Effect of Ammonium Ions on Egg Hatching and Second-Stage Juveniles of Meloidogyne incognita in Axenic Tomato Root Culture

Eggs, either dispersed or in masses, and second-stage juveniles (J2) of Meloidogyne incognita were exposed to different concentrations of ammonium ions in a nutrient agar medium upon which excised tomato roots were growing. Egg hatch and J2 penetration of the roots was slowed or inhibited at high (54 and 324 mg/liter) but not at low (1.5 and 9 mg/liter) concentrations of ammonium nitrate. The effect of ammonium on J2 appeared to be temporary and reversible. High potassium nitrate concentration (1,116 mg/liter) did not modify egg hatch or J2 penetration. There was no adverse effect from the high ammonium nitrate concentrations or an equivalent potassium nitrate concentration on root dry weight. Ammonium ions influence nematodes both directly and via plant roots and do so independently of microbial organisms.     

Influence of Photoperiod and Temperature on Migrations of Meloidogyne Juveniles

Photoperiod influences the migration of M. incognita juveniles toward tomato roots. Approximately 33% migrated vertically 20 cm in 7 days to roots when 12 h dark were alternated with 12 h light. Only 7% migrated when light was constant for 24 h. Vertical migration of M. incognita juveniles was studied at 14, 16, 18, 20, and 22 C. The migration of M. incognita juveniles begins at about 18 C and reaches its maximum at 22 C. The migration of M. hapla and M. incognita juveniles were compared at 14, 18, and 22 C. Juveniles of M. hapla were able to migrate at a lower temperature than those of M. incognita. With M. hapla, there was no significant difference in migration between 18 and 22 C.     

Cuticular Collagenous Proteins of Second-stage Juveniles and Adult Females of Meloidogyne incognita: Isolation and Partial Characterization

Cuticles isolated from second-stage juveniles and adult females of Meloidogyne incognita were purified by treatment with 1% sodium dodecyl sulfate (SDS). The juvenile cuticle was composed of three zones differing in their solubility in β-mercaptoethanol (BME). Proteins in the cortical and median zones were partially soluble in BME, whereas the basal zone was the least soluble. The BME-soluble proteins from the juvenile cuticle were separated into 12 bands by SDS-polyacrylamide gel electrophoresis and characterized as collagenous proteins based on their sensitivity to collagenase and amino acid composition. The adult cuticle consisted of two zones which were dissolved extensively by BME. The basal zone was completely solubilized, leaving behind a network of fibers corresponding to the cortical zone. The BME-soluble proteins from the adult cuticle were separated by electrophoresis into nine bands one of which constituted > 55% of the total BME-soluble proteins. All bands were characterized as collagenous proteins. Collagenous proteins from juvenile cuticles also contained glycoproteins which were absent from the adult cuticles.     

Relationship Between Morphology and Parasitism in Two Populations of Meloidogyne incognita

The reliability of morphological characters and host differential plants for distinguishing between two populations of Meloidogyne incognita was studied. Population A (originally from North Carolina) had incognita-type perineal patterns. A single egg mass subpopulation of population A had a mixture of incognita and acrita perineal patterns with 33% of the patterns atypical for either species. Population B (from Georgia) had predominantly acrita-type patterns with only about 5% atypical patterns. The head shapes of males from both populations were mainly M. incognita. On the basis of stylet length, both populations conformed to M. incognita acrita. Both populations were identified as M. incognita race 1 by reaction on the North Carolina differential hosts. Reactions on azalea and pepper gave no clear identification of the populations. We concluded that there is no relation between perineal pattern, male head shape, and parasitism of host differentials with the two populations studied.     

Influence of Environmental Factors on the Hatch and Survival of Meloidogyne incognita

The influence of soil temperature and moisture on Meloidogyne incognita (Kofoid and White) Chitwood was examined in relation to hatching and survival of second-stage juveniles (J2). Nematodes were cultured on cotton (Gossypium hirsutum L. cv. Acala SJ2) under field conditions to provide populations similar to those found in the field in late autumn. Egg masses were placed in a temperature range (9-12 C and 21 C), and hatch was measured over a period equivalent to 20 degree days > 10 C (DD10). Hatch occurred below the reported 18 C activity threshold, was restricted below 12 C, and was inhibited below 10 C. Soil moisture influence on hatch was measured by placing egg masses in Hesperia sandy loam and subjecting them to suction pressures ranging from -1.1 bars to -4 .5 bars. Suction potentials of less than -2 bars reduced hatch and less than -3 bars inhibited hatch. J2 were placed in sandy loam soil with soil moisture near field capacity, and their motility was measured over a period of 500 DD10. In the absence of a host, more than 90% of J2 became nonmotile over this period.     

Characterization of Carbohydrates on the Surface of Second-stage Juveniles of Meloidogyne spp.

Fluorescent conjugates of the lectins soybean agglutinin (SBA), Concanavalin A (Con A), wheat germ agglutinin (WGA), Lotus tetragonolobus agglutinin (LOT), and Limulus polyphemus agglutinin (LPA) bound primarily to amphidial openings and amphidial secretions of viable, preinfective second-stage juveniles (J2) of Meloidogyne incognita races 1 and 3 (Mil, Mi3) and M. javanica (Mj). No substantial difference in fluorescent lectin binding was observed among the populations examined. Binding of only LOT and LPA were inhibited in the presence of 0.1 M competitive sugar. Structural differences in amphidial carbohydrate complexes among populations of Mi 1, Mi3, and Mj were revealed by glycohydrolase treatment of preinfective J2 and subsequent labeling with fluorescent lectins. A quantitative microfiltration enzyme-linked lectin assay revealed previously undetected differences in lectin binding to nonglycohydrolase-treated J2. Freinfective J2 of Mj bound the greatest amount of SBA, LOT, and WGA, whereas J2 of Mil bound the most LPA.     

Morphological Comparison of Second-Stage Juveniles of Six Populations of Meloidogyne hapla by SEM

External morphology of second-stage juveniles of six populations of Meloidogyne hapla, hclonging to two cytological races (A and B), and one population each of M. arenaria, M. incognita, and M. javanica was compared by scanning electron microscopy (SEM). Race A of M. hapla included three facultatively parthenogenetic populations with haploid chromosome numbers of 15. 16, and 17; race B consisted of three mitotically parthenogenetic populations with somalic chromosome numhers of 45, 45, and 48. The mitotically parthenogenetic populations of M. arenaria, M. incognita, and M. javanica had 54, 41-43, and 44 chromosomes, respectively. Observations were made on head structures, lateral field, excretory pore, anal opening, and tail. Head morphology, including shape and proportion of labial disc and lips, expression of labial and cephalic sensilla, and markings on head region, was distinctly different for each species. M. hapla populations of race A were distinct from each other but showed much intrapopulatiou variation in head morphology. Populations of race B were different from those of race A and were very stable and quite similar in head morphology. Considerable inter- and intrapopulatiou variation made the structure of the lateral field, excretory pore, anal opening, and tail of little value in distinguishing species or populations. The results are discussed in relation to earlier SEM observations on the genus Helerodera.     

Growth of Isolates of Paecilomyces lilacinus and Their Efficacy in Biocontrol of Meloidogyne incognita on Tomato

The potential of 13 Paecilomyces lilacinus isolates from various geographic regions as biocontrol agents against Meloidogyne incognita, the effects of temperature on their growth, and the characterization of the impact of soil temperature on their efficacy for controlling this nematode were investigated. Maximum fungal growth, as determined by dry weight of the mycelium, occurred from 24 to 30 C; least growth was at 12 and 36 C. The best control of M. incognita was provided by an isolate from Peru or a mixture of isolates of P. lilacinus. As soil temperatures increased from 16 to 28 C, both root-knot damage caused by M. incognita and percentage of egg masses infected by P. lilacinus increased. The greatest residual P. lilacinus activity on M. incognita was attained with a mixture of fungal isolates. These isolates effected lower root-galling and necrosis, egg development, and enhanced shoot growth compared with plants inoculated with M. incognita alone.     

Ultrastructural Cytochemistry of Secretory Granules of Esophageal Glands of Meloidogyne incognita

Ultrastructural cytochemical tests for several enzymes, proteins, carbohydrates, and nucleic acids were conducted on secretory granules o£ dorsal and subventral esophageal glands of preparasitic second-stage juveniles and the dorsal gland of adult females of Meloidogyne incognita. Secretory granules in the subventral glands of juveniles stained positive for acid phosphatase. Peroxidase, DNase, RNase, cellulase, and nucleic acids were not detected in these granules. Secretory granules in the dorsal gland of adult females stained positive for peroxidase (pH 7.6) in < 50% of the tests, Acid phosphatase, β-glucuronidase, DNase, RNase, polyphenoloxidase, cellulase, and carbohydrates were not detected in dorsal gland granules in adult females. Positive staining with cobalt thiocyanate, a stain for amino groups of basic proteins, occurred in secretory granules in the dorsal gland, ribosomes, and chromatin in adult females. Ribosomes, nuclei, and secretory granules of the dorsal gland of adult females intensely stained when incubated in three reagents specific for nucleic acid.     

Production and Partial Characterization of Stylet Exudate from Adult Females of Meloidogyne incognita

Adult females of Meloidogyne incognita were excised from tomato roots and incubated in 0.04 M phosphate buffered saline, pH 7.4 for 18-72 hours to allow accumulation of stylet exudate. Twenty-four percent of the females produced exudate during the initial 18-hour incubation period; 70% of those females producing exudate initially produced additional exudate during the subsequent 54-hour incubation period. Analysis of exudate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of at least nine major protein bands. Differential staining with silver and Coomassie Brilliant Blue G-250 stains indicated that three of the bands were glycoproteins. Upon acid hydrolysis, 14 amino acids were detected in the stylet exudate. The basic amino acids lysine, histidine, and arginine comprised 21.8% of the total amino acids detected. No peroxidase activity was detected in the stylet exudates. Data presented extend and generally confirm prior work on the chemical composition of stylet exudate.     

Morphological Comparison of Head Shape and Stylet Morphology of Second-stage Juveniles of Meloidogyne Species

Head shape and stylet morphology of second-stage juveniles of one population each of M. incognita, M. javanica, M. arenaria, and M. hapla were compared by light microscopy. Excised stylets of each species were also compared by scanning electron microscopy (SEM). Differences in head morphology were observed only between M. hapla and the other three species. In SEM, differences in stylet size, shape, and relative distance of the dorsal esophageal gland orifice to the base of the stylet were evident. Differences in stylet morphology between M. incognita and M. javanica could not he detected by light microscopy, but M. arenaria and M. hapla could be distinguished from each other and from the other two species. Head shape and styler morphology of second-stage juveniles are considered useful taxonomic characters.     

Histological Responses of Four Leguminous Crops Infected with Meloidogyne incognita

Histological responses to Meloidogyne incognita infection in Rhizobium nodules of clover, horsebean, lupine, and pea were investigated. The formation of giant cells in vascular bundles of nodules and roots, and the basal connection of the nodule, were usually associated with abnormal xylem and/or deformed xylem strands. However, giant cells did not disturb or prevent the development of nodular tissues. Areas in which galls formed, wall thickness of giant cells, and number of giant cells around the nematode head varied with plant species. Ranking by gall size and giant-cell wall thickness was horsebean > lupine and pea > clover. The multinucleate condition in giant cells resulted from repeated mitoses without subsequent cytokinesis. The resulting nuclei agglomerated in irregularly shaped masses in some giant cells.     

Interaction of Meloidogyne incognita and Water Stress in Two Cotton Cultivars

A series of controlled-environment experiments were conducted to elucidate the effects of Meloidogyne incognita on host physiology and plant-water relations of two cotton (Gossypium hirsutum) cultivars that differed in their susceptibility to nematode infection. Inoculation of M. incognita-resistant cultivar Auburn 634 did not affect growth, stomatal resistance, or components of plant-water potential relative to uninoculated controls. However, nematode infection of the susceptible cultivar Stoneville 506 greatly suppressed water flow through intact roots. This inhibition exceeded 28% on a root-length basis and was similar to that observed as a consequence of severe water stress in a high evaporative demand environment. Nematodes did not affect the components of leaf water potential, stomatal resistance, transpiration, or leaf temperature. However, these factors were affected by the interaction of M. incognita and water stress. Our results indicate that M. incognita infection may alter host-plant water balance and may be a significant factor in early-season stress on cotton seedlings.     

Extremely Sensitive Thermotaxis of the Nematode Meloidogyne incognita

Eggs of the root-knot nematode Meloidogyne incognita were acclimated to 23 C. Newly hatched second-stage juveniles migrated toward higher temperatures when placed in shallow thermal gradients averaging 23 C. The threshold gradient for this response was below 0.001 C/cm, with a best estimate of 4 x 10⁻⁴ C/cm. Calculations of physical limitations on thermotaxis indicate that this sensitivity is well within the limits of what is physically possible.     

Influence of Initial Population Densities of Meloidogyne incognita on Three Chile Cultivars

The effects of Meloidogyne incognita on the Big Jim, Jalapeno, and New Mexico No. 6 chile (Capsicum annuum) cultivars were investigated in microplots for two growing seasons. All three cultivars were susceptible to M. incognita and reacted similarly to different initial populations of this nematode. Severe stunting and yield suppressions occurred at all initial M. incognita densities tested ranging from 385 to 4,230 eggs and larvae/500 cm³ soil. Regression analysis of the microplot data from a sandy loam soil showed yield losses of 31% for the 1978 season and 25% for the 1979 season for the three cultivars for each 10-fold increase in the initial population of M. incognita.     

Influence of Meloidogyne incognita on the Water Relations of Cotton Grown in Microplots

The effects of Meloidogyne incognita on the growth and water relations of cotton were evaluated in a 2-year field study. Microplots containing methyl bromide-fumigated fine sandy loam soil were infested with the nematode and planted to cotton (Gossypium hirsutum L.). Treatments included addition of nematodes alone, addition of nematodes plus the insecticide-nematicide aldicarb (1.7 kg/ha), and an untreated control. Meloidogyne incognita population densities reached high levels in both treatments where nematodes were included. Root galling, plant height at harvest, and seed cotton yield were decreased by nematode infection. In older plants (89 days after planting [DAP]), leaf transpiration rates and stomatal conductance were reduced, and leaf temperature was increased by nematode infection. Nematode infection did not affect (P = 0.05) leaf water potential in either young or older plants but lowered the osmotic potential. The maximum rate and cumulative amount of water flowing through intact plants during a 24-hour period were lower, on both a whole-plant and per-unit-leaf-area basis, in infected plants than in control plants. Application of aldicarb moderated some of the nematode effects but did not eliminate them.     

Biomass Partitioning in Tomato Plants Infected with Meloidogyne incognita

Tomato plants were inoculated with Meloidogyne incognita at initial populations (Pi) of 0, 1, 10, 50, 100, and 200 (x 1,000) eggs per plant and maintained in a growth chamber for 40 days. Total fresh biomass (roots + shoots) at harvest was unchanged by nematode inoculation with Pi of 1 x 10⁵ eggs or less. Reductions in fresh shoot weight with increasing Pi coincided with increases in root weight. Total fresh biomass declined with Pi above 1 x 10⁵ eggs, whereas total dry biomass declined at Pi above 1 x 10⁴ eggs. The greatest reduction percentages in fresh shoot biomass induced by root-knot nematodes occurred in the stem tissue, followed by the petiole + rachis; the least weight loss occurred in the leaflets. Although biomass varied among shoot tissues, the relationship between biomass of various shoot tissues and Pi was described by quadratic equations. The linear and quadratic coefficients of the equations (stem, petiole + rachis, or leaflets on Pi) did not differ among tissues when calculations were based on standardized values. Meloidogyne incognita-infected plants had thinner leaves (leaf area/leaf weight) than did uninfected plants. Reductions in leaf weight and leaf area with nematode inoculation occurred at nodes 5-15 and 4, 6-14, respectively. Losses in plant height and mass due to nematodes reflected shorter internodes with less plant mass at each node.     

Impact of Soil Texture on the Reproductive and Damage Potentials of Rotylenchulus reniformis and Meloidogyne incognita on Cotton

The effects of soil type and initial inoculum density (Pi) on the reproductive and damage potentials of Meloidogyne incognita and Rotylenchulus reniformis on cotton were evaluated in microplot experiments from 1991 to 1993. The equilibrium nematode population density for R. reniformis on cotton was much greater than that of M. incognita, indicating that cotton is a better host for R. reniformis than M. incognita. Reproduction of M. incognita was greater in coarse-textured soils than in fine-textured soils, whereas R. reniformis reproduction was greatest in a Portsmouth loamy sand with intermediate percentages of clay plus silt. Population densities of M. incognita were inversely related to the percentage of silt and clay, but R. reniformis was favored by moderate levels of clay plus silt (ca. 28%). Both M. incognita races 3 and 4 and R. reniformis effected suppression of seed-cotton yield in all soil types evaluated. Cotton-yield suppression was greatest in response to R. reniformis at high Pi. Cotton maturity, measured as percentage of open bolls at different dates, was affected by the presence of nematodes in all 3 years.     

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 ≤1.0 at both 6 weeks and 10 weeks after inoculation compared with 25.8 and 26.5 for Deltapine 16 at 6 and 10 weeks after inoculation, respectively, and 9.1 and 2.6 for Stoneville 453. Thus, the results indicate that significant advances have been made in developing improved cotton germplasm lines with the potential to produce higher yields in soils infested with R. reniformis or M. incogaita. In addition to good yield potential, germplasm lines N222-1-91 and N320-2-91 appear to possess low levels of resistance to R. reniformis and a high level of resistance to M. incognita. This germplasm combines high yield potential with significant levels of resistance to both R. reniformis and M. incognita.     

Yield-loss Models for Tobacco Infected with Meloidogyne incognita as Affected by Soil Moisture

Yield-loss models were developed for tobacco infected with Meloidogyne incognita grown in microplots under various irrigation regimes. The rate of relative yield loss per initial nematode density (Pi), where relative yield is a proportion of the value of the harvested leaves in uninfected plants with the same irrigation treatment, was greater under conditions of water stress or with high irrigation than at an intermediate level of soil moisture. The maximum rate of plant growth per degree-day (base 10 C) was reduced as nematode Pi increased when plots contained adequate water. When plants were under water stress, increasing Pi did not luther reduce the maximum rate of plant growth (water stress was the limiting factor). Cumulative soil matric potential values were calculated to describe the relationship between available water in the soil (matric potential) due to the irrigation treatments and subsequent plant growth.