Characterization of the Culturable Sporobiota of Spanish Olive Groves and Its Tolerance toward Environmental Challenges

ABSTRACT Olive agriculture presents an integral economic and social pillar of the Mediterranean region with 95% of the world’s olive tree population concentrated in this area. A diverse ecosystem consisting of fungi, archaea, viruses, protozoa, and microbial communities—the soil microbiome—plays a central role in maintaining healthy soils while keeping up productivity. Spore-forming organisms (i.e., the sporobiota) have been identified as one of the predominant communities of the soil microbiome and are known for the wide variety of antimicrobial properties and extraordinary resistance. Hence, the aim of this work was to determine the culturable sporobiota of Spanish olive orchards and characterize its phenotypic properties toward common environmental challenges. A collection of 417 heat-resistant bacteria were isolated from five Spanish olive orchards. This collective was termed the “olive sporobiota.” Rep-PCR clustering of representative isolates revealed that they all belonged to the group of Bacillus spp., or closely related species, showing a great variety of species and strains. Representative isolates showed susceptibility to common antibiotics, as well as good resistance to heavy metal exposure, with an order of metal tolerance determined as iron > copper > nickel > manganese > zinc > cadmium. Finally, we showed that the application of mineral fertilizer can in several cases enhance bacterial growth and thus potentially increase the relative proportion of the sporobiota in the olive grove ecosystem. In summary, the identification of the culturable olive sporobiota increases our understanding of the microbial diversity in Spanish olive groves, while tolerance and resistance profiles provide important insights into the phenotypic characteristics of the microbial community. IMPORTANCE Microbial communities are a key component of healthy soils. Spore-forming microorganisms represent a large fraction of this community—termed the “sporobiota”—and play a central role in creating a conducive environment for plant growth and food production. In addition, given their unique features, such as extraordinary stability and antimicrobial properties, members of the sporobiota present interesting candidates for biotechnological applications, such as sustainable plant protection products or in a clinical setting. For this, however, more information is needed on the spore-forming community of agricultural installations, ultimately promoting a transition toward a more sustainable agriculture.

biocontrol agent, while at the same time guaranteeing minimum alteration of natural soil conditions (8,22).
Given the above, in this study, we set out to determine the culturable sporobiota of Spanish olive groves. We have determined the composition of the Bacillus spp. and related species community present in these agricultural soils, as well as their resistance properties toward environmental challenges faced in large-scale agriculture. These challenges include the exposure to heavy metals, fertilizers, and antibiotic compounds, notably with a view to developing novel biocontrol agents.

RESULTS
Sample collection and isolation of the culturable olive sporobiota. To isolate Bacillus species and related spore formers from olive tree leaves and the surrounding soil, a total of five different olive orchards in Andalusia, Spain, were selected (Table 1). To specifically select spore-forming organisms, the sample was heated to 78°C for 20 min, effectively removing vegetative (heat-labile) cells, while allowing the survival of only heat-resistant organisms (e.g., endospores). In general, low numbers of bacteria were isolated from the samples, especially from exogenic and endophytic leaf samples. This was also highly dependent on the sample origin.
Identification of the culturable olive sporobiota of Spanish olive orchards. A collection of 417 isolates was obtained from five different origins, representative of the soil, leaf surface, and endophytic culturable spore-forming microbial population in Spanish olive orchards mainly isolated from La Guardia de Jaén (LGJ, 171 isolates) followed by Linares (LIN, 146 isolates), in addition to Málaga (MA, 59 isolates), Jimena (JP, 18 isolates), and Bedmar y Garcíez (BG, 23 isolates) ( Table 1). Of these isolates, 10 appeared to be unculturable after the initial isolation step, leaving 407 candidates for further analysis. (GTG) 5 Rep-PCR analysis allowed detection of clonal relationships between isolates. Results grouping isolates by origin are shown in Fig. 1 to 5. To identify isolates, 16S rRNA of representative species from each cluster were sequenced.
The collection of 168 LGJ isolates was reduced to 144 after Rep-PCR cluster analysis (Fig. 4). Rep-PCR profiling notably revealed 2 main genomic groups: a minor group (G1) consisting of 5 isolates and a major group (G2). G1 could be grouped into the subcluster G1-1 with 4 isolates mostly identified as Bacillus sp. (without species identification) and the single isolate G1-2 (UJA_LGJ_236). G2 on the other hand showed a subdivision into 4 major subgroups (G2-1 to G2-4), demonstrating a large microbial diversity of the culturable olive sporobiota from this origin. G2-3 comprises only one strain UJA_LGJ_262, whereas the largest subgroup was G2-2 followed by G2-1 and G2-4 showing bacterial strain and genus heterogeneity. In this regard, a comparatively high number of Bacillus cereus isolates (7), as well as B. thuringiensis (5), were identified in G2-2.
Finally, the cluster analysis of Rep-PCR profiles of 58 isolates derived from the MA orchard yielded 57 strains. (Fig. 5) with an overall distribution into 2 clusters (G1 and G2). Here, G2 included only 1 isolate (UJA_MA_395, Bacillus endophyticus YN14), while cluster 1 comprised the great majority of species and strains isolated from this origin. Similar to clusters described from other origins, G1 showed various subdivisions, notably into G1-1 and G1-2, the latter of which split into further subgroups. 16S rRNA sequencing data for strains representative of the smaller G1-1 cluster (8 isolates) yielded mostly Bacillus sp. without further specifying the strains. On the other hand, cluster G1-2 included a large variety of species and strains.
A small number of isolates showed a slower sporulation process with immature spores, i.e., unreleased, phase bright spores in the mother cell, as the predominant configuration. However, all isolates did demonstrate the ability to sporulate.
Screening for B. anthracis virulence plasmids. To screen for the presence of virulent B. anthracis among the olive orchard isolates, PCR analyses were performed to
Although to a lower extent than RA, the majority of tested isolates also showed resistance to cefotaxime (CTX) (66.7%), with 29.9% full and 3.4% intermediate susceptibility when exposed to this antibiotic. Interestingly, almost all isolates exhibited intermediate susceptibility to ciprofloxacin (CIP) (95.7%), while 4.3% were resistant to CIP treatment.
Tolerance of heavy metal exposure and mineral-based fertilizer treatment. Finally, the tolerance to heavy metal exposure and mineral-based fertilizer treatment was tested, to evaluate resistance properties of the culturable olive sporobiota to these substances. For this, first, MICs for heavy metals were determined for 64 isolates chosen based on their representative position within the phylogenetic trees (Table 4). MIC was defined as the minimal concentration that inhibits visible bacterial growth (23). The results indicated that isolates of the olive sporobiota were able to tolerate relatively high heavy metal loads with concentrations ranging from 0.5 mM # MIC , 25 mM. In this regard, results suggested the following order of metal tolerance for the tested isolates: iron . copper . nickel . manganese . zinc . cadmium. No particular strain patterns could be established, although in general B. cereus strains tended to display high resistance to iron and zinc, while P. megaterium appeared to mostly have high tolerance to manganese. P. simplex, on the other hand, consistently showed good tolerance toward zinc.
Second, the culturable olive sporobiota tolerance to mineral-based fertilizer treatment was evaluated. For this the same representative isolates as above were exposed to a nitrogen-phosphate-potassium (NPK) fertilizer, routinely used in olive agriculture. Bacterial growth after NPK exposure at different concentrations was evaluated in comparison to untreated controls ( Table 4). The results showed that NPK treatment at 0.1Â and 1Â of the recommended concentration (0.3 and 3 g/liter, respectively) had no negative effect on bacterial growth. In fact, treatment with 1Â NPK did improve the growth of 37.5% of Bacillus sp. (24 strains) in comparison to untreated samples. Exposure to higher-than-recommended concentrations of NPK fertilizer had a more varied effect. In this regard, 12 strains (18.8%) were susceptible to the treatment, while 4 strains (6.3%) and 9 strains (14.1%) showed increased or highly increased growth, respectively, compared to untreated samples. These data suggest that fertilizer treatment of olive orchards can also influence the growth properties of the bacterial community, which could have both positive and negative effects.  TET  ERY  AMC  AMP  CHL  CIP  RA  NFN  GEN  IPM  CTX  LIN (58)  Susceptible  51  48  49  47  47  0  2  56  57  57  26  Intermediate  1  0  5  2  0  56  1  1  0  0  3  Resistant  6  10  4  9  11  2  55  1  1  1  LGJ (

DISCUSSION
The plant microbiome is essential to maintain a healthy soil and plant environment (2). Bacillus spp. and recently reclassified species previously belonging to the genus present one of the major bacterial components of agricultural ecosystems, including olive orchards (8,24). As such, they play a central role in maintaining a healthy environment for the plant, for example by capturing nutrients and facilitating nutrient uptake, removing toxic elements, or acting as antimicrobial agents (8,22). Given the importance of these bacteria in maintaining a healthy and productive plant environment, in this study, we aimed to determine and characterize the culturable spore-forming microbiome of Spanish olive orchards collectively called the culturable olive sporobiota.
The 16S rRNA analysis showed a wide variety of Bacillus sp. and Bacillus related species present in the soil surrounding olive trees, as well as the leave surface and endophytic community of olive trees. Bacillus spp. are ubiquitously present in the environment, including soil and plants (6). Dendrogram analysis showed a great diversity and heterogeneity of spore formers present in olive agricultural soils and olive plants. Rep-PCR fingerprinting of the culturable sporobiota isolates from different origins revealed various genetic groups of each origin (between 2 and 4). It is, however, noteworthy that a variety of species were detected throughout the sample collection, irrespective of the origin. Consequently, the overall presence and distribution of Bacilli and the great diversity of different species were consistent with previous reports on microbial communities for agricultural soil samples (25). Although in general an even distribution of detected sporobiota isolates was observed in the Rep-PCR profiles, isolates from the LGJ origin (La Guardia de Jaén) showed a higher prevalence of members of the B. cereus sensu lato group (excluding virulent B. anthracis) with 15 of 24 B. cereus sensu lato isolates originating from this orchard. Like its fellow Bacillus species, B. cereus sensu lato is ubiquitously present in the environment, including soil (26), while the prevalence in this origin could be due to soil conditions particularly conducive to B. cereus sensu lato. In this regard, B. cereus sensu lato was also the most identified species group (24 isolates) in the culturable olive sporobiota (B. cereus sp. [12 isolates], B. thuringiensis [11 isolates], and B. wiedmannii [1 isolate]). Multiplex PCR analysis for virulent pXO1 and pXO2 plasmids furthermore indicated the absence of virulent B. anthracis strains among the culturable olive sporobiota (27). However, the presence of nonvirulent strains in our culturable olive sporobiota collection cannot be confidently excluded under the chosen experimental conditions (28).
In addition, we could observe slight variability in bacterial load depending on the origin. This is probably not surprising, as bacterial load depends on the soil composition and also experiences seasonal fluctuations (29).
The inclusion of a heat treatment step (78°C, 20 min) during bacterial isolation ensured that only heat-resistant, presumably spore-forming bacteria were isolated (30). The formation of endospores, conferring the ability to resist harsh environmental such as wet heat (31), was confirmed by the ability of all tested isolates to sporulate on rich sporulation media. Implicitly, this procedure also shows that all isolated strains were able to successfully undergo sporulation/germination life cycles, as in this work both dormant and vegetative cells were studied (32). Antibiotic resistance among microbial communities is a growing global concern for agriculture (33) with major European Union and international policy initiatives aiming to limit its spread (34). Hence, in this study, we also evaluated the resistance of the culturable olive sporobiota to selected antibiotics. Our results confirmed those obtained in other studies, with tested Bacillus spp. isolates being susceptible to gentamicin (99.1%), nitrofurantoin (96.6%), imipenem (94.9%), tetracycline (88.0%), erythromycin (86.3%), and chloramphenicol (CHL) (79.5%) (35). In contrast to other reports, the tested isolates also showed relatively high susceptibility to several b-lactam antibiotics, i.e., amoxicillin-clavulanic acid (83.8%) and ampicillin (80.3%), while the expected the resistance to cefotaxime (66.7%)-although to a lower extent-was detected (36,37). It would thus be interesting to test the presence/absence of b-lactamase enzymes in the bacterial genome in particular of these isolates in future studies.
In addition, and in accordance with previous studies, the tested isolates showed high resistance to rifampicin (92,3%) (36). Interestingly, and in contrast to other studies, mostly intermediate resistance to ciprofloxacin (95,7%) was detected for the tested isolates. This could potentially suggest that the members of the culturable olive sporobiota are acquiring a resistance to this antibiotic (35). It is also noteworthy that the overall trend of antibiotic resistance was also reflected when analyzed by origin. Hence, under the chosen conditions, the sporobiota origin did not influence antibiotic resistance properties of the tested representative isolates in our study.
In addition to AMR spread, heavy metal contamination is a serious challenge for commercial agriculture and food production, as the metals can be taken up by plants and ultimately enter the food chain (38). The European Union is thus constantly revising the safety of metal content in foodstuff and (if necessary) adjusting maximum permitted levels as set by Commission Regulation 1881/2006. The ability of Bacillus spp. to tolerate metal exposure, as well as absorb and remove heavy metals from the soil, thus effectively decontaminating the environment, has previously been shown (39). Hence, here we tested the olive sporobiota's tolerance to heavy metal exposure. In accordance with literature on metal resistance of Bacillus and Lactobacillus spp., isolates showed a good tolerance to heavy metals, with an order of tolerance of iron . copper . nickel . manganese . zinc . cadmium (23). This suggests that members of the culturable olive sporobiota could also potentially thrive in soils with an elevated metal load as a result of environmental conditions or anthropogenic activity (40). Accordingly, as a next step, it would be interesting to test the metal remediation capacity of the most promising species isolated in this work.
Finally, we investigated the culturable olive sporobiota's response to mineral-based fertilizer treatment, notably at low (0.1Â), recommended (1Â), and excess (10Â) concentrations. Although there is a shift toward organic farming also in olive agriculture, still many of the most commonly used fertilizers are mineral based (https://ec.europa .eu/eurostat/statistics-explained/index.php?title=Agri-environmental_indicator_-_mineral_ fertiliser_consumption, June 2022). In general, it has been shown that in addition to improving crop yields, mineral fertilizer application alters soil properties and the soil microbiome (41). In this regard, the applied fertilizers not only have beneficial effects on plant growth but also provide an extra source of nutrients to the bacterial community, consequently increasing microbial biomass and diversity (42). Findings from our study are in line with these expectations, showing an improved bacterial growth of over one third of tested olive sporobiota isolates (37.5%) and no negative effect in comparison to untreated samples at 1Â concentration.
These results indicate that mineral fertilizer treatment has a nonuniform moderately positive effect on the culturable sporobiota bacterial community in olive orchards when applied at the recommended concentration. Nevertheless, treatment at higher NPK concentrations (10Â) had a negative effect on nearly one fifth (18.8%) of the tested isolates, suggesting a reduced tolerance of the culturable olive sporobiota to high NPK concentrations.
Conclusions. In this study, we determined the culturable Bacillus spp. and related spore-forming species in Spanish olive orchards collectively called the culturable olive sporobiota. We could demonstrate that the culturable olive sporobiota mostly follows susceptibility patterns for antibiotic treatments and heavy metal tolerance, although some strains differed from the expected pattern. We also showed that treatment with mineral fertilizer does not negatively affect or can even be conducive for bacterial growth. The obtained information may prove useful for further evaluating the effect and progression of AMR spread among microbial communities, as well as heavy metal tolerance in (contaminated) soils. Here, it would be particularly interesting for future investigations to study the ability of the culturable olive sporobiota collectively or separately to absorb and remove heavy metals from the soil or the potential effect of different farming practices (organics versus nonorganic) on the Bacillus spp. and related bacterial community.
It is also conceivable that one or several of the determined inherent species could play an important role in the development of novel biotechnological applications for agricultural use. In particular, the generated library of spore formers native to olive groves could provide a unique tool for the development of novel biological plant protection products in olive agriculture, notably given that Bacillus spp. already play a central role as one of the most widely used biopesticides, as well as its ability to produce a wide range of antimicrobial peptides (17,21).

MATERIALS AND METHODS
Sampling in olive groves. The samples analyzed in this study were collected over a period of 5 months (September 2021 to January 2022) from olive groves at five different locations in Andalusia, Spain (Table 1). Briefly, soil samples were taken close to the tree trunk at approximately 10-cm depths. Equally, leaf samples corresponding to the same vertical location on the tree were taken in the middle part of the canopy. The samples were collected in sterile containers and stored at room temperature until further processing.
Isolation of heat-resistant bacteria. To remove heat-labile, non-spore-forming organisms, 1 g of soil or 2 g of leaves were resuspended in 9 mL sterile 0.85% (wt/vol) saline solution and incubated at 78°C for 20 min. Serial dilutions were prepared in 0.85% (wt/vol) saline solution and plated in triplicate on tryptic soy agar (TSA) and incubated at 30°C overnight. Representative single colonies were randomly picked and purified on nonselective medium prior to storage in 25% (vol/vol) glycerol at 220°C or 280°C for further use. To isolate endophytic Bacilli, 2 g of leaves were first sterilized by serial washing steps in sterile 0.85% saline solution, 70% (vol/vol) ethanol, and 0.85% (wt/vol) saline solution prior to resuspension in 9 mL sterile 0.85% (wt/vol) saline solution. Endophytic bacteria were physically released by treatment in a stomacher unit (1 to 2 min, setting: high). Heat-resistant microorganisms were isolated from the resulting suspensions as described above.
Molecular characterization and identification of the culturable olive sporobiota. (i) Detection of virulent Bacillus anthracis species. In a first instance, PCR was used to screen for the potential presence of virulent B. anthracis species and take necessary safety precautions, if applicable. To do so, a multiplex PCR as described by Ogawa et al. (27) was performed detecting cap and pag genes on the B. anthracis virulence plasmids pX01 and pX02, as well as targeting the chromosome of B. anthracis and B. anthracis-like species. PCR was performed from a fresh overnight colony using MyTaq Red Mix (Meridian Bioscience) following the manufacturer's instructions under the following conditions: initial denaturation at 94°C for 1 min followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 2 min, extension at 72°C for 2 min, and final extension at 72°C for 10 min. Multiplex primers and concentrations are indicated in Table 5. PCR products were visualized by agarose gel electrophoresis (1% [wt/vol]  (43). Essentially, DNA was amplified from a fresh overnight single colony using 1 mL of 100 mM (GTG) 5 primer (59-GTGGTGGTGGTGGTG-39) and MyFi polymerase (meridian Bioscience) in a final volume of 25 mL using the following conditions: initial denaturation 95°C for 3 min, followed by 30 cycles of denaturation at 90°C for 30 s, annealing step 1 at 40°C for 1 min, annealing step 2 at 40°C to 72°C for 5:20 min (ramping 0.1°C/s) and extension at 72°C for 2 min. A final extension was performed at 72°C for 8 min. Amplicons were subsequently subjected to agarose gel electrophoresis (1.8% [wt/vol] agarose in 1Â Tris-borate-EDTA buffer) for 16 h at 48 V, stained with RedSafe nucleic acid staining solution (iNtRON) and visualized using a UV transilluminator. Band profiles were analyzed using Bionumerics software, version 2.5 (Applied Maths, Kortrijk, Belgium). The Pearson product correlation coefficient and the unweighted pair group method using arithmetic averages (UPGMA) were used to group Rep-PCR patterns through cluster analysis.
(iii) Identification of the culturable olive sporobiota. Representative isolates of each profile as identified by Rep-PCR and morphological features were selected and identified by 16S rRNA gene sequencing. Bacterial cultures were grown at 37°C overnight in tryptic soy broth, washed with 0.5% (wt/ vol) saline solution, boiled for 10 min, and immediately transferred on ice. The resulting genomic DNA extracts were centrifuged, and 1 mL supernatant was used for PCR amplification with the primer pair 27f-YM/1492r (59-AGAGTTTGATYMTGGCTCAG-39/59-TACCTTGTTACGACTT-39) (44).
Purified amplicons were sequenced using the same primer pair, and partial sequences were assembled with the A plasmid editor (ApE) software (45). Bacterial species were tentatively identified through a homology search with the BLAST N algorithm (National Center for Biotechnology Information [NCBI], USA) based on the highest alignment score and the percentage of identity.
Evaluation of sporulation ability. To confirm the ability to sporulate under laboratory conditions, culturable sporobiota isolates were subjected to nutrient starvation to induce sporulation (46). Briefly, a fresh colony was streaked on 2Â SG plates and incubated at 30°C for 3 days. The ability of the isolate to form endospores was evaluated by light microscopy using a 100Â objective in a Zeiss upright light microscope. Sporulation efficiency was evaluated based on visual release of endospores from mother cells.
Antibiotic susceptibility testing. Antibiotic susceptibility of the isolated olive sporobiota species was examined by the disk diffusion method as described by the EUCAST 2021 guidelines. Essentially, bacterial suspensions were plated to form confluent lawns on Mueller-Hinton agar, and antibiotic disks were placed on the plates. The tested antibiotics were ampicillin (AMP, 10 mg), amoxicillin-clavulanic acid (AMC, 20/10 mg), cefotaxime (CTX, 30 mg), chloramphenicol (CHL, 30 mg), ciprofloxacin (CIP, 5 mg), rifampicin (RA, 5 mg), nitrofurantoin (NFN, 300 mg), erythromycin (ERY, 15 mg), gentamicin (GEN, 10 mg), imipenem (IPM, 10 mg), and tetracycline (TET, 30 mg), respectively. After 18 to 20 h incubation at 35°C, the inhibition zones were measured and compared to the breaking points of the respective antibiotics. For IPM, CIP, and ERY, Bacillus specific breakpoint values set by EUCAST (2021) were used. As there were no Bacillus-specific values available for the remaining antibiotics, the EUCAST criteria (2021) for Staphylococcus aureus were adopted for TET, GEN, RA, and CHL, whereas for AMC, AMP, NFN, and CTX, the Enterobacteriales breakpoints as defined by the Clinical and Laboratory Standards Institute (CLSI, 2020) were used as references (35).
Evaluation of resistance properties. (i) Metal tolerance. The sensitivity of selected sporobiota isolates, representative of identified strains/origins toward the heavy metals cadmium (CdSO 4 Á8/3H 2 O), copper (CuCl 2 Á2H 2 O), iron (FeSO 4 Á7H 2 O), zinc (ZnCl 2 ), nickel (NiCl 2 Á2H 2 O), and manganese (MnCl 2 Á4H 2 O) (all Sigma-Aldrich, ES) was tested using the broth microdilution method (23). A total of 180 mL Mueller-Hinton broth supplemented with the respective metal at concentrations ranging from 0 to 25 mM were distributed in each well of 96-well microtiter plates. Metal solutions were then inoculated with 20-mL bacterial overnight cultures grown in tryptic soy broth or agar at 37°C previously adjusted to a concentration of 0.5 McFarland in Mueller-Hinton Broth. Microtiter plates were incubated at 37°C under aerobic conditions, and bacterial growth was evaluated by the presence of turbidity and/or bacterial deposition at the bottom of the plate. MIC was defined as the lowest concentration of metal that inhibited visible growth. Each experiment was performed in triplicate.

SUPPLEMENTAL MATERIAL
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ACKNOWLEDGMENTS
This project received funding from the European Union's Horizon 2020 research and innovation program under Marie Skłodowska-Curie grant agreement No. 101029930.
We acknowledge the research team at the University of Jaen (EI_BIO1_2021), as well as the collaboration of olive grove owners for the sample collection (Andalusia, Spain).