Redescription of Heterodea zeae,the Corn Cyst Nematode,with SEM Observations

Heterodera zeae, the corn cyst nematode, is redescribed and illustrated with comparative details and measurements of females, cysts, and larvae from Maryland, USA; and India. Scanning electron micrographs o f specimens from the United States are also presented. Revised measurements for the larval stylet and new diadnostic characters, especially in the cyst cone, for H. zeae are given. The relationship of H. zeae to close species is discussed.     

Host Status of Five Weed Species and Their Effects on Pratylenchus zeae Infestation of Maize

The host suitability of five of the most common weed species occurring in maize (Zea mays L.) fields in South Africa to Pratylenchus zeae was tested. Based on the number of nematodes per root unit, mealie crotalaria (Crotalaria sphaerocarpa) was a good host; goose grass (Eleusine indica), common pigweed (Amaranthus hybridus), and thorn apple (Datura stramonium) were moderate hosts; and khaki weed (Tagetes minuta) was a poor host. Only the root residues of khaki weed suppressed the P. zeae infestation of subsequently grown maize. When goose grass, khaki weed, and mealie crotalaria were grown in association with maize in soil infested with P. zeae, goose grass and khaki weed severely suppressed maize root development; this resulted in a low number of nematodes per maize root system and a high number of nematodes per maize root unit. Mealie crotalaria did not restrict maize root growth and did not affect nematode densities per maize root system or maize root unit. Special attention should be given to the control of mealie crotalaria, which is a good host for P. zeae, and goose grass, which, in addition to its ability to compete with maize, is also a suitable host for P. zeae.     

Reproduction of Heterodera zeae and Its Suppression of Corn Plant Growth as Affected by Temperature

Reproduction of the corn cyst nematode (Heterodera zeae) and its effect on growth of corn (Zea mays) was studied in plant growth chambers at 24, 27, 30, 33, and 36 C. Reproduction of H. zeae increased directly with increase in temperature from 24 to 36 C. Fifteen-cm-d pots of corn seedlings inoculated with a mixture of 5,000 eggs + J2 and maintained for 8 weeks in growth chambers contained an average of 7,042 cysts + females at 36 C, but only 350 cysts + females at 24 C. Fresh weights of plants without nematodes were highest at 27 C and lowest at 36 C. Nematodes suppressed plant fresh weight by an average of 30% at 27 C and by 27% at 33 C but did not suppress plant weight at 36 C. Heterodera zeae has the highest reported temperature optimum for reproduction of any cyst nematode.     

Molecular and Morphological Characterization of the Corn Cyst Nematode,Heterodera zeae,from Greece

The corn cyst nematode Heterodera zeae was detected in soil from an organic maize field in northern Greece. In greenhouse studies, reproduction of H. zeae was detected on maize plants (Zeae mays) using soil high in organic matter; the field was under winter fallow at the time of sampling. Maize plants were grown in a greenhouse with soil from the affected field used as inoculum. Females appeared after six weeks incubation, and abundant cysts were present after 12 weeks. Morphological and molecular diagnosis confirmed the presence of H. zeae in the field. Cysts were identified on the basis of cyst shape and characteristics of the cyst terminal cone, including nature of fenestration, presence of bullae, cyst wall pattern, and fenestral diameter. Second-stage juveniles were identified by body and stylet length, the shape of stylet knobs, shape and length of the tail and hyaline tail terminus, and by the number of lateral lines. Molecular analysis included amplification of the ribosomal internal transcribed spacer regions (ITS 1&2 rDNA) 28S large ribosomal subunit (LSU) D2-D3 expansion segment, and partial 18S small ribosomal subunit (SSU). Restriction fragment length polymorphism (RFLP) of ITS rDNA exhibited several unique enzyme patterns that may be diagnostically useful for H. zeae. These findings are in agreement with prior analysis of H. zeae populations from the U.S. and India. Phylogenetic relationships inferred from ITS rDNA are congruent with previous analyses that placed H. zeae in a clade with H. turcomanica, H. salixophila and species of the Humuli group. Phylogenetic trees based upon heat shock protein (Hsp90) coding sequence were in general agreement with a prior study using the same marker. This study represents the first record of H. zeae in Greece and the second report of this nematode in Europe.     

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.     

Distribution of Field Corn Roots and Parasitic Nematodes in Subsoiled and Nonsubsoiled Soil

A field trial was conducted for 2 years in an Arredondo fine sand containing a tillage pan at 15-20 cm deep to determine the influence of subsoiling on the distribution of corn roots and plant-parasitic nematodes. Soil samples were taken at various depths and row positions at 30, 60, and 90 days after planting in field corn subsoiled under the row with two chisels and in non-subsoiled corn. At 30 and 60 days, in-row nematode population densities to 60 cm deep were not affected by subsoiling compared with population densities in nonsubsoiled plots. After 90 days, subsoiling had not affected total root length or root weight at the 20 depth-row position sampling combinations, but population densities of Meloidogyne incognita and Criconemella spp. had increased in subsoiled corn. Numbers of Pratylenchus zeae were not affected. Subsoiling generally resulted in a change in distribution of corn roots and nematodes in the soil profile but caused little total increase in either roots or numbers of nematodes. Corn yield was increased by subsoiling.     

Seasonal Population Dynamics of Selected Plant-Parasitic Nematodes on Four Monocultured Crops

Seasonal fluctuations in field populations of Meloidogyne incognita, Pratylenchus zeae, P. brachyurus, Criconemoides ornatus, Trichodorus christiei, and Helicotylenchus dihystera on monocultured corn, cotton, peanut, and soybean were determined monthly for 4 yr. Population densities of M. incognita were greater in corn and cotton plots than in peanut and soybean plots from July until January. Those of Pratylenchus spp. were greater on corn and soybean than on cotton and peanut during all months except May and June. C. ornatus populations were greater on corn and peanut than on cotton and soybean during all months. C. ornatus on corn and peanut was more numerous in July than in other months. There was no significant increase in populations of T. christiei, except on corn in June. H. dihystera was greater in cotton and soybean plots than in corn and peanut plots from August through December.     

Development of Heterodera glycines Ichinohe on Soybean,Glycine max (L.) Merr., under Gnotobiotic Conditions

The life cycle of the soybean cyst nematode, Race 3 (SCN 3), Heterodera glycines Inchinohe was determined from observations of the developmental stages on soybean Glycine max cv. Kent root explants under gnotobiotic conditions at 25 C. Approximately 51% of the second-stage larvae penetrated the root l day after inoculation (DAI). Third-stage larvae appeared 4 DAI, became sexually differentiated 5 DAI, and protruded from the root tissues 6 DAI. Fourth-stage males and females were observed 7 DAI. Ensheathcd adult males were observed at 9 DAI and exsheathed to free adults at 11 DAI. The fourth-stage female became an adult at l0 DAI, Males entwined arotmd the gelatinons sac of the female at 12 DAI and were assumed to be mating. Some males actually penetrated and were enveloped by the gelatinous sac. The female-to-male sex ratio ranged from 2.3 to 9.5:1. First- and second-stage larvae were observed in the egg 17 and 19 DAI, respectively. The life cycle of the SCN 3 was completed 21 DAI upon hatching of the eggs and emergence of second-stage larvae. The average number of eggs in the cyst body and gelatinous sac, was 210 and 187, respectively. Key words: reproduction, soybean cyst nematode, scanning electron microscopy.     

Population Dynamics,Root Penetration,and Feeding Behavior of Pratylenchus agilis in Monoxenic Root Cultures of Corn,Tomato, and Soybean

Population dynamics, rate of root penetration, and external root feeding behavior of Pratylenchus agilis (Pa) in monoxenic cultures of intact corn seedlings and root explants of corn, tomato, and soybean were studied. In descending order of suitability as hosts were I. O. Chief corn, Rutgers tomato, and Williams soybean. Soybean entries Kent, Pickett 71, PI 90763, and Essex were poor hosts. Numbers of eggs and vermiform Pa in the agar medium indicated total fecundity and host suitability. Agar, sand, or soil as support media did not appear to affect Pa root penetration, but the rate of corn root growth did. Whereas most vermiform Pa and eggs were in roots, substantial numbers appeared able to feed and complete their life cycle as ectoparasites on root epidermal cells and root hairs.     

Screening Soybean for Resistance to Heterodera glycines Ichinohe Using Monoxenic Cultures

A simple, rapid, and inexpensive method for evaluation of host-parasite interactions, based on monoxenic cultures, is described. Axenic root explants of Glycine max (L.) Merr., cultured on a holidic agar medium, were inoculated with axenic second-stage larvae of Heterodera glycines Ichinohe, Race 3. A clear separation of susceptible and resistant cultivars, based on numbers of mature female nematodes present after 3 wk at 25 C, was observed. The method described should aid researchers in the evaluation of the host response to infection by H. glycines.     

Description of Males of Heterodera zeae

The male ofHeterodera zeae, the corn cyst nematode, is described and illustrated for the first time. Specimens were obtained from a culture originating from cysts collected in Kent County, Maryland, at the site of the first known infestation of H. zeae in the United States.     

Factors Influencing Emergence of Juveniles from Cysts of Heterodera zeae

Several factors were studied to determine their effects on hatch and emergence of second-stage juveniles (J2) from cysts of Heterodera zeae. The optimum temperature for emergence of J2 from cysts of H. zeae was 30 C. No juveniles emerged from cysts at 10 or 40 C. Immersion of cysts in 4 mM zinc chloride solution stimulated 10% greater emergence of J2 than occurred in tap water controls during 28 days. Fresh corn rhizosphere leachates from 25-day and older plants growing in sand or sandy field soil stimulated 22-24% greater emergence of J2 from cysts than occurred in tap water after 28 days. Rhizosphere leachates stored for 30 days at 4 C and leachates of sand, sandy field soil, and silty field soil inhibited emergence of J2 from cysts by 7-12% compared to tap water. Rhizosphere leachates from corn plants aged 20, 30, 40, 50, or 60 days growing in sandy field soil stimulated emergence of J2 from cysts. Similar numbers of J2 emerged from cysts regardless of whether the source of cysts was field microplot cultures, greenhouse cultures, or growth chamber cultures. Fertilizing growth chamber cultures of H. zeae on corn plants resulted in a doubling of the numbers of cysts produced in the cultures, and those cysts yielded 2-3 times as many emerged J2 in hatching tests compared to cysts from similar unfertilized cultures.     

Characterization of Heterodera zeae Populations from Portugal

Three populations of the corn cyst nematode Heterodera zeae, one found in the rhizosphere of a fig tree and two infecting corn, were studied using the morphology and morphometry of cysts and second-stage juveniles, and compared with other populations. The intrapopulation and intraspecific variability are discussed. A simple and improved technique to prepare vulval cones for SEM is described. The non-specific esterase patterns of females, isolated from infected corn, were analyzed by electrophoresis in polyacrylamide gels. Two bands of esterase activity were detected. The occurrence of H. zeae is reported for the first time in Portugal and Europe.     

Temperature and the Life Cycle of Heterodera zeae

Development of the corn cyst nematode, Heterodera zeae, was studied in growth chambers at 20, 25, 29, 33, and 36 ± 1 C on Zea mays cv. Pioneer 3184. The optimum temperature for reproduction appeared to be 33 C, at which the life cycle, from second-stage juvenile (J2) to J2, was completed in 15-18 days; at 36 C, 19-20 days were required. Juveniles emerged from eggs within 28 days at 29 C and after 42 days at 25 C. Although J2 were present within eggs after 63 days at 20 C, emergence was not observed up to 99 days after inoculation. Female nematodes produced fewer eggs at 20 C than at higher temperatures.     

Cell cycle parameters and accumulation of metaphase cells in maize suspension cultures

Cell cycle parameters of maize (Zea maysL cv Black Mexican Sweet) suspension cultures and root meristem cells were determined by pulse labelling with [³H]thymidine ([³H]TdR). Total cell cycle time for the suspension cultures was 27 h; 3 h in G1, 14 h in S, 6 h in G2, 2.2 h in prophase, 1 h in metaphase, 0.1 h in anaphase, and 0.7 h in telophase. Cell cycle durations in root meristem cells of Black Mexican Sweet (BMS) corn with and without B chromosomes in vivo were 20.0 h and 18.3h, respectively. Chemical and physical methods were used successfully to accumulate mitoses in the suspension cultures; compared to the untreated control, the mitotic index of the treated cultures was increased from 4 to 23% and the frequency of metaphase cells increased dramatically from 3 to 19%.     

Identification of Cyst Nematodes of Agronomic and Regulatory Concern with PCR-RFLP of ITS1

The first internally transcribed spacer region (ITS1) from cyst nematode species (Heteroderidae) was compared by nucleotide sequencing and PCR-RFLP. European, Asian, and North American isolates of five heterodefid species were examined to assess intraspecific variation. PCR-RFLP patterns of amplified ITS1 DNA from pea cyst nematode, Heterodera goettingiana, from Northern Ireland were identical with patterns from Washington State. Sequencing demonstrated that ITS1 heterogeneity existed within individuals and between isolates, but did not result in different restriction patterns. Three Indian and two U.S. isolates of the corn cyst nematode, Heterodera zeae, were compared. Sequencing detected variation among ITS1 clones from the same individual, between individuals, and between isolates. PCR-RFLP detected several restriction site differences between Indian and U.S. isolates. The basis for the restriction site differences between isolates from India and the U.S. appeared to be the result of additional, variant ITS1 regions amplified from the U.S. isolates, which were not found in the three India isolates. PCR-RFLP from individuals of the U.S. isolates created a composite pattern derived from several ITS1 types. A second primer set was specifically designed to permit discrimination between soybean (H. glycines) and sugar beet (H. schachtii) cyst nematodes. Fok I digestion of amplified product from soybean cyst nematode isolates displayed a uniform pattern, readily discernible from the pattern of sugar beet and clover cyst nematode (H. trifolii).     

Effects and Absorption of Sethoxydim in Cell Cycle Progression of Corn (Zea mays) and Pea (Pisum sativum)

The mechanisms of selective herbicidal action of sethoxydim were investigated by using cultured root tips of corn (Zea mays L. cv Goldencrossbantam) and pea (Pisum sativum L. cv Alaska). Meristematic cells in the cultured roots were arrested in G₁ and G₂ of the cell division cycle by sucrose starvation and resumed growth and cell division (proliferation) when sucrose was provided. Corn root growth after sucrose addition was inhibited by sethoxydim at concentrations of 0.01 micromolar and greater when roots were treated in the presence of sucrose but was not inhibited at 10 micromolar sethoxydim when they were treated during sucrose starvation. Greater absorption of [¹⁴C]sethoxydim into the meristematic region of corn roots was observed when cells were in proliferative condition but not when they were arrested by sucrose starvation, whereas no greater absorption of the herbicide into pea meristems was observed in either growth condition. In the cell cycle study, greater absorption of [¹⁴C]sethoxydim into the corn root meristem was observed at a certain limited time before S (DNA synthesis) stage. The physiological effects and the greater absorption of sethoxydim clearly depended on cell cycle progression of corn root meristem, whereas fatty acid synthesis, as well as its inhibition by sethoxydim, was not associated with either cell cycle progression or greater absorption of the herbicide.     

Influence of Organic Pesticides on Nematode and Corn Earworm Damage and on Yield of Sweet Corn

Soil fumigants and nonvolatile pesticides increased growth and yield of sweet corn ''Seneca Chief'' over that of control plants in a 3-year study. Nematicide treatments increased average yields by 31% over controls, but did not significantly affect the mean weight per ear. Increase in yield was related to control of Belonolaimus longicaudatus, Trichodorus christiei and Pratylenchus zeae. Nonvolatile chemicals more effectively reduced populations of B. longicaudatus and T. christiei than did soil fumigants. Aldicarb did not control Criconemoides ornatus. All pesticides controlled P. zeae. Pesticides did not control Heliothis zea effectively.     

Studies on the Host Range,Biology, and Pathogenicity of Punctodera punctata Infecting Turfgrasses

Punctodera punctata completed its life cycle on Poa annua (annual bluegrass), P. pratensis (Merion Kentucky bluegrass), Lolium perenne (perennial ryegrass), and Festuca rubra rubra (spreading fescue). Minimum time for completion of a life cycle from second-stage juvenile to mature brown cyst was 40 days at 22-28 C. Inoculation by single juveniles indicated that reproduction was most likely by amphimixis. Infestation levels of 50 or 500 juveniles/250 cm³ soil did not affect top dry weight, root dry weight, or total dry weight of Poa annua.     

Tests for Transmission of Prunus Necrotic Ringspot and Two Nepoviruses by Criconemella xenoplax

In two of three trials, detectable color reactions in ELISA for Prunus necrotic ringspot virus (PNRSV) were observed for Criconemella xenoplax handpicked from the root zone of infected peach trees. Criconemella xenoplax (500/pot) handpicked from root zones of peach trees infected with PNRSV failed to transmit the virus to cucumber or peach seedlings. The nematode also failed to transmit tomato ringspot (TomRSV) or tobacco ringspot viruses between cucumbers, although Xiphinema americanum transmitted TomRSV under the same conditions. Plants of peach, cucumber, Chenopodium quinoa, and Catharanthus roseus were not infected by PNRSV when grown in soil containing C. xenoplax collected from root zones of PNRSV-infected trees. Shirofugen cherry scions budded on Mazzard cherry seedling rootstocks remained symptomless when transplanted into root zones of PNRSV-infected trees. Virus transmission was not detected by ELISA when C. xenoplax individuals were observed to feed on cucumber root explants that were infected with PNRSV and subsequently fed on roots of Prunus besseyi in agar cultures. Even if virus transmission by C. xenoplax occurs via contamination rather than by a specific mechanism, it must be rare.