Original Research Submitted: May 2026 / Revised: June 2026 /Accepted: June 2026 Spizaetus: Jurnal Biologi dan Pendidikan Biologi p-ISSN: 2716-151X e-ISSN: 2722-869X Isolation and Antimicrobial Activity of Endophytic Bacteria from Rhizophora mucronata Roots in the Kupang Mangrove Ecotourism Area Isolasi dan Uji Aktivitas Antimikrobia Bakteri Endofit Akar Mangrove Rhizophora mucronata di Kawasan Ekowisata Mangrove Kupang Alexander Tage*.
Chatarina Gradict Semiun.
Yulita Iryani Mamulak Study Program of Biology.
Faculty of Science and Technology.
Widya Mandira Catholic University.
Kupang, 85225.
Indonesia *Corresponding Author: alexandertage@unwira.
Abstract.
Semi-arid mangrove ecosystems harbor significant potential as sources of endophytic bacteria producing adaptive antimicrobial compounds.
This study aimed to isolate and evaluate the antimicrobial activity of endophytic bacteria from Rhizophora mucronata prop and lateral roots across three management zones .
ore, buffer, and utilizatio.
in the Kupang Mangrove Ecotourism Area.
Sampling was conducted in MarchAeApril 2026, followed by root surface sterilization and isolation on Nutrient Agar.
Phenotypic characterization was performed macroscopically, microscopically, and biochemically, while antimicrobial activity was assessed using the Kirby-Bauer disk diffusion method against Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922.
From 69 initial colonies, 18 pure isolates were obtained, with Bacillus sp.
as the dominant genus .
4%).
Notably.
Bacillus sp.
isolates ZS-04 and ZI-05 exhibited the most potent bioactivity, yielding very strong (>20 m.
inhibition zones against S.
aureus and strong .
Ae20 m.
inhibition against E.
One-way ANOVA revealed no significant zonal differences for S.
= 0.
, whereas a statistically significant zonal effect was observed for E.
= 0.
, particularly between the utilization and buffer zones.
These findings confirm that endophytic Bacillus sp.
from R.
mucronata roots, particularly isolates ZS-04 and ZI-05, produce bioactive compounds with strong, targeted antibacterial activity against Gram-positive pathogens, highlighting semi-arid mangrove endophytes as promising, stress-adapted reservoirs for novel natural antimicrobial agents.
Keywords: antimicrobial_activity.
endophytic_bacteria.
mangrove_zonation.
rhizophora_mucronata Abstrak.
Ekosistem mangrove semi-arid menyimpan potensi besar sebagai sumber bakteri endofit penghasil senyawa antimikrobia yang adaptif.
Penelitian ini bertujuan mengisolasi dan mengevaluasi aktivitas antimikrobia bakteri endofit dari akar tunjang dan lateral Rhizophora mucronata pada tiga zona pengelolaan .
nti, penyangga, dan pemanfaata.
di Kawasan Ekowisata Mangrove Kupang.
Pengambilan sampel dilakukan pada MaretAeApril 2026, diikuti sterilisasi permukaan akar dan isolasi pada media Nutrient Agar.
Karakterisasi fenotipik dilakukan secara makroskopik, mikroskopik, dan biokimia, sedangkan uji aktivitas antimikrobia menggunakan metode difusi cakram Kirby-Bauer terhadap Staphylococcus aureus ATCC 25923 dan Escherichia coli ATCC 25922.
Dari 69 koloni awal, diperoleh 18 isolat murni dengan Bacillus sp.
sebagai genus dominan .
,4%).
Secara khusus, isolat Bacillus sp.
ZS-04 dan ZI-05 menunjukkan bioaktivitas paling poten, menghasilkan zona hambat yang sangat kuat (>20 m.
terhadap S.
aureus dan kuat .
Ae20 m.
terhadap E.
Analisis ANOVA menunjukkan tidak ada perbedaan zonasi yang signifikan terhadap S.
= 0,.
, namun terdapat pengaruh zonasi yang signifikan terhadap E.
= 0,.
, khususnya antara zona pemanfaatan dan penyangga.
Hasil ini mengonfirmasi bahwa endofit Bacillus sp.
dari akar R.
mucronata, khususnya isolat ZS-04 dan ZI-05, menghasilkan senyawa bioaktif dengan aktivitas antibakteri yang kuat dan terarget terhadap patogen Gram-positif, yang menyoroti endofit mangrove semi-arid sebagai reservoir yang adaptif terhadap stres dan menjanjikan untuk pengembangan agen antimikrobia alami yang baru Kata Kunci: aktivitas_antimikrobia.
bakteri_endofit.
rhizophora_mucronata.
zona_mangrove DOI: 10.
55241/spibio.
Introduction Mangrove ecosystems are highly productive coastal habitats that play a crucial role in shoreline protection and blue carbon sequestration .
As naturally functioning ecosystems, mangrove forests harbor high biodiversity and deliver essential ecological services that sustain broader coastal and terrestrial forest ecosystems .
Indonesia encompasses approximately 3,455,628 hectares of mangrove forests distributed along its extensive archipelagic coastline, including East Nusa Tenggara Province (NTT) .
This region, characterized by semi-arid climatic conditions and extreme salinity fluctuations, supports an estimated 23,016 hectares of mangrove ecosystems.
In this region.
Rhizophora mucronata .
ocally known as bakau or red mangrov.
is the dominant species within the family Rhizophoraceae, distinguished by its characteristic prop root system.
These roots interact directly with anoxic sediments and brackish waters exhibiting fluctuating salinity, creating an extreme microenvironment that drives unique microorganisms associated with them .
mucronata has long been utilized by coastal communities in Southeast Asia as traditional medicine for treating skin infections, wounds, and fever.
Modern phytochemical studies have confirmed that extracts of R.
mucronata bark, leaves, and metabolites, including tannins, flavonoids, saponins, and terpenoids, that exhibit significant antimicrobial activity against both Gram-positive and Gram-negative pathogens .
Given that endophytic bacteria are known to synthesize bioactive compounds structurally similar to or identical with those produced by their host plants, the documented antimicrobial properties of R.
mucronata provide a strong rationale for bioprospecting its root- associated endophytes as alternative sources of novel antibacterial agents.
Endophytic bacteria that colonize plant tissues in a mutualistic association have been demonstrated to produce secondary metabolites with high biological activity in response to environmental stressors .
Extreme environmental pressures, including sediment anoxia, elevated salinity, and pronounced diurnal temperature fluctuations in semi-arid mangrove habitats, are hypothesized to selectively drive the biosynthesis of unique bioactive compounds rarely identified in wet tropical mangrove ecosystems .
Amid the global antimicrobial resistance (AMR) crisis, which threatens the clinical efficacy of conventional antibiotics, the bioprospecting of bioactive compounds from mangrove endophytes has emerged as a highly promising alternative strategy .
This imperative is particularly pressing in Indonesia, where escalating crosectoral AMR challenges underscore the critical need to discover novel antibiotics from local biodiversity.
The Kupang Mangrove Ecotourism Area is structured into three functional management zones .
ore, buffer, and utilizatio.
, which collectively represent a gradient of anthropogenic disturbance and distinct ecological parameters.
The core zone receives the highest level of protection with virtually no human access, the buffer zone functions as an ecological transition area with restricted entry, and the utilization zone experiences the highest tourist visitation and the most intensive human disturbance.
These contrasting environmental conditions are hypothesized to shape endophytic microbial communities with distinct diversity profiles and antimicrobial potential.
While documented the antimicrobial activity of endophytic bacteria isolated from various mangrove species .
, the rootassociated endophytic communities of R.
mucronata across distinct management zones in semi-arid eastern Indonesia remain unexplored.
Roots were specifically selected as the target organ in this study because they interact directly with the most extreme microenvironment within the mangrove ecosystemAinamely, anoxic and highly saline sediments.
Rootassociated growth-promoting bacteria (PGPB) have evolved under these harsh conditions to produce a diverse array of beneficial compounds, including metabolites, which not only aid in host stress tolerance but also enhance resistance against soil-borne pathogens .
Because the root system serves as the primary interface and first line of defense against environmental pathogens, endophytes colonizing the roots are theoretically and empirically expected to compared to those isolated from aboveground organs such as leaves or stems.
Furthermore, endophytic bacteria are comparable to the bioactive compounds produced by their host plants .
Given the documented antimicrobial properties of root-associated endophytes represent a highly rational and This knowledge gap underpins the hypothesis that ecological zoning shapes both the community composition and the antimicrobial potential of R.
mucronata root endophytes.
Accordingly, this study aims to isolate, identify, and comparatively evaluate the antimicrobial potential of root-associated endophytic bacteria across the three functional zones.
Preliminary findings demonstrate that these root endophytes, predominantly Bacillus sp.
, possess Specifically, isolates ZS-04 and ZI-05 exhibited strong inhibitory activity against Staphylococcus aureus .
nhibition zones >20 m.
, while activity against Escherichia coli showed significant zonal variation, highlighting the influence of ecological gradients on metabolite production.
Methods This laboratory-based experimental comparative design to assess how ecological zoning influences endophytic diversity and antimicrobial potential.
three individual trees per zone .
ore, buffer, utilizatio.
in the Kupang Mangrove Ecotourism Area (Figure .
Prop roots were targeted as they represent the primary interface with the anoxic, saline sediment environment, while the inclusion of fine lateral roots ensured the capture of the most active zones of microbial colonization and nutrient exchange.
In situ measurements of air/water temperature, salinity, and water/soil pH were recorded Samples were transported at 4Ae8AC to the UNWIRA Laboratory for Sample Collection and Environmental Profiling The research was conducted from March to April 2026.
Healthy R.
roots, specifically a combination of prop .
roots and their associated fine lateral roots, were collected from a depth of 0Ae20 cm below the sediment surface from Figure 1.
Research location map of the Kupang Mangrove Ecotourism Area.
The green, blue, and yellow shaded areas represent the core, buffer, and utilization zones, respectively.
Sampling points are marked with symbols indicating the zone: ZI (Cor.
ZP (Utilizatio.
, and ZS (Buffe.
and microscopic/biochemical profiles (Gram stain, catalase, oxidase, starch hydrolysi.
were recorded following BergeyAos Manual .
Isolation & Characterization Surface sterilization followed the protocol described by Pakaya et al.
with specific modifications tailored for mangrove root tissues.
While the original protocol utilized 1% NaOCl for 5 minutes on sponge tissues, this study adjusted the concentration and exposure time to 3% NaOCl for 3 minutes to effectively penetrate the thicker, tannin-rich cuticle of mucronata roots.
Furthermore, the rinsing step was increased to three cycles with sterile distilled water to ensure the complete removal of residual sterilizing Finally, instead of Marine Agar supplemented with ketoconazole, sterilized root fragments were plated on standard Nutrient Agar (NA) and incubated at 28AC for 48 h, optimizing conditions for mesophilic endophytic bacteria.
To verify sterilization efficacy, 100 AAL of the final rinse water was plated onto NA and the absence of microbial growth confirmed successful surface sterilization.
Macroscopic traits .
olony morphology.
Antimicrobial Activity Assay Antimicrobial activity was evaluated using the Kirby-Bauer disk diffusion method against two reference pathogen strains: Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922.
The diameter of the zone of inhibition was measured using a vernier caliper .
ccuracy: A0.
01 m.
and categorized according to the Davis-Stout criteria: very strong (>20 m.
, strong .
Ae20 m.
, moderate .
Ae10 m.
, weak (<5 m.
, and no activity .
Data Analysis Data are reported as mean A SD .
Zonal differences were analyzed via one-way ANOVA ( = 0.
with TukeyAos HSD post hoc test using IBM SPSS v25.
Result and Discussion environmental parameters were conducted concurrently with root sample collection at all sampling points across the three functional management zones.
The temperature, water temperature, water pH, soil pH, and salinity.
The recorded environmental conditions for each zone are presented in Table 1.
Results indicate a clear ecological gradient across the core, buffer, and utilization zones, as reflected by variation in temperature, pH, and salinity values at each sampling location.
These hypothesized to play a significant role in shaping the diversity of endophytic microbial communities associated with R.
mucronata roots across the respective Table 1.
Environmental conditions were recorded during root sample collection across the three
functional management zones Sampling Air Temp
Water Temp
Water
Soil
Salinity Weather
Point
(AC)
(AC)
H) .
H) (A) Core
ZI-T1
Clear
Core
ZI-T2
Clear
Core
ZI-T3
Clear
Buffer
ZP-T1
Clear
Buffer
ZP-T2
Clear
Buffer
ZP-T3
Clear Utilization ZS-T1 Clear Utilization ZS-T2 Clear Utilization ZS-T3 Clear Legend: temperature and salinity values exhibit a gradient between the core zone .
igher humidity, elevated salinit.
and the utilization zone .
igher temperature, reduced salinity attributable to surface freshwater Data represent mean values from single measurements per sampling point.
weather conditions were recorded visually at the time of collection.
Zone Environmental Conditions Measurements of environmental parameters across the three functional zones revealed a distinct ecological gradient (Table .
Air and water temperatures gradually increased from the core zone .
4Ae32.
1AC and 29.
7Ae30.
4AC)
to the utilization zone .
1Ae34.
7AC and 0Ae32.
7AC), while both water and soil pH showed a slight decline.
In contrast, salinity decreased progressively from the core zone, which exhibited relatively high values .
2Ae29.
1A), to the buffer zone .
3Ae 4A), reaching its lowest values in the utilization zone .
8Ae23.
7A).
These physicochemical differences are primarily driven by the core zone's direct marine influence and the utilization zone's exposure to higher solar radiation, tourist activity, and potential surface freshwater This environmental gradient directly correlates with both the diversity of the isolated endophytes and their antimicrobial potential, validating the zoning approach proposed in this study.
The core zone, characterized by the highest salinity and the most stable conditions, yielded the highest number of isolates .
, suggesting that the undisturbed, highsalinity environment supports a richer endophytic community.
Conversely, the utilization zone, subjected to the highest temperatures, lowest salinity, and lowest pH, produced the fewest isolates .
, likely reflecting the combined negative impacts of anthropogenic disturbance and environmental stress on microbial colonization.
Interestingly, while the core zone had the highest isolate yield, the buffer zoneAiwhich represents a transitional environment with moderate salinity and intermediate disturbanceAi harbored the most potent antimicrobial producers, specifically isolate ZS-04 with an inhibition zone of 22.
15 mm against S.
This pattern aligns with the environmental stress can selectively drive the biosynthesis of potent secondary metabolites as a competitive strategy.
Thus, the distinct physicochemical profiles of each management zone not only shape the quantitative diversity of the rootassociated significantly influence the qualitative bioactivity of the endophytic bacteria.
Isolation Characterization Phenotypic Following profiling, a total of 69 bacterial colonies were initially observed from the root samples of nine R.
mucronata across the three management zones.
After purification on Nutrient Agar (NA), 18 morphologically distinct pure isolates were successfully obtained: 7 from the core zone, 6 from the buffer zone, and 5 from the utilization zone in Figure 2 and Figure 3.
The macroscopic, characteristics of these 18 purified isolates are summarized in Table 2 and Table 3.
Figure 2.
Initial isolation of endophytic bacteria from R.
mucronata root samples on Nutrient Agar (NA) Figure 3.
Morphologically distinct pure cultures obtained following quadrant-streak purification of endophytic bacteria isolated from R.
mucronata roots Table 2.
Macroscopic colony morphology of the 18 purified endophytic bacterial isolates from R.
Isolate Code
ZI-01
ZI-02
ZI-03
ZI-04
ZI-05
ZI-06
ZI-07
ZP-01
ZP-02
Zone Core Core Core Core Core Core Core Utilization Utilization Colony Shape Circular Irregular Circular Circular Irregular Circular Circular Circular Irregular Color Elevation Margin Texture Diameter .
Creamy white
Convex
Entire
Mucoid
White
Flat
Undulate Dry Yellow
Convex
Entire
Shiny Cream
Flat
Entire
Dry Creamy white
Umbonate Lobate Mucoid
Orange Convex
Entire
Shiny White
Flat
Entire
Dry Cream
Convex
Entire
Mucoid
White
Flat
Undulate Dry Yellowish
ZP-03
Utilization Circular
Convex
Entire
Shiny ZP-04
Utilization Circular
White
Umbonate Entire
Dry ZP-05
Utilization Irregular
Cream
Flat
Lobate Mucoid
ZS-01
Buffer
Circular
Creamy white
Convex
Entire
Mucoid
ZS-02
Buffer
Circular
Yellow
Convex
Undulate Shiny ZS-03
Buffer
Irregular
White
Flat
Undulate Dry ZS-04
Buffer
Circular
Cream
Umbonate Entire
Mucoid
ZS-05
Buffer Circular Orange Convex Entire Shiny ZS-06 Buffer Irregular White Flat Entire Dry Legend: isolate code prefixes: ZI = core zone.
ZP = utilization zone.
ZS = buffer zone.
Colony diameters were measured using a digital vernier caliper .
ccuracy: A0.
01 m.
Morphological observations were recorded from pure cultures after 48 h of incubation at 28 AC on Nutrient Agar.
Table 3.
Microscopic characteristics (Gram stainin.
and biochemical assay results for the 18 purified endophytic bacterial isolates from R.
mucronata roots.
Isolate Starch Zone Gram Morphology Arrangement Catalase Oxidase Putative Genus Code Hydrolysis ZI-01 Core Rod Single Oe Bacillus sp.
ZI-02
Core Rod Chain Oe Bacillus sp.
ZI-03
Core Oe Rod Single Oe Pseudomonas sp.
ZI-04
Core Coccus Cluster Oe Oe Staphylococcus sp.
ZI-05
Core Rod Single Oe Bacillus sp.
ZI-06
Core Coccus Cluster Oe Micrococcus sp.
ZI-07
Core Oe Rod Single Oe Oe Serratia sp.
ZP-01 Utilization Rod Single Oe Bacillus sp.
ZP-02 Utilization Oe Rod Single Oe Pseudomonas sp.
ZP-03 Utilization Coccus Cluster Oe Oe Staphylococcus sp.
ZP-04 Utilization Rod Chain Oe Bacillus sp.
ZP-05 Utilization Oe Rod Single Oe Oe Enterobacter sp.
ZS-01
Buffer Rod Single Oe Bacillus sp.
ZS-02
Buffer Oe Rod Single Oe Pseudomonas sp.
ZS-03
Buffer Rod Chain Oe Bacillus sp.
ZS-04
Buffer Rod Single Oe Bacillus sp.
ZS-05
Buffer Coccus Cluster Oe Micrococcus sp.
ZS-06
Buffer Oe Rod Single Oe Oe Serratia sp.
Legend: isolate code prefixes: ZI = core zone.
ZP = utilization zone.
ZS = buffer zone.
Gram reaction: = Gram-positive.
Oe = Gram-negative.
Cell morphology: rod = Bacillus-shaped, coccus = spherical.
Cell arrangement: single = solitary cells, chain = strepto-arrangement, cluster = staphylo-arrangement.
Biochemical assays: = positive reaction.
Oe = negative Putative genus-level identification is based solely on phenotypic characteristics following Bergey's Manual of Determinative Bacteriology .
Definitive genus and species identification requires confirmatory molecular analysis .
, 16S rRNA gene sequencin.
Bacillus sp.
was the most dominant genus, accounting for 8 isolates .
distributed across all three zones.
This dominance is likely driven by the genus's exceptional adaptability to the extreme Kupang mangrove, particularly its capacity to form endospores that ensure survival under the high salinity .
p to 29.
1A) and anoxic conditions recorded in the core zone (Table Endophytic Bacillus isolates from R.
mucronata are also known to exhibit degradation of organic matter and nutrient acquisition from host tissues .
Furthermore.
Bacillus welldocumented for plant growth-promoting (PGP) traits, such as nitrogen fixation and phosphate solubilization, which foster mutualistic symbioses with the mangrove host .
While Bacillus dominated the overall isolate collection, the presence of other functionally distinct genera further highlights the metabolic diversity of this root-associated microbiome.
The detection of three Pseudomonas isolates (ZI-03.
ZP-02.
ZS-.
can be attributed to the genus's established ecological role in nutrient cycling and its adaptation to partially anaerobic conditions Pseudomonas is among the most prevalent Indonesian mangrove ecosystems, largely due to its enzymes and bioactive metabolites that enhance host plant resilience to environmental stress .
Research indicates that Pseudomonas efficiently colonizes Rhizophora root zones through chemotactic responses to organic-rich root exudates and exhibits notable heavy metal bioremediation capabilities .
, a trait particularly advantageous given the frequent metal accumulation in mangrove Despite its lower relative abundance compared to Bacillus, this genus remains consistently distributed across multiple habitat zones.
Beyond these Gram-negative rods, the endophytic community also included Gram-positive cocci, reflecting a broader spectrum of microbial adaptation to the saline The isolation of Staphylococcus sp.
and Micrococcus sp.
isolates eac.
from mucronata roots highlights the diversity of endophytic bacteria capable of thriving high-salinity Staphylococcus documented in mangrove habitats globally, suggesting they possess effective osmotic stress tolerance mechanisms that facilitate tissue colonization .
Studies position Micrococcus and Staphylococcus as key members of heterotrophic communities, decomposition in environments enriched with complex aromatic compounds from Rhizophora leaf litter .
Their presence here underscores a remarkable adaptive capacity to the unique rhizospheric microenvironment, which is dynamically influenced by tidal fluctuations .
addition to these osmotolerant cocci, a smaller fraction of the isolates belonged to genera traditionally recognized for their specific plant growth-promoting capabilities in nutrient-limited soils.
The detection of two Serratia sp.
isolates and one Enterobacter sp.
can be explained by the functional roles of these genera as plant growth-promoting bacteria (PGPB).
Serratia strains isolated from plant rhizospheres are welldocumented phytohormone production, supporting host stress-prone conditions .
Studies on mangrove endophytes indicate that Enterobacter and Serratia effectively colonize root tissues in response to chemical signaling compounds exuded by Rhizophora roots, while their capacity for non-symbiotic nitrogen fixation contributes to overall nitrogen availability .
Their presence highlights the interactions in the R.
It is important to acknowledge a key limitation of this study regarding the taxonomic identification of the isolates.
The genus-level assignments presented in Table 3 are tentative and based solely on characteristics (Gram staining, catalase, oxidase, and starch hydrolysi.
While these tests provide a useful preliminary classification following Bergey's Manual .
, they are insufficient for definitive taxonomic resolution, as biochemical profiles can frequently overlap between Without confirmatory molecular techniques, such as 16S rRNA gene sequencing or automated identification systems .
VITEK), the exact genus and species of these isolates cannot be conclusively Therefore, these phenotypic assignments should be interpreted as presumptive, and future studies should prioritize molecular identification to validate these findings and enable precise taxonomic classification of the bioactive Antimicrobial Activity Antimicrobial activity was evaluated for all 18 purified isolates using the KirbyBauer disk diffusion method against two Staphylococcus aureus and Escherichia Inhibition zone diameters were according to the Davis-Stout criteria: very strong (VS, >20 m.
, strong (S, 10Ae20 m.
, moderate (M, 5Ae10 m.
, weak (W, <5 m.
, and no activity .
Chloramphenicol .
AA.
and sterile Nutrient Broth served as positive and negative controls, respectively.
The comprehensive results are presented in Table 4.
Table 4.
Mean inhibition zones of antimicrobial activity exhibited by endophytic bacterial isolates against
Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922
Zone
Isolate Code
Putative Genus
Core
Core
ZI-01
ZI-02
Bacillus sp.
Bacillus sp.
Inhibition Zone .
20 A 0.
33 (S)
25 A 0.
40 (S)
31 A 0.
42 (S)
0 (NA)
Core
Core
Core
Core
Core
Utilization Utilization Utilization Utilization Utilization Buffer
Buffer
Buffer
Buffer
Buffer
Buffer
ZI-03
Pseudomonas sp.
19 A 0.
34 (S)
55 A 0.
35 (S)
ZI-04
Staphylococcus sp.
0 (NA)
0 (NA)
ZI-05
Bacillus sp.
29 A 0.
45 (VS)
39 A 0.
45 (S)
ZI-06
Micrococcus sp.
72 A 0.
39 (M)
0 (NA)
ZI-07
Serratia sp.
0 (NA)
0 (NA)
ZP-01
Bacillus sp.
62 A 0.
40 (S)
19 A 0.
36 (S)
ZP-02
Pseudomonas sp.
0 (NA)
0 (NA)
ZP-03
Staphylococcus sp.
86 A 0.
35 (M)
0 (NA)
ZP-04
Bacillus sp.
79 A 0.
46 (S)
32 A 0.
40 (M)
ZP-05
Enterobacter sp.
0 (NA)
0 (NA)
ZS-01
Bacillus sp.
49 A 0.
60 (S)
82 A 0.
41 (S)
ZS-02
Pseudomonas sp.
72 A 0.
39 (S)
62 A 0.
40 (S)
ZS-03
Bacillus sp.
0 (NA)
0 (NA)
ZS-04
Bacillus sp.
15 A 0.
65 (VS)
32 A 0.
40 (S)
ZS-05
Micrococcus sp.
92 A 0.
39 (M)
0 (NA)
ZS-06
Serratia sp.
0 (NA)
0 (NA)
Active isolates .
Positive Chloramphenicol .
19 A 0.
36 (S)
39 A 0.
45 (S)
AA.
Negative Sterile Nutrient Broth
(NB)
Legend: values represent mean inhibition zone diameter A standard deviation (SD) from three independent Activity categories: VS = very strong (>20 m.
S = strong .
Ae20 m.
M = moderate .
Ae10 m.
W = weak (<5 m.
NA = no activity .
Positive control: chloramphenicol disk .
AA.
control: sterile nutrient broth.
Isolate code prefixes: ZI = core zone.
ZP = utilization zone.
ZS = buffer zone.
Against the Gram-positive pathogen aureus, antimicrobial activity varied considerably among the isolates.
Notably, isolates ZS-04 and ZI-05 .
oth Bacillus sp.
demonstrated the most potent inhibitory effects, yielding mean inhibition zones of 15 A 0.
65 mm and 21.
29 A 0.
45 mm.
Based on the Davis-Stout criteria, these are categorized as very strong (VS) activity (Figure .
Several other isolates exhibited strong .
Ae20 m.
or moderate .
Ae10 m.
inhibition, while a subset showed no detectable activity.
These findings align with prior studies by Prihanto .
and Fatimah .
, who reported that endophytic bacteria isolated from mangroves possess the capacity to metabolites, such as alkaloids, flavonoids, and terpenoids, which are highly effective against Gram-positive pathogens.
While the activity against S.
aureus was broadly distributed, the response of the Gramnegative pathogen E.
coli revealed a distinctly different and more selective Consistent with this selective pattern, antimicrobial testing against E.
coli showed markedly lower overall efficacy, with 11 of the 18 isolates .
1%) exhibiting no detectable inhibition.
However, the same highly active isolates.
ZI-05 and ZS-04, also produced the largest inhibition zones against this Gram-negative bacterium, 39 A 0.
45 mm and 18.
40 mm, respectively.
Under the corrected criteria, these values fall into the strong (S) category .
Ae20 m.
(Figure .
This differential susceptibility is primarily attributed to the structural defenses of Gram-negative bacteria, specifically the outer lipopolysaccharide (LPS) membrane that acts as a formidable permeability barrier, coupled with intrinsic resistance mechanisms such as multidrug efflux pumps .
The markedly lower antimicrobial activity against E.
coli compared to S.
aureus reflects the inherent selectivity of bioactive metabolites produced by R.
mucronata endophytes.
This pattern strongly aligns with findings by Putri .
, narrow-spectrum compounds from Indonesian endophytes often exhibit greater efficacy against Grampositive pathogens.
From a therapeutic advantageous, as targeted antimicrobials can eradicate specific pathogens while hostAos microbiota.
Nevertheless, isolates ZI-05.
ZS-04, and ZI-03 demonstrated strong inhibition of E.
coli and merit further chemical and mechanistic characterization to unlock their potential against Gram- This resistance in E.
coli naturally leads to greater variability in inhibition data, which is further reflected in the statistical analysis of zonal effects.
Figure 4.
Representative inhibition zones exhibited by endophytic bacterial isolates ZS-04 (Bacillus sp.
and ZI-05 (Bacillus sp.
) against S.
Figure 5.
Representative inhibition zones exhibited by endophytic bacterial isolates ZS-04 (Bacillus sp.
and ZI-05 (Bacillus sp.
) against E.
Comparative Statistical Analysis To evaluate the influence of potential, the inhibition zone data from active isolates were subjected to one-way ANOVA ( = 0.
, preceded by ShapiroWilk normality and LeveneAos homogeneity of variance tests.
Where significant differences were detected.
TukeyAos HSD post hoc test was applied to identify specific zonal variations.
The summary of these statistical evaluations is detailed in Table 6.
Table 6.
Summary of assumption testing, one-way ANOVA, and pairwise comparisons of antimicrobial activity across functional management zones.
Post hoc .
Normality Homogeneity (ShapiroANOVA .
(LeveneAos Tes.
ZI vs ZP ZI vs ZS ZP vs ZS Wil.
Normal Homogeneous 785 .
Normal Homogeneous 045 .
Legend: ZI = Core Zone.
ZP = Utilization Zone.
ZS = Buffer Zone.
= significant.
= not significant.
The value .
= 18 for all isolates .
alue 0 was not included in the analysi.
Post hoc Tukey HSD test for E.
The number of isolates .
selected is those that have bacterial inhibition zone values.
Pathogen Descriptive analysis for S.
revealed a gradient in mean activity, highest in the buffer zone .
57 m.
, followed by the core .
94 m.
and utilization zones .
76 m.
However, one-way ANOVA indicated no statistically significant differences among the zones .
= 0.
This lack of statistical significance is largely attributed to high within-group variance, driven by the inclusion of nonactive isolates .
ero inhibitio.
and the limited sample size per zone.
Such intrazone heterogeneity reflects the inherent variability in biosynthetic capacity among endophytic isolates, influenced by genetic conditions, as observed by Annisa et al.
Furthermore, this pattern is exploration studies, such as those by Astrid .
, wherein a significant portion of isolates exhibited weak or no activity, thereby increasing data dispersion.
Despite this statistical dispersion, it is important to note that the mean inhibition zones of the active isolates across all zones consistently fall within the strong activity category .
Ae 20 m.
, with peak values exceeding 20 mm classified as very strong, confirming their biologically meaningful inhibitory In contrast to the uniform response of S.
aureus, the statistical analysis for E.
coli revealed a significant ecological gradient.
Indeed, antimicrobial activity against coli demonstrated a statistically significant effect of ecological zoning (ANOVA, p = 0.
TukeyAos HSD post hoc testing specifically identified a significant difference between the utilization and buffer zones .
= 0.
This differential statistical significance between the two pathogens underscores the impact of bacterial cell wall architecture: while the exposed peptidoglycan layer of Grampositive S.
aureus makes it generally susceptible to a wider range of compounds .
asking zonal difference.
, the complex outer membrane of Gram-negative E.
confers greater intrinsic resistance, resulting in more variable and zonesensitive responses, as highlighted by Sarmira .
Consequently, only the most potent, zone-specific metabolites can overcome this barrier, making Gramnegative pathogens more discriminative indicators of ecological gradients in endophyte bioprospecting studies.
Conclusion This study successfully isolated and characterized 18 endophytic bacterial isolates from the prop and lateral roots of Rhizophora mucronata across three distinct management zones .
ore, buffer, and utilizatio.
in a semi-arid mangrove Bacillus sp.
emerged as the dominant genus .
4%), demonstrating exceptional adaptability to extreme rhizospheric conditions.
Notably.
Bacillus
isolates ZS-04 and ZI-05 exhibited the most potent bioactivity, yielding very strong
(>20
Staphylococcus aureus and strong .
Ae20 m.
inhibition against Escherichia coli.
Statistically, while intra-zone variability masked significant zonal differences for S.
= 0.
, a significant ecological gradient was observed for E.
= .
, indicating that Gram-negative pathogens serve as more sensitive indicators of zonal variations in endophytic metabolite production.
These findings semi-arid endophytes as promising, stress-adapted reservoirs of targeted natural antibacterial However, as the current taxonomic assignments are based solely on phenotypic characteristics, future research must prioritize 16S rRNA gene sequencing for definitive taxonomic validation, coupled with LC-MS/MS-based metabolite profiling to elucidate the specific bioactive compounds responsible for this selective antimicrobial activity.
Acknowledgments The authors gratefully acknowledge Widya Mandira Catholic University for financial support through the 2025 Phase II Research Grant.
We also extend our sincere appreciation to the management of the Laboratory Unit (UPT).
Faculty of Science and Technology UNWIRA, for providing the essential laboratory facilities, reagents, and technical equipment that enabled the successful completion of this study.
References