Root Cortical Cell Spherical Bodies Associated with an Induced Resistance Reaction in Monoxenic Cultures of Meloidogyne incognita

The root-knot nematode Meloidogyne incognita was monoxenically cultured on excised roots of soybean cv. Pickett and tomato cv. Rutgers in agar media containing either 0 to 1,600 μg/ml ammonium nitrate or 0 to 100 μg/ml urea. Observations with scanning and transmission electron microscopy indicated that an elevated concentration of ammonium nitrate or urea inhibited giant cell formation and suppressed nematode development in the infected soybean roots. In the tomato roots, concentrations of ammonium nitrate above 400 μg/ml or urea above 25 μg/ml inhibited giant cell formation and nematode development. Coincident with the nitrogen concentrations that suppressed giant cell formation was the appearance of electron-dense spherical bodies in the cortical parenchyma cells of both the soybean and tomato roots. These bodies, which were 1-4 μm in diameter, appeared to form in the cytoplasm and migrate to the cell vacuole.     

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.     

Growth of Potato and Control of Pratylenchus penetrans with Oxamyl-treated Seed Pieces in Greenhouse Studies

Oxamyl was applied to both uncut and cut potato tubers in aqueous solutions of 1,000 to 32,000 μg/ml. Emergence in greenhouse pots was delayed for a day or more after soaking cut tuber pieces in 32,000 μg/ml. After 10 weeks plant growth was greater, relative to the control, when Pratylenchus penetrans-infested soil was planted with cut tubers soaked for 20 minutes in 32,000 μg/ml. Soaking for 40 minutes did not increase nematode control nor affect plant growth. Oxamyl applied to tubers at 1,000 μg/ml reduced the numbers of P. penetrans in the soil by 20% and in the roots by 35%; at 32,000 μg/ml, the numbers of P. penetrans in the soil were reduced by 73-86% and in the roots by 86-97%. The numbers of P. penetrans did not increase in the roots of plants developed from cut tubers soaked in 32,000 μg/ml over a period of 10 weeks, but numbers of lesion nematodes had begun to increase in the soil.     

Nematicidal Effects of Oxamyl Applied to Leaves of Banana Seedlings

Foliar applications of oxamyl prevented nematodes from invading roots of diploid bananas. One spray with 1,250 μg/ml was more effective than 1, 2, or 3 sprays with 625 μg/ml applied at 5-day intervals. After 3 sprays with 1,250 μg/ml, invasion may be prevented for up to 4 weeks and possibly longer. Washing roots after oxamyl treatments prevented nematicidal control. When applied to nematode-infected plants, three sprays of oxamyl decreased nematode populations in the roots.     

Penetration of 1,2-Dibromo-3-Chloropropane in a Florida Soil

Gas-liquid chromatography was used to detect movement of the nematicide, 1,2-dibromo-3-chloropropane (DBCP), in soil columns containing top- and subsoil of Astatula fine sand. Topsoil contained 1.4-1.6% organic matter and subsoil 0.20-0.25%. DBCP was applied at various rates as aqueous drenches. Depth of penetration was controlled by organic matter in topsoil and varied with the amount of water applied. Maximum DBCP penetration after 14 days was 28 cm; maximum water infiltration, 115 cm. Maximum depth of penetration was obtained with a water emulsion of 30 μg/ml of DBCP applied in 15 cm of water. DBCP applied in 5 cm of water to soils containing 2.0% and 0.125% organic matter penetrated 6 cm and 60 cm, respectively.     

Nicotine Content of Tobacco Roots and Toxicity to Meloidogyne incognita

The motility of Meloidogyne incognita second-stage juveniles (J2) and their ability to induce root galls in tomato were progressively decreased upon exposure to nicotine at concentrations of 1-100 μg/ml. EC₅₀ values ranged from 14.5 to 22.3 μg/ml, but J2 motility and root-gall induction were not eliminated at 100 μg/ml nicotine. Nicotine in both resistant NC 89 and susceptible NC 2326 tobacco roots was increased significantly 4 days after exposure to M. incognita. The increase was greater in resistant than in susceptible tobacco. Root nicotine concentrations were estimated to be 661.1-979.1 μg/g fresh weight. More M. incognita were detected in roots of susceptible than in roots of resistant tobacco. Numbers of nematodes within resistant roots decreased as duration of exposure to M. incognita was increased from 4 to 16 days. Concentrations of nicotine were apparently sufficient to affect M. incognita in both susceptible and resistant tobacco roots. Localization of nicotine at infection sites must be determined to ascertain its association with resistance.     

In-Vitro and In-Vivo Effects of Aldicarb on Survival and Development of Heterodera schachtii

Aqueous solutions of 5-500 μg/ml aldicarb inhibited hatching of Heterodera schachtii. Addition of hatching agents, zinc chloride, or sugarbeet root diffusate, to the aldicarb solutions did not decrease the inhibition of hatching. When cysts were removed from the aldicarb solufions and then treated for 4 wk in sugarbeet root diffusate, larvae hatched and emerged. Treatments of newly hatched larvae of H. schachtii with 5-100 μg/ml aldicarb depressed later development of larvae on sugarbeet (Beta vulgaris). Similar treatments with aldicarb sulfoxide had less effect on larval development, and aldicarb sulfone had no effect. Numbers of treated larvae that survived and developed were inversely proportional to concentration (0.1-5.0 μg/ml) and duration (0-14 days) of aldicarb treatments. Development of H. schachtii on sugarbeet grown in aldicarb-treated soil was inversely proportional to the concentration of aldicarb in the tested range of 0.75 - 3.0 μg aldicarb/g of soil. Transfer of nematode-infected plants to soil with aldicarb retarded nematode development, whereas transfer of plants first grownin treated soil to nematode-infested soil only slightly suppressed nematode development. Development of H. schachtii was inhibited in slices of storage roots of table beet (B. vulgaris), sugarbeet and turnip, (Brassica rapa), that had grown in soil treated with aldicarb.     

A Bioassay to Estimate Root Penetration by Nematodes

An in vitro bioassay with a 96-well microtiter plate was used to study the effect of lectins on burrowing nematode penetration of citrus roots. In each well, one 4-mm root segment, excised from the zone of elongation of rough lemon roots, was buried in 0.88 g dry sand. Addition of a Radopholus citrophilus suspension containing ca. 300 nematodes in 50 μ1 test solution completely moistened the sand in each well. The technique assured uniform treatment concentration throughout the medium. Within 16-24 hours, burrowing nematodes penetrated citrus root pieces, primarily through the cut ends. The lectins (100 μg/ml) Concanavalin A (Con A), soybean agglutinin (SBA), wheat germ agglutinin (WGA), and Lotus tetragonolobus agglutinin (LOT) stimulated an increase in penetration of citrus root segments by Radopholus citrophilus. Concentrations as low as 12.5 μg/ml Con A, LOT, and WGA stimulated burrowing nematode penetration of citrus roots. Heat denaturation of the lectins reversed their effect on penetration; however, incubation of nematodes in lectin (25 μg/ml) with 25 mM competitive sugars did not. The reason for enhanced penetration associated with lectins is unclear.     

Interaction of Vesicular-arbuscular Mycorrhizal Fungi and Phosphorus with Meloidogyne incognita on Tomato

The influence of two vesicular-arbuscular mycorrhizal fungi and phosphorus (P) nutrition on penetration, development, and reproduction by Meloidogyne incognita on Walter tomato was studied in the greenhouse. Inoculation with either Gigaspora margarita or Glomus mosseae 2 wk prior to nematode inoculation did not alter infection by M. incognita compared with nonmycorrhizal plants, regardless of soil P level (either 3 μg [low P] or 30 μg [high P] available P/g soil). At a given soil P level, nematode penetration and reproduction did not differ in mycorrhizal and nonmycorrhizal plants. However, plants grown in high P soil had greater root weights, increased nematode penetration and egg production per plant, and decreased colonization by mycorrhizal fungi, compared with plants grown in low P soil. The number of eggs per female nematode on mycorrhizal and nonmycorrhizal plants was not influenced by P treatment. Tomato plants with split root systems grown in double-compartment containers which had either low P soil in both sides or high P in one side and low P in the other, were inoculated at transplanting with G. margarita and 2 wk later one-half of the split root system of each plant was inoculated with M. incognita larvae. Although the mycoorhizal fungus increased the inorganic P content of the root to a level comparable to that in plants grown in high P soil, nematode penetration and reproduction were not altered. In a third series of experiments, the rate of nematode development was not influenced by either the presence of G. margarita or high soil P, compared with control plants grown in low P soil. These data indicate that supplemental P (30 μ/g soil) alters root-knot nematode infection of tomato more than G. mosseae and G. margarita.     

Influence of Glomus intraradices and Soil Phosphorus on Meloidogyne incognita Infecting Cucumis melo

The interaction among Glomus intraradices, Meloidogyne incognita, and cantaloupe was studied at three soil phosphorus (P) levels in a greenhouse. All plants grew poorly in soil not amended with P, regardless of mycorrhizal or nematode status. In soil amended with 50 μg P /g soil, M. incognita suppressed the growth of nonmycorrhizal plants by 84%. In contrast, growth of mycorrhizal plants inoculated with M. incognita was retarded by only 21%. A similar trend occurred in plants grown in soil with 100 μg P /g soil. Mycorrhizal infection had no effect on the degree of root-knot gall formation and did not affect the number of nematode eggs per egg mass. Mineral levels in plant shoots generally declined as soil P levels increased and were not significantly influenced by G. intraradices or M. incognita.     

Effects of Aldicarb and Its Sulfoxide and Sulfone on the Biology of Tylenchulus semipenetrans

In laboratory testing, egg hatch of Tylenchulus semipenetrans was stimulated at concentrations of 1 and 10 μg/ml aldicarb solution and inhibited at 50 and 100 μg/ml. Aldicarb was more inhibitory to egg hatch than the aldicarb sulfoxide and the aldicarb sulfone. Inhibition of hatch at the high concentration was associated with delays in the molting processes, lack of larval movement within the egg, and delays in embryonic development. Nematode motility was reduced at 10, 50, and 100 μg/ml of aldicarb and aldicarb sulfoxide solution, and at 50 and 100 μg/ml aldicarb sulfone. Male development was retarded at 10 μg/nrl and almost completely inhibited at 50 and 100 μg/ml of the three chemicals. In greenhouse tests, female development antl reproduction on roots of citrus seedlings were suppressed by aldicarb at rates of 2.6 μg/ml and completely inhibited at 10.6 μg/ml of soil solution during a 50-day experimental period. Under field conditions, there was little systemic movement of aldicarb into roots located outside treated areas. Aldicarb reduced the nematode larvae and the female adult population in the second year after the second treatment. There were no differences in egg hatch and sex ratio of citrus nematodes between treated and nontreated roots.     

Method for Establishing a Bacterial Inoculum on Corn Roots

Few bacteria from the corn rhizosphere grew in media with 50 μg of mancozeb per ml. A mancozeb-resistant Pseudomonas strain from the rhizosphere was serially subcultured in media containing mancozeb and spectinomycin until it was resistant to 175 μg of mancozeb and 850 μg of spectinomycin per ml. The population of the pseudomonad added to soil fell to low numbers in 6 days in unamended or glucose-amended soil, but its numbers exceeded 10⁵/g for at least 12 days if the soil was supplemented with mancozeb. The numbers of this organism remained small on corn roots derived from untreated, inoculated seeds, but the population was two or more orders of magnitude greater on roots derived from mancozeb-coated seeds. The abundance of the inoculum strain on the 3-cm portion of roots nearest the stem declined markedly after about 1 week, but applying urea to the foliage reduced or prevented the decline. The numbers of the pseudomonad on segments of roots 3- to 6- and 6- to 9-cm from the stem were higher on plants derived from the mancozeb-coated seeds. Applying spectinomycin to the foliage did not promote growth of the bacterium. This method is proposed as a means to establish an introduced bacterium on plant roots.     

Control of Heterodera schachtii with Foliar Application of Nematicides

Foliar applications of ethyl 4-(methylthio)-m-tolyl isopropylphosphoramidate (phenamiphos) or S-methyl 1-(dimethylcarbamoyl)-N-[(methylcarbamoyl)oxy] thioformimidate (oxamyl) retarded infection of sugarbeets by the sugarbeet nematode, Heterodera schachtii under greenhouse conditions. Maximum nematode control was obtained when treatments were applied previous to, or at the time of, inoculation of plants with the nematode. Consecutive foliar applications inhibited nematode development, with four applications giving greatest inhibition of maturation. A treatment with either phenamiphos or oxamyl at 2,000 μg/ml (ppm) resulted in the greatest increase in plant growth, and 4,000 μg/ml gave the best nematode control. A treatment of 4,000 μg/ml of either phenamiphos or oxamyl was phytotoxic. However, this was due to container confinement of the chemical since phytotoxicity at this rate has not been observed under field conditions.     

In Vitro Hatch and Survival of Heterodera glycines as Affected by Alachlor and Phenamiphos

Heterodera glycines (race 1) eggs were exposed to aqueous solutions o f selected concentrations o f the herbicide alachlor and the organophosphate nematicide phenamiphos alone and in herbicide-nematicide combinations. Phenamiphos (0.5 μg/ml) + alachlor (0.063, 0.125, or 1.0 μg/ ml) treatments increased the incidence o f juvenile hatch over that of untreated controls at 18 days. At 18 and 25 days, phenamiphos (0.5 μg/ml) treatments contained more juveniles than did phenamiphos at 1.0 μg/ml. Phenamiphos (1.0 μg/ml) alone and in combination with alachlor (1.0 μg/ ml) suppressed hatch for 21 days and juvenile survival for more than 21 days. Alachlor treatments enhanced juvenile survival compared to the untreated control at 14 and 21 days. Technical alachlor gave results similar to those of the formulated product.     

Nematicide Treatments of Turfgrass Seed

Seed treatments of improved Kentucky bluegrass and fescue cultivars with carbofuran, oxamyl, and phenamiphos dissolved in acetone reduced seedling emergence, but treatments were not extremely phytotoxic. Phenamiphos was the most toxic, particularly at the 5,000 μg/ml concentration. Fresh weight of grass clippings 35 d following planting generally was greater in treatments than in controls except for the 5,000 μg/ml phenamiphos treatments on certain cultivars. All nematicide seed treatments reduced the number of Pratylenchus penetrans subsequently recovered from Pennlawn creeping red fescue roots 4-5 wk after treatment. The infusion of nematicides into grass seed with organic solvents appears to be an effective means of reducing nematode damage to turfgrass seedling with little environmental hazard.     

Efficacy of 1,2-Dibromo-3-chloropropane for Control of Meloidogyne javanica as Influenced by Concentration,Exposure Time and Rate of Degradation

Laboratory experiments were conducted by applying 1,2-dibromo-3-chloropropane (DBCP) to sealed vials of soil infested with Meloidogyne javanica. A minimum initial concentration of 0.25 μg of DBCP/g of oven-dry soil killed all nematodes within 35 days. A concentration of 1.0 μg/g killed all nematodes within 28 days. The rate of degradation of this chemical was determined by treatment of steamed and nonsteamed dry soil in open and sealed vials. Extraction of tile chemical, followed by quantification by gas chromatography, showed approximately 100% of the amount applied recovered after 14 days in sealed vials without soil. With soil present, approximately 10% of the amount of chemical applied was recovered.     

The Hypersensitivity Reaction of Tomatoes Resistant to Meloidogyne incognita: Reversal by Cytokinins

Initiation of larval growth, induction of cell necrosis, and gall formation in the host were measured as criteria of resistance or susceptibility of tomato seedlings to the root-knot nematode, Meloidogyne incognita (Kofoid &White) Chitwood. Seedlings grown at 27 C on water agar containing additions were scored 3 or 4 days after infection.In the absence of exogenous plant growth regulatory substances, approximately 73% of larvae that entered roots of susceptible plants showed growth, none induced necrosis and nearly all induced gall formation. In roots of a resistant variety, only 4% of the larvae grew, 88% induced necrosis of host cells, and only 29% induced galls. Exogenously supplied cytokinins shifted the response of the resistant plants toward the susceptible reaction. Exogenous kinetin at 0.4 and 0.8 µmolar allowed 55 and 57% of the nematodes to grow, reduced the incidence of necrosis to 32 and 31%, and increased gall formation to 73 and 65%. Three additional cytokinins, Zeatin, 6-(γ,γ-dimethylallylamino)purine, and 6-benzylaminopurine produced effects similar to kinetin. Exogenous indoleacetic acid, gibberellic acid, adenine, guanine, uracil, thymine, cytidine, and 6-methylaminopurine neither increased the percentage of larvae which grew nor decreased the extent of host cell necrosis.     

Sensitivity of Meloidogyne incognita and Rotylenchulus reniformis to Abamectin

Avermectins are macrocyclic lactones produced by Streptomyces avermitilis. Abamectin is a blend of B_1a and B_1b avermectins that is being used as a seed treatment to control plant-parasitic nematodes on cotton and some vegetable crops. No LD₅₀ values, data on nematode recovery following brief exposure, or effects of sublethal concentrations on infectivity of the plant-parasitic nematodes Meloidogyne incognita or Rotylenchulus reniformis are available. Using an assay of nematode mobility, LD₅₀ values of 1.56 μg/ml and 32.9 μg/ml were calculated based on 2 hr exposure for M. incognita and R. reniformis, respectively. There was no recovery of either nematode after exposure for 1 hr. Mortality of M. incognita continued to increase following a 1 hr exposure, whereas R. reniformis mortality remained unchanged at 24 hr after the nematodes were removed from the abamectin solution. Sublethal concentrations of 1.56 to 0.39 μg/ml for M. incognita and 32.9 to 8.2 μg/ml for R. reniformis reduced infectivity of each nematode on tomato roots. The toxicity of abamectin to these nematodes was comparable to that of aldicarb.     

A combination of sbmA and tolC mutations in Escherichia coli K-12 Tn10-carrying strains results in hypersusceptibility to tetracycline

Previously, we demonstrated that Escherichia coli tolC mutations reduce the high-level resistance to tetracycline afforded by the transposon Tn10-encoded TetA pump from resistance at 200 μg/ml to resistance at 40 μg/ml. In this study, we found that the addition of an sbmA mutation to a tolC::Tn10 mutant exacerbates this phenotype: the double mutant did not form colonies, even in the presence of tetracycline at a concentration as low as 5 μg/ml. Inactivation of sbmA alone partially inhibited high-level tetracycline resistance, from resistance at 200 μg/ml to resistance at 120 μg/ml. There thus appears to be an additive effect of the mutations, resulting in almost complete suppression of the phenotypic expression of Tn10 tetracycline resistance.