Morphological and Molecular Identification of Globodera pallida Associated with Potato in Idaho

The identity of a newly discovered population of pale potato cyst nematode Globodera pallida associated with potato in eastern Idaho was established by morphological and molecular methods. Morphometrics of cysts and second-stage juveniles were generally within the expected ranges for G. pallida with some variations noted. The Idaho population and paratype material from Epworth, Lincolnshire, England, both showed variations in tail shape, with bluntly rounded to finely pointed tail termini. Compared to literature values for the paratypes, second-stage juveniles of the Idaho population had a somewhat shorter mean body length, and cysts had a slightly higher mean distance from the anus to the nearest edge of the fenestra. PCR-RFLP of the rDNA ITS region, sequence-specific multiplex PCR and DNA sequence comparisons all confirmed the identity of the Idaho population as G. pallida. The ITS rDNA sequence of the Idaho isolate was identical to those from York, England, and the Netherlands. Species-specific primers that can positively identify the tobacco cyst nematode Globodera tabacum were also developed, providing a new assay for distinguishing this species from G. pallida and the golden potato cyst nematode Globodera rostochiensis.     

The Relationship between Temperature and Development in Globodera ellingtonae

A new cyst nematode species, Globodera ellingtonae, was recently described from populations in Oregon and Idaho. This nematode has been shown to reproduce on potato. Because of this nematode’s close relationship to the potato cyst nematodes, G. rostochiensis and G. pallida, an understanding of the risk of its potential spread, including prediction of potential geographical distribution, is required. To determine the development of G. ellingtonae under different temperatures, we conducted growth chamber experiments over a range of temperatures (10.0°C to 26.5°C) and tracked length of time to various developmental stages, including adult females bearing the next generation of eggs. Both the time to peak population densities of G. ellingtonae life stages and their duration in roots generally increased with decreasing temperature. Regression of growth rate to second-stage (J2) and third-stage (J3) juveniles on temperature yielded different base temperatures: 6.3°C and 4.4°C for J2 and J3, respectively. Setting a base temperature of 6°C allowed calculation of the degree-days (DD6) over which different life stages occurred. The largest population densities of J2 were found in roots between 50 and 200 DD6. Population densities of J3 peaked between 200 and 300 DD6. Adult males were detected in soil starting at 300 to 400 DD6 and remained detectable for approximately 500 DD6. By 784 to 884 DD6, half of the eggs in adult females contained vermiform juveniles. Given the similarity in temperature ranges for successful development between G. ellingtonae and G. rostochiensis, G. ellingtonae populations likely could survive in the same geographic range in which G. rostochiensis now occurs.     

Comparative Responses of Globodera rostochiensis and G. pallida to Hatching Chemicals

Globodera rostochiensis and G. pallida responded similarly to hatch stimulation by potato root leachate, but proportionally more second-stage juveniles (J2s) of G. rostochiensis hatched than of G. pallida in response to picrolonic acid, sodium thiocyanate, alpha-solanine, and alpha-chaconine. Fractionation of the potato root leachate identified hatching factors with species-selective (active toward both species but stimulating greater hatch of one species than the other), -specific (active toward only one species), and -neutral (equally active toward both species) activities. In a comparison of two populations of each of the two potato cyst nematode (PCN) species, however, greater similarity in response to the individual hatching factors was observed among populations of different species produced under the same conditions than among different populations of the same PCN species. Smaller numbers of species-specific and species-selective hatching factor stimulants and hatching inhibitors than of hatching factors were resolved. In a study to determine whether the different hatching responses of the two species to the same root leachate were associated with different ratios of species-selective and species-specific hatching factors, G. rostochiensis pathotype Ro1 exhibited greater hatch than did G. pallida pathotype Pa2/3 in response to leachate from older plants (more than 38 days old), while G. pallida exhibited greater hatch in response to leachate from younger plants (less than 38 days old); the response of G. pallida pathotype Pal with respect to plant age was intermediate between the other two populations. Combined molecular exclusion-ion exchange chromatography of the root leachates from plants of different ages revealed an increase in the proportion of G. rostochiensis-specific and -selective hatching factors as the plants aged.     

Two Simple Methods for the Collection of Individual Life Stages of Reniform Nematode,Rotylenchulus reniformis

The sedentary semi-endoparasitic nematode Rotylenchulus reniformis, the reniform nematode, is a serious pest of cotton and soybean in the United States. In recent years, interest in the molecular biology of the interaction between R. reniformis and its plant hosts has increased; however, the unusual life cycle of R. reniformis presents a unique set of challenges to researchers who wish to study the developmental expression of a particular nematode gene or evaluate life stage–specific effects of a specific treatment such as RNA-interference or a potential nematicide. In this report, we describe a simple method to collect R. reniformis juvenile and vermiform adult life stages under in vitro conditions and a second method to collect viable parasitic sedentary females from host plant roots. Rotylenchulus reniformis eggs were hatched over a Baermann funnel and the resultant second-stage juveniles incubated in petri plates containing sterile water at 30°C. Nematode development was monitored through the appearance of fourth-stage juveniles and specific time-points at which each developmental stage predominated were determined. Viable parasitic sedentary females were collected from infected roots using a second method that combined blending, sieving, and sucrose flotation. Rotylenchulus reniformis life stages collected with these methods can be used for nucleic acid or protein extraction or other experimental purposes that rely on life stage–specific data.     

Heterodera raskii n. sp. (Heteroderidae: Tylenchina), a Cyst Nematode on Grass, from Hyderabad,India

Heterodera raskii n. sp. is described and illustrated from specimens collected from roots of bulb grass, Cyperus bulbosus, in Hyderabad, India. The new species belongs to the ''goettingiana'' group and differs from closely related H. cyperi by the elongate ovoid shaped cysts and females, greater fenestral length, width, vulval slit, and absence of egg sac. The stylet knob shape was round in second-stage juveniles and posteriorly sloping in females and males of H. raskii n. sp., while it was anteriorly directed in second-stage juveniles and spherical in females and males of H. cyperi.     

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.     

Image Analysis of the Growth of Globodera pallida and Meloidogyne incognita on Transgenic Tomato Roots Expressing Cystatins

An approach based on image analysis that enables rapid collection and analysis of nematode size and shape during growth is reported. This technique has been applied to assess Meloidogyne incognita and Globodera pallida during their development over 35 and 42 days, respectively, on transgenic tomato roots expressing the wild-type rice cystatin Oc-I or an engineered variant, Oc-IAD86. Morphometric values were established that subdivided enlarged saccate females from other life stages. Analysis of this data subset indicates that the size of females and the frequency with which they parasitize roots expressing a cystatin are reduced. Results also demonstrate that cystatins can influence the growth of G. pallida prior to the adult stage. Similar image analysis procedures should be generally applicable to the study of host status or erivironmental factors that influence growth rates of plant-parasitic nematodes.     

Influence of Incubation Solution on the Rate of Recovery of Pratylenchus brachyurus from Cotton Roots

The rate of recovery of Pratylenchus brachyurus from cotton roots was enhanced when the tissue was incubated in solutions containing 10 ppm ethoxyethyl mercuric chloride, 50 ppm dihydrostreptomycin sulfate, 50, 100, or 1,000 ppm diisobutylphenoxethyl dimethyl benzyl ammonium chloride, or mixtures of these compounds. Incubation in 10 or 100 ppm zinc sulfate, zinc chloride, or magnesium chloride also enhanced the rate of recovery. Incubation solutions containing 1 or 1,000 ppm zinc chloride or magnesium chloride had no influence on this phenomenon, whereas, 10,000 ppm zinc sulfate, zinc chloride, or magnesium chloride retarded the rate of recovery. A t all incubation intervals during the first 21 days after the roots were removed from soil, the P. brachyurus population consisted of approximately 25% second-stage juveniles, 44% third and fourth-stage juveniles, and 31% females. At least 88% of the second-stage juveniles and 51% of the third and fourth-stage juveniles passed through a single 325-mesh sieve, whereas, 84% of the females collected were retained on a sieve of this mesh.     

Changes in Vertical Distribution of Pratylenchus scribneri under Potato and Corn

The vertical distribution of Pratylenchus scribneri populations was monitored under irrigated corn and potato grown in loamy sand soil. population estimates were based on the number of nematodes recovered from 100-cm³ soil samples and the roots contained therein. Reproduction was assessed by counting the number of second-stage juveniles. An index of population maturity was computed to evaluate the age structure of populations. At no time were nematodes distributed uniformly among five soil depths from 0 to 37.5 cm deep. During the summer (June-September), changes in the total number of P. scribneri and the number of second-stage juveniles recovered were not consistent among the depths sampled. Early (April-June) and late (September-November) in the season, changes in the abundance, reproduction, and maturity of populations were similar among depths. The timing and pattern of increases in numbers of nematodes suggests that variation in the abundance of P. scribneri in the soil profile beneath potato and corn was caused primarily by reproduction rather than the movement of nematodes.     

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

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

Efficient Procedure for Extracting Tylenchulus semipenetrans from Citrus Roots

Investigations were undertaken to determine the suitability of sucrose and magnesium sulphate solutions and a silica colloidal suspension with centrifugation for extracting Tylenchulus semipenetrans from citrus roots. The efficiency of incubation, sodium hypochlorite, centrifugation, and maceration methods was also compared. Numbers of females recovered by centrifugation with colloidal silica were greater than those from sucrose or magnesium sulphate. Incubation, sodium hypochlorite, and centrifugation methods were satisfactory for extracting eggs, second-stage juveniles, and males, whereas the maceration-sieving method was less efficient. Combining the sodium hypochlorite method with a 15-second maceration followed by centrifugation in colloidal silica reduced the recovery of T. semipenetrans females from citrus roots.     

Life Cycle of the Golden Cyst Nematode,Globodera rostochiensis,in Quebec,Canada

In 2006, the golden cyst nematode, Globodera rostochiensis, was discovered in the province of Quebec, Canada. We report here the life cycle of G. rostochiensis under the climatic conditions of southwestern Quebec. Only one full generation was completed per year under these latitudes. On susceptible potato cv. Snowden, G. rostochiensis needed a minimum of 579 growing degree units (GDU) (base 5.9°C) to complete its life cycle and the first mature cysts were observed 42 to 63 days after planting (DAP). In soil, second-stage juveniles (J2) were first observed 14 to 21 DAP, whereas both white females on roots and males in soil appeared synchronously after 35 to 42 days. The duration of the life cycle was affected by temperature but not by soil type. A second wave of hatching systematically occurred later in the season and a second generation of males was observed during the 2011 growth season. No complete second cycle was observed before plant senescence. Climate change and later maturing cultivars/crops could allow the development of a full second generation in the future.     

Nematode Numbers and Crop Yield in a Fenamiphos-Treated Sweet Corn-Sweet Potato-Vetch Cropping System

Nematode population densities and yield of sweet corn and sweet potato as affected by the nematicide fenamiphos, in a sweet corn-sweet potato-vetch cropping system, were determined in a 5-year test (1981-85). Sweet potato was the best host of Meloidogyne incognita of these three crops. Fenamiphos 15G (6.7 kg a.i./ha) incorporated broadcast in the top 15 cm of the soil layer before planting of each crop increased (P ≤ 0.05) yields of sweet corn in 1981 and 1982 and sweet potato number 1 grade in 1982 and 1983. Yield of sweet corn and numbers of M. incognita second-stage juveniles (J2) in the soil each month were negatively correlated from planting (r = - 0.47) to harvest (r = -0.61) in 1982. Yield of number 1 sweet potato was inversely related to numbers of J2 in the soil in July-October 1982 and July-September 1983. Yield of cracked storage roots was positively related to the numbers of J2 in the soil on one or more sampling dates in all years except 1985. Some factor(s), such as microbial degradation, resistant M. incognita development, or environment, reduced the effect of fenamiphos.     

Hatching Behavior of Potato Cyst Nematodes from the Canary Islands

The present work investigated early hatching differences in naturally occuring field populations and newly reared populations of potato cyst nematodes from the Canary Islands. Hatching behavior of the two species appears to be distinct, with more juveniles hatched from G. pallida that hatch earlier and over a shorter time than G. rostochiensis. The hatching rate of 3-year-old PCN populations was more than double (mean 44.5% ñ 1) that shown by newly reared populations (mean 19.1% ñ 12.5), and those that could be classified as pathotype Pa 1 (Pa 1 and P 13) were found to hatch particularly poorly. Significant differences were also observed in the juveniles released in tap water between newly reared populations of both species, with mean hatch significantly higher for G. rostochiensis. The results are discussed in relation to the implication that these findings may have for competition between the two species of PCN in the field.     

Reproduction of Globodera rostochiensis on Transformed Roots of Solanum tuberosum cv. Desiree

Transformed roots of the susceptible potato Solanum tuberosum L. cv. Desiree were inoculated with second-stage juveniles (J2) of Globodera rostochiensis pathotype Rol. Adult males emerged after 3-4 weeks and matings with females occurred. After 8 weeks gentle pressure on the eggs of maturing females released the J2 which were viable. Because this technique enables the production of vigorously growing roots with numerous laterals, it may be suitable for obtaining a high yield of sterile potato cyst nematodes.     

Size Differences Among Root-knot Nematodes on Resistant and Susceptible Alyceclover Genotypes

The influence of plant resistance on the size of individual root-knot nematodes was determined in greenhouse experiments. Five genotypes of alyceclover were inoculated with second-stage juveniles of Meloidogyne incognita race 3 or M. arenaria race 1. Plants were harvested at selected intervals and stained for detection of the nematodes, which were dissected from the roots. Length, width, and sagittal-sectional area of each animal were measured using an image-analysis system, and areas of nematodes in all stages were compared at different times and across alyceclover lines. Nematodes feeding on roots of resistant lines were consistently smaller than those on susceptible plants, with significant differences in growth detected after the final molt. Similar results were observed with both nematode species.     

Pasteuria sp. Parasitizing Trophonema okamotoi in Florida

Two populations of Trophonema okamotoi parasitized by Pasteuria sp. were found on Liquidambar styraciflua (sweetgum) and on an unidentified tropical grass in north-central Florida. Endospores of this Pasteuria sp. attached to motile vermiform second-stage juveniles (J2) and males of T. okamotoi, but not to other developmental stages. Sporangia and new endospores were produced only inside the bodies of swollen and sedentary third- and fourth-stage juveniles and females that developed in the host roots. No egg masses were produced by infected T. okamotoi females. The endospore diameter from the tropical grass population was 4.93 μm and the central core diameter was 1.97 μm; measurements of endospores from the sweetgum populations were similar. Endospores that were collected from T. okamotoi and added to uninfected T. okamotoi and other plant-parasitic nematodes attached/to J2 of T. okamotoi but did not attach to juveniles and adults of Helicotylenchus pseudorotrustus, Pratylenchus brachyurus, or to J2 of either Meloidogyne arenaria race 1, M. incognita race 1, M. javanica, or Tylenchulus semipenetrans. Pasteuria sp. from T. okamotoi differed from the described Pasteuria species in endospore size, host preference, and rate of attachment.     

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.     

Effect of Soybean Tip Removal on Penetration and Development of Heterodera glycines

On a few occasions, soybeans with broken root tips were included in tests to evaluate resistance to Heterodera glycines. Although females developed on these plants, the numbers tended to be lower than on similarly treated intact roots. To test the possibility that removal of the root meristem affected nematode development, a culture system using pruned soybeans was devised that permitted access to the roots without disturbing the plants. Treatments included removal of 2 mm of root tip at various times ranging from 24 hours before to 10 days after inoculation, or roots left intact. In each experiment, all roots were inoculated at the same time with equal numbers of freshly hatched second-stage juveniles of Heterodera glycines. No differences in nematode development were detected in plants with root tips removed after inoculation compared to the control. When tips were removed at or before inoculation, fewer juveniles entered roots and relatively fewer nematodes developed. Penetration levels and development correlated with root tip removal such that progressively fewer nematodes entered roots and relatively greater numbers of nematodes remained undeveloped as the time interval between root tip removal and inoculation was increased.