Nematicidal Effects of Silver Nanoparticles on Root-knot Nematode in Bermudagrass

Certain nematodes are common soilborne organisms found in turfgrass in the United States that cause significant economic damage to golf course turf. One of the most prevalent plant-parasitic nematodes infesting turfgrass are root-knot nematodes (Meloidogyne spp.). Chemical treatment options for root-knot nematodes in turfgrass are limited, and there is a need for new nematicidal active ingredients to address this problem. In this study, we evaluated the use of silver nanoparticles (AgNP) as a potential nematicide in laboratory and field experiments. AgNP was synthesized by a redox reaction of silver nitrate with sodium borohydride using 0.2% starch as a stabilizer. When J2 of M. incognita were exposed to AgNP in water at 30 to 150 μg/ml, >99% nematodes became inactive in 6 hr. When turfgrass and soil composite samples infested with M. graminis were treated with 150 μg/ml AgNP, J2 were reduced in the soil samples by 92% and 82% after 4- and 2-d exposures, respectively, in the treated compared to the nontreated soil samples. Field trials evaluating AgNP were conducted on a bermudagrass (Cynodon dactylon × C. transvaalensis) putting green infested with M. graminis. Biweekly application of 90.4 mg/m² of AgNP improved turfgrass quality in one year and reduced gall formation in the roots in two years without phytotoxicity. The AgNP application did not significantly reduce the number of M. graminis J2 in plots during the growing season. The laboratory assays attested to the nematicidal effect of AgNP, and the field evaluation demonstrated its benefits for mitigating damage caused by root-knot nematode in bermudagrass.     

Management of Root-knot Nematodes by Phenamiphos Applied through an Irrigation Simulator with Various Amounts of Water

Phenamiphos (6.7 kg a.i./ha) was applied via an irrigation simulator to squash at planting (AP) and 2 weeks after planting (PP), and to corn AP and 1 week PP to manage root-knot nematodes (Meloidogyne incognita). The nematicide was applied with 0.25, 0.64, 1.27, and 1.91 cm surface water/ ha to a Lakeland sand in which the soil moisture was at or near field capacity. Based on efficacy and crop response, no additional benefits resulted when phenamiphos was applied in volumes of water greater than 0.25 crn/ha. The cost of applying each 0.25 cm of water over a hectare is approximately $1.08, or a 92% reduction in nematicide application cost over conventional methods ($13.50/ha). Low root-gall indices and high yields from squash and corn indicate more effective nematode management when phenamiphos was applied AP rather than PP. Results from this method of applying phenamiphos suggest that certain nematicides could be used as salvage alternatives when nematodes are detected in crops soon after planting. For multiple-pest management, nematicides, other compatible biocides, and fertilizers could be applied simultaneously with sprinkler irrigation.     

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.     

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.     

Effects of Selected Nematicides on Hatching of Heterodera schachtii

Aldicarb, carbofuran, fensulfothion, and phenamiphos were tested in concentrations of 1-100 μg/ml for their effects on hatching of Heterodera schachtii. Exposure of cysts to 1 μg aldicarb or carbofuran/ml stimulated hatch whereas phenamiphos and, to a lesser degree, fensulfothion inhibited hatch. Addition of aldicarb to sugarbeet root diffusate or 4 mM zinc chloride suppressed activities of these hatching agents. Transfer of cysts previously treated with aldicarb or carbofuran to zinc chloride or water rapidly initiated hatch which finally exceeded the hatch from cysts not treated with the nematicides.     

Parasitism of Xiphinema rivesi and X. americanum by Zoosporic Fungi

Living Xiphinema americanum (Xa) and X. rivesi (Xr) extracted from soil samples and stored for 1-5 days at 4 or 20 C contained aseptate fungal hyphae. The fungi directly penetrated the nematode''s cuticle from spores encysted near the head. Penetration through the stoma, vulva, or anus was rare. Catenaria anguillulae (Cat), Lagenidium caudatura (Lag), Aphanomyces sp. (Aph), and Leptolegnia sp. (Lep) were isolated into pure culture from infected nematodes. The pathogenicity of these zoosporic fungi was determined by incubating mixed freshly extracted Xa and Xr in 2% soil extract (pH = 6.7, conductivity = 48 μmhos, 20 ± 2 C) containing zoospores obtained from single-spore isolates. After 4 days, Cat, Lag, Aph, and Lep had infected 78, 18, 13, and 22%, respectively, of the nematodes. Both Xa and Xr were infected by every fungus; however, the relative susceptibility of Xa and Xr to these fungi was not determined. All noninoculated control nematodes remained uninfected and alive. In a second experiment, parasitism of Xa and Xr by Aph and Lep was increased when nematodes were incubated in 2% soil extract for 4 days before exposure to zoospores. In a third experiment, parasitism of Xa and Xr by Cat was greater in diluted saturation soil extract (conductivity = 100-400 μmhos) than in undiluted saturation extract (conductivity = 780 μmhos). Cat produced small zoospores (4-μm-d), bulbous infection hyphae, and assimilative hyphae of varying diameters in nematodes, whereas Lag, Aph, and Lep produced large zoospores (8-μm-d) and tubular, uniform infection and assimilative hyphae in nematodes.     

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.     

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.     

Nematode Control Related to Fusarium Wilt in Soybean and Root Rot and Zinc Deficiency in Corn

Nematode and disease problems of irrigated, double-cropped soybean and corn, and zinc deficiency of corn were investigated. Ethylene dibromide, phenamiphos, and aldicarb were equally effective for controlling nematodes and increasing yields of corn planted minimum-till and soybean planted in a moldboard plow prepared seedbed. The residual effects on yields of nematicides applied to the preceeding crop occurred during 3 years for soybean and 1 year for corn. Fusarium wilt symptoms of soybean that developed during 2 years of the study were less severe in all nematicide-treated plots than in control plots. Typical zinc deficiency symptoms on 30-day-old corn plants were observed during 1 year of the study in certain plots. Symptoms were not evident on plants grown on plots treated with ethylene dibromide, and only occasional plants had symptoms on plots treated with phenamiphos and aldicarb. The amount of yield response directly related to nematode control could not be determined because of the apparent interaction of nematodes on the expression of Fusarium wilt of soybean. Our study strongly indicates that the expression of Fusarium wilt of soybean and zinc deficiency in corn are influenced by nematodes and that nematicides will reduce their severity.     

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.     

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.     

Host Plant Resistance as an Alternative to Methyl Bromide for Managing Meloidogyne incognita in Pepper

Pre-plant soil fumigation with methyl bromide and host resistance were compared for managing the southern root-knot nematode (Meloidogyne incognita) in pepper. Three pepper cultivars (Carolina Cayenne, Keystone Resistant Giant, and California Wonder) that differed in resistance to M. incognita were grown in field plots that had been fumigated with methyl bromide (98% CH₃Br : 2% CCl₃NO₂ [w/w]) before planting or left untreated. Carolina Cayenne is a well-adapted cayenne-type pepper that is highly resistant to M. incognita. The bell-type peppers Keystone Resistant Giant and California Wonder are intermediate to susceptible and susceptible, respectively. None of the cultivars exhibited root galling in the methyl bromide fumigated plots and nematode reproduction was minimal (<250 eggs/g fresh root), indicating that the fumigation treatment was highly effective in controlling M. incognita. Root galling of Carolina Cayenne and nematode reproduction were minimal, and fruit yields were not reduced in the untreated plots. The root-galling reaction for Keystone Resistant Giant was intermediate (gall index = 2.9, on a scale of 1 to 5), and nematode reproduction was moderately high. However, yields of Keystone Resistant Giant were not reduced in untreated plots. Root galling was severe (gall index = 4.3) on susceptible California Wonder, nematode reproduction was high, and fruit yields were reduced (P ≤ 0.05) in untreated plots. The resistance exhibited by Carolina Cayenne and Keystone Resistant Giant provides an alternative to methyl bromide for reducing yield losses by southern root-knot nematodes in pepper. The high level of resistance of Carolina Cayenne also suppresses population densities of M. incognita.     

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.     

Resistance of Anhydrobiotic Aphelenchus avenae to Methyl Bromide Fumigation

The effect of methyl bromide (MB) was tested on active and anhydrobiotic Aphelenchus avenae. A. avenae was induced into anhydrobiosis by three different techniques. Both active and anhydrobiotic nematodes were subjected to 3,000 μ1 MB/liter air for 14 periods from 0 to 82 h. Anhydrobiotic nematodes were more resistant to fumigation than active nematodes, regardless of the technique used to induce anhydrobiosis. The percent survival decreased with increasing MB exposures (μ1 MB × h). For an LD₉₅ of 45,000-54,000 μ1/1 × h were required for active nematodes and >279,000 μ1/1 × h for anhydrobiotic nematodes.     

Evaluation of Paecilomyces lilacinus as a Biocontrol Agent of Meloidogyne javanica on Tobacco

The efficacy of the nematode parasite Paecilomyces lilacinus, alone and in combination with phenamiphos and ethoprop, for controlling the root-knot nematode Meloidogyne javanica on tobacco and the ability of this fungus to colonize in soil under field conditions were evaluated for 2 years in microplots. Combinations and individual treatments of the fungus grown on autoclaved wheat seed, M. javanica eggs (76,000 per plot), and nematicides were applied to specified microplots at the time of transplanting tobacco the first year. Vetch was planted as a winter cover crop, and the fungus and nematicides were applied again the second year to specified plots at transplanting time. The fungus did not control the nematode in either year of these experiments. The average root-gall index (0 = no visible galls and 5 = > 100 galls per root system) ranged from 2.7 to 3.9 the first year and from 4.3 to 5.0 the second in nematode-infested plots treated with nematicides. Plants with M. javanica alone or in combination with P. lilacinus had galling indices of 5.0 both years; the latter produced lower yields than all other treatments during both years of the study. Nevertheless, the average soil population densities of P. lilacinus remained high, ranging from 1.2 to 1.3 × 106 propagules/g soil 1 week after the initial inoculation and from 1.6 to 2.3 × 104 propagules/g soil at harvest the second year. At harvest the second year the density of fungal propagules was greatest at the depth of inoculation, 15 cm, and rapidly decreased below this level.     

Control of Globodera rostochiensis in Relation to Method of Applying Nematodes

Soaking potato tuber pieces for 15 min in 8,000 μg/ml of oxamyl just before planting reduced the number of Globodera rostochiensis cysts that developed on potato roots, but this treatment was phytotoxic. Five foliar applications of 1.12 kg a.i./ha of oxamyl or carbofuran at 10-day intervals beginning when 90% of the plants had emerged suppressed increase in G. rostochiensis densities. Similar foliar applications of phenamiphos were ineffective in controlling G. rostochiensis. Soil applications (in the row at planting) of aldicarb, carbofuran, phenamiphos, ethoprop, and oxamyl at 5.6 kg a.i./ha reduced the numbers of white females that developed on potato roots, but only those treatments involving aldicarb and oxamyl suppressed G. rostochiensis population increase. Combined soil and foliar treatments did not provide any advantage over soil treatment alone, as soil applications of 5.6 kg a.i./ha alone were equal to, or better than, combined soil (3.4 kg a.i./ha) and foliar (2.2 kg a.i./ha) applications in controlling G. rostochiensis.     

Effects of Crop Residue on the Persistence of Steinernema carpocapsae

We determined the effects of crop residue on the persistence of an entomopathogenic nematode, Steinernema carpocapsae. During 2 consecutive years, nematodes were applied at rates of 2.5 × 10₄ and 1.0 × 10⁵ infective juveniles/m² to small field plots planted with corn. Nematode persistence was monitored by exposing Galleria mellonella larvae to soil samples from plots with and without crop residue (approximately 75% coverage of soybean stubble). Persistence of S. carpocapsae was significantly greater in crop residue plots than in plots without residue. In crop residue plots that received the higher rate of nematode application, larval mortality did not significantly decrease during the study period (3 to 5 days) and remained above 85%. In nematode-treated plots without crop residue, however, larval mortality fell from over 96% to below 11% and 35% in the first and second trials, respectively. The increased crop residue may have benefited nematode persistence through protection from desiccation or ultraviolet light. We conclude that increased ground cover in cropping systems (e.g., due to reduced tillage) may lead to increased insect pest suppression with entomopathogenic nematodes.     

Sampling Citrus Fibrous Roots and Tylenchulus semipenetrans

Sampling precision was investigated for Tylenchulus semipenetrans juveniles and males in soil and females from roots and for citrus fibrous root mass density. For the case of two composite samples of 15 cores each, counts of juvenile and male nematodes were estimated to be within 40% of μ, at P < 0.06 (α) in orchards where $\bar{x}$ > 1,500 nematodes/100 cm³ soil. A similar level of α was estimated for measurements of fibrous root mass density, but at a precision level of 25% of μ. Densities of female nematodes were estimated with less precision than juveniles and males. Precision estimates from a general sample plan derived from Taylor''s Power Law were in good agreement with estimates from individual orchards. Two aspects involved in deriving sampling plans for management advisory purposes were investigated. A minimum of five to six preliminary samples were required to appreciably reduce bias toward underestimation of σ. The use of a Student''s t value rather than a standard normal deviate in formulae to estimate sample size increased the estimates by an average of three units. Cases in which the use of z rather than Student''s t is appropriate for these formulae are discussed.     

Applying Nematicides through an Overhead Sprinkler Irrigation System for Control of Nematodes

Phenamiphos, ethoprop, and carbofuran each at 6.7 kg a.i./ha were applied to squash, southern pea, and corn via injection into a sprinkler irrigation system. This method was then compared with a conventional application of phenamiphos and ethoprop granules spread on the soil surface and incorporated into the top 15 cm for control of Macroposthonia ornata and Meloidogyne incognita. Nematode populations in the soil and root-gall indices were lower, and yields greater, in treated than in untreated plots, but there were no significant differences between the methods of application in most comparisons.     

Growth and Energy Demand of Meloidogyne incognita on Susceptible and Resistant Vitis vinifera Cultivars

Food (energy) consumption rates ofMeloidogyne incognita were calculated on Vitis vinifera cv. French Colombard (highly susceptible) and cv. Thompson Seedless (moderately resistant). One-month-old grape seedlings in styrofoam cups were inoculated with 2,000 or 8,000 M. incognita second-stage juveniles (J2) and maintained at 17.5 degree days (DD - base 10 C)/day until maximum adult female growth and (or) the end of oviposition. At 70 DD intervals, nematode fresh biomass was calculated on the basis of volumes of 15-20 nematodes per plant obtained with a digitizer and computer algorithm. Egg production was measured at 50-80 DD intervals by weighing 7-10 egg masses and counting the number of eggs. Nematode growth and food (energy) consumption rates were calculated up to 1,000 DD based on biomass increase, respiratory requirements, and an assumption of 60 % assimilation efficiency. The growth rate of a single root-knot nematode, excluding egg production, was similar in both cultivars and had a logistic form. The maximum fresh weight of a mature female nematode was ca. 29-32 μg. The total biomass increase, including egg production, also had a logistic form. Maximum biomass (mature adult female and egg mass) was 211 μg on French Colombard and 127 μg on Thompson Seedless. The calculated total cost to the host for the development of a single J2 from root penetration to the end of oviposition for body growth and total biomass was 0.535 and 0.486 calories with a total energy demand of 1.176 and 0.834 calories in French Colombard and Thompson Seedless, respectively.