Characterization and Pathogenicity Test of Entomopathogenic Nematode Steinernema Species-Kalro

Entomopathogenic nematodes (EPNs) are worldwide soil-dwelling insect parasitic nematodes. They are potential pest bio-control agents a key component of Integrated Pest Management (IPM) programs. This study aimed to characterize and evaluate the pathogenicity of an EPN isolate from Kenya. The nematode was isolated from soils using insect bait technique and both morphological and molecular identification was performed. Efficacy of the isolate was evaluated against Tomato leafminer larvae (Tuta absoluta Meyrick.) using dose-based treatments of 0-control, 100, 150, 200, and 250 infective juveniles (IJs/ml). Morphological analysis revealed body length (L) of 835(659-987) μm and 1781 (1297-2097) μm from fresh IJs and males respectively. Males lacked a mucron. The isolate was characterized by the partial sequence length of 877 bp of the ITS region. Blastn results indicated the EPN isolate had a similarity match of 81-92% with Afro-tropical Steinernema species. It matched with Steinernema sp. (AY230186.1) from Kenya at 92% and Sri Lanka (AY230184.1). Phylogenetic analysis placed the isolate together with Steinernema sp. (AY230186.1) and (AY230184.1) with a bootstrap value of 100%. Maximum mean larval mortality (80%; 96%) was achieved 24 and 48 h post-treatment at concentration 150 IJs/ml. All nematode concentrations achieved over 50% mean mortality after 24 h period. There was a significant difference (P = 0.001) between doses 150 and 200 IJs/ml. From the study, it was concluded that the nematode isolate was Steinernema sp now referred to as Steinernema sp. Kalro (Genebank Accession MW151701). The EPN has the potential for development as a biological control agent against T. absoluta.


Introduction
Entomopathogenic nematodes (EPNs) in the families' Steinernematidae and Heterorhabditidae are pathogenic to insects (Kalia et al., 2014;Gozel & Kasp, 2015;de Brida et al., 2017), found in most soils worldwide. The EPNs free-living non-feeding stage, infective juveniles (IJs) which penetrate the insect host via body orifices or through the cuticle. Once in the hemolymph, the IJs release symbiotic bacteria that multiplies as the EPNs nourish on them and insect tissue and reproduce, killing the insect host within 24-72 hours (Kaushik & Chaubey, 2016;Caoili et al., 2018;Yooyangket et al., 2018).
The released bacteria provide pathogenicity, degrade and breakdown host tissue, and suppress the immunity of the host. These bacteria are known to produce toxic proteins (metabolites) that render EPNs lethal to insect hosts. They produce antibiotics and enzymes in addition to toxins. The mutualism of bacteria and nematodes is vital as it inhibits the development of resistance in the host insect. The nematodes complete their lifecycle within the host insect after which they exit into the soil and lie in wait for another suitable host (Poinar Jr., & Grewal, 2012;Sternberg & Dillman, 2012;Kalia et al., 2014;Gozel & Kasap, 2015).
Isolation and identification of indigenous EPNs population from their preferred conditions is a crucial step in the development of effective biological pest management. This is because such species are suited to local climatic conditions (Salvadori et al., 2012;de Brida et al., 2017;Kalia et al., 2014). Commercial use of EPNs as pest bio-agents has triggered a search for new strains and evaluation of their virulence against agricultural pests (Shapiro-Ilan et al., 2012). Most EPN species have not been known taxonomically, but tools for their identification have been developed. Nematode characterization is mainly based on morphological and morphometric characters which are limited due to a wide range of values/ratios among strains. There is, therefore, need for Deoxyribonucleic acid (DNA) sequence analysis for accurate identification (Liu & Berry, 1995). Characterization of EPNs requires study of the male tail, size and shape of spicules, body size, presence or absence of mucron, and lateral lines of infective juveniles (Nguyen, 2007;Hating et al., 2009).
Tomato (Solanum lycopersicum L.), is one of the world's most commonly and extensively grown edible fruit vegetables (Asgedom et al., 2011). Kenya is ranked 6 th in tomato production in Africa with a total production of 397,007 tonnes (FAO, 2012). However, its production is constrained mainly by insect pests among them Tomato leafminer (Tuta absoluta Meyrick.) that causes yield losses of up to 100%. The pest is mainly managed by chemical pesticides. Chemical pesticides are costly and pose environmental and food safety concerns. Their use on T. absoluta is also limited due to the pests' nature of the damage and its ability to develop insecticide-resistant strains (Haddi et al., 2012;Nicolopoulou-Stamati et al., 2016;Bala et al., 2019). Entomopathogenic nematode Sternernema feltiae has successfully been used as a biological control agent in the management of pests like leaf miner, thrips, and cutworms in carnation flowers in Kenya (PCPB, 2018). This has prompted a search for IPM options that are safe for humans, animals, and the environment. The study aimed to isolate, identify, and evaluate the potential use of nematode isolate as a biological control against T. absoluta.

Tuta absoluta Culture
Tuta absoluta life stages were collected from infested tomato farms to establish insect culture at Kenya Agricultural and Livestock Research Organisation (KALRO)-Horticulture Research Institute.

Entomopathogenic Nematode Isolate Culture
The EPN isolate was isolated from soils at KALRO-Thika and reared using the insect-baiting method as described by (Bedding & Akhurst, 1975). The soil was collected (250 gm) and 15 pre-pupa stages of Greater wax moth (Galleria mellonella) placed on the soil in a bowl. The samples were stored at room temperature of 25±2 °C and inspected for larval mortality every 24 hr. The infected G. mellonella cadavers showing typical symptoms of EPN infection were collected, cleaned in distilled water, and nematodes harvested according to White (1927). The EPN infective juvenile (IJs) were stored at 25±2 °C. The EPN culture was referred to as nematode isolate.

Morphological Identification of Nematode Isolate
The newly collected nematode isolate was reared in vivo in the pre-pupa stage of Galleria mellonella larvae. The G. mellonella cadavers were dissected on the 3 rd day to obtained 20 males of the nematode. In the 4-6 day, 20 emerging infective juveniles IJs were picked from G. mellonella cadaver.
Fresh IJs and males were killed at 50-60 °C in a water bath for 3 minutes and fixed in 2-3 drops of Triethanoalamine formalin (TAF) (Courtney et al., 1955). After 48 h, the fixed nematodes were mounted on glass slides with coverslips supported by wax to avoid flattening them sealed with nail varnish. Nematode morphology was studied according to Nguyen (2007) using a compound microscope, Leica Suit, DM 750 (Leica Microsystems Switzerland Ltd.).

Molecular Characterization of Nematode Isolate
Nematode infected G. mellonella cadavers were surface sterilized in 70% alcohol and dissected to get gravid females. The females were preserved in 50 µl of 95% alcohol and stored at 4 °C. Genomic DNA was extracted according to Razia et al. (2011), Caoili et al. (2017, protocols with modifications. The preserved nematode samples of gravid females were rehydrated in distilled water overnight (12 h). The obtained DNA was quantified and purified on a spectrophotometer and stored at -40 °C for later use.
The PCR amplification of the ITS region of the local nematode isolate was performed, in 12.5 µl of 10× of PCR master mix (Bio lab, England). The TW81 (5'-GTTTCCAGTAGGTGAACCTGC-3'), forward and AB28 (5'-ATATGCTTAAGTTCAGCGGGT-3') reverse primers were used for the partial gene amplification (Joyce et al., 1994). Thermocycler (ProFlex PCR System Applied biosystem) conditions were set at 94 °C for 5 min, 94 °C for 1min, 55 °C for 1 min, 72 °C for 2 min and 72 °C for 5 min all at 35 cycles per min. Electrophoresis of PCR products (5 µl) was run at 100 volts for an hour. The products were sequenced by Microgen, Korea.
The sequence was edited using BioEdit v 7.0.5 Sequence Alignment Editor Software (Hall, 1999). Correction of the alignment was performed manually while multiple sequence alignment was done using MUSCLE on SeaView version 4 Alignment and analysis program (Edgar, 2004;Gouy et al., 2010). A similarity search of the jas.ccsenet.org Journal of Agricultural Science Vol. 13, No. 3;2021 deduced consensus sequence of EPN from the NCBI database was then done using Basic Local Alignment Search Tool (BLASTn) at (https://blast.ncbi.nlm.nih.gov) (Altschul et al., 1990;Altschul et al., 1997).
The evolutionary relationship of the nematode isolate was compared to 16 selected blastn hits. The EPN Heterorhabditis safricana (EF88006) was used as an out-group for taxonomic comparison, Phylogenetic analysis was performed using Neighbour-Joining, Distance method on SeaView version 4 program (Edgar, 2004;Gouy et al., 2010). Branch length was estimated with 1000 bootstrap replications at a 70% threshold for relatedness for the automatically generated phylogenic tree.

Insecticidal Activity of the Nematode Isolate
Experimental infections were carried to determine the efficacy of the isolate against Tuta absoluta larvae. Nematode infective juveniles (IJs) concentration (0, 100, 150, 200, and 250) in 1 ml of nematode suspension was determined. The insect larvae were collected from tomatoes established and maintained in a screen house. On a sterile 9 cm, petri dish lined with white cotton cloth Five T. absoluta larvae were singly placed for each treatment.
The experiment was a completely randomized design (CRD) with five treatments (Control and 100,150,200,and 250 IJs in 1 ml of distilled water). The Control treatment was 1 ml of distilled water without nematodes. Each treatment with five larvae was replicated five times (N = 25). To confirm nematode pathogenicity, T. absoluta larvae cadavers were randomly selected from each treatment and dissected under the microscope. Data on larval mortality was recorded every 24 hr for two days.

Statistical Analysis
Morphometric data were analyzed using Microsoft Office Excel 2010. Data on larval mortality was subjected to analysis of variance using GenStat Software, 15 th edition. Means were separated using Fisher's protected least significant difference test at 1% significance level.

Morphology of Entomopathogenic Nematode
The

Molecular Characterization
The nematode sequence partial length of the ITS of the rDNA sequence was 877 base pairs (bp
The EPN was pathogenic against T. absoluta over time and across all the IJs doses. Pathogenicity of EPNs against T. absoluta and other economically important agricultural lepidopteran pests has been documented (Salvadori et al., 2012;Kalia et al., 2014;Gozel & Kasap, 2015;Caoili et al., 2018). There was an increase in mortality rate with an increase in IJs dose of up to 150. This indicated higher nematode efficiency at a lower concentration. The decrease in mortality at higher IJs concentration could be attributable to competition for entry points, penetration ability, and virulence of nematode in the petri dish bioassay. According to Gulzar et al. (2020), IJs penetration and virulence influence nematode pathogenicity.

Conclusions
The local nematode isolate was a Steinernema sp. EPNs, based on morphological and molecular analysis. The sequence was deposited to Gene-bank as Steinernema sp. Kalro (Accession MW151701). The EPN has significant potential as a biological control agent against T. absoluta. Further taxonomic evaluation of Steinernema sp. Kalro to species level and field trials on efficacy is recommended.