Endosymbiotic microbes from entomopathogenic nematode (EPNs) and their applications as biocontrol agents for agro-environmental sustainability

The biological diversity on planet earth is declining day by day, due to different factors such as excessive applications of pesticides. The utilization of chemical pesticides affected environment as well as microorganisms. The awareness among the peoples towards the hazards by the residual toxicity of chemical pesticides should be developed for agro-environmental sustainability. Entomopathogenic nematodes (EPNs) are the bacto-helminth parasites which show classical mutualism with the genera Xenorhabdus and Photorhabdus. The nematodes along with its endosymbiotic bacteria have a biocontrol potential which could be used to reduce chemical pesticides. Applications of bioagents have been reported and resulted in considerable reduction in pathogens. Furthermore, these bioagents are biodegradable, eco-friendly and easy to apply for protection of crops against diverse pathogenic organism. The nematode-bacterium complexes are effective against huge range of bacteria, fungi, nematodes and insects that are harmful to the crops. Along with biocontrol potential, the endosymbionts produce diverse secondary metabolic compounds, exoenzymes and toxic compounds that show antibiotic, antimycotic, nematicidal, miticidal and anticancerous properties. The present review deals with the diversity of endosymbiotic microbes from EPNs and their role in biocontrol for the agro-environmental sustainability.


Background
Entomopathogenic nematodes (EPNs) are microscopic roundworms that belong to the families Heterorhabditidae and Steinernematidae of phylum Nematoda. EPNs are beneficial nematodes that exhibit a holoparasitic mode of survival (Bhat et al. 2020). The EPNs have been reported to survive in most of environmental conditions except psychrophilic conditions of Antarctica (Hominick 2002). The EPNs from genus Steinernema and Heterorhabditis were considered deadly fatal for a number of agricultural insects (Liu et al. 2020). Globally, 17 species of genus Heterorhabditis and 100 species of genus Steinernema have been reported that are found to be lethal for insect pests (Bhat et al. 2020). These nematodes showed mutualistic associations with endosymbiotic bacterial species that live inside the nematode. A major role has been played by these bacterial endosymbionts in nutritional physiology (Feldhaar 2011). The endosymbionts Xenorhabdus and Photorhabdus reside in symbiotic Page 2 of 19 Tomar et al. Egyptian Journal of Biological Pest Control (2022) 32:80 association with EPNs Steinernema and Heterorhabditis (Kaya and Gaugler 1993). In EPNs, the third-stage juvenile (dauer juvenile) resides freely in soil with non-foraging behaviour, and carries endosymbiotic bacteria inside the body which is responsible for causing the pathogenicity in their host. Once the dauer juvenile penetrates into the host body through spiracles or through natural body openings, it releases its symbiotic bacteria inside the haemocoel of the insect. The bacterial cells duplicate and generate severe toxins that have a high insecticidal potential and can assassinate its host in less than 2 days (Adams and Nguyen 2002). The infected host dies soon after infection due to contagion. Within the insect cadaver, these bacteria nourish the EPNs and promote the growth and reproduction of EPNs. As the food availability shortens, the dauer juvenile of EPNs comes out of the cadaver and look for a new host. Applications of EPNs with their bacterial endosymbionts become a prime approach in the biocontrol sector as well as in integrated pest management. Moreover, these EPNs now become model organisms and used widely in the fields such as evolutionary biology, biological control, soil ecology as well as the bacterial symbiotic mutualism (Stock 2015). Emerging dissatisfaction due to the excessive applications of chemicals insecticides for insect pest control has increased, as they showed adverse effects on the environment and human health . Thus, it has turned out to be essential to diminish the utilization of these insecticides and replaces those using ecologically safer products in a sustainable agriculture perspective.
In recent times, these beneficial endosymbionts become a treasure trove for insecticidal compounds as well as for different bioactive compounds. The endosymbionts can decrease the chemical insecticides used in insect control and plant protection by stabilizing the environmental changes (Thakur et al. 2020(Thakur et al. , 2021Migunova and Sasanelli 2021). Undoubtedly, these endosymbionts may become a favourable substitute in increasing the biological control of numerous phyto-insect pests as well as pathogens. EPN-associated bacterial endosymbionts have a high potentiality for agricultural pest control due to the toxic compounds and proteins produced as secondary metabolites (Thakur et al. 2022a(Thakur et al. , 2022b. The present review deals with distribution, identification and culturing practices of endosymbionts associated with EPNs. The major emphasis has been laid on the bioactive compounds produced by the endosymbionts.

Entomopathogenic nematode
Since the seventeenth century or possibly prior to these EPNs were familiar (Nguyen and Smart Jr 2004) although in the nineteenth and twentieth century's vast studies on EPNs were carried out, and it was reported that EPNs were distributed worldwide. Steiner was the first to expound the EPN as Aplectana kraussei (Steinernema kraussei) from a hymenopterous sawfly (Steiner 1923), followed by Neoplectana glaseri from Popillia japonica (scarabaeid beetle) (Steiner 1929), S. feltiae, S. affinae, and S. carpocapsae were from Cydia pomonella (Weiser 1955). Mracek (2002) reported that the nematodes as most victorious organisms on earth which have been found nearly all types of habitats. In India, various investigations have been accomplished to find out the new EPNs species that resulted in the isolation of a new H. indica species from Coimbatore, Tamil Nadu (Poinar Jr et al. 1992). EPNs species were previously described as an excellent means of biocontrol for the management of agricultural insect pests. Till now, 11 EPNs species were disclosed in India including Heterorhabditis indica, H. bacteriophora, S. sangi, S. bicornutum, S. abbasi (S. thermophilum), S. siamkayai, S. carpocapsae (S. meghalayense), S. riobrave, S. glaseri, S. surkhetense, and S. hermaphroditum (S. dharanai) (Lalramnghaki 2017).

Distribution of endosymbionts of EPNs
The nematodes of families Steinernematidae and Heterorhabditidae have gained the interest of humanity towards their promising biocontrol capacity of managing insect population (Singh et al. 2022). Xenorhabdus and Photorhabdus, the Gram-negative bacteria, are endosymbionts of the dauer juvenile (IJ 3 ) phase of EPNs. The IJs can perforate the insect via natural body orifice. It liberated the endosymbionts inside the blood stream of the host upon getting entry inside the host. Inside the host blood stream, the bacterial cells reciprocated. The occurrence of huge bacterial cells in the host midgut resulted in demises of insect after 24-48 h (Kaya and Gaugler 1993). Huge numbers of Xenorhabdus and Photorhabdus have been reported as endosymbionts from Steinernema and Heterorhabditis (Tables 1, 2).

Taxonomy of endosymbionts
Bacterial groups are considered as the most diversified biological group having varied phylogeny of the organisms (Adams et al. 2006). The fossil traces of cyanobacteria were found about 2.9 billion years back, and the presence of nematodes was supposed somewhat earlier in the Cambrian radiation (Noffke et al. 2003). In the mid-Paleozoic era, it has been considered that the primogenitor of family Heterorhabditidae and Steinernematidae started to reconnoitre their symbiotic interactions with the members of Enterobacteriaceae (Poinar Jr 1993). Achromobacter nematophilus was the first endosymbiont found in mutualistic association with Neoaplectana Page 3 of 19 Tomar et al. Egyptian Journal of Biological Pest Control (2022) 32:80  carpocapsae Weiser (Poinar Jr and Thomas 1965). The characteristic features of A. nematophila did not suit with any of the previously approved genera that results in the emergence of the novel genera Xenorhabdus. This novel genus includes bacterial symbionts X. luminescens and X. nematophilus (Akhurst 1983a). A noticeable separation of X. luminescens among the other Xenorhabdus genera was based upon the phenotype as well as the genotype of the individual ) that suggest the establishment of new genera Photorhabdus having mutualistic association with the nematodes of genera Heterorhabditis (Boemare et al. 1993). The earlier classification of Xenorhabdus and Photorhabdus into separate genera is completely based upon their phenotypic characters as well as on the mechanism of symbiosis exhibited by them (Thomas and Poinar Jr 1979). After that, the taxonomy of prokaryotes was done through a multiphase prospective that include the combined information of various types of genotype and phenotypes. This multiphase perspective is further followed by molecular biology techniques including DNA-DNA hybridization and sequence analysis of 16S rRNA gene that turns out to be the foundation of the bacterial classification (Stackebrandt 2006). New species of endosymbionts were discovered by following the 98.7% relatedness concept of 16S rRNA gene, still an initial move towards identification along with the threshold of 70% in DNA-DNA hybridization and the threshold of 80% in DNA-DNA hybridization for efficiently balancing negligible criterion in the bacterial taxonomy approach (Goris et al. 2007). The gene sequencing technique 16 R-gene for classification was regarded as inappropriate because of little differences along with lateral gene transfer (LTG) (Tailliez et al. 2010). The multilocus sequence analysis (MLSA) technique has been extensively utilized for the recognition of many noval species of bacteria (Liu et al. 2017). The enhanced sequencing methodologies permit the utilization of whole genetic sequence for the identification and classification (Lee et al. 2016). A remarkable potential of genome taxonomy has been shown in the high-resolution classification of genera Photorhabdus (Machado et al. 2018).

Life history of endosymbionts
The EPNs are the organisms that cause diseases in insects. In the nematode life cycle specialized infective juvenile (IJ 3 ) is the only form that lives outside the host insect (Poinar Jr et al. 1979). Inside the intestinal tract of infective juvenile these symbiotic bacteria populate. The other juvenile stages IJ 1 and IJ 2 cannot live without their host and do not carry symbiotic bacteria in their intestinal tract. The endosymbionts (Photorhabdus and Xenorhabdus) of EPNs exhibit almost similar life cycles. The infective juvenile works as a carrier in transmitting the bacterial endosymbionts (Sicard et al. 2004). Upon entering to the insect body, nematode moves to the haemocoel cavity of the insect and released its endosymbionts. These endosymbiotic bacteria have the ability to escape from the insect immune system and are responsible for causing pathogenicity in the insects by releasing a variety of insecticidal toxins (Koppenhöfer et al. 2007).
In addition to pathogenicity against insects, these bacteria defend the insect cadaver from invading microorganisms (pathogens, competitors, and predators) by producing antimicrobial compounds and also contribute to nematode reproduction (Gulcu et al. 2012). Inside host insect nematodes undergo about 2-3 rounds of reproduction before the nutrients depleted after that infective juvenile (IJ 3 ) form way out from the insect corpse and seek for a new host (Grewal and Georgis 1999).
Photorhabus bacteria are the endosymbionts of Heterorhabditis that make a colony behind the basal bulb in the anterior portion of the intestine. They were also reported scattered in the remaining portion of the intestine (Ciche et al. 2003). The Xenorhabdus are endosymbionts of Steinernema that colonize in the specific bilobed vesicle of intestine (Martens et al. 2003). The life cycle exhibited by these bacteria is quite unique and interesting as they lived in symbiotic association with nematode and are pathogenic to harmful insects. The endosymbiotic complexes of Steinernema show a very high resemblance with Heterorhabditis and have a little variability in their life cycle that leads to the independent classification of the two genera. Endosymbiont Photorhabdus are emanated via the anterior region, i.e. mouth of nematode (Ciche et al. 2008), whereas Xenorhabdus is liberated from the posterior region, via anus, of the nematode (Sicard et al. 2004). Host immune suppressing proteins were released by Steinernema that might support their endosymbionts release (Simoes and Rosa 1998) but in Heterorhabditis, this process is still mysterious (Forst and Clarke 2002). Brillard et al. (2002) reported that both genera show haemotoxic behaviour.
The endosymbionts of both genera release certain protein toxins as well as exoenzymes that are responsible for causing septicaemia in host insects that leads to insect death (Forst and Clarke 2002). In the late infectious state, the proteinaceous toxins produced by bacteria damaged the midgut of the insect mainly the epithelium lining (Silva et al. 2002) (Figs. 1, 2). Xenorhabdus and Photorhabdus exhibit colony pleomorphism, i.e. phase variation phenomenon in which coexistence of two different variants was observed in single bacterial species. Differences in trait numbers were observed in these variants like release of antibiotics, proteins, pigment substances, lipases, and bioluminescence (Turlin et al. 2006). The endosymbiotic bacteria show two phases in their life cycle that are morphologically distinct from each other. In Xenorhabdus spp., the cells under phase I are somewhat larger, mobile with crystalline inclusion bodies and liberate secondary metabolic substances such as lipases, proteases, and some other bioactive components. Phase II cells are much smaller than phase I, immobile and can easily be recognized by staining with dyes (triphenyltetrazolium chloride and bromothymol blue). The cells in phase I condition when exposed to nutritional media having the fusion of two dyes triphenyltetrazolium chloride and bromothymol blue, they shows clearly distinct dark blue bacterial colonies with red core. But an exemption was also noticed that phase I bacterial cells do not absorb dye (bromothymol blue). It has stated that in two phases of their life cycle, the phase I bacteria was normally found in association with reproducing nematodes, while the phase II bacteria were found in the nematode infected cadaver (Turlin et al. 2006).

Culturing practices and cost effectiveness of endosymbionts
The endosymbionts Xenorhabdus and Photorhabdus can be easily isolated from the dead larval cadaver of Galleria mellonella L. infected with IJs of EPNs after 24 h. The infected cadavers should be sanitized with merthiolate (0-1% for 120-180 min) and cleaned multiple times with distilled water. After that transferred to the yeast-soy-based (YS) broth and changed into suspension with the help of tissue homogenizer.
Page 6 of 19 Tomar et al. Egyptian Journal of Biological Pest Control (2022) 32:80 The suspension should be spread over the nutrient agar (NA) containing bromothymol blue and triphenyltetrazolium chloride. The endosymbionts can be isolated using hanging drop system method. The dead cadavers should be soaked into absolute ethanol for surface cleaning and allowed to dehydrate in sterile Petri dish. With the help of a sterilized forceps, dead cadaver should be dissected and a drop of haemolymph dragged out using a sanitized loop. The loop containing haemolymph streaked over the NBT agar and should be placed at room temperature for growth (Poinar and Thomas 1966). An alternative approach for endosymbionts isolation was by directly crushing the IJs. The IJs (50-100IJ) have sterilized with thiomersal (1% for 2 h) and centrifuged. After centrifugation the pellet containing IJs should be washed several times and placed onto nutrient broth (NB) luria-bertani (LB), tryptic soy broth (TSB). The suspension should be spread over the NBT agar media or luria-bertani media and cultivated at room temperature.

Role of endosymbionts as biocontrol agents
Endosymbionts metabolism produces a wide range of secondary metabolites for potential applications in different sectors. In EPNs infected insects, these endosymbionts defend the host cadaver from the invading microbiome by the secretion of a variety of compounds possessing antibiotic activities. These metabolites are also used as biocontrol negotiators for the management of viruses, fungi, nematodes, and insects (Lulamba et al. 2021). The bioactive components produced by Xenorhabdus and Photorhabdus play an essential role in the biological transformation of host insects. Production of different kinds of antibiotics, proteases, adhesions, lipases, and haemolysins were observed from endosymbionts. Even a single Xenorhabdus strain produces multiple metabolites that act against variety of pathogenic organisms. Webster et al. (2002) recorded the productions of several antimicrobials such as β-lactam carbapenem, isopropylstilbenes from Photorhabdus species.
The discovery of such antimicrobial complexes acquires  (Tables 3, 4). Numerous bactericidal, fungicidal, entomopathogenic as well as nematode killing activities have been reported from endosymbionts (Muangpat et al. 2020). All the metabolic complexes that showed different activities against insects, parasites, fungi, and viruses were possessed a particular gene that encoded for particular traits (da Silva et al. 2020). Various strains of Xenorhabdus produces a variety of secondary metabolites that show a wide range of bioactivity, antibacterial activities, antifungal activities, nematicidal activities, insecticidal activities as well as several cytotoxic properties (Brachmann and Bode 2013) (Tables 3,  4).

Insecticidal activity
Insecticidal activities are the insect killing or insect controlling mechanisms, showed by Photorhabdus and Xenorhabdus. Nematodes penetrate the host body through naturalistic orifices and liberate the bacteria through defecation that releases compounds that suppress the insect immunity (Webster et al. 2002). They have been found to infect Cydia pomonella, Delia radicum Diaprepes abbreviates, Thrips spp., Otiorhynchus sulcatus, Phyllopertha horticola as well as the larvae of several other insect orders including dipterans and lepidopterans (Georgis et al. 2006). Several X. nematophila strains produce UnA protein that inhibits the accumulation of haemocytes which results in the formation of cover or sheath around the bacteria and nematode (Ribeiro et al. 2003). Dunphy and Webster (1991) reported that these protein and lipopolysaccharides in G. mellonella stop the cluster formation of haemocytes that hinder phenoloxidase activation which is a major tool of insect immune system (Forst et al. 1997). Eicosanoids responsible for the cellular immunity in insect's also suppressed by X. nematophila strains. They hinder the action of phospholipase A 2 (PLA 2 ) upon which the production of eicosanoids depends (Kim et al. 2018). Without eicosanoids, insects were died due to acute bloodstream infection or septicaemia. An insecticidal protein (57 kDa) produced by X. budapestensis D43 trigger the phenoloxidase cascade in G. mellonella and resulted in extreme immune responses such as productions of a high amount of quinones (Yang et al. 2012a, b). This extreme production of quinine is lethal for the insect larvae. Even     programmed cell death in insect haemocytes was also caused by several species of Xenorhabdus such as X. nematophila, X. beddingii, X. japonica, and X. kozodoii (Cho and Kim 2004). Another cytotoxic protein CyA produced from X. nematophila can destroy the insects within one or two days (Khush et al. 2002). It was reported that toxin complexes of X. nematophilus include three insecticidal proteins xptA 2 , xptB 1 , and xptC 1 that united to form a tetramer (∼1120 kDa) that attached to the outer membranes of insects and form pores in the membrane and have very high insecticidal activity against lepidopteran insects (Sheets et al. 2011). PMFI296 genes of X. nematophila produce insect killing proteins such as xptA 1 , xptA 2 , xptB 1 and xptC 1 . These proteins are highly virulent against Pieris brassicae, P. rapae, and Heliothis virescens (Sergeant et al. 2003). TccC1 toxic gene from X. nematophila is responsible for causing mortality in G. mellonella larvae (Lee et al. 2004). X. nematophila strain CBNU produces Txp40 protein which is toxic against larval stage of Plutella xylostella (Park et al. 2004). The leucine responsive protein (lrp) from X. nematophila, also possessed insect killing ability (Hussa et al. 2015). Xenocyloins produced from X. bovienii (SS-2004) also found virulent against insects and affect the haemocytic activity of insects (Proschak et al. 2014). Outer membrane vesicles (OMV) proteins GroEL homolog (∼58-kDa) of X. nematophila also exhibit oral toxicity against Helicoverpa armigera (Hb.) larvae (Joshi et al. 2008). Xenematide peptides isolated from X. nematophila also showed insect killing potential (Lang et al. 2008). Compounds xenematides and rhabdopeptides derived from X. nematophila also possess insecticidal activities as they affect the haemocytes of insects (Reimer et al. 2009). Srf ABC toxin derived from the fosmid clones of X. stockiae HN_xs01 strain also has insect killing properties. Srf ABC toxin brings G2/M at halt and causes necrobiosis or cell death in CF-203 cells (midgut cells) of H. armigera larvae (Yang et al. 2019). Chitinase protein (76-kDa) from X. nematophila strain ATCC 19061 exhibit endochitinase activity, β-N-acetylglucosaminidase and chitobiosidase activities were found highly lethal against H. armigera (Mahmood et al. 2020).
Even the cell-free filtrate of Photorhabdus spp. also exhibits insect killing properties. The genome of Photorhabdus laumondii (TT01 strain) encodes for a wide range of metabolic compounds including lipases, toxins, adhesins, proteases, and haemolysins and variety of antibiotic substances (Zamora-Lagos et al. 2018). Crude extract of P. luminescence laumondii (TT01strain) reported to cause toxicity in Bemisia tabaci (Shrestha and Lee 2012) and crude extract of P. luminescence sonorensis was effective against Helicoverpa zea. Many secondary metabolites produced by Photorhabdus also pathogenic to insects. These metabolites are anthraquinone derivatives, stilbene derivatives and genistine that are highly virulent against insects (Chalabaev et al. 2008). Anthraquinone derivatives 3-methoxychrysazine and 1,3-dimethoxy-8-hydroxy-9,10-anthraquinone, extracted from P. temperata effective against various mosquito spp. (Ahn et al. 2013). Baur et al. (1998) also describe that these anthraquinone acts as an obstacle for ants and birds. Stilbenes derived from Photorhabdus hamper the activity of phenoloxidase by interfering and interrupting the insect (Manduca sexta) immune system. This phenoloxidase is the major component of insect immune system and is responsible for melanisation (Eleftherianos and Revenis 2011). TccC3 (adenosine diphosphate (ADP)-ribosyltransferases) and TccC5 (DP-ribosylated Rho guanosine triphosphatase) toxic proteins produced by P. luminescens hinder the mechanism of phagocytosis in insect cells and intracellular actin polymerization (Lang et al. 2010). P. luminescens, also produces tyrosine based compounds, rhabduscin that suppress the insect immune system (Crawford et al. 2012).

Antifungal activity
The metabolites produced by endosymbionts have been reported to inhibit the growth of the multiple fungi. Huge numbers of research studies were carried out and being continued to measure the efficacy of endosymbionts against fungal pathogens. X. nematophila produces cyclolipopetide having lysin rich residue is highly effective against fungal pathogens including plant as well as animal fungal pathogens (Gualtieri et al. 2009). Chen et al. (1994) evaluated the inhibitory effect of X. nematophilus X. bovienii and P. luminescens against 32 fungal species and they found effectiveness against all fungal species. Even the growth of 7 major phytopathogenic fungi: Trichoderma pseudokingi, Botrytis cinerea, Mucor piriformis, Ceratocystis ulmi, Pythium coloratum, Ceratocystis dryocoetidis, and Pythium ultimum were completely suppressed by these endosymbionts. Webster et al. (1995) reported the fungicidal activities of endosymbionts Xenorhabdus and Photorhabdus. The metabolic complexes from Xenorhabdus and Photorhabdus were isolated and it is found that these compounds have defensive and fungus eliminating properties. High antimycotic effect was recorded in Sclerotinia sclerotiorum when cell free filtrate of X. szentirmaii was applied (Chacon-Orozco et al. 2020). It was reported that inside insect cadaver antimycotic substances were produced by Photorhabdus spp. that protect the cadaver and prevent the growth from invading fungal pathogen (Chen et al. 1994). Crude extract of Photorhabdus spp. was assessed against fungal phytopathogens such as Phomopsis sp., Fusi cladosporium effusum, Glomerella cingulata, Phytophthora cactorum, and Monilinia fructicola. Modest effect of endosymbionts against fungal phytopathogens was observed (Shapiro-Ilan et al. 2009). Strong fungicidal effect of Photorhabdus spp. was observed towards Moniliophthora roreri (San-Blas et al. 2012). Even several specified secondary metabolic complexes have been applied to assess their antimycotic peoperties. Li et al. (1995b) applied 3,5-dihydroxy-4-isopropylstilbene on Page 12 of 19 Tomar et al. Egyptian Journal of Biological Pest Control (2022) 32:80 fungi Aspergillus fumigatus, A. flavus, Cryptococcus neoformans, Botrytis cinerea, and Candida tropicale. Photorhabdus temperata SN259 strain produces seven metabolic complexes among which two stilbene complexes such as 3-hydroxy-2-isopropyl-5-phenethyl phenyl carbamate and 2-isopropyl-5-([E]-2-phenylethenyl) benzene-1,3-diol (syn. 3,5-dihydroxy-4-isopropyl stilbene) were applied to investigate its impact upon F. oxysporum, Rhizoctonia solani, Pythium aphanidermatum, and Exserohilum turcicum. A highly strong inhibitory effect has been observed against P. aphanidermatum (Shi et al. 2012). Transcinnamic acid isolated from P. luminescens prevents the growth of the Fusicladium effusum . P. temperata M102 derived metabolite benzaldehyde was assessed against three phytopathogens Phytophthora capsici, Corynespora cassiicola, and R. solani. Benzaldehyde shows high inhibitory effect against the fungal phytopathogen. Even Photorhabdus spp. has been reported to hinder the growth of about 32 fungal species (Ullah et al. 2014). Trans-cinnamic acid (TCA) isolated from Photorhabdus sp. hinder the growth of Colletotrichum acutatum, Colletotrichum gloeosporioides and Colletotrichum fragariae (Chen et al. 1996). TCA also inhibits the growth of F. effusum . The efficiency of cell free filtrate from P. luminescens strain VS, P. temperata and P. luminescens strain K122 was assessed against various phytopathogenic fungal species such as: Armillaria tabescens, Fusicladium carpophilum, F. effusum, Glomerella cingulata and Monilinia fructicola. It was found that these supernatants completely suppress the growth of the phytopathogenic fungi (Hazir et al. 2016). Under an in vitro inhibition test, fungi Alternaria alternate and Fusarium oxysporum sp. asparagi were treated with the crude extract of P. luminescens sp. sonorensis, that show mild effect against these fungi although the growth was retarded (Orozco et al. 2016). Secondary metabolic complexes produce by P. akhurstii exhibit fungicidal activities against Colletotrichum gloeosporioides (Tu et al. 2022).

Nematicidal activity
The cell free substrate of Photorhabdus spp. not only exhibit insecticidal, antibiotic and antimycotic activities but also possesses nematicidal properties. They have been reported to kill or manage various species of plant parasitic nematodes. Several strains of Photorhabdus spp. were evaluated against Meloidogyne incognita and Bursaphelenchus xylophilus. They were highly lethal against second-stage juveniles of M. incognita and adults as well as fourth-stage juveniles of Bursaphelenchus xylophilus (Hu et al. 1999). Crude extract of P. luminescens sonorensis CH35 strain was applied against three nematode species namely: M. incognita, Caenorhabditis elegans and S. carpocapsae. It was found that this supernatant was highly effective against M. incognita (J2), while it exhibited very low nematicidal activities against C. elegans and S. carpocapsae (Orozco et al. 2016). The metabolic substances stilbene (3,5-Dihydroxy-4-isopropylstilbene) and indole produced through P. luminescens strain MD were tested against Aphelenchoides rhytium, Bursaphelenchus spp., C. elegans and M. incognita. High nematode killing abilities were exhibited by these derivatives against fourth-stage juvenile and adult forms of three tested species; however, no nematicidal effect of these metabolites has been observed against M. incognita (J2) (Hu et al. 1999). Cell-free supernatant of Xenorhabdus spp. is highly toxic against M. incognita and showed inhibitory effects (Grewal et al. 1999). Lewis et al. (2001) worked on the interactions of S. feltiae and X. bovienii with M. incognita and found that tomato plant infected with M. incognita when treated with same rate of S. feltiae, affect egg production with lesser galls in their roots. X. bovienii also shows inhibitory effect against M. incognita.
Cell-free filtrate of X. bovienii was applied against Meloidogyne javanica and M. incognita and a moderate effect was observed (Kepenekci et al. 2018). Bi et al. (2018) reported about seven metabolites (Rhabdopeptide I-O or 1-7) from X. budapestensis SN84 that possess nematode killing properties and is found effective against M. incognita (J2). Among all seven isolated metabolites, rhabdopeptide J2 was highly effective. Xenorhabdus spp. along with neem cake and furadan was applied to control the M. incognita infestation in Grapevines. It was reported that all treatment significantly suppresses the nematodes population in grapevines (El-Deen et al. 2019).

Bioformulations produced from endosymbionts
Bioformulations are biological pesticides invented with useful microbiomes including bacteria, viruses, fungi, nematodes, and plant-based extracts as well as semiochemicals, as active component. Generally, bioformulations developments are based upon the bioresource detection, optimization, stabilization, and risk executive energy. The biopesticides have been regarded as best alternate of chemical insecticides in sustainable crop production due to their eco-friendly behaviour (Gašić and Tanović 2013). Nowadays, peoples become aware about the hazards caused by extensive use of synthetic chemical insecticides and moving towards healthy and organic foodstuff. Bioformulation applications easily tackle the insect resistance problems, provide effective protection to the crops and are an important part of integrated pest management (IPM) strategies (Elad et al. 1996). A wide range of biopesticides from entomopathogenic bacteria were available for insect pest management. These Page 13 of 19 Tomar et al. Egyptian Journal of Biological Pest Control (2022) 32:80 formulations are in the form of dry (granular) and wet products (liquid and wet powder) (Singh et al. 2014). Dust powder, seed dressing powder, water dispersible granules as well as wet powders form with dilution attributes are considered under dry formulations. Emulsions, suspoemulsions, suspension concentrates, capsule suspensions, oil dispersions, and ultra-low-volume formulations were considered under wet products/formulation (Knowles 2005). The endosymbionts Xenorhabdus and Photorhabdus exhibited insecticidal, antimycotic, bactericidal and nematicidal activities so they can be employed for bioformulation production (Namsena and Bussaman 2020). Namsena et al. (2016) reported three bioformulations of Xenorhabdus (X. stockiae PB09) in the form of liquid supernatant, cell pellet, wettable powder form and observed high mortality in mites even after storage up to 45 days at 4 °C. P. luminescens along with paraffin oil, Tween-20 and sucrose were applied directly as a spray against Pieris brassicae, and 100% larval mortality was observed within 24 h of foliar application (Mohan et al. 2003). Sodium alginate capsules were also prepared from P. luminescens akhurstii that was reported to cause 100% killing of S. litura within 48 h of its infection (Rajagopal et al. 2006). Toxicity in Prays oleae was also recorded by P. temperate, when ingested directly (Tounsi et al. 2006) (Fig. 3).

Conclusion and future prospects
Entomopathogenic nematodes (EPNs), the valuable nematodes, have been reported as the finest biocontrol agents. EPNs are considered as the best substitute of chemical insecticides due to their high potential of infecting the insects hidden even in mysterious places with high multiplication ability as well as their eco-friendly nature. In the field of biological pest management, the application of EPNs along with their bacterial symbionts becomes a popular approach of pest control. The diversified secretion systems of entomopathogenic bacteria were involved in the release secondary metabolite. These secondary metabolites are toxic proteins that possess high insecticidal potential along with high antimicrobial activities. Xenorhabdus and Photorhabdus strains produced multiple metabolites that act against a variety of organisms including, protozoans, fungi, nematodes  Tomar et al. Egyptian Journal of Biological Pest Control (2022) 32:80 insects, and even against cancerous cells. Various kinds of antibiotic were also produced from several species of symbiotic bacteria that showed the importance of these bacterial symbionts in the drug industry. There is a need of more surveys on EPNs to discover more species of endosymbiotic bacteria. The secondary metabolites, rich sources of toxic and bioactive compounds from endosymbiotic bacteria, need to be optimized and explored in the future; along with that there is a necessity of development of some by-product from these bacteria and their metabolites.