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.     

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.     

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.     

Enhanced cold tolerance of the entomopathogenic nematode Steinernema feltiae through genetic selection

Cold sensitivity of entomopathogenic nematodes severely restricts their biological control potential in some environments. We selected the SN strain of Steinernema feltiae together with its bacterial symbiont, Xenorhabdus bovenii, for improved cold tolerance by repeated passage through the wax moth Galleria mellonella larvae at 15°C. Nematode virulence (total insect mortality and speed of kill) and establishment (initiation of nematode development following penetration) were evaluated after six (= 12–24 generations) and 12 passages (= 24–36 generations). Cold selection enhanced nematode virulence at the cooler temperatures. Virulence measured as total insect-mortality at 8°C improved by 5.3- and 6.6-fold after six and 12 passages, respectively. Only small improvements (1.2–1.5-fold) were observed in speed of kill. Nematode establishment improved at all temperatures after 12 passages; the highest increase of 9-fold was observed at 8°C. Our results lend support to the hypotheses that functional traits along a continuously distributed environmental variable are genetically correlated and that the area under the fitness function is not always constant. Trade-offs in percentage mortality and speed of kill by the selected nematodes were observed at the warmer extreme after six passages of selection only. The implications of rapid changes in thermal sensitivity for economic mass-production of nematodes are discussed.     

Phoresy of the entomopathogenic nematode Steinernema feltiae by the earthworm Eisenia fetida

The free-living stage of entomopathogenic nematodes occurs in soil, and is an environmental-friendly alternative for biological control. However, their dispersal capability is limited. Earthworms improve soil characteristics, changing soil structure and influencing many edaphic organisms. Thus, earthworms could be used as vectors to introduce/disperse beneficial organisms. Nevertheless this interaction has not been studied in detail. This study presents the infectivity results of Steinernema feltiae after passing through the Eisenia fetida gut. Although entomopathogenic nematodes have no deleterious effects on earthworms, their passage through E. fetida gut seriously affected their mobility and virulence.     

Susceptibility of mushroom pests to the insect-parasitic nematodes Steinernema feltiae and Heterorhabditis heliothidis

The potential of two species of insect-parasitic rhabditid nematodes (Steinernema feltiae, Heterorhabditis heliothidis) for biological control of mushroom flies was studied in pot trials. Three Diptera that commonly infest mushroom crops were used; the larvae of Megaselia halterata (Phoridae), Heteropeza pygmaea (Cecidomyiidae) and Lycoriella auripila (Sciaridae) were all susceptible to parasitism by both nematode species. Fewer adult phorids and sciarids emerged when compost was nematode-treated and, for L. auripila, the effects of nematode applications at spawning, casing or on both occasions were compared. Casing treatments were more effective than spawning treatments; little extra benefit was gained from applying the nematodes twice. Populations of paedogenetic larvae of H. pygmaea built up rapidly in untreated compost, but were reduced when S. feltiae was applied, and were eradicated by H. heliothidis. Because they can penetrate insect cuticle, as well as natural body openings, Heterorhabditis spp. may be more suitable than Steinernema spp. for the control of mushroom fly larvae.     

Biocontrol potential of the entomopathogenic nematodes Heterorhabditis bacteriophora and Steinernema carpocapsae on cucurbit fly,Dacus ciliatus (Diptera: Tephritidae)

Entomopathogenic nematodes (EPNs) from the families Steinernematidae and Hererorhabditidae are considered excellent biological control agents against many insects that damage the roots of crops. In a regional survey, native EPNs were isolated, and laboratory and greenhouse experiments were conducted to determine the infectivity of EPNs against the cucurbit fly, Dacus ciliatus Loew (Diptera: Tephritidae). Preliminary experiments showed high virulence by a native strain of Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae) and a commercial strain of Steinernema carpocapsae Weiser (Rhabditida: Steinernematidae). These two strains were employed for further analysis while another native species, Steinernema feltiae, was excluded due to low virulence. In laboratory experiments, larvae and adult flies were susceptible to nematode infection, but both nematode species induced low mortality on pupae. S. carpocapsae had a significantly lower LC₅₀ value against larvae than H. bacteriophora in filter paper assays. Both species of EPNs were effective against adult flies but S. carpocapsae caused higher adult mortality. When EPN species were applied to naturally infested fruit (150 and 300 IJs/cm²), the mortality rates of D. ciliatus larvae were 28% for S. carpocapsae and 12% for H. bacteriophora. Both EPN strains successfully reproduced and emerged from larvae of D. ciliates. In a greenhouse experiment, H. bacteriophora and S. carpocapsae had similar effects on fly larvae. Higher rates of larval mortality were observed in sandy loam and sand soils than in clay loam. The efficacy of S. carpocapsae and H. bacteriophora was higher at 25 and 30°C than at 19°C. The results indicated that S. carpocapsae had the best potential as a biocontrol agent of D. ciliatus, based on its higher virulence and better ability to locate the fly larvae within infected fruits.     

Hybridisation and genetic selection for improving desiccation tolerance of the entomopathogenic nematode Steinernema feltiae

Shelf life of biological control products based on the entomopathogenic nematode Steinernema feltiae is rather limited. In order to prolong shelf life, the metabolism of nematodes during storage must be reduced, either by low temperature or by means of desiccation of the third stage dauer juveniles (DJs). Tolerance to desiccation is limited in S. feltiae. Their tolerance can be increased by an adaptation to moderate desiccation conditions. The objective of this study was to screen for tolerant strains among wild type populations of S. feltiae, hybridise most tolerant strains and further improve desiccation tolerance by subjection of the hybrid strain to genetic selection. Dehydrating conditions, measured as water activity (a _w-values), were produced by treating DJs with different concentrations of the polymer polyethylene glycol 600. Significant variation was recorded among 24 S. feltiae strains. The mean tolerated water activity survived by 50% of the population (WA₅₀) ranged from 0.78 to 0.93 when nematodes were not adapted to desiccation stress and from 0.66 to 0.88 when preadapted to desiccation stress. The six most desiccation tolerant strains of non-adapted and adapted nematode populations were crossed. Preadapted tolerance of hybrids was superior to the tolerance of parental strains, whereas non-adapted tolerance was not increased. The most tolerant hybrid had a WA₅₀ when preadapted of 0.67 and 0.86 when not adapted. The tolerance was lost after few reproductive cycles in the insect Galleria mellonella but was recovered again after six selection cycles with exposure to increasing stress conditions. Virulence and reproduction potential was not negatively affected by the selection. Stabilisation of the selection progress will be a major challenge to enable commercial exploitation of the genetic improvement.     

Desiccation tolerance among different isolates of the entomopathogenic nematode Steinernema feltiae (Fillipjev)

Poor storage capacity is a major constraint limiting further expansion of the use of entomopathogenic nematodes. In order to prolong shelf life, a quiescent state of the dauer juveniles (DJs) should be induced. This can be attained by means of desiccation of DJs. In this study, 24 natural isolations of Steinernema feltiae were exposed to desiccation stress in non-ionic polyethylene glycol 600. The dehydrating conditions were measured as water activity, a(w)-value. Non-adapted and adapted DJs were tested separately under a series of dehydrating conditions. The mean tolerated a(w)-value (MW50) ranged from 0.85 for the isolate NEP1 to 0.95 for FIN1, ISR5 and ITA2 when not adapted to desiccation stress and from MW50 of 0.822 for CR1 to 0.98 for ISR6 when adapted to the stress conditions. CR1 tolerated the lowest desiccation stress at an a(w)-value for the most tolerant 10% of the population (MW10) at 0.65 when DJs had been adapted to stress. No significant differences were recorded between all isolates in non-adapted DJs populations MW10 was compared. No correlation between tolerance under non-adapted and adapted conditions were found. Most tolerant isolates will now be used for cross-breeding and subsequent genetic selection to enhance desiccation tolerance.     

Populations of the phorid fly Megaselia halterata on an experimental mushroom unit over four years

Populations of phorid flies on an experimental mushroom unit were sampled simultaneously in both spawn-running and mushroom cropping-houses by sticky traps. Comparison between these populations showed that each reached a maximum about August and that the rate of increase, as measured by the ratio between them, varied from 22 × (at the beginning of February and in mid-August to 115 × (at the beginning of June and the end of December). Population growth within a crop was assessed from the peaks plotted from weekly catches. Three such peaks could be seen, starting from initial numbers which were high, on average, at casing but with little increase after the seventh week of cropping. However, population growth within crops, found by pooling data for different seasons, showed logistic increases though slopes varied from low, for May to June-spawned crops, to high for October spawning. Wide variations occurred in the proportion of the total flies represented in the initial catches in the first wk after casing. Partial regressions were examined for the numbers caught in cropping-houses on those taken in spawn-running rooms throughout the year. Catches for crops spawned during June to September gave lower slopes for high cropping-house populations. Spawn-running room catches showed a linear relation with outdoor 9.00 a.m. B.S.T. temperature and cropping-house numbers a logistic relation, a better fit obtaining with temperatures at spawning rather than 5 wk later during cropping. Catches during spawn-running were correlated with those during subsequent cropping and these with catches made during the previous crop on the unit, irrespective of site. Since it was bimodal, rate of increase between spawn-running and cropping-houses did not correlate with outdoor temperature but revealed some very high catches in winter. Many flies were trapped in the cropping-houses early in the year when flies were absent from spawn-running rooms. Evidence for movements within farms is discussed, especially with respect to overwintering of the phorid, which validates routine trapping of both populations.     

Efficacy of the entomopathogenic nematode, Steinernema feltiae, against Bemisia tabaci in relation to plant species

Abstract:  The use of infective juveniles (IJs) of the entomopathogenic nematode, Steinernema feltiae , to control the immature stages of the tobacco whitefly, Bemisia tabaci (Gennadius) (Hom., Aleyrodidae), on a range of host plants was investigated. Foliar applications of S. feltiae (10 000 IJs/ml) were made to tomato ( Lycopersicon esculentum ), cucumber ( Cucumis sativus ), poinsettia ( Euphorbia pulcherrima ), chrysanthemum ( Dendranthema spp.) and verbena ( Verbena hybrida ) infested with second instar B. tabaci , to determine whether efficacy was influenced by plant species. The effect of the adjuvants Agral, Triton X-100, methylcellulose, glycerol and spraying oil on the level of pest mortality was also assessed using two selected host plants, tomato and verbena. Following nematode application B. tabaci mortalities of 32, 28, 22 and 22% were recorded on tomato, cucumber, verbena and chrysanthemum, respectively, but a lower mean mortality was noted for whitefly feeding on poinsettia (10%). Mortality of B. tabaci on tomato and verbena was significantly increased by the addition of either Triton X-100 or Agral to the spray suspension. The use of Triton X-100 raised the mortality level to 63 and 37% on tomato and verbena, respectively, while 50 and 27% mortality followed the use of Agral on the two hosts. With the exception of glycerol no phytotoxic effects were observed by the adjuvants when applied to all five host plant species used in this study. The potential for use of the entomopathogenic nematode, S. feltiae , as a non-chemical alternative control measure for B. tabaci immatures is discussed.     

Field efficacy of the entomopathogenic nematode Steinernema feltiae against the Mediterranean flat-headed rootborer Capnodis tenebrionis

The Mediterranean flat-headed rootborer, Capnodis tenebrionis (L.) (Coleoptera: Buprestidae), is an economically important pest of stone fruit and seed fruit in Mediterranean areas. The potential control of the entomopathogenic nematode Steinernema feltiae (Filipjev) (strain Bpa), isolated from a dead C. tenebrionis larva, was tested in a cherry tree orchard in Ullastrell, Barcelona (Spain). Nematode infective juveniles (IJs) were applied by drench and injection. In both the treatments, a rate of 1 million IJs was applied per tree every week during 4 or 8 weeks, with a total dose of 4 × 10⁶ IJs/tree and 8 × 10⁶ IJs/tree. Number, stage and localization of insects in each tree trunk were recorded. In both the experiments, S. feltiae significantly reduced the population of C. tenebrionis providing control ranging from 88.3% to 97%. No significant differences were recorded between the different treatments. Persistence of nematodes was recorded until 6 weeks after application. Results indicate that the application of S. feltiae (Bpa) provides adequate control of C. tenebrionis in cherry trees.     

Effects of Application Rates of Steinernema feltiae (Nematoda: Steinernematidae) on Biological Control of the Mushroom Fly Lycoriella auripila (Diptera: Sciaridae)

The potential of Steinernema feltiae for the biological control of Lycoriella auripila was tested in commercial mushroom‐growing conditions. The nematodes, applied at rates of 1.5, 3, 6 or 12 x 10 ⁶ infective juveniles per 34 kg tray of spawn‐run compost, were mixed into the casing material before it was spread over the compost surface. When compared with untreated control trays, any rate of nematode application significantly reduced fly emergence. Insecticides significantly reduced mushroom yields; nematodes significantly increased them. At a rate of 3 x 10 ⁶ infectives/tray S. feltiae elicited mean total increases in the weight and numbers of mushrooms produced of 8% and 11% respectively. The nematodes also reduced the incidence of mushrooms spoiled by tunnelling sciarid larvae. The early decline in the numbers of nematodes persisting in casing was a trend that was reversed later, when evidence was obtained that S. feltiae was recycling in insects that had been killed. When applied at a rate of 3 ‐10⁶infectives/tray of compost S. feltiae should provide reliable and cost‐effective biological control of L. auripila.     

Comparative assessment of the efficacy of entomopathogenic nematode species at reducing western corn rootworm larvae and root damage in maize

The western corn rootworm (Diabrotica virgifera virgifera LeConte, Coleoptera: Chrysomelidae) is an invasive maize (Zea mays L.) pest in Europe. Crop yield is significantly impacted by the feeding of all three larval instars on maize roots, making them prime targets for control measures. Therefore, the control efficacy of three entomopathogenic nematodes (EPNs), Steinernema feltiae (Filipjev), Heterorhabditis bacteriophora Poinar, and H. megidis Poinar, Jackson and Klein (Nematoda: Rhabditida), was studied in four field plot experiments in southern Hungary in 2005 and 2006. All EPN species significantly reduced D. v. virgifera independently, whether applied as a row spray with a solid stream into the soil at sowing or onto the soil along maize rows in June. When applied at maize sowing, H. bacteriophora was more effective at reducing D. v. virgifera (81%) than H. megidis (49%) and S. feltiae (36%). When applied in June, H. bacteriophora and H. megidis were more effective at reducing D. v. virgifera (around 70%) than S. feltiae (32%). All tested EPN species significantly reduced damage on maize roots independently, whether they were applied at sowing or in June. Damage, however, was not totally prevented. The use of H. bacteriophora for the development of a biological control product for inundative releases against D. v. virgifera larvae is suggested.     

Susceptibility and behavioral responses of the dampwood termite Zootermopsis angusticollis to the entomopathogenic nematode Steinernema carpocapsae

Termites exploit microbially rich resources such as decayed wood and soil that are colonized by potentially pathogenic and parasitic fungi, bacteria, viruses, and nematodes. In colonies composed of thousands of individuals, the risk of infection among nestmates is significant, and individual and social behavior could involve various adaptations to resist disease and parasitism. Here we show that the dampwood termite Zootermopsis angusticollis (Hagen) exhibits a dosage dependent susceptibility to the soil nematode Steinernema carpocapsae (Weiser) (Mexican strain) and that this social insect significantly alters its behavior in response to this entomopathogenic roundworm. Relative to their baseline behavior, termites exposed to infective juveniles increased the frequency and duration of allogrooming and vibratory displays as well as two other novel behaviors, abdominal tip-raising and self-scratching. Whereas the first two behaviors likely reflect general adaptations to reduce susceptibility to a variety of pathogens and parasites, the latter behaviors might be specific to nematodes because they have never been observed in Z. angusticollis in any other pathogenic context. Our results support the hypotheses that behavioral responses in termites are important in the control of pathogenic and parasitic microorganisms and that termite susceptibility is socially mediated.     

Developmental temperature effects on five geographic isolates of the entomopathogenic nematode Steinernema feltiae (Nematoda: Steinernematidae).

The development of five geographic isolates of Steinernema feltiae at 5, 8, 10, 15, 20, 25, and 28 degrees C in wax moth, Galleria mellonella, larvae was examined. The isolates were from Mediterranean (Sinop from Turkey, SN from France, and Monterey from California), subtropical (Rafaela from Argentina), and tropical (MG-14 from Hawaii) regions. All isolates caused 100% mortality of wax moth larvae and developed and produced progeny between 8 and 25 degrees C. At 28 degrees C, mortality was 100%, but no progeny was observed. The highest infective juvenile production was observed at 15 degrees C for all isolates. In general, the tropical isolate, MG-14, had the lowest production of infective juveniles. The time of emergence of the infective juveniles from the host cadaver showed some differences among isolates, with the Sinop isolate having the earliest emergence time from cadavers at 15 degrees C (10 days) and 20 degrees C (8 days). At 25 degrees C, the infective juveniles of the Sinop, SN, and Rafaela isolates emerged from the cadavers from 5 to 7 days. Time of host death by all isolates showed no differences at 8, 10, 15, 20, and 28 degrees C. At 25 degrees C for all isolates (except the MG-14), shorter times to host death were observed. Host death occurred at 12 days at 8 degrees C, 9 to 11 days at 10 degrees C, 4 to 5 days at 15 degrees C, 3 days at 20 degrees C, and 2 days at 25 and 28 degrees C. For penetration efficiency, the Sinop, SN, and Rafaela isolates penetrated their hosts at 5, 8, and 10 degrees C. Penetration of the infective juveniles was consistently high for all isolates at 15, 20, 25, and 28 degrees C, but it was significantly lower for the MG-14 isolate at 15, 25, and 28 degrees C. No progeny production occurred at 28 degrees C, but nematode penetration did occur with the MG-14 isolate having significantly lower penetration than the other isolates. When nematodes were produced at 8, 15, and 23 degrees C in wax moth larvae, all isolates had infective juveniles with longer body lengths at 8 degrees C followed by 15 and 23 degrees C. To further verify body length at the different temperatures, beet armyworm, Spodoptera exigua, larvae and dog-food agar medium were used, respectively, for in vivo and in vitro culture of the Sinop isolate. Infective juvenile body length showed the same trends, with the longest being at 8 degrees C and decreasing in length from 15 to 23 degrees C. The data suggest that quality of food for the nematode and temperature (that is, developmental time) influence the body length of the infective juvenile.     

Biological control of sciarid and phorid pests of mushroom with predatory mites from the genus Hypoaspis(Acari: Hypoaspidae) and the entomopathogenic nematode Steinernema feltiae

In small-scale experiments, the predatory mites, Hypoaspis aculeifer (Canestrini) and H. miles Berlese, applied at 700 mites m(-2), and the entomopathogenic nematode, Steinernema feltiae (Filipjev) applied at 3 x 10(-6) nematodes m(-2) controlled sciarids and phorids in mushroom compost and casing substrates. For both mite species, earliest application to the growing substrate following sciarid infestation reduced sciarid emergence. In contrast, later application of each biological control agent provided more effective control of phorid emergence. The behaviour of adult mites suggested that H. aculeifer were more positively geotactic than H. miles although both species could penetrate compost and casing substrates to a depth of 2-12 cm. A majority of S. feltiae nematodes resided at a depth of 2-4 cm in both substrate types. Independent application of H. aculeifer provided more comprehensive control of sciarids and phorids than the other biological agents studied, owing to its better dispersal within compost and casing, and ability to attack larvae of differing ages.     

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.