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.     

Monoclonal Antibodies to the Esophageal Glands and Stylet Secretions of Heterodera glycines

Three monodonal antibodies (MAbs) that bound to secretory granules within the subventral esophageal glands of second-stage juveniles (J2) of the soybean cyst nematode (SCN), Heterodera glycines, were developed from intrasplenic immunizations of a mouse with homogenates of SCN J2. Two MAbs to the secretory granules within subventral glands and one MAb to granules within the dorsal esophageal gland of SCN J2 were developed by intrasplenic immunizations with J2 stylet secretions. Stylet secretions, produced in vitro by incubating SCN J2 in 5-methoxy DMT oxalate, were solubilized with a high pH buffer and concentrated for use as antigen. Three of the five MAbs specific to the subventral esophageal glands bound to stylet secretions from SCN J2 in immunofluorescence and ELISA assays. Two of these three MAbs also bound to secretory granules within both the dorsal and subventral esophageal glands of young SCN females. All five of the subventral gland MAbs bound to the subventral glands of Heterodera schachtii and one bound to the subventral glands of Globodera tabacum, but none bound to any structures in Meloidogyne incognita or Caenorhabditis elegans.     

Ultrastructure of Esophageal Gland Secretory Granules in Juveniles of Heterodera glycines

Ultrastructural observations of the feeding sites of soybean cyst nematode juveniles 3 days after inoculation of soybean roots revealed the presence of feeding tubes in the host cell syncytium. Feeding tubes, which were extruded from the stylet tips, were formed by products of secretory granules that originated in the dorsal esophageal gland and accumulated in the ampulla of the gland extension. Granules traversing the space between the gland cell and the ampulla were regulated in their movement by two sets of sphincter-like muscles located anterior and posterior to the metacorpus pump chamber. Sections through the sphincter muscles revealed obliquely arranged fibers, which in a contracted mode caused microtubules in the gland extension to be tightly packed and devoid of granules.     

Effects of Monoclonal Antibodies,Cationized Ferritin,and Other Organic Molecules on Adhesion of Pasteuria penetrans Endospores to Meloidogyne incognita

The incidence of adhesion of Pasteuria penetrans endospores to Meloidogyne incognita second-stage juveniles (J2) was studied after pretreatment of the latter with monoclonal antibodies (MAb), cationized ferritin, and other organic molecules in replicated trials. Monoclonal antibodies developed to a cuticular epitope of M. incognita second-stage juveniles gave significant reductions in attachment of P. penetrans endospores to treated nematodes. MAb bound to the entire length of J2 except for the area of the lateral field, where binding was restricted to the incisures. Since reductions in attachment with MAb treatment were modest, it is uncertain if these results implicated a specific surface protein as a factor that interacted in binding of the endospore to the nematode cuticle. Endospore attachment was decreased following treatment of the nematode with the detergents sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB). Endospore attachment to live nematodes was significantly greater than attachment to dead nematodes. Attachment rates of three P. penetrans isolates to M. incognita race 3 varied between isolates. The effects of neuraminidase, pronase, pepsin, trypsin, lipase, and Na periodate on endospore attachment were inconsistent. The cationic dye alcian blue, which binds sulfate and carboxyl groups on acidic glycans, had no consistent effect on endospore attachment. The incidence of endospore attachment was significantly lower but modest, at best, for nematodes that were treated with cationized ferritin alone or cationized ferritin following monoclonal antibody. The lack of consistency or extreme reduction in most experiments suggests that attachment of P. penetrans spores to M. incognita is not specified by only one physico-chemical factor, but may involve a combination of at least two physico-chemical factors (including surface charge and movement of the J2). This points to a need for analysis of combined or factorial treatment effects.     

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.     

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.     

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.     

Effects of Resistance in Phaseolus vulgaris on Development of Meloidogyne Species

Use of resistant Phaseolus vulgaris germplasm has a potential role in limiting damaging effects of Meloidogyne spp. on bean production. Effects of two genetic resistance systems in common bean germptasm on penetration and development of Meloidogyne spp. were studied under growth room conditions at 22°C to 25°C. Nemasnap (gene system 1) and G1805 (gene system 2) were inoculated with second-stage juveniles (J2) of M. incognita race 2 and M. arenaria race 1, respectively; Black Valentine was used as the susceptible control. Up to 7 days after inoculation, there were no differences in numbers of M. incognita J2 penetrating roots of Black Valentine and Nemasnap; subsequently, more nematodes were present in Black Valentine roots (P < 0.05). More nematodes reached advanced stages of development in Black Valentine than in Nemasnap roots (P < 0.05). Total numbers of M. arenaria were greater in Black Valentine than in G 1805 roots from 14 days after inoculation (P < 0.05). Advanced stages of development occurred earlier and in greater numbers in Black Valentine plants than in G1805 plants. In these studies, resistance to M. incognita race 2 and M. arenaria race 1 in bean germplasm, which contain gene system 1 and gene system 2, respectively, was expressed by delayed nematode development rather than by differential penetration compared with susceptible plants.     

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.     

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.     

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.     

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.     

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.     

The Immunofluorescent Localization of Subventral Pharyngeal Gland Epitopes of Preparasitic Juveniles of Heterodera glycines Using Laser Scanning Confocal Microscopy

Laser scanning confocal microscopy (LSCM) was used to localize the reactivity of a monoclonal antibody (Sv2) that binds to the subventral pharyngeal glands of preparasitic juveniles of Heterodera glycines. The greater resolution, magnification, and image analysis of LSCM compared with conventional epifluorescent microscopy enabled Sv2 binding to be localized much more precisely to the periphery of the secretory granules. A linear increase of about 55% in fluorescent intensity was found over a 23-μm length of subventral pharyngeal gland just distal to the terminal ampullae. LSCM is a rapid and effective technique for precise immunolocalization of epitopes.     

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.     

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.     

Esterase and Malate Dehydrogenase Phenotypes in Portuguese Populations of Meloidogyne Species

Nonspecific esterases and malate dehydrogenases of 1-5 females from 40 root-knot nematode populations from Portugal were analyzed by electrophoresis in 0.4-mm-thick polyacrylamide gels. Fourteen major bands of esterase activity were detected, corresponding to 10 distinct phenotypes, Meloidogyne javanica and M. hapla had distinct species-specific phenotypes. Two phenotypes occurred in M. arenaria. The most variability was found among M. incognita populations. Of the remaining two phenotypes, one was associated with M. hispanica and the other belonged to a new species. Three malate dehydrogenase phenotypes were discerned on the basis of particular combinations of the eight main bands of activity found. As previously found, esterases were more useful than malate dehydrogenases in identification of the major Meloidogyne species. The host plant had no effect on the nematode esterase or malate dehydrogenase phenotypes.     

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.     

Origin of a Meloidogyne incognita Surface Coat Antigen

The surface coat (SC) of plant nematodes is thought to originate either from the living hypodermis or from secretory glands associated with the excretory system or nervous system. In this study, we investigated the origin of the SC of Meloidogyne incognita by immunolocalization with a monoclonal antibody raised against the surface coat of the preparasitic juveniles (J2). Under the electron microscope, strong labeling was found on the cuticular surface and in the rectal dilation of the J2, while labeling was absent in other parts of the nematode, including the hypodermis, excretory system, nervous system, and digestive system. Because the rectal glands are known to be the origin of the gelatinous egg matrix produced by adult females of Meloidogyne, we also examined sections of mature females from monoxenic cultures of Arabidopsis thaliana. Labeling of the female occurred in the rectal glands and in the gelatinous matrix exuded from the anus. At the ultrastructural level, gold particles were mainly deposited in multivesicular bodies that appeared to be associated with the Golgi bodies of the rectal glands. Our results suggest that at least one component of the J2 SC originates from the rectal gland cells and that the SC of the J2 shares common epitopes with the gelatinous egg matrix of mature females.     

Interaction between Meloidogyne incognita and Hoplolaimus columbus on Davis Soybean

Greenhouse and laboratory experiments were performed to determine if an interaction exists between Meloidogyne incognita and Hoplolaimus columbus on Davis soybean. Greenhouse tests were performed with three population levels of M. incognita and H. columbus (0, 1,500, 6,000/1.5-liter pot) separately and in all combinations. Dry root weight (DRT) declined nonlinearly and dry shoot weight (DST) declined linearly with respect to increasing initial populations of M. incognita and H. columbus. When the two nematode species were added to the soil together, the amount of DRT and DST suppression by one species was dependent on the initial level of the concomitant species. The final root population of M. incognita or H. columbus declined linearly with increasing initial population density of the concomitant species. H. columbus suppressed M. incognita populations in the soil nonlinearly, but M. incognita had no effect on H. columbus.