Dağ Keçilerinden İzole Edilen Anaerobik Gut Fungusların Moleküler Tanımlanması

Yıl 2025, Cilt: 11 Sayı: 2, 604 - 615, 29.12.2025

Cemal Elinç , Bülent Kar

https://doi.org/10.29132/ijpas.1664332

Öz

Anaerobik Gut funguslar (AGF) Neocallimastigomycota filumunda bulunmaktadır, bu fungusların özellikle otoburların sindirim sisteminde, yemlerin tüketiminde temel koloni olduğundan dolayı önemli rol oynadığı bilinmektedir. AGF’lar salgıladıkları lig-noselülozik enzimler yardımıyla bitki dokularını parçalayarak sindirilmesini sağla-maktadırlar. Bu enzimler son yıllarda gıda ve yem sanayinde de kullanılmaktadır. Bu açıdan farklı ruminant hayvanlardan izole edilen ve tanımlaması yapılan funguslar değerli olmaktadır. Bu çalışmada Tunceli yöresinde yaban hayatta bulunan dağ keçilerinden fungus izolasyonu yapılmış, morfolojik ve moleküler olarak tanımlanmıştır. Moleküler tanımlamada yaklaşık 1500 bç uzunluğundaki ITS bölgesi kullanılmıştır. İzole edilen 9 fungustan Orpinomiyces sp. (AGF-5) Feramyces sp.(AGF-8,10) ve Neocallimastix sp. (AGF-1,2,3,4,7,11) cinslerine ait olduğu belir-lenmiştir. Yabani ruminant hayvanlardaki mikrobiyal popülasyonun belirlenmesi için daha fazla çalışmaya ihtiyaç bulunmaktadır.

Anahtar Kelimeler

Anaerobik Gut fungi , Dağ keçileri , PCR , ITS

Destekleyen Kurum

TÜBİTAK 2209-A Üniversite Öğrencileri Araştırma Projeleri Destekleme Programı

Proje Numarası

1919B012220256

Molecular Identification of Anaerobic Gut Fungi Isolated from Mountain Goats

Öz

Anaerobic Gut Fungi (AGF) are found in the Neocallimastigomycota phylum, and it is known that these fungi play an important role, especially in the digestive system of herbivores, as they are the main colonies in the consumption of feed. AGFs provide digestion by breaking down plant tissues with the help of lignocellulosic enzymes they secrete. These enzymes have also been used in the food and feed industry in recent years. In this respect, fungi isolated and identified from different ruminant animals are valuable. In this study, fungi were isolated from mountain goats living in the wild in the Tunceli region and identified morphologically and molecularly. The ITS region of ap-proximately 1500 bp was used in molecular identification. It was determined that 9 isolated fungi belonged to the genera Orpinomyces sp. (AGF-5), Feramyces sp. (AGF-8,10) and Neocallimastix sp. (AGF-1,2,3,4,7,11). Further studies are needed to determine the microbial population in wild ruminant animals.

Anahtar Kelimeler

Anaerobik Gut fungi , Mountain Goats , fungus , PCR , ITS

Proje Numarası

1919B012220256

Kaynakça

• [1] T.J. Hackmann ve J.N. Spain. “Invited review: ruminant ecology and evolution: perspectives useful to ruminant livestock research and production”, J Dairy Sci, 93:1320–1334, 2010, doi: 10.3168/jds.2009 2071
• [2] J.E. Decker vd., “Resolving the evolution of extant and extinct ruminants with high-throughput phylogenomics”, Proc Natl Acad Sci, USA 106:18644–18649, 2009, doi: 10.1073/pnas. 0904691106
• [3] R. Heller, P. Frandsen, E.D. Lorenzen ve H.R, Siegismund, “Are there really twice as many bovid species as we thought? ”, Syst Biol 62:490–493, 2013, doi: 10.1093/sysbio/syt004
• [4] R.J. Gruninger vd., “Anaerobic fungi [Phylum Neocallimastigomycota]: advances in unders-tanding their taxonomy, life cycle, ecology, role and biotechno logical potential”, FEMS Microbiol Ecol, 90:1–17, 2014, doi: 10.1111/ 1574-6941.12383
• [5] M. Yan vd., “Interrogating the viral dark matter of the rumen ecosystem with a global virome database” Nat Commun 14:5254, 2023, doi: 10.1038/ s41467-023-41075-2
• [6] M.B. Couger vd., “Transcriptomic analysis of lignocellulosic biomass degradation by the anaerobic fungal isolate Orpinomyces sp. strain C1A”, Biotechnol Biofuels, 8:208, 2015, doi: 10.1186/s13068-015-0390-0
• [7] L.H. Hagen vd., “Proteome specialization of anaerobic fungi during ruminal degradation of recalcitrant plant fiber” ISME J, 15:421–434, 2021, doi: 10.1038/s41396-020-00769-x
• [8] N.H. Youssef vd., “The genome of the anaerobic fungus Orpinomyces sp. strain C1A reveals the unique evolutionary history of a remarkable plant biomass degrader” Appl Environ Microbiol 79:4620–4634, 2013, doi: 10.1128/AEM.00821-13
• [9] R. Elliott vd., “The influence of anaerobic fungi on rumen volatile fatty acid concentrations in vivo,” J Agric Sci 109:13–17, 1987, doi: 10.1017/ S0021859600080928
• [10] S.A. Huws vd., “ Addressing global ruminant agricultural challenges through understanding the rumen microbiome: past, present, and future” Front Microbiol 9:2161, 2018, doi: 10.3389/fmicb.2018.02161
• [11] U. Comlekcioglu vd., “Effects of carbon sources on enzyme production of Neocallimastix sp. and Orpinomyces sp.” Kafkas Univ Vet Fak Derg. 18: 799–806, 2012
• [12] M. S. Elshahed vd., “ Characterization and rank assignment criteria for the anaerobic fungi [Neocallimastigomycota]”. International Journal of Systematic and Evolutionary Microbi-ology, 72[7], 005449, 2022.
• [13] F. C. Yazdıç vd., “Anaerobik funguslarda hidrojenozomlar: Hidrojen üreten organel-ler”. Mantar Dergisi, 12(2), 190-208. 2021.
• [14] D. A. Wubah, M. S. Fuller ve D. E. “Akin Resistant body formation in Neocallimastix sp., an anaerobic fungus from the rumen of a cow, ” Mycologia, 83(1), 40-47. (1991).
• [15] S. S. Calkins vd., “Development of an RNA interference (RNAi) gene knockdown protocol in the anaerobic gut fungus Pecoramyces ruminantium strain C1A,” PeerJ, 6, e4276. 2018.
• [16] C.A. Hooker vd., “A genetic engineering toolbox for the Lignocellulolytic anaerobic gut fungus Neocallimastix frontalis,” ACS Synth. Biol, 12, 1034–1045. doi: 10.1021/ acssyn-bio.2c00502. (2023).
• [17] J.K. Henske vd., “Biomass-degrading enzymes are catabolite repressed in anaerobic gut fungi,” AICHE J. 64, 4263–4270. doi: 10.1002/aic.16395, (2018a).
• [18] R. J. Gruninger vd., “Anaerobic Fungi [Phylum Neocallimastigomycota]: Advances in Understanding Their Taxonomy, Life Cycle, Ecology, Role and Biotechnological Potential, ” FEMS microbiology ecology, 90(1), 1-17.(2014).
• [19] J. E. Edwards vd., “PCR and omics based techniques to study the diversity, ecology and biology of anaerobic fungi: insights, challenges and opportunities,” Frontiers in microbiology, 8, 1657. (2017).
• [20] R.A. Hanafy, B. Johnson, M.S. Elshahed ve N.H. Youssef, “Anaeromyces contortus, sp. nov., a new anaerobic gut fungal species [Neocallimastigomycota] isolated from the feces of cow and goat,” Mycologia, 110[3], 502-512. (2018).
• [21] A. Joshi, vd., “Liebetanzomycespolymorphus gen. et sp. nov., a new anaerobic fungus [Neocallimastigomycota] isolated from the rumen of a goat,” MycoKeys, (40),89. (2018).
• [22] C. J. Pratt, E. E. Chandler, N. H. Youssef ve M. S. Elshahed, “Testudinimyces gracilis gen. nov, sp. nov. and Astrotestudinimyces divisus gen. nov, sp. nov., two novel, deep-branching anaerobic gut fungal genera from tortoise faeces,” International Journal of Systematic and Evolutionary Microbiology, 73(5), 005921. (2023).
• [23] C. G. Orpin, “Studies on the rumen flagellate Sphaeromonas communis,” Microbiology, 94(2), 270-280. (1976).
• [24] A. Karaman, H. Yücel, K. Ekinci ve S. Cömertpay, “Comparing Cellulotic Enzyme Ac-tivities of Neocallimastix sp. in Orpin’s and Menke’s Media,” Mantar Dergisi, 13(1), 55-61. (2022).
• [25] C. Orpın, “Taxonomy, biology, and distribution in nature. anaerobic fungi: biology, ecology, and function (М),” NY: Marcel Dekker Inc, 6-19. (1994).
• [26] R. A. Hanafy vd., “Taxonomy of the anaerobic gut fungi [Neocallimastigomycota]: a review of classification criteria and description of current taxa,” International journal of systematic and evolutionary microbiology, 72(7), 005322. (2022).
• [27] M.P. Bryant,“Commentary on the Hungate technique for culture of anaerobic bacteria,” Am J Clin Nutr;25:1324–1328. (1972).
• [28] R.E. Hungate,“A roll tube method for cultivation of strict anaer obes,” Meth Micro-biol;3:117–132. (1969)
• [29] S. Calkins, N.C. Elledge, R.A. Hanafy, M.S. Elshahed ve N. A. Youssef, “Fast and reliable procedure for spore collection from anaerobic fungi: Application for RNA uptake and long- term storage of isolates,” J Microbiol Methods;127:206–213. (2016).
• [30] K.V. Solomon, J.K. Henske, M.K. Theodorou ve M.A. O’Malley,“Robust and effective methodologies for cryopreservation and DNA extraction from anaerobic gut fungi,” Anae-robe 2016;38:39–46. (2016).
• [31] R. Nagpal, A.K. Puniya, J.P. Sehgal ve K. Singh, “Survival of anaerobic fungus Caecomyces sp. in various preservation methods: a comparative study,” Mycoscience;53:427–432. (2012).
• [32] Y.W. Ho ve D.J.S. Barr, “Classification of anaerobic gut fungi from herbivores with emp-hasis on rumen fungi from Malaysia”. Mycologia;87:655–677. (2018)
• [33] C.G. Orpin “The rumen flagellate Piromonas communis: its life- history and invasion of plant material in the rumen,” J Gen Micro biol;99:107–117. (1977)
• [34] C.G. Orpin “Isolation of cellulolytic phycomycete fungi from the caecum of the horse” J Gen Microbiol;123:287–296. (1981).
• [35] K.N. Joblin “Isolation, enumeration, and maintenance of rumen anaerobic fungi in roll tubes,” Appl Environ Microbiol;42:1119–1122. (1981).
• [36] I.B. Heath, T. Bauchop ve R.A. Skipp, “Assignment of the rumen anaerobe Neocallimastix frontalis to the Spizellomycetales [Chytridiomycetes] on the basis of its polyflagellate zo-ospore ultrastructure,” Can J Bot;61:295–307. (1983).
• [37] D.J.S. Barr, “The phylogenetic and taxonomic implications of flagellar rootlet morphology among zoosporic fungi” Biosystems;14:359–370. (1981).
• [38] J.J. Gold, I.B. Heath ve T. Bauchop, “Ultrastructural description of a new chytrid genus of caecum anaerobe, Caecomyces equi gen. nov., sp. nov., assigned to the Neocallimasticaceae,” Biosystems;21:403–415. (1988).
• [39] D.J. Barr, H. Kudo, K.D. Jakober ve K.J. Cheng,“Morphology and development of rumen fungi: nneocallimastix sp., ppiromyces communis, and oorpinomyces bovis gen. nov., sp. nov.” Can J Bot ;67:2815–2824. (1989).
• [40] A. Breton vd.,“Anaeromyces mucronatus nov. gen., nov. sp. a new strictly anaerobic rumen fungus with polycentric thallus,” FEMS Microbiol Lett;70:177–182. .(1990).
• [41] E. Ozkose, B.J. Thomas, D.R. Davies, G.W. Griffith ve M.K. Theodorou, “Cyllamyces aberensis gen. nov. sp. nov., a new anaerobic gut fungus with branched sporangiophores isolated from cattle,” Can J Bot, 79:666–673. (2001)
• [42] R.A. Hanafy, M.S. Elshahed, A.S. Liggenstoffer, G.W. Griffith ve N.H. Youssef, “Peco-ramyces ruminantium, gen. nov., sp. nov., an anaerobic gut fungus from the feces of cattle and sheep,” Mycologia, 109:231–243. (2017).
• [43] S.S. Dagar vd., “A new anaerobic fungus [Oontomyces anksri gen. nov., sp. nov.] from the digestive tract of the Indian camel [Camelus drom edarius],” Fungal Biol, 119:731–737. (2015)
• [44] T.M. Callaghan vd.,“Buwchfawromyces eastonii gen. nov., sp. nov.: a new anaerobic fungus [Neocallimastigomycota] isolated from buffalo faeces,” MC;9:11–28. (2015)
• [45] R.A. Hanafy vd.,“Seven new Neocallimastigomycota genera from wild, zoo- housed, and domesticated herbivores greatly expand the taxonomic diversity of the phylum,” Mycolo-gia;112:1212–1239. (2020).
• [46] R.A. Hanafy, M.S. Elshahed ve N.H. Youssef, “Feramyces austinii, gen. nov., sp. nov., an anaerobic gut fungus from rumen and fecal samples of wild Barbary sheep and fallow deer,” Mycologia;110:513–525. (2018).
• [47] R.A. Hanafy, N.H. Youssef ve M.S. Elshahed, “Paucimyces polynucleatus gen. nov, sp. nov., a novel polycentric genus of anaerobic gut fungi from the faeces of a wild blackbuck ante-lope,” Int J Syst Evol Microbiol;71:004832. (2021).
• [48] M. Stabel vd.,“Aestipascuomyces dupliciliberans gen. nov, sp. nov., the first cultured rep-resentative of the uncultured SK4 clade from aoudad sheep and alpaca,” Microorganisms, 8:1734. (2020).
• [49] A. Ranganathan vd.,“ Utilizing anaerobic fungi for two- stage sugar extraction and biofuel production from lignocellulosic biomass,” Front Microbiol,8:635. (2017).
• [50] E.T. Hillman vd.,“Hydrolysis of lignocellulose by anaerobic fungi produces free sugars and organic acids for two- stage fine chemical production with Kluyveromyces marxianus,” Bi-otechnol Prog;37:e3172. (2021).
• [51] C.L. Swift vd.,“Anaerobic gut fungi are an untapped reservoir of natural products,” Proc Natl Acad Sci USA;118:e2019855118. (2021).
• [52] H. Huuki vd.,“Long-term effects of early-life rumen microbiota modulation on dairy cow production performance and methane emissions,” Front Microbiol 13:983823. https://doi.org/10.3389/ fmicb.2022.983823(2022).
• [53] C.H. Meili vd.,“Patterns and determi nants of the global herbivorous mycobiome,” Nat Commun 14:3798. doi: 10.1038/s41467-023-39508-z (2023).
• [54] C. H. Meili vd.,“Diversity and community structure of anaerobic gut fungi in the rumen of wild and domesticated herbivores,” Applied and Environmental Microbiology, 90(2), e01492-23. [2024].
• [55] C.G. Orpin “The rumen flagellate Piromonas communis: its life- history and invasion of plant material in the rumen,” J Gen Micro biol;99:107–117. (1977)