The Indiscriminate Chemical Makeup of Secondary Metabolites Derived from Endophytes Harvested from Aloe barbadensis Miller in South Africa’s Limpopo Region

The efficacy of 23 bacterial isolates obtained from surface-sterilized stems and leaves of three medicinal plants (Aloe barbadensis Miller, Artemisia afra, and Moringa oleifera) was investigated in an endeavour to prevent the growth of Mycobacterium bovis using the cross-streak method. Endophytes were isolated by incubating sterile plant materials on nutrient agar at 30 °C for 5 days. Two isolates showing activity were subsequently utilized to produce the extracts. Whole-genome sequencing (WGC) was used to identify the isolates. Secondary metabolites produced after 7 days of growth in nutrient broth were harvested through extraction with ethyl acetate. The extracts were chemically profiled using gas chromatography–high resolution time-of-flight mass spectrometry (GC–HRTOF-MS). NCBI BLAST search results revealed that the isolated endophytes belonged to the Pseudomonas and Enterobacter genera, based on WGC. Two endophytes, Aloe I4 and Aloe I3–I5 from Aloe barbadensis, exhibited potency based on the cross-streak method. The metabolite profiling of the selected endophytes identified 34 metabolites from Aloe I4, including ergotamine, octadecane, L-proline and 143 other metabolites including quinoline and valeramide, which inhibit microbial quorum sensing. These findings suggest that bacterial endophytes from medicinal plants, particularly Aloe barbadensis, hold promise as sources of antimycobacterial agents for human health applications.


Introduction
The healthcare sector has become greatly concerned with the global growing resistance to antimicrobial and chemotherapeutic medications in recent years.There is a pressing need to explore and create novel anticancer and antimicrobial agents [1].As a result, there has been a renewed passion for investigating microorganisms as a potential repository of new discoveries.This is based on the remarkable success of bacterial compounds in the production of effective antimicrobial agents, anticancer drugs, and agricultural pesticides [2].As an alternative to antibiotics, bacterial bioactive chemicals may play a role in microbe-to-microbe and microbe-to-host reactions, according to a growing body of research [3].
The use of medicinal plants to address human health problems dates back many years, and the benefits of medicinal plants in the health care system are undeniable.Plants are important and reliable reservoirs of bioactive compounds, as evidenced by their prolonged and continuous use in addressing the pressing issues in human health [4].Aloe barbadensis Miller, commonly known as aloe vera, Artemisia afra, known as wormwood, and Moringa oleifera, generally named drumstick tree, are three important medicinal plants used in South Africa to address various clinical conditions.Found in tropical and temperate regions, aloe vera is traditionally used for the treatment and prevention of various ailments such as sunburns, wounds, skin disorders, diabetes, ulcers, and other conditions [5].Recent pharmacological data indicated that Aloe vera displayed anticancer action, digestive protective activity, and antimicrobial properties [6].Artemisia afra, on the other hand, is commonly used as an infusion for the treatment of malaria and ailments such as colds, fever, influenza, sore throats, pneumonia, and many other ailments [7,8].The antibacterial activity of wormwood against Mycobacterium tuberculosis has been documented [9].Moringa Oleifera is another important medicinal plant harbouring bioactive substances in its seeds, leaves, flowers, and pods that is used to alleviate symptoms such as joint pains, malnutrition, and headaches, with many applications in the food sector [10].Moringa is considered an effective agent against hypocholesterolaemia and hypolipidemia and has also shown antimicrobial properties against various human pathogens including E. coli and S. aureus [11].Medicinal plants, like any other plants, are generally susceptible to abiotic and biotic stress conditions.To manage and control these attacks, plants often draw benefit from their symbiotic relationship with microorganisms found in their tissues [12].Endophytes (bacteria and fungi) are suitable suppliers of bioactive chemicals that do not only benefit these plants but have also found use in disease control.
Bacterial endophytes reside within plants' internal tissues without any notable signs of negative effects on the host.Endophytes have characteristics which are beneficial to their host plants, such as promoting development, maintaining protection against insects and pests, exhibiting antimicrobial properties against plant pathogens, and aiding in regulating stress.They facilitate global nutrient cycling and the differentiation between host health and disease states [13].They also serve as valuable sources of biotechnologically significant molecules, with numerous biocompatible and therapeutic substances.These organisms function as reservoirs for distinct bioactive metabolites, including alkaloids, phenolic acids, steroids, and tannins, with antibacterial, insecticidal, and anticancer properties, [14] to name a few.
Secondary metabolites play a crucial role in mediating microbial interactions in natural environments, exhibiting a wide range of activities which can span from competitive to cooperative dynamics.Instances of such interactions encompass the synthesis of bioactive secondary metabolites.It is worth noting that these metabolites exhibit variable degrees of specificity, wherein certain metabolites exhibit a broad-spectrum effect by targeting a large range of molecular targets, while others exhibit a narrow-spectrum effect by targeting a more limited scope of molecular targets [4].The exploration of endophytic secondary metabolites with bioactive properties has gained increasing attention, owing to their various bioactive capabilities and wide range of structural categories.Currently, only a few plants have been studied to explore their endophytic biodiversity and ability to produce bioactive secondary metabolites.In effect, every medicinal plant species on the planet is a habitat for multiple varieties of endophytic bacteria.Endophytes can undertake species-specific interactions and co-evolve with their plant hosts.
The best-known medicinal usage of Aloe barbadensis Miller, a succulent plant which thrives in dry and subtropical climates, is in Ayurvedic, homoeopathic and allopathic medicine.Throughout history, diverse nations have used aloe vera extensively for various purposes.These include using aloe vera to decrease perspiration, administering the plant orally to manage diabetes, and employing the plant to alleviate a variety of gastrointestinal disorders.It has been utilized for managing burn injuries, minor lacerations, genital herpes, and eczema.The leaves of this medicinal plant are abundant in vitamins, minerals, natural sugars, enzymes, amino acids, and various bioactive molecules.These molecules possess soothing, aperient, anti-inflammatory, antioxidant, antimicrobial, antihelmenthic, antifungal, aphrodisiac, antiseptic and cosmetic properties.Given its healing and rejuvenating properties, cosmetic companies use this plant widely.This study aimed to screen, identify, and characterize the secondary metabolites associated with two bacterial endophytes originating from native medicinal plants in Limpopo, South Africa.

Results
In this study, twenty-three (23) bacterial endophytes were isolated from the stems and leaves of the abovementioned medicinal plants.Seven (7) bacterial endophytes were isolated from Moringa oleifera, nine (9) from Artemisia afra and seven (7) from Aloe vera.The isolates were then screened for antimicrobial properties, and the chemical profiles of biologically active isolates were studied and are presented below.

Antimicrobial Susceptibility of Mycobacterium bovis
The cross-streak method used to measure antimicrobial activity revealed that M. bovis was sensitive to secondary metabolites produced by some bacterial isolates.Of the 23 endophytic bacteria tested, 21 showed no activity against the test organism M. bovis, while two isolates, Aloe I4 and Aloe I3-I5, showed clear areas of inhibition against M. bovis (Figure 1).

Results
In this study, twenty-three (23) bacterial endophytes were isolated from the stems and leaves of the abovementioned medicinal plants.Seven (7) bacterial endophytes were isolated from Moringa oleifera, nine (9) from Artemisia afra and seven (7) from Aloe vera.The isolates were then screened for antimicrobial properties, and the chemical profiles of biologically active isolates were studied and are presented below.

Antimicrobial Susceptibility of Mycobacterium bovis
The cross-streak method used to measure antimicrobial activity revealed that M. bovis was sensitive to secondary metabolites produced by some bacterial isolates.Of the 23 endophytic bacteria tested, 21 showed no activity against the test organism M. bovis, while two isolates, Aloe I4 and Aloe I3-I5, showed clear areas of inhibition against M. bovis (Figure 1).An inhibitory zone of 5 mm was recorded with isolate Aloe I4, and no growth of M. bovis was observed in the Petri dish treated with Aloe I3-I5 (Figure 2).The complete absence of growth or total inhibition noted here could be attributed to the chemical profile of the isolate.An inhibitory zone of 5 mm was recorded with isolate Aloe I4, and no growth of M. bovis was observed in the Petri dish treated with Aloe I3-I5 (Figure 2).The complete absence of growth or total inhibition noted here could be attributed to the chemical profile of the isolate.
Our findings corroborate similar observations in the literature [14].Previous studies have proven that endophytes produce active compounds which exhibit antibacterial, antifungal, antiviral, antisuppressant, and antioxidant properties [15].According to Lertcanawanichakul and Sawangnop [16], the cross-streak method is preferred over the agar well diffusion method, but it can have drawbacks as the margins of the inhibition zones are usually indistinct, making it difficult to obtain quantitative data.This was experienced in this study; however, with the clear zones/areas observed on the plates containing Aloe I4 and Aloe I3-I5, compared to the other 21 isolates (Figure 2), it could be deduced that they have antimicrobial activity against Mycobacterium bovis.Other methods, such as the gel well diffusion method, could be employed to further characterize the isolates' bioactivity against test species.Endophytes and medicinal plants produce vital secondary metabolites which could be used for human benefit.Our findings corroborate similar observations in the literature [14].Previous studies have proven that endophytes produce active compounds which exhibit antibacterial, antifungal, antiviral, antisuppressant, and antioxidant properties [15].According to Lertcanawanichakul and Sawangnop [16], the cross-streak method is preferred over the agar well diffusion method, but it can have drawbacks as the margins of the inhibition zones are usually indistinct, making it difficult to obtain quantitative data.This was experienced in this study; however, with the clear zones/areas observed on the plates containing Aloe I4 and Aloe I3-I5, compared to the other 21 isolates (Figure 2), it could be deduced that they have antimicrobial activity against Mycobacterium bovis.Other methods, such as the gel well diffusion method, could be employed to further characterize the isolates' bioactivity against test species.Endophytes and medicinal plants produce vital secondary metabolites which could be used for human benefit.

Isolation and Characterization of Endophytes
Surface sterilization aids in the removal of potential pollutants (such as soil) and the promotion of internal microorganismal growth [17].As no bacterial colonies were seen on any of the control plates, the surface sterilization of all plant parts was deemed successful.Various colonies were observed on the plates, with pigmentation varying between white, cream white, yellow, pinkish, and pale yellow.The endophytic isolates varied in texture between viscid, pasty, moist, mucoid, and dry.In this study, twenty-three (23) bacterial endophytes were isolated from the stems and leaves of the abovementioned medicinal plants.Seven (7) bacterial endophytes were isolated from Moringa oleifera, nine (9) from Artemisia afra, and seven (7) from Aloe vera.Our findings corroborate those of Strobel [18], who reported the successful isolation of endophytes from plant parts such as roots, leaves, and stems of Rhyncholacis penicillata.Microscopy analysis revealed thirteen (13) Gram-negative rods and ten (10) Gram-positive rods and cocci isolates.Host plant species, maturity, territorial and ecological distribution, season of sample collection, exterior sterilization method, and growth conditions are all variables which can impact the diversity and dispersion of bacterial endophytes in plants [19].Several factors influence the distribution and variety of bacterial endophytes in plants, including host plant species, age, plant tissue type, geographical and habitat distribution, sampling season, surface sterilization method, growth media, and culture conditions [19].The morphological characteristics mentioned here are insufficient for successfully identifying and characterizing bacterial endophytes or species, so molecular techniques such as whole-genome sequencing (WGS) were used to fully identify the species in terms of phylogeny, morphology, virulence, and other important factors [20].

Isolation and Characterization of Endophytes
Surface sterilization aids in the removal of potential pollutants (such as soil) and the promotion of internal microorganismal growth [17].As no bacterial colonies were seen on any of the control plates, the surface sterilization of all plant parts was deemed successful.Various colonies were observed on the plates, with pigmentation varying between white, cream white, yellow, pinkish, and pale yellow.The endophytic isolates varied in texture between viscid, pasty, moist, mucoid, and dry.In this study, twenty-three (23) bacterial endophytes were isolated from the stems and leaves of the abovementioned medicinal plants.Seven (7) bacterial endophytes were isolated from Moringa oleifera, nine (9) from Artemisia afra, and seven (7) from Aloe vera.Our findings corroborate those of Strobel [18], who reported the successful isolation of endophytes from plant parts such as roots, leaves, and stems of Rhyncholacis penicillata.Microscopy analysis revealed thirteen (13) Gram-negative rods and ten (10) Gram-positive rods and cocci isolates.Host plant species, maturity, territorial and ecological distribution, season of sample collection, exterior sterilization method, and growth conditions are all variables which can impact the diversity and dispersion of bacterial endophytes in plants [19].Several factors influence the distribution and variety of bacterial endophytes in plants, including host plant species, age, plant tissue type, geographical and habitat distribution, sampling season, surface sterilization method, growth media, and culture conditions [19].The morphological characteristics mentioned here are insufficient for successfully identifying and characterizing bacterial endophytes or species, so molecular techniques such as wholegenome sequencing (WGS) were used to fully identify the species in terms of phylogeny, morphology, virulence, and other important factors [20].

Molecular and Phylogenetic Identification of Aloe I4 and Aloe I3-I5
Whole-genome sequence (WGS) analysis has become an important tool for determining bacterial relationships and is commonly utilized for the identification of microorganisms.It is used frequently to determine phylogenetic relationships between organisms [21].De novo genome assemblies were used in this project for the molecular and phylogenetic identification of the isolates.According to the NCBI BLAST results for the Aloe I4 and Aloe I3-I5 gene sequences, the isolates belong to two different bacterial genera: Pseudomonas and Enterobacter.The WGS results of the isolates (Tables 1 and 2) were submitted to the NCBI database for genome sequences.Enterobacter sp.I4, the whole genome shotgun project, was deposited on GenBank (https://DDBJ/ENA/GenBank (accessed on 27 March 2022)) and assigned a Bio-sample number SAMN26660210 and Bio-project number PRJNA816151 under the accession JALBUO000000000.Pseudomonas sp.I3-I5, the whole genome shot-gun project, was deposited on GenBank (https://DDBJ/ENA/GenBank (accessed on 27 March 2022)) and assigned a Bio-sample number SAMN26660159 and Bio-project number PRJNA816147 under the accession CP093940.According to Menpara and Sumitra [22], dominating endophytes, particularly in medicinal plants, belong to genera such as Enterobacter, Acinetobacter, Staphylococcus, and Pseudomonas.The two bacterial endophytes identified in this project belonged to the Enterobacter and Pseudomonas genera.The BLAST results showed that the bacterial endophyte Aloe I4 has 100% similarity to several Enterobacter species, but the top hit was Enterobacter cloacae ATCC 13047.Other species included Enterobacter mori LMG 25706, Enterobacter bugandensis EB-247, and Enterobacter dykesii EIT.It also showed 92% similarity to Enterobacter hormaechei subsp.And Enterobacter Xiangfangensis LMG 27195.Two outgroup species that are closely related to Enterobacter were also revealed, namely Klebsiella quasipneumoniae 01A030 and Kluyvera cryocrescens NBRC 102467 (Figure 3).Enterobacter cloacae (E.cloacae) can be found in numerous places, such as sewage, soil, and food [23].E. cloacae, E. aerogenes and E. sakazakii are species that are commonly present in clinical materials.Enterobacter colonies are either non-pigmented or yellow pigmented [24].In this project, the colonies of Aloe I4 appeared yellow pigmented.Due to the 100% phylogenetic similarity of Aloe I4 and Enterobacter cloacae ATCC 13047, it was assigned the strain name Enterobacter cloacae I4.
Pseudomonas species are frequently found in plants and have been isolated from a variety of plant parts and tissues [25].Isolate Aloe I3-I5 showed 99% similarity to Pseudomonas fulva DSM 17717 and Pseudomonas parafulva DSM 17004.It also showed 85% similarity to Pseudomonas sichuanensis WCHPS060039, 70% similarity to Pseudomonas reidholzensis CCOS 865, and 63% similarity to Pseudomonas putida NBRC 14164 (Figure 4).Because Aloe I3-I5 showed a very close relation to Pseudomonas fulva DSM 17717, it was assigned a strain name of Pseudomonas fulva I3-I5.Pseudomonas species are frequently found in plants and have been isolated from a variety of plant parts and tissues [25].Isolate Aloe I3-I5 showed 99% similarity to Pseudomonas fulva DSM 17717 and Pseudomonas parafulva DSM 17004.It also showed 85% similarity to Pseudomonas sichuanensis WCHPS060039, 70% similarity to Pseudomonas reidholzensis CCOS 865, and 63% similarity to Pseudomonas putida NBRC 14164 (Figure 4).Because Aloe I3-I5 showed a very close relation to Pseudomonas fulva DSM 17717, it was assigned a strain name of Pseudomonas fulva I3-I5.

Chemical Profile of Aloe vera I3-I5 and Aloe vera I4
Plants and endophytes produce bioactive chemical substances which could have industrial applications, including pharmaceuticals, cosmetics, agriculture, and food and beverages [26].In this project, GCMS was used to identify secondary metabolites produced by endophytic isolates with antimicrobial activity against Mycobacterium bovis.Both isolates, namely, Pseudomonas fulva I3-I5 and Enterobacter cloacae I4, produced a variety of

Chemical Profile of Aloe vera I3-I5 and Aloe vera I4
Plants and endophytes produce bioactive chemical substances which could have industrial applications, including pharmaceuticals, cosmetics, agriculture, and food and beverages [26].In this project, GCMS was used to identify secondary metabolites produced by endophytic isolates with antimicrobial activity against Mycobacterium bovis.Both isolates, namely, Pseudomonas fulva I3-I5 and Enterobacter cloacae I4, produced a variety of active compounds, with functional groups ranging across alkanes, esters, alcohols, ketones, and other organic compounds.
Hexadecane was one of the active compounds produced by both isolates and has been proven to have antifungal, antibacterial, and antioxidant properties [27].Symptoms of human tuberculosis caused by M. bovis infection include fever, headaches, heavy sweat, coughing, abdominal pain, weight loss, and diarrhoea [28].According to [29], ergotamine has vital properties such as antibacterial and antifungal activity.It is also employed in pharmaceuticals for the treatment of fever, headaches, and migraine.Fever is one of the symptoms of TB, and this active compound in isolation could be employed and incorporated in the treatment of TB.Pyrollo was isolated from ethyl acetate extracts of Pseudomonas fulva I3-I5 and Enterobacter cloacae I4, which is used in anti-inflammatory drugs, antibiotics and antitumour agents [30].Methyl alcohol was extracted from Enterobacter cloacae I4 and is known to possess antimicrobial properties.It is commonly employed in pharmaceuticals to produce cholesterol, as well as antibiotics such as streptomycin and vitamins and hormones.
Another active compound extracted from Pseudomonas fulva I3-I5 and Enterobacter cloacae I4 with bioactive properties was octacosane, which is used in the synthesis of proteins [31].L-proline was identified in both isolates; it is known to have antibacterial and antifungal properties and is used in pharmaceutical preparations such as injections [32].Most of the active compounds extracted in this study, such as bifenthrin, bumetrizole, nonanal, metolachlor, and quinolone, have pharmaceutical applications but could also have other biotechnological significances, such as uses in cosmetics, pesticides, and detergents, as well as in food and beverages.This study proves the significance of employing medicinal plants and endophytes in drug discovery and development, to eradicate or decrease the rising microbial resistance taking place globally.Endophyte isolation and the investigation of their active compounds is gaining traction worldwide due to their vital health properties [33].

Susceptibility Test of Mycobacterium bovis against Endophytic Bacteria
The cross-streak technique was used to assess the antimycobacterial potency of the 23 isolates against Mycobacterium bovis following the method of Okudo and Wallis [34].In brief, Muller-Hinton agar (MHA) (Sigma-Aldrich, Johannesburg, South Africa, BCBZ7677) plates were prepared following manufacturer guidelines, and 20 mL of the medium was allowed to solidify in Petri dishes.A line was drawn in the centre to divide the plates into twos at the back of each plate for the inoculation of the 23 isolated endophytes.Perpendicularly to this line, two other lines were drawn for the inoculation of M. bovis.Using an inoculation loop, a loop full of each of the 23 bacterial isolates were streaked throughout the central line on the Petri dishes containing the solidifies Muller-Hinton agar and incubated at 37 • C for 3 days for maximum growth.Then, afterwards, the plates were cross streaked with a loop full of test organism (M.bovis) at a 90 • angle (on the two lines drawn prior) to the bacterial isolates and further incubated for 5 days at 37 • C to allow for antimicrobial activity.
Endophytic bacteria were isolated from plant components (fresh, healthy, and diseasefree stems and leaves) immediately after therapeutic plants were collected.First, all medicinal plant parts (stems and leaves) were washed under running tap water to eliminate contaminants such as soil and dust debris.Plant components were sliced into small segments and treated with 5% Tween 20 for 5 min while vigorously shaking them, then rinsed with distilled water (dH 2 O) several times [25].The next step was disinfecting the plant components with 70% ethanol for 1 min, after which components were cleansed five times with dH 2 O to remove any ethanol residue.They were then treated with 1% sodium hypochlorite and rinsed five times with dH 2 O, and the final wash was plated on nutrient agar (NA) as a control.The plant components' outer surfaces were removed, and they were ground in phosphate-buffered saline (PBS).Before being spread out on nutrient agar plates, all macerated components were serially diluted to a concentration of 10 −3 [25].The plates were incubated at 30 • C for 5 days with the controls, with bacterial growth measured daily.After the incubation period, various colonies were selected and sub-cultured on nutrient agar to yield pure isolates.Pure bacterial isolates were kept at −80 • C with 50 percent glycerol on a 1 mL glycerol-1 mL broth medium ratio overnight [25].
The whole-genome sequencing (WGS) was conducted using PacBio technology by Inqaba Biotec in Pretoria, South Africa.The newly generated genome sequences were compared to the most closely related bacterial species using the Basic Local Alignment Search Tool (BLAST) on the NCBI platform (https://blast.ncbi.nlm.nih.gov,accessed on 30 March 2022).

Processing of Samples for Metabolite Profiling
Following the modified approach proposed by Xu et al. [35] and Daji et al. [36], centrifuge tubes were employed to combine 10 mL of 100% methanol with 1 g of the extracts.Using Scientech 704 from Labotech in Johannesburg, South Africa, samples were vortexed before an ultrasonic-aided extraction was carried out for one hour at 4 • C. Subsequently, samples underwent centrifugation at 3500× g revolutions per minute (rpm) at a temperature of 4 • C for 5 min using an Eppendorf 5702R centrifuge (Merck, Johannesburg, South Africa).The liquid portion was filtered using filter sheets with a pore size of 0.45 micrometres and transferred into dark amber vials.The extraction process was performed in triplicate for each sample.

Analysis Using GC-HRTOF-MS
To achieve a reproducible result, the mass optimization of the instrument (Leco Pegasus GC-HRTOF-MS, St. Joseph, MI, USA) was performed and passed the preceding analysis.The extracts were introduced into the GC-HRTOF-MS system (Gerstel GmbH & Co. KG, Mülheim (Ruhr), Germany).The system was fitted with a column, with dimensions of 30 m in length, 0.25 millimetres in internal diameter, and a film thickness of 0.25 micrometres.A sample of 1 microliter was introduced into the gas chromatographymass spectrometry (GC-MS) system at a flow rate of 1 millilitre per minute, with helium serving as the carrier gas.The input temperature was set at 250 • C, while the transfer line temperature was set at 225 • C. The initial setting of the oven was set at 70 • C for a duration of 30 s. Subsequently, the temperature was decreased at a rate of 10 • C per minute until it reached 10 • C. It was then increased to 150 • C and maintained for a period of 2 min.Following this, the temperature was once again decreased at a rate of 10 • C per minute until it reached 10 • C. Finally, the temperature was raised to 330 • C for a duration of 180 s.The experimental settings suggested for the analysis of MS data included acquiring 13 spectra per second, a mass-to-charge ratio range of 30-1000 m/z, electron impact at an energy of 70 eV, and maintaining the ion source temperature at 250 • C and the extraction frequency at 1.25 Hz.Three sets of triplicate samples were subjected to three injections each, resulting in a total of nine injections per sample.The data were analysed with ChromaTOF ® program.All uses of active compounds and applications were gathered from NCBI PubChem database, which may be found at [37].

Figure 1 .
Figure 1.Bar graph showing the antimicrobial properties of the isolated bacteria endophytes from medicinal plants against Mycobacterium bovis.Twenty-one isolates showed no inhibition and two isolates showed inhibition, with isolate Aloe I3/5 exhibiting total inhibition.(0): no inhibition, (10): inhibition observed, (100): complete inhibition or no growth of M. bovis at all.

Figure 1 .
Figure 1.Bar graph showing the antimicrobial properties of the isolated bacteria endophytes from medicinal plants against Mycobacterium bovis.Twenty-one isolates showed no inhibition and two isolates showed inhibition, with isolate Aloe I3/5 exhibiting total inhibition.(0): no inhibition, (10): inhibition observed, (100): complete inhibition or no growth of M. bovis at all.

Figure 2 .
Figure 2. Photograph of the cross-streak method showing the antimicrobial activity of isolate Aloe I4 (left) with a 5 mm area of inhibition (A: Aloe I4, B: M. bovis; C: Zone of inhibition) and Aloe I3/5 (right) showing no growth of M. bovis after 5 days of incubation (A: Aloe I3-I5; B: M. bovis).

Figure 2 .
Figure 2. Photograph of the cross-streak method showing the antimicrobial activity of isolate Aloe I4 (left) with a 5 mm area of inhibition (A: Aloe I4, B: M. bovis; C: Zone of inhibition) and Aloe I3/5 (right) showing no growth of M. bovis after 5 days of incubation (A: Aloe I3-I5; B: M. bovis).

Figure 3 .
Figure 3. Enterobacter cloacae I4 phylogenetic tree based on de novo genome assembly analysis.The numbers beneath the branches represent bootstrap support levels based on 100 replications.

Figure 3 .
Figure 3. Enterobacter cloacae I4 phylogenetic tree based on de novo genome assembly analysis.The numbers beneath the branches represent bootstrap support levels based on 100 replications.

Figure 4 .
Figure 4. Pseudomonas fulva I3-I5 phylogenetic tree based on de novo genome assembly analysis.The numbers beneath the branches represent bootstrap support levels based on 100 replications.

Figure 4 .
Figure 4. Pseudomonas fulva I3-I5 phylogenetic tree based on de novo genome assembly analysis.The numbers beneath the branches represent bootstrap support levels based on 100 replications.

Table 1 .
De novo genome assembly summary metrics for Aloe I4.Primary contigs represent pseudo haplotype assemblies, while haplotigs represent fully phased and assembled regions of the genome.

Table 2 .
De novo genome assembly summary metrics for Aloe I3-I5.Primary contigs represent pseudo haplotype assemblies, while haplotigs represent fully phased and assembled regions of the genome.