Penapisan Bakteri Penghambat Fusarium yang Diisolasi dari Cairan Kantung Semar (Nepenthes sp.)
The genus Fusarium sp. is a pathogenic fungal for many cultivated plants. The bacteria isolated from monkey cup (Nepenthes sp.) fluid possess the ability to produce hydrolytic enzyme, such as chitinase which can be utilized to inhibit the growth of mycelia of pathogenic fungi. The aims of this study are to isolate bacteria from monkey cup liquid, to test their abilities to produce protease, chitinase, and cellulase, as well as their abilities to inhibit Fusarium. The bacteria were isolated using serial dilution method on Reasoner’s 2A agar medium. Enzymatic activities of bacterial isolates were determined by inoculating them on tested medium supplemented with casein protein, chitin, and cellulose, whereas their antifungal activities were assayed using a direct confrontation method between tested bacterial isolates and pathogenic fungal on Malt Extract Agar medium. Molecular identification of bacteria with antifungal activity was performed by analyzing the 16S rRNA gene sequences. Isolation process of bacteria from monkey cup fluid resulted in 99 bacterial isolates with the ability to produce either protease, chitinase, and/or cellulose enzymes. A total of 37 bacterial isolates were capable of producing at least two hydrolytic enzymes. Antifungal assay of those bacteria showed that as many as 25 isolates have the ability to inhibit the growth of Fusarium sp. Based on the analysis of 16S rRNA gene sequences revealed that those isolates were closely related to three Burkholderia species, namely B. arboris, B. contaminans, and B. rijonensis.
Keywords: antifungal, Burkholderia, chitinase, cellulaseN, epenthes, protease
Adhikari M, Negi B, Kaushik N, Adhikari A, Al-Khedairy AA, Kaushik NK, Choi EH. 2017. T-2 mycotoxin: toxicological effects and decontamination strategies. Oncotarget. 8(20): 3393333952. https:// doi.org/10.18632/oncotarget.15422
Amini J, Agapoor Z, Ashengroph M. 2016. Evaluation of Streptomyces spp. against Fusarium oxysporum f. sp. ciceris for the management of chickpea wilt. Journal of Plant Protection Research. 56(3): 257264. https:// doi.org/10.1515/jppr-2016-0038
Bhore SJ, Komathi V, Kandasamy KI. 2013. Diversity of endophytic bacteria in medicinally important Nepenthes species. Journal of Natural Science, Biology and Medicine. 4(2): 431434. https://doi.org/ 10.4103/0976-9668.117022
Bubici G, Kaushal M, Prigigallo MI, Gomez-Lama Cabanaz C, Mercado-Blanco J. 2019. Biological control agents against fusarium wilt of banana. Frontiers in Microbiology. 10: 133. https:// doi.org/10.3389/fmicb.2019.01290
Buch F, Rott M, Rottloff S, Paetz C, HIlke I, Raessler M, Mithofer A. 2013. Secreted pitfall-trap fluid of carnivorous Nepenthes plants is unsuitable for microbial growth. Annals of Botany. 111(3): 375383. https://doi.org/10.1093/aob/mcs287
Chan XY, Hong KW, Yin WF, Chan KG. 2016. Microbiome and biocatalytic bacteria in monkey cup (Nepenthes pitcher) digestive fluid. Scientific Reports. 6(1): 110. https://doi.org/10.1038/ srep20016
Chou LY, Clarke CM, Dykes GA. 2014. Bacterial communities associated with the pitcher fluids of three Nepenthes (Nepenthaceae) pitcher plant species growing in the wild. Archives of Microbiology. 196(10): 709717. https://doi.org/ 10.1007/s00203-014-1011-1
Cordova-Kreylos AL, Fernandez LE, Koivunen M, Yang A, Flor-Weiler L, Marrone PG. 2013. Isolation and characterization of Burkholderia rinojensis sp. nov., a non-Burkholderia cepacia complex soil bacterium with insecticidal and miticidal activities. Applied and Environmental Microbiology. 79(24): 76697678. https://doi.org/10.1128/AEM.02365-13
Dinolfo MI, Castañares E, Stenglein SA. 2017. Fusarium-plant interaction: state of the art-a review. Plant Protection Science. 53(2): 6170. https://doi.org/10.17221/182/2015-PPS
Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research. 32(5): 17921797. https://doi.org/10.1093/nar/gkh340
Eilenberg H, Pnini-Cohen S, Rahamin Y, Sionov E, Segal E, Carmeli S, Zilberstein A. 2010. Induced production of antifungal naphthoquinones in the pitchers of the carnivorous plant Nepenthes khasiana. Journal of Experimental Botany. 61(3): 911922. https://doi.org/10.1093/jxb/erp359
Elshafie HS, Camele I, Racioppi R, Scranol L, Lacobellis NS, Bufo SA. 2012. In vitro antifungal activity of Burkholderia gladioli pv. agaricicola against some phytopathogenic fungi. International Journal of Molecular Sciences. 13(12): 1629116302. https://doi.org/10.3390/ijms1312 16291
Frankowski J, Lorito M, Scala F, Schmid R, Berg G, Bahl H . 2001. Purification and properties of two chitinolytic enzymes of Serratia plymuthica HRO-C48. Archives of Microbiology. 176(6): 421426. https://doi.org/10.1007/s002030100347
Gilbert J, Fernando WGD. 2004. Epidemiology and biological control of Gibberella zeae /Fusarium graminearum. Canadian Journal of Plant Pathology. 26(4): 464472. https://doi.org/10.1080/0706 066040 9507166
Hall TA. 1999. BIOEDIT: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/ NT. Nucleic Acids Symposium Series. 41(41): 9598. https://doi.org/10.22202/ bc.2015.v1i1.1523
Hidayat Y. 2015. Isoalsi bakteri penghasil antibiotika dari cairan kantong semar (Nepenthes spp.) cagar alam lembah harau Sumatera Barat. Bioconcetta. 1(1): 2031.
Jeun YC, Park KS, Kim CH, Fowler WD, Kloepper JW. 2004. Cytological observations of cucumber plants during induced resistance elicited by rhizobacteria. Biological Control. 29(1): 3442. https://doi.org/ 10.1016/ S1049-9644(03)00082-3
Kanokratana P, Mhuanthong W, Laothanacareon T, Tangphatsornruang S, Eurwilaichitr L, Kruetreepradit T, Mayes S, Champreda V. 2016. Comparative study of bacterial communities in Nepenthes pitchers and their correlation to species and fluid acidity. Microbial Ecology. 72(2): 381393. https://doi.org/10.1007/s00248-016-0798-5
Kimura M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution. 16(2): 112120. https://doi.org/10.1007/BF01731581
Kiran T, Asad W, Siddiqui S, Ajaz M, Rasool SA. 2015. Industrially important hydrolytic enzyme diversity explored in stove ash bacterial isolates. Pakistan Journal of Pharmaceutical Sciences. 28(6): 20352040.
Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution. 35(6): 15471549. https://doi.org/10. 1093/ molbev/msy096
Lane DJ. 1991. 16S/23S rRNA sequencing. Stackebrandt E, Goodfellow M, Ed. In: Nucleic acid techniques in bacterial systematics. Chinchester (UK): John Wiley and Sons Ltd. p. 115175
Lemanceau P, Alabouvette C. 1991. Biological control of fusarium diseases by fluorescent Pseudomonas and non-pathogenic Fusarium. Crop Protection. 10(4): 279286. https://doi.org/10.1016/0261-2194(91)90006-D
Liang YL, Zhang Z, Wu M, Wu Y, Feng JX. 2014. Isolation, screening, and identification of cellulolytic bacteria from natural reserves in the subtropical region of China and optimization of cellulase production by Paenibacillus terrae ME27-1. BioMed Research International. 2014: 113. https:// doi.org/10.1155/ 2014/512497
Lin Z, Falkinham III JO, Tawfik KA, Jeffs P, Bray B, Dubay G, Cox JE, Schmidt EW. 2012. Burkholdines from Burkholderia ambifaria: antifungal agents and possible virulence factors. Journal of Natural Products. 75(9): 15181523. https://doi.org/10. 1021/ np300108u
Lu SE, Novak J, Austin FW, Gu G, Ellis D, Kirk M, Wilson-Stanford S, Tonelli M, Smith L. 2009. Occidiofungin, a unique antifungal glycopeptide produced by a strain of Burkholderia contaminans. Biochemistry. 48(35): 83128321. https://doi.org/ 10.1021/bi900814c
Maryani N, Lombard L, Poerba YS, Subandiyah S, Crous PW, Kema GHJ. 2019. Phylogeny and genetic diversity of the banana fusarium wilt pathogen Fusarium oxysporum f. sp. cubense in the Indonesian centre of origin. Studies in Mycology. 92: 155194. https://doi.org/10.1016/j.simyco. 2018.06.003
McCormick SP, Stanley AM, Stover NA, Alexander NJ. 2011. Trichothecenes: from simple to complex mycotoxins. Toxins. 3(7): 802814. https:// doi.org/10.3390/toxins3070802
Meliah S, Kusumawati DI, Ilyas M. 2020. Preliminary study of myxobacteria as biocontrol agents for panama disease pathogen, tropical race 4 Fusarium odoratissimum. In IOP Conference Series: Earth and Environmental Science. 457(1): 012060 https://doi.org/10.1088/1755-1315/457/1/012060
Mithöfer A. 2011. Carnivorous pitcher plants: insights in an old topic. Phytochemistry. 72(13): 16781682. https://doi.org/10.1016/j.phytochem.2010.11.024
Moran JA, Clarke CM, Hawkins BJ. 2003. From carnivore to detritivore? Isotopic evidence for leaf litter utilization by the tropical pitcher plant Nepenthes ampullaria. International Journal of Plant Sciences. 164(4): 635639.
Morohoshi T, Oikawa M, Sato S, Kikuchi N, Nato N, Ikeda T. 2011. Isolation and characterization of novel lipases from a metagenomic library of the microbial community in the pitcher fluid of the carnivorous plant Nepenthes hybrida. Journal of Bioscience and Bioengineering. 112(4): 315320. https://doi.org/10.1016/j.jbiosc.2011.06.010
Park JY, Oh SA, Anderson AJ, Neiswender J, Kim JS, Kim YC. 2011. Production of the antifungal compounds phenazine and pyrrolnitrin from Pseudomonas chlororaphis O6 is differentially regulated by glucose. Letters in Applied Microbiology. 52(5): 532537. https://doi.org/10. 1111/ j.1472-765X.2011.03036.x
Ploetz RC. 2006. Fusarium-induced diseases of tropical perennial crops fusarium wilt of banana is caused by several pathogens referred to as Fusarium oxysporum f.sp cubense. Symposium: Fusarium-Induced Diseases of Tropical Perennial Crops. 96(6): 653656. https://doi.org/10.1094/ PHYTO-96-0653
Prapagdee B, Kuekulvong C, Mongkolsuk S. 2008. Antifungal potential of extracellular metabolites produced by Streptomyces hygroscopicus against phytopathogenic fungi. International Journal of Biological Sciences. 4(5): 330337 https:// doi.org/10.7150/ijbs.4.330
Reasoner DJ, Geldreich EE. 1985. A new medium for the enumeration and subculture of bacteria from potable water. Applied and Environmental Microbiology. 49(1): 17. https://doi.org/10.1128/ AEM.49.1.1-7.1985
Saitou N, Nei M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular biology and evolution. 4(4): 406425. https://doi.org/10.1093/oxfordjournals.molbev.a040454
Santana MF, Queiroz MV. 2015. Transposable elements in fungi: a genomic approach. Scientific Journal of Genetics and Gene Therapy. 1(1): 1216. https://doi.org/10.17352/sjggt.000003
Sari W, Wiyono S, Nurmansyah A, Munif A, Poerwanto R. 2018. Keanekaragaman dan patogenisitas Fusarium spp. asal beberapa kultivar pisang. Jurnal Fitopatologi Indonesia. 13(6): 216228. https:// doi.org/10.14692/jfi.13.6.216
Srinivas C, Devi DN, Murthy KN, Mohan CD, Lakshmeesha TR, Singh B, Kalagatur NK, Niranjana RR, Hashem A, Alqarawi AA, Tabassum B, Abd Allah EF, NAyaka C, Srivastava RK. 2019. Fusarium oxysporum f.sp. lycopersici causal agent of vascular wilt disease of tomato: biology to diversity-a review. Saudi Journal of Biological Sciences. 26(7): 13151324. https://doi.org/ 10.1016/ j.sjbs.2019.06.002
Sultan MZ, Park K, Lee SY, Park JK, Varughhese T, Moon SS. 2008. Novel oxidized derivatives of antifungal pyrrolnitrin from the bacterium Burkholderia cepacia K87. Journal of Antibiotics. 61(7): 420425. https://doi.org/10.1038/ja.2008.58
Sun JB, Peng M, Wang YG, Zhao PJ, Xia QY. 2011. Isolation and characterization of antagonistic bacteria against fusarium wilt and induction of defense related enzymes in banana. African Journal of Microbiology Research. 5(5): 509515.
Takahashi K, Athauda SB, Matsumoto K, Rajapakshe S, Kuribayashi M, Kojima M, Kubomura-Yoshida N, Iwamatsu A, Shiabata C, Inoue H. 2005. Nepenthesin, a unique member of a novel subfamily of aspartic proteinases: enzymatic and structural characteristics. Current Protein & Peptide Science. 6(6): 513525. https://doi.org/10.2174/138920 305774933259
Takeuchi Y, Chaffron S, Salcher MM, Shimizu-Inatsugi R, Kobayashi MJ, Diway B, von Mering C, Pernhaler J, Shimizu KK. 2015. Bacterial diversity and composition in the fluid of pitcher plants of the Genus Nepenthes. Systematic and Applied Microbiology. 38(5): 330339. https://doi.org/ 10.1016/j.syapm.2015.05.006
Vanlaera E, LiPuma JJ, Baldwin A, Henry D, De Brandt E, Mahenthiralingam E, Speert D, Dowson C, Vandamme P. 2008. Burkholderia latens sp. nov., Burkholderia diffusa sp. nov., Burkholderia arboris sp. nov., Burkholderia seminalis sp. nov., and Burkholderia metallica sp. nov., novel species within the Burkholderia cepacia complex. International Journal of Systematic and Evolutionary Microbiology. 58(7): 15801590. https://doi.org/10. 1099/ ijs.0.65634-0
Wang XQ, Liu AX, Guerrero A, Liu J, Yu XQ, Deng P, Ma L, BAird SM, Smith L, Li XD, Lu SE. 2016. Occidiofungin is an important component responsible for the antifungal activity of Burkholderia pyrrocinia strain Lyc2. Journal of Applied Microbiology. 120(3): 607618. https:// doi.org/10.1111/jam.13036
Yolanda H, Makashinda IM, Aprilia M, Sanjaya N, Gunawan H, Dewi R. 2015. Nepenthes rafflesiana pitcher liquid has antifungal activity against Candida spp. Mycoses. 33(2): 8390.
Yoon SH, Ha SM, Kwon S, Lim J, Kim Y, Seo H, Chun J. 2017. Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. International Journal of Systematic and Evolutionary Microbiology. 67(5): 16131617. https://doi.org/10.1099/ijsem.0.001755
Authors who publish with this journal agree to the following terms:
- Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.
- Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.
- Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access).