Morphology of Females and Cysts of Globodera tabacum tabacum,G. t. virginiae,and G. t. solanacearum (Nemata: Heteroderinae)

Detailed morphological comparisons with light and scanning electron microscopy were made of white females and cysts of several isolates of Globodera tabacum sspp. tabacum (GTT), virginiae (GTV), and solanacearum (GTS). Observations focused on body shape, anterior region including head shape, lip pattern, stylet morphology, and the terminal area in females; and body shape and terminal area of cysts. The most useful characters to separate the three subspecies were forms of the female body, cyst, stylet knobs, tail region, perineal tubercles, anal-fenestral ridge patterns, and the distinctiveness of the anus. GTT is characterized by having round females and cysts, sharply back sloped stylet knobs, clumped perineal tubercles in the vulval region, tight parallel ridges in the cyst anal-fenestral region, and a uniformly conoid tail region. GTV is characterized by its ovoid to ellipsoid female and cyst shape, the "Dutch shoe" shape of the dorsal stylet knob, the more dispersed perineal tubercles, a maze-like pattern of ridges in the anal-fenestral region, and an indistinct anus. GTS is characterized by its ovoid to ellipsoid female and cyst shape, moderately backward sloped stylet knobs, more widely separated ridges, a distinct anus, and a usually crescent shaped tail region. Much variability in shape and patterns is visible among all the isolates of the different subspecies. Tubercles in the neck, as well as bullae, are reported, and their taxonomic value is discussed.     

Morphology of Second-stage Juveniles and Males of Globodera tabacum tabacum,G. t. virginiae,and G. t. solanacearum (Nemata: Heteroderinae)

Morphological comparisons with light microscopy and scanning electron microscopy were made among second-stage juveniles (J2) and males of several isolates of the three subspecies of the tobacco cyst nematode complex, Globodera tabacum sspp. tabacum, virginiae, and solanacearum. Observations focused on the anterior region, (including head shape, lip pattern, and stylet morphology) and the tail region (including tail shape in J2 and spicules in males). The three subspecies could not be separated on the basis of any of these characters.     

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.     

Glomus fasciculatum,a Weak Pathogen of Heterodera glycines

The occurrence ofchlamydospores of Glomus fasciculatum (Gf) within cysts of the soybean cyst nematode, Heterodera glycines, and the effects of vesicular-arbuscular mycorrhizae on nematode population dynamics and soybean (Glycine max) plant growth were investigated. Chlamydospores occupied 1-24% of cysts recovered from field soil samples. Hyphae of Missouri isolate Gfl penetrated the female nematode cuticle shortly after she ruptured the root epidermis. Convoluted hyphae filled infected eggs, and sporogenesis occurred within infected eggs. G. microcarpum, G. mosseae, and two isolates of Gf were inoculated with H. glycines on plants of ''Essex'' soybeans. Each of the two Gf isolates infected about 1% of the nematode eggs in experimental pot cuhures. The Gfl isolate decreased the number of first-generation adult females 26%, compared with the nonmycorrhizal control. The total numbers of first-generation plus second-generation adult females were similar for both Gf isolates and 29-41% greater than the nonmycorrhizal control. Soybean plants with Gf and H. glycines produced more biomass than did nonmycorrhizal plants with nematodes, but only Gfl delayed leaf senescence.     

Hatch and Reproduction of Globodera tabacum tabacum in Response to Tobacco,Tomato, or Black Nightshade

The effects of broadleaf tobacco, tomato, and black nightshade on juvenile hatch and reproduction of Globodera tabacum tabacum were determined in laboratory and greenhouse experiments. Root exudates from nightshade stimulated greater egg hatch than those from either ''Rutgers'' tomato or ''86-4'' tobacco. Hatch was greater at higher proportions of root exudates for all three plant species. Root exudates from plants greater than 3 weeks old stimulated more hatch than younger plants. No regression relationships existed between plant age and nematode batch. In other experiments, hatch from eggs in cysts was higher for tomato and nightshade after 10 weeks in greenhouse pots compared to tobacco and bare soil. Numbers of second-stage juveniles in eggs in cysts produced from a previous generation on the same host were highest on nightshade and less on tomato and tobacco. Cysts of variable age recovered from field soil had increased hatch in both root exudates or water compared to recently produced cysts from plants in growth chambers. Globodera t. tabacum may be subject to both host and environmentally mediated diapause.     

Effects of Inoculum Density and Egg Age on Establishment of Globodera rostochiensis Populations

The establishment of Globodera rostochiensis Rol populations was examined under greenhouse conditions. The probability of G. rostochiensis population establishment was calculated from the number of plants that produced new cysts with viable eggs following inoculation with various numbers of eggs of different ages. Probability of population establishment was positively correlated with inoculum density but was not affected by the age of eggs used in these experiments. The probability of G. rostochiensis establishment ranged from 5% at densities of 2 eggs/pot to 100% at densities of 25 eggs/pot or greater. At densities of 3 eggs/pot and beyond, there was no correlation between inoculum density and the number of viable eggs/new cyst. Also, the number of plants that produced new cysts was a function of inoculum density and not age of eggs. Juveniles from eggs 1 year old or older were equally as infective as were those from eggs in newly developed cysts (4 months old).     

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.     

Life Cycle and Mating Behavior of Belonolaimus longicaudatus in Gnotobiotic Culture

The life cycle of Belonolaimus longicaudatus was observed in vitro on excised roots of Zea mays. Roots were cultured on Gamborg''s B5 medium in petri dishes with 1.5% agar adjusted to pH 5.8 and incubated at 28 °C in darkness. Second-stage juveniles (J2) fed on the roots and started the second molt (M2) to the third-stage juveniles 2 days after inoculation (DAI). The third molt (M3) to the fourth-stage juveniles occurred 7 DAI, followed by the fourth molt (M4) to males 13 DAI or to females 14 DAI. Nematode gender differences were observed by the end of the fourth molt. The first male appeared 15 DAI and the first female 17 DAI, after which mating occurred. Males were attracted to females, and mating was observed. Mating was required for reproduction. Fertilized females began to lay eggs 19 DAI and continued egg laying without the further presence of males during a 90-day observation. All of the eggs hatched. Unfertilized females rarely laid eggs, and none of the eggs were able to hatch. Feeding took place between each molt and before egg deposition occurred. The first-stage juveniles molted in the eggs 4 days after deposition, and J2 hatched from eggs 5 days after egg deposition. The life cycle from J2 to J2 was completed in 24 days.     

Genetics of Burley and Flue-Cured Tobacco Resistance to Globodera tabacum tabacum

Genotypes of burley (cultivars B-21 and B-49), flue-cured (line VA-81 and cultivar PD-4), and Connecticut broadleaf (cultivar C9) tobacco (Nicotiana tabacum) resistant (R) or susceptible (S) to the tobacco cyst nematode Globodera tabacum tabacum were crossed. F1 progeny of burley and susceptible broadleaf were selfed and backcrossed to produce additional progeny for evaluation of resistance in greenhouse experiments. Plants without adult female nematodes visible (×10 magnification) on the root surface 6 weeks after inoculation were classified as resistant, whereas those plants in which one or more females were evident were classified as susceptible. Segregation ratios for progeny of resistant and susceptible plants were not different from 3:1 and 1:1 for F2 (F1 × F1) and BC1 (F1 × S) lines, respectively, indicating that resistance in burley to G. t. tabacum is conferred by a single, dominant gene. Segregation ratios for resistance in crosses between nematode-resistant burley and flue-cured tobacco (F1 and F2 progeny) and between burley-flue-cured hybrids and broadleaf BC1 (F1 × S) and BC2 (BC1 × S) progeny were consistent with the assumption that resistance to G. t. tabacum in burley and flue-cured tobacco is conferred by the same or closely linked single, dominant gene(s).     

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.     

Shade Tobacco Yield Loss and Globodera tabacum tabacum Population Changes in Relation to Initial Nematode Density

Field microplot experiments were conducted from 1987 to 1992 to determine the relationship between fresh weight leaf yield of shade tobacco (Nicotiana tabacum) and initial density of Globodera tabacum tabacum (encysted J2 per cm³ soil). Initial nematode densities of 0.1 to 1,097 J2/cm³ soil were negatively correlated with leaf yield, total shoot weight, and normalized plant height 5 to 6 weeks after transplanting (r = -0.73, -0.73, and -0.52, respectively). Nonlinear damage functions were used to relate initial G. t. tabacum densities to the yield and shoot weight data. The model described leaf yield losses of < 5 % for initial nematode densities of less than 100 J2/cm³ soil. Densities above 100 J2 resulted in yields decreasing exponentially to a maximum yield loss of >40% at 500 to 1,000 J2/cm³ soil. A similar initial density tolerance threshold relationship was observed for total shoot weight. No threshold effect was evident for standardized plant height, which was a poor predictor of leaf yield. Globodera tabacum tabacum population increase over a growing season was described by a linear relation on a log/log plot (R² = 0.73).     

Description of Globodera ellingtonae n. sp. (Nematoda: Heteroderidae) from Oregon

A new species of cyst nematode, Globodera ellingtonae, is described from soil collected from a field in Oregon. Second-stage juveniles (J2) of the species are characterized by body length of 365-515 μm, stylet length of 19-22.5 μm, basal knobs rounded posteriorly and pointed anteriorly, tail 39-55 μm, hyaline tail terminus 20-32.5 μm, and tail tapering uniformly but abruptly narrowing and constricted near the posterior third of the hyaline portion, ending with a peg-like, finely rounded to pointed terminus. Cysts are spherical to sub-spherical, dark to light brown and circumfenestrate and cyst wall pattern is ridge-like with heavy punctations. Males have a stylet length of 21-25 μm and spicule length of 30-37 μm with a pointed thorn-like tip. Females have a stylet length of 20-22.5 μm, one head annule and labial disc, heavy punctations on the cuticle, and short vulval slit 7.5-8 μm long. Morphologically this new, round-cyst species differs from the related species G. pallida, G. rostochiensis, G. tabacum complex and G. mexicana by its distinctive J2 tail, and by one or another of the following: shorter mean stylet length in J2, females and males; number of refractive bodies in the hyaline tail terminus of J2; cyst morphology including Granek’s ratio; number of cuticular ridges between the anus and vulva; and in the shape and length of spicules in males. Its relationship to these closely related species are discussed. Based upon analysis of ribosomal internal transcribed spacer (ITS) sequences, G. ellingtonae n. sp. is distinct from G. pallida, G. rostochiensis, G. tabacum and G. mexicana. Bayesian and Maximum Parsimony analysis of cloned ITS rRNA gene sequences indicated three clades, with intraspecific variability as high as 2.8%. In silico analysis revealed ITS restriction fragment length polymorphisms for enzymes Bsh 1236I, Hinf I, and Rsa I that overlap patterns for other Globodera species.     

Morphological and Molecular Characterization of Meloidogyne mayaguensis Isolates from Florida

The discovery of Meloidogyne mayaguensis is confirmed in Florida; this is the first report for the continental United States. Meloidogyne mayaguensis is a virulent species that can reproduce on host cultivars bred for nematode resistance. The perineal patterns of M. mayaguensis isolates from Florida show morphological variability and often are similar to M. incognita. Useful morphological characters for the separation of M. mayaguensis from M. incognita from Florida are the male stylet length values (smaller for M. mayaguensis than M. incognita) and J2 tail length values (greater for M. mayaguensis than M. incognita). Meloidogyne mayaguensis values for these characters overlap with those of M. arenaria and M. javanica from Florida. Enzyme analyses of Florida M. mayaguensis isolates show two major bands (VS1-S1 phenotype) of esterase activity, and one strong malate dehydrogenase band (Rm 1.4) plus two additional weak bands that migrated close together. Their detection requires larger amounts of homogenates from several females. Amplification of two separate regions of mitochondrial DNA resulted in products of a unique size. PCR primers embedded in the COII and 16S genes produced a product size of 705 bp, and amplification of the 63-bp repeat region resulted in a single product of 322 bp. Nucleotide sequence comparison of these mitochondrial products together with sequence from 18S rDNA and ITS1 from the nuclear genome were nearly identical with the corresponding regions from a M. mayaguensis isolate from Mayaguez, Puerto Rico, the type locality of the species. Meloidogyne mayaguensis reproduced on cotton, pepper, tobacco, and watermelon but not on peanut. Preliminary results indicate the M. mayaguensis isolates from Florida can reproduce on tomato containing the Mi gene. Molecular techniques for the identification of M. mayaguensis will be particularly useful in cases of M. mayaguensis populations mixed with M. arenaria, M. incognita, and M. javanica, which are the most economically important root-knot nematode species in Florida, and especially when low (<25) numbers of specimens of these species are recovered from the soil.     

Broadleaf Tobacco Yield Loss in Relation to Initial Globodera tabacum tabacum Population Density

Field microplot experiments were conducted from 1995 to 1998 to determine the relationship between fresh shoot weight of stalk-cut broadleaf and shade-grown cigar wrapper tobacco types (Nicotiana tabacum L.) and initial density of Globodera tabacum tabacum second stage juveniles (J2) per cm³ soil. Total shoot weight was negatively correlated with initial nematode densities of 12.3 to 747.3 J2/cm³ soil (r = -0.53 and -0.70 for broadleaf and shade-grown tobacco, respectively). Nonlinear damage functions were used to relate initial G. t. tabacum densities to shoot weight. The models described shoot weight losses of less than 14% or 39% for broadleaf and shade tobacco, respectively, at G. t. tabacum densities below 50 J2/cm³ soil. Total shoot weights were reduced by 40% and 60% of uninfested plots as preplant nematode densities approached maximum levels (>600 J2/cm³ soil) for broadleaf and shade tobacco, respectively. Globodera t. tabacum population increase over a growing season was described by a linear relation on a log/log plot (R² = 0.07 and 0.61 for broadleaf and shade, respectively). These experiments demonstrate that G. t. tabacum can directly reduce shoot weight of stalk-cut broadleaf tobacco. Broadleaf is more tolerant to nematode infection than shade tobacco, as shade tobacco shoot weight reductions were greater at the same initial nematode densities in the same years.     

Dormancy of Heterodera glycines in Missouri

A 2-year study was conducted in field microplots to determine the relative importance of soybean phenology and soil temperature on induction of dormancy in Heterodera glycines in Missouri. Four near-isogenic soybean lines differing for maturity date were planted in microplots infested with a race 5 isolate of H. glycines. Soil temperature was monitored at a depth of 15 cm. Eggs of H. glycines, extracted from cysts collected monthly from each microplot, were used in hatching tests and bioassays to determine dormancy. Egg hatching and second-stage juvenile (J2) infectivity rates decreased sharply from their highest levels in midsummer (July-August) to a low level by October of each year and remained low (< 10% hatching and < 0.2 J2/cm root) until May or June of the following year. The patterns of numbers of females and eggs in the bioassays were similar. The decreases were not related to soil temperature and did not differ consistently among soybean isolines. The monophasic changes in all nematode responses with peak midsummer rates suggest that H. glycines produces one primary generation per year in central Missouri. Changes in hatching rates and the timing of minimum and maximum rates suggested that H. glycines eggs exhibit more than one type of dormancy.     

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.     

Wheat Germ Agglutinin Bound to the Outer Cuticle of the Seed Gall Nematodes Anguina agrostis and A. tritici

The presence of wheat germ agglutinin (WGA) on the cuticular surface of the seed gall nematodes Anguina agrostis and Anguina tritici was demonstrated, and the nature of its binding was examined. Crude extracts from the cuticles of A. tritici agglutinated human red blood cells, and only N-acetylglucosamine (GlucNAc) inhibited the agglutination. Distribution of the lectin was visualized by treating live infective juveniles (J2) with rabbit anti-WGA antibody and staining with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG. The lectin bound to the outer cuticular surface of the whole body wall. Pretreatment with GlucNAc oligomers did not reduce the fluorescence created by the anti-WGA-WGA binding, indicating at least a partial nonspeciflc adhesion of the WGA to the nematode surface. Proteolytic enzyme pretreatments diminished the fluorescence, whereas lipase and periodate pretreatments increased the fluorescence. Adult females and males were labeled only on the head and tail, whereas eggs were not labeled at all. It was concluded that the WGA on the J2 cuticle originates from the host.     

Ribosomal DNA Comparisons of Globodera from Two Continents

Ribosomal DNA (rDNA) sequence data were compared for five species of Globodera, including G. rostochiensis, G. pallida, G. virginiae, and two undescribed Globodera isolates from Mexico collected from weed species and maintained on Solanum dulcamara. The rDNA comparisons included both internal transcribed spacers (ITS1 and ITS2), the 5.8S rRNA gene, and small portions of the 3'' end of the 18S gene and the 5'' end of the 28S gene. Phylogenetic analysis of the rDNA sequence data indicated that the two potato cyst nematodes, G. pallida and especially G. rostochiensis, are closely related to the Mexican isolates, whereas G. virginiae is relatively dissimilar to the others and more distantly related. The data are consistent with the thesis that Mexico is the center of origin for the potato cyst nematodes.     

Effects of Initial Nematode Density on Population Dynamics of Globodera rostochiensis on Resistant and Susceptible Potatoes

The influence of resistant and susceptible potato cultivars on Globodera rostochiensis population density changes was studied at different nematode inoculum levels (Pi) in the greenhouse and field. Soil in which one susceptible and two resistant cultivars were grown and fallow soil in pots was infested with cysts to result in densities of 0.04-75 eggs/cm³ soil. A resistant cultivar was grown in an infested field with Pi of 0.7-16.7 eggs/cm³ soil. Pi was positively correlated with decline of soil population densities due to hatch where resistant potatoes were grown in the greenhouse and in the field but not in fallow soil. However, Pi was not correlated with in vitro hatch of G. rostochiensis cysts in water or potato root diffusate. Under continuous culture o f a resistant cultivar, viable eggs per cyst declined 60-90% per plant growth cycle (4 weeks) and the number of cysts containing viable eggs had decreased by 77% after five cycles. The rate of G. rostochiensis reproduction on both resistant and susceptible cultivars was negatively correlated with Pi. These data were used to predict the effect of resistant and susceptible potato cultivars on G. rostochiensis soil population dynamics.