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.     

Genotypic Differentiation of Meloidogyne Populations by Detection of Restriction Fragment Length Difference in Total DNA

Detection of EcoRI restriction fragment length differences in repetitive DNA sequences permitted the rapid diagnosis, by genotype, of randomly selected populations of Meloidogyne incognita, Races 1, 2, 3, and 4; M. javanica; M. arenaria, Races 1 and 2; and M. hapla, Races A and B.     

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.     

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.     

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.     

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.     

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.     

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.     

Meloidogyne incognita and Tomato Response to Thiamine,Ascorbic Acid, L-arginine,and L-glutamic Acid

The influence of solutions of ascorbic acid, thiamine, L-arginine, and L-gtutamic acid on egg hatch, juvenile survival, and development and reproduction of Meloidogyne incognita in susceptible and resistant tomatoes was studied. Maximum inhibition of egg hatch occurred at 2,000, 4,000, and 2,000 ppm for ascorbic acid, L-arginine, and L-glutamic acid, respectively. Larval survival was significantly reduced by concentrations of 2,000 ppm ascorbic acid and 1,000 ppm of L-arginine. Maximum inhibition of egg hatch and mortality of juveniles was achieved at a concentration of 4,000 ppm of ascorbic acid and L-arginine. L-glutamic acid and thiamine had respective moderate and minimal toxic effects. Foliar sprays of ascorbic acid, L-arginine, or L-glutamic acid suppressed the numbers of root galls, females, and egg masses on the susceptible tomato cultivar Tropic. Ascorbic acid and L-arginine had highly significant effects when applied to foliage before inoculation with nematodes. Thiamine had little effect. All sprays suppressed the numbers of root galls and females in roots of the resistant cultivar VFN8 when treatments were applied before inoculation. They were not, however, effective as post-inoculation treatments. Growth of a susceptible cultivar was improved by post-inoculation and pre-inoculation treatments when compared with the control plants which had neither nematode infection nor chemical treatment. No positive growth response to chemical treatment was seen in resistant control plants.     

Characterization of Anionic Peroxidases in Tomato Isolines Infected by Meloidogyne incognita

Changes in peroxidase activity during nematode infection were studied using root extracts of tomato near-isogenic lines differing in resistance to Meloidogyne incognita. Total peroxidase activity increased slightly in crude extracts of four susceptible isolines but doubled in two resistant lines, Monita and Motaci. Nematode infection enhanced levels of both p-phenylenediamine-pyrocatechol oxidase and syringaldazine oxidase 7 days after inoculation, especially in resistant lines. This elevated peroxidase activity in resistant isolines was caused by an increase in anionic peroxidase activity. These enzymes, which likely are involved in lignification, were isolated and purified from tomato isolines by ammonium sulfate precipitation, high performance ion-exchange chromatography, and gel electrophoresis. The purified anionic peroxidase extracts contained an electrophoretic band with Rf 0.51 that was present in extracts of infected but not uninfected roots.     

Variability in Reproduction of Four Races of Meloidogyne incognita on Two Cultivars of Soybean

Variability in the reproduction of the four races ofMeloidogyne incognita on the soybean cuhivars Pickett 71 and Centennial was studied in growth chamber experiments. Analysis of variance in the number of eggs produced by the races 6 weeks after the plants had been inoculated with 5,000 eggs of each race revealed that the nematode race by soybean cultivar interaction was highly significant (P = 0.001). Races 1, 3, and 4 produced from about 5,000 to 15,000 eggs per root system on Pickett 71 and only from about 300 to 600 eggs per root system on Centennial. In contrast, race 2 produced about 8,000 eggs per root system on Centennial and about 1,200 eggs per root system on Pickett 71. In a second experiment, in which the plants were inoculated with 2,000 second-stage juveniles, race 1 and race 2 produced about 13,000 and 3,000 eggs per root system, respectively, on Pickett 71 and about 600 and 10,000 eggs per root system, respectively, on Centennial. The results suggest that M. incognita resistance in soybean is race-specific.     

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.     

Variability in Reproduction of Four Populations of Meloidogyne incognita on Six Cultivars of Cotton

A range of virulence levels was found in four populations of Meloidogyne incognita collected from cotton fields of the Punjab region of Pakistan. The most virulent population was associated with development of larger gall size, larger giant cell formation and improved success of juveniles transitioning into reproducing adults. The most virulent nematode population, MI-78, emanated from cotton cultivar NIAB-78. This cotton cultivar also possessed the greatest level of resistance to the three other nematode populations evaluated in this study. The source of plant resistance was not evident during root penetration by second-stage juveniles (J2), but became apparent as nematode feeding was attempted. Although one other cotton cultivar, CIM-506, could also be designated as showing a level of resistance, none of the other cultivars reduced any nematode stage by more than 75% of that achieved on the best host. These data provide an example of a single cotton cultivar that could have short-term utility in field settings. The data also provide insight for future cotton breeding programs.     

Penetration and Development of Meloidogyne incognita on Roots of Resistant Soybean Genotypes

Meloidogyne incognita penetration and development were studied in roots of highly resistant (PI 96354, PI 417444), resistant (Forrest), and susceptible (Bossier) soybean genotypes. Although more second-stage juveniles (J2) had penetrated roots of PI 96354 and PI 417444 than roots of Forrest and Bossier by 2 days after inoculation, fewer J2 were present in roots of PI 96354 at 4 days after inoculation. Juvenile development in all genotypes was evident by 6 days after inoculation, with the highest number of swollen J2 present in roots of Bossier. At 16 days after inoculation, roots of PI 96354 had 87%, 74%, and 53% fewer J2 than were present in roots of Bossier, Forrest, and PI 417444, respectively. Differential emigration of J2, not fewer invasion sites, was responsible for the low number of nematodes in roots of the highly resistant PI 96354. Some 72% of the J2 penetrating the roots of this genotype emerged within 5 days after inoculation, whereas 4%, 54%, and 83% emerged from roots of Bossier, Forrest, and PI 417444, respectively. Penetration of roots of PI 96354 decreased the ability of J2 emerging from these roots to infect other soybean roots.     

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.     

Induction of Isoperoxidases in Resistant and Susceptible Tomato Cultivars by Meloidogyne incognita

Isoperoxidases were detected in resistant Rossol and susceptible Roma VF tomato roots uninfected and infected by Meloidogyne incognita. Syringaldazine, guaiacol, p-phenylenediamine-pyrocatechol (PPD-PC), and indoleacetic acid (IAA) were used as substrates, and the corresponding peroxidative activities were detected either in cytoplasmic or in cell wall fractions, except for IAA oxidase, which was measured in soluble and microsomal fractions. Isoperoxidase activities and cellular locations were induced differently in resistant and susceptible cultivars by nematodes. Nematode infestation markedly enhanced syringaldazine oxidase activity in cell walls of the resistant cultivar. This isoperoxidase is involved in the last step of lignin deposition in plants. Conversely, the susceptible cultivar reacted to M. incognita infection with an increase in cytoplasmic PPD-PC oxidase activity, which presumedly is involved in ethylene production; no changes in cell wall isoperoxidases were observed. IAA oxidase was inhibited in susceptible plants after nematode inoculation, whereas in resistant plants this activity increased in the soluble fraction and decreased in the microsomal fraction.     

Identification of the Chromosome Carrying the Factor for Resistance to Meloidogyne incognita in Tobacco

To identify the chromosome carrying the factor for resistance to Meloidogyne incognita in tobacco, crosses were made between resistant tobacco ''NC95'' as pollen parent and each of the 12 tobacco monosomics (A-L) representative of the Tomentosae half of the Nicotiana tabacum chromosome complement. Of the F₁ seedlings, 927 plants were grown for observation. From these, 223 plants were selected as possible monosomics on the basis of morphological characteristics. These plants were self-pollinated, and the resulting F₂ plants were inoculated with both M. incognita acrita and M. incognita incognita. Sixteen F₂ populations, derived from the haplo-G monosome, were completely resistant. All of the F₂ populations derived from the other 11 monosomic crosses segregated into a 3:1 (resistant:susceptible) ratio. These results indicate that the factor for resistance to M. incognita is located on the G chromosome of N. tabacum. This is the first report establishing the N. tabacum chromosome that carries the factor for root-knot resistance. The results are consistant with our earlier evidence that M. incognita resistance in tobacco is derived from N. tomentosa, a species in the section Tomentosae of the subgenus Tabacum, genus Nicotiana. The other 12 chromosomes of N. tabacum have affinities with N. sylvestris, section Alatae, subgenus Petunoides, genus Nicotiana.     

Localization of Cuticular Binding Sites of Concanavalin A on Caenorhabditis elegans and Meloidogyne incognita

Utilizing a Concanavalin A (Con A)-hemocyanin conjugate, the majority of cuticular Con A binding sites were shown to be localized on the head region of Caenorhabclitis elegans and Meloidogyne incognita. Secretions which apparently emanated from the amphids and inner labial papillae did not label.     

Identification of msp1 Gene Variants in Populations of Meloidogyne incognita Using PCR-DGGE

Effectors of root-knot nematodes are essential for parasitism and prone to recognition by adapted variants of the host plants. This selective pressure initiates hypervariability of effector genes. Diversity of the gene variants within nematode populations might correlate with host preferences. In this study we developed a method to compare the distribution of variants of the effector gene msp1 among populations of Meloidogyne incognita. Primers were designed to amplify a 234-bp fragment of msp1. Sequencing of cloned PCR products revealed five msp1 variants from seven populations that were distinguishable in their reproduction on five host plants. A protocol for denaturing gradient gel electrophoresis (DGGE) was developed to separate these msp1 variants. DGGE for replicated pools of juveniles from the seven populations revealed ten variants of msp1. A correlation between the presence of a particular gene variant and the reproductive potential on particular hosts was not evident. Especially race 3 showed substantial variation within the population. DGGE fingerprints of msp1 tended to cluster the populations according to their reproduction rate on pepper. The developed method could be useful for analyzing population heterogeneity and epidemiology of M. incognita.     

Effect of Meloidogyne incognita on Plant Nutrient Concentration and Its Influence on the Physiology of Beans

Phaseolus vulgaris plants, 3, 8, 11, and 13 days old, were inoculated with 0, 2,000, 4,000, or 8,000 second-stage Meloidogyne incognita larvae and maintained under controlled conditions. The photosynthetic rate and the shoot and root concentration of K, Ca, Mn, Fe, Cu, and Zn were determined by destructive assay at 1-27-day intervals and by nondestructive assay of leaves, stems, and roots at 27 or 28 days after inoculation. In the destructive assay, the concentration of the elements in the plant tissues did not change until 1 week after inoculation. Thereafter, the trend was mostly decreasing for shoot K and Fe and increasing in the root, whereas Ca had the opposite trend in the shoots. Manganese, Cu, and Fe showed variable trends. Generally, the concentration of K and Mn increased, whereas Ca and Fe decreased, with duration of infection in all treatments. Zinc and Cu decreased in the highest nematode treatments. The overall elemental content generally decreased with level of infection from 1 week after inoculation. Photosynthetic rate based on shoot K concentration significantly decreased with level of infection. In most of the nondestructive assays, the concentrations of shoot K, Zn, and Mn decreased, whereas Ca increased with increasing nematode treatment. One of the first effects of the nematode on host physiology appears to be a change in concentration of nutrient elements in the host plant.