The future of nematode management in cotton.

The importance of plant-parasitic nematodes as yield-limiting pathogens of cotton has received increased recognition and attention in the United States in the recent past. This paper summarizes the remarks made during a symposium of the same title that was held in July 2007 at the joint meeting of the Society of Nematologists and the American Phytopathological Society in San Diego, California. Although several cultural practices, including crop rotation, can be effective in suppressing the populations of the important nematode pathogens of cotton, the economic realities of cotton production limit their use. The use of nematicides is also limited by issues of efficacy and economics. There is a need for development of chemistries that will address these limitations. Also needed are systems that would enable precise nematicide application in terms of rate and placement only in areas where nematode population densities warrant application. Substantial progress is being made in the identification, characterization and mapping of loci for resistance to Meloidogyne incognita and Rotylenchulus reniformis. These data will lead to efficient marker-assisted selection systems that will likely result in development and release of nematode-resistant cotton cultivars with superior yield potential and high fiber quality.

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.

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.

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

Cell growth inhibition by the multifunctional multivalent zinc-finger factor CTCF.

The 11-zinc finger protein CCTC-binding factor (CTCF) employs different sets of zinc fingers to form distinct complexes with varying CTCF- target sequences (CTSs) that mediate the repression or activation of gene expression and the creation of hormone-responsive gene silencers and of diverse vertebrate enhancer-blocking elements (chromatin insulators). To determine how these varying effects would integrate in vivo, we engineered a variety of expression systems to study effects of CTCF on cell growth. Here we show that ectopic expression of CTCF in many cell types inhibits cell clonogenicity by causing profound growth retardation without apoptosis. In asynchronous cultures, the cell-cycle profile of CTCF-expressing cells remained unaltered, which suggested that progression through the cycle was slowed at multiple points. Although conditionally induced CTCF caused the S-phase block, CTCF can also arrest cell division. Viable CTCF-expressing cells could be maintained without dividing for several days. While MYC is the well-characterized CTCF target, the inhibitory effects of CTCF on cell growth could not be ascribed solely to repression of MYC, suggesting that additional CTS-driven genes involved in growth-regulatory circuits, such as p19ARF, are likely to contribute to CTCF-induced growth arrest. These findings indicate that CTCF may regulate cell-cycle progression at multiple steps within the cycle, and add to the growing evidence for the function of CTCF as a tumor suppressor gene.

Physical and functional interaction between two pluripotent proteins, the Y-box DNA/RNA-binding factor, YB-1, and the multivalent zinc finger factor, CTCF.

CTCF is a unique, highly conserved, and ubiquitously expressed 11 zinc finger (ZF) transcriptional factor with multiple DNA site specificities. It is able to bind to varying target sequences to perform different regulatory roles, including promoter activation or repression, creating hormone-responsive gene silencing elements, and functional block of enhancer-promoter interactions. Because different sets of ZFs are utilized to recognize different CTCF target DNA sites, each of the diverse DNA.CTCF complexes might engage different essential protein partners to define distinct functional readouts. To identify such proteins, we developed an affinity chromatography method based on matrix-immobilized purified recombinant CTCF. This approach resulted in isolation of several CTCF protein partners. One of these was identified as the multifunctional Y-box DNA/RNA-binding factor, YB-1, known to be involved in transcription, replication, and RNA processing. We examined CTCF/YB-1 interaction by reciprocal immunoprecipitation experiments with anti-CTCF and anti-YB-1 antibodies, and found that CTCF and YB-1 form complexes in vivo. We show that the bacterially expressed ZF domain of CTCF is fully sufficient to retain YB-1 in vitro. To assess possible functional significance of CTCF/YB-1 binding, we employed the very first identified by us, negatively regulated, target for CTCF (c-myc oncogene promoter) as a model in co-transfection assays with both CTCF and YB-1 expression vectors. Although expression of YB-1 alone had no effect, co-expression with CTCF resulted in a marked enhancement of CTCF-driven c-myc transcriptional repression. Thus our findings demonstrate, for the first time, the biological relevance of the CTCF/YB-1 interaction.

Tolerance to Rotylenchulus reniformis and Resistance to Meloidogyne incognita Race 3 in High-Yielding Breeding Lines of Upland Cotton.

Field experiments in 1992 and 1994 were conducted to determine the effect of Rotylenchulus reniformis, reniform nematode, on lint yield and fiber quality of 10 experimental breeding lines of cotton (Gossypium hirsutum) in untreated plots or plots fumigated with 1,3-dichloropropene. Controls were La. RN 1032, a germplasm line possessing some resistance to R. reniformis, and Stoneville 453, a cultivar that is susceptible to reniform nematode. Several breeding lines produced greater lint yields than Stoneville 453 or La. RN 1032 in both fumigated and untreated plots. Average lint yield suppression due to R. reniformis for six of the 10 breeding lines was less than half of the 52% yield reduction sustained by Stoneville 453. In growth chamber experiments, R. reniformis multiplication factors for La. RN 1032 and breeding lines N222-1-91, N320-2-91, and N419-1-91 were significantly lower than on Deltapine 16 and Stoneville 453 at 6 weeks after inoculation. R. reniformis populations increased by more than 50-fold on all entries within 10 weeks. In growth chambers, the breeding lines N220-1-92, N222-1-91, and N320-2-91 were resistant to Meloidoglyne incognita race 3; multiplication factors were

Resistance to Meloidogyne incognita Race 3 and Rotylenchulus reniformis in Wild Accessions of Gossypium hirsutum and G. barbadense from Mexico.

Forty-six accessions of G. hirsutum and two of G. barbadense were examined for resistance to Meloidogyne incognita race 3 and Rotylenchulus reniformis in environmental growth chamber experiments, with the objective of finding new sources of resistance. Only the G. barbadense accessions, TX-1347 and TX-1348, supported significantly less reproduction by R. reniformis than the susceptible control, Deltapine 16 (USDA accession SA-1186). However, they were highly susceptible to M. incognita race 3. The G. hirsutum accessions TX-1174, TX-1440, TX-2076, TX-2079, and TX-2107 had levels of resistance to M. incognita race 3 as great as or greater than those of Clevewilt 6 and Wild Mexican Jack Jones, which are the primary sources of resistance to M. incognita race 3 in the most resistant breeding lines. No accession was as resistant as the highly resistant line Auburn 623 RNR (SA-1492). Resistant accessions were from the Mexican coastal states of Campeche, Quintana Roo, Tabasco, Veracruz, and Yucatan. Populations of R. reniformis from Alabama, Mississippi, Louisiana, and Texas, and of M. incognita race 3 from Mississippi, Texas, and California, had similar reproductive rates on resistant genotypes. Thus, new sources of resistance to M. incognita race 3 but not to R. reniformis were identified in wild accessions of G. hirsutum from southern Mexico.

Repulsion of Meloidogyne incognita by Alginate Pellets Containing Hyphae of Monacrosporium cionopagum, M. ellipsosporum, or Hirsutella rhossiliensis.

The responses of second-stage juveniles (J2) of Meloidogyne incognita race 3 to calcium alginate pellets containing hyphae of the nematophagous fungi Monacrosporiura cionopagum, M. ellipsosporum, and Hirsutella rhossiliensis were examined using cylinders (38-mm-diam., 40 or 72 mm long) of sand (94% <250-mum particle size). Sand was wetted with a synthetic soil solution (10% moisture, 0.06 bar water potential). A layer of 10 or 20 pellets was placed 4 or 20 mm from one end of the cylinder. After 3, 5, or 13 days, J2 were put on both ends, on one end, or in the center; J2 were extracted from 8-ram-thick sections 1 or 2 days later. All three fungal pellets were repellent; pellets without fungi were not. Aqueous extracts of all pellets and of sand in which fungal pellets had been incubated were repellent, but acetone extracts redissolved in water were not. Injection of CO (20 mul/minute) into the pellet layer attracted J2 and increased fungal-induced mortality. In vials containing four randomly positioned pellets and 17 cm(3) of sand or loamy sand, the three fungi suppressed the invasion of cabbage roots by M. javanica J2. Counts of healthy and parasitized nematodes observed in roots or extracted from soil indicated that, in the vial assay, the failure of J2 to penetrate roots resulted primarily from parasitism rather than repulsion. Data were similar whether fungal inoculum consisted of pelletized hyphae or fungal-colonized Steinernema glaseri. Thus, the results indicate that nematode attractants and repellents can have major or negligible effects on the biological control efficacy of pelletized nematophagous fungi. Factors that might influence the importance of substances released by the pellets include the strength, geometry, and duration of gradients; pellet degradation by soil microflora; the nematode species involved; and attractants released by roots.

Optimal Release Rates for Attracting Meloidogyne incognita, Rotylenchulus reniformis, and Other Nematodes to Carbon Dioxide in Sand.

Movement of vermiform stages of Meloidogyne incognita, Rotylenchulus reniformis, Ditylenchus phyllobius, Steinernema glaseri, and Caenorhabditis elegans in response to carbon dioxide was studied in 40- and 72-mm-long cylinders of moist sand inside 38-mm-d acrylic tubes. Meloidogyne incognita, R. reniformis, and S. glaseri were attracted to CO when placed on a linear gradient of 0.2%/cm at a mean CO concentration of 1.2%. When CO was delivered into the sand through a syringe needle at flow rates between 2 and 130 mul/minute, the optimal flow rate for attracting M. incognita and R. reniformis was 15 mul/minute, and maximal attraction of the two species from a distance of 52 mm was achieved after 29 and 40 hours, respectively. After 24 hours, a total CO volume of 20 cm(3) was sufficient to induce 96% of all M. incognita introduced to move into the half of the cylinder into which CO was delivered and more than 75 % to accumulate in the 9 cm(3) of sand volume nearest the source. Results indicate it may be possible to use a chemical or biological source of CO to attract nematodes to nematicide granules or biocontrol agents.

Movement of Five Nematode Species through Sand Subjected to Natural Temperature Gradient Fluctuations.

Temperature gradient fluctuations that occur naturally as a result of heating and cooling of the soil surface were reproduced within 15-cm-d, 15-cm-long acrylic tubes filled with moist sand. Sunny and rainy periods during the late summer in eastern Texas were simulated. Five ecologically different nematode species were adapted to fluctuating temperatures for 20-36 hours at a simulated depth of 12.5 cm before being injected simultaneously into the centers of tubes at that depth. When heat waves were propagated horizontally to eliminate gravitational effects, the movement of Ditylenchus phyllobius, Steinernema glaseri, and Heterorhabditis bacteriophora relative to the thermal surface was rapid and largely random. However, Rotylenchulus reniformis moved away from and Meloidogyne incognita moved toward the thermal surface. When heat waves were propagated upward or downward, responses to temperature were the same as when propagated horizontally, irrespective of gravity. The initial direction of movement 1.5 hours after introduction to 20-era-long tubes at five depths at five intervals within a 24-hour cycle indicated that M. incognita moved away from and R. reniformis moved toward the temperature to which last exposed. Differences in movement of the five species tested relative to gravity appeared related to body length, with the smallest nematodes moving downward and the largest moving upward.

Meloidogyne incognita and Rotylenchulus reniformis and Associated Soil Textures from Some Cotton Production Areas of Texas.

The incidence of Meloidogyne incognita and Rotylenchulus reniformis on cotton was determined in 1989-92 from 1,089 soil samples collected from 31 counties that account for nearly 60% of the 2.2 million hectares planted to cotton in Texas. Meloidogyne incognita was commonly found in the Southern High Plains and Brazos River Valley regions of Texas (57% and 34%, respectively, of samples) but was found in less than 8% of samples from the Central Blacklands, Coastal Bend, Low Plains, or the Upper Gulf Coast regions. Rotylenchulus reniformis was widely distributed in the Brazos River Valley (24% of samples) and found occasionally in the Upper Gulf Coast (8% of samples). Meloidogyne incognita was found only rarely in soils with greater than 40% clay content, whereas Rotylenchulus reniformis was frequently found in finely textured soils but was less common in soils with greater than 40% sand content. In samples infested with M. incognita or R. reniformis, population densities of these species were at least 10-fold greater than population densities of other plant-parasitic species present in the sample. Root-knot and reniform nematodes were not found together in high population densities (>100 individuals/500 cm(3)) in the same sample.

Counting nematodes with a microplate reader.

The feasibility of counting plant-parasitic nematodes in aqueous suspensions by measuring light transmittance through aqueous suspensions with an ELISA microplate reader was explored. Absorbance readings for eggs or vermiform stages of three species were linearly related (R(2) > 0.99) to concentrations between 0 and 10,000 nematodes/ml. Coefficients of variation ranged from 12-23%, depending on the species and developmental stage used. The method, therefore, was at least as accurate as direct counts of nematodes in aliquots on a microscope and more than 100 times as fast. The method should have direct application in research programs on plant resistance to nematodes, nematode population dynamics, and nematode behavior.

Carbon Dioxide and Temperature Gradielits in Baermann Funnel Extraction of Rotylenchulus reniformis.

Vermiform Rotylenchulus reniformis were anesthetized in water by 10-40% CO but were fully motile for 24 hours in water below 5% CO. When air containing 2.5% CO was blown onto agar, nematodes accumulated at the point of highest CO concentration. Nematodes also accumulated when chilling (0.2-1 C) of agar by the gas flow at the accumulation point was offset with heat from a fiber optic. In Baermann funnels containing R. reniformis in silt loam and sandy clay loam soils, CO in funnel water increased during 24 hours from 0 to ca. 1%; more CO accumulated below the soil layer than above. Bubbling air with 2.5% CO into water below soil in covered funnels increased the CO gradient and increased nematode extraction, whereas bubbling air without CO below soil purged CO from the water and decreased nematode extraction. Manipulation of CO within funnels usually increased extraction by only 30% and never by more than 3-fold. Controlling temperature gradients consistently increased extraction by 2-30-fold.

Survey of Current Distribution of Rotylenchulus reniformis in the United States.

The reniform nematode, Rotylenchulus reniformis, has been reported from all Gulf Coast states, Arkansas, Hawaii, North Carolina, and South Carolina. Experts in 11 states identified the counties or parishes where the nematode is found and provided information regarding associated soil parameters, climate, crops, and crop management. Rotylenchulus reniformis has been detected in 187 counties and parishes of the southeastern United States and is most widespread in Louisiana, Mississippi, Alabama, Florida, and Georgia. In every state except Florida and Hawaii, economically damaging soil populations were associated with continuous cotton production. Other crops considered to be damaged by R. reniformis were soybean, tobacco, several vegetables, and pineapple (Hawaii). There was no consistent relationship between the nematode's presence and soil texture, soil pH, rainfall, or irrigation regime. However, certain respondents associated damage from the nematode primarily with silty or clay soils (Texas, Hawaii, Florida, and Georgia) or with silty soils with exceptionally tow pH (Hawaii and Louisiana).

Accelerated movement of nematodes from soil in baermann funnels with temperature gradients.

Baermann funnels were modified to eliminate or reverse the small temperature gradient (1-2 C/cm) across the soil layer that normally results from water evaporation. Effects of modifications on extraction efficiency were examined at various ambient temperatures and after overnight adaptation of three nematode species at 20 and 30 C. Extraction of Meloidogyne incognita from sandy loam, Tylenchulus semipenetrans from sandy clay loam, and Rotylenchulus reniformis from silt was greatly accelerated simply by covering funnels to prevent evaporation. In most cases, covering increased the nematodes extracted by 10-100 times after 5.5-48 hours. Faster and more efficient extraction of R. reniformis occurred over a wide range of ambient temperature (18-29 C). Effects of ambient temperature and temperature gradient direction on Baermann funnel extraction of R. reniformis were partly inconsistent with the behavior of R. reniformis in agar. Nematodes in agar moved toward cold at some ambient temperatures and toward heat at other temperatures. They always appeared to move toward cold on Baermann funnels. Differences were not attributable to blockage of gas exchange by covers. In agar and in funnels, the patterns of response to ambient temperature were shifted in the direction of the storage temperature.

Characterization and partial purification of attractants for nematodeOrrina phyllobia from foliage ofSolanum elaeagnifolium.

An unknown attractant for the nematodeOrrina phyllobia was extracted with water from foliage ofSolanum elaeagnifolium. Stability, solubility, ionic character, and Chromatographie purification were investigated using a bioassay based on nematode aggregation in agar. Activity was nonvolatile, dialyzable, heat stable below 60 °C, and partially lost within 30 min at 100 °C. Activity was unchanged from pH 5 to 12, but was entirely lost at pH 2. Loss of activity at low pH did not appear to result from direct effects of pH on nematode behavior and was partially recovered by readjustment to pH 7. The attractive factor was most soluble in water and appeared to be cationically but not anionically exchangeable. Activity appeared to Chromatograph as several compounds by high-performance liquid chromatography employing reverse phase C18 and amine-bonded columns. Various known compounds that are common toSolanum spp. or that attract other nematodes were unattractive. Extraction ofS. elaeagnifolium foliage specifically for solanaceous glycoalkaloids using methods developed forS. tuberosum did not yield an attractive product.

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.

Geographical Distributions of Rotylenchulus reniformis, Meloidogyne incognita, and Tylenchulus semipenetrans in the Lower Rio Grande Valley as Related to Soil Texture and Land Use.

A survey was conducted over a 22-year period to evaluate the influence of soil texture and land use on the geographical distributions of Rotylenchulus reniformis, Meloidogyne incognita, and Tylenchulus semipenetrans in the lower Rio Grande valley. The distributions of R. reniformis and M. incognita were related to soil texture, whereas T. semipenetrans occurred wherever host plants were present regardless of soil texture. The incidence of M. incognita was greatest in elevated sandy loams and moderately well-drained silts of modern flood terraces of the Rio Grande river. Rotylenchulus reniformis occurred predominantly in clay silts and clays of ancient flood terraces. Clay loams and sandy clay loams of the central, irrigated portion of the lower Rio Grande valley appeared favorable for M. incognita and R. reniformis. Differences between the geographical distributions of these two species could not be attributed to host crops.

Effects of Cyanide Ion and Hypoxia on the Volumes of Second-Stage Juveniles of Meloidogyne incognita in Polyethylene Glycol Solutions.

Changes in the volumes of second-stage juveniles of Meloidogyne incognita were monitored in aqueous solutions of polyethylene glycol supplemented with dilute balanced salts. At key points within a 48-hour cycle of fluctuating water potential, nematodes were placed under hypoxic conditions or exposed to the respiratory inhibitor, sodium cyanide, to detect any respiration-dependent process that regulates volume. Aerobic respiratory arrest at -500 kPa induced pronounced water loss, lateral and dorsoventral collapse of the body wall, and abnormal failure to shorten longitudinally. Durations of hypoxia that were innocuous in dilute solutions were lethal during 500 kPa increases and decreases in water potential; the same water potential changes under aerobic conditions had no effect on viability. Data are consistent with the hypothesis that respiration is essential to survive water potential changes.