Fungi Associated with Females and Cysts of Heterodera glycines in a Florida Soybean Field

Fungal colonization was determined for females and cysts of Heterodera glycines on soybean roots or in rhizosphere soil from a Florida soybean field. A total of 1,620 females and cysts were examined in 1991, and 1,303 were examined in 1992. More than 35 species of fungi were isolated from females and cysts. The frequency of fungi colonizing white and yellow females was low, but a high frequency of fungi was encountered in brown cysts, which increased with time of exposure of the cysts to the soil. No single fungal species predominated in the nematode females or cysts in this field. Rarely was a female or cyst colonized by more than one fungus. The common fungi isolated from the females and cysts were Neocosmospora vasinfecta, Fusarium solani, Fusarium oxysporum, Dictyochaeta coffeae, Dictyochaeta heteroderae, Pyrenochaeta terrestris, Exophiala pisciphila, Gliocladium catenulatum, Stagonospora heteroderae, and a black yeast-like fungus. The communities of common fungal species isolated from cysts in several regions in the southeastern United States appear to be similar.     

Temperature-based Prediction of Egg-Mass Production by Meloidogyne incognita

A mturation-rate relationship for Meloidogyne incognita on Lycopersicon esculentum ''Rutgers'' was derived and used to estimate harvest dates for maximmn egg hatch from laboratory cultures at ambient temperatures. Daily maturation increments were totaled (nematode maturation total, NMT) and correlated with hatch from isolated white, yellow, and amber egg masses. Hatch per mass fluctuated periodically from ca. 1.0 NMT, when egg masses were first visible, to 2.5 NMT by which time plants showed stress. Maximum yields from white and yellow masses occurred, with a shorter than expected periodicity, at 1.5-1.8 and 2.1-2.2 NMT. White masses decreased from 90% of the total masses at 1.0 NMT to 5% at 2.5 NMT, as the proportion of yellow and amber masses increased concomitantly. Harvested masses per gram of root varied from 97 to 276; hatch per gram of root, 11,000 to 86,000.     

Temperature Effects on Race Determination in Heterodera glycines

Currently there are 16 possible races for Heterodera glycines, and these are differentiated based on ability of a nematode population to develop on a set of four differential soybean genotypes. Because results are based on numbers of nematode females that develop to a specific stage rather than on the reproductive capability of these females, race determinations based on female indices may not represent results obtained after several reproductive cycles of H. glycines. Counting numbers of eggs and juveniles, and then developing corresponding indices, would allow reproduction to be considered in making race determinations. Our objectives were to compare the present race identification scheme for H. glycines based on female indices with those using egg and juvenile indices and to examine the effect of temperature on race designations using female, egg, and juvenile indices. Race designations for H. glycines populations from two locations in Illinois were determined at 20, 27, and 30 °C in a water bath. The numbers of females, eggs, and juveniles (at 19 days) were recorded, and an index based on each life stage was calculated. Race determinations based on female, egg, or juvenile indices were inconsistent when conducted at 20 °C, which demonstrates that this temperature is not suitable for identifying races of H. glycines. However race designations at 27 and 30 °C were consistent for all three indices. This indicates that counting females, eggs, or juveniles should be equally reliable when race determinations are conducted at these two temperatures, and choice of method would depend on investigator preference or research objective.     

FMRFamide-like Immunoactivity in Heterodera glycines (Nemata: Tylenchida)

Material antigenically related to the neuromodulatory peptide FMRFamide was detected and examined in preparations of the soybean cyst nematode, Heterodera glycines, and in the free-living nematodes Caenorhabditis elegans and Panagrellus redivivus. FMRFamide-related peptides were quantified by an enzyme-linked immunosorbent assay. Specific activities were remarkably similar among all of the vermiform members of the three species. FMRFamide-related peptide immunoactivity was present in both sexes and all stages of H. glycines examined. The highest specific activity was present in second-stage juveniles and in males, and the lowest in white and yellow females. Total FMRFamide-related peptide level per individual was highest in brown females, with 90% of the activity associated with the eggs. Peptide levels in these eggs and in second-stage juveniles were comparable and increased in adults, especially in females. Chromatographic analysis of FMRFamide-related peptide preparations from H. glycines juveniles, C. elegans, and P. redivivus revealed distinct qualitative differences between the infective plant parasite and the free-living nematodes.     

Influence of Rhizoctonia solani on Egg Hatching and Infectivity of Rotylenchulus reniformis

The effects of culture filtrates of Rhizoctonia solani and root exudates of R. solani-infected cotton (Gossypium hirsutum) seedlings on hatching of eggs and infectivity of females of Rotylenchulus reniformis were evaluated in an attempt to account for the enhanced nematode reproduction observed in the presence of this fungus. Crude filtrates of R. solani cultures growing over sterile, deionized distilled water did not affect egg hatching. Exudates from roots of cotton seedlings increased hatching of R. reniformis eggs over that observed in water controls. Exudates from cotton seedling roots not infected or infected with R. solani did not differ in their effect on egg hatching. However, infection of cotton seedlings by reniform females was increased in the presence of R. solani, resulting in the augmented egg production and juvenile population densities in soil observed in greenhouse studies.     

Reproduction of Virulent Isolates of Meloidogyne incognita on Susceptible and Mi-resistant Tomato

The reproductive potential of natural and laboratory-selected Meloidogyne incognita isolates virulent against the tomato Mi resistance gene, all derived from a single egg-mass, were compared when the nematodes were inoculated on susceptible and resistant tomato. Fewer second-stage juveniles (P = 0.01) of the two virulent populations selected under laboratory conditions matured to females on the resistant tomato compared to the susceptible cultivar. In contrast, no differences were found between the number of egg masses produced on the resistant versus the susceptible tomato by the two natural virulent isolates. No clear general trends concerning the fecundity of the females could be inferred from the comparative analysis of the numbers of eggs per egg mass x tomato cultivar combination. These observations suggested that the genetic changes induced under environmentally controlled nematode growth might be different from those occurring in natural Mi-resistance breaking biotypes grown without environmental control.     

Ditylenchus destructor in Hulls and Seeds of Peanut

The potato rot nematode, Ditylenchus destructor Thorne, is reported for the first time in hulls and seeds of peanut. The populations found differed from D. dipsaei and D. myceliophagus in habitat, number of lateral incisures, shape of tail tip, and length of postvulval sac. Infected hulls had brown necrotic tissue at the point of connection with the peg, and a black discoloration appeared first along the longitudinal veins. Infected seeds were usually shrunken, and testae and embryos had a yellow to brown or black discoloration. Of 877 seed samples graded "damaged" from all major peanut producing areas of South Africa, 73% were infected.     

Life Cycle of Steinernema scapterisci Nguyen &Smart, 1990

The life cycle of Steinernema scapterisci Nguyen and Smart, 1990 consists of an egg stage, four juvenile stages, and an adult stage (male and female). The cycle from IJ (third stage infective juveniles) to IJ may proceed by one of two routes. If the nutrient supply is sufficient and the population is not overcrowded, the IJ develop to adult males and females of the first generation. Most eggs from these adult females hatch and the juveniles develop through each life stage to become adult males and females of the second generation. Eggs produced by these females develop to IJ. This cycle takes 8-10 days (long cycle) at 24 C. If the nutrient supply is insufficient or if overcrowded, the IJ develop to adult males and females of the first generation, and eggs produced by the females develop directly to IJ. This cycle takes 6-7 days (short cycle). The nematode is less tolerant of lower temperatures and more tolerant of higher temperatures than are other species of the genus. The sex ratio is influenced by temperature. At 15 and 24 C, females constituted 54% and 60% of the population, respectively, but at 30 C females constituted 47% of the population.     

Lipid Composition of Cyst Stages of Globodera rostochiensis

Lipid compositional analysis was conducted on the white, yellow, and brown cyst stages of Globodera rostochiensis (golden cyst nematode). Triacylglycerols were the largest lipid fraction in all stages examined, ranging from 55-75% of total lipid. Ethanolamine phosphoglycerides and choline phosphoglycerides were present in high amounts in all cyst fractions, with a total phospholipid content of 20%, 14.7%, and 12.8% in the white, yellow, and brown cyst stages, respectively. Sterols, steryl esters, sphingomyelin, and cardiolipin were found in minor amounts in all three cyst stages and showed greater changes than other classes of lipids relative to cyst stage. The fatty acid compositions of the three cyst stages were similar. Eicosenoic acid (20:1) and arachidonic acid (20:4) were found in higher concentrations than other fatty acids in all cyst preparations; vaccenic acid (18:1) occurred at the third highest concentration. More than 78% of total fatty acids were unsaturated at all cyst stages, and more than 60% were of C20 or longer chain length. The lipid profile of all three cyst stages is consistent with invertebrate adaptation to low-temperature environments.     

Degree-day Models for Predicting Egg Hatch and Population Increase of Criconemella xenoplax

A degree-day model was derived to predict egg hatch for Criconemella xenoplax. Eggs collected from gravid females were incubated in distilled water at constant temperatures of 10-35 C. Sixty-six percent of all eggs hatched between 13 and 32 C, and 42% hatched at 10 C. All eggs aborted above 32.5 C. Between 25 and 32 C, 8.5 ± 0.5 days were required for egg hatch. Degree-day requirement for egg hatch at 10-30 C was estimated to be 154 ± 5 with a base of 9.03 ± 0.04 C. This base of 9 C was adopted in studies of the relationship between degree-days and nematode population increase on Prunus seedlings grown 9-11 weeks in a greenhouse. Degree-day accumulations were based upon daily averages from maximum and minimum air temperatures. Ratios of final to initial population densities exhibited an exponential pattern in relation to degree-day accumulations with proportionate doubling increment of 0.100 ± 0.049 every 139 ± 8 degree-days. These results provide a means of predicting nematode population increase under greenhouse conditions and a basis for choosing sampling intervals when evaluating nematode multiplication.     

Comparisons of Female and Egg Assays to Identify Rotylenchulus reniformis Resistance in Cotton

More plants can be screened for reniform nematode resistance each year if the time involved can be shortened. In this study, the hypothesis that female counts are as efficient as egg counts in identifying resistant genotypes was tested. In two greenhouse experiments Gossypium genotypes which varied from resistant to susceptible to reniform nematode (Rotylenchulus reniformis) were compared to a susceptible control cultivar. Infested field soil served as the inoculum source for the first experiment, and vermiform stages extracted from greenhouse cultures were used to infest soil in the second experiment. Six replicates of each genotype were harvested 25 d after planting and swollen females were counted. The remaining plants were harvested 35 d after planting and eggs extracted from the roots were counted. Processing and counting times recorded in the first experiment were similar for both assessment methods, but 10 additional days were required for egg-based assessment. Contrast analyses showed that assessments based on females per gram of root were equivalent to assessments based on eggs per gram of root for the five genotypes tested in the first experiment and for an expanded set of 13 genotypes tested in the second experiment. The results indicated that either life stage can be used to screen for resistance.     

Flow Cytometric Analysis and Sorting of Heterodera glycines Eggs

A nondestructive technique was developed to characterize and separate eggs of soybean cyst nematode, Heterodera glycines, by developmental stage using flow cytometry. Eggs from cysts cultured on susceptible soybean roots were suspended in 0.1% xanthan gum or 59% sucrose and loaded into either a Coulter EPICS 752 or EPICS 753 flow cytometer. Eggs were analyzed and sorted according to forward angle and 90° light scatter, flow cytometric parameters that are relative measures of object size and granularity, respectively. Mature eggs containing vermiform juveniles were less granular and slightly larger than eggs in earlier stages of embryogeny, allowing for separation of mature eggs from immature eggs. The effectiveness of flow cytometric sorting was evaluated by comparing the developmental stages of subpopulations of unsorted and sorted eggs. Of a subpopulation of unsorted eggs, 62% contained vermiform juveniles, whereas 85 to 95% of sorted subpopulations of larger, less granular eggs contained vermiform juveniles. Suspending H. glycines eggs in 0.1% xanthan gum or 59% sucrose for flow cytometric analysis had no effect on subsequent egg hatch in vitro. This technique is an efficient and effective means to collect large, relatively homogeneous quantities of H. glycines eggs in early or late embryogeny, and would likely be useful for analyzing and sorting eggs of other nematode species for use in developmental, genetic, or physiological research, or for identification and collection of parasitized eggs.     

Sterol Composition and Ecdysteroid Content of Eggs of the Root-knot Nematodes Meloidogyne incognita and M. arenaria

Free and esterified sterols of eggs of the root-knot nematodes Meloidogyne incognita races 2 and 3 and M. arenaria race 1 were isolated and identified by gas-liquid chromatography-mass spectrometry. The major sterols of eggs of each race were 24-ethylcholesterol (33.4-38.8% of total sterol), 24-ethylcholestanol (18.3-25.3%), 24-methylcholesterol (8.6-11.7%), 24-methylcholestanol (7.7-12.5%), and cholesterol (4.6-11.6%). Consequently, the major metabolic transformation performed by Meloidogyne females or eggs upon host sterols appeared to be saturation of the sterol nucleus. The free and esterified sterols of the same race did not differ appreciably, except for a slight enrichment of the steryl esters in cholesterol. Although the sterol composition of Meloidogyne eggs differed from that of other life stages of other genera of plant-parasitic nematodes, the three Meloidogyne races could not be distinguished from each other by their egg sterols. Ecdysteroids, compounds with hormonal function in insects, were not detected by radioimmunoassay in the Meloidogyne eggs either as free ecdysteroids or as polar conjugates.     

Low-Temperature Scanning Electron Microscope Observations of the Meloidogyne incognita Egg Mass: The Gelatinous Matrix and Embryo Development

The root-knot nematode Meloidogyne incognita was cultured monoxenically on excised tomato roots. Galls and egg masses were observed daily using a light microscope. Two phases were distinguished in the gelatinous matrix of the egg mass: a translucent, amorphous material on the surface of the egg mass and a denser, layered phase in which nematode eggs were deposited. Egg masses were also cryofixed, fractured, and observed as frozen, hydrated specimens on a cold stage in a scanning electron microscope (SEM). In the SEM, the layered phase appeared as a meshwork of fibrils that became more loosely associated as the gelatinous matrix aged: Small pearl-like bodies were observed along the fibers of gelatinous matrix. The egg shell surface and several stages of embryo development, including the one-cell stage, initial cleavages, blastula, gastrula, tadpole stage, elongation, and molt of the first-stage juvenile within the egg shell, were observed and photographed with this technique. The developmental events observed were consistent with those described in other nematode species with different techniques.     

Growth and Energy Demand of Meloidogyne incognita on Susceptible and Resistant Vitis vinifera Cultivars

Food (energy) consumption rates ofMeloidogyne incognita were calculated on Vitis vinifera cv. French Colombard (highly susceptible) and cv. Thompson Seedless (moderately resistant). One-month-old grape seedlings in styrofoam cups were inoculated with 2,000 or 8,000 M. incognita second-stage juveniles (J2) and maintained at 17.5 degree days (DD - base 10 C)/day until maximum adult female growth and (or) the end of oviposition. At 70 DD intervals, nematode fresh biomass was calculated on the basis of volumes of 15-20 nematodes per plant obtained with a digitizer and computer algorithm. Egg production was measured at 50-80 DD intervals by weighing 7-10 egg masses and counting the number of eggs. Nematode growth and food (energy) consumption rates were calculated up to 1,000 DD based on biomass increase, respiratory requirements, and an assumption of 60 % assimilation efficiency. The growth rate of a single root-knot nematode, excluding egg production, was similar in both cultivars and had a logistic form. The maximum fresh weight of a mature female nematode was ca. 29-32 μg. The total biomass increase, including egg production, also had a logistic form. Maximum biomass (mature adult female and egg mass) was 211 μg on French Colombard and 127 μg on Thompson Seedless. The calculated total cost to the host for the development of a single J2 from root penetration to the end of oviposition for body growth and total biomass was 0.535 and 0.486 calories with a total energy demand of 1.176 and 0.834 calories in French Colombard and Thompson Seedless, respectively.     

Overwintering Stages of Meloidogyne incognita in Vitis vinifera

The overwintering of Meloidogyne incognita in and around Vitis vinifera cv. French Colombard roots was studied in a naturally infested vineyard at the Kearney Agricultural Center, in a growth chamber, in inoculated vines in microplots at the University of California, Davis, and in a greenhouse. Infected roots were sampled at intervals from onset of vine dormancy until plants accumulated about 800 degree days (DD - base 10 C). Embryogenesis within eggs, classified as less than or more than 16 cells and fully differentiated, and numbers of juveniles (second to fourth stage) and preovipositional and mature (egg-laying) adult stages in roots were determined. All stages were present at the onset of dormancy. Juveniles and immature females were not recovered during the dormant period. Mature females and eggs were always present in roots, although the number of mature females generally decreased with time after onset of dormancy. In contrast, in a greenhouse experiment that accumulated comparable DD without the host plant going through dormancy, the number of mature females increased. After bud break, the number of eggs per female increased and all nematode stages were found in host roots. Eggs in all stages of embryogenesis were observed at all times of sampling, indicating that females overwinter and are capable of laying eggs when conditions improve in the spring and need to be considered in nematode management decisions.     

Competition between the Plant-parasitic Nematodes Pratylenchus neglectus and Meloidogyne chitwoodi

In experiments on competition between Pratylenchus neglectus and Meloidogyne chitwoodi in barley, the species that parasitized the roots first inhibited penetration by the latter species. Prior presence of P. neglectus impeded the development of M. chitwoodi. Pratylenchus neglectus reduced egg production, final population levels, and reproductive index of M. chitwoodi. The reduction was linearly related to initial population densities of P. neglectus. Initial population densities of M. chitwoodi had no effect on final population levels of P. neglectus. Carbon assimilation by barley plants was reduced when either nematode species was present alone, but not when both were present together. Both nematode species assimilated lower amounts of carbon when present together than when present alone. A split-root experiment demonstrated that translocatable chemicals were not involved in the competition between the two species.     

A Technique for Evaluating Heterodera glycines Development in Susceptible and Resistant Soybean

A technique was developed to evaluate Heterodera glycines development in susceptible and resistant soybean. Roots of 3-day-old soybean were exposed to infective juveniles of H. glyci.nes in sand for 8 hours followed by washing and transfer to hydroponic culture. The cotyledons and apical meristem were removed and plants were maintained under constant light, which resulted in a dwarfed plant system. After 15 or 20 days at 27 C, nematodes were rated for development. Emerged males were sieved from the culture water and females were counted directly from the roots. Nematodes remaining in the roots were rated for development after staining and clearing the tissues. The proportion of nematodes at each stage of development and the frequency of completed molts for each stage were calculated from these data. This technique showed that resistance to H. glycines was stage related and did not affect males and females equally in all resistant hosts. The resistance of plant introduction PI 209332 primarily affected development of third and fourth-stage juveniles; ''Pickett'' mainly affected second and third-stage juveniles, whereas PI 89772 affected all stages. Male development was markedly affected in PI 89772 and ''Pickett'' but not in PI 209332.     

Influence of Temperature on Multiplication and Egg Hatching of Longidorus africanus

Longidorus africanus multiplication on tomato was highest at 29 °C. Few nematodes were recovered after 6 weeks at soil temperatures of 35 °C or below 23 °C. The time to egg hatching was shortest and the percentage of eggs hatching was highest at 29 °C. The minimum temperature and the heat sum above this temperature required for egg development were calculated to be 14.3 °C and 94.08 degree-days, respectively. The thermal times required for egg development by L. africanus and L. elongatus were nearly identical. For both species the product of the base temperature and the heat sum was near constant, and at a temperature of 22.3 °C the rates of egg development were equal.     

In Vitro Culture and Feeding Behavior of Belonolaimus longicaudatus on Excised Zea mays Roots

A greenhouse population of the sting nematode, Belonolaimus longicaudatus, obtained from an infested golf course in California''s Coachella Valley, was surface-decontaminated and cuhured on excised roots of Zea mays supported by Gamborg''s B5 medium. At 26-27 °C the females laid eggs, and newly emerged juveniles of the second generation completed three molts within 29 days after egg deposition. Sixty days after inoculation with 60 females and 40 males, an average of 529 nematodes and 83 eggs were recovered from the culture. The feeding process consisted of probing, stylet penetration, ingestion, and stylet retraction. Feeding seemed to be necessary before egg deposition or molting occurred. The sting nematode was observed feeding exclusively as an ectoparasite and preferably at the region of cell division and elongation. Vigorous feeding by many nematodes usually caused discoloration of root tips and termination of growth.