Histopathology and Host Range Studies of the Redwood Nematode Rhizonema sequoiae

Second-stage larvae of Rehizonma sequoiae Cid del Prado Vera et al. tunnel through the cortex of the redwood Sequoia sempervirens (D. Don) Endl. root to the vascular tissue where each developing female induces a single ovoid or occasionally spherical giant cell with a single ovoid to spherical nucleus containing one to four enlarged nucleoli. Nematode tunnels are filled with a gel material and often contain second-stage larvae and males. There is tissue necrosis around females, and cortical tissue is destroyed after infection by many second-stage larvae. R. sequoiae females developed to maturity on S. sempervirens, Acer macrophyllum Pursh, AInus rhombifolia Nutt., Libocedrus decurrens Torr, Pseudotsuga menziesii (Mirb.) Franco, and Sequoiadendron giganteum (Lindl.) Decne. In the Marin County, California, forest mature females were also found naturally infecting Lithocarpus densiflorus (Hook &Arn.) Rehd., Umbellularia californica (Hook &Arn.) Nutt., and Arbutus menziesii Pursh.     

Post-infection Development and Morphology of Meloidogyne cruciani

The development and life stages of Meloidogyne cruciani on tomato was studied at 28 C. Roots of 2-wk-old ''Rutgers'' tomato seedlings were exposed to inoculum for 24 h, rinsed, and the seedlings repotted. No major changes in juvenile development were observed prior to 8 days after inoculation. At 11 days the second-stage juvenile had enlarged considerably. The genital primordium had not yet asumed the V-shape characteristic of developing females, but the presence of rectal glands identified the juveniles as females. At this time (11 days), two additional, previously undescribed esophageal lobes were first observed; they were adjacent to the dorsal and subventral glands. After molting from second to third stage, the stylet cone, shaft, and the lumen of the stylet knobs are shed and remain attached to the second-stage cuticle. The excretory duct of the third-stage juveniles was directed anteriorly from the excretory pore of the second-stage cuticle and appear attached to the body wall of the third-stage juveniles opposite the procorpus. At 19 days after inoculation, the last molt took place. The adult female possessed a new stylet, a large five-gland esophagus, a prominent excretory system ending in a unicellular gland and a fully developed reproductive system.     

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.     

Scanning Electron Microscope Study of the Root-knot Nematode (Meloidogyne incognita) on Tomato Root

This study examines the types of structural information that can be gained by utilizing the scanning electron microscope (SEM) and a cryofracture technique to examine the host-parasite interaction. Roots of tomato, Lycopersicon esculentum cv. Marglobe, were cultured aseptically and inoculated with the root-knot nematode, Meloidogyne incognita. Twenty-four hours to four weeks after inoculation, developing galls were removed from the cultures and processed for SEM observation. The cryofracture technique was used to reveal internal structural features within the developing galls. The results illustrate structural details concerning penetration of the roots, differentiation of syncytia, and development of the nematodes beginning with the second-stage larvae and ending with adult egg-laying females.     

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.     

Post-Infection Development and Histopathology of Meloidogyne incognita in Resistant Cotton

The numbers of Meloidogyne incognita larvae which migrated from cotton roots declined over a 16-day period, but the difference in numbers migrating from resistant and susceptible cultivars was not significant. Larvae penetrated susceptible roots, matured, and reproduced within 14 days following inoculation, whereas nematode development in the resistant roots was greatly retarded. Three types of histological responses were observed in infected, resistant roots, and these correlated with the degree of nematode development. Some galls were examined which contained only fragments of nematodes; others contained no detectable traces of developing larvae. Formation of druses in galls, but not in healthy tissue, was noted in both cultivars 20 days after inoculation. Massive invasion of roots resulted in deep longitudinal fissures of root cortex.     

Histopathology of Root-knot Nematode (Meloidogyne incognita) Infection on White Yam (Dioscorea rotundata) Tubers

White yam tissues naturally and artificially infected with root-knot nematodes were fixed, sectioned, and examined with a microscope. Infective second-stage juveniles of Meloidogyne incognita penetrated and moved intercellularly within the tuber. Feeding sites were always in the ground tissue layer where the vascular tissues are distributed in the tubers. Giant cells were always associated with xylem tissue. They were thin walled with dense cytoplasm and multinucleated. The nuclei of the giant cells were only half the size of those found in roots of infected tomato plants. Normal nematode growth and development followed giant cell formation. Females deposited eggs into a gelatinous egg mass within the tuber, and a necrotic ring formed around the female after eggs had been produced. Second-stage juveniles hatched, migrated, and re-infected other areas of the tuber. No males were observed from the tuber.     

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.     

Effects of Oxamyl on the Citrus Nematode,Tylenchulus semipenetrans,and on Infection of Sweet Orange

Foliar sprays of 4 μg/ml oxamyl on sweet orange trees in a greenhouse slightly depressed the number of Tylenchulus semipenetrans larvae obtained from roots and soil, but similar treatments were not effective in two orchards. Soil drench treatments decreased the number of citrus nematode larvae obtained from roots or soil of citrus plants grown itt a greenhouse and in orchards. Exposure to 5-10 μg/ml of oxamyl in water was lethal to only a few second-stage larvae treated 10 days, and many second-stage larvae in 2.0 μg/ml oxamyl recovered motility when transferred to fresh water. Aqueous solutions of 50 and 100 μg/ml of oxamyl were toxic to citrus nematode larvae. Additional observations indicate that oxamyl interfered with hatch of citrus nematode larvae and was nematistatic and/or protected sweet orange roots from infection. Oxamyl degraded at different rates in two soils. The number of citrus nematode larvae that infected and developed on sweet orange roots was increased by an undetermined product of the degradation of oxamyl in soil, water, and possibly within plants. This product apparently was translocated in roots.     

Development of Meloidogyne chitwoodi on Wheat

Postinfection development of Meloidogyne chitwoodi from second-stage juveniles (J2) to mature females and egg deposition on ''Nugaines'' winter wheat required 105, 51, 36, and 21 days at 10, 15, 20, and 25 C. At 25 C, the J2 induced cavities and hyperplasia in the cortex and apical meristem of root tips with hypertrophy of cortical and apical meristem cell nuclei, 2 and 5 days after inoculation. Giant cells induced by late J2 were observed in the stele 10 days after inoculation. Clusters of egg-laying females were common on wheat root galls 25 days after inoculation. Juveniles penetrated wheat roots at 4 C and above, but not at 2 C, when inoculum was obtained from cultures grown at 20 C, but no penetration occurred at 4 C when inoculum was stored for 12 hours at 4 C before inoculation. In northern Utah, J2 penetrated Nugaines wheat roots in the field in mid-May, about 5 months after seedling emergence. M. chitwoodi eggs were first observed on wheat roots in mid-July when plants were in blossom. Only 40% of overwintered M. chitwoodi eggs hatched at 25 C.     

Effect of Temperature on Growth, Development and Reproduction of Meloidogyne hapla in Lettuce

Temperature was an important factor in growth, development and reproduction of Meloidogyne hapla in lettuce. Growth, as measured by increase in diameter of females, was not appreciably different at the intermediate (21.1 C night and 26.7 C day) and high (26.7 C night and 32.2 C day) temperature regimes, but was considerably less at the low temperature regime (15.5 C night and 21.1 C day) than at the two higher temperature regimes. Second-stage female larvae developed into adults 14 days after inoculation at the high, 18 days at the intermediate and 34 days at the low temperature regime. Eggs were observed 20 days after inoculation at the high, 26 days at the intermediate and 54 days at the low temperature regime. Number of eggs and larvae after 6 weeks was greater at the high than at the intermediate temperature regime and no eggs or larvae occurred at the low temperature regime during the observed 6 weeks.     

Inhibition of Syncytia Formation and Root-knot Nematode Development on Cultures of Excised Tomato Roots

Two different defined growth media were used to culture aseptically the root-knot nematode, Meloidogyne incognita, on excised roots of tomato, Lycopersicon esculentum cv ''Marglobe.'' One of these media, STW, was a formulation by Skoog, Tsui, and White and the other, MS, a formulation by Murashige and Skoog. From 1 through 4 weeks, inoculated tissues were fractured to observe root infection, giant-cell formation, and nematode development with the scanning electron microscope (SEM). Four weeks after inoculation, the fresh weights of roots and developmental stages of nematodes were recorded. SEM observations indicated that roots cultured on the STW medium had normal growth and infection sites with galls that supported the development of mature females by 4 weeks. Roots cultured on the MS medium were less vigorous and had infection sites with galls containing only one to four syncytialike cells that did not support the development of mature females. Eighty percent of the larvae infecting roots cultured on the MS medium failed to develop into mature females. To determine which factor(s) affected root growth and nematode development, inoculated and uninoculated roots were grown on media consisting of different combinations of the organic and inorganic fractions of the STW and MS formulations. These experiments indicated that the organic fraction of STW was essential for normal root growth; however, the inorganic fraction of MS inhibited normal gall formation and nematode development. Further testing of the inorganic fractions revealed that the high concentration of ammonium nitrate in the MS medium was a factor that inhibited giant-cell formation and nematode development.     

The invasion and development of the cereal cyst-nematode, Heterodera avenae in the roots of autumn-and spring-sown cereals

Observations were made at 2 or 4 wk intervals from December to harvest on all stages of Heterodera avenae in winter oats growing on infested land. Second-stage larvae were present in all soil samples except on 5 and 20 July. Invasion and development of larvae was slow during winter. The nodal and seminal roots of winter oats were both heavily invaded by the nematode; larvae which invaded seminal roots tended to become male whereas those in nodal roots tended to become female. There was a small second invasion in August. Females were first observed on the roots of winter oats on 17 May, 214 days after the crop was sown and 62 days after the first fourth-stage larva was observed. The nodal roots of spring barley contained few H. avenae larvae whereas these roots were heavily invaded in winter wheat and oats. In spring barley the nodal roots were developing in June and July when few second-stage larvae were in the soil whereas in winter oats and wheat the nodal roots were growing rapidly in April when larvae were most numerous, and so were heavily invaded.     

Infectivity of Pratylenchus penetrans on Alfalfa

The infectivity of Pratylenchus penetrans on alfalfa seedlings cv. Du Pulls was studied. The dense root-hair zone was the preferred zone of penetration by females, males, and third-stage larvae. A lesion initially appeared as a water-soaked area at the root surface, becoming yellow and elliptical as the nematode entered the cortex, with dark-brown cells later appearing in the centre as the nematode fed. At 20 C, females penetrated roots earlier, faster, and in greater numbers than either males or third-stage larvae. Females penetrated roots at temperatures from 5 to 35 C, with maximum penetration between 10 and 30 C, while males and third-stage larvae penetrated roots only between 10 and 30 C with maximum penetration a t 20 C. Penetration of roots by females, males, and third-stage larvae increased after storage of 5 C for 35 days, but decreased after storage of 140 days or more. Combinations of the three life stages in pairs neither enhanced nor inhibited penetration of roots by individual life stages; males were not attracted to females. Increasing inoculum density up to 20 nematodes/seedling did not affect penetration.     

Effect of Meloidogyne incognita on Plant Nutrient Concentration and Its Influence on the Physiology of Beans

Phaseolus vulgaris plants, 3, 8, 11, and 13 days old, were inoculated with 0, 2,000, 4,000, or 8,000 second-stage Meloidogyne incognita larvae and maintained under controlled conditions. The photosynthetic rate and the shoot and root concentration of K, Ca, Mn, Fe, Cu, and Zn were determined by destructive assay at 1-27-day intervals and by nondestructive assay of leaves, stems, and roots at 27 or 28 days after inoculation. In the destructive assay, the concentration of the elements in the plant tissues did not change until 1 week after inoculation. Thereafter, the trend was mostly decreasing for shoot K and Fe and increasing in the root, whereas Ca had the opposite trend in the shoots. Manganese, Cu, and Fe showed variable trends. Generally, the concentration of K and Mn increased, whereas Ca and Fe decreased, with duration of infection in all treatments. Zinc and Cu decreased in the highest nematode treatments. The overall elemental content generally decreased with level of infection from 1 week after inoculation. Photosynthetic rate based on shoot K concentration significantly decreased with level of infection. In most of the nondestructive assays, the concentrations of shoot K, Zn, and Mn decreased, whereas Ca increased with increasing nematode treatment. One of the first effects of the nematode on host physiology appears to be a change in concentration of nutrient elements in the host plant.     

Effects of Oxime Carbamate Nematicides on Development of Heterodera schachtii on Sugarbeet

Treatment of sugarbeet, Beta vulgaris L., with aldicarb, aldicarb sulfoxide, or aldicarb sulfone 10 days after plants were inoculated with Heterodera schachtii prevented development of the nematode, but second-stage larvae penetrated the roots. These chemicals had no measurable effects on nematodes in plants treated 15 days after inoculation. The tests established that soil treatments of aldicarb are directly or indirectly lethal to larvae developing within roots of sugarbeet. Heterodera schachtii failed to develop on root slices of red table beet grown in soil treated with aldicarb or aldicarb sulfoxide. Similar treatment of plants with aldicarb sulfone or oxamyl did not affect subsequent development of H. schachtii on root slices of treated plants.     

An ultrastructural study of the invasion of Ascaris suum larvae by neutrophils

Neutrophils are capable of penetrating the cuticle of the second-stage larvae of the nematode Ascaris suum. The integrity of the internal tissues of the larvae is then destroyed by cytoplasmic extensions of the neutrophils.     

Penetration and Development of Meloidogyne incognita on Roots of Resistant Soybean Genotypes

Meloidogyne incognita penetration and development were studied in roots of highly resistant (PI 96354, PI 417444), resistant (Forrest), and susceptible (Bossier) soybean genotypes. Although more second-stage juveniles (J2) had penetrated roots of PI 96354 and PI 417444 than roots of Forrest and Bossier by 2 days after inoculation, fewer J2 were present in roots of PI 96354 at 4 days after inoculation. Juvenile development in all genotypes was evident by 6 days after inoculation, with the highest number of swollen J2 present in roots of Bossier. At 16 days after inoculation, roots of PI 96354 had 87%, 74%, and 53% fewer J2 than were present in roots of Bossier, Forrest, and PI 417444, respectively. Differential emigration of J2, not fewer invasion sites, was responsible for the low number of nematodes in roots of the highly resistant PI 96354. Some 72% of the J2 penetrating the roots of this genotype emerged within 5 days after inoculation, whereas 4%, 54%, and 83% emerged from roots of Bossier, Forrest, and PI 417444, respectively. Penetration of roots of PI 96354 decreased the ability of J2 emerging from these roots to infect other soybean roots.     

Meloidogyne aquatilis n. sp (Nematoda:Meloidogynidae) from Spartina pectinata with a Key to the Canadian Species of Meloidogyne

A new root-knot nematode, Meloidogyne aquatilis n. sp., attacking the roots of Spartina pectinata Link growing in the Ottawa River is described and illustrated. Meloidogyne aquatilis is distinguished from M. graminis by the light brown body color and by the absence of perineal lateral fields in the female. The male differs by the shorter stylet and by the hemizonid being separated by 7-9 annules from the excretory pore. The second-stage juveniles are also recognized by the 7-9-annule gap between the hemizonid and excretory pore and by the shorter tail with a disc-like subterminal tail structure, lower b ratio value, and inflated rectum. A key to the root-knot nematode species of Canada based on females, males, and juveniles is provided. The type host of Dolichodera fluvialis Mulvey and Ebsary, 1980, Spartina pectinata Link, is reported for the first time.     

Development of Thelastoma bulhoesi (Oxyurata: Thelastomatida) and the Effect of Thiabendazole on the Unembryonated Egg

The embryological and postembryological development of Thelastoma bulhoesi was determined. Initial cleavage was into unequal cells and occurred within 1-2 hours at 25 C. Cell division was holoblastic but no true morula is formed. Gastrulation occurred at approximately 48 hours by epibolic synectic mechanisms. First-stage larvae were fully developed at 96 hours. The molt to second-stage larvae was initiated in the egg and was completed at hatching. Second-stage larvae were first observed in the host at 11 hours postinfection, third-stage larvae at 18 hours, and fourth-stage larvae at 192 hours. Adult female worms were observed at 32 days. Thiabendazole, in even the lowest concentrations, inhibited the developmem of unembryonated ova.