Isolation and evaluation of bacteria associated with entomopathogenic nematode Heterorhabditis spp. against the spider mite, Tetranychus truncatus Ehara (Acari: Tetranychidae)

Entomopathogenic nematodes (EPNs) belonging to the Heterorhabditis spp. harbour symbiotically associated bacteria which are toxic to a wide range of insect pests. Isolation, purification, characterization and mass multiplication of such bacteria will be a promising strategy in the management of the pests. This study was carried out to isolate the EPN from different locations, isolate and purify the bacterial colonies, characterize the bacteria through morphological and molecular strategies and to test the efficacy of different bacteria in the control of polyphagous Tetranychus truncatus Ehara mites. EPNs were isolated from soil samples at 11 localities of Kerala State, India, and used to infect the Galleria mellonella L. larvae. Bacteria associated with the haemolymph of the infected larvae were isolated, which on NBTA medium have produced circular to irregular, entire, opaque and smooth colonies. Sequence characterization of the 16S rRNA revealed nine isolates namely: one symbiotic bacterium Photorhabdus luminescens, two Pseudomonas aeruginosa, five Ochrobactrum sp. and one Stenotrophomonas maltophilia. Phylogenetic analysis using the sequences has further confirmed the bacterial identity. Evaluation of the cell suspension (CS) and cell-free supernatant (CFS) of P. luminescens, P. aeruginosa and Ochrobactrum sp. for their adulticidal and ovicidal efficiencies on T. truncatus had identified significant adulticidal effects by P. luminescens, followed by P. aeruginosa. After 96 h of treatment, P. luminescens at 108 cells/ml resulted in a significantly higher mortality rate of adult mites (64.00 and 60.67%, respectively, for CFS and CS), compared to that resulted by P. aeruginosa (38.67 and 33.33%). Results of this study showed that P. luminescens associated with the EPN Heterorhabditis spp. is a promising biocontrol agent for T. truncatus.


Background
Entomopathogenic nematodes (EPNs) belonging to the families Steinernematidae and Heterorhabditidae are obligate insect parasites which can be used as potential biocontrol agents against important soil dwelling pests (Lacey and Georgis 2012). The bacteria symbiotically associated with EPNs are toxic to a wide range of insect

Open Access
Egyptian Journal of Biological Pest Control *Correspondence: deepu.mathew@kau.in pests (Guo et al. 1999). Nematodes protect the bacteria from the host's immune system and help in their transportation by forming a special vesicle in the infective L3 juvenile stage, as in case of Steinernematidae, or retain throughout the intestine as in case of Heterorhabditidae. The bacteria help in maintaining ideal conditions for the reproduction of the nematodes by releasing some of the nutrients. They also release antimicrobial substances, which prevent the growth of other bacteria (Boemare et al. 1996).
Photorhabdus luminescens is the main bacterial species symbiotically associated with the EPN Heterorhabditis (Boemare et al. 1996). Pathogenicity of this bacterium against insect pests has been well documented by earlier scientists (Bowen et al. 1998). Additionally, few studies report the pathogenicity of this bacterium to mite species (Kulkarni et al. 2017). However, many other bacteria are also associated with EPNs (Babic et al. 2000). Entomopathogenicity of some of these bacteria associated with Heterorhabditis spp. has also been reported (Salgado-Morales et al. 2019).
Spider mites (Tetranychidae) are serious sucking pests of many agricultural and horticultural crops. Among the spider mites, Tetranychus truncatus Ehara (Acari: Tetranychidae) is a major species infesting economically important crops of Kerala State, India (Bennur et al. 2015). In this study, bacteria associated with the EPNs Heterorhabditis spp. were evaluated under laboratory conditions for their bio-efficacy against T. truncatus.

Collection of soil samples
Soil samples were collected randomly from 11 localities of Kerala State, India. From each locality, 500 g soil was collected from 15 cm depth, sealed and labelled in polybags and brought to the laboratory.

Isolation of Heterorhabditis from the soil
Following the baiting technique (Bedding and Akhurst 1975), EPNs were isolated from the soil samples. Larvae of greater wax moth, Galleria mellonella L. (Lepidoptera: Pyralidae) obtained from the culture maintained by All India Network Project on Agricultural Acarology (AIN-PAA), Kerala Agricultural University, India, were used for the isolation and maintenance of EPNs. Four to five healthy last-instar larvae of G. mellonella were released separately into plastic containers of 250 ml size, containing the soil samples collected from different locations. The soil samples were moistened before transferring to the plastic container to maintain adequate moisture content. Mouth of the container was covered with muslin cloth, placed upside down and stored at room temperature. Infected larvae which turned to brick red colour and died within 24-48 h, indicating infection by the EPNs were picked and washed thoroughly with distilled water and transferred to White's trap (White 1927) and kept in dark at 28 °C. For the multiplication and maintenance of EPNs, infective juveniles (IJs) released from the cadavers along the sides of the Petri plate were collected, stored in vials and used as stock for infecting the Galleria larvae. To get infected, four to five larvae were released into Petri plate lined with filter paper, moistened with IJ suspension (1 ml), incubated at 28 °C in a BOD incubator and observed for mortality. The dead cadavers were used for the isolation of EPNs-associated bacteria.

Isolation of bacteria from the EPNs
Bacteria were isolated from the EPNs infected cadavers and inoculated on NBTA plates. Within 24-48 h of death, the cadavers were collected and surface sterilized with 70% ethanol under aseptic conditions. The cadavers were dissected with a sterile blade, and a drop of haemolymph was streaked on Petri plates containing 10-20 ml of NBTA media (peptone 5 g, beef extract 3 g, NaCl 5 g, bromothymol blue dye 0.025 g, 2,3,5 triphenyl-tetrazolium chloride (TTC) 0.04 g, agar 15 g and distilled water 1000 ml) using a sterile inoculation loop (Woodring and Kaya 1988). Same medium was used for isolation, purification and maintenance of the bacteria. The Petri plates were then sealed tightly by using parafilm and kept in BOD incubator for 24-48 h at the optimum temperature of 28 °C and dark conditions and observed for colony proliferation. Individual pure colonies were selected, streaked on NBTA plates and subcultured continuously to get pure colonies of uniform size and morphology.

Identification of the bacteria
Bacteria associated with EPNs were studied for their cultural characters and 16S rRNA sequences. The colony characters such as shape, colour, edge, margin, elevation and surface as well as change in the colour of media, were recorded. The isolates were also subjected to Gram's reaction.
For molecular characterization, 16S rRNA gene was amplified and sequenced through colony PCR on fresh cultures of 24-48 h. Reaction mixture (50 μl) was prepared by adding EmeraldAmp GT PCR Master mix (25 μl), water (23 μl), forward and reverse primers (1 μl each) and bacterial culture was added by picking a single colony with tooth pick. Universal primer sequences (27f/1492r) 5′ AGA GTT TGA TCC TGG CTC AG 3′ (forward) and 5′ ACG GCT ACC TTG TTA CGA CTT 3′ (reverse) were used (Mulla et al. 2017). Thermal program followed had initial denaturation at 94 °C for 4 min, followed by 35 cycles consisting of denaturation at 94 °C for 30 s, annealing at 55.7 °C for 45 s retrieved from GenBank. Phylogenetic tree was constructed by neighbour joining method with 500 bootstrap replications.

Bioassay of EPN-associated bacteria against Tetranychus truncatus
Bacterial cells as well as cell-free supernatant (CFS) of five bacterial isolates (one isolate of P. luminescens, two isolates of P. aeruginosa and two isolates of Ochrobactrum) were evaluated independently against the eggs and adults of T. truncatus at 10 4 , 10 5 , 10 6 , 10 7 and 10 8 cells/ ml concentrations. The Pseudomonas and Photorhabdus isolates were selected because of their high pathogenicity against some of the insect pests (Salgado-Morales et al. 2019). Since Ochrobactrum spp. is reported to be pathogenic (Fu and Liu 2019) and non-pathogenic (Babic et al. 2000) to insects, its isolates were also included. The emerging multidrug-resistant opportunistic pathogen Stenotrophomonas (Brooke 2012) was excluded from the study. Experiments were laid out in completely randomized design with 26 treatments including control and three replications per treatment. For the ovicidal assay, ten gravid females of T. truncatus were transferred on the leaf bit of mulberry (5 × 5 cm 2 each), placed in a Petri plate lined with wet cotton and allowed for laying eggs. After 24 h, the mites were removed and 25 eggs were retained per leaf bit. For adulticidal assay, three leaf bits with 25 gravid female mites were used for each treatment. Sterile liquid broth of NBT (150 ml) was taken in a conical flask and a loop full of fresh bacterial culture was added aseptically, sealed and maintained in an orbital shaker at 200 rpm for 24-48 h and used as mother culture. Serial dilution, plating and colony counting method were followed to assess the initial concentration of the mother culture. Based on the initial concentration, 10 4 , 10 5 , 10 6 , 10 7 and 10 8 cell/ml were prepared by serial dilution. Ten millilitre each of these dilutions was centrifuged at 4000 rpm for 20 min for the preparation of cell suspension and cell-free supernatant and sprayed separately on leaf bits with egg and adult mites, using a hand atomizer (2 ml/leaf bit). Per cent mortality was recorded by observing under a stereo binocular microscope (LEICA EZ4 HD) at 24, 48, 72 and 96 h of spraying.

Results
Eleven isolates of EPNs Heterorhabditis spp. were isolated from the soil samples by baiting method. The infective juvenile suspension prepared from the EPNs isolates ( Fig. 1A) was used for infecting healthy Galleria larvae ( Fig. 1B-D). Bacteria were isolated from the infected cadavers (Fig. 1E) and cultured on NBTA media. When haemolymph from the infected cadavers of G. mellonella was streaked on NBTA plates, bacterial colonies developed within 24-48 h. Eleven bacteria were isolated from the EPN-infected Galleria cadavers and coded based on the locality from where soil was collected for EPN isolation (Table 1).

Cultural characterization of the bacteria
The colony characteristics of the isolates KL1 and KT1 were similar on NBTA medium (Table 2), with circular shape, irregular edges, opaque, flat and smooth surface. Similarly, the isolates MP1, MT1, FR1, EKM1, HI1, HQ1 and HS1 were similar, but differed from the isolates, KL1 and KT1 in colour of the colony. The colour varied from red (FR1, MT1, HI1) to pinkish red (HS1, MP1, EKM1, HQ1). Elevation of the colonies varied from flat (KL1. KT1, FR1, EKM1), low convex (MP1, HS1) to raised (MT1, CF1, HI1, HQ1, WH1). Isolates WH1 and CF1 were similar, producing dark red colonies with white margin and the media turned from yellowish to blue (Fig. 1F). Circular to irregular, opaque, raised, smooth colonies were produced. Under 100X oil immersion microscope, the cells appeared red in colour (Fig. 1G) and were identified as Gram's negative. However, the shape of the cell varied among the isolates from short to long rods.

Molecular characterization
Using the universal primers, 16S rRNA gene from eleven isolates was amplified (Fig. 2) and sequenced. Out of the eleven isolates, only nine isolates were identified, as the sequencing information for the remaining two isolates (WH1 and MT1) was incomplete even after repeated sequencing. Of the nine isolates identified, isolates KL1 and KT1 were identified as Pseudomonas aeruginosa, while the isolate HQ1 was identified as Stenotrophomonas maltophilia ( The phylogenetic analysis of 16S rRNA gene from the nine isolates along with 47 related accessions from the GenBank has resulted in a phylogenetic tree with two clusters (Fig. 3). Cluster A had subcluster A1

Bioassay of the EPN-associated bacteria against Tetranychus truncatus
Five bacterial isolates (CF1-P. luminescens, KL1-P. aeruginosa strain 1, KT1-P. aeruginosa strain 2, HI1-Ochrobactrum sp. strain 1 and FR1-Ochrobactrum sp. strain 2) were evaluated for their biocontrol efficacy. Though the isolates had non-significant ovicidal action (Fig. 4), substantial adulticidal effects were recorded (Fig. 5, Additional file 1: Tables S1 and S2). The highest egg mortality of 30.67% was observed at 96 h, in the treatment with 10 8 cells/ml of P. luminescens. In adulticidal treatments, no mortality was observed up to 24 h but after 48 h, mortality rate had increased. The P. luminescens isolate was significantly superior over other isolates in both CS and CFS forms. At 96 h, treatment with 10 8 cells/ml of P. luminescens recorded significantly higher mortality of 60.67%. The same treatment recorded 64.00% mortality in its CFS form. This was followed by the other treatments of P. luminescens at 10 7 cells/ml (52.00% in CS and 56.00% in CFS), 10 5 cells/ml (50.67% in CS and 53.33% in CFS), 10 6 cells/ml (49.33% in CS and 52.00% in CFS) and 10 4 cells/ml (49.33% in CS and 48.00% in CFS). Among treatments of P. aeruginosa, the highest mortality of 38.67% in CFS and 33.33% in CS was recorded in the treatment with 10 8 cells/ml of strain 2 (KT1 isolate). No significant mortality rate was observed in any of the treatments of Ochrobactrum spp.

Discussion
In this study, nine bacterial isolates were isolated from the haemolymph of EPNs (Heterorhabditis spp.) infected G. mellonella cadaver. This included one species of symbiotic bacteria, P. luminescens and eight non-symbiotic/ associated bacteria namely two isolates of P. aeruginosa, five isolates of Ochrobactrum and one isolate of Stenotrophomonas maltophilia. Photorhabdus is the predominant genus of entomopathogenic bacteria, which is a natural symbiont of the EPN, Heterorhabditis spp. (Boemare et al. 1993). It is Gram negative and bioluminescent. The bacterium supports growth of the nematode in the association, but is a deadly pathogen to most of the insect pests (Clarke 2008). They reside in the gut of the nematodes and get released into the insect haemocoel soon after the EPN infects the insect and kill them within 24-48 h. Other than the symbionts, few associated bacteria also reside within the system of Heterorhabditis. Bacterial species such as Alcaligenes aquatilis, Alcaligenes faecalis, Enterococcus mundtii, Pseudomonas protegens, Serratia nematodiphila, Serratia marcescens and Stenotrophomonas maltophilia were previously isolated from the Heterorhabditis infected Galleria cadaver (Ruiu et al. 2017).
Ochrobactrum is a Gram-negative opportunistic bacterium (Brucellaceae) which mostly occurs singly. This bacterium is related to Brucella, Phyllobacterium, Rhizobium and Agrobacterium. Occurrence of natural dixenic association between the bacterial symbiont P. luminescens and the bacterium Ochrobactrum sp. in Heterorhabditis species was reported by Babic et al. (2000). In this study, five isolates of Ochrobactrum were found in association with the EPN-infected Galleria. Akhurst (1982) reported the antibiotic properties of the Xenorhabdus sp. (now Photorhabdus) which inhibits the growth of other microorganisms. However, Aujoulat et al. (2019) observed the ability of Ochrobactrum sp. to grow well even in the presence of P. luminescens due of its resistance to the antibiotics produced by the Photorhabdus.
Pseudomonas includes disease-causing opportunistic bacteria. P. aeruginosa is a free living, Gram-negative   et al. Egyptian Journal of Biological Pest Control (2022) 32:87 bacterium having extensive metabolic diversity. The Stenotrophomonas spp., formerly isolated as P. maltophilia, is an emerging multidrug-resistant opportunistic pathogen. Ruiu et al. (2017) isolated S. maltophilia from the Heterorhabditis infected Galleria cadaver.
In the phylogenetic tree, the sequences of Ochrobactrum spp. and Stenotrophomonas spp. formed two subclades indicating the divergence of these genera from a common ancestry. In clade 2, Photorhabdus and Pseudomonas subclustered, indicating the evolutionary relationship of the two species. Velasco et al. (1998) reported the similarity of O. anthropi and O. intermedium with Photorhabdus luminescens subsp. akhurstii. All the subclades have further branched into subclusters within the species, indicating significant inter-specific diversity.
Efficacy of the symbiotic bacterium P. luminescens against different insect pests was reported by several workers (Kumar et al. 2014). However, studies on the efficacy of this bacterium on mites are limited. Due to the virulent properties and the ability to infect a wide range of insect hosts, it is a promising candidate for agricultural use as a mass-produced biological control agent (Gerdes  In this study, adult mortality rate was higher by P. luminescens treatment, compared to P. aeruginosa and Ochrobactrum. At the highest concentration of 10 8 cells/ml, P. luminescens has resulted in 60.67% mortality. Mortality was negligible with the other isolates. This clearly showed that P. luminescens is more effective against T. truncatus than the other bacteria. The EPN-associated bacteria: Ochrobactrum as well as P. aeruginosa were also reported to possess insecticidal activity against few pests. Salgado-Morales et al. (2019) assessed the pathogenicity of P. luminescens HIM3 and P. aeruginosa NA04 isolated from Heterorhabditis indica against G. mellonella and few other insect pests. Both the isolates were found to be highly virulent to G. mellonella with nearly complete mortality after 24 h. P. luminescens was virulent to both of the insect hosts but the pathogenicity of P. aeruginosa varied with the hosts. They also noticed that a high concentration of P. aeruginosa was required for infecting Tenebrio molitor. P. aeruginosa recorded only (10%) mortality of Diatraea magnifactella Dyar after 36 h of treatment, but mortality was complete with P. luminescens. In the present study, mortality of T. truncatus was less when treated with P. aeruginosa compared to P. luminescens. The bacterium Ochrobactrum tritici isolated from the EPN Oscheius chongmingensis was reported to cause 93.33% mortality of G. mellonella (Fu and Liu 2019). But Ochrobactrum isolates did not exhibit appreciable activity against T. truncatus in this study.

Conclusions
This study identified a potential isolate of P. luminescens with significant adulticidal action against T. truncatus. Techniques for mass-multiplying P. luminescens are to be standardized for utilization of this bacterial isolate in mite pest management.