Extremely Sensitive Thermotaxis of the Nematode Meloidogyne incognita

Eggs of the root-knot nematode Meloidogyne incognita were acclimated to 23 C. Newly hatched second-stage juveniles migrated toward higher temperatures when placed in shallow thermal gradients averaging 23 C. The threshold gradient for this response was below 0.001 C/cm, with a best estimate of 4 x 10⁻⁴ C/cm. Calculations of physical limitations on thermotaxis indicate that this sensitivity is well within the limits of what is physically possible.     

Damage Functions for Meloidogyne arenaria on Peanut

Microplot experiments were conducted in 1989 and 1990 to determine the relationship between yield of peanut (Arachis hypogaea) and inoculum density ofMeloidogyne arenaria race 1. Nine inoculum densities were used, ranging from 0-200 eggs/100 cm³ soil (1989) or from 0-100 eggs/100 cm³ (1990), and each density was replicated 10 times. In 1989, higher final densities (mean of 1,171 juveniles [J2]/100 cm³ soil) were obtained in plots inoculated with 0.5 to 50 eggs/100 cm³ soil than in plots inoculated with 100 to 200 eggs/100 cm³ (313 J2/100 cm³ soil). In 1990, final densities of M. arenaria reached high levels (≥ 1,111 J2/100 cm³ soil) in all inoculated plots. Pod yield and dry weight of foliage at harvest were negatively correlated (P ≤ 0.05) with inoculum density in both seasons. In 1989, the relationship between pod weight (y) and initial density (x) was described by Seinhorst''s equation, with y = 0.088 + 0.91(0.90)⁽x⁻¹⁾ and r² = 0.826. In 1990, the relationship was y = 0.22 + 0.78(0.97)⁽x⁻¹⁾ and r² = 0.794. These equations suggest tolerance limits of approximately 1 egg/100 cm³ soil, which may require specialized methods, such as bioassay, for detection.     

Transport of Palmitic Acid Across the Tegument of the Entomophilic Nematode Romanomermis culicivorax

Romanomermis culicivorax juveniles, dissected out of Aedes aegypti larvae 7 days after infection, were incubated under controlled conditions in isotonic saline containing ¹⁴C-U-palmitic acid to investigate the nature of the transport mechanism(s) used by the nematode for transcuticular uptake of palmitic acid. Net uptake of the isotope by the nematode was of a logarithmic nature with respect to time. Uptake of palmitic acid was accomplished by a combination of diffusion and a mediated process which was substrate saturable and competitively inhibited by myristic and stearic acids. Both 2,4-dinitrophenol and ouabain inhibited uptake of palmitic acid and thus supported the hypothesis that the carrier system is of the active transport variety and is coupled to a Na⁺K⁺ ATPase pump.     

Plant Protection with Inorganic Ions

Gradients of salts of the specific ion repellents for Meloidogyne incognita -- NH₄⁺, K⁺, Cl⁻, and NO₃⁻ -- have been demonstrated to shield tomato roots from infestation in soil. The strategy of these greenhouse experiments was to interpose a salt barrier in a soil column between the plant roots and the nematodes. The relative effectiveness of the salts as a barrier to infective second-stage juveniles in a sandy loam was NH₄NO₃, NH₄Cl > KNO₃ > KCl. Some of these ions are beneficial to plant growth, and the results suggest that a new environmentally tolerable means of plant protection is possible.     

Comparison of Two Steinernematid Species for Control of the Root Weevil Diaprepes abbreviatus

Steinernema carpocapsae Weiser All strain was compared to Steinernema riobravis Cabanillas, Poinar, and Raulston for control of the root weevil, Diaprepes abbreviatus (L.), in the laboratory and in potted citrus. In the laboratory bioassay, D. abbreviatus larvae were exposed to 30, 60, and 120 nematodes/cm³ in sand. Insect mortality 1 week after application was greater (P ≤ 0.05) for S. riobravis than for S. carpocapsae in the laboratory bioassay. In the greenhouse bioassay, D. abbreviatus larvae were exposed to 3 and 9 nematodes per cm³ of soil in potted citrus. Again, at each rate, mortality was greater (P ≤ 0.05) in pots treated with S. riobravis than in pots treated with S. carpocapsae. The results of this study suggest that S. riobravis is a better biological control agent against D. abbreviatus larvae in potted plants than S. carpocapsae.     

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

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

Effects of Temperature on Development of Heterodera glycines on Glycine max and Phaseolus vulgaris

Soybean cyst nematode resistant ''Fayette'' and susceptible ''Williams 79'' soybeans (Glycine max) and resistant ''WIS (RRR) 36'' and susceptible ''Eagle'' snap beans (Phaseolus vulgaris) were used in determining the effects of host and temperature on the development, female production, sex ratios, and host response to Heterodera glycines. Temperatures were maintained constant at 16, 20, 24, 28, and 32 C using water-filled tanks. The most rapid development and greatest female production occurred between 20 and 28 C. The equation DS = 5(10⁻⁶)x²y² - 3(10⁻⁴)x²y - 2.8(10⁻³)x² - 1.94(10⁻²)y² + 0.4288x + 1.0220y - 12.7185, where DS = developmental stage, X = time, and Y = temperature, predicted the developmental stage of the nematode and accounted for 84% of the variation. Male : female ratios did not differ within this range and were generally less than one. At all temperatures the resistant soybean produced the greatest number of necrotic responses to H. glycines infection, followed by the resistant snap bean. The susceptible soybean and snap bean produced the fewest necrotic responses.     

Use of Cellulose Acetate Electrophoresis in the Taxonomy of Steinernematids (Rhabditida,Nematoda)

A steinernematid nematode was isolated from soil samples collected near St. John''s, Newfoundland, Canada. On the basis of its morphometry and RFLPs in ribosomal DNA spacer, it was designated as a new strain, NF, of Steinernema feltiae. Cellulose acetate electrophoresis was used to separate isozymes of eight enzymes in infective juveniles of S. feltiae NF as well as four other isolates: S. feltiae Umeå strain, S. feltiae L1C strain, Steinernema carpocapsae All strain, and Steinernema riobravis TX strain. Based on comparisons of the relative electrophoretic mobilities (μ) of the isozymes, one of the eight enzymes (arginine kinase) yielded zymograms that were distinctive for each of the isolates, except for the Umeå and NF strains of S. feltiae, which had identical banding patterns. Four enzymes (fumarate hydratase, phosphoglucoisomerase, phosphoglucomutase, and 6-phosphogluconate dehydrogenase) yielded isozyme banding patterns that were characteristic for all isolates, except for the L1C and NF strains of S. feltiae, which were identical. Two enzymes (aspartate amino transferase and glycerol-3-phosphate dehydrogenase) yielded zymograms that permitted S. carpocapsae All strain to be discriminated from the other four isolates, while the remaining enzyme (mannose-6-phosphate isomerase) was discriminatory for S. riobravis TX strain. Except for one enzyme, the isozyme banding pattern of the NF isolate of S. feltiae was the same as in the L1C strain, isolated 13 years previously from Newfoundland. Cellulose acetate electrophoresis could prove invaluable for taxonomic identification of isolates of steinernematids, provided that a combination of enzymes is used.     

Effect of Meloidogyne incognita and Importance of the Inoculum on the Yield of Eggplant

The relationship between population densities of race 1 of Meloidogyne incognita and yield of eggplant was studied. Microplots were infested with finely chopped nematode-infected pepper roots to give population densities of 0, 0.062, 0.125, 0.25, 0.50, 1, 2, 4, 8, 16, 32, 64, and 128 eggs and juveniles/cm³ soil. Both plant growth and yield were suppressed by the nematode. A tolerance limit of 0.054 eggs and juveniles/cm³ soil and a minimum relative yield of 0.05 at four or more eggs and juveniles/cm³ soil were derived by fitting the data with the equation y = m + (1 - m)z^P⁻T. Maximum nematode reproduction rate was 12,300. Hatch of eggs from egg masses in water or from sodium hypochlorite dissolved egg masses was similar (41% and 39%), but egg viability was significantly greater from egg masses in water (58%) than from sodium hypochlorite dissolved egg masses (12%) after 4 weeks. Greater numbers of nematodes were collected from roots of tomatoes from soil infested with entire egg masses than from tomato roots from soil infested with egg masses dissolved by sodium hypochlorite.     

Influence of Soil pH and Oxygen on Persistence of Steinernema spp.

Survival of infective juveniles of Steinernema carpocapsae and Steinernema glaseri gradually declined during 16 weeks of observation as the tested soil pH decreased from pH 8 to pH 4. Survival of both species of Steinernema dropped sharply after 1 week at pH 10. Survival or S. carpocapsae and S. glaseri was similar at pH 4, 6, and 8 during the first 4 weeks, but S. carpocapsae survival was significantly greater than S. glaseri at pH 10 through 16 weeks. Steinernema carpocapsae and S. glaseri that had been stored at pH 4, 6, and 8 for 16 weeks, and at pH 10 for 1 or more weeks were not infective to Galleria mellonella larvae. Steinernema carpocapsae survival was significantly greater than that of S. glaseri at oxygen:nitrogen ratios of 1:99, 5:95, and 10:90 during the first 2 weeks, and survival of both nematode species declined sharply to less than 20% after 4 weeks. Survival of both nematode species significantly decreased after 8 weeks as the tested oxygen concentrations decreased from 20 to 1%, and no nematode survival was recorded after 16 weeks. Steinernema carpocapsae pathogenicity was significantly greater than that of S. glaseri during the first 2 weeks. No nematode pathogenicity was recorded at oxygen concentrations of 1, 5, and 10% after 2 weeks and at 20% after 16 weeks.     

不同糖源及糖水平对大菱鲆糖代谢酶活性的影响

采用34双因素实验设计, 以初始质量为(8.060.08) g的大菱鲆幼鱼(Scophthalmus maximus L.)为对象, 研究在饲料中添加3种糖源(葡萄糖、蔗糖和糊精)及4个水平(0、5%、15%、28%)对大菱鲆肝脏糖酵解关键酶己糖激酶(HK)、葡萄糖激酶(GK)、磷酸果糖激酶(PFK)、丙酮酸激酶(PK)和糖异生关键酶磷酸烯醇式丙酮酸羧激酶(PEPCK)、1, 6-二磷酸果糖酶(FBPase)活性的影响。结果表明: 饲料糖添加量从0升高到15%时, 大菱鲆的糖酵解酶GK和PK活性随饲料葡萄糖或糊精含量的增加而增加; 当饲料中葡萄糖或糊精含量为28%时, GK和PK活性有下降的趋势。3种糖源的4个添加水平对HK和PFK活性均无显著影响(P 0.05)。添加不同水平的葡萄糖对大菱鲆糖异生途径的PEPCK活性无显著影响(P 0.05), 但在饲料中葡萄糖添加量为5%时显著促进了FBPase活性(P 0.05), 当葡萄糖添加量升高为15%或28%时, FBPase活性与对照组无显著差异(P 0.05)。糊精作为饲料糖源时抑制了大菱鲆肝脏FBPase和PEPCK的活性, 而添加不同水平的蔗糖对FBPase和PEPCK活性的影响均不显著(P 0.05)。总的来说, 从大菱鲆幼鱼肝脏糖代谢角度而言, 在饲料中添加15%的葡萄糖或糊精时, 可以有效促进大菱鲆肝脏糖酵解能力; 较添加葡萄糖, 糊精在促进大菱鲆肝脏糖酵解的同时对糖异生存在一定程度的抑制。蔗糖作为饲料糖源时, 仅在添加量为28%时显著促进糖酵解酶GK活性, 糖酵解其他酶活性以及糖异生酶活性均不受蔗糖水平的显著影响。       

Cryopreservation of Steinernema carpocapsae and Heterorhabditis bacteriophora

A method for the cryopreservation of third-stage infective juveniles (IJ) of Steinernema carpocapsae and Heterorhabiditis bacteriophora was developed. Cryoprotection was achieved by incubating the nematodes in 22% glycerol (S. carpocapsae) or 14% glycerol (H. bacteriophora) for 24 hours, followed by 70% methanol at 0 C for 10 minutes. The viability of S. carpocapsae frozen in liquid nitrogen as 20 μl volumes spread over cover slip glass was > 80%. Survival of H. bacteriophora frozen on glass varied from 10 to 60% but was improved to > 80% by replacing the glass with filter paper. Cryopreservation and storage of 1-ml aliqots of S. carpocapsae IJ resulted in > 50% survival after 8 months; pathogenicity was retained and normal in vitro development took place. Trehalose and glycerol levels increased and glycogen levels decreased during incubation of S. carpocapsae IJ in glycerol. Normal levels of trehalose, glycerol and glycogen were restored during post freezing rehydration.     

Response of Steinernema carpocapsae Infective Juveniles to the Plasma of Three Insect Species

Exsheathed infective juveniles of Steinernema carpocapsae All strain were attracted to the plasma of three species of insects in agar plate bioassays. Plasma of Pieris rapae crucivora, Spodoptera litura, and Agrotis segetum attracted 88.6%, 80.4%, and 64.4%, respectively, of Steinernema carpocapsae juveniles added to plates. Autoclaved plasma of S. litura larvae attracted more juveniles than saline controls, but less than nonautoclaved plasma. The active agent passed through a 14,000 MW dialysis membrane.     

Stage-specific Population Development and Fecundity of Paratrichodorus minor

A conceptual model of the life cycle of Paratrichodorus minor consisting of the egg stage, four juvenile stages, and the adult stage was proposed. Development of an individual from one stage to the next was described by a probability distribution defined by the mean length of time spent in the stage and the standard deviation associated with the mean duration. Experiments were conducted to estimate stage durations, stage-specific survivorships, and a fecundity rate for females. Eggs hatched on agar plates at a mean time of 53.3 ± 7.3 degree-days using a basal threshold of 10 C (DD₁₀) with a range of 40-64 DD₁₀ after deposition. Forty-five percent of the eggs observed ultimately hatched. Of the eggs that died, 44% died before the nematode form could be observed in the egg and 56% died after movement had been observed. First generation population peaks following inoculation with first-stage juveniles occurred at 28 DD₁₀ for second-stage juveniles, 67 DD₁₀ for third-stage juveniles, 109 DD₁₀ for fourth-stage juveniles, and 143 DD₁₀ for adults. Adult males are rare and were never observed in these studies. The fecundity rate was 0.784 eggs/(female-DD₁₀⁻¹), but the maximum length of the egg-laying period was not determined. The minimum egg-laying period was 73-113 DD₁₀, and minimum egg production was 57-86 eggs per female. The preovipositional period for adult females was estimated to be 79 DD₁₀. In the presence of a host, total population numbers increased, but in the absence of a host, the population declined to 33 % of the initial level after 300 DD₁₀.     

Impact of a Nematode-parasitic Fungus on the Effectiveness of Entomopathogenic Nematodes

The impact of the nematode-parasitic fungus Hirsutella rhossiliensis on the effectiveness of Steinernema carpocapsae, S. glaseri, and Heterorhabditis bacteriophora against Galleria mellonella larvae was assessed in the laboratory. The presence of Hirsutella conidia on the third-stage (J3) cuticle of S. carpocapsae and H. bacteriophora interfered with infection of insect larvae. Conidia on the J3 cuticle of S. glaseri and on the ensheathing second-stage cuticle of H. bacteriophora did not reduce the nematodes'' ability to infect larvae. The LD₅₀ values for S. carpocapsae, S. glaseri, and H. bacteriophora in sand containing H. rhossiliensis were not different from those in sterilized sand when Galleria larvae were added at the same time as the nematodes. However, when Galleria larvae were added 3 days after the nematodes, the LD₅₀ of S. glaseri was higher in Hirsutella-infested sand than in sterilized sand, whereas the LD₅₀ of H. bacteriophora was the same in infested and sterilized sand. Although the LD₅₀ of S. carpocapsae was much higher in Hirsutella-infested sand than in sterilized sand, the data were too variable to detect a significant difference. These data suggest that H. bacteriophora may be more effective than Steinernema species at reducing insect pests in habitats with abundant nematode-parasitic fungi.     

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.     

Effect of Exsheathment on Motility and Pathogenicity of Two Entomopathogenic Nematode Species

The effect of sheath loss on motility and pathogenicity of the entomopathogenic nematodes, Heterorhabditis bacteriophora and Steinernema carpocapsae, was examined using both naturally and chemically exsheathed (desheathed) infective juveniles. Exsheathed S. carpocapsae showed increased motility on agar compared to sheathed nematodes. The presence of a host increased motility threefold in all S. carpocapsae treatments. These results suggest that activation of S. carpocapsae host finding may result from sheath loss in addition to host stimuli. Desheathed H. bacteriophora were significantly less motile than the sheathed or exsheathed groups. The decreased motility may be due to adverse effects of the chemical treatment for desheathment. Sheath loss did not affect the pathogenicity of either species.     

Effects of Enzymes,Chemicals, and Tempertaure on Steinernema carpocapsae Attraction to Host Plasma

Migration of exsheathed infective juveniles of Steinernema carpocapsae to plasma of the host insect Spodoptera litura was not affected by treatments with the lectins concanavalin A, soybean agglutinin, or wheat germ agglutinin; with the enzymes neuraminidase, α-mannosidase, lipase, pronase, or phospholipase C; or with cetyl trimethylammonium bromide or spermidine. Treatment with sodium metaperiodate or sodium hypochlorite inhibited nematode attraction towards insect plasma; numbers of randomly wandering nematodes increased. Nematode migration towards the source of attraction was unaffected by temperatures below 33 C but was impaired at 35 and 37 C. The adverse effect of 5 mM and 10 mM NaIO₄ on migratory behavior was reversed 24 hours after rinsing with buffered saline. The effect of NaOCl on nematode behavior was slightly reversible at concentrations of 0.2 and 0.4% (v/v) but apparently irreversible at 0.6 and 1.0%. The effect of heat treatment at 35 and 37 C was reversible.     

Biological Control of Soil Pests by Mixed Application of Entomopathogenic and Fungivorous Nematodes

In greenhouse experiments, massive application of the fungivorous nematode, Aphelenchus avenae, in summer at 26-33 C (1 x l0⁵ nematodes/500 cm³ autoclaved soil) or in autumn at 18-23 C (5 x 10⁴ nematodes/500 cm³ autoclaved soil) suppressed pre-emergence damping-off of cucumber seedlings due to Rhizoctonia solani AG-4 by 67% or 87%, respectively. Application of 2 x l0⁵ A. avenae to sterilized soil infested with R. solani caused leafminer-like symptom on the cotyledons, which did not occur in mixed inoculations with the entomopathogenic nematode, Steinernema carpocapsae. When 1 x 10⁶ A. avenae were applied 3 days before inoculation with 100 Meloidogyne incognita juveniles, gall numbers on tomato roots were reduced to 50% of controls. Gall numbers also were suppressed by S. carpocapsae (str. All). Reduction in gall numbers was no greater with mixed application of A. avenae and S. carpocapsae than with application of single species, even though twice the number of nematodes were added in the former case. These nematodes were positively attracted to tomato root tips. Aphelenchus avenae suppressed infection of the turnip moth, Agrotis segetum, but not the common cutworm, Spodoptera litura, by S. carpocapsae.     

Effects of Potassium Fertilization and Pratylenchus penetrans on Yield and Potassium Content of Red Clover and Alfalfa

Red clover and alfalfa were inoculated with Pratylenchus penetrans and grown in an Alberry sandy loam soil to which potassium (K⁺) was added at seeding at 0, 41.5, 83, and 166 μg/g. In one experiment with alfalfa, additional K⁺ was added after each forage cut to replace that which was removed. Nematode populations were not consistently affected by K⁺ fertilization. Nematode infection stunted red clover and alfalfa and resulted in lower yields at all K⁺ levels, except for alfalfa at the lowest K⁺ level. Nematode infection had no effect on taproot yields. However, it resulted in lower rootlet yields from red clover at all K⁺ levels, lower rootlet yields from alfalfa only at the highest K⁺ level in one experiment, and lower rootlet yields at all but the lowest K⁺ level in a second experiment, potassium fertilization enhanced yield of red clover and alfalfa. Yield increases were smaller from increased K⁺ fertilization in nematode-infested soil than in noninfested soil. Pralylenchus penetrans had little effect on the K⁺ content of red clover or alfalfa. The stunting of plants from nematode infection resulted in less K⁺ being removed from the soil.