Sterol Composition of the Corn Cyst Nematode,Heterodera zeae,and Corn Roots

Sterols from free sterol and steryl ester fractions from Heterodera zeae and from total lipids of Zea mays roots were analyzed by gas-liquid chromatography (GLC) and by GLC-mass spectrometry. The major free sterols of H. zeae were 24-ethylcholesterol (54.4% of total free sterol), 24-ethylcholesta-5,22-dien-3β-ol (13.3%), 24-methylcholesterol (12.5%), and cholesterol (7.2%). The same four sterols comprised 34.6%, 7.2%, 30.3%, and 18.6%, respectively, of the esterified sterols of H. zeae. Corn root sterols included 46.6% 24-ethylcholesta-5,22-dien-3β-ol, 16.7% methylcholesterol, 16.4% cycloartenol, 12.7% 24-ethylcholesterol, and 0.5% cholesterol. The sterol 24-composition of H. zeae differed greatly from that of the only other cyst nematode previously investigated, Globodera solanacearum.     

Host Suitability and Response of Asparagus Cultivars to Meloidogyne Species and Races

The host-parasite relationships of asparagus and Meloidogyne spp. were examined under greenhouse and microplot conditions. Meloidogyne species and races differed greatly in their ability to reproduce on asparagus seedlings. Meloidogyne hapla generally failed to reproduce, and M. javanica, M. arenaria race 1, and M. incognita race 3 reproduced poorly, with a reproduction factor (Rf = final population/initial population) usually < 1.0. Only M. arenaria race 2 and M. incognita races 1 and 4 reproduced consistently on all asparagus cultivars tested (Rf typically 1-11). No effect of M. incognita race 4 on host growth was detected. Meloidogyne arenaria race 2 and M. incognita race 1 had slight negative effects (5-10%) on plant and root growth.     

Penetration and Post-infectional Development and Reproduction of Meloidogyne arenaria Races 1 and 2 on Susceptible and Resistant Soybean Genotypes

Penetration, post-infectional development, reproduction, and fecundity of Meloidogyne arenaria races 1 and 2 were studied on susceptible (CNS), partially resistant (Jackson), and highly resistant (PI 200538 and PI 230977) soybean genotypes in the greenhouse. The ability to locate and invade roots was similar between races, but more juveniles penetrated roots of susceptible CNS than the resistant genotypes. At 10 days after inoculation, 56% and 99% to 100% of race 1 second-stage juveniles were vermiform or sexually undifferentiated in CNS and the resistant genotypes, respectively. In contrast, only 2%, 42%, 44%, and 62% of race 2 juveniles had not initiated development in CNS, Jackson, PI 200538, and PI 230977, respectively. By 20 days after inoculation, 88% to 100% of race 2 nematodes in roots of all genotypes were females, whereas only 25% and 1% of race 1 were females in CNS and the resistant genotypes, respectively. For all four genotypes, race 1 produce 85% to 96% fewer eggs per root system 45 days after inoculation than race 2. At 45 days after inoculation race 2 produced more eggs on CNS than the other genotypes.     

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.     

Repeated Sampling to Determine the Precision of Estimating Nematode Population Densities

The first phase of this study involved repeated samplings of five fields using composite samples of 10, 20, 40, and 80 soil cores, to determine the precision of nematode assays. The second phase focused on randomly selecting two and four 2-ha subunits (data on Meloidogyne spp.) of 24 fields ranging from 6 to 40 ha and computing the precision of estimated means for these numbers ofsubunits versus the general field mean (based on all 2-ha subunits). Average numbers of nematodes from most samples containing Meloidogyne spp., Heterodera glycines, Helicotylenchus dihystera, Scutellonema brachyurum, and (or) Hoplolaimus galeatus were within 50% of the overall means. Coefficient of variation (CV) values were generally lower for 40 cores than for 10, 20, and 80 cores per sample. When data for all nematodes and fields were combined, this value was lowest for 40 and 80 cores. The CV values were higher for Meloidogyne spp. than for H. glycines. Means of two samplings increased the probability of obtaining numbers nearer the mean for that field than numbers from a single composite sample. For the second phase, population estimates of Meloidogyne spp. based on four 2-ha subunits generally were closer to field means than were those for two subunits. Sampling precision with these subunits diminished greatly in large fields with variable soils and (or) mixed cropping histories. Either two or four subunits gave population estimates within 3-20% of the field mean in most instances. The mean man hours required for sampling ca. 2-ha parcels of 4-20-ha fields was 0.54 hours.     

Comparisons of Isozyme Phenotypes in Five Meloidogyne spp. with Isoelectric Focusing

Meloidogyne incognita race 1, M. javanica, M. arenaria race 1, M. hapla, and an undescribed Meloidogyne sp. were analyzed by comparing isozyme phenotypes of esterase, malate dehydrogenase, phosphoglucomutase, isocitrate dehydrogenase, and α-glycerophosphate dehydrogenase. Isozyme phenotypes were obtained from single mature females by isoelectric focusing electrophoresis. Of these five isozymes, only esterase and phosphoglucomutase could be used to separate all five Meloidogyne spp.; however, the single esterase electromorphs were similar for M. incognita and M. hapla. Yet when both nematodes were run on the same gel, differences in their esterase phenotypes were detectable. Isozyme phenotypes from the other three isozymes revealed a great deal of similarity among M. incognita, M. javanica, M. arenaria, and the undescribed Meloidogyne sp.     

Optimum Initial Inoculum Levels for Evaluation of Resistance in Tomato to Meloidogyne spp. at Two Different Soil Temperatures

The effects of Meloidogyne incognita or M. javanica at five initial inoculum levels of 20, 100, 200, 1,000, and 2,000 eggs and infective juveniles per seedling on ''Floradade,'' ''Nemarex,'' ''Patriot,'' and ''PI 129149-2(sib)-5'' tomatoes maintained at 25 or 32.5 C were studied. The number of egg masses on roots of the susceptible cultivar Floradade was similar for both species of root-knot nematodes at either 2.5 or 32.5 C soil temperatures. At 25 C, very low numbers of egg masses were produced by both species of root-knot nematodes on Nematex, Patriot, and Lycopersicon peruvianum PI 129149-2(sib)-5. At 32.5 C, the best inoculum level for assessing resistance in these tomato genotypes was 200 eggs and infective juveniles per seedling. With 28 days of incubation, this temperature and inoculum level produced quantitative differences in resistance for both species of Meloidogyne.     

Nematode Interactions with Weeds and Sugarcane Mosaic Virus in Louisiana Sugarcane

Weeds did not appear to serve as reservoirs for phytophagous Louisiana sugarcane nematode populations except for Criconemella spp., Meloidogyne spp., Tylenchorhynchus annulatus, and total phytophagous nematode densities were lower on weed-stressed cane and were accompanied by reduced accumulations of free cysteine, proline, and 13 other free amino acids in sugarcane. A significant weed-virus interaction for sugarcane free cysteine accumulation was detected; T. annulatus populations were highly correlated (r = 0.59, P ≤ 0.001) with the weed-induced and virus-induced changes in free cysteine. Sugarcane nematodes interacted differently with the weed and virus stresses and changes in host plant stress-related free amino acid concentrations.     

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.     

Parasitic Variability of Meloidogyne incognita Populations on Susceptible and Resistant Cotton

Root gall induction and egg production by the four recognized host races and two cytological races of Meloidogyne incognita were compared on cotton Gossypium hirsutum cvs. Deltapine 16 (root-knot susceptible) and Auburn 634 (highly resistant). The 12 nematode populations included in the study were from various parts of the world. No population increases occurred on the highly resistant cultivar. After 45 days, populations of host races 1 and 2 induced slight root galling on both cuhivars with only limited reproduction. Host race 4 populations induced moderate root galling with higher reproduction on Deltapine 16 than that of race 1 or race 2 populations. Host race 3 populations induced severe root galling with population density increases of 7-30-fold. In a complementary study, 24 cotton cultivars or breeding lines were compared for suitability as hosts for a typical population of M. incognita race 3. The poorest hosts, ''Aubnru 623,'' ''Auburn 634,'' and ''McNair 220,'' yielded fewer eggs after 45 days than were added initially. The best hosts - ''M-8.'' ''DES 24-8,'' ''McNair 235,'' and ''Coker 20l'' - yielded > 5 times as many eggs as were added initially.     

Penetration Rates by Second-stage Juveniles of Meloidogyne spp. and Heterodera glycines into Soybean Roots

The rates of soybean root penetration by freshly hatched second-stage juveniles (J2) of Meloidogyne arenaria, M. hapla, M. incognita, M. javanica, and Heterodera glycines races 1 and 5 were examined over a period of 1 to 240 hours. Heterodera glycines entered roots more quickly than Meloidogyne spp. Penetration by most nematodes was accomplished within 48 hours. The increases in penetration after 48 hours were insufficient to warrant further assessments. Penetration of J2 into roots of soybean seedfings in a styrofoam container was as good or better than in a clay pot. Thus, rapid and accurate root-penetration assessments can be made at 48 hours after inoculation.     

Sterols and related metabolites from five species of sponges

Free sterol fractions were isolated from the marine sponges Phyllospongia madagascarensis, Scalarispongia sp., Oceanapia sp., Monanchora clathrata and studied by GLC, GLC–MS, and spectroscopy NMR. P. madagascarensis and Scalarispongia sp. contained common Δ⁵-sterols; cholesterol was shown to be a main sterol of both the sponges. Oceanapia sp. contained stanols and minor Δ⁵-sterols with 24R-24,25-methylene-5α-cholestan-3β-ol as a main constituent. Many free sterols from M. clathrata were Δ⁷-series compounds, and latosterol was a main sterol. Δ⁴-3-Ketosteroids and Δ⁵-sterol esters were found in the Antarctic sponge Haliclona sp., but free sterols were practically absent except for trace amount of cholesterol. A chemotaxonomic application of sterols in relation to the genera Phyllospongia, Oceanapia and the family Crambeidae is provided. The known cases of the absence of sterols in sponges and probable reasons of the phenomenon are discussed.     

Occurrence of Pasteuria-like Organisms on Selected Plant-Pamsitic Nematodes of Pineapple in the Hawaiian Islands

Soils from 320 sites representing diverse undisturbed habitats from five Hawaiian Islands were assessed for occurrence of Pasteuria-like organisms. Mean annual rainfall at sites ranged from 125-350 cm, elevation from 69-2,286 m, and annual mean temperature from 12-24 C. Seven different natural communities were represented: wet lowland, mesic lowland, wet montane, mesk montane, dry montane, mesic subalpine, and dry alpine. Pasteuria spp. in a soil sample was detected by baiting with infective stages of Helicotylenchus dihystera, Meloidogyne javanica, Pratylenchus brachyurus, and Rotylenchulus reniformis, followed by cultivation of the nematodes on pineapple plants for 10-11 months. All nematode baits except R. reniformis were readily recovered from the soil samples. A sample was considered Pasteuria-positive if at least 5 % of the nematode specimens showed endospore attachment. Thirteen percent of all samples were positive for Pasteuria-like organisms. The frequencies of association between Pasteuria spp. and Meloidogyne, Helicotylenchus, or Pratylenchus species were 52%, 24%, and 24%, respectively. Positive samples were more prevalent on the older islands of Kauai and Oahu (75%), in lowland communities (61%), and in areas with introduced vegetation (60%). More than 27% of the positive samples were associated with plant species in a few selected families that included Meliaceae and Myrtaceae. Occurrence of Pasteuria spp. seemed to be positively associated with mean annual rainfall or temperature, but negatively associated with elevation.     

Response of Resistant Soybean Plant Introductions to Meloidogyne arenaria Races 1 and 2

Resistant plant introductions, PI 230977 and PI 200538, and partially resistant Jackson and susceptible CNS were evaluated for seed yield in response to races 1 and 2 of Meloidogyne arenaria. Initial soil population densities (Pi) of the nematode were 0, 31, 125, and 500 eggs/100 cm³ soil. At the highest Pi, yield suppressions of CNS, Jackson, PI 230977, and PI 200538 were 55, 28, 31, and 29%, and 99, 86, 66, and 58% for races 1 and 2 compared with uninfested controls. Numbers of second-stage juveniles (J2) present in roots 14 days after planting increased as Pi increased, but did not differ between the two races. At the highest Pi, fewer race 1 (40-57%) and race 2 (53-68%) J2 were present in roots of the plant introductions than in roots of Jackson. Soil population densities of race 1 J2 at 135 days after planting were 83-89% lower on the resistant genotypes than on CNS. These numbers did not differ for race 2. Reproductive factors were considerably higher for race 2 compared to race 1 for all genotype by Pi combinations, except for CNS at the highest Pi.     

Stage-specific Differences in Lectin Binding to the Surface of Anguina tritici and Meloidogyne incognita

The occurrence and distribution of several lectin binding sites on the outer surfaces of eggs, preparasitic second-stage juveniles (J2), parasitic second-stage juveniles (PJ2), females, and males of two tylenchid nematodes, Anguina tritici and Meloidogyne incognita race 3, were compared. In both species, a greater variety of lectins bound to the eggs than to other life stages; lectin binding to eggs was also more intense than it was to other life stages. Species-specific differences also occurred. More lectins bound to the amphids or amphidial secretions of M. incognita J2 than to the amphids or amphidial secretions of A. tritici J2. Lectins also bound to the amphids or amphidial secretions of adult male and female A. tritici, but binding to the cuticle occurred only at the head and tail and was not consistent in all specimens. Canavalia ensiformis and Ulex europaeus lectins bound specifically to the outer cuticle of M. incognita. Several other lectins bound nonspecifically. Oxidation of the cuticle with periodate under mild conditions, as well as pretreatment of the nematodes with lipase, markedly increased the binding of lectins to the cuticle of A. tritici J2 but not, in most cases, to M. incognita J2 or eggs of either species.     

Cytogenetic Status of Meloidogyne (Hypsoperine) spartinae in Relation to Other Meloidogyne Species

Four populations of Meloidogyne spartinae from the coast of North and South Carolina were identical cytogenetically. Fourteen rod-shaped chromosomes were present in oogonia and spermatogonia, whereas seven bivalents were observed in oocytes and spermatocytes. There were no distinguishable sex chromosomes. Chromosome behavior was similar to that of other Meloidogyne species. A slight deviation in morphology of prometaphase bivalents was attributed to an increase in frequency of chiasmata that may be associated with the obligatorily amphimictic reproduction of this nematode. The anatomy of the oviduct-spermatotheca region and most cytogenetic features studied suggested that M. spartinae can be regarded as a root-knot nematode. Its position in the genus Meloidogyne or Hypsoperine can be decided by taxonomists. Its small chromosome number (n = 7) compared to the larger number (n = 13-19) of other Meloidogyne species suggests that, cytologically, M. spartinae stands closer to the ancestral form from which the prescent day root-knot nematodes have evolved.     

Optimization of Mitochondrial DNA-based Hybridization Assays to Diagnostics in Soil

Nucleic acid hybridization among root-knot nematode mitochondrial DNAs can be used to identify several Meloidogyne species. Research was initiated to optimize mitochondrial DNA-based molecular diagnostics for the demanding environments likely to be encountered in field isolates. DNA hybridization using reconstituted DNA-soil mixtures revealed a loss of assay sensitivity ranging from 34% to 92% with four agronomic soils tested. This problem was alleviated by the addition of exogenously added DNA. Variation in nematode egg lysis procedures also affected hybridization efficiency, with NaOC1 treatment most effective at disrupting Meloidogyne eggs. These optimized conditions permit detection of mtDNA released from one to five Meloidogyne eggs using standard nucleic acid hybridization procedures.     

Mode of Parasitism of Meloidogyne and Other Nemaiode Eggs by Dactylella oviparasitica

Hyphae of Dactylella oviparasitica proliferated rapidly through MeIoidogyne egg masses, and appressoria formed when they contacted eggs. The fungus probably penetrated egg shells mechanically, although chitinase production detected in culture suggested that enzymatic penetration was also possible. In soil, D. oviparasitica invaded egg masses soon after they were deposited on the root surface and eventually parasitized most of the first eggs laid. Occasionally the fungus grew into Meloidogyne females, halting egg production prematurely. The fungus parasitized eggs in the gelatinous matrix or eggs freed from the matrix and placed on agar or in soil. Specificity in nematode egg parasitism was not displayed, for D. oviparasitica parasitized eggs of four Meloidogyne spp., Acrobeloides sp., Heterodera schachtii, and Tylenchulus semipenetrans. In tests in a growth chamber, parasitism by D. oviparasitica suppressed galling on M. incognita-infected tomato plants.     

Root Tissue Response of Two Related Soybean Cultivars to Infection by Lectin-treated Meloidogyne spp.

Treatment of second-stage juveniles (J2) of Meloidogyne incognita race 1 and M. javanica with soybean agglutinin, Concanavalin A, wheat germ agglutinin, Lotus tetragonolobus agglutinin, or Limax flavus agglutinin or the corresponding competitive sugars for each of these lectins did not alter normal root tissue response of soybean cultivars Centennial and Pickett 71 to infection by M. incognita race 1 or M. javanica. Giant cells were frequently induced in Centennial and Pickett 71 roots 5 and 20 days after inoculation of roots with untreated J2 of a population of M. incognita race 3. Treatment of J2 of M. incognita race 3 with the lectins or carbohydrates listed above caused Centennial, but not Pickett 71, root tissue to respond in a hypersensitive manner to infection by M. incognita race 3. Penetration of soybean roots by J2 of Meloidogyne spp. was strongly inhibited in the presence of 0.1 M sialic acid. Treatment of J2 with sialic acid was not lethal to nematodes, and the inhibitory activity of sialic acid was apparently not caused by low pH. These results suggest that carbohydrates may influence plant-nematode interactions.     

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.