Potential of two indigenous strains of entomopathogenic nematodes, (Steinernema feltiae and Heterorhabditis bacteriophora) against the tomato leafminer (TLM), Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), under laboratory and greenhouse environmental bioassay

The tomato leafminer (Tuta Absoluta) is an important pest of tomato crops which have features like; high reproductive potential, Invasive species are major threats to agronomic and natural ecosystems. Based on the pathogenicity of strains, only two isolates effectively show larvicidal activity. The native isolate was obtained from soil samples, collected from Rajgarh, Hamachi Pradesh, India. Petri dish bioassay use nematodes S. feltiae HR1 and H. bacteriophora HR2 species dose (0, 10, 20, 40, 80, 160/ IJs/cm 2 ). The (%) 1st instar larval mortality was ranged from 24.15 to 85.38%. 2nd instar from 29.87 to 90.00%. 3rd instar from 24.15 to 90.00%. 4th instar from 31.53 to 90.00% and pupae stage mortality was ranged from 31.53 to 85.38%. Larvicidal activity after 48-and 72-hours exposure, the S. feltiae and H. bacteriophora (1.0, 1.30, 1.60, 1.90, 2.20/IJs/cm 2 ) showed potent larvicidal activity with LC 50, LC 75 and LC 90 of all instars and pupa show high mortality. The strain inhibits the larval and pupal development 48 to 72 hr exposer time with LC 50 range from 05.42 to 23.67, LC 75 20.29 to 83.12, LC 90 16.52 to 98.89. Green house test is using the seam isolate of EPNs (HR1, HR2) on foliar application it caused by signi�cant mortality results. These studies demonstrate the challenge for invasive species. The local indigenous strains of EPNs (S. feltiae HR1, H. bacteriophora HR2) as a good biocontrol agent against invasive pest of T. absoluta.


Introduction
Invasive species are important threats to the ecological and agricultural systems.Tomato leafminer (TLM), Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), is native to South America.Outside its native place, the pest was detected for the rst time in discovered in Spain in 2006 (Urbaneja et al., 2008), and since it quickly attacked 80 countries including India (CABI, 2018;CABI, 2020).The insect feeds on leaves, fruits, shoots, and apical shoots tomatoes can also cause 100 percent damage to plants in the absence of regulatory measures (Urbaneja et al., 2012;Balal et al., 2016;Biondi and Desneux, 2019).In India, the emergence of this insect was rst carved with tomatoes in Pune and Bangalore in 2014 (Sridhar et al., 2014;Shashank et al., 2015), and since then it has spread to almost every part of the country tomato planting (Sridhar et al., 2014;Kalleshwaraswamy et al., 2015;Shashank et al., 2015;Balal et al., 2016;Sharma and Gavkare, 2017).In Himachal Pradesh, T. absoluta was rst acquired in 2015 tomatoes in Nauni, Solan (Sharma and Gavkare, 2017).On problemsolving has emerged as a major threat to tomatoes in various parts of the state in open spaces and poly-houses.Previous reports indicate that in recent areas, T. absoluta quickly acquired a serious insect condition without drug abuse (Bielza, 2010;Desneux et al., 2010).Tuta absoluta control in particular based on pesticides and sometimes up to 14 times Insecticides while growing were needed (Campos et al., 2017;Han et al., 2019;Abbas et al., 2021;Waiba et al., 2021).
Therefore, this insect is di cult to control chemically because the larvae live inside the mines and fruits where it is di cult to nd pesticides.Biotic energy is high, spread speed again the ability to increase resistance to pesticides keeps pests at the bay challenge (Desneux et al., 2010;Ingegno et al., 2013;Roditakis et al., 2013;Roditakis et al., 2015).Invasive new invasive insect, tomato pinworm / small leaf, T. Absoluta was rst recorded in Pune in a tomato plant that grew in a eld and a thousand houses in 2014 and in southwestern India (Karnataka state) (ICAR 2015), and to our knowledge, there are no reports of this pest in eastern and northern and eastern India.Subsequently, the pest was reported on a farmer's farm in major tomato growing districts including Himachal Pradesh.
Entomopathogenic nematodes (EPNs) from the families Heterorhabditidae and Steinernematidae are soil organisms that bind to natural insects (Kaya and Gaugler 1993).These nematodes have evolved into interactions with bacteria in the genera Photorhabdus associated with Heterorhabditis spp., It is carried in the intestines of infectious hairs (IJs) (Bird and Akhurst 1983;Arthurs et al., 2004;Silva et al., 2002;Kasi et al., 2021).Xenorhabdus is connected to Steinernema spp.and is con ned to a speci c skin within the intestines of IJs.Nematodes receive their treatment by tracking insects (Lewis et al., 2006;Mansour and Biondi, 2021).After IJs have found a culprit, they infect it with an ori ce such as the mouth, anus or edges or by entering the cuticle (especially in Heterorhabditis spp.).As soon as the IJs enter the host, they break down their outer cuticle (Sicard et al., 2004;Ben Husin and Port, 2021) and begin to absorb the hemolymph, causing the de ant to be released by extraction (Steinernema spp.)Or recurrence (Heterorhabditis spp.) (Martens et al., 2004;Martens and Goodrich-Blair 2005).The structure of nematodes -bacteria kill the host between 24 -48h through septicemia or toxemia (Dowds and Peters 1971;Forst and Clarke 2002;Ndereyimana et al., 2020).Bacteria reminiscent of nematodes, which appear as IJs from extinct insect cadaver in search of new transmitters (Poinar 1990).
More than 100 species of EPNs have been identi ed worldwide (about 80% by steinernematid) and at least 13 of these species have been marketed (Shapiro-Ilan et al., 2014;Ndereyimana et al., 2019).In general, the natural violence against various insect species varies between EPN species.In addition, the differences between the types of EPNs in terms of demand management and tolerance of environmental conditions such as temperature and humidity may determine the eld performance of EPNs (Martens et al., 2004;Noug, 2021).EPNs have been widely used in the control of economically important pests living in different habitats (Grewal et al., 2005;Susurluk, 2008).However, the formation of EPNs to delay the extinction or addition of supplements to increase leaf coverage and persistence of IJs have improved the use of EPNs in insect-infested animals (Williams and Walters 2000;Arthurs et al., 2004;Head et al., 2004;Kasi et al., 2021;Ben Husin and Port, 2021).
The objective of the present study was to provide fundamental information necessary for the utilization of indigenously isolated EPNs as biological control agents.The study dealt with 2 nematode species such as S. feltiae and H. bacteriophora and their pathogenicity against Tuta absoluta under laboratory and greenhouse conditions.

Materials And Methods
A T. absoluta colony was maintained on tomato plants under greenhouse conditions.The colony had been established from larvae collected in September 2020 from the vegetable science department form the tomato greenhouse in UHF, HP, India, that used S. feltiae and H. bacteriophora (Poinar) for the pest's management.
Rearing of tomato pinworm, Tuta absoluta TLM larvae and pupae were collected in a place that holds full heat greenhouse.T. Absoluta was raised in a nursery greenhouse (Roditakis et al., 2013), at 26 ± 2 ° C, 60 ± 10% RH, and L: D 8:16 photoperiod.These insects are grown in wooden cages covered with 80 mesh organdy cloth on tomato plants (S.Lycopersicum L.).Adults are fed 10% of the sugar solution in the oviposition cage.

Source of entomopathogenic nematodes
Two isolates of S. feltiae and H. bacteriophora were used in this study.The native isolate was obtained from soil samples, collected from Rajgarh, Hamachi Pradesh, India, using G. mellonella larvae as nematode traps.This isolate was cultured based on the method describe (Woodring and Kaya 1988) at 21 ± 1 °C on the last instar larvae of G. mellonella.Infective juveniles (IJs) that emerged during the rst ten days were collected from white traps stored at 4 °C in distilled water for up to 14 days (Woodring and Kaya 1988).The nematodes were acclimatized at room temperature for about 30 min before being used in the experiments.

Effect of Nematode Concentration
Bioassays was performed in a petri dish (9 cm).Each unit is lled with 20 grams of sandy loam (Table 1).Soil moisture was adjusted to 7% (w / w).IJs were applied evenly on the sterilized sand surface at 0, 5, 10, 20, 40, 80, and 160 IJs / cm2 in 1 ml of distilled water.Final sand moisture 10% (w / w).Ten t in each instar.The containers were kept at room temperature for 1 h before placing the entire larvae on the soil surface.There are four replicas for each concentration.Under controlled conditions the containers were kept in the growth chamber for 72 h, after which the larvae were separated from the surface through a ne sieve and held individually under controlled conditions until adulthood.Three days later, 25% of the dead larvae were randomly selected and dissected under a stereomicroscope to con rm nematode infection.The experiment was performed twice.

Larvicidal activity
Each nematode species was added at different concentrations (1.00, 1.30, 1.60, 1.90, 2.20/ mL -1 ) into the 9 cm petri dish in triplicate with 2 ml of dechlorinated sterile water and 60 larvae of tested Tuta absoluta strains.The larvae were provided with young tomato leaves.One Petri plate without EPNs suspension was used as a control.After 24, 48, and 72 hr the number of dead larvae was calculated.The strains that killed more than 50% of the larvae were considered pathogenic (Morton and Garcia-del-Pino 2009;Kasi et al., 2021).Two nematode isolates were examined quantitatively for larvicidal activity against T. absoluta, using various concentrations of EPNs suspensions.The infected larvae were observed under a stereo zoom microscope for each concentration at 72 h exposure time.

Study Site Selection
The experimental farm is located at an elevation of 1260 m above mean sea level with 30 o 52'N latitude and 77 o 11'E longitude with East-West orientation of poly-house which represents the mid-hill zone of Himalayas, HP, India.This is an ideal location for poly-house with various features like single door, side, and top vent, drip irrigation, fogging facility, and internal shading with 50 % green agro UV stabilized shade net.

Experimental material and layout plan
The tomato germplasm commercial market, F 1 Hybrid (BSS-816) were evaluated under a naturally ventilated poly-house having a 200 m 2 area.The experiment was conducted in a randomized block design (RBD) with four replications inside the modi ed naturally ventilated polyhouse (25 m × 10 m).Ten plants of the hybrid were planted at a spacing of 70 × 30 cm and trained on two stems in each replication.

Treatments
Table 1 Treatments used in the field experiment to control Tuta absoluta (M.) Water Untreated (Control)

Statistical Analysis
Insect mortality was control-corrected (Abbott 1925) and Arcsine transformed when required to meet assumptions of normality and homogeneity of variances.
In all experiments, control-corrected mortality was subjected to one-factor analysis of variance (ANOVA) 100 -% mortality in the control The corrected percent mortality data thus obtained for different concentrations of Tuta absoluta (M.) at different concentrations were subjected to probit analysis as per the method given (Finney 1971).Concentration-mortality response data was conducted.Also, LSD (P< 0.05) values were calculated to differentiate means among treatments.

Results
Petri dish bioassay of Tuta absoluta life stages bioassay of T. absoluta (1 st instar) The results revealed that the 1 st instars larvae of the tomato leafminer were highly susceptible to two nematode species tested.After 72 h of treatment, there was an increase in (%) larval mortality in all treatments.The (%) larval mortality was ranged from 24.15 to 85.38 % (Fig 1 Lethal Concentration are calculated by Probit Methods. Significant differences from control are indicated (**P < 0.01; *P < 0.05).
The mortality was determined through different concentrations for both 48 and 72 h exposure.The mortality rate depends on the concentration and exposure time.However, the highest mortality range was observing H. bacteriophora treatment at very low concentrations for 48 and 72 h.Even though S. feltiae showed slow mortality in 48 and 72 h exposure time, they restrained the larval development at the early pupal stage.

E cacy of EPNs against T. absoluta in condition
Data pertaining to survival population of tomato leafminer Tuta absoluta on tomato one day before and 3 rd ,7 th 10 th and 14 th days after rst spray was presented in (Table 2).The mean survival population of T. absoluta larvae one day before spray was ranged from 5.77 to 7.36 larvae/ plant.The pre-treatment data was found to be non-signi cant indicating the uniformity population of pest throughout the experimental plot.At 14 th days of rst spray slight increase in the larval population was observed in all the treated plots.The mean number survival populations of larvae were ranged from 1.31 to 2.13 larvae/plant.In untreated control plot it was recorded highest 4.09 larvae/plant.The treatment with H. bacteriophora at 1,00,000 IJ's/cm 2 (1.31 larvae/plant) was found be consistently effective against T. absoluta followed by H. bacteriophora at 2,00,000 IJ's/cm 2 (1.42 larvae/plant) which were found to be equally effective.
Thus, overall results on e cacy indicated that H. bacteriophora at 1,00,000 IJ's/cm 2 (1.78 larvae/plant) was the most effective compared to other treatments in reducing the survival population of T. absoluta.The treatment with S. feltiae at 1,00,000 IJ's/cm 2 (1.97 larvae/plant) was the next best effective treatment.
Followed by H. bacteriophora at 2,00,000 IJ's/cm 2 (2.00 larvae/plant) were next in order of e cacy.At 14 th days of second spray slight increase in the larval population was observed in all the treated plots.The mean number survival populations of larvae were ranged from 1.18 to 1.51 larvae/plant.In untreated control plot it was recorded highest 4.24 larvae/plant.The treatment with H. bacteriophora at 2,00,000 IJ's/cm 2 (1.18 larvae/plant) was found be consistently effective against T. absoluta followed by S. feltiae at 2,00,000 IJ's/cm 2 (1.38 larvae/plant) which were found to be equally effective.Treatment with H.
Thus, overall performance of the various treatments after third spray found that treatment with H. bacteriophora at 4,00,000 IJ's/cm 2 proved to be consistently most effective and superior over rest of the treatments and recorded the lowest larval population (1.17 larvae/ plant) compared to 4.48 larvae/plant recorded in untreated control.Treatment with H. bacteriophora at 2,00,000 IJ's/cm 2 (1.34 larvae/plant), was next best treatment in the order of e cacy.Signi cant differences do not exist among the treatments with S. feltiae at 2,00,000 IJ's/cm 2 (1.60 larvae/plant) and S. feltiae at 4,00,000 IJ's/cm 2 (1.65 larvae/plant) were found on par with each other.Whereas, the treatments with S. feltiae at 1,00,000 IJ's/cm 2 (1.83 larvae/plant), and H. bacteriophora at 2,00,000 IJ's/cm 2 (2.25 larvae/ plant) were found moderately effective in reducing the surviving T. absoluta population.The overall results of the present investigation after three spraying presented in (Table 20.4) revealed that among the entomopathogenic agents H. bacteriophora at 2,00,000 IJ's/cm 2 proved to be consistently effective against T. absoluta by recording a least larval population (1.64 Larvae/plant).The treatment with H. bacteriophora at 4,00,000 IJ's/cm 2 and S. feltiae at 1,00,000 IJ's/cm 2 , S. feltiae at 2,00,000 IJ's/cm 2 also showed better results against T. absoluta.

Discussion
In this study it is evident that S. feltiae and H. bacteriophora were able to kill Tuta absoluta larvae at various concentrations namely 1,00,000, 2,00,000 and 4,00,000 IJs/ cm 2 .Tuta absoluta larvae mortality increased with increase in concentrations.In this case the highest concentration tested (4,00,000 IJs/ cm 2 ) achieved the highest mortality under poly-house conditions.This mortality increase with increase in concentration can be attributed to large population of symbiotic bacteria released by EPNs when they penetrate the larvae as reported by (Eleftherianos et al. 2010;Han et al., 2019).Steinernematid IJs retain Xenorhabdus symbionts within an intestinal vesicle, while Photorhabdus cells stick together in the anterior part of the Heterorhabditids gut and releases them upon invasion of an insect host (Dillman et al. 2012;Lankin et al., 2021).
The third instar larvae were used; other studies revealed that EPNs were able to nd and kill all the four larval instars inside or outside the leaf galleries (Batalla-Carrera et al., 2010;Van Damme et al., 2016;kasi et al., 2021).Found that T. absoluta larval stage was the most vulnerable to EPNs (Batalla-Carrera et al., 2010;Ben Husin and Port, 2021).They thus emphasized the necessity to apply EPNs on the above-ground part of the tomato plant to ensure effective control of this pest using the most suitable isolates against a particular pest in a given environment (Abbas et al., 2021;Mansour and Biondi, 2021).
The ability of EPNs to reach and kill T. absoluta larvae in leaf galleries was also reported (Batalla-Carrera et al., 2010;Van Damme et al., 2016;Kamali et al., 2018).The local EPN isolates were able to kill T. absoluta and had even been found to be effective against white grubs in Rwanda (Kajuga et al., 2018;Ndereyimana et al., 2019), while it was not easy to nd EPNs which can kill them (Laznik et al., 2015).Although EPNs live naturally in soil, different researchers found that they can be used on above-ground parts of the plant to control effectively the pests living in cryptic habitats like in leaf galleries (Batalla-Carrera et al., 2010;Garcia-del-Pino et al., 2013;Lankin et al., 2021), which concurs with the results of the present study under laboratory conditions.
The IJs multiply quickly and kill the host 24 -72 h after infection (Gozel and Kasap 2015;Van Damme et al., 2016;Kasi et al., 2021).This was veri ed in the present study where all local EPN isolates caused between 53.3 and 96.7 % mortality just within the rst 24 h after inoculation, while in 72 h, they all had caused between 96.3 and 100 % mortality.This is not the case of other entomopathogens like entomopathogenic fungi, which require 3-5 days or even more time to kill their host (Reda and Hatem 2012;Abbas et al., 2021).
Different pathogenicity levels displayed by the studied EPNs agree with other studies, using different EPN isolates (Gozel and Kasap 2015;Van Damme et al., 2016;Han et al., 2019).This underlines the necessity of EPNs screening and selection as emphasized (Sharma et al., 2011;Biondi et al., 2018), in a view to boost their e cacy of EPNs.The highest e cacy of local EPN isolates than the exotics could be explained by the fact that these EPNs (Yan et al., 2016;Kasi et al., 2021;Mansour and Biondi, 2021) and they might be more adapted to the local conditions than the exotics, which were isolated in a completely different environment.These results agree with the earlier ndings where locally isolated biological control including EPNs, performed better than exotics (Lima et al., 2017;Nouh, 2021).The larvae of T. absoluta present susceptibility to EPNs, unlike their pupae, which present tolerance and thus a low mortality rate of less than 20% caused by EPNs (Batalla-Carrera et al., 2010;Garcia-del-Pino et al., 2013;Ben Husin and Port, 2021;Kasi et al., 2021).
The results of this study revealed that local EPN isolates were able to nd and kill T. absoluta larvae stages inside the leaf galleries under laboratory conditions and their e cacy increased with exposure time.The e cacy of local indigenous EPN isolates was signi cantly superior to that of the exotic species.This is the rst study carried out in Rajghar on the good biocontrol potential of indigenous strains (HR1 and HR2) EPNs against T. absoluta.The results of this study form the basis for further research.High EPN e cacy obtained under laboratory conditions cannot easily be extrapolated to eld e cacy.
Therefore, future eld experiments on tomato crops are justi ed to fully determine the potential of local EPN isolates against T. absoluta in Himachal Pradesh conditions.

Figures
Figures

Table 2 .
Efficacy of entomopathogenic nematode against Tuta absoluta in tomato crop (overall effect of three sprays).
*Figures in parentheses are transformed values Öx+ 0.5