pH and temperatures optimation activity of crude chitinase of oil palm endophytic bacteria Bacillus cereus which have antagonistic properties on pathogenic fungi Ganoderma boninense

Endophytic bacteria is promised solution to suppress basal stem rot disease caused by fungus Ganoderma boninense. The antifungal activities of selected isolate endophytic bacteria from oil palm plantation in South Kalimantan was studied. Bacillus cereus was tested for its antifungal activities of crude chitinase and secondary metabolites against the growth of Ganoderma boninense mycelium with dual cultured method. The results were showed that chitinase had the largest inhibition zone (18.5%) against growth inhibition of fungi Ganoderma boninense’s mycelium. A series of optimation assays of chitinase activity were conducted at pH 3 to 8 with 0.5 interval, and temperature at 30 to 70°C, with 5°C interval. Specific chitinase activities was measured using the colorimetric method. The highest specific chitinase activity significantly at 1.66393±0.04807 mU/μg (95% confidence level) at pH 5.5 and temperature 45°C.


Introduction
Indonesia as the country with the largest palm oil plantations in the world, faced some problems.One of them is stem rot disease caused by pathogenic fungus Ganoderma boninense.The case studies from a few numbers of disease, that caused by pathogenic fungi of Ganoderma species in many natural palmoil environments, were showed that the disease was possibly can be exterminated by a biocontrol agent.Therefore, many studies have been conducted to control Ganoderma using biocontrol agents as the main objective [1].The study had been held by [2] indicated that Penicillium can be used to compete against Ganoderma and it was reduced the chance of Ganoderma colonising the roots of palm oil plants.Also, another promising antagonistic agent have been found such as Trichoderma [3] which was showed an antagonistic activity against in vitro Ganoderma boninense.
There are also endophytic bacteria, which can live in plant tissues of the host plants without showing the symptom's diseases [4,5].Some endophytic bacteria are exhibit an antagonistic property topathogenic fungi that attack their host [6,7].They have been isolated from a variety of hosts, either wildand within agriculture and plantations, such as woody plants [8], bananas [9] and grass [10].Therefore, endophytic bacteria are relatively novel in biocontrol disease of research.[11] in her research was successfully identified endophytic bacteria of Bacillus cereus which has antagonistic properties against Ganoderma boninense in oil palm plants.The studies of bioactive compounds which involves in antagonistic process have not been carried out yet.The large number of endophytic bacterias, from the gram-positive to gram-negative group, will be helping their host to fight 1243 (2023) 012017 IOP Publishing doi:10.1088/1755-1315/1243/1/012017 2 the plant pathogens by means of fungal and antifungal cell wall lysis activity [12].Some endophytic bacterias such as Serratia spp., Burkholderia spp., Pseudomonas spp., Bacillus spp., and Fusarium spp., which were found to induce the resistance systems of plants and showed the biological completeness such as antibiotic activity and lysis [6,7].Research by [13] was pointed that there are the best chitinolytic bacterias have been isolated from the soil successfully and their antagonistic properties to all fungal pathogens have been tested.Chitinolytic bacterias are chitinase-producing bacteria, which has the type of chitin lysis enzyme, a sugar polymer that makes up of the fungal cell wall.
Therefore, this study has been conducted to examine the presence of antifungal chitinase and metabolite activities in selected endophytic bacterias, that have antagonistic properties against the pathogenic fungus Ganoderma boninense.These endophytic bacterias were isolated from oil palm of PT.Agro Menara Rahmat oil palm plantation in South Kalimantan.Data from this study will be useful for further studies about the application of selected antagonistic endophytic isolates.

Material
The material that used in this study were selected endophytic bacterial isolates from palm oil tissue (roots, stems, leaf fronds, and fruit) of PT.Agro Menara Rahmat, South Kalimantan that has been identified, Bacillus cereus.Ganoderma boninense fungi culture from the Mycology Laboratory-PAU, School of Life Science and Technology, Bandung Institute of Technology.

Metabolite and chitinase enzyme productions
Selected bacteria were grown on Nutrient Broth (NB) media.The culture was incubated for 24 hours and shakes at 30qC 125 rpm.Next, 5% of the starter culture was inoculated into Production Medium (MPB), then incubated for 4 days at 30qC and shakes at 150 rpm.After incubation time completed, the culture was centrifuged at 5,000 rpm (2935 x g) at 4qC for 30 minutes until supernatant of extracellular metabolites and deposits was obtain.
The active compound contained in the supernatant had isolated with organic solvent solutions, including ethyl acetate (EtOAc) in a ratio (1:1).Extraction has been done twice.First, 1,000 mL supernatant was extracted with 500 mL EtOAc, after shaked and left for a while, it would be formed two layers, the upper layer of EtOAc solution was taken, and the water solution was extracted once againwith 500 mL EtOAc.Afterward, the extraction would be resulted of 1,000 mL of EtOAc and 1,000mL of water.Furthermore, the EtOAc solution was dried with an evaporator, in order to obtain the dried ofEtOAc precipitate and it was named precipitate (A).
Water layer residue of it was extracted again using saturated n-Butanol (BuOH) (water layer: nbutanol = 2: 1).The extraction also done twice.First, 1,000 mL of water layer was extracted with 250 mL BuOH, after that the BuOH layer had been taken, then water layer was extracted with 250 mL BuOH, after that the BuOH layer had been taken, then water layer was extracted once again with 250 mL of BuOH, so in the end it would produce 500 mL BuOH layer.Next, the solution was dried with an evaporator to get the dry precipitate of BuOH layer and its given name was precipitate (B).The BuOH layer was dried in an evaporator, which BuOH layer was added with aquades (4:1) before it put into the evaporator [14].
The production of crude chitinase enzymes were carried out by prepared colloidal chitin in advance with the method of [15].20 grams of chitin shell (Sigma) were dissolved in 180 mL 10 N HCl, then stirred for 1.5-2 hours at room temperature.Chitin suspension would be formed and it was soaked in 2L distilled water, then left overnight.The precipitate which was formed had been collected and drained.The sediment was washed four times with tap water and 3 times with aquades.At the end of washed, pH measurements were taken.If the pH reaches 5.5-6, the drained chitin colloid could be storedat 4qC.The content of chitin in the suspension could be determined by weighed 10 mL of the chitin suspension that had been dried at 80qC, so a constant weight would be obtained.
Selected isolates were grown in 20 mL liquid chitin medium with 0.2% (w/v) chitin.Growth conditions were included inoculum concentration (starter) 5% (v/v), pH 6.8, temperature 30qC, incubation time 72 hours and had been shake with a speed of 120 rpm.The starter's medium which had been used was the same as the production medium and as much as 1 x 107/mL of bacterias were inoculated into it.The starter was incubated at 30qC for 24 hours.The enzyme was harvested with centrifugation of the cultivation medium at 4,000 rpm (1,878 x g), for 20 minutes at 4qC.The supernatantresulted was a crude enzyme and for further testing it could be stored at 0qC.

Enzymatic activity of cell-free culture filtrate
A qualitative assay of the crude chitinase activity was carried out to confirm chitinase production from selected isolates using the cell-free culture filtration diffusion method in the chitin-water agar medium as described [18].A total of 100 L cell-free culture filtrate was dropped in the holes of 8 mm diameter on agar medium which contained 0.2% chitin, and incubated for 4 days at room temperature.Chitinase activity was characterized with the presence of clear zone that formed around the holes in the chitinwater agar medium.Sterile aquades was used as controls.

The antifungal activity of crude chitinase extract and metabolites
Crude extracts of metabolites and cell-free culture filtrates were subjected to a dual culture test with grown Ganoderma boninense in the middle of a Petri dish, 7 mm in diameter, on a PDA medium.At the final phase of mycelium growth, a 5 mm sterile disk paper was placed, which already dropped with100 mL metabolite extract and sterile distilled water as the control.The culture had been grown for 7 days, then the growth of mycelium was observed.

pH and temperature optimation of crude chitinase activity
Chitinase enzyme optimization in this experiment was included the optimum temperature and pH characterization of the enzyme.Each character of parameters was carried out through the chitinase enzyme activity test.The temperature variation of the test was carried out at 30-70qC at 5qC intervals in the water bath, while the variation of the enzyme's pH was tested at pH 3-8, with an interval of 0.5.Preparation of a pH solution for low pH (3-5.5) was used 50 mM citrate buffer, whereas for high pH (6-8) was used 50 mM phosphate buffer.
Total of 1 mL crude enzymes from isolates and 1 mL of chitin substrate [1% colloidal chitin (w/v) in a buffer solution with a variation of pH 3-8 and incubated at temperature variations (30-70qC) for 60 minutes.The reaction was stopped by heated the mixture in boiled water for 15 minutes and then centrifuged at 3,000 rpm (1057 x g) for 20 minutes.Chitinase activity was calculated based on the difference between the levels of N-acetyl glucosamine (GlcNAc) which released in the treatment with the GlcNAc level contained in the crude enzymes that was not treated (control).The GlcNAc produced would be analyzed by colorimetry utilised a spectrophotometer with the Reissig method of Jeuniaux [19].One unit of chitinase activity has been defined by the amount of enzyme which freeing of 1 mmol GlcNAc / hour.Meanwhile, the specific activity of chitinase is 1 U/μg protein.Determination amount of dissolved protein has been supporting by Lowry method [20].Specific Chitinase activity is determined using equation (1):

Metabolite and chitinase enzyme productions
Crude chitinase enzyme production had been done used the colloidal chitin substrate in a Minimal Medium from selected isolates [16,17].This cell-free culture filtrate was confirmed qualitatively with the chitinolytic enzyme content used the diffusion method on the chitin water agar medium [18].A clear zone was observed in the treatment hole (Figure 1).
The clear zone showed in the treatment hole was the effect of colloidal chitin dissolution due to the activity of chitinolytic enzymes [21] in the filtrate which secreted on the culture medium.Research [22] pointed that liquid culture containing chitin as a carbon source would be resulting the maximum production of metabolites which degrade chitin.The study also indicated that bacterial growth was relatively good on chitin-containing mediums, it has been suspected due to its ability which is the degradation of chitin polymer complexes for its growth.
The crude enzymes found in cell-free culture filtrate from chitin-minimal colloid media are called chitinolytic enzymes, as described in the research of [23].He showed that the chitinolytic enzyme has been produced by Aspergillus sp. in a chitin-minimal colloidal medium could be hydrolyze β-chitin, αchitin, and colloidal chitin.
Research by [24] indicated that the extracellular protein of Chromobacterium violaceum which extracted from the minimal liquid medium that added the colloidal chitin, contained chitinase with cutting the endo-mode chitin polymer and chitobiosidase (exokitinase).Chitinase produces GlcNAc monomers, while chitobiosidase produces dimeric (GlcNAc) 2. [25] also showed that the enzymechitobiosidase was present in the cell-free culture of B. cereus in the minimal colloidal chitin medium.
Research by [24] also exhibited that culture in a minimal medium which added glucose, sucrose or LB as a carbon source did not indicate the presence of chitinolytic enzymes.This research showed that colloidal chitin or a simple chitin derivative product was needed to produce chitinase.In previous studies, colloidal chitin was tested with partial hydrolysis techniques used 10N HCl and showed that it could induce various types of chitinolytic enzymes, including N-acetylglucosaminidase, endokitinase, and chitobiosidase in Aeromonas caviae species [26], E. agglomerants [27], and B. cereus [28] and in the Trichoderma harzianum fungi species [29].The three chitinolytic enzymes are including in the classification of the chitinase enzyme.
In addition to the colloidal chitin substrate, the factors that able optimize chitinase production in Trichoderma hazarnum as shown by [29], including incubation time, incubation temperature, substrate concentration, inoculum size, medium pH, type of chitin substrate, and nitrogen sources.Maximum chitinase production in a medium containing chitin has also been reported in the study of Bhushan [30].

Antifungal activity test of chitinolytic enzymes & organic extract
Crude extract of chitinase and secondary metabolites with a concentration of 0.3 g/mL, then dropped to 100 mL of sterile disk paper, and planted on the edge of a petri dish in PDA culture.Ganoderma boninense was placed in the middle of a PDA culture using a 7 mm paper hole that was grown for 7 days, to observe the antifungal activity of the extract against mycelium growth.The results showed that there were no inhibitory zones in the control and metabolite extracts, inhibitory zones were observed in crude chitinolytic enzyme extracts (Figure 2), with inhibition zones of 18.5% (Table 1).Crude chitinase enzyme extracted in this study has antifungal activity.It was thought that its antifungal activity was derived from the enzyme chitinase and other chitin degrading enzymes which be found in the filtrate from the culture medium of the minimal chitin.The chitinase enzyme will be degrades the chitin polymers which contain in the cell walls of Ganoderma boninense fungi.The chitinase enzyme has been produced by inducing of colloidal chitin substrate as the only carbon source in the chitinase production medium.
[31] research showed that chitin supplementation able to increase the biocontrol of pathogenic fungi that cause rot disease by chitinolytic bacteria, whereas the application of bacterial cells as such or merely the application of chitin does not show any effect on disease levels.Chitin supplementation hasbeen needed to induce extracellular chitinase production by B. cereus CRS 7 which can degrade the pathogenic cell wall material and inhibit its growth.Control disease of partially pure chitinase in form of prophylactic spray B.cereus CRS 7 bacteria is supporting the main action of chitinolysis in improving The similar results in the study of Harighi et al., [32] showed the existence of inhibitory zones in the growth of R.solani around the hole which contained the chitinase enzyme Chi42 in the PDA medium, whereas in other holes that contained sodium acetate buffer without enzymes did not indicate the presence of inhibitory zones.It was suspected that the enzyme chitinase plays a role in the cell wall lysis of R.solani.It also has been supported by the further research on the effects of 60 units of pure enzymes which lysis the cell walls of R.solani fungi that has been incubated for two days, then observed under the microscope.From this research, it has been be observed that crude chitinase extract able to inhibit the growth of Ganoderma boninense mycelium around the hole.
Other studies also mentioned the presence of other hydrolytic enzymes which produced from medium contained colloidal chitin as a carbon source.Research by [33] on Paenibacillus chitinolitycusJK2 showed the activities of cellulase and xylanase on medium contained chitin as a carbon source, whereas the cellulase and xylanase activities were not shown on medium which added specific substrates, such as CMC and xylan.It was speculated that the production of xylanase and selualse wererelated to chitinase production and the presence of chitin in the medium.This assumption also issued the possibility of regulating these enzyme genes as a group (cluster).[33] expressed that their hypothesis must be investigated furthermore with purification and characterization of enzymes.It is known that cellulase and xylanase are the hydrolysing enzymes of cellulose and hemicellulose polysaccharides, which are the materials containing in plant cell walls [34,35].
Meanwhile, the paper disc that was dropped of metabolites did not show any inhibitory zone on the growth of mycelium Ganoderma boninense (Figure 2).It was presumed that the extracted metabolitehad not contain the antibiotic agent to inhibit the growth of the Ganoderma boninense mycelium.Other studies have shown the antifungal activity of 2,4-diethylfloroglusinol extracted from the organic solvent ethyl acetate in bacteria has been isolated from Salamander skin [36].Van Loon & Bakker [37] in their research said that the endophytic bacteria have ability to antagonize pathogens by means of competition, antibiotic production, or secretion of lysis enzymes, or its combinations.The selected isolates in this study were appearently to tend producing secretion of their lysine enzyme than the production of antibiotics in the antagonistic nature of Ganoderma boninense pathogenic fungi.
It is also suspected that the bacteria secreted the sort of elicitor molecule which useful in antagonistic properties indirectly.The elicitor will be activated if it is in a plant cell (in situ) by induction.Various species of Bacillus strains can induce the plant diseases resistance and then following of root colonization [38].Bacillus mechanisms to inhibit the growth hormones such as auxin production [39], increase phosphorus absorption [39], biocontrol ability [40], and induction of resistance systems (ISR) [41].

pH and temperature optimization of chitinase activity
Generally, chitinase activity was appeared to increase proportionally at 30qC-45qC and then decrease at 50qC-70qC (Figure 3).The temperature ranges for chitinase activity in this study were quite broad, from 40qC-60qC.Chitinase activity was showed similar pattern as pH ranges under each temperature condition.Chitinase activity was increased proportionally from pH 3 to pH 5.5 and decreased at pH 6, then continued with proportional increase until pH 8. Specific chitinase activity was highest at pH 5.5 and 45qC with two replications.
Research of [42] was given evidenced also, that Chi 46 chitinase activity from Chaetomium globosum yeast had the highest value at 45qC and stable at 35-65qC.Crude chitinase from Paenibacillus chitinolyticus JK2 also indicated that the highest activity was occurred at 37qC and had a relative activity above 90% in the temperature range 40-60qC31.The Bacillus licheniformis had a stable chitinase activity in temperature range of 50-70qC [43].
In case the relative chitinase activity was measured against the highest activity point at 45qC and pH 5.5, it could be seen that at pH 5.5 the chitinase relative activity pattern was started to increase from 30qC-45qC, then decreases at temperature of 55qC-70qC.Relative activity at 45qC had reached 100%.The relative activity of chitinase had valued above 60% at the temperatures between 40-60qC (Figure 4).These results were similar to the studies of [33] which showed that the highest relative activity of crude chitinase from Paenibacillus chitinolytics JK2 was reached at 37qC, and approached 90% at 50qC.Specific chitinase activity toward pH range treatment had relatively similar pattern in each temperature condition, which was proportionally increased from pH 3 to pH 5.5.Then decreased precisely at pH 6, followed with increased proportionally until pH 8 (Figure 5).The highest specific chitinase activity was at pH 5.5 at 45qC which was around 1.66393 mU/mg (Figure 4), then decreased at pH 6, continued of proportional increased until pH 8 at 1.21272 mU/mg, that was the second highest point.Therefore, specific chitinase activity experiment was carried out at 45qC from pH 5.5 to pH 10 and made with three repetitions, then observed the pH which the chitinase activity began to declined.In Figure 5 it could be seen that the highest chitinase activity was at pH 5.5, then decreased at pH 6 and increased proportionally toward pH 8, then decreases again until pH 10.
Chitinase activity had two optimum points, that was at pH 5.5 and pH 8. Similar results were shown in the research of [33] signified that the crude chitinase activity of Paenibacullus chitinolyticus JK2 had a double optimum point at pH 5 and 7, which showed relative activity reaching 90% at pH 4 and 65% at pH 8.
The existence of two optimum points on chitinase activity in this study was suggested, that the crude extracts from cell-free culture filtrate derived from minimal medium contained chitin, had produced more than one type of chitinolytic enzyme, so the chitinase activity had generated two optimum points.This hypothesis needs to be proven by purifying the enzyme and its characterizing.
The similar case was shown in research of [44] which exhibited the chitinase from B. subtilis had two optimum pHs, at pH 6 and pH 11, it was meant to indicated the presence of two isoforms.Research [45] showed that three types of pure chitinase (ChiA, ChiB, and ChiC1 from Serratia marcescens appeared to have optimum activity in different pH ranges.ChiB and ChiC1 represented thehigher activity at lower pH than high pH, whereas ChiA maintained its activity higher than 80% from its maximum point between pH 4 and 11.Many bacterias such as Aeromonas sp., Pseudomonas aeruginosa K-187, and S. griseus HUT 6037 able to synthesize several different types of chitinase [46,47,48].

Conclusion
Antifungal properties were observed in the crude extract of the enzyme chitinase.The inhibition zone had been produced by the crude chitinase enzyme filtrate was 18.5%, while the inhibition zone which produced of secondary metabolite filtrate was 0%.Specific chitinase activity of selected isolate culture filtrate had the highest value at pH 5.5 and temperature 45qC, 1.66393 ± 0.04807 mU/mg.

Figure 1 .
Figure 1.Chitin Dissolution due to Chitinolytic Activity of Bacillus cereus Isolates on Chitin Agar Medium.(A) Treatment and (B) Control.

Figure 2 .
Figure 2. Antifungal Activity Tests of Crude Metabolite Extracts on Growth of Mycelium Ganoderma boninense

Figure 4 .
Figure 4. Relative Activity of Chitinase's to temperature at pH 5.5.
Statistical analysis to discover the specific chitinase activity in crude chitinase extract would be tested by univariate analysis (ANOVA) in a completely randomized design (CRD) at 95% confidence level.If there was obvious difference, then proceed with the Post Hoc Test DMRT (Duncan's Multiple Range Test) at 95% confidence level.Statistical tests were carried out using the Statistical Product and Service Solution (SPSS) statistical program for Window release 16.

Table 1 .
Inhibitory Zones of Metabolite Crude Extract (*) Crude extract of chitinolytic enzymes (cell-free culture filtrate from chitin minimal-colloid medium)