Antioxidant Enzymes in Phytoparasitic Nematodes

Presence of different antioxidant enzymes, such as superoxide dismutase (SOD), catalase, and ascorbate, p-phenilendiamine-pyrocathecol (PPD-PC), o-dianisidine, and guaiacol isoperoxidases, was shown in the phytoparasific nematode species Meloidogyne incognita, M. hapla, Globodera rostochiensis, G. pallida, Heterodera schachtii, H. carotae, and Xiphinema index. The activity of the enzymes tested differed among the life stages examined. SOD was present in cysts but was not detected in Meloidogyne egg masses. Catalase activity of Meloidogyne females was higher than that of preparasitic stages and cyst-nematode females. For the first time, ascorbate peroxidase was found to occur commonly in phytoparasitic nematodes, with the highest activity in the invading life-stages. In all the life stages examined, the antioxidant enzyme activities of M. hapla were markedly higher than those of M. incognita. Glutathione peroxidase was not found in the species examined.     

Major Sperm Protein Genes from Globodera rostochiensis

Three genes in the major sperm protein (MSP) gene family from the potato cyst nematode Globodera rostochiensis were cloned and sequenced. In contrast to the absence of introns in Caenorhabditis elegans MSP genes, these genes in G. rostochiensis contained a 57 nucleotide intron, with normal exon-intron boundaries, in the same relative location as the intron in Onchocerca volvulus. The MSP genes of G. rostochiensis had putative CAAT, TATA, and polyadenylation signals. The predicted G. rostochiensis MSP gene product is 126 amino acids long, one residue shorter than the products in the other species. The comparison of MSP amino acid sequences from four diverse nematode species suggests that O. volvulus, Ascaris suum, and C. elegans may be more closely related to each other than they are to G. rostochiensis.     

Plant-parasitic Nematode Acetylcholinesterase Inhibition by Carbamate and Organophosphate Nematicides

The sensitivity of acetylcholinesterases (ACHE) isolated from the plant-parasitic nematodes Meloidogyne arenaria, M. incognita, and Heterodera glycines and the free-living nematode Caenorhabditis elegans to carbamate and organophosphate nematicides was examined. The AChE from plant-parasitic nematode species were more sensitive to carbamate inhibitors than was AChE from C. elegans, but response to the organophosphates was approximately equivalent. The sulfur-containing phosphate nematicides were poor inhibitors of nematode acetylcholinesterase, but treatment with an oxidizing agent greatly improved inhibition. Behavioral bioassays with living nematodes revealed a poor relationship between enzyme inhibition and expression of symptoms in live nematodes.     

Dehydrogenases,Acid and Alkaline Phosphatases,and Esterases for Chemotaxonomy of Selected Meloidogyne,Ditylenchus, Heterodera and Aphelenchus spp.

Various taxonomically useful profiles of four dehydrogenases (lactate, malate, glucose-6-phosphate, and a-glycerophosphate) and three hydrolases (acid and alkaline phosphatase and esterase) were detected in whole nematode homogenates of Meloidogyne javanica, M. hapla, M. incognita, M. arenaria, Ditylenchus dipsaci, D. triformis, Heterodera glycines, and Aphelenchus avenae. The enzyme profiles were stable in populations cultured on several different hosts. A tentative enzymically-determined phylogeny of Meloidogyne is given.     

Comparative Disc-Electrophoretic Protein Analyses of Selected Meloidogyne,Ditylenchus, Heterodera and Aphelenchus spp.

Disc-electrophoretic separation of soluble proteins from whole nematode homogenates yielded band profiles useful for distinguishing selected species of Meloidogyne and Ditylenchus, and the genera Heterodera, and Aphelenchus. Certain protein bands were common to all the species of Meloidogyne, whereas other bands were specific. Meloidogyne spp. and Heterodera glycines shared some protein similarities, but other genera differed distinctly. Protein profiles of Meloidogyne spp. were not significantly altered by the host on which the nematode was cultured.     

Broad Meloidogyne Resistance in Potato Based on RNA Interference of Effector Gene 16D10

Root-knot nematodes (Meloidogyne spp.) are a significant problem in potato (Solanum tuberosum) production. There is no potato cultivar with Meloidogyne resistance, even though resistance genes have been identified in wild potato species and were introgressed into breeding lines. The objectives of this study were to generate stable transgenic potato lines in a cv. Russet Burbank background that carry an RNA interference (RNAi) transgene capable of silencing the 16D10 Meloidogyne effector gene, and test for resistance against some of the most important root-knot nematode species affecting potato, i.e., M. arenaria, M. chitwoodi, M. hapla, M. incognita, and M. javanica. At 35 days after inoculation (DAI), the number of egg masses per plant was significantly reduced by 65% to 97% (P < 0.05) in the RNAi line compared to wild type and empty vector controls. The largest reduction was observed in M. hapla, whereas the smallest reduction occurred in M. javanica. Likewise, the number of eggs per plant was significantly reduced by 66% to 87% in M. arenaria and M. hapla, respectively, compared to wild type and empty vector controls (P < 0.05). Plant-mediated RNAi silencing of the 16D10 effector gene resulted in significant resistance against all of the root-knot nematode species tested, whereas R_Mc1(blb), the only known Meloidogyne resistance gene in potato, did not have a broad resistance effect. Silencing of 16D10 did not interfere with the attraction of M. incognita second-stage juveniles to roots, nor did it reduce root invasion.     

Susceptibility of Several Common Subtropical Weeds to Meloidogyne arenaria,M. incognita,and M. javanica

Experiments were conducted in the greenhouse to assess root galling and egg production of three root-knot nematode species, Meloidogyne arenaria, M. incognita, and M. javanica, on several weeds common to Florida agricultural land. Weeds evaluated were Amaranthus retroflexus (redroot pigweed), Cyperus esculentus (yellow nutsedge), Eleusine indica (goosegrass), Portulaca oleracea (common purslane), and Solanum americanum (American black nightshade). Additionally, although it is recommended as a cover crop in southern regions of the U.S., Aeschynomene americana (American jointvetch) was evaluated as a weed following the detection of root galling in a heavy volunteer infestation of an experimental field in southeastern Florida. Weeds were propagated from seed and inoculated with 1000 nematode eggs when plants reached the two true-leaf stage. Tomato (Solanum lycopersicum ‘Rutgers’) was included as a positive control. Aeschynomene americana and P. oleracea roots supported the highest number of juveniles (J₂) and had the highest number of eggs/g of root for all three species of Meloidogyne tested. However, though P. oleracea supported very high root levels of the three nematode species tested, its fleshy roots did not exhibit severe gall symptoms. Low levels of apparent galling, combined with high egg production, increase the potential for P. oleracea to support populations of these three species of root-knot nematodes to a degree that may not be appropriately recognized. This research quantifies the impact of P. oleracea as a host for M. arenaria, M. incognita, and M. javanica compared to several other important weeds commonly found in Florida agricultural production, and the potential for A. americana to serve as an important weed host of the three species of root-knot nematode tested in southern regions of Florida.     

Evaluation of 31 Potential Biofumigant Brassicaceous Plants as Hosts for Three Meloiodogyne Species

Brassicaceous cover crops can be used for biofumigation after soil incorporation of the mowed crop. This strategy can be used to manage root-knot nematodes (Meloidogyne spp.), but the fact that many of these crops are host to root-knot nematodes can result in an undesired nematode population increase during the cultivation of the cover crop. To avoid this, cover crop cultivars that are poor or nonhosts should be selected. In this study, the host status of 31 plants in the family Brassicaceae for the three root-knot nematode species M. incognita, M. javanica, and M. hapla were evaluated, and compared with a susceptible tomato host in repeated greenhouse pot trials. The results showed that M. incognita and M. javanica responded in a similar fashion to the different cover cultivars. Indian mustard (Brassica juncea) and turnip (B. rapa) were generally good hosts, whereas most oil radish cultivars (Raphanus. sativus ssp. oleiferus) were poor hosts. However, some oil radish cultivars were among the best hosts for M. hapla. The arugula (Eruca sativa) cultivar Nemat was a poor host for all three nematode species tested. This study provides important information for chosing a cover crop with the purpose of managing root-knot nematodes.     

Role of Nematodes,Nematicides, and Crop Rotation on the Productivity and Quality of Potato,Sweet Potato,Peanut, and Grain Sorghum

The objective of this experiment was to determine the effects of fenamiphos 15G and short-cycle potato (PO)-sweet potato (SP) grown continuously and in rotation with peanut (PE)-grain sorghum (GS) on yield, crop quality, and mixed nematode population densities of Meloidogyne arenaria, M. hapla, M. incognita, and Mesocriconema ornatum. Greater root-gall indices and damage by M. hapla and M. incognita occurred on potato than other crops. Most crop yields were higher and root-gall indices lower from fenamiphos-treated plots than untreated plots. The total yield of potato in the PO-SP and PO-SP-PE-GS sequences increased from 1983 to 1985 in plots infested with M. hapla or M. arenaria and M. incognita in combination and decreased in 1986 to 1987 when root-knot nematode populations shifted to M. incognita. The total yields of sweet potato in the PO-SP-PE-GS sequence were similar in 1983 and 1985, and declined each year in the PO-SP sequence as a consequence of M. incognita population density increase in the soil. Yield of peanut from soil infested with M. hapla increased 82% in fenamiphos-treated plots compared to untreated plots. Fenamiphos treatment increased yield of grain sorghum from 5% to 45% over untreated controls. The declining yields of potato and sweet potato observed with both the PO-SP and PO-SP-PE-GS sequences indicate that these crop systems should not be used longer than 3 years in soil infested with M. incognita, M. arenaria, or M. hapla. Under these conditions, these two cropping systems promote a population shift in favor of M. incognita, which is more damaging to potato and sweet potato than M. arenaria and M. hapla.     

Using FAME Analysis to Compare, Differentiate, and Identify Multiple Nematode Species

We have adapted the Sherlock^® Microbial Identification system for identification of plant parasitic nematodes based on their fatty acid profiles. Fatty acid profiles of 12 separate plant parasitic nematode species have been determined using this system. Additionally, separate profiles have been developed for Rotylenchulus reniformis and Meloidogyne incognita based on their host plant, four species and three races within the Meloidogyne genus, and three life stages of Heterodera glycines. Statistically, 85% of these profiles can be delimited from one another; the specific comparisons between the cyst and vermiform stages of H. glycines, M. hapla and M. arenaria, and M. arenaria and M. javanica cannot be segregated using canonical analysis. By incorporating each of these fatty acid profiles into the Sherlock^® Analysis Software, 20 library entries were created. While there was some similarity among profiles, all entries correctly identified the proper organism to genus, species, race, life stage, and host at greater than 86% accuracy. The remaining 14% were correctly identified to genus, although species and race may not be correct due to the underlying variables of host or life stage. These results are promising and indicate that this library could be used for diagnostics labs to increase response time.     

Meloidogyne partityla on Pecan Isozyme Phenotypes and Other Host

Meloidogyne sp. from five pecan (Carya illinoensis) orchards in Texas were distinctive in host range and iszoyme profiles from common species of Meloidogyne but were morphologically congruent with Meloidogyne partityla Kleynhans, a species previously known only in South Africa. In addition to pecan, species of walnut (Juglans hindsii and J. regia) and hickory (C. ovata) also were hosts. No reproduction was observed on 15 other plant species from nine families, including several common hosts of other Meloidogyne spp. Three esterase phenotypes and two malate dehydrogenase phenotypes of M. partityla were identified by polyacrylamide gel electrophoresis. Each of these isozyme phenotypes was distinct from those of the more common species M. arenaria, M. hapla, M. incognita, and M. javanica.     

Population Dynamics of Meloidogyne incognita,M. arenaria,and Other Nematodes and Crop Yields in Rotations of Cotton,Peanut, and Wheat Under Minimum Tillage

Wheat, cotton, and peanut were arranged in three cropping sequences to determine the effects of fenamiphos (6.7 kg a.i./ha) and cropping sequence on nematode population densities and crop yields under conservation tillage and irrigation for 6 years. The cropping sequences included a wheat winter cover crop each year and summer crops of cotton every year, peanut every year, or cotton rotated every other year with peanut. The population densities of Meloidogyne spp. and Helicotylenchus dihystera were determined monthly during the experiment. Numbers of M. incognita increased on cotton and decreased on peanut, whereas M. arenaria increased on peanut, and decreased on cotton; both nematode species remained in moderate to high numbers in plots of wheat. Root damage was more severe on cotton than peanut and was not affected by fenamiphos treatment. The H. dihystera population densities were highest in plots with cotton every summer, intermediate in the cotton-peanut rotation, and lowest in plots with peanut every summer. Over all years and cropping sequences, yield increases in fenamiphos treatment over untreated control were 9% for wheat, 8% for cotton, and 0% for peanut. Peanut yields following cotton were generally higher than yields following peanut. These results show that nematode problems may be manageable in cotton and peanut production under conservation tillage and irrigation in the southeastern United States.     

Host Suitability of Selected Hybrids,Varieties and Inbreds of Corn to Populations of Meloidogyne spp.

Rates of reproduction of root-knot nematodes on corn varied with Meloidogyne species, with different populations of certain species, and with corn cultivars. M. arenaria, M. incognita and M. javanica reproduced at varying rates on all corn cultivars tested. None of the three selections of M. hapla reproduced on corn. Most of the Meloidogyne populations increased more rapidly on ''Coker'' and ''Pioneer'' hybrids than on ''McNair'' hybrids or on open-pollinated varieties or inbreds. Nematodes often reduced root growth, but the differences within given nematode-cultivar treatments were not usually significant. Root growth of ''Coker 911,'' which supported a high rate of reproduction, was affected less than ''Pioneer 309B'' which supported a low rate of nematode reproduction.     

DNA Complexity of the Root-knot Nematode (Meloidogyne spp.) Genome

Cot curves derived from renaturation kinetics of sheared denatured DNA indicated that the genome of six populations representing the four most common root-knot nematode species (Meloidogyne incognita, M. arenaria, M. javanica, and M. hapla) is composed of 20% repetitive and 80% nonrepetitive sequences of DNA. Cot curves were almost identical, indicating that all populations had a haploid genome of approximately the same size. Calculations from an average Cot curve gave an estimate of 0.51 x 108 nucleotide base pairs for the haploid genome of the four Meloidogyne species. This genome is about 12-13 times larger than the genome of the E. coli strain used as a control.     

Diversity of Meloidogyne spp. on Musa in Martinique,Guadeloupe, and French Guiana

Ninety-six isolates of Meloidogyne species collected from banana fields from Martinique, Guadeloupe, and French Guiana, were examined using esterase (Est) and malate dehydrogenase (Mdh) phenotypes. Adult females identified as M. arenaria, M. incognita, M. javanica, M. cruciani, M. hispanica, and Meloidogyne sp. showed species-specific phenotypes only for the esterase enzymes. Intraspecific variability among isolates of M. arenaria, M. incognita, and M. javanica was detected using Est and Mdh. Perineal patterns were used as a complementary tool together with enzyme characterization and were essential for checking the morphological consistency of the identification. The major species of M. arenaria and M. incognita were detected at 61.9% and 34.3% of the total number of isolates, respectively, and the other minor species at 3.8%. The mixed Meloidogyne species were detected in 45.1% of the samples. Genetic analysis was conducted using RAPD markers, which alone or in combination provided reliable polymorphisms both between and within species. RAPD analysis of the data resulted in clustering of species and isolates congruent with esterase phenotype characterization. The intraspecific variability in M. incognita and in M. arenaria represented 14.9% and 61.6% of the amplified polymorphic fragments, respectively. This high level of variation in M. arenaria isolates may indicate multiple origins for populations classified as M. arenaria or more than one species inside the same group, but more detailed morphological and DNA studies will be necessary to test this hypothesis.     

Host Range and Ecology of Isolates of Pasteuria spp. from the Southeastern United States

Isolates of Pasteuria penetrans were evaluated for ecological characteristics that are important in determining their potential as biological control agents. Isolate P-20 survived without loss of its ability to attach to its host nematode in dry, moist, and wet soil and in soil wetted and dried repeatedly for 6 weeks. Some spores moved 6.4 cm (the maximum distance tested) downward in soil within 3 days with percolating water. The isolates varied greatly in their attachment to different nematode species and genera. Of five isolates tested in spore-infested soil, three (P-104, P-122, B-3) attached to two or more nematode species, whereas B-8 attached only to Meloidogyne hapla and B-I did not attach to any of the nematodes tested. In water suspensions, spores of isolate P-20 attached readily to M. arenaria but only a few spores attached to other Meloidogyne spp. Isolate P-104 attached to all Meloidogyne spp. tested but not to Pratylenchus scribneri. Isolate B-4 attached to all species of Meloidogyne and Pratylenchus tested, but the rate of attachment was relatively low. Isolate P-Z00 attached in high numbers to M. arenaria when spores were extracted from females of this nematode; when extracted from M. javanica females, fewer spores attached to M. arenaria than to M. javanica or M. incognita.     

Interactions between Meloidogyne incognita,M. hapla,and Pratylenchus brachyurus in Tobacco

In a greenhouse pot experiment on the pathogenicity and interactions of Meloidogyne incognita, M. hapla and Pratylenchus brachyurus on four cultivars o f tobacco the cultivars ''Hicks'' and ''NC 2326'' were susceptible to each nematode and "NC 95'' and ''NC 2512'' resistant only to M. incognita.Mean heights of susceptible plants were depressed but fresh weight of tops did not differ significantly. Meloidogyne spp. increased fresh weight of susceptible (but not the resistant) roots.Reproduction of M. incognita was decreased in the presence of P. brachyurus in one case. M. hapla reproduction was less with either of the other nematodes in five out of eight cases. In 12 combinations involving P. brachyurus, reproduction of this species was depressed in seven, not affected in four and increased in one.Mechanisms involved in associative interactions were not identified but appeared to be indirect and to involve individual host-nematode responses.     

Suitability of Zucchini and Cucumber Genotypes to Populations of Meloidogyne arenaria,M. incognita,and M. javanica

The host suitability of five zucchini and three cucumber genotypes to Meloidogyne incognita (MiPM26) and M. javanica (Mj05) was determined in pot experiments in a greenhouse. The number of egg masses (EM) did not differ among the genotypes of zucchini or cucumber, but the eggs/plant and reproduction factor (Rf) did slightly. M. incognita MiPM26 showed lower EM, eggs/plant, and Rf than M. javanica Mj05. Examination of the zucchini galls for nematode postinfection development revealed unsuitable conditions for M. incognita MiPM26 as only 22% of the females produced EM compared to 95% of the M. javanica females. As far as cucumber was concerned, 86% of the M. incognita and 99% of the M. javanica females produced EM, respectively. In a second type of experiments, several populations of M. arenaria, M. incognita, and M. javanica were tested on zucchini cv. Amalthee and cucumber cv. Dasher II to assess the parasitic variation among species and populations of Meloidogyne. A greater parasitic variation was observed in zucchini than cucumber. Zucchini responded as a poor host for M. incognita MiPM26, MiAL09, and MiAL48, but as a good host for MiAL10 and MiAL15. Intraspecific variation was not observed among the M. javanica or M. arenaria populations. Cucumber was a good host for all the tested populations. Overall, both cucurbits were suitable hosts for Meloidogyne but zucchini was a poorer host than the cucumber.     

Effect of Carbamate,Organophosphate, and Avermectin Nematicides on Oxygen Consumption by Three Meloidogyne spp.

Second-stage juveniles (I2) of Meloidogyne arenaria consumed more oxygen (P ≤ 0.05) than M. incognita J2, which in turn consumed more than M. javanica J2 (4,820, 4,530, and 3,970 μl per hour per g nematode dryweight, respectively). Decrease in oxygen consumption depended on the nematicide used. Except for aldicarb, there was no differential sensitivity among the three nematode species. Meloidogyne javanica had a greater percentage decrease (P ≤ 0.05) in oxygen uptake when treated with aldicarb, relative to the untreated control, than either M. arenaria or M. incognita. Meloidogyne javanica J2 had a greater degree of recovery from fenamiphos or aldicarb intoxication, after subsequent transfer to water, than did M. incognita. This finding may relate to differential sensitivity among Meloidogyne spp. in the field. Degree of respiratory inhibition and loss of nematode motility for M. javanica after exposure to the nematicides were positively correlated (P ≤ 0.05).     

Host Suitability of the Olive Cultivars Arbequina and Picual for Plant-Parasitic Nematodes

Host suitability of olive cultivars Arbequina and Picual to several plant-parasitic nematodes was studied under controlled conditions. Arbequina and Picual were not suitable hosts for the root-lesion nematodes Pratylenchus fallax, P. thornei, and Zygotylenchus guevarai. However, the ring nematode Mesocriconema xenoplax and the spiral nematodes Helicotylenchus digonicus and H. pseudorobustus reproduced on both olive cultivars. The potential of Meloidogyne arenaria race 2, M. incognita race 1, and M. javanica, as well as P. vulnus and P. penetrans to damage olive cultivars, was also assessed. Picual planting stocks infected by root-knot nematodes showed a distinct yellowing affecting the uppermost leaves, followed by a partial defoliation. Symptoms were more severe on M. arenaria and M. javanica-infected plants than on M. incognita-infected plants. Inoculation of plants with 15,000 eggs + second-stage juveniles/pot of these Meloidogyne spp. suppressed the main height of shoot and number of nodes of Arbequina, but not Picual. Infection by each of the two lesion nematodes (5,000 nematodes/pot) or by each of the three Meloidogyne spp. suppressed (P < 0.05) the main stem diameter of both cultivars. On Arbequina, the reproduction rate of Meloidogyne spp. was higher (P < 0.05) than that of Pratylenchus spp.; on Picual, Pratylenchus spp. reproduction was higher (P < 0.05) than that of Meloidogyne spp.