Biological Control of the Phytoparasitic Nematode Mesocriconema xenoplax on Peach Trees

Seven fluorescent Pseudomonas spp. capable of inhibiting reproduction of Mesocriconema xenoplax have been isolated from soil sites that suppress both nematode multiplication and Peach Tree Short Life (PTSL). One of these seven strains, Pseudomonas sp. BG33R, inhibits M. xenoplax multiplication in vivo and egg hatch in vitro. Mesocriconema xenoplax populations on peach seedlings inoculated with BG33R and planted into soil-solarized field plots remained at or below the economic threshold for nematicide treatment in South Carolina for nearly 18 months. Soil solarization alone induced a shift toward a microbial community that was suppressive to nematode multiplication. Additionally, five Tn5 mutants of BG33R, lacking the ability to kill eggs, have been generated. The Tn5 insertion site in each mutant has been cloned and sequenced. DNA sequence analysis has revealed a high degree of homology to several genes of interest because of their potential involvement in the production of the egg-kill factor. These Tn5 egg-kill negative mutants also no longer produce protease or salicylic acid while producing nearly twice the amount of fluorescent siderophore as the wild type parent.     

Dynamics of Concomitant Populations of Meloidogyne incognita and Criconemella xenoplax on Peach

The interaction between Meloidogyne incognita and Criconemella xenoplax on nematode reproduction and growth of Lovell peach was studied in field microlots and the greenhouse. Meloidogyne incognita suppressed reproduction of C. xenoplax in both field and greenhouse experiments. Tree growth, as measured by trunk diameter, was reduced (P ≤ 0.05) in the presence of M. incognita as compared with C. xenoplax of the uninoculated control trees 26 months following inoculation. A similar response regarding dry root weight was also detected in greenhouse-grown seedlings after 5 months. The presence of C. xenoplax did not affect Lovell tree growth. A synergistic effect causing a reduction (P ≤ 0.05) in tree growth was recorded 26 and 38 months following inoculation. The presence of M. incognita increased levels of malonyl-1-aminocyclopropane-1-carboxylic acid content in leaves of trees grown in field microplots 19 months after inoculaoon. Meloidogyne incognita appears to be a more dominant parasite than C. xenoplax on Lovell peach.     

Influence of Criconemella xenoplax and Pruning Time on Short Life of Peach Trees

Influences of Criconemella xenoplax and pruning dates were studied in field microplots with ''Nemaguard'' peach cuttings on a site not previously planted to peaches. Trees with or without C. xenoplax were pruned beginning in December 1984 or March 1985. Peach tree short life (PTSL) did not occur in the absence of C. xenoplax. PTSL occurred earlier in December-pruned than in March-pruned inoculated trees. Results confirm that "old" peach sites are not required for PTSL to occur. Pruning Nemaguard and ''Lovell'' greenhouse-grown seedlings reduced the root mass of both stocks and stimulated Nemaguard, but not Lovell, shoot regrowth. Numbers of C. xenoplax per gram of dry root were greater on pruned than on unpruned seedlings.     

Peach Leaf Senescence Delayed by Criconemella xenoplax

Fall annual leaf senescence of peach was delayed in the field and in microplots in the presence of Criconemella xenoplax. Soil from the rhizosphere of orchard trees with greener leaves had ca. 2.5 × more nematodes than soil around trees in a more advanced state of fall senescence. In microplots, monoclonal antibody enzyme immunoassay (EIA) of leaf cytokinins indicated that concentration of zeatin riboside-like substances and chlorophyll content were greater in leaves of trees growing in nematode-infested soil than in trees in uninfested soil. EIA also indicated the presence of substances resembling trans-zeatin, zeatin riboside, dihydrozeatin, and dihydrozeatin riboside-like substances in whole body homogenates of C. xenoplax. Levels of zeatin-like substances were present in the nematode in greater levels than the other related substances.     

Compatibility of Soil Amendments with Entomopathogenic Nematodes

The impact of inorganic and organic fertilizers on the infectivity, reproduction, and population dynamics of entomopathogenic nematodes was investigated. Prolonged (10- to 20-day) laboratory exposure to high inorganic fertilizer concentrations inhibited nematode infectivity and reproduction, whereas short (1-day) exposures increased infectivity. Heterorhabditis bacteriophora was more sensitive to adverse effects than were two species of Steinernema. In field studies, organic manure resulted in increased densities of a native population of Steinernema feltiae, whereas NPK fertilizer suppressed nematode densities regardless of manure applications. Inorganic fertilizers are likely to be compatible with nematodes in tank mixes and should not reduce the effectiveness of nematodes used for short-term control as biological insecticides, but may interfere with attempts to use nematodes as inoculative agents for long-term control. Organic manure used as fertilizer may encourage nematode establishment and recycling.     

Biological Control of the Pecan Weevil,Curculio caryae (Coleoptera: Curculionidae), with Entomopathogenic Nematodes

Steinernema carpocapsae (Weiser) strain A11, S. feltiae (Filipjev) strain SN, and Heterorhabditis bacteriophora Poinar strains HP88 and Georgia were tested for their efficacy as biological control agents of the pecan weevil, Curculio caryae (Horn), in pecan orchard soil-profile containers under greenhouse conditions. Percentage C. caryae parasitism by S. carpocapsae and H. bacteriophora strain HP88 and Georgia was consistently poor when applied either prior to or following C. caryae entry into the soil, suggesting that these nematode species and (or) their enterobacteria are poor biological control agents of weevil larvae. Soil taken 21 days following application of S. carpocapsae or H. bacteriophora strain HP88 induced a low rate of infection of Galleria mellonella larvae, whereas soil that had been similarily treated with H. bacteriophora strain Georgia induced a moderate rate of infection. Percentage C. caryae parasitism by S. feltiae was consistently low when applied following C. caryae entry into the soil and was inconsistent when applied as a barrier prior to entry of weevil larvae into the soil. Soil taken 21 days following application of S. feltiae induced a high rate of infection of G. mellonella larvae.     

Use of Entomopathogenic Nematodes to Suppress Meloidogyne incognita on Greenhouse Tomatoes

Tomato seedlings in a growth chamber were inoculated with 150 Meloidogyne incognita eggs and 25 infective juveniles (IJ)/cm² of Steinernema feltiae, S. riobrave, or Heterorhabditis bacteriophora. With the exception of seedling roots treated with H. bacteriophora, all seedlings treated with entomopathogenic nematodes had fewer M. incognita juveniles inside roots and produced fewer eggs than the control seedlings. Tomato plants in the greenhouse were infested with 4,000 M. incognita eggs and treated 2 weeks before, 1 week before, at the same time, 1 week after, or 2 weeks after with 25 or 125 IJ/cm² of S. feltiae, S. riobrave, or H. bacteriophora. Plants with pre- and post-infestation applications of S. feltiae or S. riobrave suppressed M. incognita. Plants treated with H. bacteriophora 1 week before and at the time of infestation suppressed M. incognita. Increasing the rate of H. bacteriophora and S. feltiae from 25 to 125 IJ/cm² improved M. incognita suppression.     

Development Rates in Entomopathogenic Nematodes: Infected Hosts vs. Aqueous Suspension

Rearing conditions have been shown to affect several aspects of entomopathogenic nematode biology, including dispersal behavior and infectivity. The present study explores the differences in development rate of Heterorhabditis bacteriophora and Steinernema carpocapsae when infective juveniles (IJ) were collected in water using the standard White trap method vs. natural emergence from cadavers into sand. We exposed Galleria mellonella to IJ entompopathogenic nematodes treated in one of three ways: collected in a White trap, allowed to emerge directly into sand, or collected in a White trap and treated with a cadaver homogenate. When S. carpocapsae IJ were allowed to emerge from cadavers directly into sand and then allowed to infect new hosts, they developed into adults at a faster rate than IJ that were collected with White traps. The difference in development was not due to differential infection rates. No difference in development stages was detected amount the same H. bacteriophora treatments.     

Effect of Initial Population Density of Criconemella xenoplax on Reducing Sugars,Free Amino Acids,and Survival of Peach Seedlings over Time

Percentage of mortality, growth suppression, and changes in free amino acid and reducing sugar content in root and (or) stem tissue of Nemaguard peach seedlings were studied in the greenhouse in relation to time and eight different initial population densities (Pi) of Criconemella xenoplax. After 90 and 180 days, free amino acid content in root tissue significantly increased with increasing nematode numbers. Suppression of root volume, dry root and stem weight, height increase, plant survival, and content of reducing sugars in root tissue were detected at 180 and 270 days and following pruning. All criteria were negatively correlated with nematode Pi. Changes in growth, metabolic parameters, and survival percentage were attributed to Pi density of C. xenoplax, duration of the experiment, and nematode reproduction rate.     

Entomopathogenic Nematodes for Control of Insect Pests Above and Below Ground with Comments on Commercial Production

Entomopathogenic nematodes (EPNs) have been utilized in classical, conservation, and augmentative biological control programs. The vast majority of applied research has focused on their potential as inundatively applied augmentative biological control agents. Extensive research over the past three decades has demonstrated both their successes and failures for control of insect pests of crops, ornamental plants, trees and lawn and turf. In this paper we present highlights of their development for control of insect pests above and below ground. The target insects include those from foliar, soil surface, cryptic and subterranean habitats. Advances in mass-production and formulation technology of EPNs, the discovery of numerous efficacious isolates/strains, and the desirability of reducing pesticide usage have resulted in a surge of commercial use and development of EPNs. Commercially produced EPNs are currently in use for control of scarab larvae in lawns and turf, fungus gnats in mushroom production, invasive mole crickets in lawn and turf, black vine weevil in nursery plants, and Diaprepes root weevil in citrus in addition to other pest insects. However, demonstrated successful control of several other insects, often has not lead to capture of a significant share of the pesticide market for these pests.     

Field Application of Entomopathogenic Nematodes for Control of Delia radicum in Collards

Control of Delia radicum (cabbage maggot) in field collards (Brassica oleracea) was compared after one or two applications of entomopathogenic nematodes, Steinernema carpocapsae (All strain) and Heterorhabditis bacterophora (HP88 strain), a single application of granular chlorpyrifos, and a water-only treatment. Nematodes were applied with a sprayer during the egg stage of first-generation D. radicum, and chlorpyrifos was hand placed around collard stems during the same period. A second nematode application was made 10 days later. Chlorpyrifos treatment resulted in fewer puparia per plant, less root damage and higher yield than all other treatments, including the control. Collard yield from nematode-treated beds did not differ from controls. These data indicate that, under these field conditions, the species or strains of entomopathogenic nematodes tested did not reduce the number of active cabbage maggots, nor did they prevent collard root damage.     

Effects of Mesocriconema xenoplax on Vitis vinifera and Associated Mycorrhizal Fungi

Previous surveys of vineyards had indicated that Mesocriconema xenoplax was present in 85% of vineyards in western Oregon, but yields were not depressed in established vines. Microplot studies were initiated in 1997 in a Willamette Valley vineyard to determine the impact of M. xenoplax on vine establishment. Plots were infested with 0.03, 0.6, and 3.0 M. xenoplax g^-1 soil and planted with self-rooted Chardonnay and Pinot Noir vines. In November 2000, four growing seasons after planting, pruning weights, fine root weights, and fruit yield of vines planted in infested soil were reduced by 58%, 75%, and 33%, respectively, relative to control vines (planted in noninfested soil). In 1998 with ca 2000 degree-day base 9 °C accumulation, population densities increased 32-fold and 44-fold on 1-year-old Chardonnay and Pinot Noir vines, respectively. Nematode population dynamics and pruning data suggested that the carrying capacity of vines in microplots was 5 to 8 M. xenoplax g^-1 soil. In November 2000, more than 80% of the fine root length was colonized by arbuscular mycorrhizal fungi in all treatments. The frequency of fine roots containing arbuscules (the site of nutrient transfer between plant and fungus), however, was depressed from 5% to 65% in plants infested initially with M. xenoplax as compared to controls. Competition for photosynthate within the root system is proposed as a possible mechanism by which nematodes suppressed arbuscule frequency.     

Influence of Salinity on Survival and Infectivity of Entomopathogenic: Nematodes

Exposure to NaC1, KCI, and CaCl₂ affected the entomopathogenic nematodes Heterorhabditis bacteriophora and Steinernema glaseri differently. Survival, virulence, and penetration efficiency of S. glaseri were not affected by these salts. At high concentrations, however, all three salts inhibited its ability to move through a soil column and locate and infect a susceptible host. Calcium chloride and KCl had no effect on H. bacteriophora survival, penetration efficiency, or movement through a soil column, but moderate concentrations of these salts enhanced H. bacteriophora virulence. NaCl, however, adversely affected each of these parameters at high salinities (>16 dS/m). Salt effects on S. glaseri are attributed solely to interference with nematode host-finding ability, whereas the NaCl effects on H. bacteriophora are attributed to its toxicity and possibly to interference with host-finding behavior.     

Tests for Transmission of Prunus Necrotic Ringspot and Two Nepoviruses by Criconemella xenoplax

In two of three trials, detectable color reactions in ELISA for Prunus necrotic ringspot virus (PNRSV) were observed for Criconemella xenoplax handpicked from the root zone of infected peach trees. Criconemella xenoplax (500/pot) handpicked from root zones of peach trees infected with PNRSV failed to transmit the virus to cucumber or peach seedlings. The nematode also failed to transmit tomato ringspot (TomRSV) or tobacco ringspot viruses between cucumbers, although Xiphinema americanum transmitted TomRSV under the same conditions. Plants of peach, cucumber, Chenopodium quinoa, and Catharanthus roseus were not infected by PNRSV when grown in soil containing C. xenoplax collected from root zones of PNRSV-infected trees. Shirofugen cherry scions budded on Mazzard cherry seedling rootstocks remained symptomless when transplanted into root zones of PNRSV-infected trees. Virus transmission was not detected by ELISA when C. xenoplax individuals were observed to feed on cucumber root explants that were infected with PNRSV and subsequently fed on roots of Prunus besseyi in agar cultures. Even if virus transmission by C. xenoplax occurs via contamination rather than by a specific mechanism, it must be rare.     

Competition Between Tylenchorhynchus annulatus and Mesocriconema xenoplax on Grain Sorghum as Influenced by Macrophomina phaseolina

Greenhouse experiments were conducted to examine competition between Tylenchorhynchus annulatus and Mesocriconema xenoplax on grain sorghum roots that were colonized by the fungus Macrophomina phaseolina or free from fungus colonization. An incomplete factorial treatment design consisted of two levels of M. phaseolina (0 or 10 colony-forming units/g soil) and 12 T. annulatus:M. xenoplax ratios: 1,000:0; 750:0; 500:0; 250:0; 0:0; 0:250; 0:500; 0:750; 0:1,000; 750:250; 500:500; and 250:750. Plants were harvested after 105 days. Despite similar feeding habits, competition between these ectoparasitic nematode species was limited. Tylenchorhynchus annulatus was more susceptible to antagonism by M. xenoplax than the reverse, but susceptibility depended on initial inoculum ratio. Root colonization by M. phaseolina reduced competitive effects of T. annulatus on M. xenoplax but not the reverse. Both nematode species reduced shoot dry weight but only T. annulatus reduced root dry weight. Both plant weight parameters were reduced by M. phaseolina.     

Effect of Application Method on Fitness of Entomopathogenic Nematodes Emerging at Different Times

The entomopathogenic nematode species Steinernema feltiae and Heterorhabditis bacteriophora were compared for survival and infectivity of infective juveniles (IJ) collected with a standard White trap (i.e., emerging from hosts and accumulating in water) and later applied to sand (treatment A) to IJ allowed to emerge from hosts into sand (treatment C). Percentage IJ survival and infectivity was compared between treatments for S. feltiae IJ that emerged between days 1 to 3 and days 4 to 6. For H. bacteriophora, percentage IJ survival and infectivity was compared between treatments only for infective juveniles that emerged between days 4 to 6. For S. feltiae IJ percentage survival and infectivity decreased with time (P ≤ 0.05) and was greater (P ≤ 0.05) for IJ from treatment C than for IJ from treatment A. For H. bacteriophora IJ percentage survival decreased (P ≤ 0.05) and percentage infectivity increased (P ≤ 0.05) with time. While percent survival was higher (P ≤ 0.05) for treatment C than for A, percent infectivity was not different between treatments.     

Competition Between Entomopathogenic and Free-Living Bactivorous Nematodes in Larvae of the Weevil Diaprepes abbreviatus

Field and laboratory experiments were conducted to determine the degree to which free-living, bactivorous nematodes (FLBN) are able to competitively displace entomopathogenic nematodes (EPN) from insect cadavers. Two hundred larvae of the insect Diaprepes abbreviatus were buried at regular intervals during 2 years in experimental plots that were untreated or treated twice annually with Steinernema riobrave. Larvae were recovered after 7 days, and nematodes emerging from cadavers during the next 30 days were identified. The monthly prevalence of FLBN was directly related to that of S. riobrave (r = 0.38; P = 0.001) but was not related to the prevalence of the endemic EPN, S. diaprepesi, Heterorhabditis zealandica, H. indica, or H. bacteriophora (r = 0.02; P = 0.80). In a second experiment, treatment of small field plots with S. riobrave increased the prevalence of insect cadavers in which only FLBN were detected compared to untreated controls (30% vs. 14%; P = 0.052), and increased numbers of FLBN per buried insect by more than 10-fold. In the laboratory, sand microcosms containing one D. abbreviatus larva were treated with (i) the FLBN, Pellioditis sp.; (ii) S. riobrave; (iii) S. riobrave + Pellioditis; or (iv) neither nematode. Insect mortality was higher in the presence of both nematodes (57%) than when S. riobrave was alone (42%) (P = 0.01). An average of 59.2 Pellioditis sp. g^-1 insect body weight emerged in the presence of S. riobrave, whereas 6.2 nematodes g^-1 insect were recovered in the absence of the EPN (P = 0.01). Pellioditis sp. reduced the number of S. riobrave per cadaver by 84%; (P = 0.03), and per available insect by 82% (P = 0.001), compared to S. riobrave alone. Population size of S. diaprepesi was not affected by Pellioditis sp. in experiments of the same design. Faster development (P = 0.05) and nutrient appropriation within the insect cadaver by S. diaprepesi compared to S. riobrave may increase the fitness of the former species to compete with Pellioditis sp. The results of these studies demonstrate the potential of FLBN to regulate population densities of EPN and to dampen estimates of EPN-induced mortality of insect pests in the field.     

Association of Criconemella xenoplax and Fusarium spp. with Root Necrosis and Growth of Peach

Criconemella xenoplax, Fusarium solani, and F. oxysporum caused necrosis of Nemaguard peach feeder roots in greenhouse tests. Root necrosis was more extensive in the presence of either fungus than wtih C. xenoplax alone. Shoot growth and plant height were less for plants inoculated with F. oxysporum or F. solani than for plants inoculated with the fungi plus C. xenoplax. Neither synergistic nor additive effects on root necrosis or plant growth occurred between C. xenoplax and the fungal pathogens.     

Population Characteristics and Dosage Trajectory Analysis for Mesocriconema xenoplax in California Prunus Orchards

The Mesocriconema xenoplax population increased exponentially in a newly planted peach orchard. The rate of increase was greater on Nemaguard than on Lovell rootstock and was reduced by postplant nematicides. Population levels were more stable in an established almond orchard on Nemaguard rootstock. All life stages of the nematode were present year round; lower ratios of juveniles to adults in summer suggested adverse effects of temperature and dry soil. Also in summer, there was a smaller proportion of the population in the upper 30 cm of soil than at greater depths. Nematode dosage, average nematode density multiplied by accumulated degree-days (physiological time) of the sampling interval, was useful in quantifying nematode stress on trees and as an indicator of the nematode management effectiveness. The annual trajectory of the nematode dosage could be determined by two samplings, one in spring and one in fall. A nematode predator, the parasitic fungus Hirsutella rhossiliensis, did not regulate ring nematode populations in the newly planted orchard; a recovery period was necessary for increase in the prevalence of parasitism.     

Optimization of Inoculation for In Vivo Production of Entomopathogenic Nematodes

Entomopathogenic nematodes are potent biopesticides that can be mass-produced by in vitro or in vivo methods. For in vivo production, consistently high infection rates are critical to efficiency of the process. Our objective was to optimize in vivo inoculation of Steinernema carpocapsae and Heterorhabditis bacteriophora in Galleria mellonella and Tenebrio molitor by determining effects of inoculation method, nematode concentration, and host density. We found immersing hosts in a nematode suspension to be approximately four times more efficient in time than pipeting inoculum onto the hosts. The number of hosts exhibiting signs of nematode infection increased with nematode concentration and decreased with host density per unit area. This is the first report indicating an effect of host density on inoculation efficiency. We did not detect an effect of nematode inoculum concentration on nematode yield per host or per gram of host. Yield was affected by host density in one of the four nematode-host combinations (S. carpocapsae and T. molitor). We conclude that optimization of inoculation parameters is a necessary component of developing an in vivo production system for entomopathogenic nematodes.