Simple In Vivo Production and Storage Methods for Steinernema carpocapsae Infective Juveniles

Methods are described for standardized in vivo production, rapid harvest, and storage, in a concentrated form, of infective juveniles of the entomopathogenic nematode, Steinernema carpocapsae Mexican strain Kapow selection. Nematodes were stored in nematode wool configurations, consisting of mats of intertwined infective juveniles. Freshly harvested nematodes are readily available in adequate quantities for laboratory and small-scale field evaluations as well as cottage industry production.     

Improved Nematode Extraction from Carrot Disk Culture

Radopholus spp. were reared in carrot tissue culture via established procedures, with slight modification. Several plant tissue maceration enzymes and flotation media (salts and sucrose) were evaluated with regard to nematode toxicity and extraction efficiency. Best extraction of viable nematodes and eggs was attained when carrot tissue infested with Radopholus citrophilus or R. similis was macerated with a mixture of 0.50% driselase and 0.50% cellulysin, w/v each, with 2.5 ml of enzyme solution based for each gram of carrot tissue. Maceration slurries containing carrot tissue and nematodes were maintained in open flasks on a rotary shaker (175 rpm) at 26 C for 24 hours. Nematodes and eggs were extracted from resultant culture slurries by flotation with MgSO₄-7H₂0 (sp gr 1.1). A protocol is presented to extract large quantities of viable burrowing nematodes and their eggs from carrot disk cultures.     

Effect of Single and Interplantings on Pathogenicity of Pratyenchus penetrans and P. neglectus to Alfalfa and Crested Wheatgrass

Alfalfa is a host of Pratylenchus penetrans and P. neglectus, whereas crested wheatgrass is a host of P. neglectus but not of P. penetrans. In a 120-day greenhouse experiment at 24 ñ 3 C, P. neglectus inhibited the growth of ''Lahontan'' alfalfa and ''Fairway'' crested wheatgrass. There were no differences in persistence and plant growth of alfalfa and crested wheatgrass, or reproduction of P. neglectus, in single plantings of alfalfa (AO) or crested wheatgrass (CWO), or in interplanted alfalfa and crested wheatgrass (ACW) treatments. On alfalfa, P. penetrans inhibited growth and reproduced more than did P. neglectus. Inhibition of plant growth and reproduction of P. penetrans was greater on alfalfa in AO than in ACW treatments. Pratylenchus penetrans did not reproduce on crested wheatgrass, but inhibited growth of crested wheatgrass in interplanted treatments and was avirulent in single planted treatments. Results were similar in a controlled growth chamber experiment at 15, 20, 25, and 30 C. Both nematode species inhibited alfalfa growth at all temperatures, and P. penetrans was more virulent than was P. neglectus to alfalfa at all temperatures and treatments. Plant growth inhibition and reproduction of P. penetrans on alfalfa in single and interplanted treatments were similar at 15-20 C, but were greater in single than in interplanted treatments at 25-30 C. Pratylenchus penetrans was avirulent to crested wheatgrass in the single planted treatments at all temperatures, but inhibited growth of crested wheatgrass in interplanted treatments at 20-30 C. Plant growth and reproduction of P. neglectus on crested wheatgrass was similar in single and interplanted treatments at 20-30 C and 15-30 C, respectively.     

Overwintering and Cold Tolerance of the Mermithid Filipjevimermis leipsandra

Overwintering survival of Filipjevimermis leipsandra postparasitic adults ranged from 12 to 15% over 2 years. Nematodes recovered from biweekly samples oviposited within a week when exposed to warm temperatures. More nematodes were recovered from the lower portions of soil cores than from the upper. In laboratory tests of cold tolerance, the LD₅₀ at -5 C was 5.5 hours, whereas exposure to -2 C for as long as 96 hours resulted in no mortality.     

Anhydrobiotic Coiling of Nematodes in Soil

Nematodes of three genera (Acrobeloides sp., Aphelenchus avenae, and Scutellonema brachyurum) were induced to coil and enter anhydrobiosis in drying soil of two types: sandy loam and loamy sand. Coiling was studied in relationship to soil moisture characteristics. Coiling and the physiological state of anhydrobiosis occurred before the water in sandy soils reached a water potential of -15 bars. Coiling was maximum at 3-6 bars, depending on the soil type and nematode species. It appeared that induction of coiling and anhydrohiosis were determined by the physical forces exerted by the water film surrounding the nematode, which, for these three species, was 6-9 monomolecular layers of water, rather than the % moisture and relative humidity of the soil per se.     

Cryopreservation of Pratylenchys spp.

Abstract: Optimal conditions for cryopreserving of populations of root lesion nematode (Pratylenchus spp.) were determined. Nematode survival was achieved using glycerol pre-treatments in the range of 14-17% (w/w). Increasing duration of the incubation in glycerol (up to 5 days) before immersion in liquid nitrogen significantly influenced nematode survival The highest mean survival for P. thornei was 76% after incubation in glycerol for 5 days. Nematodes were able to reproduce and infect carrot disc cultures after cryopreservation. This technique has valuable applications for long-term germplasm storage and maintenance of genetic lines.     

Granular Formulations of Steinernema carpocapsae (strain All) (Nematoda: Rhabditida) with Improved Shelf Life

Shelf life (nematode survival) of Steinernema carpocapsae (strain All) nematodes at 21 C in "Pesta" granules, made by a pasta-like process, was increased from 8 to 26 weeks by incorporating low concentrations of formaldehyde. Pesta samples containing an average of 427,000 nematodes/g were prepared with wheat flour (semolina or bread flour), kaolin, bentonite, peat moss, nematode slurry, and formaldehyde (0-1.4% w/w) and were dried to a water content of 23.6-26.9%. Nematodes emerged from Pesta (S. carpocapsae) granules when placed in water or on moist filter paper. Incorporation of 0.2% w/w formaldehyde (nominal; 0.05% by analysis) was optimum for increasing nematode survival in semolina-based Pesta, and also inhibited fungal growth on the granules. Bread flour Pesta samples prepared by formaldehyde addition to the nematode slurry prior to dough preparation, rather than by addition to a mixture of dry ingredients, had longer shelf life. Nematodes recovered from granules made with 0.2% formaldehyde and stored 20 weeks at 21 C caused 100% mortality of wax moth (Galleria mellonella) larvae.     

Interactions Among Selected Endoparasitic Nematodes and Three Pseudomonads on Alfalfa

Meloidogyne hapla, Pratylenchus penetrans, and Helicotylenchus dihystera, reduced the growth of ''Saranac AR alfalfa seedlings when applied at concentrations of 50 nematodes per plant. All except P. penetrans reduced seedling growth when applied at 25 per seedling. M. hapla reduced growth when applied at 12 per seedling. Nematodes interacted with three pseudomonads to produce greater growth reductions than were obtained with single pathogens, suggesting synergistic relationships. Ditylenchus dipsaci, applied at 25 or 50 nematodes per seedling, reduced plant weight compared with weights of control plants, but did not interact with test bacteria. All of the nematodes except D. dipsaci produced root wounds which were invaded by bacteria.     

Spatial Patterns of Entomopathogenic Nematodes in Microcosms: Implications for Laboratory Experiments

Laboratory microcosms were used to: i) measure the effects of soil moisture on survival of Steinernema riobravis and ii) investigate the suitability of using microcosms to study motility and survival of these nematodes. Nematodes recovered from soil contained in petri dishes declined by more than 95% during 7 days, whereas nematodes recovered from the inner surfaces of dishes increased 35-fold. After 7 days in dishes, >20 times as many nematodes were recovered from dish surfaces than from soil. Nematodes exhibited a negative geotropism; greater numbers of nematodes were recovered from the lid surfaces than from the surfaces of dishes. Survivorship of nematodes in soil in plastic centrifuge tubes was somewhat greater than in petri dishes, and fewer nematodes ascended above the soil line in tubes than dishes. Downward migration of nematodes was inversely related to soil column diameter, possibly due to relatively unimpeded movement along container surfaces. An assay was developed by which nematodes were rinsed from the inner surfaces of centrifuge tubes into the soil. The resulting slurry was then processed on Baermann trays to recover motile nematodes. Nematode survival in soil in centrifuge tubes was higher at soil moistures between 2-4% than at lower (0.5-1.0%) and higher (4.0-12.0%) moisture levels. Survival of S. riobravis may be enhanced by quiescence induced by moisture deficits.     

Cephalobus litoralis: Biology and Tolerance to Desiccation

Cephalobus litoralis (Akhtar, 1962) Andrássy, 1984 reproduced parthenogenetically and completed its life cycle in 72-90 hours. Each female deposited 200-300 eggs. The nematodes showed synchronized movements in the rhythms of the anterior parts of the body. The nematodes were coiled when dried in culture medium or in slowly evaporating water droplets on the tops of culture plates, but in pellets they assumed irregular postures. Nematodes in pellets stored at high humidity could be reactivated after storage for 28 days.     

Effects of Environmental Factors and Cultural Practices on Parasitism of Alfalfa by Ditylenchus dipsaci

Cool humid weather enhanced development and reproduction of Ditylenchus dipsaci in alfalfa in laboratory and field studies in Utah. Relative humidity and nematode reproduction were positively correlated (P < 0.05), whereas air temperature and nematode reproduction were negatively correlated (P < 0.05). The greatest number of nematodes per gram of alfalfa tissue was found in nondormant Moapa alfalfa tissue at St. George during April, whereas the greatest numbers of nematodes were found in dormant Ranger alfalfa in June at West Jordan and Smithfield. There was 100% invasion of both resistant Lahontan and susceptible Ranger alfalfa plants at soil moisture levels of 61-94% field capacity. Fall burning of alfalfa to control weeds reduced, and spring burning increased, the incidence of invaded plants, nematodes per gram of plant tissue, and the mortality of susceptible Ranger (P < 0.01) and Moapa (P < 0.01) alfalfa plants over that of plants in nonburned control plots. Fall burning also reduced and spring burning increased the incidence of invaded plants (P < 0.05), but had no influence on nematodes per gram of plant tissue or the mortality of resistant Lahontan and Nevada Synthetic XX alfalfa over those of plants in control plots.     

Biological Relationship of Meloidogyne hapla Populations to Alfalfa Cultivars

Greenhouse and growth chamber studies were established to determine if there are pathological and physiological differences among Meloidogyne hapla populations from California (CA), Nevada (NV), Utah (UT), and Wyoming (WY) on alfalfa cultivars classified as resistant or susceptible to root-knot nematodes. In the greenhouse, plant survival was not consistent with resistance classifications. While all highly resistant Nevada Synthetic germplasm (Nev Syn XX) plants survived inoculation with all nematode populations, two cultivars classified as moderately resistant (''Chief'' and ''Kingstar'') survived (P ≤ 0.05) inoculation with M. hapla populations better than did ''Lobo'' cultivar, which is classified as resistant. Plant growth of Nev Syn XX was suppressed by only the CA population, whereas growth of the other alfalfa cultivars classified as M. hapla resistant or moderately resistant was suppressed by all nematode populations. Excluding Nev Syn XX, all alfalfa cultivars were severely galled and susceptible to all nematode populations. Except for Nev Syn XX, reproduction did not differ among the nematode populations on alfalfa cultivars. Nev Syn XX was not as favorable a host to CA as were the other cultivars; but, it was a good host (reproductive factor [Rf] = 37). Temperature affected plant resistance; the UT and WY populations were more pathogenic at 15-25 C, and CA was more pathogenic at 30 C. Nev Syn XX was susceptible to all nematode populations, except for CA, at only 30 C, and all other alfalfa cultivars were susceptible to all nematode populations at all temperatures.     

Evaluation of Cultivars,Experimental Lines and Plant Introduction Collection of Sainfoin for Resistance to Meloidogyne hapla Chitwood

Stands of several cultivars and experimental lines of sainfoin (Onobrychis viciifolia) were severely reduced (92% average loss) in a field naturally infested with Meloidogyne hapla. Stands of two alfalfa cultivars included in the test were unaffected. In studies conducted in the greenhouse with plants inoculated at the time of seeding, average mortality was 55% for sainfoin entries and 7% for Ladak alfalfa. Little mortality occurred when plants were inoculated after establishment. Three months after inoculation, all sainfoin entries were heavily galled (range of 3.3-3.7 on a scale of 1-4) while roots of Ladak were only slightly galled (rating of 1.6). Intermating of plants selected in the field plots for resistance to M. hapla showed a slight increase in resistance. Of the 147 plant introduction lines tested in the greenhouse, none were resistant to M. hapla.     

A Nematicide Seed Treatment to Control Ditylenchus dipsaci on Seedling Alfalfa

Three nematicides were evaluated as seed treatments to control the alfalfa stem nematode (Ditylenchus dipsaci) on seedling alfalfa. Alfalfa seeds were soaked for 10 hours in a 0.5% (formulated by weight) concentration of either carbofuran, phenamiphos or oxamyl in acetone with no adverse effect on seed germination. All three treatments decreased nematode damage and increased survival of ''Ranger'' (susceptible) and ''Lahontan'' (resistant) alfalfa plants, when seeds were planted in soil infested with D. dipsaci. Mean live plant counts after 6 weeks in the untreated control, acetone alone, carbofuran, phenamiphos, and oxamyl treatments, respectively, were 4.3, 6.3, 19.0, 19.8, and 19.0 for Lahontan and 4.5, 1.5, 18.5, 19.3, and 18.0 for Ranger from 20 seeds/pot. Nematicide seed treatments resulted in significantly healthier Ranger alfalfa plants 4 months after planting. The combination of seed treatment and host resistance may provide a means of establishing alfalfa in an alfalfa monocropped system where soil populations of D. dipsaci are high.     

Pinewood Nematode,Bursaphelenchus xylophilus,Associated with Red Pine, Pinus resinosa,in Western Maryland

Red pines Pinus resinosa in Garrett and Allegany counties, Maryland, were examined during 1982-84 to determine distribution of the pinewood nematode, Bursaphelenchus xylophilus, within and among trees. Approximately 25-year-old (younger) and 47-year-old (older) trees were subdivided into the following categories: 1) trees with mostly green needles; 2) trees with mostly reddish-brown needles; 3) trees lacking needles but with bark intact; 4) trees lacking both needles and bark; and 5) trees with chlorotic, bleached-green needles. Bursaphelenchus xylophilus was found infecting 68% of younger red pines and 77% of older red pines. Nematodes were not evenly distributed in trees within any given tree decadence category or in trees of the same age. Nematodes were recovered from 20% of wood samples from trunks and primary and secondary branches in younger pines and from 15 % of older red pines. On the basis of tree decadence category, the highest incidence of infection in younger trees (31%) was in bleached-green needled trees (category 5), whereas in older trees the highest infection (25%) occurred in green needled trees (category 1). At both sites trunks were infected more often than branches.     

Efficacy of Oxamyl Coated on Alfalfa Seed with a Polymer Sticker in Pratylenchus and Meloidogyne Infested Soils

A polymer sticker was used as a coating in which oxamyl was applied to seeds of alfalfa cultivar Saranac for the control of Pratylenchus penetrans and Meloidogyne hapla. The sticker, diluted 1:1 (sticker:water) to 1:5, delayed seedling emergence during the first 4 days after planting. By day 13, however, emergence from all sticker treatments was comparable to the control. Shoot growth of seedlings at day 21 was less than that of the control only from seeds coated with a 1:1 dilution; root growth and nodulation were not affected. Sticker-coated seeds absorbed 30-58% as much water in 3.5 hours as was absorbed by uncoated seeds. Oxamyl concentrations of 40-160 mg/ml in a 1:5 sticker : water mixture had no adverse affect on seedling emergence, growth, and nodulation over 3 weeks. Oxamyl at 160 mg/ml was more effective against P. penetrans than M. hapla. Growth of alfalfa in P. penetrans-infested soil was greater than that of the control in each sampling for 11 weeks. The reduction of number of P. penetrans in soil and roots moderated slowly over 11 weeks from 90% to 60%. Shoot and root growth of alfalfa from oxamyl-coated seed in M. hapla-infested soil were greater than those of the control for 7 and 11 weeks, respectively. The reduction in the number of M. hapla in the soil and roots changed from 80% at 7 weeks to 15% at 11 weeks.     

Differential Effects of Pratylenchus neglectus Populations on Single and Interplantings of Alfalfa Grasses

The invasion by three different Utah populations of Pratylenchus neglectus (UTI, UT2, UT3) was similar in single and interplantings of ''Lahontan'' alfalfa and ''Fairway'' crested wheatgrass at 24 ñ 3 °C. Population UT3 was more pathogenic than UT1 and UT2 on both alfalfa and crested wheatgrass. Inoculum density was positively correlated with an invasion by P. neglectus. Invasions by UT3 at all initial populations (Pi) exceeded that of UT1 and UT2 for both single and interplanted treatments. The greatest reductions in shoot and root weights of alfalfa and crested wheatgrass were at a Pi of 8 P. neglectus/cm³ soil. Pi was negatively correlated with alfalfa and crested wheatgrass shoot and root growth and nematode reproduction. The reproductive factor (Rf) for UT3 exceeded that of UT1 and UT2 in single and interplantings at all inoculum levels. There were no differences in Rfin the Utah populations in single or interplantings. A nematode invasion increased with temperature and was greatest at 30 °C. Population UT3 was more pathogenic than UT1 and UT2 and reduced shoot and root growth at all soil temperatures. Populations UT1 and UT2 reduced shoot and root growth at 20-30 °C. Soil temperature was negatively correlated with shoot and root growth and positively correlated with nematode reproduction. Reproduction of UT3 exceeded that of UT1 and UT2 at all soil temperatures.     

Interaction of Ditylenchus dipsaci and Meloidogyne hapla on Resistant and Susceptible Plant Species

Numbers ofDitylenchus dipsaci or Meloidogyne hapla invading Ranger alfalfa, Tender crop bean, Stone Improved tomato, AH-14 sugarbeet, Yellow sweet clover, and Wasatch wheat from single inoculations were not significantly different from numbers by invasion of combined inoculations. D. dipsaci was recovered only from shoot and M. hapla only from root tissue. Combined inoculations did not affect reproduction of either D. dipsaci or M. hapla. D. dipsaci suppressed shoot growth of all species at 15-30 C, and M. hapla suppressed shoot growth of tomato, sugarbeet, and sweet clover at 20, 25, and 30 C. There was a positive correlation (P < 0.05) between shoot and root growth suppression by D. dipsaci on all cultivars except wheat at 20 C and tomato at 30 C. M. hapla suppressed (P < 0.05) root growth of sugarbeet at 20-50 C and wheat at 30 C. Growth suppression was synergistic in combined inoculations of sweet clover shoot growth at 15 C and root growth at 20-30 C, wheat root growth at 15 and 20 C, and tomato root growth at 15-30 C (P < 0.05) D. dipsaci invasions caused mortality of alfalfa and sweet clover at 15-30 C and sugarbeet at 20-30 C. Mortality rates of alfalfa and sweet clover increased synergistically (P < 0.05) from combined inoculations.     

Histopathology of Ditylenchus destructor on Peanut

The time and mode of entry, and development of Ditylenchus destructor in peanut were studied in field and greenhouse experiments. Few nematodes were present in the cortex of the roots. At 90-120 days after planting, D. destructor was observed in the exocarp at the base of the pod near the point of connection with the peg. The peg was invaded from this primary infection site. The endocarp of the hull was usually penetrated through openings at the base of the mesocarp and sometimes at the pod apex. Numerous D. destructor were present in the testa and the vascular bundles. Nematodes were found in the embryo but not in the cotyledons. The histopathology of D. destructor closely resembles that of the peanut testa nematode, Aphelenchoides arachidis Bos.     

Pathological Relationship of Ditylenchus dipsaci and Fusarium oxysporum f. sp. medicaginis on alfalfa

Ditylenchus dipsaci and Fusarium oxysporum f. sp. medicaginis synergistically affected the mortality and plant growth of Ranger alfalfa, a cultivar susceptible to stem nematode and Fusarium wilt. The nematode-fungus relationship had an additive effect on mortality and plant growth of Lahontan (nematode resistant and Fusarium wilt susceptible) and of Moapa 69 (nematode susceptible and Fusarium wilt resistant). Mortality rates were 13, 16, 46, and 49% for Ranger; 4, 18, 26, and 28% for Lahontan; and 19, 10, 32, and 30% for Moapa 69 inoculated with D. dipsaci, F. oxysporum f. sp. medicaginis, and simultaneously and sequentially with D. dipsaci and F. oxysporum f. sp. medicaginis, respectively. Shoot weights as a percentage of uninoculated controls for the same treatments were 52, 84, 26, and 28%, for Ranger; 74, 86, 64, and 64% for Lahontan; and 50, 95, 44, and 39% for Moapa 69. Plant growth suppression was related to vascular bundle infection and discoloration of alfalfa root tissue. Disease severity and plant growth of alfalfa did not differ with simultaneous or sequential inoculations of the two pathogens. Fusarium oxysporum f. sp. medicaginis affected alfalfa growth but not nematode reproduction.