Effect of Temperature on the Embryogenesis of Geographic Populations of Rotylenchulus reniformis

The effect of temperature on the embryonic development of three populations of reniform nematode (Rotylenchulus reniformis) from the southeastern United States was studied. The development of eggs from single-cell stage to eclosion of second-stage juvenile was monitored at 20, 25, 30, and 35°C. All populations completed embryogenesis in 7 days at 25°C. The greatest differences among populations in time to completion of embryogenesis were observed at 20 and 35°C. Results at the intermediate temperatures (25 and 30°C) were similar for the three populations. The optimal temperature for embryogenesis was calculated to be 31.4°C for the population from Alabama, 28.4°C for the one from Mississippi, and 37.5°C for the one from South Carolina.     

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.     

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.     

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.     

Extraction of Root-associated Meloidogyne incognita and Rotylenchulus reniformis

A technique based on physical maceration of root tissue was developed to extract vermiform and swollen stages of Meloidogyne incognita and Rotylenchulus reniformis. Experiments conducted on soybean and tomato evaluated the efficiency of method (stir, grind), NaOC1 concentration (0%, 0.5%), and duration (lx, 2x) on extraction of nematodes and eggs from 60-day-old populations. Root-associated populations of R. reniformis were considerably lower than those of M. incognita, so development of the method focused on the latter. Grinding liberated more nematodes than stirring, but the reverse was true for egg extraction. Among grinding treatments, a duration of 10 seconds in 0.5% NaOCl provided the most efficient extraction of nematodes and eggs. Among stirring treatments, a duration of 10 minutes in 0.5% NaOCl provided the most efficient extraction of eggs. These techniques were compared on soybean roots 30 days older than those on which the procedures were first evaluated, with consistent results.     

Effects of Soil Solarization on Rotylenchulus reniformis in the Lower Rio Grande Valley of Texas

Soil solarization was evaluated for control of Rotylenchulus reniformis in the lower Rio Grande Valley of Texas. In field experiments, solarization significantly reduced soil nematode population densities 0-15 cm deep and increased yields of lettuce and cowpea. The length of time required for 90% mortality of nematodes in soil heated under controlled conditions in the laboratory varied from 25 hours to less than 1 hour between 41 and 47 C. Daily exposures of nematode-infested soil to lethal temperatures for sublethal time periods had a cumulative lethal effect. In water, vermiform stages required up to 10 days to recover from sublethal thermal stress. Eggs were similar to juveniles in their sensitivity to high temperatures. Lethal time-temperatures under controlled conditions were in general agreement with field results.     

Vertical Distribution of Rotylenchulus reniformis in Cotton Fields

The possible impact of Rotylenchulus reniformis below plow depth was evaluated by measuring the vertical distribution of R. reniformis and soil texture in 20 symptomatic fields on 17 farms across six states. The mean nematode population density per field, 0 to 122 cm deep, ranged from 0.4 to 63 nematodes/g soil, and in 15 fields more than half of the R. reniformis present were below 30.5 cm, which is the greatest depth usually plowed by farmers or sampled by consultants. In 11 fields measured, root density was greatest in the top 15 cm of soil; however, roots consistently penetrated 92 to 122 cm deep by midseason, and in five fields in Texas and Louisiana the ratio of nematodes to root-length density within soil increased with depth. Repeated sampling during the year in Texas indicated that up to 20% of the nematodes in soil below 60 cm in the fall survived the winter. Differences between Baermann funnel and sugar flotation extraction methods were not important when compared with field-to-field differences in nematode populations and field-specific vertical distribution patterns. The results support the interpretation that R. reniformis below plow depth can significantly impact diagnosis and treatment of cotton fields infested with R. reniformis.     

Suppression of Rotylenchulus reniformis on Cotton by the Nematophagous Fungus ARF

The reniform nematode, Rotylenchulus reniformis Linford &Oliveira, has become a serious threat to cotton (Gossypium hirsutum L.) production in the United States during the past decade. The objective of this study is to isolate fungi from eggs of R. reniformis and select potential biological control agents for R. reniformis on cotton. Soil samples were collected from cotton fields located in Jefferson County, Arkansas. Eight genera of fungi were included in the 128 fungal isolates obtained, and among them were five strains of the nematophagous fungus ARF. The mtDNA RFLP pattern, colony growth characteristics, and pathogenicity indicate the five ARF isolates represent one described strain and one new strain. Light and electron microscopic observations suggest ARF is an active parasite of R. reniformis, with parasitism ranging from 48% to 79% in in vitro tests. Three greenhouse experiments demonstrated ARF successfully suppressed the number of reniform nematodes during the first and second generation of the nematode. Reductions in numbers of R. reniformis on the roots for the seven application rates of 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% ARF were 87%, 92%, 94%, 96%, 97%, 98%, and and 98%, respectively.     

Suppression of Rotylenchulus reniformis 122-cm Deep Endorses Resistance Introgression in Gossypium

Nine sources of resistance to Rotylenchulus reniformis in Gossypium (cotton) were tested by measuring population density (Pf) and root-length density 0 to 122 cm deep. A Pf in the plow layer less than the autumn sample treatment threshold used by consultants was considered the minimum criterion for acceptable resistance, regardless of population density at planting (Pi). Other criteria were ample roots and a Pf lower than on the susceptible control, as in pot studies. In a Texas field in 2001 and 2002, no resistant accessions had Pf less than the control but all did in microplots into which nematodes from Louisiana were introduced. An environmental chamber experiment ruled out nematode genetic variance and implicated unknown soil factors. Pf in field experiments in Louisiana, Mississippi, and Alabama were below threshold for zero, six and four of the accessions and above threshold in the control. Gossypium arboreum A2–87 and G. barbadense GB-713 were the most resistant accessions. Results indicate that cultivars developed from these sources will suppress R. reniformis populations but less than in pots in a single season.     

Interrelationships of Rotylenchulus reniformis with Rhizoctonia solani on Cotton

The interrelationships between reniform nematode (Rotylenchulus reniformis) and the cotton (Gossypium hirsutum) seedling blight fungus (Rhizoctonia solani) were studied using three isolates of R. solani, two populations of R. reniformis at multiple inoculum levels, and the cotton cultivars Dehapine 90 (DP 90) and Dehapine 41 (DP 41). Colonization of cotton hypocotyl tissue by R. solani resulted in increases (P ≤ 0.05) in nematode population densities in soil and in eggs recovered from the root systems in both 40- and 90-day-duration experiments. Increases in soil population densities resulted mainly from increases in juveniles. Enhanced reproduction of R. reniformis in the presence of R. solani was consistent across isolates (1, 2, and 3) of R. solani and populations (1 and 2) and inoculum levels (0.5, 2, 4, and 8 individuals/g of soil) of R. reniformis, regardless of cotton cultivar (DP 90 or DP 41). Severity of seedling blight was not influenced by the nematode. Rhizoctonia solani caused reductions (P ≤ 0.05) in cotton growth in 40- and 90-day periods. Rotylenchulus reniformis reduced cotton growth at 90 days. The relationship between nematode inoculum levels and plant growth reductions was linear. At 90 days, the combined effects of these pathogens were antagonistic to plant growth.     

Effects of Acibenzolar-S-Methyl Application to Rotylenchulus reniformis and Meloidogyne javanica

Effects of acibenzolar-s-methyl, an inducer of systemic acquired resistance in plants, on Rotylenchulus reniformis and Meloidogyne javanica in vitro and in vivo were determined. A single foliar application of acibenzolar at 50 mg/liter (5 ml of solution per plant) to 7-day-old cowpea or soybean seedlings decreased R. reniformis and M. javanica egg production by 50% 30 days after inoculation. The mechanism of acibenzolar on plant-parasitic nematodes was then investigated. Acibenzolar at 50 to 200 mg/liter did not affect movement of R. reniformis and M. javanica or penetration of second-stage juveniles (J2) of M. javanica on cowpea. However, M. javanica development was slowed and fecundity was reduced in plants treated with acibenzolar. On average, 50% of J2 that penetrated acibenzolar-treated cowpeas developed into mature females with eggs, whereas the other 50% exhibited arrested development. The number of eggs per egg mass was 450 in water-treated cowpeas, whereas the number declined to 250 in acibenzolar-treated plants. Acibenzolar may be responsible for stimulating the plants to express some resistance to the nematodes.     

Rotylenchulus reniformis below Plow Depth Suppresses Cotton Yield and Root Growth

Damage to cotton by Rotylenchulus reniformis below plow depth was evaluated in a sandy clay loam soil at Weslaco, Texas. In December 1999, 14 holes on 51-cm centers were dug 91 cm deep along the planting bed and adjacent furrow and 2 ml of 1,3-dichloropropene was placed 91, 61, and 30 cm deep as each hole was refilled and packed. This technique eliminated 96%, 81%, and 74% of R. reniformis down to 107 cm at distances 0, 25, and 51 cm laterally from the point of application (P ≤ 0.05), whereas chisel fumigation at 168 liters/ha 43 cm deep reduced nematode numbers only in the top 61 cm (P ≤ 0.001). Manual placement of fumigant increased yield 92%; chisel fumigation increased yield 88% (P ≤ 0.005). A second experiment in February 2001 placed fumigant 43 or 81 cm deep, or at both 43 and 81 cm. Holes alone had no significant effect on nematode density at planting, midseason or harvest, on root length density at midseason, or on cotton lint yield. Fumigant at 43 cm reduced nematode numbers above fumigant application depth at planting 94% (P ≤ 0.02), at midseason 37% (P ≤ 0.09), and at harvest 0%, increasing yield 57% (P ≤ 0.002). Fumigant at 81 cm reduced nematode numbers above fumigant application depth at planting 86% (P ≤ 0.02), at midseason 74% (P ≤ 0.02), and at harvest 48% (P ≤ 0.01), increasing yield 53% (P ≤ 0.002). Fumigating at both 43 and 81 cm reduced nematode numbers above 90 cm 94% at planting and 79% at midseason, increased midseason root-length density 14-fold below 76 cm, and doubled yield (P ≤ 0.02 in all cases).     

Histological Observations of Rotylenchulus reniformis on Gossypium longicalyx and Interspecific Cotton Hybrids

Observations on the development of reniform nematode (Rotylenchulus reniformis) on roots of Gossypium longicalyx, G. hirsutum, and two interspecific hybrids derived from them were made by light microscopy. Gossypium longicalyx is reported to be immune to reniform nematode, but the mechanism(s) for resistance are unknown. Penetration of G. longicalyx roots by female nematodes was confirmed, and incipient swelling of the females, indicating initiation of maturation of the reproductive system, was observed. Female maturation occurred up to the formation of a single embryo inside the female body but not beyond this point. In both hybrids, development was inhibited but progressed further than in the immune parent. Reactions ranged from highly compatible, with the formation of active syncytia and full development of females, to incompatible with little or no development of the female. Compatible plants showed characteristic hypertrophied cells, enlarged nuclei, dense cytoplasm, and partial dissolution of cell walls, whereas incompatible plant reactions included lignification of the cells adjacent to the nematode head, or the complete collapse and necrosis of the cells involved. The need to characterize reactions and to carefully select among the plants descended from the hybrids during the introgression process, as well as the importance of combining the results of reproduction tests with histological observation of the plant-nematode interactions, is discussed.     

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.     

Relationship between Meloidogyne incognita and Rotylenchulus reniformis as Influenced by Soybean Genotype

The effect of soybean genotype on competition between Meloidogyne incognita race 2 (Mi) and Rotylenchulus reniformis (Rr) was evaluated in greenhouse and microplot replacement series experiments. Soil in pots containing seedlings of ''Davis'' (susceptible to Mi) or ''Buckshot 66'' (resistant to Mi) was infested with 1,000 vermiform individuals in the following Mi:Rr ratios: 0:0, 100:0, 75:25, 50:50, 25:75, or 0:100. After 91 days, the relative nematode yields (number of nematodes in mixed culture divided by the number in nonmixed culture) of each species were calculated based on soil and root nematode populations expressed as nematodes per gram of dry root tissue. To define the relationship between the two species, calculated relative nematode yields were compared with a theoretical noncompetition model using lack-of-fit regression. In the greenhouse, Mi populations on ''Davis'' were stimulated in the presence of Rr. In microplots, low Mi and Rr population densities likely resulted from severe galling and destruction of feeder roots that probably occurred early in the season. Enhanced susceptibility to Mi was not observed on ''Buckshot 66'', which remained resistant to Mi even when colonized by Rr. Host resistance is a key factor in determining the nature of the relationship between Mi and Rr.     

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.     

Sensitivity of Meloidogyne incognita and Rotylenchulus reniformis to Abamectin

Avermectins are macrocyclic lactones produced by Streptomyces avermitilis. Abamectin is a blend of B_1a and B_1b avermectins that is being used as a seed treatment to control plant-parasitic nematodes on cotton and some vegetable crops. No LD₅₀ values, data on nematode recovery following brief exposure, or effects of sublethal concentrations on infectivity of the plant-parasitic nematodes Meloidogyne incognita or Rotylenchulus reniformis are available. Using an assay of nematode mobility, LD₅₀ values of 1.56 μg/ml and 32.9 μg/ml were calculated based on 2 hr exposure for M. incognita and R. reniformis, respectively. There was no recovery of either nematode after exposure for 1 hr. Mortality of M. incognita continued to increase following a 1 hr exposure, whereas R. reniformis mortality remained unchanged at 24 hr after the nematodes were removed from the abamectin solution. Sublethal concentrations of 1.56 to 0.39 μg/ml for M. incognita and 32.9 to 8.2 μg/ml for R. reniformis reduced infectivity of each nematode on tomato roots. The toxicity of abamectin to these nematodes was comparable to that of aldicarb.     

Nematicide Efficacy,Root Growth,and Fruit Yield in Drip-irrigated Pineapple Parasitized by Rotylenchulus reniformis

A 3-year field trial near Kunia, Oahu, Hawaii, was conducted to evaluate four nematicide treatments for efficacy against Rotylenchulus reniformis in drip-irrigated pineapple (Ananas comosus L. (Merr.)). The treatments were (A) preplant fumigation with 1,3-dichloropropene (1,3-D) (336 liter/ ha) and postplant drip application of fenamiphos (3.4 kg/ha) with restricted irrigation, (B) preplant 1,3-D only, weekly irrigation, (C) 1,3-D fenamiphos, weekly irrigation, and (D) postplant fenamiphos only, weekly irrigation. Fenamiphos was applied at 3-month intervals for 1 year after planting in three treatments. Although nematode populations increased in all treatments 1 year after planting, no differences in fruit yield were detected among treatments in the first (plant crop) harvest 19 months after planting. In the second (ratoon) crop (33 months after planting) significant yield differences, larger fruit size, and greater root biomass were obtained in the dual nematicide treatments. Root biomass increased continuously throughout the crop cycle, was greatest near the drip line, and showed a shallow depth distribution (30-40 cm). Rotylenchulus reniformis populations and fenamiphos concentrations were negatively correlated in soil profiles taken 13 months after planting. In the absence of postplant fenamiphos applications, nematode numbers were positively correlated with root biomass.     

Host Suitability of Soybean Cultivars and Breeding Lines to Reniform Nematode in Tests Conducted in 2001

Reproduction of reniform nematode Rotylenchulus reniformis on 139 soybean lines was evaluated in a greenhouse in the summer of 2001. Cultivars and lines (119 total) were new in the Arkansas and Mississippi Soybean Testing Programs, and an additional 20 were submitted by C. Overstreet, Louisiana State Extension Nematologist. A second test of 32 breeding lines and 2 cultivars from the Clemson University soybean breeding program was performed at the same time under the same conditions. Controls were the resistant cultivars Forrest and Hartwig, susceptible Braxton, and fallow infested soil. Five treatment replications were planted in sandy loam soil infested with 1,744 eggs and vermiform reniform nematodes, grown for 10 weeks in 10 cm-diam.- pots. Total reniform nematodes extracted from soil and roots was determined, and a reproductive factor (final population (Pf)/ initial inoculum level (Pi)) was calculated for each genotype. Reproduction on each genotype was compared to the reproduction on the resistant cultivar Forrest (RF), and the log ratio [log₁₀(RF + 1) is reported. Cultivars with reproduction not significantly different from Forrest (log ratio) were not suitable hosts, whereas those with greater reproductive indices were considered suitable hosts. These data will be useful in the selection of soybean cultivars to use in rotation with cotton or other susceptible crops to help control the reniform nematode and to select useful breeding lines as parent material for future development of reniform nematode resistant cultivars and lines.