Characterization of chitinolytic bacteria isolated from Ipomea pes caprae

Chitin has substantial benefits in several sectors including agriculture. In the plant protection field, chitin can promote the growth of certain bacteria which has possibly controlled the spread of fungal pathogens. Thus, chitinolytic bacteria producing chitinase enzymes can inhibit the growth of fungi. The purpose of this study was to examine the morphological and physiological characteristics of chitinolytic bacteria which collected from Ipomea pes caprae in Makassar and its ability to produce chitinolytic enzymes on medial culture. The method started to grow the isolates on the nutrient agar which were added to colloidal chitin. Furthermore, the bacteria were identified by its color, form, elevation and edge as well as tested its physiological characteristics with KOH 3% test and catalase test. In addition, bacterial chitinolytic index was carried out by using the disc diffusion method. The results showed that each isolate had yellowish white and grayish white colors; morphological characters: round and irregular colony shapes, colony edges smooth and choppy as well as umbonature and convex elevations. The physiological test results indicate that all isolates had a positive response in the catalase test, one of the isolates reacted positively to the gram test and the others reacted negatively to the test. Each isolate had chitinolytic activity with indices of 0.10, 0.09, 0.70, and 0.11.


Introduction
The primary component of fungal cell walls is chitin [1].Apparently, chitinolytic bacteria can create chitinase enzymes that impede the growth of fungus.Multiple forms of rhizobacteria, such as Bacillus subtilis, which not only promote plant growth but also shield plants from Fusarium oxysporum infection [2].Several species, such as Streptomyces, Serratia, Vibrio, and Bacillus, contain the majority of chitinolytic activity [3].
The widespread geographical distribution of the Ipomea pes caprae in tropical coastal areas represents one of the greatest opportunities for coastal communities.This plant is commonly found along the coast, particularly on sand tongues, and plays a significant role in the coastal environment [4].I. pes caprae has the ability to grow in sand or coastal locations, indicating that it can survive in minimal nutritional circumstances as long as there is sufficient sunlight.The presence of microorganisms in the rhizosphere of plants may also influence the growth of I. pes caprae.The activity of bacteria isolated from the roots of I. pes caprae in inhibiting the growth of L. pseudotheobromae have reported in the previous study [5].
Lasiodiplodia pseudotheobromae is a pathogen that affects a wide range of woody perennial crops and ornamental plants, causing various diseases such as root rot, damping-off, leaf spots, twig blight, cankers, stem-end rot, gummosis, branch dieback, and pre-and post-harvest fruit rots [6].The pathogen has a relatively broad range of hosts among cultivated and wild woody plant species [7].Previous studies have demonstrated the effectiveness of chitinolytic bacteria in inhibiting the growth of L. pseudotheobromae in the dual culture method [5].The current research aims to characterize chitinolytic bacteria morphologically and physiologically, as well as to investigate the potential of bacterial substrates to inhibit the growth of L. pseudotheobromae.

Preparation of colloidal chitin
Using a modified method [8], colloidal chitin was produced by combining 10 g of chitin with 200 ml of concentrated HC1, stirring for 2 hours, and then incubating at 4°C for 24 hours.The incubated solution was subsequently filtered with glass wool, mixed with cold sterile distilled water, and the pH was adjusted to 7.0 with NaOH.20 minutes were spent centrifuging the solution at 8,000 rpm to separate the precipitate.After rinsing the precipitate with sterile distilled water, it was centrifuged at 8000 rpm for an additional 20 minutes.The precipitate is stored in the refrigerator in the form of colloidal chitin.

Isolation of chitinolytic bacteria
Chitinolytic bacteria were isolated from I. pes caprae roots collected from the coast of Tanjung Bayang, Makassar.5 g of I. pes caprae root was weighed and diluted with 10 ml of sterile distilled water.A mortar is used to crush a mixture of distilled water and I. pes caprae roots, which is then allowed to stand for a few minutes or 2 hours at room temperature [9].The bacterial suspension was made into a series of dilutions of 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , and 10 -6 , with each dilution containing 90 ml of distilled water [10].Drigalski was used to spread 500 µl of 1 ml of suspension from each dilution series onto CCA medium in a petri plate.72 hours of incubation were carried out at 33˚C.Chitinolytic isolates were distinguished by the presence of a clear zone (halo) surrounding the colony [11].

Morphological and physiological characterization
The bacteria that were recovered as a result of the purification process were studied in order to identify their morphological and physiological properties.As part of the morphological observations, the shape of the bacterial colonies, their color, the heights of the colonies, and the edges of the colonies were observed [12].The physiological state of chitinolytic bacteria is characterized through a series of phases, including the 3% KOH test and the Catalase Test.
2.3.1.3% KOH test.3% KOH was used to determine the gram response of the bacterial isolates that were tested to the test.The test is conducted by placing 1-2 ose of the analyzed bacterial isolates on a glass slide and then applying 1-2 drops of a 3% solution of potassium hydroxide on the glass slide.The gram reaction was considered negative if the mixture had a slimy consistency, whereas a positive result was recorded if the mixture did not have a slimy consistency [12].

Catalase Test.
For the catalase test, H 2 O 2 solution was applied.The test is conducted by putting 1-2 ose of the tested bacterial isolates on a glass slide, followed by 1-2 drops of H2O2 solution.If air bubbles are produced, the catalase reaction is regarded as positive; if air bubbles are not created, the reaction is deemed negative.

Chitinolytic activity test
Disc diffusion with three replications was utilized to select bacteria.One bacterial culture loop was produced in liquid medium (1% colloidal chitin) and incubated at 180 rpm and 30 °C for 24 hours.Using sterile tweezers, liquid cultures of each bacterial isolate were inoculated onto sterile paper discs (d = 9 mm, Whattman No.1) and placed on agar media (1% colloidal chitin).The chitinolytic index was then calculated by measuring the clear zone and bacterial colonies after eight days of incubation at 30 °C.
For the chitinolytic index, the clear zone created around the colony indicating chitinolytic activity was measured.The following equation was used to determine the chitinolytic index (IK) value [13]: where: ∆Y : The magnitude of the chitinolytic index Y1 : Colony diameter Y2 : The diameter of the clear zone is added to the diameter of the colony

Antifungal activity test of chitinolytic bacterial filtrate
The filtrate culture production method adheres to the modified method suggested [14].One ose of inoculum from each inhibited chitinolytic bacterium was added to 100 mL of Nutrient Broth (NB) medium and incubated at room temperature for 24 hours.The medium suspension was centrifuged for 20 minutes at 3800 rpm to extract the supernatant filtrate.The process output was filtered using a syringe filter with a 0.45 mm membrane pore size.The culture filtrate was then utilized to combine the medium.The remaining filtrate cultures were refrigerated for storage.At 50℃, 5 mL, 10 mL, and 15 mL of endophytic bacterial filtrate cultures were combined with 95, 90, and 85 mL of sterile PDA medium (5%, 10%, and 15% dilutions, respectively).A 0.5 mm section of the pathogenic fungus was inserted in the middle of the medium.Three times were performed for each concentration.As a control, L. pseudotheobromae was cultivated on medium without the addition of culture filtrate.The samples were then cultured at room temperature for two days (the control filled the petri dish) and 12 hours of observations were made [14].The percentage of inhibition of the bacterial filtrate is calculated based on the following equation.
where: d1 = colony diameter of the control pathogenic fungus (cm) d2 = colony diameter of treated pathogenic fungus (cm) Using a two-factor factorial design with three replications, the experiment was examined.SPSS version 23 was used to analyze the acquired data.Duncan's test, with a 5% significance level, was then applied to treatments that differed significantly.The outcomes of the antagonist bacteria and test fungi were then inspected and recorded under a microscope.

Characterization of chitinolytic bacteria
The bacteria were streaked on NA medium using the quadrant scratch method and then incubated for three days.After the bacteria were observed morphologically, physiological tests were then carried out.Following are the results of the morphological and physiological characterization of chitinolytic bacteria.Furthermore, three of the four examined isolates were gram-negative, while the other isolate was gram-positive.

Chitinolytic bacteria activity
Chitinolytic activity was indicated by the formation of a hydrolysis zone or clear zone around the bacterial colonies in medium containing colloidal chitin at 12 days after incubation (days).Figure 1 shows that all isolates have chitinolytic activity.The isolates coded IPaR1 and IPaR4 after being infiltrated by bacteria produced large colonies compared to the isolates coded IpA2 and IpA3.The IPpA3 Isolate Code can produce a clear zone after the 12th day of observation.
The highest average chitinolytic index was found in the IPaR2 isolate code, where the average index was 0.70, while the lowest chitinolytic index was found in the IPaR4 isolate code, which was 0.09.The average observation can be observed in table 3 below.

Chitinolytic bacteria filtrate inhibitory activity
The inhibition activity of bacteria was observed and the diameter was measured.Observations were made every 12 hours until the control fungus colonies filled the petri dish.The results of the diameter of the fungus colony can be seen in table 4. From the data above, at 12 hours of observation the IPaR1 isolate code was not significantly different from the concentration treatment, while the other isolate codes were significantly different from the concentration.The highest and lowest diameters in each observation were found in the IPpA2 isolate code with concentrations of 5% and 15%.The development of the diameter of the L. pseudotheobromae fungus colony was relatively fast where during ± 48 hours of observation, the fungus colony that served as a control had filled the petri dish.

General discussion
The presence of chitinolytic bacteria was shown by the formation of a clear zone in a media containing colloidal chitin-modified chitin.In the process of extracting chitinolytic bacteria for use as the primary component in artificial media mixtures, colloidal chitin is widely utilized by researchers.Chitinolytic bacteria are capable of degrading chitin since they produce chitinase enzymes [11].Colloidal chitin was found to be an excellent substrate for assessing chitinase activity.Chitinase-producing bacteria carry out extracellular hydrolysis of substrates, meaning that enzymes are made within the cell, but are discharged (secreted) onto the growing medium (substrate), hence to obtain chitinase-producing microbes, an enzyme-inducing substrate is required [15].
Chitinase's physiological function is dependent on its source.Generally, chitinase is stimulated in plants by stress factors such as chitin-containing pathogen infection.Chitinase is known to play a role in the germination of spores, the formation of hypha and their branching, and the development of 1230 (2023) 012105 IOP Publishing doi:10.1088/1755-1315/1230/1/0121056 mycelium in fungus and other organisms containing chitin in their cell walls or other structures [16,17].Fungal cell walls often contain chitin, glucans, mannans, and glycoproteins [18].
Chitinolytic bacteria may be gram positive or gram negative.The results of the study demonstrated that the gram-positive isolate IPpA3 and the gram-negative isolates were both capable of hydrolyzing colloidal chitin substrates in medium, as indicated by the existence of a clear zone around the bacterial colonies.Chitinolytic microorganisms can be selected for existence by degrading chitin agar media, which can be observed by clear zones surrounding bacterial colonies [15].B. cereus [19], B. circulans [20], Serratia marcescens [21], and various species of bacteria from the genera Artrobacter, Bacillus, Cedecea, Comamonas, Pantoea, and Pseudomonas have been reported to produce chitinase enzymes [22].
In experiments utilising the dual culture approach, the antagonism between bacteria and fungus on PDA media did not demonstrate important impact.This may be caused to the high glucose content of the medium, which is unfavorable for bacterial growth.Chasanah et al. [23] conducted an experiment to determine the effect of a glucose-containing medium, which revealed that the test bacteria did not develop chitinolytic enzymes.The most efficient carbon source for bacteria is glucose, hence isolates grown on these substrates utilize glucose for growth and metabolism.This leads to the creation of nonreduced carbohydrate-degrading enzymes, such as chitinolytic enzymes [24].Gluconeogenesis is hindered by catabolite suppression when glucose or other carbohydrate carbon sources are available.Sugar composed of various monomers is exposed to acid or enzyme activity, simple monomers are released into the medium and then utilized for physiological purposes [25].
The dual culture antagonism between chitinolytic bacteria and Fusarium oxysporum that continued to develop inhibited the growth of Fusarium oxysporum mold due to the presence of the chitinase enzyme, which plays a role in degrading chitin in fungi [11].The chitinase enzyme is created inductively, meaning that it will only be made if an inducer containing chitin is available.The result of chitin degradation, in the form of the molecule N acetyl D glucosamine, is then used as a source of energy for bacteria, allowing them to multiply more rapidly and cover the first generated clean zone.
The mechanism of antagonism in the four bacterial isolates acted most likely as highly active mycoparasites.Mycoparasitic consists of four stages: chemotropic growth in response to a chemical stimulus, recognition between chitinolytic bacterial strains and specific host plant pathogens, attachment and entanglement of bacteria on the target pathogen hypha, and finally, degradation of the pathogen's cell wall [17].
The interaction between chitinolytic bacteria and chitin cell-walled fungi is advantageous for the bacteria but damaging for the fungi.Bacteria produce bioactive chemicals that can cause structural harm to fungi.In the presence of hydrolytic enzymes, such as chitinase in chitinolytic bacteria, chitin, a component of fungal cell walls, can be degraded.Chitin in the cell wall of mycelium is destroyed by chitinolytic bacteria, and the degradation products are used as a source of nutrition for bacterial development.The fungus' growth is hindered because its mycelium cannot form properly [11].
According to the research, the isolated bacteria contain bioactive chemicals that can inhibit the growth of fungi.This is seen in micrographs of fungi grown on a substrate containing bacterial filtrate.The hypha of the fungus appears inflated in this image.In general, antifungal chemicals produced by bacteria result in abnormal growth of hypha (malformations), shown by swelling and shortening of the hypha, which prevents the hypha from developing normally [22].
The hypha of the L. pseudotheobromae exhibit aberrant expansion or proliferation.This is because the antibiosis activity of chitinolytic bacteria limits fungal growth.The pathogenic fungus fusarium in which the fusarium hyphae swelled because antibiotic compounds produced by bacteria could enter pathogenic cells and cause protoplasmic dissolution [22].In addition, lysis was observed in fungal hypha, which was caused by chitinolytic bacteria releasing chitinase enzymes that can degrade the cell walls of pathogenic fungus [26].
Chitinolytic bacteria that produce chitinase are beneficial in waste management, particularly waste containing chitin [13], as biocontrol agents and for medical and industrial purposes [17].Chitinolytic bacteria can also be utilized as biological control against insect pests [3].In agriculture, chitinolytic 1230 (2023) 012105 IOP Publishing doi:10.1088/1755-1315/1230/1/0121057 bacteria are used as antagonistic agents against pathogenic fungus.Chitinolytic bacteria have a powerful antagonistic effect on pathogenic fungus with hyperparasitism mechanisms and their antibiotics.Certain bacterial chitinolytic enzymes are poisonous to pathogenic fungi that cause disease in cultivated plants, but not to other microbes in soil and host plants [17,27].

Figure 1 .
Figure 1.Activity of chitinolytic bacterial isolates.Arrows left and right indicate clear zones of chitinase enzyme activity produced by bacteria.

Table 3 .
Bacterial chitinolytic index after 12 days of incubation.The highest chitinolytic index was found in the IPpA2 code and was significantly different from the other isolates.The following is a comparison of the significance of the percentage of the chitinolytic index based on Duncan's advanced test with a 95% confidence level.1230 (2023) 012105

Table 4 .
Observation of the diameter of the fungus colonies in the bacterial filtrate inhibition test.
Note: numbers followed by the same letter are not significantly different according to Duncan's test at the 5% level.The letters (p,q,r) compare the concentration to the bacteria.