Glycoprotein Characterization of the Gelatinous Matrix in the Root-knot Nematode Meloidogyne javanica

Proteinaceous components of freshly formed gelatinous matrix (GM) of the root-knot nematode Metoidogyne javanica were analyzed. Under reducing conditions, the prominent protein fragments had molecular weights of 26 to 66 kDa and 150 to >200 kDa, and most were glycosylated. Most of the fragments were digested by proteinase K, and fewer by trypsin. The lectins soybean agglutinin (SBA), Ulex europaeus agglutinin, and wheat germ agglutinin labeled the higher molecular weight bands (i.e., >200 kDa). SBA labeled additional protein fractions between 26 and 66 kDa. Although Bandeiraea simplicifolia lectin and Concanavalin A did not label bands on the Western blot, they did label the GM in the dot blot technique. Analysis of amino acids and amino sugars in the GM revealed an unusually high amount of ammonia and galactosamine moieties.     

Composition of High Molecular Weight Excretions-Secretions from Infective Larvae of Meloidogyne javanica

Infective larvae (J2) of Meloidogyne javanica were incubated in distilled water for up to 14 days, and their high molecular weight (> 1,000 daltons) excretions-secretions (ES) were isolated and partially characterized. The ES consisted of a mixture of proteins, glycoproteins, and proteoglycans or polysaccharides as revealed by differential staining on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and compositional analysis. Carbohydrate, with approximately equal amounts of neutral monosaccharides and hexosamines, was the major constituent of the ES, with only low levels of protein detected. Acidic sugar residues, including sialic acids, were not detected.     

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.     

Meloidogyne javanica on Peanut in Florida

A mixed population of Meloidogyne arenaria race 1 and M. javanica race 3 is reported on peanut from a field in Levy County, Florida. Confirmation of M. javanica on peanut is based on esterase and malate dehydrogenase isozyme patterns resolved on polyacrylamide slab gels following electrophoresis, and perineal patterns. Up to 29% of 290 individual females collected from peanut roots in the field in autumn 2002 showed a typical esterase J3 phenotype for M. javanica. This is the third report of M. javanica infecting peanut in the United States.     

Glucuronidase Expression in Transgenic Tobacco Roots with a Parasponia Promoter on Infection with Meloidogyne javanica

The expression of a g-us reporter gene linked to a Parasponia andersonii hemoglobin promoter has been studied in transgenic tobacco plants after infection by Meloidogyne javanica. Transgenic roots were harvested at different times after nematode inoculation, and stained histochemically for expression of the gus gene. During the early stages of infection (0-2 weeks) there was little expression in giant cells, in contrast to other cells of the root. In later stages of infection (3-6 weeks) there was strong gus expression in giant cells, with virtually no expression in other cells of the root. The Parasponia hemoglobin promoter therefore appears to direct down-regulation of linked genes on induction of giant cells, but up-regulation in mature giant cells. This reflects different metabolic activities in the giant cells depending on their stage of development. The Parasponia hemoglobin promoter may respond to oxygen tension in giant cells. This suggests that oxygen tension may be limited in the metabolically active giant cells that are associated with egg-laying females.     

Meloidogyne javanica Parasitic on Peanut

Peanut fields in four governorates of Egypt were surveyed to identify species of Meloidogyne present. Fourteen populations obtained from peanut roots were all identified as M. javanica based on perineal patterns, stylet and body lengths of second-stage juveniles, esterase phenotypes, and restriction fragment length polymorphisms of mtDNA. Three of 14 populations, all from contiguous fields in the Behara governorate, had individuals with a unique two-isozyme esterase phenotype. All populations of M. javanica tested on peanut had levels of reproduction on the M. arenaria-susceptible peanut cultivar Florunner that were not different from M. arenaria (P = 0.05), and had lower levels of reproduction on the M. arenaria-resistant genotype TxAG-7 than on Florunner (P = 0.05). Reproduction of the five Egyptian populations of M. javanica tested was lower on root-knot nematode resistant tomato cultivars Better Boy and Celebrity than on the root-knot nematode susceptible cultivar Rutgers (P = 0.05). These data are evidence that some populations of M. javanica are parasitic on peanut and that the peanut and tomato genotypes resistant to M. arenaria are also resistant to these populations of M. javanica.     

Reproduction and Development of Meloidogyne incognita and M. javanica on Guardian Peach Rootstock

Guardian peach rootstock was evaluated for susceptibility to Meloidogyne incognita race 3 (Georgia-peach isolate) and M. javanica in the greenhouse. Both commercial Guardian seed sources produced plants that were poor hosts of M. incognita and M. javanica. Reproduction as measured by number of egg masses and eggs per plant, eggs per egg mass, and eggs per gram of root were a better measure of host resistance than number of root galls per plant. Penetration, development, and reproduction of M. incognita in Guardian (resistant) and Lovell (susceptible) peach were also studied in the greenhouse. Differences in susceptibility were not attributed to differential penetration by the infectivestage juveniles (J2) or the number of root galls per plant. Results indicated that M. incognita J2 penetrated Guardian roots and formed galls, but that the majority of the nematodes failed to mature and reproduce.     

Reproduction of Meloidogyne javanica on Plant Roots Genetically Transformed by Agrobacterium rhizogenes

Reproduction of Meloidogyne javanica was compared on several Agrobacterium rhizogenes-transformed root cultures under monoxenic conditions. M. javanica reproduced on all transformed roots tested; however, more females and eggs were obtained on potato and South Australian Early Dwarf Red tomato than on bindweed, Tropic tomato, lima bean, or carrot. Roots that grew at moderate rates into the agar and produced many secondary roots supported the highest reproduction. Numbers of females produced in cultures of transformed potato roots increased with increasing nematode inoculum levels, whether inoculum was dispersed eggs or juveniles. Females appeared smaller, produced fewer eggs, and were found in coalesced galls at the higher inoculum levels. The ratio between the final and initial population decreased sharply as the juvenile inoculum increased. The second-stage juvenile was preferred to dispersed eggs or egg masses for inoculation of tissue culture systems because quantity and viability of inoculum were easily assessed. Meloidogyne javanica reared on transformed root cultures were able to complete their life cycles on new transformed root cultures or greenhouse tomato plants.     

Morphological Comparison of Three Host Races of Meloidogyne javanica

A morphological and morphometric comparison using light microscopy and scanning electron microscopy was made of six populations of Meloidogyne javanica belonging to three host races (infective on pepper, peanut, or noninfective on both). The variability of certain morphological characters was studied within these populations, and the reliability of these taxonomic traits was evaluated for usefulness in species identification. The most useful diagnostic characters of M. javanica were head and stylet morphology of males and stylet morphology and perineal patterns of females. Males have an offset head region, usually lacking annulations, and a distinct, narrow head cap with slightly raised labial disc. The stylet has a cone markedly wider than the shaft at the junction and large, transversely ovoid knobs that are offset from the shaft. Females have a robust stylet with a dorsally curved cone and large, transversely ovoid knobs. Perineal patterns are oval to squarish in shape, usually with coarse, broken striae and with conspicuous lateral lines. The host races could not be differentiated on a morphological basis.     

Resistance of Interspecific Arachis Breeding Lines to Meloidogyne javanica and an Undescribed Meloidogyne Species

Resistance to a peanut-parasitic population of Meloidogyne javanica and an undescribed Meloidogyne sp. in peanut breeding lines selected for resistance to Meloidogyne javanica was examined in greenhouse tests. The interspecific hybrid TxAG-7 was resistant to reproduction of Meloidogyne javanica, M. javanica, and Meloidogyne sp. An Meloidogyne javanica-resistant selection from the second backcross (BC) of TxAG-7 to the susceptible cultivar Florunner also was resistant to M. javanica but appeared to be segregating for resistance to the Meloidogyne sp. When reproduction of M. javanica and Meloidogyne javanica were compared on five BC4F3 peanut breeding lines, each derived from Meloidogyne javanica-susceptible BC4F2 individuals, all five lines segregated for resistance to M. javanica, whereas four of the lines appeared to be susceptible to Meloidogyne javanica. These data indicate that several peanut lines selected for resistance to Meloidogyne javanica also contain genes for resistance to populations of M. javanica and the undescribed Meloidogyne sp. that are parasitic on peanut. Further, differences in segregation patterns suggest that resistance to each Meloidogyne sp. is conditioned by different genes.     

Sensitive PCR Detection of Meloidogyne arenaria, M. incognita, and M. javanica Extracted from Soil

We have developed a simple PCR assay protocol for detection of the root-knot nematode (RKN) species Meloidogyne arenaria, M. incognita, and M. javanica extracted from soil. Nematodes are extracted from soil using Baermann funnels and centrifugal flotation. The nematode-containing fraction is then digested with proteinase K, and a PCR assay is carried out with primers specific for this group of RKN and with universal primers spanning the ITS of rRNA genes. The presence of RKN J2 can be detected among large numbers of other plant-parasitic and free-living nematodes. The procedure was tested with several soil types and crops from different locations and was found to be sensitive and accurate. Analysis of unknowns and spiked soil samples indicated that detection sensitivity was the same as or higher than by microscopic examination.     

Effect of Gamma-irradiation and Heat on Root-knot Nematode,Meloidogyne javanica

Effects of gamma-irradiation on the root-knot nematode Meloidogyne javanica were investigated. A dose of 7.5 kGy killed all second-stage juveniles (J2) within 1 day after treatment. Egg hatch was completely inhibited at 6.25 kGy. A bioassay on tomato measuring galling and egg production was used to determine the infectivity of irradiated J2 and J2 hatched from irradiated eggs. The J2 and eggs irradiated with a dose of 4.25 kGy did not induce galls or reproduce on tomato plants. When nematodes were exposed to combined irradiation and heat treatment, no synergistic effect on J2 or eggs was measured. Heat treatment at 49° C for 10 minutes or 20 minutes without irradiation immobilized J2 and prevented egg development. Irradiation rates needed to kill or incapacitate M. javanica were high and may be impractical as a quarantine measure.     

Host Status of Crotalaria juncea,Sesamum indicum,Dolichos lablab,and Elymus glaucus to Meloidogyne javanica

Reproduction of Meloidogyne javanica on Crotalaria juncea PI 207657 and cv. Tropic Sun, Sesamum indicum, Dolichos lablab, and Elymus glaucus was assessed using a root-gall index, a reproductive index obtained by dividing the final population of juveniles (J2) in soil by the initial J2 population (Pf/Pi), and the number of J2 per gram of root recovered from roots by mist chamber extraction. Lycopersicon esculentum (cv. UC 204 C) was included as a susceptible host. The root-gall index and soil reproductive index were poor indicators of the host status of our test plants as compared with mist chamber extraction of J2 from roots. Lycopersicon esculentum had a mean root-gall index of 7.8. Some plants of S. indicum and E. glaucus had a few galls and other plants had none, with mean root-gall indices of 1.6 and 0.8, respectively. No galls were observed in C. juncea and D. lablab. Lycopersicon esculentum had the highest mean soil Pf/Pi value (mean = 1.93), while in C. juncea and some replicates of S. indicum no soil J2 were found. Even though some replicates had no galls, all replicates supported nematode reproduction. The mean numbers of J2 per gram root after 5 days of mist extraction were 447.7, 223.3, 165.5, 96.9, 42.3, and 41.9 for D. lablab, L. esculentum, E. glaucus, S. indicum, and C. juncea PI 207657 and cv. Tropic Sun, respectively. Accurate assessment of nematode resistance was influenced by sampling time and the nematode extraction technique used. Individual plants of both C. juncea and S. indicum supported nematode reproduction to some extent; however, both C. juncea and S. indicum have potential as cover crops to reduce M. javanica numbers.     

Effects of Acibenzolar-S-Methyl Application to Rotylenchulus reniformis and Meloidogyne javanica

Effects of acibenzolar-s-methyl, an inducer of systemic acquired resistance in plants, on Rotylenchulus reniformis and Meloidogyne javanica in vitro and in vivo were determined. A single foliar application of acibenzolar at 50 mg/liter (5 ml of solution per plant) to 7-day-old cowpea or soybean seedlings decreased R. reniformis and M. javanica egg production by 50% 30 days after inoculation. The mechanism of acibenzolar on plant-parasitic nematodes was then investigated. Acibenzolar at 50 to 200 mg/liter did not affect movement of R. reniformis and M. javanica or penetration of second-stage juveniles (J2) of M. javanica on cowpea. However, M. javanica development was slowed and fecundity was reduced in plants treated with acibenzolar. On average, 50% of J2 that penetrated acibenzolar-treated cowpeas developed into mature females with eggs, whereas the other 50% exhibited arrested development. The number of eggs per egg mass was 450 in water-treated cowpeas, whereas the number declined to 250 in acibenzolar-treated plants. Acibenzolar may be responsible for stimulating the plants to express some resistance to the nematodes.     

Suppression of Meloidogyne incognita and M. javanica by Pasteuria penetrans in Field Soil

The role of Pasteuria penetrans in suppressing numbers of root-knot nematodes was investigated in a 7-year monocuhure of tobacco in a field naturally infested with a mixed population of Meloidogyne incognita race 1 and M. javanica. The suppressiveness of the soil was tested using four treatments: autoclaving (AC), microwaving (MW), air drying (DR), and untreated. The treated soil bioassays consisted of tobacco cv. Northrup King 326 (resistant to M. incognita but susceptible to M. javanica) and cv. Coker 371 Gold (susceptible to M. incognita and M. javanica) in pots inoculated with 0 or 2,000 second-stage juveniles of M. incognita race 1. Endospores of P. penetrans were killed by AC but were only slightly affected by MW, whereas most fungal propagules were destroyed or inhibited in both treatments. Root galls, egg masses, and numbers of eggs were fewer on Coker 371 Gold in MW, DR, and untreated soil than in AC-treated soil. There were fewer egg masses than root galls on both tobacco cultivars in MW, DR, and untreated soil than in the AC treatment. Because both Meloidogyne spp. were suppressed in MW soil (with few fungi present) as well as in DR and untreated soil, the reduction in root galling, as well as numbers of egg masses and eggs appeared to have resulted from infection of both nematode species by P. penetrans.     

Meloidogyne incognita Inoculum Source Affects Host Suitability and Growth of Yellow Nutsedge and Chile Pepper

Meloidogyne incognita (Mi) reproduction and host plant responses in chile pepper (Capsicum annuum) and yellow nutsedge (Cyperus esculentus = YNS) to three sources of inoculum obtained by rearing a single Mi population on chile, YNS, and tomato were evaluated in two factorial greenhouse experiments. The interactive effects of Mi inoculum source and crop-weed competition were determined. In the absence of YNS competition, chile growth was reduced less by Mi inoculum from chile than by inoculum from YNS or tomato. When YNS was present, chile root weight was not affected and shoot weight increased with Mi initial inoculation, regardless of inoculum source. Chile plants inoculated with Mi from tomato exhibited double the nematode reproduction observed with inoculum from chile or YNS. With chile present, Mi reproduction on YNS was nearly three times greater with inoculum from tomato, but reproduction was similar among inoculum sources when chile was absent. Reductions in YNS root mass due to competition from chile failed to reduce the total number of Mi eggs produced on YNS plants. Differences in total Mi reproduction among inoculum sources were not attributable to differences in root growth or plant competition. This study illustrates the influence of Mi-YNS interactions and previous hosts on severity of Mi infection.     

Control of Meloidogyne javanica by Formulations of Inula viscosa Leaf Extracts

Inula viscosa is a perennial plant that is widely distributed in Mediterranean countries. Formulations of I. viscosa extracts were tested for their effectiveness in control of Meloidogyne javanica in laboratory, growth chamber, microplot, and field experiments. Oily pastes were obtained by extraction of dry leaves with a mixture of acetone and n-hexane or n-hexane alone, followed by evaporation of the solvents. Emulsifiable concentrate formulations of the pastes killed M. javanica juveniles in sand at a concentration of 0.01% (paste, w/w) or greater and reduced the galling index of cucumber seedlings as well as the galling index and numbers of nematode eggs on tomato plants in growth chamber experiments. In microplot experiments, the hexane-extract formulation at 26 g paste/m² reduced nematode infection on tomato plants in one of two experiments. In a field experiment, a reduction of 40% in root galling index by one of two formulations was observed on lettuce plants. The plant extracts have potential as a natural nematicide, although the formulations need improvement.     

Effects of Fluctuating Temperatures and Different Host Plants on Development of Pasteuria penetrans in Meloidogyne javanica

Greenhouse and growth room experiments were conducted to investigate the effect of host plant in relation to different nematode inoculum levels, and temperature fluctuations on the development of Pasteuria penetrans. Host plant affected the development of P. penetrans indirectly through its effect on nematode development. Endospores collected from Meloidogyne javanica females reared on different hosts did not show any differences in subsequent attachment and infectivity. The numbers of endospores produced per infected female were reduced with increasing numbers of females parasitizing okra and tomato roots. Fluctuating temperatures retarded the development of P. penetrans. The life cycle of the parasite was completed faster at approximately constant temperatures close to 30 °C than when the temperature fluctuated away from 30 °C. The temperature of irrigation water did not affect the duration of life cycle of P. penetrans.     

Pathogenic Variability Among Isolates of Meloidogyne javanica on Capsicum annum

Meloidogyne javanica isolates were collected from nine districts of Uttar Pradesh. These isolates showed pathogenic variability when inoculated on the pepper cultivars California Wonder and Suryamukhi Green. Meloidogyne javanica that infected Suryamukhi Green but not California Wonder were designated as pepper race 1 and the populations that infected both the cultivars were designated pepper race 2. Race 1 was more frequent than race 2 in Almora, Pauri Garhwal, Basti, Gorakhpur, and Deoria, whereas race 2 was more frequent than race 1 in the Dehradun, Farrukhabad, Hardoi, and Sitapur districts. The overall frequencies were 70% and 30% for race 1 and race 2, respectively, in the study area.