Nematicidal, Acaricidal and Plant Growth-Promoting Activity of Enterobacter Endophytic Strains and Identification of Genes Associated with These Biological Activities in the Genomes

In the present study, the nematicidal and acaricidal activity of three Enterobacter endophytic strains isolated from Mimosa pudica nodules was evaluated. The percentages of mortality of Enterobacter NOD4 against Panagrellus redivivus was 81.2%, and against Nacobbus aberrans 70.1%, Enterobacter NOD8 72.4% and 62.5%, and Enterobacter NOD10 64.8% and 58.7%, respectively. While against the Tyrophagus putrescentiae mite, the mortality percentages were 68.2% due to Enterobacter NOD4, 64.3% due to Enterobacter NOD8 and 77.8% due to Enterobacter NOD10. On the other hand, the ability of the three Enterobacter strains to produce indole acetic acid and phosphate solubilization, characteristics related to plant growth-promoting bacteria, was detected. Bioinformatic analysis of the genomes showed the presence of genes related to IAA production, phosphate solubilization, and nitrogen fixation. Phylogenetic analyzes of the recA gene, phylogenomics, and average nucleotide identity (ANI) allowed us to identify the strain Enterobacter NOD8 related to E. mori and Enterobacter NOD10 as E. asburiae, while Enterobacter NOD4 was identified as a possible new species of this species. The plant growth-promoting, acaricidal and nematicidal activity of the three Enterobacter strains makes them a potential agent to include in biocontrol alternatives and as growth-promoting bacteria in crops of agricultural interest.


Introduction
Agricultural pests, due to phytopathogenic nematodes and dust mites, cause economic losses in crops of great importance [1]. Some species of phytopathogenic nematodes such as Pratylenchus spp., Ditylenchus spp., Psilenchus spp., and the false root-knot nematode Nacobbus aberrans, together with Meloidogyne spp., (the latter made up of 98 species, including M. chitwood, M. incognita, M. hapla, M. javanica and M. arenaria), are responsible for causing economic losses of between 12 to 20% in the crops where they occur [2]. Another organism that causes problems in agricultural production, causing considerable damage and losses in food products of plant and animal origin, is the dust mite T. putrescentiae, since they can carry fungal spores and pathogenic bacteria for humans. The T. putrescentiae mite is a vector of pathogenic bacteria of Klebsiella spp., Candida albicans and Staphylococcus which affect humans, and are present in the mucosa and parts of the skin [3].
Plants are responsible for selecting their microbiome to have beneficial bacterial colonizers designated as plant growth-promoting bacteria (PGPB). These produce different compounds such as auxins, antibiotics, and organic acids that are related to growth promotion, as well as chitinase, protease and cellulase enzymes that are related to biocontrol, health and plant development processes [4][5][6][7][8]. The ability of endophytic bacteria to colonize the interior of plant tissues gives them an advantage in avoiding the competition present in the rhizosphere environment, and allows them to achieve a close relationship with the plant [6,9,10]. The Enterobacter genus comprises species that have been reported to promote plant growth due to their multiple growth-promoting activities. Some strains that have been reported are: E. asburiae PDA 134 isolated from date palms [11], E. cloacae isolated from citrus and corn plants [12,13] and E. asburiae from sweet potato [14]. The strain Enterobacter sp., P23 has also been reported as a growth promoter under conditions of abiotic stress such as salinity, due to its high ACC deaminase activity, extreme pH, high temperature and in the presence of a wide variety of pesticides traditionally applied in rice, peanut and corn crops [15,16].
On the other hand, different species of the genus Enterobacter have been evaluated against phytoparasitic nematodes of agricultural pests. In this context, in a study by Zhao et al. [17], the nematicidal activity of E. ludwigii AA4 was reported in 98.3 and 98.6% against the pine nematode Bursaphelenchus xylophilus, considering E. ludwigii AA4 as a biocontrol agent of the pine nematode (Pinus sylvestris) infection. Regarding root-knot nematodes, a study by Oh et al. [18] reported the in vitro nematicidal activity of E. asburiae against eggs and J2 larvae of M. incognita. The results were obtained at a concentration of 10% of the treatment against the eggs, and 53.7% and 98.2% against J2 larvae in a time of two to seven days post-treatment.
Considering the characteristics of the species of the genus Enterobacter, the objective of this work is to analyze the genome sequences of three strains isolated from Mimosa pudica nodules to identify the genes that are associated with the characteristics that promote plant growth, nematicidal activity and acaricide, as well as its phylogenetic relationship with other species of the genus Enterobacter.

Genome Assembly and Annotation
The analysis of the genomes by means of the average nucleotide identity (ANI) was obtained by comparing the sequences of the genomes against the genomes deposited in GenBank.

Phylogenomic Analysis
The sequences of the recA genes were analyzed using the BLASTn algorithm and showed similarity with the species of the Enterobacter genus. Phylogenetic trees were constructed using the recA genes. Phylogenomic analysis shows that the NOD4 strain may be a new species of this genus. The NOD8 strain is phylogenetically related to the E. mori species, while the NOD10 strain is related to the E. asburiae species (Figure 1). This is also observed with the BLAST results, where a 99% similarity with the aforementioned species was obtained.  gene  4378  4403  4198  tRNA  77  80  80  rRNA  17  19  12  tmRNA  1  1  1  othetical proteins  1064  958  965  h functional assignments  3314  3445  3233

Phylogenomic Analysis
The sequences of the recA genes were analyzed using the BLASTn algorithm and showed similarity with the species of the Enterobacter genus. Phylogenetic trees were constructed using the recA genes. Phylogenomic analysis shows that the NOD4 strain may be a new species of this genus. The NOD8 strain is phylogenetically related to the E. mori species, while the NOD10 strain is related to the E. asburiae species (Figure 1). This is also observed with the BLAST results, where a 99% similarity with the aforementioned species was obtained. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.1689)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 39.76% sites). The tree is drawn to scale, with branch lengths Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.1689)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 39.76% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 18 nucleotide sequences. All positions containing gaps and missing data were eliminated. There was a total of 879 positions in the final dataset. Evolutionary analyses were conducted in MEGA X.

Phosphate Solubilization Activity
The three strains of Enterobacter had the ability to solubilize phosphate, Enterobacter sp. NOD4 solubilizes 66.9 µg/mL, while E. mori NOD8, 69.2 µg/mL, and E. asburiae NOD10, 67.4 µg/mL at five days. The statistical analysis does not show a significant difference (ANOVA p < 0.05) in phosphate solubilization between strains (Figure 2A). Phosphate solubilization can also be observed through the formation of halos in Pikovskaya PVK agar culture medium ( Figure 2B).
in the final dataset. Evolutionary analyses were conducted in MEGA X.

Phosphate Solubilization Activity
The three strains of Enterobacter had the ability to solubilize phospha sp. NOD4 solubilizes 66.9 μg/mL, while E. mori NOD8, 69.2 μg/mL, a NOD10, 67.4 μg/mL at five days. The statistical analysis does not show a ference (ANOVA p < 0.05) in phosphate solubilization between strains (Fig phate solubilization can also be observed through the formation of halos PVK agar culture medium ( Figure 2B).

Determination of Auxin Production
The production of indoles by bacteria is a mechanism for promoting This characteristic was detected in Enterobacter sp. NOD 4 producing 101.9 NOD8, 103.9 μg/mL and E. asburiae NOD10 52.1 μg/mL ( Figure 3). In the ysis of the indole production by the strains, a significant difference was fo p < 0.05) in the NOD 4 and NOD 8 strains, with respect to the NOD 10 stra

Determination of Auxin Production
The production of indoles by bacteria is a mechanism for promoting plant growth. This characteristic was detected in Enterobacter sp. NOD 4 producing 101.9 µg/mL, E. mori NOD8, 103.9 µg/mL and E. asburiae NOD10 52.1 µg/mL ( Figure 3). In the statistical analysis of the indole production by the strains, a significant difference was found (ANOVA p < 0.05) in the NOD 4 and NOD 8 strains, with respect to the NOD 10 strain.

Siderophore Production
The Enterobacter sp. NOD4, E. mori NOD8 and E. asburiae NOD10 were inoculated in the CAS-CAA medium, where they formed a yellow halo around the colonies indicating the production of siderophores. The three strains were positive ( Figure 4).

In Vitro Evaluation of Enterobacter Strains against P. redivivus and N. aberrans
The results of the evaluation of the strains of Enterobacter sp. NOD4 at a concentration of 1 × 10 9 cell/mL against the free-living nematode P. redivivus showed a mortality of 81.2%, E. mori (NOD8) of 72.4% and E. asburiae (NOD10) of 64.8% at 24 h ( Table 2). The strain of Enterobacter sp. NOD4 against the nematode N. aberrans showed a mortality of 70.1%, E. mori (NOD8) of 62.5% and E. asburiae (NOD10) of 58.7% at 24 h (Table 3).

Treatment
Concentration %Mortality

Siderophore Production
The Enterobacter sp. NOD4, E. mori NOD8 and E. asburiae NOD10 were inoculated in the CAS-CAA medium, where they formed a yellow halo around the colonies indicating the production of siderophores. The three strains were positive ( Figure 4).

Siderophore Production
The Enterobacter sp. NOD4, E. mori NOD8 and E. asburiae NOD10 were inoculated the CAS-CAA medium, where they formed a yellow halo around the colonies indicati the production of siderophores. The three strains were positive ( Figure 4).

In Vitro Evaluation of Enterobacter Strains against P. redivivus and N. aberrans
The results of the evaluation of the strains of Enterobacter sp. NOD4 at a concentration of 1 × 10 9 cell/mL against the free-living nematode P. redivivus showed a mortality of 81.2%, E. mori (NOD8) of 72.4% and E. asburiae (NOD10) of 64.8% at 24 h ( Table 2). The strain of Enterobacter sp. NOD4 against the nematode N. aberrans showed a mortality of 70.1%, E. mori (NOD8) of 62.5% and E. asburiae (NOD10) of 58.7% at 24 h (Table 3).

In Vitro Evaluation of Enterobacter against the Mite T. putrescentiae
The results of the in vitro evaluation of Enterobacter against the mite T. putrescentiae are described below. The strain of Enterobacter sp. NOD4 at a concentration of 1 × 10 9 cell/mL against the T. putrescentiae mite showed a mortality of 68.2%, the E. mori strain (NOD8) at the same concentration of 1 × 10 9 cell/mL showed a mortality of 64.3%. E. asburiae (NOD10) at the same concentration of 1 × 10 9 cell/mL, 79.8% was obtained, and ivermectin at a concentration of 2.5 mg/mL, 99.3% mortality at 24 h (Table 4).

Bioinformatic Analysis of Genomes for the Detection of Genes Involved in Plant Growth-Promoting Activities and Biocontrol
In the genome of the E. asburiae NOD10 strain, the aspC gene was identified, encoding an enzyme that catalyzes the conversion of indole-3-pyruvic acid to indole-3-acetaldehyde. This is later converted into IAA by the enzyme aldehyde dehydrogenase (aldB), that was also detected in the genome of E. asburiae NOD10 (Table 5). Other genes that were detected in Enterobacter sp. NOD4, E. mori NOD 8 and E. asburiae NOD10 are nirD which codes for the minor subunit of the enzyme nitrite reductase, narI which codes for the alpha subunit of the enzyme nitrate reductase, nasR which regulates the assimilation of nitrates and nitrites, and narX that detects the presence of nitrites and nitrates, the amtB gene that codes for an ammonium transporter, and the nif J gene that codes for pyruvate-flavodoxinoxidoreductase. All of these genes are involved in nitrogen fixation processes (Table 5). Thus, the glnK gene is involved in the regulation of nitrogen metabolism. In the bioinformatic analysis of the Enterobacter strains, the pstS gene that codes for the phosphatebinding protein and the phoU gene that is involved in the regulation of phosphate assimilation were also detected ( Table 5). The entH gene was also identified in the bioinformatic analysis as a corrector in the synthesis of the enterobactin siderophore (Table 5).

Discussion
In the present study, biotechnological characteristics of interest were detected such as the production of indoles and phosphate solubilization, as well as the production of siderophores related to biocontrol activity. Nematicidal (P. redivivis and N. aberrans) and acaricidal (T. putrecentiae) activity was also detected in the three Enterobacter strains. In the bioinformatic analysis of the genomes sequenced in this work, genes involved in the solubilization processes of phosphate, indoles and siderophores were detected.
Phylogenetic analysis using the recA gene of the three isolates showed that they belong to the genus Enterobacter. Consistently, phylogenomic inference suggests that the Enterobacter NOD8 strain is related to E. mori, while the Enterobacter NOD10 strain is related to E. asburiae. However, the Enterobacter NOD4 strain is grouped externally from the E. asburiae-sichuanensis and E. bundangensis-kobei complexes. This could be an indication that the NOD4 isolate is a new species of Enterobacter. These results were confirmed with the analysis of the genomes using average nucleotide identity (ANI), which is a similar statistic of the genomes that allows us to identify them at the species level [19,20]. Where the cutoff to consider 96% as a species is established by the ANI analysis, two strains meet this criteria Enterobacter NOD8 and Enterobacter NOD10 to be considered as Enterobacter mori and Enterobacter asburiae species, respectively, while the Enterobacter NOD4 strain has an ANI of 84%, so it can be considered a new species [21].
In the E. asburiae HK169 strain, nematicidal activity against the root-knot nematode M. incognita (eggs and J2) was reported Oh et al. [18]. In this work, the nematicidal activity of the strains E. asburiae NOD10, E. mori NOD8 and Enterobacter sp. NOD4 against the free-living nematode P. redivivus and the plant parasite N. aberrans (J2). The study carried out by Zhao et al. [17] reported that the E. ludwigii AA4 strain has activity against the pine nematode B. xylophilus and identified the sdaB gene that codes for L-serine hydrolase that is involved in nematicidal activity.
The biocontrol of the three strains of Enterobacter was not only limited to the nematode N. aberrans, it also had activity against the dust mite T. putrescentiae, being the first report of the species E. mori NOD8, E. asburiae NOD10 and Enterobacter sp. NOD4 against this nematode. Additionally, the nematode P. redivivus was used as a study model to evaluate the nematicidal activity of the species E. mori NOD8, E. asburiae NOD10, and Enterobacter sp. NOD4. These previous results can be considered for future field applications, considering the environmental impact of these organisms.
On the other hand, a study by Hall et al. [22] reported the isolation of Comamonas as endosymbionts of the Psoroptes ovis mite for biocontrol. Saccà and Lodesani [23] reported Lactobacillus kunkeei, Bacillus thuringiensis, Bifidobacterium asteroides and Acetobacteraceae isolated from healthy honey bees and dead varroa mites, which had acaricidal activity against Varroa destructor. Finally, the strain B. amyloliquefaciens W1 and Burkholderia rinojensis were reported to have acaricidal activity against Tetranychus urticae [24,25].
Other characteristics of biotechnological interest detected in these Enterobacter strains was the ability to solubilize phosphate, where the pstS gene related to this process was identified. This phosphate-solubilizing activity has been reported in the E. ludwigii GAK2 strain and in the Enterobacter sp. 49 [8,26]. Indole Acetic Acid (IAA) production activity was also detected in E. mori NOD8, E. asburiae NOD10 and Enterobacter sp. NOD4. This activity has been reported in E. ludwigii GAK2 and Enterobacter sp. 49 [8,26]; the aspC gene was detected by bioinformatic analysis in the genome of the strain E. asburiae NOD10. The ability to produce siderophores could be observed in the three Enterobacter strains. This ability related to biocontrol has been reported in the E. roggenkampii ED5 strain, and is related to the biocontrol of phytopathogenic fungi [27]. Another study by Solanki et al. [28] reported the production of siderophores by the Enterobacter sp. strain, which is related to the biocontrol of Rhizoctonia solani. In the genomes of the three Enterobacter strains the entH gene was identified, which is involved as a corrector in the synthesis of the siderophore enterobactin. Sánchez et al. [29] reported that Enterobacter strains detected the production of indole acetic acid, solubilization of phosphate and siderophores, as well as the detection of nif H genes.
Although the nitrogen production tests were not carried out in the different strains, the bioinformatic analysis allowed the detection of genes involved in nitrogen metabolism, such as the nif J genes involved in nitrogen fixation, the narX gene in the detection of nitrates and nitrite, the nirD gene that encodes the minor subunit of the enzyme nitrite reductase involved in the reduction of nitrates to nitrites, as well as the amtB gene related to ammonium transport (Table 5). Guo et al. [27] reported the genes involved in plant growth-promoting characteristics in the E. roggenkampii ED5 genome. Ludueña et al. [8] reported the genes involved in the production of siderophores, indole acetic acid and phosphate solubilization in the Enterobacter sp. J49.

DNA Extraction, Library Preparation, and Sequencing
Genomic DNA was extracted from freshly cultivated cells of the strains using the ZR Fungal/Bacterial DNA Kit kitTM (Zymo Research (Tustin, California, USA)), according to the manufacturer's instructions. The libraries for sequencing were performed with de Nextera XT ® (Heslin Rothenberg Farley & Mesiti P.C. (Albany, New York, USA)) protocol following the manufacturer's recommendations. The samples were fragmented obtaining short chains of DNA~500 bp. The resulting fragments go through an adapter ligation process (index), and were subsequently amplified following the cycling conditions specified in the NexteraXT protocol. At the end of this process, fragment purification was performed using the Agencourt Ampure XB beads commercial system (Beckman Coulter™ (Brea, California, USA)). Once the libraries were obtained, their quality was verified and analyzed by capillary electrophoresis using the 2100 Bioanalyzer (Agilent Technologies Inc. (Santa Clara, California, USA)). Finally, the samples were analyzed on the Illumina MiSeq highthroughput sequencing platform with paired ends.

Phylogenetics and Phylogenomics
From the phylogeny carried out with the recA gene, we selected the closest and most representative genomes of the sequenced isolates to obtain a complete nucleotide level database of 8 species of Enterobacter and one of Klebsiella as an outgroup. We used GET_HOMOLOGUES and GET_PHYLOMARKERS, a pipeline designed to identify highquality markers to estimate robust genome phylogenies from the orthologous clusters, or the pan-genome matrix (PGM). A ML species tree was estimated from the concatenated set of top-ranking alignments at the DNA or protein levels, using either FastTree or IQ-TREE [32].

Phosphate Solubilization
The ability to solubilize phosphate was qualitatively determined by inoculating the strains in PY liquid medium with 0.14 mM CaCl 2 and incubating at 28 • C for 24 h with shaking at 200 rpm to obtain a pre-inoculum. Bacterial cultures were centrifuged and adjusted to 0.2 OD 600 nm. They were seeded in triplicate in NBRIP culture medium (glucose, 1%; Ca 3 (PO 4 ) 2 , 0.5%; (NH 4 ) 2 SO 4 , 0.01%; MgSO 4 7H 2 O, 0.025%; KCl, 0.02%; MgCl 2 ·6H 2 O, 0.5%; Congo red, 2.5 mg/mL agar, 1.8%) and incubated at 28 • C for seven days. After this period, the sizes of the halos around the colonies were measured [33]. Inorganic phosphate concentration was quantitatively measured in liquid culture using NBRIP medium without agar and Congo red. The strains were inoculated at 0.2 OD 600 nm and cultured for ten days at 28 • C at 200 rpm. Samples were taken every 48 h, and after centrifugation of the samples, the concentration of phosphate in the supernatant was measured by the method of Rodríguez and Fraga [34]. The phosphate concentration was expressed as 50 µg/mL.

Determination of Auxin Production
To determine the production of indoleacetic acid (IAA), the strains were grown in liquid NFB medium (composition in g/L: malic acid, 5; K 2 HPO 4

Siderophore Production Assays
Siderophore production was determined by the method described by Schwyn and Neilands [36]. CAS-CAA (Chrome azurol (100 mM) and S-casamino acids) agar plates were inoculated with the isolates at 28 • C for 12 days. Orange halos formed around the colonies on blue agar, and were considered indicative of siderophore production.

Panagrellus Redivivus
A strain of the free-living nematode P. redivivus (mixed populations) was used by Dr. Roberto-de-Lara of the Autonomous Metropolitan University (UAM, Xochimilco, Mexico in 2009). The nematodes were grown in plastic containers using commercial oat flakes and water as a substrate. The oat flakes and water were mixed and sterilized in a microwave oven [37]. The nematodes were transferred to the substrate. The containers were covered with an aluminum lid with a mesh window of fine cloth to allow oxygenation. The cultures were maintained at room temperature (25-30 • C). After one week, the population of nematodes increased considerably [38].

Tyrophagus Putrescentiae
Populations of the T. putrescentiae mite were used, whose breeding stock is found in the National Center for Disciplinary Research in Animal Health and Safety, INIFAP, Jiutepec, Morelos. The specimens were isolated in 2013 in the town of San Juan Tlacotenco, Morelos, Mexico. The population is maintained by feeding them the free-living nematode P. redivivus (Goodey) for reproduction. The mites are transferred to plates with sterile agar in order to obtain monocultures of mites, and are kept at room temperature (28 ± 2 • C) under dark conditions [38].

Experimental Design
A completely randomized design was used, which consisted of four repetitions and two replicates for each treatment. The treatments for each bioassay were Enterobacter NOD4, NOD8 and NOD10 at a concentration of 1 × 10 9 cell/mL, as a negative control distilled water was used in the tests against the mite T. putrescentiae and the phytoparasite N. aberrans. As a positive control, Nematrol plus ® , a commercial nematicide at a concentration of 6 mg/mL for N. aberrans was used. In the case of P. redivivus and T. putrescentiae, ivermectin, a commercial anthelmintic, was used at a concentration of 5 mg/mL, and distilled water was used as a negative control in the P. redivivus bioassays.
For the in vitro tests with P. redivivus and N. aberrans, 96-well microtiter plates were used. In each well 50 µL of distilled water with 100 nematodes and 50 µL of Enterobacter (NOD4, NOD8 and NOD10) were added. They were subsequently incubated in a humid chamber at 28 • C, and the nematicidal activity was evaluated at 48 h. For the in vitro bioassays of T. putrescentiae, 24-well microtiter plates were used. In each well, 10 mites were added with 50 µL of Enterobacter (NOD4, NOD8 and NOD10). They were subsequently incubated in a humid chamber at 28 • C, and after 72 h the readings were made with a stereoscope (4× and 10×).

Statistical Analysis
The data obtained were normalized using the arcsine square root transformation and analyzed as a completely randomized design. Means were compared using Tukey's test (using the R ® environment). A value of p ≤ 0.05 was considered significant [41].

Conclusions
With the results obtained in the present study, the strains of Enterobacter sp. NOD4, E. mori NOD8 and E. asburiae NOD10, can be alternative biocontrols against the nematode N. aberrans and the mite T. putrescentiae. The production of IAA and phosphate solubilization were also detected, which are characteristics that promote plant growth and which can be used in soils composed of insoluble phosphate. Therefore, they are candidates for the biocontrol of nematodes and mites in crops, as well as promoting plant growth in crops of agricultural interest.