Enhanced Production of Ligninolytic Enzymes by Mangrove Fungal Endophytes Co-cultured with Pathogenic and Beneficial Fungi
Keywords:
antagonism, beneficial fungi, co-culture, fungal interaction, lignin-degrading enzymesAbstract
Mangrove ecosystems host diverse fungal endophytes with potential ligninolytic activity, yet their functional roles under competitive stress remain underexplored. This study assessed 30 mangrove-derived fungal endophytes (MFE) for their ability to produce key ligninolytic enzymes — laccase (Lac), manganese peroxidase (MnP), and lignin peroxidase (LiP) — in single- and dualculture with the pathogenic Fusarium oxysporum and beneficial Trichoderma afroharzianum. Enzyme production was evaluated using qualitative colorimetric assays and quantified through potency index (PI), a measure of enzyme efficiency relative to colony growth. Initial screening revealed that enzyme production was species-specific, with only one isolate, Schizophyllum commune RmLE-P12, producing all three enzymes. Notably, Nigrospora and Penicillium isolates exhibited strong Lac and MnP activity, respectively, and with high PI values designating them as hyper-ligninolytic strains. Under co-culture conditions, enzyme expression and growth rates varied. Some MFE showed enhanced enzyme production in response to competition or antagonistic interaction, while others exhibited reduced or suppressed activity. Interaction assays identified six types of fungal interactions, with growth halts near contact (Type C) and challenge species overgrowth (Type E) being most prevalent. Antagonism indices indicated that T. afroharzianum exerted a stronger inhibitory effect than F. oxysporum. This comprehensive analysis highlights the species-dependent ligninolytic capabilities of MFE and reveals dynamic enzyme regulation under biotic stress.
References
Abro MA, Sun X, Li X, Jatoi GH, Guo LD. 2019 – Biocontrol potential of fungal endophytes against Fusarium oxysporum f. sp. cucumerinum causing wilt in cucumber. The Plant Pathology Journal 35(6), 598. Doi 10.5423/PPJ.OA.05.2019.0129
Ali MIA, Khalil N, Abdelghany M. 2012 – Biodegradation of some polycyclic aromatic hydrocarbons by Aspergillus terreus. African Journal of Microbiological Research 6, 3783–3790.
Apurillo CCS, Cai L, dela Cruz TEE. 2019 – Diversity and bioactivities of mangrove fungal endophytes from Leyte and Samar, Philippines. Philippine Science Letters 12, 33–48.
Bitacura JG, Jacob JKS, dela Cruz TEE. 2024 – Isolation and identification of fungal endophytes associated with leaves of Rhizophora mucronata Lamk. Acta Manilana 72, 1–17. Doi 10.53603/actamanil.72.2024.sger2382
Bouanaka H, Bellil I, Harrat W, Boussaha S, et al. 2021 – On the biocontrol by Trichoderma afroharzianum against Fusarium culmorum responsible of fusarium head blight and crown rot of wheat in Algeria. Egyptian Journal of Biological Pest Control 31(1), 68. Doi 10.1186/s41938-021-00416-3
Chowdhary P, More N, Yadav A, Bharagava RN. 2019 – Ligninolytic Enzymes: An Introduction and Applications in the Food Industry. In Kuddus M (Ed.), Enzymes in Food Biotechnology (pp. 181–195). Academic Press. Doi 10.1016/B978-0-12-813280-7.00012-8
Corrêa RCG, Rhoden SA, Mota TR, Azevedo JL, et al. 2014 – Endophytic fungi: Expanding the arsenal of industrial enzyme producers. Journal of Industrial Microbiology and Biotechnology 41(10), 1467–1478. Doi 10.1007/s10295-014-1496-2
Cruz EM, dela Cruz TEE. 2024a – Plant growth-promoting and protective potential of root fungal endophytes associated with a pioneer grass, Saccharum spontaneum L., from a lahar-affected area. Asian Journal of Mycology 7(2), 132–147. Doi 10.5943/ajom/7/2/9
Cruz EM, dela Cruz TEE. 2024b – Diversity and phylogeny of root fungal endophytes associated with lahar-grown pioneering grass Saccharum spontaneum L. Philippine Journal of Science 153 (6A), 2127–2151.
Čvančarová M, Moeder M, Filipová A, Cajthaml T. 2015 – Biotransformation of fluoroquinolone antibiotics by ligninolytic fungi – Metabolites, enzymes and residual antibacterial activity. Chemosphere 136, 311–320. Doi 10.1016/j.chemosphere.2014.12.012
dela Cruz TEE, Din HJF, Aril-dela Cruz JV. 2021 – Microbes for Sustainable Agriculture: Isolation and identification of beneficial soil- and plant-associated microorganisms. SEARCA Professorial Chair Lecture Monograph No. 6. SEARCA, Los Baños, Laguna, Philippines.
De Paula NM, da Silva K, Brugnari T, Haminiuk CWI, et al. 2022 – Biotechnological potential of fungi from a mangrove ecosystem: Enzymes, salt tolerance and decolorization of a real textile effluent. Microbiological Research 254, 126899. Doi 10.1016/j.micres.2021.126899
Dhakar K, Kooliyottil R, Joshi A, Pandey A. 2015 – Simultaneous production of ligninolytic enzymes by a temperature and pH tolerant strain of Aspergillus niger under different cultural conditions. Indian Journal of Biotechnology 14, 81–86.
Hasanin MS, Darwesh OM, Matter IA, El-Saied H. 2019 – Isolation and characterization of non-cellulolytic Aspergillus flavus EGYPTA5 exhibiting selective ligninolytic potential. Biocatalysis and Agricultural Biotechnology 17, 160–167. Doi 10.1016/j.bcab.2018.11.012
Jacob JKS, Witzel K, dela Cruz TEE. 2023 – Comparative diversity and functional traits of fungal endophytes in response to elevated mineral content in a mangrove ecosystem. Journal of Fungi 9(12), 1186. Doi 10.3390/jof9121186
Kaur H, Kapoor S, Sharma S. 2018 – An efficient method for qualitative screening of ligninolytic enzyme potential of Ganoderma lucidum. International Journal of Current Microbiology and Applied Sciences 7(8), 2442–2459. Doi 10.20546/ijcmas.2018.708.247
Krishnamurthy YL, Naik BS. 2017 – Endophytic Fungi Bioremediation. In Maheshwari DK, Annapurna K (Eds.), Endophytes: Crop Productivity and Protection. (Vol. 2, pp. 47–60). Springer International Publishing. Doi 10.1007/978-3-319-66544-3_3
Kumar A, Chandra R. 2020 – Ligninolytic enzymes and its mechanisms for degradation of lignocellulosic waste in environment. Heliyon 6(2), e03170. Doi 10.1016/j.heliyon.2020.e03170
Kumar V, Prasher IB. 2021 – Ligninolytic enzymes production by endophytic fungus Diaporthe phaseolorum (Desm.) Sacc. under the influence of different carbon and nitrogen sources. Studies in Fungi 6(1), 531–542. Doi 10.5943/sif/6/1/43
Kunjadia PD, Sanghvi GV, Kunjadia AP, Mukhopadhyay PN, Dave GS. 2016 – Role of ligninolytic enzymes of white rot fungi (Pleurotus spp.) grown with azo dyes. SpringerPlus 5(1), 1487. Doi 10.1186/s40064-016-3156-7
Lunardelli Negreiros de Carvalho P, de Oliveira Silva E, Aparecida Chagas-Paula D, Honorata Hortolan Luiz J, Ikegaki M. 2016 – Importance and implications of the production of phenolic secondary metabolites by endophytic fungi: A Mini-Review. Mini Reviews in Medicinal Chemistry 16(4), 259–271.
Manavalan T, Manavalan A, Heese K. 2015 – Characterization of lignocellulolytic enzymes from white-rot fungi. Current Microbiology 70(4), 485–498. Doi 10.1007/s00284-014-0743-0
Martinho V, dos Santos Lima LM, Barros CA, Ferrari VB, Passarini MRZ, et al. 2019 – Enzymatic potential and biosurfactant production by endophytic fungi from mangrove forest in Southeastern Brazil. AMB Express 9(1), 130. Doi 10.1186/s13568-019-0850-1
Moron LS, Lim YW, dela Cruz TEE. 2018 – Antimicrobial activities of crude culture extracts from mangrove fungal endophytes collected in Luzon Island, Philippines. Philippine Science Letters 11, 28–36.
Morón-Ríos A, Gómez-Cornelio S, Ortega-Morales BO, Rosa-García SD, et al. 2017 – Interactions between abundant fungal species influence the fungal community assemblage on limestone. PLOS ONE 12(12), e0188443. Doi 10.1371/journal.pone.0188443
Naraian R, Pandey RK, Patel Y, Singh VK. 2013 – Variable expression and regulation of genes encoding peroxidase: Multiple applications. In Gupta VK, Tuohy MG, Sharma GD, Gaur S (Eds.), Applications of Microbial Genes in Enzyme Technology (pp. 269–286). Nova Science.
Novotný Č, Svobodová K, Erbanová P, Cajthaml T, et al. 2004 – Ligninolytic fungi in bioremediation: Extracellular enzyme production and degradation rate. Soil Biology and Biochemistry 36(10), 1545–1551. Doi 10.1016/j.soilbio.2004.07.019
Oses R, Valenzuela S, Freer J, Baeza J, Rodríguez J. 2006 – Evaluation of fungal endophytes for lignocellulolytic enzyme production and wood biodegradation. International Biodeterioration & Biodegradation 57(2), 129–135. Doi 10.1016/j.ibiod.2006.01.002
Patel HK, Kalaria RK, Vasava DK, Bhalani HN. 2022 – Chapter 10 - Ligninolytic enzymes: A promising tool for bioremediation of wastewater. In Shah MP, Rodriguez-Couto S, Kapoor RT (Eds.), Development in Wastewater Treatment Research and Processes (pp. 221–242). Elsevier. Doi 10.1016/B978-0-323-85657-7.00002-X
Pham VHT, Kim J, Chang S, Chung W. 2022 – Biodegradation of methylene blue using a novel lignin peroxidase enzyme producing bacteria, named Bacillus sp. React3, as a promising candidate for dye-contaminated wastewater treatment. Fermentation 8(5), 190. Doi 10.3390/fermentation8050190
Plácido J, Capareda S. 2015 – Ligninolytic enzymes: A biotechnological alternative for bioethanol production. Bioresources and Bioprocessing 2(1), 23. Doi 10.1186/s40643-015-0049-5
Pothiraj C, Kanmani P, Balaji P. 2006 – Bioconversion of lignocellulose materials. Mycobiology 34(4), 159–165. Doi 10.4489/MYCO.2006.34.4.159
Ramirez CSP, Notarte KIR, dela Cruz TEE. 2020 – Antibacterial activities of mangrove leaf endophytic fungi from Luzon Island, Philippines. Studies in Fungi 5(1), 320–331. Doi 10.5943/sif/5/1/14
Schulz B, Boyle C. 2006 – What are Endophytes? In Schulz BJE, Boyle CJC, Sieber TN (Eds.), Microbial Root Endophytes (Vol. 9, pp. 1–13). Springer Berlin Heidelberg. Doi 10.1007/3-540-33526-9_1
Sornakili A, Thankappan S, Sridharan AP, Nithya P, et al. 2020 – Antagonistic fungal endophytes and their metabolite-mediated interactions against phytopathogens in rice. Physiological and Molecular Plant Pathology 112, 101525. Doi 10.1016/j.pmpp.2020.101525
Team RC. 2013 – R: A language and environment for statistical computing. R Foundation for Statistical Computing. https://cir.nii.ac.jp/crid/1370857669939307264 (Accessed on 21.03.2024)
Torres JMO, dela Cruz TEE. 2013 – Production of xylanases by mangrove fungi from the Philippines and their application in enzymatic pretreatment of recycled paper pulps. World Journal of Microbiology and Biotechnology 29(4), 645–655. Doi 10.1007/s11274-012-1220-1
Weiland-Bräuer N. 2021 – Friends or foes - Microbial interactions in nature. Biology 10(6), 496. Doi: 10.3390/biology10060496.
Wickham H. 2016 – Data Analysis. In H. Wickham, Ggplot2 (pp. 189–201). Springer International Publishing. Doi 10.1007/978-3-319-24277-4_9
Yang C, Sun J, Wu Z, Jiang M, Li D, Wang X, et al. 2023 – FoRSR1 is important for conidiation, fusaric acid production, and pathogenicity in Fusarium oxysporum f. sp. ginseng. Phytopathology 113(7), 1244–1253. Doi 10.1094/PHYTO-10-22-0372-R
Zhao X, Hou D, Xu J, Wang K, Hu Z. 2022 – Antagonistic activity of fungal strains against Fusarium crown rot. Plants 11(3), Article 3. Doi 10.3390/plants11030255
