Mass-culture technique of a South African <i>Heterorhabditis bacteriophora</i> isolate, using <i>in vitro</i> liquid culture

Entomopathogenic nematodes (EPNs) as bio-control have been reported as conventional and intensified method in insect pest management. In the present investigation, a survey was carried out in district Sirmaur to detect the presence of EPNs. In total, 110 soil samples were collected from the rhizospheric soil of fruit plantations and among them 35 samples possess EPNs. Based on morphological analysis, all the isolated EPNs were identified as Heterorhabditis bacteriophora. The insecticidal potential of local isolate of EPN H. bacteriophora strain EUPT-S26 was evaluated against two different larval instars (3rd and 4th instars) of Pieris brassicae (cabbage butterfly) under the laboratory conditions. Different concentrations of nematode inoculums @ 30 infective juveniles (IJs), 60 IJs, 90 IJs, 120 IJs and 150 IJs/ml along with control were applied against both the instars. The rate of pathogenicity was recorded upto 96 h. It was observed that both the instars of P. brassicae were highly susceptible for H. bacteriophora strain EUPT-S26 infection. The inoculum level of 150 IJs was causing highest mortality to both 3rd and 4th larval instars at 96 h of exposure. The data recorded from the present study shows lethal concentration LC50=29.13 IJs (95% FL=21.92–38.71) in 3rd instars and LC50=21.62 IJs (95% FL=16.06–29.10) against 4th instar larvae after 96 h of nematodes exposure. It was concluded from the present investigation, that the local isolates of H. bacteriophora strain EUPT-S26 showed significant mortality among both the larval instars that keeps on enhanced with the increase in nematode inoculum concentration and exposure time. Further studies are required to explore their insecticidal potential against other insect pests in the field condition also.

Antagonists and defense mechanisms of entomopathogenic nematodes and their mutualistic bacteria

Impact of egg yolk type in liquid culture production of Steinernema carpocapsae JAP1 and their virulence

Entomopathogenic nematodes (EPNs) of the family Steinernematidae are effective biological control agents against important pest insects. The in vitro liquid culture method of mass production is used to produce EPNs of high quantity, quality and with reduced cost by upscaling. The first step in liquid mass production is the use of shake flasks to obtain monoxenic infective juvenile (IJ) inoculum for optimisation purposes, followed by upscaling to a desktop fermenter. This study was undertaken to assess the impact of the addition of agar and glucose to the culture medium, as well as assessing the impact of bacterial cell density inoculum on IJ recovery and yield. Steinernema jeffreyense was cultured in 250 ml Erlenmeyer flasks, with the mutualistic bacterium Xenorhabdus khoisanae. In this study the impact of four different agar and glucose concentrations showed negligible impact on nematode recovery and yield. Different initial bacterial inoculum densities inoculated to the culture medium showed a low inoculum density of 2 % (6 × 106 cells) of the bacteria culture to be the optimal inoculum concentration. A bacterial growth curve for X. khoisanae in tryptic soy broth, showed 40–44 h to be the optimum time for introduction of the initial nematode inoculum into the complex medium.

Infectivity and post-infection development were investigated at 25°C for infective juveniles (IJs) which originated via endotokia matricida in hermaphrodites and/or female adults of the entomopathogenic nematodes, Heterorhabditis bacteriophora, Steinernema glaseri and S. carpocapsae. The IJs spontaneously emerging out of larval cadavers of Galleria mellonella were designated as normal IJs and used as comparison. Nematode invasion was the most prompt and numerous for normal IJs, followed by IJs produced via endotokia matricida in the 1st, 2nd, and 3rd generation adults of the three nematode species examined. Post-infection development and reproduction of nematodes also occurred more promptly and numerously when inoculation was made with normal IJs. The insecticidal activity of IJs originating from endotokia matricida was inferior to that of normal IJs which retained a significantly higher density of symbiotic bacteria than the former IJs of the respective nematode species. The IJs reproduced in and emerged out of host cadavers showed similar pathogenicity and bacterial retention, irrespective of the origin of the IJs used as inocula.

Entomopathogenic nematodes (EPNs) are commonly used biocontrol agents of insect pests, with a wide range of commercially available isolates targeting specific pests. New isolates are, however, required to improve pest control across a wider range of environmental conditions for target pests, including emerging threats. We assessed the effect of temperature on survival and virulence of 17 Australian isolates of five EPN species (Heterorhabditis bacteriophora, Heterorhabditis indica, Heterorhabditis marelatus, Heterorhabditis zealandica and Steinernema feltiae) against larvae and pupae of the Queensland fruit fly, Bactrocera tryoni. All isolates still infected and killed larvae after infective juveniles (IJ) had been kept without insect hosts at 15 °C, 25 °C or 30 °C for two weeks, indicating their potential to remain viable under field conditions. However, the mean LD50 value ranged from 35 to 150 and was generally lower at 15 °C than at 25 °C and 30 °C. Similarly, after IJs had been kept at 25 °C for 1–3 weeks without insect hosts, all isolates infected B. tryoni larvae, with mean LD50 values ranging from 25 to 144. Interestingly, 15 isolates infected and killed B. tryoni pupae after one week, with a mean LD50 value between 130 and 209, but only two isolates after two weeks, with a mean LD50 value between 229 to 209. No pupal mortality was seen after three weeks. In absence of hosts, EPNs survived longer at 15 °C and 25 °C than at 30 °C. Complete EPN mortality occurred after nine weeks at 30 °C, and after 18 weeks at 15 °C and 25 °C, except for some survival in one S. feltiae isolate (Sf.ECCS). Overall, six isolates of H. indica (Hi.HRN2, Hi.LMI2, Hi.QF6), H. bacteriophora (Hb.HIE), H. zealandica (Hz.NAR1) and S. feltiae (Sf.ECCS) performed best and need further testing as potential biocontrol agents against B. tryoni under semi-field and field conditions.

Caribbean fruit fly, also known as Caribfly or Anastrepha suspensa , is a major tephritid pest of guavas. A virulent entomopathogenic nematode (EPN) species was investigated to suppress the fruit-to-soil stages of Caribflies, which are also attacked by the koinobiont parasitoid Diachasmimorpha longicaudata in south Florida. The main objective was to develop a feasible and cost-effective EPN-application method for integrated pest management (IPM) of Caribfly to improve guava production. Naturally infested guavas were treated with increasing Heterorhabditis bacteriophora infective juvenile (IJ) concentration or rate (0, 25, 50, …, 1,600 IJs cm -2 ) in field trials to measure the optimum IJ rate and then examine sensitivity of producing guavas to inclusion of Heterorhabditis bacteriophora in Caribfly IPM plans. Relative survival of Caribfly in treatments significantly decreased with increasing IJ rate from 0 to 100 IJs cm -2 . Similarly, probability of observing large numbers of parasitoid wasps ( Diachasmimorpha longicaudata ) in EPN treatments significantly declined with increasing IJ rate (0-100 IJs cm -2 ), even though the non-target effects of Heterorhabditis bacteriophora on relative survival of Diachasmimorpha longicaudata could not be determined because of few emerging parasitoid wasps. Optimum suppression (⩾ 60%) of Caribfly was consistently achieved at 100 IJs cm -2 or 17,500 IJs fruit -1 . Profitability analysis showed that Heterorhabditis bacteriophora can be included in Caribfly IPM tactics to produce guavas. Costs of EPNs in Caribfly IPM are minimized if Heterorhabditis bacteriophora is strategically applied by spot treatment of fruit. Repayment of costs of EPN-augmentation by spot treatments appears achievable by recovering 5.71% of the annual yield losses (⩾1,963 kg ha -1 ≈ US$ 8,650 ha -1 ), which are largely due to Caribfly infestation. Hectare-wide EPN-augmentation (or broadcasting) method requires more fruit recovery than the total annual yield losses to repay its high costs. Profitability of guava production in south Florida will not be very sensitive to marginal costs of the spot treatment method, when compared to the field-wide broadcasting of Heterorhabditis bacteriophora .

Entomopathogenic nematodes (EPNs) of the families Heterorhabditidae and Steinernematidae are efficient biological control agents against important insect pests. In vitro liquid culture production technology is a key factor in the success of implementing EPNs as a biological control agent. One of the first steps of in vitro mass culture is to use shake flasks to obtain nematode inoculum for optimising and upscaling to desktop and industrial fermenters. This study was the first attempt on the in vitro liquid mass culture of a local South African isolate, Steinernema jeffreyense, in 250 ml Erlenmeyer flasks, together with their mutualistic bacteria, Xenorhabdus khoisanae. After the successful in vitro production of S. jeffreyense-inoculum, different parameters for optimizing infective juvenile (IJ) recovery (developmental step when the IJ moult to initiate the life cycle) and yield, were investigated. This includes the effect of the volume of liquid medium in the flasks, two different orbital shakers setups and the initial IJ inoculum density. With 30 ml of liquid medium the mean percentage recovery of IJ after six days was 86%, with a yield of 121,833 IJ ml−1 after 14 days, in comparison to 75% and 99,875 IJs ml−1 respectively when 50 ml of liquid medium was used. No significant difference was found between IJ recovery and yield, using different orbital shakers setups. Among the three inoculum concentrations tested (1000, 2000 and 3000 IJ ml−1), the lowest concentration gave the highest IJ recovery and yield. Pathogenicity of IJs cultured in vitro was higher than those cultured in vivo.

A total of 27 entomopathogenic nematode (EPN) strains originally isolated from different cotton fields were characterized in laboratory experiments for their virulence, reproductive potential and environmental tolerance. The EPN strain collections included 16 Steinernema carpocapsae (SC), three Steinernema siamkayai (SS), one Steinernema monticolum (SM), and seven Heterorhabditis bacteriophora (HB). Their virulence was tested against cotton bollworms such as the American bollworm Helicoverpa armigera, the spotted bollworm Earias vittella and the cotton leafworm Spodoptera litura. Larvae of H. armigera, E. vittella and S. litura were susceptible to all the tested EPN species/strains with significant differences among EPN species/strains. The most virulent strains were APKS2 (SC), TRYH1 (HB) KKMH1 (HB) and KKMH2 (HB) on H. armigera (91.9–93.5% mortality); KKMS1 (SC), APKS2 (SC), TRYH1 (HB), KKMH1 (HB), KKMH2 (HB) and APKH1 (HB) on E. vittella (92.7% mortality); and APKS2, TRYH1, KKMH1, KKMH2 and KKMH3 on S. litura (92.7% mortality). The results of the invasion rate assay indicated that the EPN strains more virulent against the target host had greater invasion rates. In the multiplication assay, KKMH1and OCMS1 (SC) produced a greater number of infective juveniles (IJs) (32.1–32.4 x 1000 IJs/ cadaver) in Carcyra cephalonica. Test for tolerance to heat at 40°C for 2 h revealed that KKMH1, TRYH1, KKMH2, KKMS1 and APKS2 were highly tolerant (>85% survival). IJs exposed to ambient room conditions (27–29°C; 65–70% r.h.) for 2 h showed that APKS2, OCMS1 and KKMS1 were more tolerant (68–69% survival) of rapid desiccation than others. APKS2, KKMS1 and KKMH1 showed better survival (70–73%) in slow desiccation assay when exposed to 25°C with 97% r.h. for 72 h, followed by 25°C with 93% r.h. for 48 h. The H. bacteriophora KKMH1 and S. carpocapsae APKS2 performed best in nine traits out of ten tested, followed by H. bacteriophora TRYH1, which performed best for six traits. It is suggested that the EPN strains KKMH1 and APKS2 could be deployed for a cotton bollworm management program after testing their performance under field conditions.

Summary The virulence of entomopathogenic nematodes (EPN) Steinernema carpocapsae, S. feltiae and Heterorhabditis bacteriophora was evaluated against the diamondback moth, Plutella xylostella. The results revealed that diamondback moth mortality was affected by its developmental stage. For both Steinernema species, diamondback moth larval mortality peaked at 18 infective juveniles (IJ) larva−1; similar results were recorded for H. bacteriophora, with mortality peaking at 20 IJ larva−1. Mortality of pre-pupa exposed to Steinernema species increased up to 35 IJ pre-pupa−1; in S. feltiae a decreasing trend was recorded at higher concentrations of IJ. A negative correlation was recorded between LC50 and ln ET values; S. carpocapsae appeared as the most virulent EPN against larvae (6.5 IJ larva−1) and H. bacteriophora was an effective EPN against pre-pupae (6.5 IJ pre-pupa−1). EPN virulence at dose levels was evaluated by plotting LC50 against ln exposure time, and in the majority of data sets deviations from a linear model were observed and data were statistically fitted by a two-stage phase.

Nonfeeding infective juvenile (IJ) entomopathogenic nematodes (EPNs) are used as biological agents to control soil-dwelling insects, but poor storage stability remains an obstacle to their widespread acceptance by distributors and growers as well as a frustration to researchers. Age is one factor contributing to variability in EPN efficacy. We hypothesized that age effects on the infectiousness of IJs would be evident within the length of time necessary for IJs to infect a host. The penetration behavior of "young" (<1-wk-old) and "old" (2- to 4-wk-old) Heterorhabditis bacteriophora (GPS 11 strain), Steinernema carpocapsae (All strain), and Steinernema feltiae (UK strain) IJs was evaluated during 5 "exposure periods" to the larvae of the wax moth, Galleria mellonella. Individual larvae were exposed to nematode-infested soil for exposure periods of 4, 8, 16, 32, and 64 hr. Cadavers were dissected after 72 hr, and the IJs that penetrated the larvae were counted. Larval mortality did not differ significantly between 72- and 144-hr "observation periods," or points at which larval mortality was noted, for any age class or species. However, age and species effects were noted in G. mellonella mortality and nematode penetration during shorter time periods. Initial mortality caused by S. carpocapsae and H. bacteriophora IJs declined with nematode age but increased with S. feltiae IJ age. Young S. carpocapsae IJs penetrated G. mellonella larvae at higher rates than old members of the species (27-45% vs. 1-4%). Conversely, old S. feltiae IJs had higher penetration rates than young IJs (approximately 8 to 57% vs. 4 to approximately 31%), whereas H. bacteriophora IJs had very low penetration rates regardless of age (3-5.6%). Our results show that the effect of age on IJ infectiousness can be detected in IJs aged only 2 wk by a 4-hr exposure period to G. mellonella. These results have important implications for storage and application of EPNs and suggest the possibility of shortening the time required to detect nematodes in the soil.

Effect of insect cadaver desiccation and soil water potential during rehydration on entomopathogenic nematode (Rhabditida: Steinernematidae and Heterorhabditidae) production and virulence

Efficacy of carboxymethyl cellulose as an inert water-soluble carrier for formulation of entomopathogenic nematodes, Heterorhabditis bacteriophora and Steinernema carpocapsae

Effects of the nematode numbers tested and jumping distance on the jumping behavior of Steinernema abbasi (Sa), and selection of its searching ability strains through generations were investigated in this study. When tested different numbers of Sa with 45 mm distance from the lid of an experimental tray, their jumping percentages were significantly different between the groups with 1 x 105 or 5 x 104 infective juveniles (IJs) (56.4 and 40.6%) and 1 x 104 or 5 x 103 IJs (2.9 and 0.1%). With different distance (21, 33, 45, 57 mm) from the lid, the percentages of jumping IJs were 25-33.1 % in 21 to 45 mm, but only 15.5% in the 57 mm. The LT50 values of Spodoptera litura 5th instar larvae inoculated with various IJs having different jumping abilities at 15 IJs/0.5 ml/insect were 22.1 to 23.7 h, whereas those of IJs without jumping behavior were significantly longer at 26.5 h. However, all treatments could cause 100% host mortality. At 30 IJs/ml/insect, the LT50 values tested with IJs showing different jumping abilities were 22.1 to 23.7 h, but prolonged to 26.5 h in those without jumping behavior. When compared with Sa0, the IJs subjected to five rounds of selection produced a 2-fold increase in successful host-finding against Galleria mellonella, while those of 8 or 12 round selection increased to 3 and 6 fold, respectively. Distribution of nematodes with positive moving ratios were 40.6 in Sa0 and 52.7% in Sa5 at 6 cm from the host insect, 25.7% in Sa0 and 54.8% in Sa8 at 9 cm, and 26.4% in Sa0 and 61.1% in Sa12 at 12 cm. The percentages of Sa0 and Sa12 (12 cm) succeeded in host-finding against G. mellonella were significantly different at 4.6 and 15.4%, respectively, while the ratios against S. litura were not significantly different at 0.8 and 3.2%, respectively. When inoculated with 200 IJs/ml/insect, the shortest LT50 of S. litura 5th instar larvae was 20.9 h in Sa5 at 6 cm vertical distance in the soil, all treatments could cause 95% mortality. When increased the distance from the host from 6 cm to 9 cm, only Sa8 could reach 70% mortality, its LT50 being 41.6 h. However, the mortalities of other treatments were less than 50%. Comparing different searching strategies, the percentages of positive movement of ambusher, Steinernema carpocapsae (Sc); intermediate, Sa0, and cruiser, Steinernema arenarium (Sare), were 40.7, 26.4 and 0.7%, respectively, but the negative movement of Sare at 95% was much higher than others. If the nematodes were provided with S. litura as their host, the positive movement proportion in Sare increased to 10.9%. Percentage of positive movement in Sa12 (12 cm) was 61.1%, being the best strain showing searching ability in all treatments. By observing the jumping behavior of IJs in the non-selected parental strain (Sa0) and those after 5, 8, 12 rounds of selection, the highest percentage of jumping was 40.8% in Sa5 (6 cm), while the lowest was 10% in Sa12 (12 cm), indicating that selection of horizontal host-finding could not reflect the vertical jumping behavior of the same strain. A well host-finding strain of S. abbasi was discovered in this study, and its jumping and host-finding behaviors were observed. These results could possibly be useful for the pest control using entomopathogenic nematodes.

Evaluation of different sponge types on the survival and infectivity of stored entomopathogenic nematodes