In vitro antimicrobial activity of the crude extract of the endophytic bacterium Pseudomonas aeruginosa (SS93) isolated from Sapindus saponaria L.

. Endophytes colonize the interior of plant tissues without causing any damage to their hosts. The plant Sapindus saponaria L., popularly known as ‘ sabão-de-soldado ’ , presents a diversified endophytic microbiota and also medicinal properties. Endophytic microorganisms may produce secondary metabolites with different biotechnological properties. The present study aimed to evaluate the in vitro antibacterial and antifungal capacity of the crude extract of secondary metabolites produced by the endophytic bacteria P. aeruginosa SS93 isolated from S. saponaria leaves. The metabolites extract was obtained using the organic solvent ethyl acetate, and the antimicrobial activities were tested against six pathogenic bacteria ( Enterococcus faecalis [ATCC 29212], Pseudomonas aeruginosa [ATCC 27853], Shigella flexneri [ATCC 12022], Salmonella enterica [CCCD a016], Escherichia coli [ATCC 25922], and Staphylococcus aureus [ATCC 25923]), and pathogenic fungi ( Fusarium oxysporum , Glomerella sp., Sphaceloma sp., Fusarium solani , Maniliophtora perniciosa , and Sclerotinia sclerotiorum ), by agar diffusion method. In the antibacterial assay, the best results were obtained against E. faecalis and S. aureus, where the formation of inhibition halos was observed in all tested concentrations, especially at 500 and 700 µg mL -1 . Positive inhibitory activity against phytopathogenic fungi was observed, with the highest inhibition recorded against F. oxysporum (61.1%), followed by Sphaceloma sp. (55.7%), M. perniciosa (35.6%), F. solani (34.4%), and Glomerella sp. (30.4%).

The medicinal properties of certain plants may be related to the metabolites that are produced by endophytic microorganisms (Azevedo et al., 2002).The plant Sapindus saponaria L., belonging to the Sapindaceae family and popularly known as 'sabão-de-soldado', presents a diversified endophytic microbiota and also medicinal properties (Garcia et al., 2012;Ataides et al., 2018;Santos et al., 2019).Some endophytes isolated from S. saponaria leaves have already shown important biological activities such as the production of extracellular enzymes, control of phytopathogens (Santos et al., 2019), and the production of secondary metabolites with antioxidant properties (Polli et al., 2020).
In this work, we evaluated the antibacterial and antifungal capacity of the crude ethyl acetate extract containing secondary metabolites produced by the endophytic bacteria P. aeruginosa SS93 isolated from S. saponaria leaves, using the agar diffusion method.

Microorganisms
The bacterium P. aeruginosa (SS93) isolated as endophyte from healthy leaves of S. saponaria L. ) and the pathogenic fungi (Fusarium oxysporum, Glomerella sp., Sphaceloma sp., Fusarium solani, Maniliophtora perniciosa, and Sclerotinia sclerotiorum) also belong to the CMEA.The pathogenic bacteria were also grown in TSB medium (Tryptic Soy Broth) (24 hours at 37ºC), while the fungi were cultured on Potato Dextrose Agar (PDA) at 28ºC for 7 days.

Obtaining crude ethyl acetate extract
A total of 4 liters of culture medium was distributed into 16 Erlenmeyer flasks, each with a capacity of 500 ml, containing 250 ml of the medium in each flask.An aliquot of 500 µL of the adjusted endophytic bacterial solution at 0.880 (600 nm), measured using a spectrophotometer, was inoculated into Erlenmeyer flasks containing 250 mL of TSB medium (Tryptic Soy Broth).Then, the flasks were incubated at 28°C for 72 hours with 110 rpm orbital shaking.Subsequently, the cultures were centrifuged at 2,600 g for 15 min.The pellet formed was discarded and the supernatant was used for the extraction of the metabolites.
The supernatant was partitioned using the organic solvent ethyl acetate (3 × 70 mL) in a separating funnel.The organic fraction was concentrated under reduced pressure in a rotary evaporator, as described by Polonio et al. (2015).

Antibacterial activity
The pathogenic bacteria were grown for 24 hours in TSB medium and subsequently adjusted to a concentration of 1.5 x 10 8 colony-forming units mL -1 , using the McFarland 0.5 scale.The bacterial suspension (100 μL) was then evenly spread on Petri dishes containing TSA medium (Trypticase Soy Agar), using a Drigalsky's handle.Subsequently, 5 discs of Whatman filter paper No. 4 (Ø 5mm) were inserted in each plate, which were then impregnated with the crude extract of the metabolites previously diluted in Methanol (MeOH) at different concentrations (0, 100, 300, 500, and 700 µg mL -1 ).
For the positive control, we used discs inoculated with 20 µg of TETREX ® tetracycline (Bristol-Myers-Squibb), and the negative control was performed only with 10 µL of MeOH for comparison with the treatments.The plates were incubated at 37ºC for 24 hours.The bactericidal activity was evaluated by the formation of inhibition halos, measuring the distance of the halos (mm) from the edge of the filter paper discs to the edge of the halos.The results were then compared to the controls, consisting of tetracycline and MeOH.The experiment was conducted in triplicate, and the average halo sizes in millimeters for each treatment were subjected to Analysis of Variance.Subsequently, a statistical comparison was performed using the Tukey test with a significance level of p<0.05.The software Sisvar 5.3 was used for these analyses (Ferreira, 2011).

Antifungal activity
The pathogenic fungi were previously grown on PDA for 7 days at 28°C.Subsequently, disks (Ø 6mm) of the mycelium from these colonies were inoculated on fresh PDA plates, and a sterile filter paper disc (Ø 5mm) with 10 µL of the metabolic extract at 700 µg mL -1 was inoculated at the opposite pole, at a distance of 4 cm from each other.The tests were performed in triplicate, along with the positive control containing the commercial fungicide Cernonil WP (IHARA), 700 µg and Frowncide 500 SC (IHARA), with a dilution of 10 -1 (Control 1), and the negative control, only with MeOH (Control 2).
To assess the initial percentage growth inhibition index (Im%), the mycelial growth area (in cm²) of the phytopathogens was measured using ImageJ software v1.46r.The mycelial growth area values of the pathogens in each treatment and control (in triplicate) were used to calculate the Im% using the formula Im% = (1 -MT/MC) x 100, where MT represents the area of each triplicate treatment in cm², and MC represents the average area of the triplicate control in cm² (Oliveira et al., 2020).Statistical analysis was performed by comparing the mean mycelial growth areas of the treatments with the controls using the Skott-Knott test (p>0.05),with the assistance of the statistical software Sisvar v.5.3 (Ferreira, 2011).

Results and discussions
The production of metabolites by microorganisms has long been known and explored (Strobel, 2003;Schulz & Boyle, 2005;Brader et al., 2014;Oliveira et al. 2020).Most of the compounds produced by fungi or bacteria act by inhibiting the growth of other microorganisms.The production of these metabolites may be influenced by biotic and abiotic factors and could be related to the host's physiological situation (Azevedo et al., 2002).
Many bacteria species, including P. aeruginosa, can produce molecules that may be used as control substances for pathogenic fungi and bacteria (Raaijmakers, Vlami, & Souza, 2002;El-Sheshtawy & Doheim, 2014).Cultivated plants are subject to attack by several phytopathogenic microorganisms impairing their final yield.As an alternative to reduce the use of chemical compounds in agriculture, the use of endophytes as biological control agents may contribute to more eco-friendly agricultural practices (Oliveira et al., 2020).
Acta Scientiarum.Biological Sciences, v. 45, e65524, 2023 Pseudomonas aeruginosa produces various secondary metabolites with significant biological activity.Among these metabolites, pyocyanin (Abdelaziz, Kamer, & Al-Monofy, 2023;Kassob & Hummadi, 2023) and aeruginolysin exhibit potent antimicrobial activity against bacteria and fungi.Pyocyanin, a blue-green phenazine pigment, has been shown to inhibit the growth of a wide range of microorganism's species (Abdelaziz et al., 2023).Aeruginolysin, on the other hand, is a pore-forming toxin that can lyse target cells (Andrejko et al., 2019).These metabolites have been studied for their potential use in the development of new antibiotics.According to a study by Solecka, Zajko, Postek, & Rajnisz (2012), these secondary metabolites can be used as a basis for the development of novel antibiotic drugs.
In this work, we observed that the antifungal activity of the crude extract of the endophytic strain against phytopathogens fungi showed positive inhibitory activity against F. oxysporum (61.1%), followed by Sphaceloma sp.(55.7%),M. perniciosa (35.6%),F. solani (34.4%), and Glomerella sp.(30.4%).However, for S. sclerotiorum, the crude extract showed no statistically significant inhibitory activity (Table 2, Figure 2).Ziedan and El-Mohamedy (2008) demonstrated the interaction between a P. fluorescens strain and the phytopathogen F. oxysporum by scanning electron microscopy.As a result, these authors found a deterioration on the cell wall of the pathogen, which might be related to the secondary metabolites produced by the bacterium.This mechanism of action may occur by antagonism.Some species of Pseudomonas can produce metabolites with antifungal activity, especially against Fusarium and Sclerotinia pathogens (Zhou, Zhao, & Dai, 2014).Pseudomonas species may be isolated from different niches and hosts, which suggests great adaptability and a promising source of biologically active secondary metabolites (Bano & Musarrat, 2003;Anand & Kulothungan, 2010;Verhagen, Trotel-Aziz, Jeandet, Baillieul, & Aziz, 2011;Gupta, Panwar, & Jha, 2013;Janek, Łukaszewicz, & Krasowska, 2013;Zhou et al., 2014).Besides, the capacity of endophytes associated with S. saponaria to produce secondary metabolites with biotechnological properties of interest (Polli et al., 2020), reinforce the importance of studies that explore more endophytic isolates from this plant.This approach aims to select strains with potential for application in different areas, such as in the health and agriculture fields, similar to the characteristics found in this work with the endophyte SS93.against methanol, commercial fungicide, and metabolites from SS93 respectively.

Conclusion
Our research findings suggest that the endophytic bacterium P. aeruginosa SS93 has the ability to produce antibacterial and antifungal compounds, making it a promising candidate for the isolation and production of pharmaceutical molecules with antibacterial properties.Specifically, these compounds show efficacy against E. faecalis and S. aureus, making them valuable for the pharmaceutical industry.Furthermore, they exhibit potential in agriculture for controlling phytopathogens such as F. oxysporum and Sphaceloma sp.It is important to continue studying these metabolites, not only to identify the chemical composition of the extract, but also to conduct isolated antimicrobial evaluations.
in the same column do not differ statistically according to the Skott-Knott test (p <0.05).

Figure 1 .
Figure 1.Antimicrobial activity of the crude extract of secondary metabolites from the endophytic strain SS93 (Pseudomonas aeruginosa) against pathogenic bacteria.(A), (B) Enterococcus faecalis against the SS93 metabolite and antibiotic Tetracycline respectively; (C), (D) Salmonella enterica against the SS93 metabolite and antibiotic Tetracycline respectively; (E), (F) Staphylococcus aureus against the SS93 metabolite and antibiotic Tetracycline respectively.

Table 1 .
Activity of the crude extract of secondary metabolites of the endophytic strain SS93 (Pseudomonas aeruginosa) against different pathogenic bacteria.