Infectivity of Steinernema spp. (Nematoda: Steinernematidae) to Adult Litter Beetles, Alphitobius diaperinus (Coleoptera: Tenebrionidae) in the Laboratory

Three species of Steinernema, S. carpocapsae, S. feltiae, and S. scapterisci consisting of 12 different strains, were tested for their infectivity towards adults of the litter beetle Alphitobius diaperinus. Of the five most promising nematode strains, LC₅₀ values ranged from 1.5 to 77.0 nematodes per host in the filter paper assays. Assays in poultry litter material revealed LC₅₀ values to be 5.8 and 14.6 nematodes per host for the Mexican S. carpocapsae strain and Pye S. feltiae strain.     

Compatibility of Steinernema feltiae (Nematoda: Steinernematidae) with Pesticides and Plant Growth Regulators Used in Glasshouse Plant Production

Compatibility of the entomopathogenic nematode, Steinernema feltiae Filipjev with 17 different pesticides and three plant growth regulators, recommended for drench application in glasshouses, was evaluated. The products tested were two biofungicides, Streptomyces griseoviridis (Mycostop) and Trichoderma harzianum (Rootshield), nine chemical fungicides, iprodione (Chipco 26GT), thiophanate-methyl (Fungo Flo 4.5F), azoxystrobin (Heritage), fludioxonil (Medallion), flutolanil (Prostar), mefenoxam (SudDue Maxx 21.3ME), PCNB (Terraclor 400F), triflumizole (Terraguard), and etridiazole (Terrazole), two bioinsecticides, spinosad (Conserve SC) and Bacillus thuringiensis subsp. israelensis (Gnatrol), three chemical insecticides diflubenzuron (Adept IGR), acephate (Orthene), and fenoxycarb (Precision 25WP), one herbicide, clethodim (Envoy) and three plant growth regulators, ancymidol (A-Rest), paclobutrazol (Bonzi), and uniconazole-P (Sumagic). Infective juvenile nematodes were exposed to each product at the highest recommended concentration in 24-well plates at 22°C. Observations on the viability and infectivity of the nematodes were made at 4, 24, and 72 h after exposure. We found that S. feltiae is compatible with the majority of the tested formulations with no loss in viability and infectivity up to 24 h of exposure. The viability of S. feltiae was more than 80% in all the products even after 72 h of exposure. Three pesticide formulations, Prostar (1%), Gnatrol (9%), and Terrazole (10%) decreased the viability of S. feltiae significantly within 24 h compared to the controls. However, during the 24 to 72 hr incubation period, eight pesticides affected the viability of S. feltiae with Gnatrol and Terrazole causing the highest decrease (17% and 15%, respectively). Only Terrazole decreased the infectivity of S. feltiae to Galleria mellonella larvae compared to the control when tested after 24 h exposure. At 72 hr, Orthene and Terrazole caused significant decrease in the infectivity of S. feltiae (10% and 15%, respectively) and Gnatrol caused a significant increase in the infectivity (11%) compared to the control. Our results suggest that S. feltiae can be tank-mixed and applied in combination with all the tested formulations, except Terrazole.     

Latent infection: a low temperature survival strategy in steinernematid nematodes

1. Entomopathogenic nematodes penetrate and kill Galleria mellonella within 48 h at optimal temperatures.

2. Low temperature induces infection latency, preventing host death until optimal conditions resume.

3. Infected Galleria survived 25 days at 5°C. On transfer to 25°C, 100% and 12.5% of Steinernema carpocapsae and Steinernema riobravis infected larvae died within 72 h.

4. Infective juvenile penetration decreased with decreasing temperature; declining from 49.7 and 49.3 nematodes/host at 25°C to 6.3 and 0.25 nematodes/host at 5°C for S. carpocapsae and S. riobravis, respectively.

5. Latent infection occurs, albeit infrequently, due to low host penetration at low temperature.

Differential susceptibility of subterranean termite castes to entomopathogenic nematodes

We examined the attachment and infectivity of two entomopathogenic nematode species, Steinernema carpocapsae and Heterorhabditis indica, on soldiers and workers in two subterranean termite species, Coptotermes formosanus and C. vastator. In attachment tests with S. carpocapsae, more nematodes attached to soldiers of C. formosanus and C. vastator in the absence of workers compared to soldiers that were in the presence of workers. In tests with soldiers alone, workers alone, and mixture of equal numbers of workers and soldiers, soldiers in the mixed groups had lower mortality than soldiers alone after 1 and 4 days for both termite species. Exposure of small groups of either termite species to S. carpocapsae resulted in higher mortality of soldiers after 1 and 4 days post exposure. Mortality in soldiers alone exposed to H. indica was not significant compared to mortality in mixed groups at 1 day exposure, but was significant after 4 days. In concentration-mortality tests, a significant two-way interaction existed between nematode concentration and termite caste for C. formosanus exposed to S. carpocapsae for 1 and 4 days. A significant effect of nematode concentration was found for C. vastator exposed to H. indica for 1 day. This termite species had lower mortality when exposed to H. indica after 1 and 4 days compared to C. formosanus. At 4 days post exposure to H. indica, the effects of nematode concentration and termite caste were significant. Steinernema carpocapsae caused higher mortality than H. indica, but mortality of workers was higher when exposed to H. indica. Soldiers of both species experienced rapid mortality when exposed to S. carpocapsae, whereas soldiers of C. vastator experienced lower mortality when exposed to H. indica. Thus, our results show that when soldiers alone or workers alone are exposed to the nematodes, there is a differential susceptibility of soldiers and workers to nematode infection with soldiers being more susceptible than workers. In a mixed group of soldiers and workers or workers alone, it appears that grooming behavior of the workers serves as a mechanism to reduce nematode infection of soldiers and workers. The reason for this differential response to nematode infection is that soldiers do not exhibit grooming behavior.     

Comparative study of the effect of selected agrochemical products on Steinernema feltiae (Rhabditida: Steinernematidae)

The effect of three neurotoxic insecticides, three photosynthetic inhibitor herbicides and three enzymatic inhibitor herbicides on infective juveniles (IJs) of Steinernema feltiae Rioja (native) and ENTONEM® (commercial) strains were evaluated after a 48-h exposure at field tank concentrations and overnight treatment in mQ-water, using Spodoptera littoralis as target. Nematode survival was not affected by acetyl-cholinesterase inhibitors chlorpyrifos and pirimicarb, although chlorpyrifos seriously reduced their virulence. Both nematode strains showed differential sensitivity to cypermethrin, which affects the sodium channels of the nerve membrane, with the ENTONEM® strain being more tolerant than Rioja strain. However, these chemicals showed a strong sublethal effect on the nematode reproductive potential, limiting seriously their possible recycling in the field. Herbicides showed differential toxic effects on nematode survival. The commercial strain was tolerant to enzymatic inhibitor herbicides, whereas tribenuron and chlorsulfuron reduced Rioja strain survival. However, photosynthetic inhibitor herbicides severely affected survival of both nematode strains, with the Rioja strain being more sensitive. Sublethal effects on both nematode strains were observed only after exposition to terbutryn+chlortoluron+triasulfuron, increasing the time to kill insect larvae. These results are useful to optimize EPN dosages and to estimate their field recycling.     

Evaluation of the nematodes Steinernema feltiae and Thripinema nicklewoodi as biological control agents of western flower thrips Frankliniella occidentalis infesting chrysanthemum

In greenhouse studies, we evaluated a commercial formulation of the entomopathogenic nematode Steinernema feltiae and the inoculative release of the thrips-parasitic nematode Thripinema nicklewoodi against western flower thrips (WFT), Frankliniella occidentalis Pergande infesting potted chrysanthemums. Foliar sprays of S. feltiae applied at 1.25-2.5×10³ IJ mL^-1 and 1000 - 2000 L ha^-1 at 3-day intervals alone (targeting feeding stages) or in combination with soil applications (simultaneously treating non-feeding stages in the soil at the same rates) decreased but did not provide adequate control of thrips in flowering plants artificially infested with a dense population. Similar nematode treatments applied for four to five applications at 6-day intervals in two batches of initially clean chrysanthemums failed to prevent unacceptable damage to flowers and leaves from a dense natural infestation within the greenhouse. Although some IJ survived up to 48 h within flowers and flower buds, few nematode-infected thrips (larvae and adults) were recovered. In studies with T. nicklewoodi (which is not amenable for mass production), the inoculative releases of two parasitized hosts per plant enabled the nematode to become established within existing WFT populations under greenhouse conditions. However, relatively poor transmission and slow speed of kill (nematode primarily suppresses populations through host sterilization) prevented low level inoculations being effective over a single crop cycle. Further studies showed that transmission of T. nicklewoodi persisted for nine host generations, infected up to 83% of adult thrips and provided long-term suppression of discrete caged populations, but only after uneconomically high thrips densities had been reached.     

Infection of Anastrepha ludens (Diptera: Tephritidae) larvae by Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae) under laboratory and field conditions

Laboratory and field experiments were performed to determine the efficiency of the entomopathogenic nematode Heterorhabditis bacteriophora against third instar larvae of the tephritid fruit fly Anastrepha ludens. Infection was affected by low (6%) and high (12-24%) soil moisture; the highest prevalence of infection was observed at 9% moisture. LC₅₀ values were estimated under laboratory conditions at densities of 0.16, 0.26 and 0.64 larvae/cm³ of sand in containers of different depths (2, 5 and 8 cm) at 10% moisture, and larval ages (third instar, early versus late stadium). Third instar A. ludens were significantly more susceptible to infection early in the stadium than late in the stadium, irrespective of host density (LC₅₀ ∼15 infective juvenile nematodes/cm² soil surface). Infection of late stadium third instars was significantly reduced at low density. Application of 115 and 345 infective juvenile nematodes/cm² (representing one and three times the laboratory LC₅₀ at the lowest host density, respectively), in experimental plots in a commercial mango orchard, resulted in 46.7% (range of SE: 45.2-48.1) and 76.1% (SE: 74.8-77.3) infection, respectively. We conclude that H. bacteriophora merits further study as a natural enemy of Anastrepha spp. in tropical regions of the Americas.     

The Compatibility of Control Agents used for the Control of the South American Leafminer, Liriomyza huidobrensis

The compatibility of infective juveniles (IJs) of the entomopathogenic nematode, Steinernema feltiae, the parasitic wasps Diglyphus isaea and Dacnusa sibirica, and chemical insecticides to control larval stages of the South American leafminer, Liriomyza huidobrensis, on leafy salad crops was investigated. In intact leaf mines, leafminer larvae already parasitized by D. isaea which had developed to the larval or pupal stage, and D. sibirica in the larval stage were subsequently also infected by S. feltiae following a foliar application. This reduced the potential of the wasps to survive until the adult stage. In similar tests foliar applied chemical insecticides reduced the survival to adulthood of D. sibirica and their host larvae. However, a soil drench of imidaclorpid did not cause a significant reduction in the number of D. sibirica which survived the treatment and developed to adult emergence. Tests to investigate the utilisation of parasitoids following foliar applications of nematodes found that adult D. isaea did not discriminate between the healthy and nematode-infected leafminer larvae for host feeding, although 98% of eggs laid by the female wasps were deposited alongside healthy larvae.     

Effect of combination treatment with entomopathogenic fungi Beauveria bassiana and Nomuraea rileyi (Hypocreales) on Spodoptera litura (Lepidoptera: Noctuidaeae)

In biocontrol of insect pests, efficacy of treatment with multiple pathogens has not been frequently investigated but may have potential for effective management. The possible advantage of a combination treatment with two entomopathogenic fungi - Beauveria bassiana and Nomuraea rileyi - was assessed in laboratory bioassays on second instar Spodoptera litura. From among the fungal isolates of an epizootic population, two isolates of each fungus differing in virulence to S. litura were chosen, one highly virulent and the other with low virulence. The bioassays were carried out at either a continuous temperature of 25±1°C or at a temperature cycle of 32±2°C 8 h/21±2°C 16 h to mimic the field temperatures during the epizootic. Treatments with the two fungi were done both simultaneously and sequentially. In combination treatments at 25±1°C, in all isolate combinations, a majority of the larvae showed N. rileyi induced mycosis; the percentage mortality and speed of kill of insects in these treatments was similar to the N. rileyi isolate used in the combination treatments. At the temperature cycle of 32±2°C 8 h/21±2°C 16 h, in all combination treatments, all the dead insects exhibited B. bassiana mycosis; the mortality pattern was similar to the B. bassiana isolate used in the combination treatments. The adverse effect of high temperature on virulence of N. rileyi was however, not evident in in vitro growth assays. Combination treatment with both fungi did not have a synergistic effect on insect mortality.     

Biocontrol Potential of Entomopathogenic Nematodes Against Winter Moths (Operophtera brumata and O. fagata) (Lepidoptera: Geometridae) Infesting Urban Trees

Infectivity and biocontrol potential of entomopathogenic nematodes against winter moths (Operophtera brumata and O. fagata)pupating in the soil were examined in laboratory, semi-field and field conditions. A pilot experiment conducted in the field showed that Steinernema feltiae was completely ineffective against pupae of these moths in the soil. Subsequent laboratory tests revealed that none of the tested species (i.e. S. feltiae, S. affinae, S. carpocapsae, Heterorhabditis megidis and H. bacteriophora) could colonise the pupae, while mature larvae descending to the soil for pupation and prepupae were highly susceptible to nematode infection. No differences were observed between O. brumata and O. fagata in susceptibility to nematodes. In laboratory experiments H. megidis applied at 1.5×10⁵infective juveniles (IJ) m^-2infected almost 100% of insects exposed for 6 days in the soil. It was significantly more infective than H. bacteriophora (73-77%) and Steinernema species (29-50%). H. megidis was also highly effective in semi-field conditions when applied at an even lower dose, i.e. 10⁵IJ m^-2. After a 45-day experiment, only 3% of insects descending for pupation survived in the soil pre-treated with this species. This was significantly less than in soil with S. feltiae (43%) and control treated with water only (59%). Very high efficacy of H. megidis and a relatively easy method for its field application through ground spraying gives some promise for environmentally safe and successful biological control of winter moths during their pupation in the soil. The low application rate required and recycling in the host could be additional advantages for economic and long lasting protection of high value trees, particularly those in urban parks and forests.