Efficacy of different entomopathogenic nematode isolates, against the peach fruit fly, Bactrocera zonata (Saund.) (Diptera: Tephritidae)

Background The invasive peach fruit fly (PFF), Bactrocera zonata (Saunders) (Diptera: Tephritidae), is a native of South‑ east Asia. Entomopathogens like nematodes, bacteria, viruses and fungi have been shown to be effective as a biologi‑ cal control agent against B. zonata . Evaluation the efficacy of different entomopathogenic nematode isolates (EPNs) belonged to the two families (Steinernematidae and Heterorhabditidae); ( Steinernema carpocapsae (AII), S. carpocap-sae (EGAZ10), Heterorhabditis bacteriophora (HP88) and H. indica


Background
The peach fruit fly (PFF), Bactrocera zonata (Saunders, 1841) (Diptera: Tephritidae), is an invasive pest species, native to Southeast Asia.It was first recognized as a new pest of guava and mango in the northern region of Egypt in 1998 (El-Minshawy et al. 1999).According to Hashem et al. (2007), B. zonata has a wide variety of hosts, including fruits and vegetables.It spreads quickly in great Page 2 of 9 Sallam et al. Egyptian Journal of Biological Pest Control (2024) 34:12 numbers throughout Egyptian Governorates.Due to the favorable climate in Egypt, the PFF lives in Egypt and developed into a significant pest over the past ten years and began infesting a variety of fruit hosts, including citrus, mango, peach, fig, guava, apricot, and apple.Additionally, it targets various foods as secondary hosts, including tomato, pepper, and eggplant (Ghanim 2009).
Use of insecticides as an only method to control pests in fruits and vegetables has caused environmental pollution and hygienic problems that represent a risk for human and animals (Gallo 2007).In comparison with pesticides, biological management is less cost-effective and less harmful to the environment (Rizvi et al. 2009).According to Dias et al. (2018), natural enemies, parasitoids, predators, and pathogens are frequently used in biological management because they attack pests.Entomopathogens are natural microorganisms that can be found in a variety of ecosystems infecting different stages of the insect hosts.It has been demonstrated that pathogens like nematodes, bacteria, viruses and fungi are viable biological control agents against B. zonata (Bilal et al. 2021).Rashad et al. (2015) suggested that EPNs and fungi can be used as alternative tools of pesticides for controlling B. zonata after validating protocols of field conditions.Some tephritid species have been observed to be mortally affected by steinernematid and heterorhabditis species.Different phases of B. zonata may be infected by EPNs, according to (Mahmoud et al. 2016).
The objective of this study was to evaluate the efficacy of different EPN isolates: Steinernema carpocapsae (AII), S. carpocapsae (EGAZ10), Heterorhabditis bacteriophora (HP88) and H. indica (EGAZ2) belonged to two families (Steinernematidae and Heterorhabditidae) against the full-grown B. zonata larvae under laboratory, semifield and field conditions.Also, to evaluate the influence of combination for whether applying two EPN strains at (semi-field and field experiment); it was synergistic, additive or antagonistic effects.

Insect culture
The stock colony of PFF used in the present study was reared in cages measured (35 × 30 × 30 cm) at the Biological Control Department, Plant Protection Research Institute, Agricultural Research Center (ARC)-Giza, Egypt.Rearing was carried out under greenhouse conditions of 25 ± 2 °C and 55-65% R.H. and the photoperiod ranged 14-16:8-10 L: D. on artificial diet according to (Hosni et al. 2011 andRashad et al. 2015).
For mass culturing of the tested nematode species, S. carpocapsae (AII); S. carpocapsae (EGAZ10); H. bacteriophora (HP88) and H. indica (EGAZ2), strains were propagated at the Physiology Laboratory, of PPRI, Giza, Egypt in vivo using larvae of the greater wax moth, Galleria mellonella (Linnaeus, 1758) as a host according to (Shamseldean et al. 2009).Rearing of G. mellonella was acquired from infested bee hives and reared in jars according to Metwally et al. (2012).Fifty last instar larvae of G. mellonella were placed in Petri-dishes; each contains two filter papers moistened with water suspension of nematodes at concentration of (30.000IJs/2 ml. of distilled water).After 10-15 days of infection, the host cadavers were transferred to nematode-collecting dishes according to White traps method (White 1927) to collect the infective juveniles (IJs).The emerging juveniles of S. carpocapsae (at day 10 post-infection), and, H. bacteriophora (at day 15 post-infection) were harvested daily and then stored in plastic bottles (15 × 15 × 10 cm) containing distilled water at 8-10 and 15 °C until used.

Pathogenicity of the EPN species against B. zonata full-grown larvae under laboratory conditions
Three different species (four isolates) of EPNs: S. carpocapsae (AII), S. carpocapsae (EGAZ10), H. bacteriophora (HP88) and H. Indica (EGAZ2) were suspended in distilled water (d.w.) at the concentrations of 250, 500, 1000, 1500 and 2000 IJs/ml d.w.against B. zonata fullgrown larvae.Plastic cups measuring (5.5 × 4 cm) filled with 25 gm.mixed soil (clay 50% + beach sand 50%), supplied with ten B. zonata full-grown larvae, treated with a suspension of nematode species, and then covered with a borer plastic lid.Control experiment was conducted by placing larvae on soil moistened with 1 ml.distilled water only.Four replicates (10 larvae/replicate) were used for each treatment.The mortality was estimated three days post treatments.The experiments were carried out at 25 ± 2 °C and R. H. (60-70% and 20% soil moisture).

Semi-field experiment
The semi-field experiment was performed under trees (uncontrolled conditions) of citrus orchard at ARC-Giza, Egypt, as a preliminary test to examine the effect of the combination of EPN species.The experimental design consisted of six trees for two EPNs treatments and one another tree for control using a total of seven trees.The experiment was applied under trees canopies.The combined nematodes suspension concentrations (combination between the two tested steinernematids or between those species of heterorhabditis) occurred for 1000, 2000, and 4000 IJs/ml.at maximum temperature (Max.T.) (28.85 °C); minimum temperatures (Min.T.) (19.71 °C) and R.H. (81.71%).Climatic conditions data were obtained from (Cairo International Airport Station) weather underground site (https:// www.wunde rgrou nd.com).
Four replicates were conducted for each concentration and distributed under every selected tree of the trial orchards; the accumulative mortality rate was estimated after ten days post treatments.The soil sample was taken using a spoon.The amount of the orchard soil was weighted at a rate of 25 gm and placed in a piece of muslin (to prevent larvae from escaping and attacking insects in the soil, especially ants) supplied with 20 fullgrown larvae/replicate.The pieces of muslin were tied with a piece of thread (Fig. 1).The time recorded during the experiment was 5 p.m., the muslin piece containing infected PFF full-grown larvae was buried 5 cm in the soil under the tree canopy and the control treatment was buried without nematodes.The experiment was irrigated every two days by a water hand sprayer in the 0.5 m 2 area surrounding each treatment.

Field experiment
Based on the initial screening of the previous four nematode strains, the most efficient steinernematid and heterorhabditis strains were selected for additional evaluations.The experiment was carried out in plastic cans measured (19 × 14 × 8 cm) (Fig. 2) filled with 200 gm soil (moisten soil) under four citrus trees canopies which maximum temperature (30.85 °C), minimum Fig. 1 A piece of muslin contained infected full-grown larvae of Bactrocera zonata Fig. 2 Cans contained Bactrocera zonata full-grown larvae in the field experiment at ARC, Giza Governorate temperature (18.14 °C) and R.H. (88.00%), at the ARC, Giza, Egypt.The combined EPN species (S. carpocapsae (AII) + H. bacteriophora (HP88)) were treated at a concentration of 3000 IJs/ml.The EPNs applied for each test mixed with soil and water, which always kept at 55 ± 5% of the soil weight.In this experiment, every 20 fullgrown larvae/replicate were placed in the plastic cans and covered with stockings to prevent escaping larvae and attacking by ants then tied with rubber bands.Part of the bottom of the cans was buried in the soil.Four replicates were used where each treatment placed next to its control.The numbers of dead larvae were recorded after 48 h, and the emergence of adults was recorded after 8 days post treatment.
The dead larvae (Fig. 3) and pupae for laboratory, semifield and field experiments were incubated in white trap for collecting the infective juveniles of EPNs.
To find out if combining two EPN species would have an antagonistic, additive, or synergistic effect on B. zonata full-grown larvae, laboratory, semi-field and field experiments were carried out.
The combined action of the mixtures was expressed as a Co-toxicity factor, estimated by the equation of Mansour et al. (1966) as follows: where: Observed % mortality is the mortality of individuals treated by the combination.Expected % mortality is the sum of mortalities of each material when used singly.These factors were used to differentiate the results into 3 categories.The interaction result was assessed as, 'synergism' when Co-toxicity factor values were greater than or equal to 20; 'addition' when Co-toxicity factor values were between − 20 and + 20; and 'antagonism' when Cotoxicity factor values were less than or equal to − 20.

Statistical analysis
Obtained data were analyzed according to Finney (1971).Percentage mortality was plotted versus the corresponding concentrations probit lines, and the median lethal concentration LC 50 was determined for established Co − Toxicity factor = Observed % mortality − Expected % mortality Expected % mortality X100 Fig. 3 Infected Bactrocera zonata full-grown larvae with entomopathogenic nematodes regression lines using Ehab soft LDP line software to calculate probit analysis (www.Ehabs oft.com/ LDP line).
Lower and upper limits were determined when the (g) factor was less than 0.4.Data obtained for significant differences were analyzed using the Analysis of Variance one way (ANOVA) technique, and the means were separated using Fisher's least significant difference (LSD) test (p = 0.05 level).

Laboratory experiments
Data  ), respectively, at 3 days of treatments (Table 2).Data pointed that steinernematid strains were more effective against B. zonata full-grown larvae than heterorhabditis strains at 25 ± 2 °C and R.H. (60-70% and 20% soil moisture).Also, Table 1 showed that there were significant differences between mortality percentages at different concentrations.

Semi-field experiment
The combined concentrations of 1000, 2000 and 4000 IJs/ml for the two tested steinernematid (S. carpocapsae (AII) & S. carpocapsae (EGAZ10)) or those of heterorhabditis (H.bacteriophora (HP88) & H. indica (EGAZ2)) strains were used, in semi-field conditions at the ARC, Giza, Egypt.Data in Table 3 revealed that the combination of steinernematid strains was more effective than the combination of heterorhabditis strains.Direct relationship between the mortality percentages at different concentrations was recorded.Data in Table 4 revealed an antagonistic effect of the combined steinernematids, S. carpocapsae (AII) and S. carpocapsae (EGAZ10) where Co-toxicity factor value is (− 67.6).Also, H. bacteriophora (HP88) and H. indica (EGAZ2) in combination has a Co-Toxicity factor value (− 66.6).Despite the increase in the proportion of B. zonata death in the combined treatments for all the tested EPNs, the type of interaction was antagonism.

Field experiment
According to the previous trials, the field experiment was conducted.A nematode suspension was used at concentration of 3000 IJs/ml for the combined S. carpocapsae (AII) and H. bacteriophora (HP88) strains, under field conditions of maximum temperature (30.85 °C), minimum temperature (18.14 °C) and R.H. (88%).Results revealed that the combination of the two different strains of EPNs (S. carpocapsae (AII) + H. bacteriophora (HP88)) recorded a mortality percentage of 97.5%.On the other hand, the combined EPNs S. carpocapsae (AII) and H. bacteriophora (HP88) strains recorded an antagonistic effect, where the Co-toxicity factor value was (− 42.6) (Table 4).
Table 1 Accumulative mortality percentages of Bactrocera zonata full-grown larvae with different concentrations under laboratory conditions at day 3 post experiment

Discussion
Results indicated that all tested nematodes had variable modes of bactericidal action in the B. zonata host, resulting in markedly antagonistic effects after strains' combination.The previous results revealed that steinernematid strains were more effective against B. zonata full-grown larvae than heterorhabditis strains.Also, there were significant differences between mortality percentages at different concentrations when EPNs were separately tested.Combination of the steinernematid or heterorhabditis strains was more effective on the target pest.Data revealed the presence of an antagonistic effect due to the combinations of (S.  (2021) who reported that the mortality percentage increased as IJs concentration increased.Rashad et al. (2015) demonstrated that both S. carpocapsae and S. riobrave were superior to H. bacteriophora in contact treatment against B. zonata full-grown larvae; and infectivity among those tested nematode strains was found significantly different.Steinernematid strains, also referred to as "ambushers," were more successful than heterorhabditis strains and more effective against soil-dwelling insects, as reported by Attalla and Eweis (2002).In field tests, Steinernema and Heterorhabditis species have been employed to control pest insects that emerge from fruit and burrow into the soil to pupate, with differing degrees of success.For example, fruit flies Rhagoletis indifferens and Anastrepha ludens   (Diptera: Tephritidae) (Toledo et al. 2005) and several other species reviewed by Dolinski and Lacey (2007).Soil properties such as moisture level, pH, organic matter content, texture, and others, affect EPNs dispersal and potential to find a host (Stuart et al. 2015).Third instar larvae of several tephritid flies were reported to be susceptible to EPNs (Langford et al. 2014).Similarly, S. carpocapsae and S. feltiae were found to be more effective species under laboratory, semi-field, and field conditions against the European cherry fruit fly larvae where the mortalities were 88, 78, and 88%, respectively, while no mortality of pupae was observed (Köppler et al. 2004).On the other hand, at field temperature of 30 °C, Heterorhabditis species have a very strong ability to signal their maximal degree of pathogenicity according to El Khoury et al. (2018).These results are different from one insect to another may be due to some reasons such as insect behavior or insect physiological or/and the competition between the used two nematode strains or species.It was reported that combining nematodes species might be more effective in the field, because of the strongest concentration effect and highest propagation rate of the EPNs (O'Callaghan et al. 2014).The effect of mutualistic EPNs Xenorhabdus and Photorhabdus is gram-negative bacteria that can produce several secondary metabolites, including antimicrobial compounds.Otherwise, the nematodes or bacteria themselves make significant contributions to pathogenesis within the insect, poisoning effect and may be due to the presence of some physical or chemical reactions in the insect hemolymph (Lewis and Clarke 2012) and (Chang et al. 2019).

Conclusion
All EPNs experiments showed the efficacy of foreign nematodes than the local ones against B. zonata fullgrown larvae and increasing mortality with concentration increase.Also, combination of the two highly effective imported strains gave satisfied results especially in the field experiment.

Entomopathogenic
Values followed by the same letter in the same raw are not significantly different at P = 0.05 in Table1showed the mortality percentages of B.

Table 2
Toxicity of EPN strains against Bactrocera zonata full-grown larvae under laboratory conditions