Isolation of Chitinolytic Bacteria as Biocontrol Agent for Pathogenic Fungi on Cocoa Fruit in Sumbermanjing District, Malang
Cahyaning Sulistyantini
*, Irfan Mustafa, Yoga Dwi Jatmiko
Department of Biology, Faculty of Mathematics and Natural Sciences, University of Brawijaya, Malang, Indonesia Abstract
The productivity of cocoa plants in Sumbermanjing District is lower than in other parts of Malang Regency. Diseases like pathogenic fungi are to blame for the low output of cocoa plants. This research aimed to find chitinolytic bacteria that were superior at degrading chitin and limiting the growth of pathogenic fungi. These bacteria were isolated from cocoa garden soil and shrimp shell dumps. The bacteria from soil samples taken from the shrimp shell dump in Ujungpangkah District and the cocoa gardens in Sumbermanjing District were cultivated on an agar medium supplemented with colloidal chitin. The pathogenic fungi were discovered from decaying cocoa pods from Sumbermanjing District cocoa plantations. An antagonist test was used to gauge the inhibitory power, and a semi-quantitative test was used to measure the chitinolytic index. The similarity of 16S rDNA sequences allowed for the identification of superior bacterial isolates. The T2S1 isolates of chitinolytic bacteria had the highest levels of pathogenic fungi inhibition (25.14%, 20.85%, and 25.24%). With a 16S rDNA sequence similarity of 99.77%, Bacillus subtilis C10 was identified as the bacterium T2S1.
Keywords: Bacillus subtilis, Pathogenetic fungi, Cocoa, Chitinase, 16s-rDNA.
INTRODUCTION1
One of the plantation products, particularly in rural areas, that has a significant economic significance in boosting the local economy is cocoa. The government began promoting the cocoa plant as one of the primary commodities in 1980 [1]. After Ghana and the Republic of Côte d'Ivoire, Indonesia was the third largest cocoa producer. There was 1,774,303.97 ha of cocoa plantations in Indonesia in 2014 [2]. Around 3,883 acres of cocoa plantations are located in Malang Regency, and 3,053 tons of cocoa are produced annually. A total of 365 tons of cocoa are built on 361 acres of plantations in the Sumbermanjing District. Comparing Malang Regency to other locations, cocoa production is still lower [3]. Diseases brought on by fungi are why cocoa plants produce so little. Phytophthora megakarya is one of the fungi that can cause fruit rot in cocoa plants [4].
Chitinolytic bacteria is one of the biocontrols which can be applied to eliminate the pathogenic fungi of cocoa plants. Chitinolytic bacteria can break down and degrade the chitin that makes up the fungal cell wall. These bacteria can inhibit the growth of pathogenic fungi in plants.
Chitinolytic bacteria have strong antagonistic activity against pathogenic fungi. [5]. Pathogenic fungi on black pod rot of cocoa called Phytophthora palmivora mycelia grew more
*Correspondence Address:
Cahyaning Sulistyantini
E-mail : [email protected]
Address : Dept. Biology, University of Brawijaya, Veteran Malang, 65145
slowly due to the presence of the bacteria Bacillus subtilis. The inhibition was 37.73% - 100%. It convinced that they were potentially beneficial microorganisms for future sustainable crop disease management strategies [6].
In Indonesia, utilizing biological control agents using chitinolytic bacteria that can prevent the formation of pathogenic fungi on cocoa trees is an interesting approach since it is considered one of the safest strategies. This research aimed to find chitinolytic bacteria that were superior at degrading chitin and preventing the formation of pathogenic fungi as a biocontrol alternative to chemical fungicides. These bacteria were obtained from soil from cocoa gardens and dirt from shrimp shell dumps.
MATERIAL AND METHODS Soil Sampling
Soil samples were obtained from Ringin Kembar Village, Sumbermanjing District, Malang Regency, and from a shrimp shell waste disposal site in Sidorejo Village, Ujungpangkah District, Gresik Regency. Three selected plants with coordinate locations in Sumbermanjing District, plant 1: S”08°16’14.3” E”112°43’.0”; plant 2:
S”08°16’15.0” E”112°43’42.9” and plant 3:
S”08°16’14.5” E”112°43’43.2”. Three selected points with coordinate locations in Ujungpangkah District, location 1: S”06°54’13.0”
E”112°29’23.8”; location 2: S”06°54’15.8”
E”112°29’26.5” and location 3: S”06°54’14.9”
E”112°29’27.2”. Next, for each location, soil samples were taken from three points.
Pathogenic fungi were obtained from rotting
cocoa fruits from cacao plants in Ringin Kembar Village, Sumbermanjing District, Malang Regency.
Production of Colloidal Chitin
Chitin from PT. Surindo Biotech was used in this process. A 200 mL of strong HCl (37%
concentration) was used to dissolve 20 g of chitin (shrimp shell flakes). The chitin flakes were then agitated for two hours at 30°C and 700 rpm until they had dissolved in HCl and formed a thick, creamy colloid. Once the colloid and ethanol had dissolved and become white with a thick texture, 1000 ml of 100% ethanol was added and swirled at 1000 rpm, 30°C. The colloid's pH was then returned to neutral (6.5–7) using 1 M NaOH, and centrifugation was carried out for 10 minutes at 8000 rpm, 30°C. The supernatant was discarded, and the pellet was dried in an oven at 50°C for 48 – 72 hours to form a powder [7].
Chitinolytic Bacteria Isolation
Following serial dilutions of 25 g soil sample in 225 mL of 0.85% NaCl solution from 10-2 to 10-8. A petri dish containing chitin agar medium was inoculated with 100 µL of the sample from each dilution, and the dish was cultured for seven days at room temperature. The medium contained (g.L-1): 15 agar, 10 colloidal chitin, 1 tryptone, 0.5 yeast extract, 1 NaCl, 0.1 MgSO4.7H2O, 0.5 KH2PO4, and 1 K2HPO4 (pH 7). The total plate count (TPC) method was used to determine the colony abundance. The serial dilution method and spread plate were used to purify colonies with various morphologies. After seven days of incubation at room temperature, the medium was used to isolate individual colonies from neighboring colonies [8].
Crude chitinase enzyme was from the same medium without agar. The cell cultures were collected after seven days. The cultures were then centrifuged at 8400 rpm for 10 min to obtain the crude extract of extracellular chitinase enzyme in supernatant [6].
Semi-Quantitative Test of Chitin Degradation Potential
This test aimed to evaluate the clear zone performance from each colony using disk diffusion method. As much as 30 µL of all bacteria cultures with similar densities were poured onto the 6 mm filter paper disks on top of the chitin agar medium. Three paper disks were placed in one petri dish as a test. The plates were incubated at room temperature for seven days.
The plates were incubated at room temperature in seven days to measure the index of clear zone
forming. The isolates with the highest clear zone index were chosen for the following stage. The chitinolytic index formula [9]:
𝐶𝐼 =𝐷𝐶𝑍 𝐶𝐷 Description:
CI = Chitinolytic Index (cm) DCZ = Diameter of Clear Zone (cm) CD = Colony Diameter (cm)
Isolation of Fruit Rot Pathogenic Fungi
To obtain a tiny portion of the fruit peel, the peel of the cocoa pod with pod rot symptoms was cut into slices. After that, sterile distilled water was used to rinse the fruit peel. The fruit peel was then moved to a petri dish and sliced into roughly 1 x 1 cm in size pieces. The prepared petri dish was filled with three cocoa pod shell pieces, and PDA media with chloramphenicol was added. The petri dish was then incubated at room temperature for seven days. After further purification, a single colony was produced using the single spore method, which was then separated from the other colonies after being incubated for seven days at room temperature.
The collected fungi isolates were identified based on the microscopic structure and colony shape [10].
Postulate Koch Test
The Postulate Koch test was spreading pure cultures of the fungi to healthy cocoa pods of the same size to ascertain the pathogenic potential of isolates. Next, observation was made to see if the fruit displays the same symptoms. Five equal pieces were cut lengthwise from two identical cocoa pods. Each component was put in a sterile container, and added to the petri dish 10 mL of sterile distilled water until all the fungi were flooded. Then 100 µL was taken and lightly injected into the skin after being massaged into the skin surface. Then incubated for seven days [11].
Antagonist Test against Pathogenic Fungi Two wells with a diameter of 5 mm and a separation of 3 cm were created after the PDA media had cooled. Prepared crude chitinase enzyme extract (six isolates) was placed into soft PDA (90% agar) wells and let to diffuse for an hour at room temperature. Using sterile distilled water, a conidial fungi suspension was created by drenching a seven-day-old fungi culture. A concentration approximately 1103 conidia.mL-1 was used for this solution. Following the enzyme's diffusion, 10 µL of this suspension was
added to the other wells. Seventy-two hours were then spent incubating at 30°C room temperature. The inhibition percentage was determined using the formula above [12]:
𝐼 % = 𝐴 − 𝐵 𝐴 × 100 Description:
I = Percentage of inhibition (%)
A = Diameter of the fungi colony in the control (cm) B = Diameter of a fungi colony with treatment (cm)
The inhibition parameter data were analyzed for variance using one-way ANOVA (p≤0.05). If there is a significant difference, then proceed with the Tukey test.
Identification of Bacteria Based on 16S rDNA The Zymo Kit was used for chromosomal DNA isolation for selected isolates. Chromosomal DNA was isolated from superior isolates. Using a PCR (Polymerase Chain Reaction) Thermocyler Amplitron® with universal primers 27f (5' AGA GTT TGA TCC TGG CTC AG) and 1492r (5' GGT TAC CTT GTT ACG ACT T) and a PCR program, the following bacterial 16S rDNA sequences were amplified: Pre-Denaturation 94oC 5 minutes, Denaturation 94°C 30 seconds, Annealing 55°C 30 seconds, Extension 72°C 1.5 minutes and final extension 72°C 5 minutes [13]. An ABI 3130 Automatic Sequencer Analyzer was used to sequence amplicons. Sequencer Scanner V.1 was used to modify the 16S rDNA sequences, and BioEdit V.7.2's CAP Contig Assembly was used to join them. The NCBI BLASTN tool was used, the 16S rDNA sequence was BLASTed, and the nucleotide sequence was established using GenBank [14]. The MEGA V.6 ClustalW Multiple Alignment tool was used to align the isolated 16S rDNA sequences and reference strains. The Neighbor-Joining (NJ) approach was used to build and infer the phylogenetic tree, with bootstrapping 1000 times [13].
RESULTS AND DISCUSSION
Semi-Quantitative Test of Chitin Degradation Potential
The formation of a clear zone surrounding the bacterial colonies proved that the isolates could enzymatically break down chitin in an agar medium. From 6 samples, the semi-quantitative chitin degradation potential test revealed superior bacteria (Table 1).
Bacterial isolation and selection on chitin media revealed that varying levels of bacterial growth were present, as indicated by the
chitinolytic index. Variations in bacterial adaptability to the medium or environment bring this on. It was understood that bacteria can manufacture chitinase on the selective solid medium by creating a clear zone surrounding the colony. The specific region will also be more significant; the more chitin is destroyed, the more enzymes are produced [15].
Table 1. Semi-quantitative test results for chitin degradation potential
Isolate Sample Chitinolytic index (cm)
T1S1 Cacao 3.78
T1S2 Cacao 3.74
T1S3 Cacao 3.25
T2S1 Shrimp 1.81
T2S2 Shrimp 1.48
T2S3 Shrimp 1.43
Identification of Pathogenic Fungi
Five fungi isolates were obtained due to the isolation of pathogenic fungi, and the Postulate Koch (Table 2) test was then conducted. The Postulate Koch test involves inoculating isolates made during isolation on healthy fruit. The objective of this test was to confirm that the pathogenic fungi isolates identified are harm healthy cocoa pods.
Table 2. Postulate koch test
Isolates Pathogens of Cocoa Fruit
J1 +
J2 -
J3 +
J4 -
J5 +
The findings led to the isolation of three pathogenic fungi isolates from a total of five pathogenic fungi, which were then named Phytophthora sp., Fusarium sp., and Gloeosporeum sp. (Fig. 1). When viewed macroscopically, Phytophthora sp. colonies have a rounded form, a cotton-like texture, irregular edges, radial lines, and a white color [16]. The mycelium of Fusarium sp. is initially white, but as it ages, it turns cream or pale yellow, and in some cases, it even turns slightly pinkish purple. The mycelium creates branches and is insulated.
Several Fusarium sp. isolates will produce blue or red pigments in the medium. [17]. Gloeosporium sp. has white, circle-shaped, and irregular colony margins as its macroscopic traits. From the bottom surface, there are dark patches to be seen. At the same time, the bacilli-like conidia that are widely dispersed throughout the hyphae are microscopic characteristics. Conidiophores are short, colorless, unbranched, and
uninsulated. Hyaline hyphae and septum are developed automatically at the ends of the conidiophores, where the conidia are [18].
Isolates Pictures
J1
J3
J5
Figure 1. Macroscopic fungi, J1 (Phytophthora sp.), J3 (Fusarium sp.), and J5 (Gloeosporeum sp.) Antagonist Test against Pathogenic Fungi
The antagonist test findings revealed that the bacterial isolate T2S1 was more effective at suppressing pathogenic fungi. The strong inhibition of each pathogenic fungi is evidence of this. The inhibition of T2S1 bacteria on J1, J3, and J5 is depicted in Figure 2. as 25.14%, 20.85%, and 25.24%, respectively.
According to the semi-quantitative test of chitin breakdown capacity, the T1S1 bacterial isolate had a 3.78 cm chitinolytic index, the highest. However, the bacterial isolate T2S1
showed a chitinolytic index of 1.81 cm in the superior bacterial antagonist test, which was able to block three pathogenic fungi. This variation is thought to be caused by the various kinds of chitinase produced.
Figure 2. Inhibition by superior bacteria in fungi. Fungi:
J1, J3, J5. Bacteria: T1S1, T1S2, T1S3, T2S1, T2S2, T2S3.
The hydrolysis activity of chitin from bacterial isolates can vary depending on the type of chitinase used. Compared to ChiB and ChiC, ChiA is reportedly able to hydrolyze chitin at the fastest rate. More quickly than ChiC, ChiB can hydrolyze chitin. ChiA is most effective at breaking down solid or powdered [19].
The highest bacteria (cacao) with chitinolytyc index had ChiA chitin type. This type can hydrolyze chitin fast in coloidal chitin media [19].
But in antagonist test, chitin type of the bacteria (Bacillus Genus) was ChiB. In reference, ChiA can fast to hydrolyze chitin in fungi than ChiB [20].
Identification of Superior Chitinolytic Bacteria Based on the similarity of 16S rDNA sequences, chitinolytic bacteria with the potential to act as biocontrol agents for pathogenic fungi were discovered phylogenetically. Bacillus subtilis C10 was recognized as the isolate T2S1 with a 99.77%
similarity, as shown in Figure 3.
Figure 3. Phylogeny tree that showed T2S1 isolate and references bacterial strains based on 16S rDNA sequence according to Neighbor-joining algorithm.
Gram-positive Bacillus subtilis possesses rod- shaped cells that are 0.3-2.2 x 1.2-7.0 m in size [21]. By suppressing the growth of dangerous fungi, B. subtilis can produce antifungal substances such as subtilin, bacitracin, bacilli, and bacillomyxin [22]. Bacillus subtilis, which may create antimicrobials and promote plant development, is one of the biocontrol agents used to combat illness. Commercial items made from this bacterium strain have been widely produced. B. subtilis capacity to inhibit certain plant diseases and its rhizobacteria that promote plant growth are further benefits (PGPR) [23].
CONCLUSION
T2S1 was the superior chitinolytic bacterial isolate with the most significant potential to deter pathogenic fungi. Bacillus subtilis C10 was identified as the T2S1 isolate with 99.77%
similarity. This research requires an enzyme activity test as a further test to improve the criteria for bacteria inhibiting pathogenic fungi.
REFERENCES
[1] Susanto, F.X. 2010. Tanaman kakao budidaya pengolahan hasil. Kanisius.
Yogyakarta.
[2] Direktorat Jenderal Perkebunan. 2015.
Statistik Perkebunan Indonesia 2014- 2016. Direktorat Jenderal Perkebunan.
[3] Dinas Tanaman Pangan, Hortikultura, dan Perkebunan. 2018. Statistik Perkebunan Kabupaten Malang 2018. Direktorat Jenderal Perkebunan. Kabupaten Malang.
[4] Akrofi, A.Y., I. Amoako-Atta, M. Assuah, E.K.
Asare. 2015. Black pod disease on cacao (Theobroma cacao, L) in Ghana: spread of Phytophthora megakarya and role of economic plants in the disease epidemiology. Crop Prot. 72. 66-75.
[5] Wu, M.L., Y.C. Chuang, J.P. Chen, C.S. Chen, M.C. Chang. 2001. Identification and characterization of the three chitin-binding domains within the multidomain chitinase Chi92 from Aeromonas hydrophila JP101.
Appl. Environ. Microbiol. 67(11). 5100-5106.
[6] Wibowo, A., T. Joko, S. Subandiyah, K.
Kageyama. 2020. Antagonistic potential of endophytic bacteria against Phytophthora palmivora causing black pod rot disease on Cacao (Theobroma cacao L.) in Indonesia. Plant Pathol. J. 19(1). 22-41.
[7] Faramarzi, M.A., M. Fazeli, M.T. Yazdi, S.
Adrangi, K.J. Al-Ahmadi, N. Tasharrofi, F.A.
Mohseni. 2009. Optimization of cultural conditions for production of chitinase by a
soil isolate of Massilia timonae.
Biotechnology. 8(1). 93-99.
[8] Asif, T., U. Javed, S.B. Zafar, A. Ansari, S.A.U.
Qader, A. Aman. 2019. Bioconversion of colloidal chitin using novel chitinase from Glutamicibacter uratoxydans exhibiting anti-fungal potential by hydrolyzing chitin within fungal cell wall. Waste Biomass Valorization. 11. 1-15.
[9] Nafiah, H., S. Pujiyanto, B. Raharjo. 2017.
Isolasi dan uji aktivitas kitinase isolat bakteri dari Kawasan Geotermal Dieng. Bioma:
Berkala Ilmiah Biologi. 19(1). 22-29.
[10] Alwakeel, S.S. 2013. Molecular identification of isolated fungi from stored apples in Riyadh, Saudi Arabia. Saudi J. Biol.
Sci. 20(4). 311-317.
[11] Hafsah, S., Z. Zuyasna. 2013. Uji patogenisitas beberapa isolat penyakit busuk buah kakao asal Aceh dan evaluasi efektivitas metode inokulasi. Jurnal Agrista.
17(1). 42-48.
[12] Nurcihan, H., A. Ilgaz, D. Basaran. 2011.
Antimicrobial potential of Xylaria polymorpha (Pers.) Grev. Afr. J. Microbiol.
Res. 5(6). 728-730.
[13] Setia, I.N. 2018. Eksplorasi bakteri diazotrof rhizosfer jeruk keprok (Citrus reticulata L.) varietas batu 55 sebagai agen plant growh promoting rhizobaceria (PGPR). Master Thesis. Universitas Brawijaya. Malang.
[14] Soeka, Y.S., Sulistiani. 2011. Seleksi, karakterisasi, dan identifikasi bakteri penghasil kitinase yang diisolasi dari Gunung Bromo Jawa Timur. Jurnal Natur Indonesia. 13(2). 155-161
[15] Suryani, W. 2016. Isolasi dan identifikasi mikroorganisme kitinolitik asal limbah cairan rumen sapi serta optimasi produksi enzim kitinase. Bachelor Thesis. Universitas Halu Oleo. Kendari.
[16] Banasuru, G. 2015. Isolasi dan karakterisai morfologi phytophthora palmivora pada buah kakao pada perkebunan kakao di Konawe Selatan, Sulawesi Tenggara.
Bachelor Thesis. Universitas Halu Oleo.
Kendari.
[17] Feti, R.R., L. Soesanto, E. Mugiastuti. 2010.
Kajian mekanisme antagonis Pseudomonas fluorencens P60 terhadap Fusarium oxysporum f. sp. lycopersici pada tanaman tomat in Vivo. J. HPT Tropika. 10(2). 108- 115.
[18] Afriyeni, Y., N. Nasir, Jumjunidang. 2013.
Jenis-Jenis jamur pada pembusukan buah
kakao (Theobroma cacao, L.) di Sumatera Barat. Jurnal Biologi Universitas Andalas.
2(2). 124-129.
[19] Lehninger. 2012. Principles of biochemistry.
W.H. Freeman. USA.
[20] Sato, Y., Y. Araki, 2008. Identification of inducers for chitinase B (ChiB) production in Bacillus cereus CH and estimation of its induction mechanism. J. Environ.
Biotechnol. 8. 119-121.
[21] Aini, F.N., S. Sukamto, D. Wahyuni, R.G Suhesti, Q. Ayyunin. 2013. Penghambatan
pertumbuhan Colletotrichum
gloeosporioides oleh Trichoderma harzianum, Trichoderma koningii, Bacillus subtilis, Pseudomonas fluorescens. Jurnal Pelita Perkebunan. 29(1). 44-52.
[22] Montealegre, J.R., R. Reyes, L.M. Perez, R.
Herrera, P. Silva, X. Besoain. 2003. Selection of bioantagonistic bacteria to be used in biological control of Rhizoctonia solani in tomato. Electron. J. Biotechnol. 6(2). 116–
127.
[23] Wartono, G., H.M. Kikin. 2014. Efektivitas formulasi spora Bacillus subtilis B12 sebagai agen pengendali hayati penyakit hawar daun bakteri pada tanaman padi. Penelitian Pertanian Tanaman Pangan. 34(1). 21-28.