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.     

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.     

Survival of Heterodera zeae in Soil in the Field and in the Laboratory

Eggs and (or) second-stage juveniles (J2) inside cysts of Heterodera zeae survived over winter in the field with no detectable mortality at all six depths to 30 cm from which soil samples were collected between corn stubble in the row at 4-8-week intervals. Few or no free J2 were recovered from soil collected in January-April from the top 5 cm, but some were recovered at all samplings from soil collected at greater depths. Emergence of J2 from cysts and numbers of females developing on corn roots in bioassays of cysts increased substantially between January and April. Cyst numbers in a fallow area of the corn field did not decline at any depth to 30 cm during 20 months. Free soil J2, J2 emerged from cysts, and females from the bioassay of cysts were highest at the first soil sampling in July after 10 months of fallow; numbers of nematodes in all three categories declined thereafter, but a few were still detectable after 20 months of fallow. Some cysts were still being recovered after 51 months from naturally infested field soil stored moist in the laboratory at 2 C and 24 C. Females were produced in the bioassays of cysts recovered from soil stored for 38 months at 24 C and for 32 months at 2 C. No free J2 were recovered from soil after 1 month of storage at -18 C, but even after 7 months storage J2 emerged from cysts that were recovered and many females developed in bioassays of those cysts.     

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).     

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.     

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.     

Genetic Analysis of Esterase Polymorphism in the Soybean Cyst Nematode, Heterodera glycines

The genetic basis of esterase polymorphism in Heterodera glycines was investigated through controlled matings and analysis of F₁ and F₂ progeny. Three nematode lines, each fixed for a different esterase phenotype, were isolated and purified through repeated directional selection and inbreeding. Each phenotype was characterized by its distinct pair of closely spaced bands of esterase activity. Single-female single-male crosses were conducted according to a modified agar-plate mating technique. F₁ progeny were homogeneous, exhibiting both parental esterase phenotypes (codominant heterozygotes) but no hybrid bands. Approximately 1,500 F₂ progeny segregated in a 1:2:1 ratio for expression of the esterase phenotypes of the female parental line, the heterozygote, and the male parental line. Apparently the three esterase phenotypes correspond to three codominant alleles of a single esterase locus. Reciprocal crosses gave similar results, suggesting no maternal inheritance.     

Nonhost Root Penetration by Soybean Cyst Nematode

A total of 66 plants in 50 species were inoculated with eggs and juveniles of soybean cyst nematode, Heterodera glycines. Roots were stained and observed for penetration and development of the nematode. Twenty-six plants were not penetrated; twenty-three were penetrated, but there was no development of the nematode; eight were penetrated with some nematode development; two were penetrated and had considerable nematode development, but few nematodes, if any, matured; and seven were penetrated with many nematodes maturing. The penetration of nonhosts may imply some susceptibility and that populations eventually would build up on the penetrated plants. Plants not penetrated may be useful as rotation plants because no reproduction would occur.     

Cereal Cyst Nematode (Heterodera avenae) on Oats. II. Early Root Development and Nematode Tolerance

The effect of Heterodera avenae infestation on early seminal and lateral root growth was examined in four oat genotypes differing in tolerance to H. avenae. Recently emerged seminal roots were inoculated with a range of H. avenae larval densities, then transferred a hydroponic system to remove the effect of later nematode penetration on root development. Intolerance to H. avenae was assessed in terms of impairment of seminal root extension resulting in fewer primary lateral roots emerging from the seminal root below the zone of juvenile penetration. Tolerant plants infested with H. avenae had longer lateral root systems than infested intolerant plants. The decline in lateral root growth below the penetration zone was partly offset by increased growth above. This did not contribute to tolerance, however, as there were no differences between cultivars for this feature. Nematodes induced earlier nodal root emergence in all cultivars. Nodal root development was most advanced on the most tolerant cultivar.     

Attempt to Select a Cyst Nematode Population on Soybean Plant Introduction 437654

A method of selecting soybean cyst nematode (Heterodera glycines Ichinohe) on segregating soybean progeny was evaluated for developing a population capable of reproducing on PI 437654. Direct selection on PI 437654 was not possible, since no cysts developed on it. Cysts were selected for 12 nematode generations on F₃ and F₄ plants of Forrest x PI 437654. No cysts of the selected population were produced on PI 437654, but more males were produced on it by the selected population than by the base population. The number of cysts on Forrest and other soybean lines considered to have some of the same genes for resistance increased with selection as expected. The increase in number of males on these other lines with some of the same genes for resistance as Forrest was greater than anticipated, indicating that these lines may have some of the same genes as PI 437654.     

Redescription of Heterodera fici (Nematoda: Heteroderidae) with SEM Observations

Heterodera fici is redescribed and illustrated with comparative details and revised measurements and diagnostic characters of the females, males, cysts, and juveniles from Maryland and Pakistan. This species is in the "schachtii group" (cysts lemon shaped, with bullae, and ambifenestrate) but the fenestrae in some cysts, presumab!y young ones, are small and widely spaced, appearing bifenestrate. It is most closely related to H. schachtii, H. glycines, and H. cajani but differs from these species especially in having cysts with small, scattered bullae and weakly developed underbridge; and males with four small nipples on tail tip. Scanning electron microscopy (SEM) observations of the specimens are also presented. The relationship of this species to closely related forms is discussed.     

Description and SEM Observations of a New Species of Cyst Nematode Heterodera goldeni (Nematoda: Heteroderidae) Attacking Panicum coloratum in Egypt

A cyst nematode, Heterodera goldeni n. sp., is photographed and described from Qasabagrass roots (Panicum coloratum L.) in Alexandria, Egypt. It is characterized in having second-stage juveniles with body length of 546 µm (450-612), stylet length of 22.6 µm (22-23.5) with anchor-shaped knobs, lateral field with 3 lines, tail 60-75 µm, hyaline tail terminus 38.4 µm (33-43); cysts are lemon-shaped, dark to light brown with an extensive sub-crystalline layer covering the entire cyst, cuticular midbody pattern zig-zag, cysts ambifenestrate, well-developed underbridge with finger-like projections, bullae present, vulva slit measuring 44-48 µm long. Males are absent, and females have heavy punctations on the cuticle. Its relationship to H. graminophila described from Florida and Louisiana and H. leuceilyma described from Florida are discussed. The present known distribution is restricted to Alexandria, Egypt. Its economic importance in rangeland grasses and cultivated crops such as rice is not known.     

Field Efficacy of Verticillium lecanii,Sex Pheromone,and Pheromone Analogs as Potential Management Agents for Soybean Cyst Nematode

A soybean cyst nematode sex pheromone (vanillic acid), chemical analogs of the pheromone, and the fungus Verticillium lecanii were applied in alginate prills (340 kg/ha) to microplots and small-scale field plots as potential management agents for Heterodera glycines on soybean. In 1991 microplot tests, treatment with V. lecanii, vanillic acid, syringic acid plus V. lecanii, or vanillic acid plus V. lecanii lowered midseason cyst numbers compared with the untreated susceptible cultivar control, autoclaved V. lecanii treatment, or aldicarb treatment, At-harvest cyst numbers were lowest with V. lecanii and with vanillic acid treatments. Aldicarb treatment reduced midseason cyst numbers in 1992. There were no differences among seed yields either year. In the field trials, numbers of cysts were reduced one or both years with aldicarb, ferulic acid, syringic acid, vanillic acid, or 4-hydroxy-3-methoxybenzonitfile treatments, or with a resistant cultivar, compared to an untreated susceptible cultivar. Highest yields were recorded after treatment with 4-hydroxy-3-methoxybenzonitrile (1991), methyl vanillate (1992), and aldicarb (1992). These studies indicate that some chemical analogs of vanillic acid have potential for use in soybean cyst nematode management schemes.     

Suppression of Cyst Nematode by Natural Infestation of a Nematophagous Fungus

Penetration of cabbage roots by Heterodera schachtii was suppressed 50-77% in loamy sand naturally infested with the nematophagous fungus Hirsutella rhossiliensis. When Heterodera schachtii was incubated in the suppressive soil without plants for 2 days, 40-63% of the juveniles had Hirsutella rhossiliensis spores adhering to their cuticles. Of those with spores, 82-92% were infected. Infected nematodes were killed and filled with hyphae within 2-3 days. Addition of KCl to soil did not increase infection of Heterodera schachtii by Hirsutella rhossiliensis. The percentage of infection was lower when nematodes were touched to two spores and incubated in KCl solution than when nematodes naturally acquired two spores in soil.     

Demonstration of Multiple Mating in Heterodera glycines with Biochemical Markers

Controlled crosses of Heterodera glycines were carried out by placing one o r more virgin females of known esterase phenotype on an agar plate and adding, at various time intervals, one or more males of different esterase phenotypes. Progeny (second-stage juveniles) of such crosses were propagated on soybeans, and 30 days later young females were subjected to electrophoretic analysis to determine their esterase phenotype. Esterase phenotypes that represented the heterozygous state of the maternal and paternal genomes confirmed the hybrid nature of the progeny and identified their male parent. When each of 74 females was given the opportunity to mate successively with two males of different esterase phenotypes, 43 mated with a single male and 31 mated with both males. One female mated with three males, i.e., with a male of its own population (sib mating) and the two males provided for the cross. Inseminated females could mate for a second time soon after, or as late as 24 hours after, their first mating. When single males were given the opportunity to mate with many females, about equal numbers of them inseminated zero, one, two, or three females. In greenhouse tests, 12 females were given the opportunity to mate with many males of three different esterase phenotypes. Two females mated with one and possibly more males of the same phenotype, and 10 females mated with males of two different esterase phenotypes. In conclusion, multiple mating appears to be a common behavior of males and females of H. glycines.     

DNA Restriction Fragment Length Polymorphism in Races of the Soybean Cyst Nematode,Heterodera glycines

Restriction endonuclease digests of total DNA from races 3, 4, and 5 of the soybean cyst nematode, Heterodera glycines, have been analyzed on agarose gels. DNA fragment patterns of race 4 were completely different from those patterns obtained for races 3 and 5 by all eight restriction enzymes tested. Differences in long and short restriction DNA fragments generated by the enzyme Msp I or its isoschizomer, Hpa II, were detected between race 3 and 5 digestion profiles. Rapid DNA isolation followed by its digestion with either Msp I or Hpa II enzymes and visualization of repetitive DNA fragments in agarose gels provided a diagnostic assay for the populations of the three races examined in this study.     

Effect of Initial Nematode Density on Managing Globodera rostochiensis with Resistant Cultivars and Nonhosts

Cropping systems in which resistant potato cultivars were grown at different frequencies in rotation with susceptible cultivars and a nonhost (oats) were evaluated at four initial nematode population densities (Pi) for their ability to maintain Globodera rostochiensis at a target level of <0.2 egg/cm³ of soil. At a Pi of 0.1 to 1 egg/cm³ of soil, cropping systems with 2 successive years of a resistant cultivar every 3 years of potato production reduced and maintained G. rostochiensis at <0.2 egg/cm³ of soil. At a Pi of 1 to 4 eggs/cm³ of soil, 2 successive years of a resistant cultivar followed by 1 year of oats for every 4 years of production were necessary to reduce and maintain G. rostochiensis populations at <0.2 egg/cm³ of soil. At a Pi greater than 4 eggs/cm³ of soil, 2 successive years of a resistant cultivar plus 1 year of oats reduced G. rostochiensis densities to <0.2 egg/cm³ of soil, but the population increased above that density after cropping 1 year to a susceptible cultivar. The numbers of cysts and eggs per cyst in the final population (Pf) of G. rostochiensis were influenced by initial density and the frequency of growing a susceptible cultivar in a cropping system. The lowest number of cysts and eggs per cyst in the final G. rostochiensis population occurred with a cropping system consisting of 2 successive years of a resistant cultivar followed by oats with a susceptible cultivar grown the fourth year of production.     

Response of Soybean in Cyst Nematode-Infested Soils at Three Soil Water Regimes

Large pot (2 years) and field experiments (1 year) were conducted to determine the response of susceptible soybean Glycine max (L.) Merr. cultivars (Essex and Hutcheson) grown in soybean-cyst-nematode (SCN), Heterodera glycines-infested soils at three soil water regimes. The soil water regimes were irrigation whenever soil water potential ([psi]s) 0.30-m deep was i) -30 kPa (I-30) or ii) - 50 kPa (I-50), and iii) no irrigation. Cyst nematode levels in the pot experiment were either 0 or 20,000 second-stage juveniles (J2) per pot. The field experiment was conducted on soil naturally infested with a population of 145 to 475 cysts L⁻¹ of soil. All growth parameters studied were drastically affected in the presence of SCN under nonirrigated conditions for the large pot tests; however, SCN did not influence growth parameters in the field experiment. Seed yield was lowest in the no irrigation treatment when all treatments were compared in both the pot and field experiments. The infested no irrigation treatment in the pot experiment had the lowest yield among soil water treatments.     

SEM Observations on the Marine Nematode Dracognomus simplex (Gerlach, 1954) Allen and Noffsinger, 1978 (Draconematidae: Prochaetosomatinae)

The free-living marine nematode Dracognomus simplex (Gerlach, 1954) Allen &Noffsinger, 1978 was studied by scanning electron microscopy (SEM). The morphology of males and females is described and illustrated in detail. In addition to the typical and modified adhesion tubes, a new type of posterior adhesion tube was discovered. A neotype is proposed for Dracognomus simplex, and D. simplex sensu Decraemer &Gourbault, 1986 is renamed as Dracognomus americanum n. sp. Additionally, a key toward the Dracognomus species is proposed.