Glucuronidase Expression in Transgenic Tobacco Roots with a Parasponia Promoter on Infection with Meloidogyne javanica

The expression of a g-us reporter gene linked to a Parasponia andersonii hemoglobin promoter has been studied in transgenic tobacco plants after infection by Meloidogyne javanica. Transgenic roots were harvested at different times after nematode inoculation, and stained histochemically for expression of the gus gene. During the early stages of infection (0-2 weeks) there was little expression in giant cells, in contrast to other cells of the root. In later stages of infection (3-6 weeks) there was strong gus expression in giant cells, with virtually no expression in other cells of the root. The Parasponia hemoglobin promoter therefore appears to direct down-regulation of linked genes on induction of giant cells, but up-regulation in mature giant cells. This reflects different metabolic activities in the giant cells depending on their stage of development. The Parasponia hemoglobin promoter may respond to oxygen tension in giant cells. This suggests that oxygen tension may be limited in the metabolically active giant cells that are associated with egg-laying females.     

Use of Cucumis metuliferus as a Rootstock for Melon to Manage Meloidogyne incognita

Root-knot nematode-susceptible melons (Cantaloupe) were grown in pots with varying levels of Meloidogyne incognita and were compared to susceptible melons that were grafted onto Cucumis metuliferus or Cucurbita moschata rootstocks. In addition, the effect of using melons as transplants in nematode-infested soil was compared to direct seeding of melons in nematode-infested soil. There were no differences in shoot or root weight, or severity of root galling between transplanted and direct-seeded non-grafted susceptible melon in nematode-infested soil. Susceptible melon grafted on C. moschata rootstocks had lower root gall ratings and, at high nematode densities, higher shoot weights than non-grafted susceptible melons. However, final nematode levels were not lower on the grafted than on the non-grafted plants, and it was therefore concluded that grafting susceptible melon on to C. moschata rootstock made the plants tolerant, but not resistant, to the nematodes. Grafting susceptible melons on C. metuliferus rootstocks also reduced levels of root galling, prevented shoot weight losses, and resulted in significantly lower nematode levels at harvest. Thus, C. metuliferus may be used as a rootstock for melon to prevent both growth reduction and a strong nematode buildup in M. incognita-infested soil.     

Control of Larval Northern Corn Rootworm. (Diabrotica barberi) with Two Steinernematid Nematode Species

The entomogenous nematodes Steinerema feltiae and S. bibionis did not penetrate the roots of corn, Zea mays, to infect larval northern corn rootworm (NCR), Diabrotica barberi, feeding within. Laboratory bioassays against first instar NCR indicated that S. feltiae, Mexican strain (LD₅₀ = 49 nematodes/insect) is more virulent than S. bibionis (LD₅₀ = 100). Numbers of NCR larvae in a grain corn crop were reduced by both nematode species applied at corn seeding time at the rate of 10,000 infective-stage juveniles per linear meter of corn row. The chemical insecticide fonofos provided significantly better control than either nematode species.     

Susceptibility of the Plum Curculio,Conotrachelus nenuphar,to Entomopathogenic Nematodes

The plum curculio, Conotrachelus nenuphar, is a major pest of pome and stone fruit. Our objective was to determine virulence and reproductive potential of six commercially available nematode species in C. nenuphar larvae and adults. Nematodes tested were Heterorhabditis bacteriophora (Hb strain), H. marelatus (Point Reyes strains), H. megidis (UK211 strain), Steinernema riobrave (355 strain), S. carpocapsae (All strain), and S. feltiae (SN strain). Survival of C. nenuphar larvae treated with S. feltiae and S. riobrave, and survival of adults treated with S. carpocapsae and S. riobrave, was reduced relative to non-treated insects. Other nematode treatments were not different from the control. Conotrachelus nenuphar larvae were more susceptible to S. feltiae infection than were adults, but for other nematode species there was no significant insect-stage effect. Reproduction in C. nenuphar was greatest for H. marelatus, which produced approximately 10,000 nematodes in larvae and 5,500 in adults. Other nematodes produced approximately 1,000 to 3,700 infective juveniles per C. nenuphar with no significant differences among nematode species or insect stages. We conclude that S. carpocapsae or S. riobrave appears to have the most potential for controlling adults, whereas S. feltiae or S. riobrave appears to have the most potential for larval control.     

Temperature Effects on Heterorhabditis megidis and Steinernema carpocapsae Infectivity to Galleria mellonella

The effect of temperature on the infection of larvae of the greater wax moth, Galleria mellonella, by Heterorhabditis megidis H90 and Steinernema carpocapsae strain All, was determined. For both species, infection, reproduction, and development were fastest at 20 to 24 °C. Infection by both H. megidis and S. carpocapsae occurred between 8 and 16 °C; however, neither species reproduced at 8 °C. Among the nematodes used in experiments at 8 °C, no H. megidis and very few S. carpocapsae developed beyond the infective juvenile stage. Compared with H. megidis, S. carpocapsae invaded and killed G. mellonella larvae faster at 8 to 16 °C. By comparing invasion rates, differences in infectivity between the two nematode species were detected that could not be detected in conventional petri dish bioassays where mortality was measured after a specified period. Invasion of G. mellonella larvae by H. megidis was faster at 24 than at 16 °C.     

Symptomless Resistance of Alfalfa to Meloidogyne incognita acrita

Penetration, development and migration of the cotton root-knot nematode, Meloidogyne incognita acrita, in resistant and susceptible alfalfa varieties was compared. Larvae entered both resistant and susceptible plants in approximately the same numbers. After 3 to 4 days, the number of larvae in resistant roots decreased sharply until at 7 days fewer than 5 larvae/seedling and no nematode development could be found. In susceptible roots, larvae became sedentary and developed normally; egg production began as early as 18 days after penetration of the host.     

Are Pathogenesis-Related Proteins Induced by Meloidogne javanica or Heterodera avenae lnvasion?

Changes in root- and leaf-soluble proteins were investigated in tomato after invasion by the root-knot nematode Meloidogyne javanica, or in barley and wheat after invasion by the cereal cyst nematode Heterodera avenae. Infection of susceptible tomato plants by M. javanica did not cause any change in the soluble-protein composition of leaves or roots compared with uninoculated plants at an early infection stage. No pathogenesis-related proteins (chitinase, glucanase, or P-14) were induced in the leaf apoplast. Changes in leaf proteins were not observed after invasion of wheat cultivars by H. avenae, whereas, in barley, a few changes in intercellular leaf proteins were recorded in resistant cultivars. These changes, however, were not the same among different H. avenae-resistant cultivars. Protein changes were found at an early stage of infection in barley and wheat roots infected with H. avenae, but no difference was found between resistant and susceptible cultivars.     

Pathogenicity and Interaction of Three Nematode Species on Six Bermudagrasses

The ring nematode (Criconemoides ornatus), stunt nematode (Tylenchorhynchus martini), and sting nematode (Belonolaimus Iongicaudatus) reproduced readily on six bermudagrasses (Common, ''U-3'', ''Tufcote'', ''Continental'', ''Tiffine'', and ''Tifdwarf''). Populations of a single nematode species influenced the population development of a second and third parasitic nematode species on a particular host plant. Activity of most nematodes adversely affected reproduction of other nematode species in mixed cultures. Generally, the number of fibrous roots produced by plants decreased as the number of nematode species in the treatments increased. Tifdwarf bermudagrass appeared to be more tolerant to C. ornatus and T. martini than other grasses tested.     

Host Status of Crotalaria juncea,Sesamum indicum,Dolichos lablab,and Elymus glaucus to Meloidogyne javanica

Reproduction of Meloidogyne javanica on Crotalaria juncea PI 207657 and cv. Tropic Sun, Sesamum indicum, Dolichos lablab, and Elymus glaucus was assessed using a root-gall index, a reproductive index obtained by dividing the final population of juveniles (J2) in soil by the initial J2 population (Pf/Pi), and the number of J2 per gram of root recovered from roots by mist chamber extraction. Lycopersicon esculentum (cv. UC 204 C) was included as a susceptible host. The root-gall index and soil reproductive index were poor indicators of the host status of our test plants as compared with mist chamber extraction of J2 from roots. Lycopersicon esculentum had a mean root-gall index of 7.8. Some plants of S. indicum and E. glaucus had a few galls and other plants had none, with mean root-gall indices of 1.6 and 0.8, respectively. No galls were observed in C. juncea and D. lablab. Lycopersicon esculentum had the highest mean soil Pf/Pi value (mean = 1.93), while in C. juncea and some replicates of S. indicum no soil J2 were found. Even though some replicates had no galls, all replicates supported nematode reproduction. The mean numbers of J2 per gram root after 5 days of mist extraction were 447.7, 223.3, 165.5, 96.9, 42.3, and 41.9 for D. lablab, L. esculentum, E. glaucus, S. indicum, and C. juncea PI 207657 and cv. Tropic Sun, respectively. Accurate assessment of nematode resistance was influenced by sampling time and the nematode extraction technique used. Individual plants of both C. juncea and S. indicum supported nematode reproduction to some extent; however, both C. juncea and S. indicum have potential as cover crops to reduce M. javanica numbers.     

In Vitro Culture of Subanguina picridis in Acroptilon repens Callus,Excised Roots,and Shoot Tissues

The knapweed nematode Subanguina picridis is a foliar parasite that is of interest as a biological weed control agent of Russian knapweed. Attempts were made to culture the nematode in callus, excised roots and in shoots derived from roots of Russian knapweed. In callus tissues, the nematode developed from second-stage juvenile to adult but failed to reproduce; it developed only to the fourth stage in excised roots. However, S. picridis was successfully cultured in vitro in shoots derived from roots. The nematode induced galls on the leaves, petioles, and shoot apices and developed and reproduced inside the galls. Gibberellic acid increased the development rate of the nematode and promoted the formation of males. This is the first gnotobiotic culture of a nematode used for biological weed control.     

Histology of the Interactions of Paecilomyces lilacinus with Meloidogyne incognita on Tomato

Excised tomato roots were examined histologically for interactions of the fungus Paecilomyces lilacinus and Meloidogyne incognita race 1. Root galling and giant-cell formation were absent in tomato roots inoculated with nematode eggs infected with P. lilacinus. Few to no galls and no giant-cell formation were found in roots dipped in a spore suspension of P. lilacinus and inoculated with M. incognita. Numerous large galls and giant cells were present in roots inoculated only with M. incognita. P. lilacinus colonized the surface of epidermal cells as well as the internal cells of epidermis and cortex. The possibility of biological protection of plant surfaces with P. lilacinus against root-knot nematodes is discussed.     

Histological Observations of Rotylenchulus reniformis on Gossypium longicalyx and Interspecific Cotton Hybrids

Observations on the development of reniform nematode (Rotylenchulus reniformis) on roots of Gossypium longicalyx, G. hirsutum, and two interspecific hybrids derived from them were made by light microscopy. Gossypium longicalyx is reported to be immune to reniform nematode, but the mechanism(s) for resistance are unknown. Penetration of G. longicalyx roots by female nematodes was confirmed, and incipient swelling of the females, indicating initiation of maturation of the reproductive system, was observed. Female maturation occurred up to the formation of a single embryo inside the female body but not beyond this point. In both hybrids, development was inhibited but progressed further than in the immune parent. Reactions ranged from highly compatible, with the formation of active syncytia and full development of females, to incompatible with little or no development of the female. Compatible plants showed characteristic hypertrophied cells, enlarged nuclei, dense cytoplasm, and partial dissolution of cell walls, whereas incompatible plant reactions included lignification of the cells adjacent to the nematode head, or the complete collapse and necrosis of the cells involved. The need to characterize reactions and to carefully select among the plants descended from the hybrids during the introgression process, as well as the importance of combining the results of reproduction tests with histological observation of the plant-nematode interactions, is discussed.     

Effectiveness of Steinernema spp. and Heterorhabditis bacteriophora against Popillia japonica in the Azores

Steinernema carpocapsae (Breton strain), S. glaseri, and Heterorhabditis bacteriophora were evaluated for their potential to control immature stages of the Japanese beetle, Popillia japonica, on Terceira Island (the Azores). In bioassays carried out at temperatures higher than 15 C, S. glaseri and H. bacteriophora caused 100% mortality of larvae, whereas S. carpocapsae caused 56% larval mortality. At temperatures slightly below 15 C, only S. glaseri remained effective. In field plots, in September, S. glaseri and S. carpocapsae reduced larval populations by 91% and 44%, respectively, when applied at the rate of 10⁶ nematodes/m². In April, S. glaseri caused 31% reduction in numbers of larvae, but S. carpocapsae was ineffective. In colder months (November-February) neither steinernematids nor H. bacteriophora reduced larval populations. Increasing the application rate from 10⁶ to 5 x 10⁶ infective stage S. glaseri per m² increased efficacy from 63% to 79% mortality.     

Reproductive Variability of Field Populations of Meloidogyne spp. on Grape Rootstocks

Variability in penetration, development, and reproduction of two resistance-breaking field pathotypes (pt.) of Meloidogyne arenaria, M. incognita, and a population of mixed Meloidogyne spp. virulent to grape hosts were compared on two resistant Vitis rootstocks ''Freedom'' and ''Harmony'' in separate tests. ''Cabernet Sauvignon'' was included as a susceptible host to all four nematode populations. Secondstage juveniles (J2) of the mixed population failed to penetrate Freedom roots. By contrast, 6% of J2 in the M. incognita population penetrated Freedom roots but did not develop beyond the swollen J2 stage. The two resistance-breaking populations of M. arenaria differed in their virulence except on susceptible roots of Cabernet Sauvignon. More J2 of M. arenaria pt. Freedom penetrated Freedom roots and reached adult stage than did M. arenaria pt. Harmony. Later life stages of M. arenaria pt. Freedom occurred earlier and in greater numbers in Harmony roots than did M. arenaria pt. Harmony. Reproduction of M. arenaria pt. Freedom was greater in Freedom and Harmony roots than M. arenaria pt. Harmony. Thus, one population of M. arenaria is highly virulent and the other is moderately virulent.     

Effect of Soil Moisture and a Surfactant on Entomopathogenic Nematode Suppression of the Pecan Weevil, Curculio caryae

Our overall goal was to investigate several aspects of pecan weevil, Curculio caryae, suppression with entomopathogenic nematodes. Specifically, our objectives were to: 1) determine optimum moisture levels for larval suppression, 2) determine suppression of adult C. caryae under field conditions, and 3) measure the effects of a surfactant on nematode efficacy. In the laboratory, virulence of Heterorhabditis megidis (UK211) and Steinernema carpocapsae (All) were tested in a loamy sand at gravimetric water contents of negative 0.01, 0.06, 0.3, 1.0, and 15 bars. Curculio caryae larval survival decreased as moisture levels increased. The nematode effect was most pronounced at –0.06 bars. At –0.01 bars, larval survival was ≤5% regardless of nematode presence, thus indicating that intense irrigation alone might reduce C. caryae populations. Overall, our results indicated no effect of a surfactant (Kinetic) on C. caryae suppression with entomopathogenic nematodes. In a greenhouse test, C. caryae larval survival was lower in all nematode treatments compared with the control, yet survival was lower in S. carpocapsae (Italian) and S. riobrave (7–12) treatments than in S. carpocapsae (Agriotos), S. carpocapsae (Mexican), and S. riobrave (355) treatments (survival was reduced to approximately 20% in the S. riobrave [7–12] treatment). A mixture of S. riobrave strains resulted in intermediate larval survival. In field experiments conducted over two consecutive years, S. riobrave (7–12) applications resulted in no observable control, and, although S. carpocapsae (Italian) provided some suppression, treatment effects were generally only detectable one day after treatment. Nematode strains possessing both high levels of virulence and a greater ability to withstand environmental conditions in the field need to be developed and tested.     

Influence of Nonhost Plants on Population Decline of Rotylenchulus reniformis

The influence of Chloris gayana, Crotalaria juncea, Digitaria decumbens, Tagetes patula, and a chitin-based soil amendment on Hawaiian populations of Rotylenchulus reniformis was examined. Chloris gayana was a nonhost for R. reniformis. The nematode did not penetrate the roots, and in greenhouse and field experiments, C. gayana reduced reniform nematode numbers at least as well as fallow. Tagetes patula was a poor host for reniform nematode and reduced reniform nematode numbers in soil better than did fallow. Crotalaria juncea was a poor host for R. reniformis, and only a small fraction of the nematode population penetrated the roots. Crotalaria juncea and D. decumbens reduced reniform nematode populations at least as well as fallow. A chitin-based soil amendment, applied at 2.24 t/ha to fallow soil, did not affect the population decline of reniform nematode.     

Nemtaode-Vector Relationships in the Pine Wilt Disease System

Pinewood nematode, Bursaphelenchus xylophilus, is the causal agent of pine wilt disease in North America and Japan. Dispersal stage dauer larvae are transported to new host trees on the body surface and within the tracheal system of several beetle species. Worldwide, 21 species of Cerambycidae, 1 genus of Buprestidae, and 2 species of Curculionidae are known to carry pinewood nematode dauer larvae upon emerging from nematode-infested trees. Five species of cerambycids in the genus Monochamus are known to transmit dauer larvae to new host trees, four North American species and one Japanese species. Primary transmission to healthy trees occurs through beetle feeding wounds on young branches. Secondary transmission to stressed trees or recently cut logs occurs through Monochamus oviposition sites.     

Enhancement of Cylindrocladium crotalariae Root Rot by Meloidogyne arenaria (Race 2) on a Peanut Cultivar Resistant to Both Pathogens

Two populations of Meloidogyne arenaria (race 2, incompatible on peanut) enhanced development of Cylindrocladium black rot (CBR) on CBR-resistant peanut cv. NC 3033 in greenhouse factorial experiments. Nematode populations 256 and 486 (0, 10³, 10⁴ eggs per 15-cm pot) were tested in all combinations with Cylindrocladium crotalariae (0, 0.5, 5, 50 microsclerotia per cm³ of soil). Root-rot index increased in the presence of either population. Positions but not slope values of inoculum density-disease curves were changed by both populations, indicating increased efficiency of microsclerotia when peanuts were grown in the presence of these nematodes. Although little or no reproduction occurred with either nematode population on NC 3033, larvae of 256 and 486 penetrated roots. Meloidogyne arenaria 486 did not induce root galls and was not snccessful in establishing feeding sites. Meloidogyne arenaria 256 produced a few very small eliptical galls and had a range of success in establishing a feeding site, varying from no giant cell development to large giant cell with production of a few eggs.     

Susceptibility of the Carrot Weevil (Coleoptera: Curculionidae) to Steinernema feltiae,S. bibionis,and Heterorhabditis heliothidis

Larvae, pupae, and adults of the carrot weevil (Listronotus oregonensis) were infected and killed by the three entomophagous nematodes (Steinernema feltiae, S. bibionis, and Heterorhabditis heliothidis) under controlled conditions. Third-stage larvae were more susceptible than pupae or adults. S. feltiae and S. bibionis were the most aggressive nematode species, causing larval mortality after 24-48 hours in both continuous and 2-hour contact with nematode suspension. The nematodes multiplied sufficiently in all insects at all stages of development; however, production of infective-stage larvae per host cadaver was variable.     

Histological Responses of Four Leguminous Crops Infected with Meloidogyne incognita

Histological responses to Meloidogyne incognita infection in Rhizobium nodules of clover, horsebean, lupine, and pea were investigated. The formation of giant cells in vascular bundles of nodules and roots, and the basal connection of the nodule, were usually associated with abnormal xylem and/or deformed xylem strands. However, giant cells did not disturb or prevent the development of nodular tissues. Areas in which galls formed, wall thickness of giant cells, and number of giant cells around the nematode head varied with plant species. Ranking by gall size and giant-cell wall thickness was horsebean > lupine and pea > clover. The multinucleate condition in giant cells resulted from repeated mitoses without subsequent cytokinesis. The resulting nuclei agglomerated in irregularly shaped masses in some giant cells.