Comparative Assessment of biological (Nematoda: Steinernema feltiae) and chemical methods of control for the mushroom fly Lycoriella auripila (Diptera: Sciaridae)

The sciarid fly Lycoriella auripila is the major pest of mushrooms cultivated in the UK. Its larvae, which are capable of damaging the crop at all stages of production, may cause severe yield losses and can only be controlled with chemical pesticides. An indigenous isolate of the insect‐parasitic nematode Steinemema feltiae was tested as a biological control agent and its effects compared with two commonly used insecticides, diazinon and diflubenzuron. The timing of application of nematodes was found to affect their efficacy. When applied to compost during spawning, nematodes did not significantly reduce fly emergence, but they did if applied at casing when they were almost as effective as diflubenzuron. Diazinon incorporated into compost did not reduce fly emergence and was also the only treatment that did not lower the incidence of mushrooms spoiled by tunnelling of the larvae of L. auripila. When compared with untreated control plots those treated both with diazinon and diflubenzuron showed significant mean losses in yield of 10% in total weight and 17% in total numbers of mushrooms picked. In contrast, when S. feltiae was applied at casing significant mean increases in yield of 7% and 19%, respectively, were attained. Infective nematodes persisted well in casing, very few were found on sporophores.     

Control of the mushroom pests Lycoriella auripila (Diptera: Sciaridae) and Megaselia halterata (Diptera: Phoridae) by Steinernema feltiae (Nematoda: Steinernematidae) in field experiments

The nematode Steinernema feltiae (Nematoda: Steinemematidae) was tested for its ability to control two main mushroom pests i.e. the sciarid Lycoriella auripila (Diptera: Sciaridae) and the phorid Megaselia halterata (Diptera: Phoridae) in growing-rooms filled with spawned compost. A clear difference between female and male sciarid control was observed. A nematode application 1 day after casing preceded by an application 1 day before casing on the compost caused an almost complete control (97%) of the F1-generation of female sciarids. The F2-generation of females was similarly controlled (95%) by an application 7 days after casing. A dosage of 1 × 10⁶nematodes m^-2was found to be equally effective as higher dosages. Diflubenzuron remained active throughout entire the cropping period with high sciarid mortality rates varying from 72% to 99%. Phorid control was variable and seemed to depend on the presence of sciarids. In one occasion the control rate of F2-generation phorid larvae was 75% and was possibly caused by the presence of new infective juvenile nematodes recycled in F2-generation sciarid larvae. Diflubenzuron did not significantly reduce phorid numbers.     

Selective breeding of Steinernema feltiae (Filipjev) (Nematoda: Steinernematidae) for improved efficacy in control of a mushroom fly,Lycoriella solani Winnertz (Diptera: Sciaridae)

A method of selecting a Steinernema feltiae strain that is effective against a mushroom fly, Lycoriella solani, is described in detail. The pest control efficacy of the selected nematode strain was evaluated and compared with the efficacy of two unselected strains. The selection procedure was designed to give preference to nematode individuals with the greatest ability (1) to search effectively for the target insect larvae in their natural habitat, (2) to infect them shortly after application and (3) to reproduce in their haemocoel. Thirty‐four rounds of selection achieved a 4‐fold improvement in nematode ability to find and parasitize third‐ and fourth‐instar larvae of the pest in the mushroom substrate. In 24‐h laboratory experiments, mortality of the insect caused by nematode juveniles rose from 22.5%, recorded for the original unselected isolate, to 92.5% for the selected strain. In a 51‐day experiment conducted on a mixed age mushroom house population of L. solani, the enhanced pest control ability of the selected strain was detected shortly after nematode application and remained high throughout the experimental period. During the first 4 weeks of the trial the selected nematode strain was significantly better than both unselected strains and caused 91.1–92.7% reduction of the fly emergence from the mushroom substrate. No difference was observed between the efficacy of the selected nematodes applied at 1 × 10⁶ and 3 ×10⁶ infective juveniles per m², while the unselected strains performed significantly better at the higher concentration. All the nematodes examined showed good persistence in the mushroom casing apparently due to recycling in the insect host.     

Comparative effects of Steinernema feltiae (Nematoda: Steinernematidae) and insecticides on yield and cropping of the mushroom Agaricus bisporus

In a mushroom crop (Agaricus bisporus) affected by a very low level of sciarid fly (Lycoriella auripila) infestation, the effects of an indigenous isolate of insect-parasitic nematode (Steinernema feltiae) and of two commonly used insecticides (diazinon and diflubenzuron) were studied. When compared with untreated plots, nematodes applied to the casing had no adverse effects on mushroom yields whereas insecticides decreased yields. At a rate of 3 × 10⁶ infective juveniles per tray (surface area = 0.56 m²), S. feltiae elicited increases of 28.5% and 19% in the mean total numbers and weights of mushrooms respectively. Treatment only with diflubenzuron resulted in 14.6% and 6% reductions in mean total numbers and weights of mushrooms, respectively; treatment with both diazinon and diflubenzuron caused 18.5% and 9.4% losses. Application of nematodes generally reduced the mean weight per mushroom whereas insecticides increased it; nematodes delayed the onset of mushroom production (first flush) whereas diflubenzuron delayed the third and fourth flushes. Nematode contamination of sporophores was minimal when S. feltiae was applied at casing. Although their numbers declined with time, the nematodes persisted, in the casing layer, throughout the cropping period of seven weeks. It is concluded that yield benefits associated with nematode application can result mainly from nematode effects on A. bisporus and not solely from suppression of a damaging pest population.     

Evaluation of a genetically selected strain of Steinernema feltiae against the mushroom sciarid Lycoriella mali

The control potential of a strain (ScP) of Steinernema feltiae genetically selected against the mushroom sciarid Lycoriella solani, was evaluated for the management of L. mali. Trials were conducted at two commercial mushroom farms with high and low levels of fly infestation. The efficacy of the ScP strain was compared with the SN strain of S. feltiae and the chitin synthesis inhibitor, diflubenzuron. At low densities of L. mali, the two strains did not differ in efficacy and caused 85–94% reduction in fly populations. At high fly densities with a mixed infestation of the phorid fly, Megaselia halterata, the ScP stain caused 56–83% reduction in L. mali populations whereas the SN strain caused 51–73% reduction. Two doses of the improved strain (0.5 times 10⁶ and 1.0 times 10⁶ infective juveniles per m² cropping area) did not differ in efficacy from diflubenzuron which caused 80% reduction in L. mali populations. The lower dose (0.5 times 10⁶) of the SN strain was less effective than diflubenzuron. The ScP strain had a major advantage over the SN strain in persistence in casing material. Nematodes had no significant effect on mushroom yields.     

The development of Steinernema feltiae (Nematoda Steinernematidae) in the sciarid fly Bradysia paupera (Diptera: Sciaridae)

There was only one generation of Steinernema feltiae in Bradysia paupera. Infection occurred after 3 h, adults developed 27 h after invasion and new infective juveniles (IJ) were produced after 48 h. Stunted females were produced in B. paupera larvae and in other small hosts and these stunted females produced small IJs. The small IJs were capable of infecting hosts and normal sized Us were produced in succeeding generations in Galleria mellonella.     

Location of immature stages of the mushroom insect pest Megaselia halterata in mushroom-growing medium

Experiments were conducted to examine the location of oviposition by the phorid fly Megaselia halterata (Wood) (Diptera: Phoridae) in uncased and cased compost. Clearly, a majority of the gravid females choose oviposition sites directly after entering the top layer of the compost. In uncased compost, 60% of all adults emerged from the top of four compost layers of equal thickness. When the compost was covered by a casing layer which was still uncolonized by Agaricus bisporus, oviposition was further concentrated in the top compost layer. In this situation, 91% of all adults emerged from the top compost layer whereas only 1.5% emerged from the casing. When the casing layer was colonized by mushroom mycelium, 45% of all adults emerged from the casing layer and 53% emerged from the top compost layer. Further concentration in the top compost layer and the casing layer occurred as a result of upward migration of larvae. When compost was cased after oviposition, up to 43% of all adults emerged from the casing layer. We concluded that in the control of phorid infestations with insect pathogenic nematodes, applications in uncased compost can be restricted to the upper compost layer. When compost and casing are filled simultaneously, nematode applications in the casing layer only could be considered.     

The use of chemicals, antagonists, repellents and physical barriers for the control of Lycoriella auripila (Diptera: Sciaridae), a pest of the cultivated mushroom Agaricus bisporus

The sciarid, Lycoriella auripila, is a serious pest of commercial mushroom production. A series of trials demonstrated that the use of early, specifically-targeted, treatments of insecticides and/or antagonists and repellents, which distance treatment time from crop harvest, have the potential to play a useful part in the control of initial and subsequent generations of this pest. Of the treatments examined, those involving a drench treatment of the compost at filling (before pasteurisation) proved to be the most effective. Cyromazine and diflubenzuron were the most active insecticides tested, with cyromazine achieving a superior level of control of the initial infestation. Repellents and antifeedants were also effective, with calcium oxalate and sinapic acid both achieving about 50% control when applied at filling. Treatments applied later during the production cycle, unless in combination with a treatment at filling, were progressively less effective at controlling both the initial sciarid infestation and later generations of larvae. Multiple treatments caused greater reductions in fly populations than did the single treatments and continued to do so throughout the cropping cycle, the greatest reduction in the initial generation (79%) occurring with a triple treatment of cyromazine. With the exception of some diflubenzuron treatments, those that were effective resulted in increases in yield. The use of a physical paper barrier caused significant increases in both fly numbers and total yield.     

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.     

Effects of Caenorhabditis elegans (Nematoda: Rhabditidae) on yield and quality of the cultivated mushroom Agaricus bisporus

Thirteen species of saprobic rhabditid nematodes (11 genera) were identified from samples of compost and casing material collected from mushroom farms in the British Isles. Caenorhabditis elegans, the most frequently found saprobe, was mass-produced monoxenically and its effects on the cultivated mushroom, Agaricus bisporus (strain U3) were studied. C. elegans did not multiply in well-prepared, pasteurised, spawned compost, whereas casing material proved to be a highly suitable environment for its reproduction. An initial casing inoculum of 10⁶ nematodes/crate of compost (7.5 kg), caused a significant reduction in mushroom yield. Losses in total mushroom yields of 11%, 20% and 26% were caused by initial inoculum rates of 10⁶, 10⁷and 2 × 10⁷ nematodes/crate, respectively. Yields were negatively correlated with the initial nematode inoculation level and regression equations were derived. The nematode treatments caused fewer mushrooms to be produced and an absence of the usual distinctive flushing patterns. C. elegans caused considerable deterioration in mushroom quality and characteristic distortion of mushrooms. Individual sporophores were mis-shapen, notched and had brown or violet coloured grills. Up to 3.8%, 6.7% and 10.8% of total weight and 3.5%, 5.4% and 8% of total numbers of mushrooms were distorted at the three highest nematode inoculum rates tested. Weights and numbers of distorted mushrooms were positively correlated with the initial nematode population. C. elegans commonly colonised sporophores.     

Effects of Caenorhabditis elegans (Nematoda: Rhabditidae) on the spread of the bacterium Pseudomonas tolaasii in mushrooms (Agaricus bisporus)

The effects of mass-produced saprobic rhabditid nematodes, Caenorhabditis elegans on the spread of the bacterial blotch pathogen, Pseudomonas tolaasii , were studied in mushroom growth chambers. C. elegans significantly reduced the intensity of blotch on sporophores. Repeated isolations of the bacterial flora from the gut of C. elegans recovered from mushroom sporophores during cropping, revealed the presence of Pseudomonas fluorescens biovar reactans . All the isolates of P. fluorescens biovar reactans isolated from nematodes were antagonists of P. tolaasii .
C. elegans produced much larger populations in monoxenic cultures with P. fluorescens biovar reactans than with P. tolaasii . It is suggested that as C. elegans selects P. fluorescens biovar reactans rather than P. tolaasii as a food substrate it probably spreads the antagonist in the mushroom crop and may contribute to the control of bacterial blotch.     

Comparison of the Efficacy of Entomopathogenic Nematodes for the Biological Control of the Mushroom Pests Lycoriella auripila (Sciaridae) and Megaselia halterata (Phoridae)

The efficacy of different species of entomopathogenic nematodes was tested against larvae of the mushroom phorid Megaselia halterata (Diptera: Phoridae) and the mushroom sciarid Lycoriella auripila (Diptera: Sciaridae). Sciarid larvae originating from infestations in casing soil during colonization by Agaricus bisporus were almost completely controlled by applications of Steinernema feltiae to the casing soil. When larvae originated from infestations in freshly spawned compost, they could be controlled by compost applications halfway through spawnrunning and by very early casing treatments. The control of phorids in compost was maximally 31% when nematodes were mixed within the infested compost at a concentration of 3 106 nematodes/m2. Only slightly higher reduction rates were obtained at higher concentrations. The control of phorids was more promising in the infested casing layer, in which S. carpocapsae was most successful. At concentrations of 6 and 15 106 nematodes/m2 this species obtained reduction rates of 65 and 73% respectively when it was applied 3 days after the end of the infestation period. These concentrations are, however, too high for practical application.     

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.     

Effectiveness of entomopathogenic nematodes Steinernema feltiae and Heterorhabditis bacteriophora on the Colorado potato beetle Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae) under laboratory and greenhouse conditions

In laboratory and greenhouse studies, the invading ability, virulence, and mortality caused by Stinernema feltiae and Heterorhabditis bacteriophora were compared. After one and two days of exposure to either nematode species, the mortality of Colordo potato beetle (CPB) Leptinotarsa decemlineata larvae at different instars, third and fourth, was recorded and the number of nematodes invading cadavers was more than the number of nematodes inside the larvae at the late last instar (one day before pre-pupa). Two concentrations, 250 and 500 IJs/dish, infective juvenile nematodes/0.5 ml were tested on different CPB larval instar. S. feltiae was more effective, with fourth instar rather than third and late last instar. On the other hand, H. bacteriophora showed a very weak effect with L. decemlineata. Also it was clear that S. feltiae was more effective and faster than H. bacteriophora: more than 70% of larvae were killed within 24 hours compared with H. bacteriophora which killed 40% of larvae within 48–72 hours. A significant difference in invading efficiency was observed with concentration 2500 IJs/pot in the greenhouse test. The number of adult females found in the cadavers of L. decemlineata larvae was always higher than the number of males. Foliage application of S. feltiae and H. bacteriophora resulted in a significant reduction of the number of damaged leaves and a lower index of damage compared with that in the control. We conclude that S. feltiae has significant potential and can help in the management of the Colorado potato beetle.     

Effect of Soil Depth and Moisture on the Vertical Distribution of Steinernema riobrave (Nematoda: Steinernematidae)

The effect of soil moisture on the distribution of Steinernema riobrave in a sand column was determined. Larvae of Pectinophora gossypiella were used to detect S. riobrave infective juveniles (IJ) in each 2.5-cm section of 30-cm-long soil columns. Soil moisture was determined for each section and related to the numbers of nematodes recovered from infected insect baits. Infective juveniles of S. riobrave applied on the sand column surface showed some degree of positive geotaxis. IJ in soil columns with a consistent moisture gradient grouped in the upper 12.7 cm within a water potential range of ¯40 to ¯0.0055 MPa (2% to 14% moisture). Nematodes in sand columns that were gradually dehydrating moved down the soil column, aggregating on the 28th day between 15-23 cm in depth. Nematode redistribution over time allowed IJ to remain within a water potential range of ¯0.1 to ¯0.012 MPa (5.2% to 9.5% moisture).     

Steinernema carpocapsae (Rhabditida: Steinernematidae) as a biological control agent against the fungus Gnat Bradysia agrestis (Diptera: Sciaridae) in propagation houses

A recently introduced fungus gnat, Bradysia agrestis, has caused serious problems in Korean propagation houses where vegetable seedlings are produced for transplant into the fields. Although chemical insecticides are available against this pest, alternate control measures are needed. A Korean isolate of the entomopathogenic nematode, Steinernema carpocapsae Pocheon strain, was tested against this insect in the laboratory and propagation house. In the laboratory, S. carpocapsae affected oviposition, with the untreated females laying an average of 121±25 eggs, whereas the treated females averaged 7±2 eggs. The infectivity of S. carpocapsae to the fungus gnat was affected by the developmental stage and temperature, with highest mortality observed with the third and fourth instars and pupal stage. Nematode mortality in the second instar fungus gnat ranged between 23 and 35%, but showed no significant differences among the temperatures tested. The egg and first instar were not infected by the nematode. In nematode dispersal studies, adult female fungus gnats alone dispersed S. carpocapsae from the nematode-treated area to the control area at a higher rate than male and female gnats or male gnats alone. In the propagation house experiments with watermelon seedlings, no significant difference was observed in fungus gnat larval reduction at S. carpocapsae concentrations of 5, 10, or 20 infective juveniles (IJs)/g of soil at 7, 14, and 21 days after treatment. In comparison with the control, the S. carpocapsae treatments significantly reduced B. agrestis larval numbers. When the watermelon seed was treated with S. carpocapsae at sowing, the larval density of B. agrestis was significantly reduced, that is, the number of B. agrestis larvae ranged from four to eight and from five to eight in the nematode-treated plots compared with 26 and 30 in the control plots on the 17th and 34th day post-treatment, respectively. In the chemical insecticide treatments, diflubenzuron and chlorpyrifos were significantly more effective than S. carpocapsae and diazinon in reducing larval populations of the fungus gnat. Our data show that, although some of the chemical insecticides were more effective than S. carpocapsae Pocheon strain, the nematode was still an effective tool for management of the fungus gnat larvae and in protecting seedlings from damage in propagation houses.     

Morphological and Ecological Characterization of Steinernema feltiae (Rhabditida: Steinernematidae) Rioja Strain Isolated from Bibio hortulanus (Diptera: Bibionidae) in Spain

A new strain of Steinernema feltiae (Rhabditida: Steinernematidae) was isolated in La Rioja (Spain) from larvae of Bibio hortulanus (Diptera: Bibionidae). A comparative morphometric analysis of this new strain and four additional S. feltiae isolates was performed. Although significant differences in morphometric measurements were observed, PCR-RFLP profiles and sequence analysis of the ITS region of rDNA confirmed the identity of the new strain as A2 RFLP type of S. feltiae. A comparative morphometric study among nematodes from three hosts, Galleria mellonella (Lepidoptera: Pyralidae), Spodoptera littoralis (Lepidoptera: Noctuidae) and B. hortulanus, was conducted. Ecological characterization of the Rioja isolate was performed in G. mellonella larvae. Larval mortality was 75.3 and 78.12% in penetration and sand column assays, respectively, and the percentage of penetrating infective juveniles was 12.0 and 2.8% in these assays. Larval mortality in the one-on-one bioassay was 4.2%, and in exposure-time bioassays, it was 50% at 11.25 hours. Relationships between morphometric characteristics and host mortality are discussed for this new strain of entomopathogenic nematode.     

Effects of Culture Method and Formulation on the Virulence of Steinernema riobrave (Rhabditida: Steinernematidae) to Diaprepes abbreviatus (Coleoptera: Curculionidae)

The Diaprepes root weevil, Diaprepes abbreviatus, is a pest of vegetables, ornamental plants, sugarcane, and citrus in Florida and the Caribbean. The entomopathogenic nematode, Steinernema riobrave, can reduce larval populations of D. abbreviatus substantially. Efficacy of entomopathogenic nematodes, however, may be affected by culture method and formulation. Using D. abbreviatus as the host, we compared the efficacy of two commercial S. riobrave formulations, a liquid and a waterdispersible granule (WDG), with each other and with in vivo produced S. riobrave. Nematodes in the commercial formulations were produced in vitro through liquid fermentation; the in vivo nematodes were cultured in Galleria mellonella and applied in aqueous suspension. Laboratory experiments measured nematode virulence in plastic cups containing soil and seventh-eighth instar D. abbreviatus. One laboratory experiment was conducted using only fresh nematodes (less than 5 days old); another experiment included WDG nematodes that were stored for 25 days at 10 °C. Two field experiments were conducted in which nematodes were applied either to potted citrus (containing D. abbreviatus larvae) placed beneath mature citrus trees or to soil directly beneath the tree. In the latter experiment, efficacy was determined by measuring mortality of caged D. abbreviatus larvae that were buried beneath the soil surface prior to application. Mortality of D. abbreviatus treated with nematodes ranged from 80-98% and 50-75% in laboratory and field experiments, respectively. In all experiments, we did not detect any significant effects of culture method or formulation.