Phylogeny and Molecular Study of Some Entomopathogenic Rhizobacteria Isolated from Two Regions in Algeria

Corresponding Author: Oulebsir-Mohandkaci Hakima Laboratoire de Consevation et Valorisation des Ressources Biologiques (VALCORE), Faculté des Sciences, Université M’Hamed Bougara de Boumerdès, Avenue de l’indépendance, Boumerdès 35 000, Algeria Tell: 00213772501934 Email: mohandkacihakima@yahoo.fr Abstract: The use of bacteria in the control of insect pests is a form of biological control whose practice is still not widespread. It is in this context that the present work falls. It concerns the isolation, characterization and identification of local bacterial strains for the purpose of their use in the control of certain pests. Indeed, 20 bacteria were isolated from soil cultivated in the region of Boumerdes (center of Algeria) with a total of 21 bacterial strains isolated from Adrar region (Desert Algerian). After carrying out the efficacy tests against 2 insect pests; Migratory locust (Locusta migratoria) and wax moth (Galleria mellonella), 8 potentially interesting strains were identified based on their genetic traits. Molecular characterization of these strains was performed by isolation of DNA, PCR and sequencing of the 16S rRNA gene, followed by phylogenetic analysis. The rDNA16S sequences of the 8 strains named B1, B2, B3, B4, B5, B6, H1 and H2 were recorded in the EMBL/EBI database and their phylogenetic analysis revealed that they belong to the genera Bacillus, Pseudomonas, Enterobacter and Delftia with a very high percentage of similarity with Bacillus thuringiensis strains (NR_043403) (99%) for isolate B1, Bacillus weihenstephanensis (NR_024697) (99%) for isolate B2, Pseudomonas fragi (JCM5420) (99%) for isolates B3 and B4, Bacillus thuringiensis (CMBLBT-5) (99%) for isolate B4, Enterobacter ludwigii (EN-119) for B5, Bacillus thuringiensis (4916) (99%) for isolate H1 and Delftia lacustris (R-54734) (100%) for isolate H2.


Introduction
Due to the use of a large amount of chemical products, continuous loss of soil fertility puts pressure on farmers toward more crop production in a sustainable manner. Therefore, it requires adapting an eco-friendly, sustainable and cost-effective approach for agricultural practices without arising environmental issues .
Indeed, the soil is a reservoir of numerous microorganisms critical for the sustainable functioning of natural and managed ecosystems (Sharma et al., 2019). Several natural rhizobacteria inhabiting the rhizospheric soil exist, their application reduces the utilization of chemical fertilizers, pesticides and other artificial growth regulators that cause severe health and environmental issues, soil infertility, water pollution and biodiversity losses .
Multiple species of entomopathogenic bacteria may be isolated from this part of soil, they are the enemies of pest-insects, whose utility in agroecosystems has been studied since decades. These entomopathogens spend significant time period in soil, either as saprotrophs, resting spores or dormant endospores (Oulebsir-Mohandkaci et al., 2016;Sharma et al., 2019).
Among the key insect-pest in Algeria, Locusta migratoria (Orthoptera-Acrdidae) and Galleria mellonela (Lepidoptera-Pyralidae) exhibit different damage to agriculture and are distributed in all the country (Oulebsir-Mohandkaci et al., 2016;Benzina et al., 2017). With increased awareness towards the environment, biological methods to control these two pests such as biopesticides based on entomopathogenic have been receiving greater attention as alternatives to chemicals pesticides (Jaronski, 2010).

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To facilitate the isolation of entomopathogenic strains from soil, insect baiting by Galleria mellonella or the 'Galleria-bait method' (Zimmermann 1986;Benzina et al., 2017), is the most renowned methodology. The migratory locust was also used for this. The main advantage of the insect baiting method is that only entomopathogens are obtained selectively amongst other soil microbes (Vega et al. 2012;Sharma et al., 2018).

Sol Sampling
The soil samples were taken from Adrar region in the Algerian desert at the rhisosphere layer of the date palm (Tamietti and Pramotton, 1990). Other samples were taken from the region of Boumerdes located in the central part of northern Algeria, from rhisosphere layer of wheat (Fig. 1).

Isolation and Phenotypic Characterization of Bacterial Isolates
Ordinary nutrient agar (GN) medium, pH 7.2 was used for isolation bacterial strains. 0.1 mL of 10-1 to 10-5 dilutions was seeded on the surface of isolation medium. The bacterial colonies obtained after incubation at 32°C for 48 h underwent purification. The obtained strains were then stored at -20°C using glycerol (20%, v/v) as cryo-protector.
Identification of bacterial genera was based primarily on the study of several macroscopic characters (the colonies type) and microscopic examinations (the form of bacterial cells and their mobility). The characterization of isolates needed physiological traits (highlighting the respiratory type, presence and position of spores and type of Gram) and biochemical (catalase, oxidase, nitrate reductase, levane sucrase, arginine di hydrolase, Voges Proskauer reaction (VP), degradation of certain substrates, gelatin, starch, casein, urea, indole, sorbitol, mannitol, citrate, tartrate and the production of lipase) (Bergey et al., 1974;Dong and Cai, 2001;Guiraud, 2003).

Molecular Identification
The molecular study concerns the 8 best performing strains, those which gave the best results on the migratory locusts for the strains isolated from Adrar and on the wax moth for the strains isolated Boumerdes.
The 16S rRNA gene was amplified in three overlapping fractions with the following universal bacterial primers:

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The Forward primers (FOR) were tailed by the M13-20FOR (CTGTAAAACGACGGCCAGT) primer whereas the Reverse ones (REV) by the M13-26REV (AGGAAACAGCTATGACCATG). Two colony PCR trials were performed per sample and per fragment and the obtained amplicons were run on agarose gel, cleaned-up and Sanger sequenced. Sanger sequencing was performed using the BigDye v3.1 chemistry on 3730XL machines (Applied Biosystems; www.appliedbiosystems.com). All obtained amplicons were sequenced from the forward and reverse directions using the universal primer-tailing of the 16S amplification primers. The sequences were base called status with the software program Sequencing Analysis v.5.2 (Applied Biosystems; www.appliedbiosystems.com). All generated chromatograms were assembled per sample using the TraceEditPro software (Ridom Bioinformatics; http://www.ridom.de/traceedit/).
The consensus sequences were analyzed by human expert for base calling errors and IUPAC code was applied for any mixed position (originating from the presence of several copies of the 16S rRNA gene for one species). The primer sequences chromatograms were manually trimmed and the resulting consensus sequences were exported in FASTA format. The obtained FASTA consensus sequences were analyzed by similarity searches with BlastN/Megablast against the nonredundant nucleotide database of GenBank (NCBI GenBank; http://blast.ncbi.nlm.nih.gov). Further on, the degree of statistical support for the branches was determined by 1000 bootstrap replicates. The maximum likelihood trees were estimated by using the rapid bootstrap option (100 replicates) (Tamura et al., 2011).

Results
Phenotypic Characterization of Isolates 20 bacterial strains were isolated from soil in the region of Boumerdes (coastal region located in the center of Algeria) with a total of 21 bacterial strains isolated from Adrar region (Sahara Desert, Algeria). Appearance of the colonies obtained on agar was very variable. The isolates were aerobic, mobile and with a positive catalase. Microscopic observation permitted their division into two groups, the first consisted of long rods, Gram positive and form a propertied sporal shape and the second contained short Gram negative rods and which non sporulating.
The isolates carrying the B1 and B2 codes provide well-isolated colonies, broad, smooth, flat, circulars, cream color with irregular edges. Observation under the light microscope, has shown that the two strains are Gram positive and are long stick shaped with square end.
Malachite green staining revealed that both isolates have a spore form. The spore has an oval shape nondeforming center position. Moreover, the two isolates B1 and B2 have shown positive results for the test of catalase, nitrate reductase and mannitol-mobility. The results of the pyruvic acid derivatives appeared positive for the Voges Proskauer reaction and the two isolates show a positive response for the hydrolysis of starch, gelatin and casein.
This isolates have all the cultural characteristics of the genus Bacillus already described by Brossard and Terry (1984) and Euzeby (2007).
Cultural characteristics on GN of the strains B3, B4 and B6 showed generally bacterial colonies whose contour was with a regular staining often beige cream. The elevation was convex for all strains with a glossy smooth appearance and varying diameters 1 to 2 mm after 24 h of culture. The bacterial strain B5 had smooth medium-sized colonies with an irregular edge. Moreover, the strains B5 and B6 showed positive results for some tests like nitrate reductase and mannitol. The strain B3 showed a positive result for the tests of mannitol and citrate. However, almost all strains B3, B4, B5 and B6 expressed negative results towards hydrolysis test of indole.

Molecular Characterization
In the present study, three bacterial genera Bacillus, Pseudomonas and Enterobacter were selected where the main members of the entomopathogenic bacteria belong to these genera (Greathead et al., 1994).
The 16S rDNA sequences of the three strains B1, B2 and B5 were obtained with 1504, 760 and 1504 nucleotides and stored in the database EMBL/EBI under reference accession numbers HE799656, HE805963 and HF911367, respectively. Sequence analysis of two strains B1 and B2 has shown they are affiliated to the genus Bacillus (the family of Bacillaceae) showing very high sequence identity (99%) with the strains Bacillus thuringiensis (NR_043403), Bacillus cereus AM7 (JQ435684), Bacillus weihenstephanensis (NR_024697.1) and Bacillus mycoides (NR_036880). Otherwise, the strain B5 showed a high sequence identity (99%) with the closely related type strains of Bacillus species, B. anthrancis strain Ames, B. cereus strain HN and B. thuringiensis strain CMBLBT-5), which indicated that this strain can belong to one of these species. Moreover, the strains H1 isolated from the region of Boumerdes is identified as Bacillus strain H1 (LT547812) with a percentage of similarity (99%) with Bacillus thuringiensis strain Se13 (KX959986) ( Table 1).

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The rDNA 16S sequences of the named strains B3, B4 and B6 were recorded in the EMBL/EBI data bank under the accession numbers HF911369, HF911366 and HF911368, respectively. The phylogenic analysis showed that the strains B3 and B4 had a sequence identity higher than 98% with those of the genus Pseudomonas. A significant similarity (based on partial 16RNAr sequence) for possible species relatedness (99%) was found with the validly described species P. fragi strain JCM5420, P. syringae isolate Lz4W and P. psychrophila strain HA-4. The distance matrix indicated that strains B3 and B4 might belong to one of these species (Table 2). Further, the strain B6 was similarly closed to the species Enterobacter ludwigii strain EN-119 and E. aerogenes strain MDAZTVIIIR105a with 99% of sequence identity. The strain H2 from the northern Algeria is identified as Delftia strain H2 (LT547813), with 100% similarity to Delftia lacustris strain: 6bw_2 (LC191530) ( Table 2).
Finally, the four strains are in the same cluster as that of Bacillus thuringiensis (NR_043403) and Bacillus cereus AM7 (JQ435684), the most widely used entomopathogenic bacteria in the world. The other strains are distributed in different clusters (Fig. 2).

Discussion
Bacteria represent the most commercially successful entomopathogenic microbial group, with most commercialized insecticides containing Gram-positive bacteria in the Bacillaceae family (De Bortoli and Jurat-Fuentes, 2019).
In fact our study allowed us to isolate, 20 bacteria from soil cultivated in the region of Boumerdes and with a total of 21 bacterial strains from the Adrar region. After carrying out the efficacy tests against 2 insect pests; Locusta migratoria and Galleria mellonella, 8 potentially interesting strains were identified based on their genetic traits. Partial sequencing of ARN16S. allowed to affiliate 4 strains to the genus Bacillus, 2 strains to the genus Pseudomonas, 1 strain to the genus Enterobacter and one strain to the genus Delfetia. The first 3 genera are known by their entomopathogenic effect (Greathead et al., 1994) but for the last one no study has demonstrated its insecticidal efficacy.
Entomopathogenic spore forming bacteria, most notably Bacillus thuringiensis (Bt), are the most widely used Microbial Pest Control Agents (MPCA). The broad spectrum of susceptible hosts, production on artificial media and ease of application using conventional equipment have resulted in widespread use against several insect pests in crops, forest and aquatic habitats (Lacey and Siegel, 2000).
Indeed, the majority of bacteria of the genus Bacillus live in the soil or they persist with their spores. These are bacteria from land who derive their nutritional needs from an organic matter, nitrogen and minerals present in the soil (Brossard and Terry, 1984;Ashnaei et al., 2009).
Presence of Pseudomonas bacteria in the rhizosphere can be explained by their rapid growth, ubiquitous nature and ability to adapt to various environmental conditions, which was reported in many studies (Bossis et al., 2000). The facility of in vitro culture and the ability of some species of this genus of bacteria to produce antibiotics and siderophores in case of iron deficiency confer special characters in the biological control (Mamoun and Olivier, 1989).
Finally, the complete 16S rDNA sequence analysis and DNA: DNA hybridizations are suggested for an extensive study for the identification of the obtained isolated strains.

Conclusion
As part of our study, sustainable use of rhizobacteria has been suggested to be an eco-friendly and cost-effective approach which increases crop yields and directly or indirectly protects plant from pathogens and the most redoubtable pests including the two pests having been the subject of our study, L. migratoria and G. mellonela.