossypium hirsutum), a species which has no naturally occurring resistance against this nematode. To identify sources of reniform nematode resistance in species closely related to upland cotton, 222 G. arboreum accessions from the U.S. germplasm collection were evaluated in repeated growth chamber experiments. In initial screenings, root infection was measured 4 wks after inoculation. The 15 accessions supporting the fewest infections (PI 529992, PI 615755, PI 615766, PI 615788, PI 615848, PI 615856, PI 615950, PI 615977, PI 615991, PI 616008, PI 616016, PI 616062, PI 616126, PI 616159, and A2 553) were evaluated again in confirmation tests lasting 8 wk. The combined totals of nematodes extracted from soil and eggs extracted from roots were analyzed. All 15 accessions tested supported significantly smaller reniform nematode populations than the susceptible controls (G. hirsutum cultivar Deltapine 16 and G. arboreum accession PI 529729). Nine accessions (PI 529992, PI 615755, PI 615766, PI 615788, PI 615856, PI 615950, PI 615991, PI 616008, and PI 616159) supported reniform nematode populations comparable to the resistant control (G. arboreum accession PI 615699), and accession PI 615848 had significantly fewer reniform nematodes than the resistant control. Cotton breeders would benefit from introgressing the newly identified resistance from these accessions into their upland cotton improvement programs.

Water Stress and Foliar Boron Application Altered Cell Wall Boron and Seed Nutrition in Near-Isogenic Cotton Lines Expressing Fuzzy and Fuzzless Seed Phenotypes.

Our previous research, conducted under well-watered conditions without fertilizer application, showed that fuzziness cottonseed trait resulted in cottonseed nutrition differences between fuzzy (F) and fuzzless (N) cottonseed. Under water stress conditions, B mobility is further limited, inhibiting B movement within the plant, affecting seed nutrition (quality). Therefore, we hypothesized that both foliar B and water stress can affect B mobility, altering cottonseed protein, oil, and mineral nutrition. The objective of the current research was to evaluate the effects of the fuzziness seed trait on boron (B) and seed nutrition under water stress and foliar B application using near-isogenic cotton lines (NILs) grown in a repeated greenhouse experiment. Plants were grown under-well watered conditions (The soil water potential was kept between -15 to -20 kPa, considered field capacity) and water stress conditions (soil water potential between -100 and -150 kPa, stressed conditions). Foliar B was applied at a rate of 1.8 kg B ha(-1) as H3BO3. Under well-watered conditions without B the concentrations of seed oil in N lines were higher than in F lines, and seed K and N levels were lower in N lines than in F lines. Concentrations of K, N, and B in leaves were higher in N lines than in F lines, opposing the trend in seeds. Water-stress resulted in higher seed protein concentrations, and the contribution of cell wall (structural) B to the total B exceeded 90%, supporting the structural role of B in plants. Foliar B application under well-watered conditions resulted in higher seed protein, oil, C, N, and B in only some lines. This research showed that cottonseed nutrition differences can occur due to seed fuzziness trait, and water stress and foliar B application can alter cottonseed nutrition.

Cottonseed protein, oil, and mineral status in near-isogenic Gossypium hirsutum cotton lines expressing fuzzy/linted and fuzzless/linted seed phenotypes under field conditions.

Cotton is an important crop in the world and is a major source of oil for human consumption and cotton meal for livestock. Cottonseed nutrition (seed composition: protein, oil, and minerals) determines the quality of seeds. Therefore, maintaining optimum levels of cottonseed nutrition is critical. Physiological and genetic mechanisms controlling the levels of these constituents in cottonseed are still largely unknown. Our previous research conducted under greenhouse conditions showed that seed and leaf nutrition differed between fuzzless and fuzzy seed isolines. Therefore, the objective of this research was to investigate the seed fuzz phenotype (trait) effects on seed protein, oil, N, C, S, and minerals in five sets of near-isogenic mutant cotton lines for seed fuzz in a 2-year experiment under field condition to evaluate the stability of the effect of the trait on seed nutrition. The isolines (genotypes) in each set differ for the seed fuzz trait (fuzzless/linted seed line, N lines, and fuzzy/linted seed line, F lines). Results showed that seed protein was higher in the fuzzy genotype in all sets, but seed oil was higher in fuzzless genotype in all sets. The concentrations of seed Ca and C were higher in all fuzzless genotypes, but N, S, B, Fe, and Zn were higher in most of the fuzzy genotypes. Generally, minerals were higher in leaves of F lines, suggesting the translocation of minerals from leaves to seeds was limited. The research demonstrated that fiber development could be involved in cottonseed composition. This may be due to the involvement of fiber development in carbon and nitrogen metabolism, and the mobility of nutrients from leaves (source) to seed (sink). This information is beneficial to breeders to consider fuzzless cottonseed for potential protein and oil use and select for higher oil or higher protein content, and to physiologists to further understand the mobility of minerals to increase the quality of cottonseed nutrition for food and feed.

Postinfection Development of Rotylenchulus reniformis on Resistant Gossypium barbadense Accessions.

The reniform nematode (Rotylenchulus reniformis) causes significant cotton (Gossypium hirsutum) losses in the southeastern United States. The research objective was to describe the effects of two resistant G. barbadense lines (cultivar TX 110 and accession GB 713) on development and fecundity of reniform nematode. Nematode development and fecundity were evaluated on the resistant lines and susceptible G. hirsutum cultivar Deltapine 16 in three repeated growth chamber experiments. Nematode development on roots early and late in the infection cycle was measured at set intervals from 1 to 25 d after inoculation (DAI) and genotypes were compared based on the number of nematodes in four developmental stages (vermiform, swelling, reniform, and gravid). At 15, 20, and 25 DAI, egg production by individual females parasitizing each genotype was measured. Unique reniform nematode developmental patterns were noted on each of the cotton genotypes. During the early stages of infection, infection and development occurred 1 d faster on susceptible cotton than on the resistant genotypes. Later, progression to the reniform and gravid stages of development occurred first on the susceptible genotype, followed by G. barbadense cultivar TX 110, and finally G. barbadense accession GB 713. Egg production by individual nematodes infecting the three genotypes was similar. This study corroborates delayed development previously reported on G. barbadense cultivar TX 110 and is the first report of delayed infection and development associated with G. barbadense accession GB 713. The different developmental patterns in the resistant genotypes suggest that unique or additional loci may confer resistance in these two lines.

Identification of Rotylenchulus reniformis Resistant Glycine Lines.

Identification of resistance to reniform nematode (Rotylenchulus reniformis) is the first step in developing resistant soybean (Glycine max) cultivars that will benefit growers in the mid-South region of the United States. This study was conducted to identify soybean (G. max and G. soja) lines with resistance to this pathogen. Sixty-one wild and domestic soybean lines were evaluated in replicated growth chamber tests. Six previously untested soybean lines with useful levels of resistance to reniform nematode were identified in both initial screening and subsequent confirmation tests: released germplasm lines DS4-SCN05 (PI 656647) and DS-880 (PI 659348); accession PI 567516 C; and breeding lines DS97-84-1, 02011-126-1-1-2-1 and 02011-126-1-1-5-1. Eleven previously untested moderately susceptible or susceptible lines were also identified: released germplasm lines D68-0099 (PI 573285) and LG01-5087-5; accessions PI 200538, PI 416937, PI 423941, PI 437697, PI 467312, PI 468916, PI 594692, and PI 603751 A; and cultivar Stafford (PI 508269). Results of previously tested lines evaluated in the current study agreed with published reports 69.6% of the time for resistant lines and 87.5% of the time for susceptible lines. Soybean breeders may benefit from incorporating the newly identified resistant lines into their breeding programs.

Comparison of whole-genome amplifications for microsatellite genotyping of Rotylenchulus reniformis

Currently, a large number of microsatellites are available for Rotylenchulus reniformis (reniform nematode); however, two barriers exist for genotyping samples from different geographical areas. The limited amount of nucleic acids obtained from single nematodes which would require their multiplication to obtain enough DNA for testing; and the strictly regulated transport of live samples and multiplication in greenhouse for being a plant pathogen. Whole-genome amplification (WGA) of samples consisting of one and five dead gravid females with their associated egg masses was successfully performed on disrupted tissue using three commercial kits. DNA yield after WGA ranged from 0.5 to 8 ug and was used to test 96 microsatellite markers we previously developed for the reniform nematode. The results were compared to those of fingerprinting the original population (MSRR03). Out of 96 markers tested, 71 had amplicons in MSRR03. Using WGA of single gravid females with their associated egg masses, 86-93 percent of the alleles found on MSRR03 were detected, and 87-88 percent of the alleles found on MSRR03 when using WGA of samples composed of five gravid females with their associated egg masses as template. Our results indicate that reniform nematode samples as small as a single gravid female with her associated egg mass can be used in WGA and direct testing with microsatellites, giving consistent results when compared to the original population.

Molecular and Morphological Characterization and Biological Control Capabilities of a Pasteuria ssp. Parasitizing Rotylenchulus reniformis, the Reniform Nematode.

Rotylenchulus reniformis is one of 10 described species of reniform nematodes and is considered the most economically significant pest within the genus, parasitizing a variety of important agricultural crops. Rotylenchulus reniformis collected from cotton fields in the Southeastern US were observed to have the nematode parasitic bacterium Pasteuria attached to their cuticles. Challenge with a Pasteuria-specific monoclonal antibody in live immuno-fluorescent assay (IFA) confirmed the discovery of Pasteuria infecting R. reniformis. Scanning and transmission electron microscopy were employed to observe endospore ultrastructure and sporogenesis within the host. Pasteuria were observed to infect and complete their life-cycle in juvenile, male and female R. reniformis. Molecular analysis using Pasteuria species-specific and degenerate primers for 16s rRNA and spoII, and subsequent phylogenetic assessment, placed the Pasteuria associated with R. reniformis in a distinct clade within established assemblages for the Pasteuria infecting phytopathogenic nematodes. A global phylogenetic assessment of Pasteuria 16s rDNA using the Neighbor-Joining method resulted in a clear branch with 100% boot-strap support that effectively partitioned the Pasteuria infecting phytopathogenic nematodes from the Pasteuria associated with bacterivorous nematodes. Phylogenetic analysis of the R. reniformis Pasteuria and Pasteuria spp. parasitizing a number of economically important plant parasitic nematodes revealed that Pasteuria with different host specificities are closely related and likely constitute biotypes of the same species. This suggests host preference, and thus effective differentiation and classification are most likely predicated by an influential virulence determinant(s) that has yet to be elucidated. Pasteuria Pr3 endospores produced by in vitro fermentation demonstrated efficacy as a commercial bionematicide to control R. reniformis on cotton in pot tests, when applied as a seed treatment and in a granular formulation. Population control was comparable to a seed-applied nematicide/insecticide (thiodicarb/imidacloprid) at a seed coating application rate of 1.0 x 10(8) spores/seed.

Identification and genomic location of a reniform nematode (Rotylenchulus reniformis) resistance locus (Ren ari) introgressed from Gossypium aridum into upland cotton (G. hirsutum).

In this association mapping study, a tri-species hybrid, [Gossypium arboreum x (G. hirsutum x G. aridum)(2)], was crossed with MD51ne (G. hirsutum) and progeny from the cross were used to identify and map SSR markers associated with reniform nematode (Rotylenchulus reniformis) resistance. Seventy-six progeny (the 50 most resistant and 26 most susceptible) plants were genotyped with 104 markers. Twenty-five markers were associated with a resistance locus that we designated Ren(ari) and two markers, BNL3279_132 and BNL2662_090, mapped within 1 cM of Ren(ari). Because the SSR fragments associated with resistance were found in G. aridum and the bridging line G 371, G. aridum is the likely source of this resistance. The resistance is simply inherited, possibly controlled by a single dominant gene. The markers identified in this project are a valuable resource to breeders and geneticists in the quest to produce cotton cultivars with a high level of resistance to reniform nematode.

Microsatellites reveal genetic diversity in Rotylenchulus reniformis populations.

Rotylenchulus reniformis is the predominant parasitic nematode of cotton in the Mid South area of the United States. Although variable levels of infection and morphological differences have been reported for this nematode, genetic variability has been more elusive. We developed microsatellite-enriched libraries for R. reniformis, produced 1152 clones, assembled 694 contigs, detected 783 simple sequence repeats (SSR) and designed 192 SSR-markers. The markers were tested on six R. reniformis cultures from four states, Texas, Louisiana, Mississippi and Georgia, in the USA. Based on performance we selected 156 SSR markers for R. reniformis from which 88 were polymorphic across the six reniform nematode populations, showing as the most frequent motif the dinucleotide AG. The polymorphic information content of the markers ranged from 0.00 to 0.82, and the percentage of multiallelic loci of the isolates was between 40.9 and 45.1%. An interesting finding in this study was the genetic variability detected among the three Mississippi isolates, for which 22 SSR markers were polymorphic. We also tested the level of infection of these isolates on six cotton genotypes, where significant differences were found between the Texas and Georgia isolates. Coincidentally, 62 polymorphic markers were able to distinguish these two populations. Further studies will be necessary to establish possible connections, if any, between markers and level of pathogenicity of the nematode. The SSR markers developed here will be useful in the assessment of the genetic diversity of this nematode, could assist in management practices for control of reniform nematode, be used in breeding programs for crop resistance, and help in detecting the origin and spread of this nematode in the United States.

Detection of Suppressiveness against Rotylenchulus reniformis in Soil from Cotton (Gossypium hirsutum) Fields in Texas and Louisiana.

Rotylenchulus reniformis is a major problem confronting cotton production in the central part of the cotton belt of the United States of America. In this study, the hypothesis that natural antagonists in some cases are responsible for unusually low densities of the nematode in certain fields was tested by assaying soils from 22 selected fields for the presence of transferable agents in pots containing cotton plants. In one field, soil from four different depth ranges was tested. In the first of two types of assays, 1 part nematode infested soil was added to 9 parts test soil that was left untreated or autoclaved before mixing; this mixture was used to fill pots. In the second type of assay, 1 part test soil was added to 9 or 19 parts pasteurized fine sand, and nematodes were introduced in aqueous suspension. In three experiments representing both types of assay, transferable or autoclavable agent(s) from four fields in South Texas suppressed nematode populations by 48, 78, 90 and 95%. In one experiment, transferable agents in five fields in Louisiana suppressed populations from 37 to 66%. Identification and evaluation of these agents for biological control of R. reniformis merits further study.

Comparisons of Female and Egg Assays to Identify Rotylenchulus reniformis Resistance in Cotton.

More plants can be screened for reniform nematode resistance each year if the time involved can be shortened. In this study, the hypothesis that female counts are as efficient as egg counts in identifying resistant genotypes was tested. In two greenhouse experiments Gossypium genotypes which varied from resistant to susceptible to reniform nematode (Rotylenchulus reniformis) were compared to a susceptible control cultivar. Infested field soil served as the inoculum source for the first experiment, and vermiform stages extracted from greenhouse cultures were used to infest soil in the second experiment. Six replicates of each genotype were harvested 25 d after planting and swollen females were counted. The remaining plants were harvested 35 d after planting and eggs extracted from the roots were counted. Processing and counting times recorded in the first experiment were similar for both assessment methods, but 10 additional days were required for egg-based assessment. Contrast analyses showed that assessments based on females per gram of root were equivalent to assessments based on eggs per gram of root for the five genotypes tested in the first experiment and for an expanded set of 13 genotypes tested in the second experiment. The results indicated that either life stage can be used to screen for resistance.