Behavior of Tethered Meloidogyne incognita

The tethered-nematode technique was adapted for use with second-stage juveniles of Meloidogyne incognita. The data demonstrate that M. incognita exhibits the same patterns of behavior as adults of the free-living nematode, Caenorhabditis elegans. The principal differences are that M. incognita is slower and less regular in its behavior than C. elegans. The frequency of normal waves is about 0.2 Hz; that of reversal waves is about 0.06 Hz. Reversal bouts last about 1 minute. In response to a change in NaCl concentration, M. incognita modulates the probability of initiating a reversal bout in the same manner as C. elegans except that it responds more slowly and is repelled instead of attracted.     

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.     

Cold Hardening of Meloidogyne hapla Second-stage Juveniles

The effect of previous exposure to low temperatures on freezing tolerance was determined for second-stage juveniles of Meloidogyne hapla. Juveniles in 5% polyethylene glycol 20,000 were exposed to 0-24 C for 12-96 hours, and then freezing tolerance was assessed by freezing samples at -4 C for 24 hours, thawing, and determining survival. Freezing tolerance was inversely related to prefreeze temperatures of 4-24 C. Prefreeze exposure to 4 C resulted in fourfold greater freezing tolerance than did exposure to 24 C. Mortality occurred during prefreeze exposure to 0 C. Most of the increase in freezing tolerance at 4 C occurred during the first 12 hours. In soil, prefreeze exposure to 4 C resulted in greater freezing tolerance than did prefreeze exposure to 24 C.     

Root Penetration by Meloidogyne incognita Juveniles Infected with Bacillus Penetrans

Bacillus penetrans inhibited penetration by Meloidogyne incognita second-stage juveniles (J2) into tomato roots in the laboratory and greenhouse. Spores from this Florida population of B. penetrans attached to J2 of M. javanica, M. incognita, and M. arenaria. A greater proportion of J2 of M. javanica were infected than were J2 of either M. incognita or M. arenaria, and a greater number of spores attached to M. incognita than to M. arenaria.     

Effect of Soil Texture on the Distribution and Infectivity of Neoaplectana glaseri (Nematoda: Steinernematidae)

The vertical migration of infective juveniles of Neoaplectana glaseri applied to the soil surface or introduced 16 cm below the soil surface was studied in pure silica sand, coarse sandy loam, silty clay loam, and clay. The number of juveniles that migrated and infected wax moth pupae placed in the soil decreased as the proportion of clay and silt increased. The majority of nematodes moved downwards 2-6 cm from the surface, but some penetrated to a depth of 14 cm in pure silica sand and coarse sandy loam. In pure silica sand and coarse sandy loam, nematodes introduced 16 cm below the soil surface were able to infect wax moth pupae located at depths of 0-4 cm and 28-32 cm. Nematodes showed a greater tendency to disperse downwards from the point of application. Movement of the nematode was least in clay soil and limited in silty clay loam soil. The number of migrating nematodes was greatest when wax moth pupae were present.     

Effects of Soil Texture on the Interaction Between Rhizoctonia solani and Meloidogyne incognita on Cotton Seedlings

Soils containing 60, 75, and 90% coarse particles (sand plus coarse silt) were prepared by dilution of a field soil with 246μm (60-mesh) silica sand. As the coarse-particle content of the soils increased, the synergistic interaction between Meloidogyne incognita and Rhizoctonia solani on cotton seedlings increased. Increasing the coarse-particle content of the soil also increased damage from the nematode alone and slightly increased soreshin damage from R. solani alone.     

Effect of Soil Texture on the Distribution and Infectivity of Neoaplectana carpocapsae (Nematoda: Steinernematidae)

The vertical migration of N. carpocapsae infective juveniles applied to the soil surface or introduced 14 cm below the soil surface was studied in four different soil types (pure silica sand, coarse sandy loam, silty clay loam, and clay). The percentage of juveniles able to migrate and infect wax moth pupae placed in the soil decreased as the percentage of clay and silt increased. Most nematodes placed on the soil surface remained within 2 cm of the surface, but some penetrated to a depth of 10 cm in pure silica sand and coarse sandy loam to infect pupae. Some pupae at the same depth were also infected with nematodes in silty clay loam soil. In pure silica sand and coarse sandy loam, nematodes introduced 14 cm below the soil surface were able to infect wax moth pupae located between 4 and 24 cm. Movement was least in clay soil and limited in silty clay loam. Nematodes showed a tendency to disperse upwards from the point of application. In all cases the number of migrating nematodes was greatest when wax moth pupae were present.     

Isolation of Subcellular Granules from Second-Stage Juveniles of Meloidogyne incognita

Subcellular granules from the second-stage (preparasitic) juveniles of root-knot nematode Meloidogyne incognita were isolated by isopycnic centrifugation on Percoll. The granules had an apparent density of 1.13 g/cm³. The relative specific activity of acid phosphatase in the granule extract was 8.4. Acid phosphatase activity was also detected histochemically in the subventral gland granules. Electron microscopy and malate dehydrogenase activity indicated that contamination of granules by mitochondria was negligible. Electrophoresis of the granule extract in the presence of sodium dodecyl sulfate showed 15-20 major protein bands.     

Effect of Meloidogyne incognita on Selected Forest Tree Species

Four or five growth stages of 14 forest tree species were tested for susceptibility to Meloidogyne incognita at five inoculum levels. Responses ranged from the highly susceptible ''China fir'' to immune ''Taiwania''. Even highly susceptible species became increasingly tolerant at later growth stages, thus root-knot appears to be a greater problem in nurseries than in established forests. Heavily suberized cells which restricted nematode development was the predominant host response in Norway spruce, and in the jack, scotch, and Virginia pines. Adult females in jack and scotch pine, which elicited a minimum of suberized tissue, were found adjacent to infection sites showing maximum suberization which indicates that resistance can be highly localized and variable within an individual host. A few gravid females, but no giant cells, were observed in these two species.     

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.     

Effect of Crotalaria juncea Amendment on Squash Infected with Meloidogyne incognita

Two greenhouse experiments were conducted to examine the effect of Crotalaria juncea amendment on Meloidogyne incognita population levels and growth of yellow squash (Cucurbita pepo). In the first experiment, four soils with a long history of receiving yard waste compost (YWC+), no-yard-waste compost (YWC-), conventional tillage, or no-tillage treatments were used; in the second experiment, only one recently cultivated soil was used. Half of the amount of each soil received air-dried residues of C. juncea as amendment before planting squash, whereas the other half did not. Crotalaria juncea amendment increased squash shoot and root weights in all soils tested, except in YWC+ soil where the organic matter content was high without the amendment. The amendment suppressed the numbers of M. incognita if the inoculum level was low, and when the soil contained relatively abundant nematode-antagonistic fungi. Microwaved soil resulted in greater numbers of M. incognita and free-living nematodes than frozen or untreated soil, indicating nematode-antagonistic microorganisms played a role in nematode suppression. The effects of C. juncea amendment on nutrient cycling were complex. Amendment with C. juncea increased the abundance of free-living nematodes and Harposporium anguillulae, a fungus antagonistic to them in the second experiment but not in the first experiment. Soil histories, especially long-term yard waste compost treatments that increased soil organic matter, can affect the performance of C. juncea amendment.     

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.     

Predicting Damage of Meloidogyne incognita on Watermelon

Quantitative growth response of watermelon (Citrullus lanatus) sensitive to Meloidogyne incognita is poorly understood. Determination of soil population densities of second-stage juveniles (J2) of M. incognita with Baermann funnel extraction often is inaccurate at low soil temperatures. In greenhouse experiments, three sandy soils were inoculated with dilution series of population densities of eggs or J2 of M. incognita and planted in small containers to watermelon ‘Royal Sweet’ or subjected to Baermann funnel extraction. After five weeks of incubation in the greenhouse bioassay plants in egg-inoculated soils, gall numbers on watermelon roots related more closely to inoculated population densities than J2 counts after Baermann funnel extraction. In April 2004, perpendicularly-inserted tubes (45-cm diameter, 55-cm deep) served as microplots where two methyl bromide-fumigated sandy soils were inoculated with egg suspensions of M. incognita at 0, 100, 1,000 or 10,000 eggs/100 cm³ of soil in 15-cm depth. At transplanting of 4-week old watermelon seedlings, soils were sampled for the bioassay or for extraction of J2 by Baermann funnel. In the Seinhorst function of harvested biomass in relation to nematode numbers, decline of biomass with increasing population densities of M. incognita was accurately modeled by the inoculated eggs (R² = 0.93) and by the counts of galls on the bioassay roots (R² = 0.98); but poorly by J2 counts (R² = 0.68). Threshold levels of watermelon top dry weight to M. incognita were 122 eggs/100 cm³ soil, 1.6 galls on bioassay roots, or 3.6 J2/100 cm³ of soil. Using the bioassay in early spring for predicting risk of nematode damage appeared useful in integrated pest management systems of watermelon.     

Winter Survival of Meloidogyne incognita in Six Soil Types

Winter survival of Meloidogyne incognita in six soil types (Fuquay sand, Norfolk loamy sand, Portsmouth loamy sand, muck, Cecil sandy clay loam, and Cecil sandy clay) was determined in microplots at one location from November 1981 to May 1982 and from November 1982 to March 1983. Survival, based on second-stage juveniles (J2) of M. incognita, from November 1981 until May 1982 ranged from 1% in the muck soil to 6% in a Cecil sandy clay loam, but survival rates were much higher the next year following a winter with higher average temperatures. Survival rates of J2 from November to March ranged from 20 to 40% the first winter and from 38 to 87% the second. Soil type did not have a striking effect on the overwintering capabilities ofM. incognita. There were no differences between clay and sand soils, whereas survival of J2 in the muck tended to be lower than in the mineral soils.     

Attachment of Pasteuria penetrans Endospores to the Surface of Meloidogyne javanica Second-stage Juveniles

Pasteuria penetrans spore adhesion to Meloidogyne javanica second-stage juveniles (J2) was examined following several different pretreatments of the latter. The detergents sodium dodecyl sulfate and Triton X-100, the carbohydrates fucose and α-methyl-D-mannoside, and the lectins concanavalin A and wheat germ agglutinin reduced spore attachment. Spores exposed to M. javanica surface coat (SC) extract exhibited decreased adherence to the J2 surface. Second-stage juveniles that had been treated with antibodies recognizing a 250-kDa antigen of J2 SC extract had fewer spores attached to their surfaces, as compared to nontreated J2, except in the head region. This inhibition pattern was similar to that of antibody-labelling on M. javanica J2 as observed by electron microscopy. It is suggested that several SC components, such as carbohydrate residues, carbohydrate-recognition domains, and a 250-kDa antigen, are involved in P. penetrans spore attachment to the surface of M. javanica.     

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.     

Effects of Soil Compaction and Meloidogyne incognita on Cotton Root Architecture and Plant Growth

The effects of a soil hardpan and Meloidogyne incognita on cotton root architecture and plant growth were evaluated in microplots in 2010 and 2011. Soil was infested with M. incognita at four different levels with or without a hardpan. The presence of a hardpan resulted in increased plant height, number of main stem nodes, and root fresh weight for cotton seedlings both years. Meloidogyne incognita decreased height and number of nodes for seedlings in 2010. Nematode infestation increased seedling root length and enhanced root magnitude, altitude, and exterior path length in 2010. This was also the case for root length and magnitude in 2011 at lower infestation levels suggesting compensatory growth. A hardpan had no consistent effect on these root parameters but increased root volume in both years. A hardpan hastened crop maturity and increased the number of fruiting branches that were produced, while M. incognita infection delayed crop development and reduced plant height and number of bolls. Both M. incognita infection and a hardpan reduced taproot length and root dry weight below the hardpan in both years. Root topological indices under all the treatments ranged from 1.71 to 1.83 both years indicating that root branching followed a herringbone pattern. The techniques for characterizing root architecture that were used in this study provide a greater understanding of changes that result from disease and soil abiotic parameters affecting root function and crop productivity.     

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.     

Interactions Between Meloidogyne incognita and Pratylenchus brachyurus on Soybean

Interactions among Meloidogyne incognita, Pratylenchus brachyurus, and soybean genotype on plant growth and nematode reproduction were studied in a greenhouse. Coker 317 (susceptible to both nematodes) and Gordon (resistant to M. incognita, susceptible to P. brachyurus) were inoculated with increasing initial population densities (Pi) of both nematodes individually and combined. M. incognita and P. brachyurus individually usually suppressed shoot growth of both cultivars, but only root growth on Coker 317 was influenced by a M. incognita × P. brachyurus interaction. Reproduction of both nematodes, although dependent on Pi, was mutually suppressed on Coker 317. P. brachyurus reproduced better on Gordon than on Coker 317 but did not affect resistance to M. incognita. Root systems of Coker 317 were split and inoculated with M. incognita or P. brachyurus or both to determine the nature of the interaction. M. incognita suppressed reproduction of P. brachyurus either when coinhabiting a half-root system or infecting opposing half-root systems; however, P. brachyurus affected M. incognita only if both nematodes infected the same half-root system.