Microdochium bolleyi is a fungal endophyte of cereals and grasses proposed as an ideal model organism for studying plant-endophyte interactions. A qPCR-based diagnostic assay was developed to detect M. bolleyi in wheat and Brachypodium distachyon tissues using the species-specific primers MbqITS derived from the ITS of the ribosomal gene. Specificity was tested against 20 fungal organisms associated with barley and wheat. Colonization dynamics, endophyte distribution in the plant, and potential of the seed transmission were analyzed in the wheat and model plant B. distachyon. The colonization of plants by endophyte starts from the germinating seed, where the seed coats are first strongly colonized, then the endophyte spreads to the adjacent parts, crown, roots near the crown, and basal parts of the stem. While in the lower distal parts of roots, the concentration of M. bolleyi DNA did not change significantly in successive samplings (30, 60, 90, 120, and 150 days after inoculation), there was a significant increase over time in the roots 1 cm under crown, crowns and stem bases. The endophyte reaches the higher parts of the base (2-4 cm above the crown) 90 days after sowing in wheat and 150 days in B. distachyon. The endophyte does not reach both host species' leaves, peduncles, and ears. Regarding the potential for seed transmission, endophyte was not detected in harvested grains of plants with heavily colonized roots. Plants grown from seeds derived from parental plants heavily colonized by endophyte did not exhibit any presence of the endophyte, so transmission by seeds was not confirmed. The course of colonization dynamics and distribution in the plant was similar for both hosts tested, with two differences: the base of the wheat stem was colonized earlier, but B. distachyon was occupied more intensively and abundantly than wheat. Thus, the designed species-specific primers could detect and quantify the endophyte in planta.

Department of Botany Faculty of Science Palacký University in Olomouc Olomouc Czech Republic

Department of Experimental Biology Faculty of Science Masaryk University Brno Czech Republic

Department of Plant Pathology Agrotest Fyto Ltd Kroměříž Czech Republic

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Chen J, Sharifi R, Khan MSS, Islam F, Bhat JA et al.. Wheat Microbiome: Structure, Dynamics, and Role in Improving Performance Under Stress Environments. Front Microbiol. 2022; 12:821546. doi: 10.3389/fmicb.2021.821546 . PubMed DOI PMC

Sharon O, Sun X, Ezrati S, Kagan-Trushina N, Sharon A. Transmission Mode and Assembly of Seed Fungal Endophyte Communities in Wheat and Wheat Wild Relatives. Phytobiomes J. 2023; 7:113–124. 10.1094/PBIOMES-11-22-0084-R. DOI

Bacon CW, White J, editors. Microbial Endophytes. 1st ed. CRC Press; 2000. Available from: 10.1201/9781482277302. vol. 2000. DOI

Rodriguez RJ, White JF Jr, Arnold AE, Redman RS. Fungal endophytes: diversity and functional roles. New Phytol. 2009; 182:314–330. doi: 10.1111/j.1469-8137.2009.02773.x PubMed DOI

Petrini O. Fungal Endophytes of Tree Leaves. in Microbial Ecology of Leaves (eds. Andrews JH, Hirano SS). Springer New York; 1991. p. 179–197.

Delaye L, García-Guzmán G, Heil M. Endophytes versus biotrophic and necrotrophic pathogens—are fungal lifestyles evolutionarily stable traits? Fungal Diversity. 2013; 60:125–135. 10.1007/s13225-013-0240-y. DOI

Sánchez Márquez S, Bills GF, Domínguez Acuña L, Zabalgogeazcoa I. Endophytic mycobiota of leaves and roots of the grass Holcus lanatus. Fungal Diversity. 2010; 41:115–123. 10.1007/s13225-009-0015-7. DOI

Photita W, Lumyong S, Lumyong P, McKenzie EHC, Hyde KD. Are some endophytes of Musa acuminata latent pathogens? Fungal Divers. 2004; 16: 131–140. http://hdl.handle.net/10722/223070.

Stergiopoulos I, Gordon TR. Cryptic fungal infections: the hidden agenda of plant pathogens. Front Plant Sci. 2014; 5:506. doi: 10.3389/fpls.2014.00506 . PubMed DOI PMC

Brader G, Compant S, Vescio K, Mitter B, Trognitz F, Ma L-J, et al.. Ecology and Genomic Insights into Plant-Pathogenic and Plant-Nonpathogenic Endophytes. Annu. Rev. Phytopathol. 2017; 55:61–83. doi: 10.1146/annurev-phyto-080516-035641 . PubMed DOI

Hardoim PR, van Overbeek LS, Berg G, Pirttilä AM, Compant S, Campisano A, et al.. The hidden world within plants: ecological and evolutionary considerations for defining functioning of microbial endophytes. Microbiol Mol Biol Rev. 2015; 79:293–320. doi: 10.1128/MMBR.00050-14 . PubMed DOI PMC

Fadiji AE, Babalola OO. Elucidating Mechanisms of Endophytes Used in Plant Protection and Other Bioactivities With Multifunctional Prospects. Front Bioeng Biotechnol. 2020; 8:532550. doi: 10.3389/fbioe.2020.00467 PubMed DOI PMC

Lata R, Chowdhury S, Gond SK, White JF Jr. Induction of abiotic stress tolerance in plants by endophytic microbes. Lett Appl Microbiol. 2018; 66:268–276. doi: 10.1111/lam.12855 . PubMed DOI

Bleša D, Matušinský P, Sedmíková R, Baláž M. The potential of Rhizoctonia-like fungi for the biological protection of cereals against fungal pathogens. Plants. 2021; 10:349. 10.3390/plants10020349. PubMed DOI PMC

Ghimire SR, Charlton ND, Bell JD, Krishnamurthy YL, Craven KD. Biodiversity of fungal endophyte communities inhabiting switchgrass (Panicum virgatum L.) growing in the native tallgrass prairie of northern Oklahoma. Fungal Diversity. 2011; 47:19–27. 10.1007/s13225-010-0085-6. DOI

Xia Y, Sahib MR, Amna A, Opiyo SO, Zhao Z, Gao YG. Culturable endophytic fungal communities associated with plants in organic and conventional farming systems and their effects on plant growth. Sci Rep. 2019; 9:1669. doi: 10.1038/s41598-018-38230-x PubMed DOI PMC

McKinnon AC. Plant tissue preparation for the detection of an endophytic fungus in planta. Methods Mol Biol. 2016; 1477:167–173. doi: 10.1007/978-1-4939-6367-6_13 PubMed DOI

Groppe K, Boller T. PCR assay based on a microsatellite-containing locus for detection and quantification of Epichloë endophytes in grass tissue. Appl Environ Microbiol. 1997; 63:1543–50. doi: 10.1128/aem.63.4.1543-1550.1997 . PubMed DOI PMC

Doss RP, Clement SL, Kuy S-R, Welty RE. A PCR-Based Technique for Detection of Neotyphodium Endophytes in Diverse Accessions of Tall Fescue. Plant Dis. 1998; 82:738–740. doi: 10.1094/PDIS.1998.82.7.738 . PubMed DOI

Zhou Y, Bradshaw RE, Johnson RD, Hume DE, Simpson WR, Schmid J. Detection and quantification of three distinct Neotyphodium lolii endophytes in Lolium perenne by real time PCR of secondary metabolite genes. Fungal Biol. 2014; 118:316–324. 10.1016/j.funbio.2014.01.003. PubMed DOI

Kelemu S, Dongyi H, Guixiu H, Takayama Y. Detecting and differentiating Acremonium implicatum: developing a PCR-based method for an endophytic fungus associated with the genus Brachiaria. Mol. Plant Pathol. 2003; 4:115–118. 10.1046/j.1364-3703.2003.00157.x. PubMed DOI

Ernst M, Neubert K, Mendgen KW, Wirsel SGR. Niche differentiation of two sympatric species of Microdochium colonizing the roots of common reed. BMC Microbiol. 2011; 11:242. 10.1186/1471-2180-11-242. PubMed DOI PMC

Ma WK, Siciliano SD, Germida JJ. A PCR-DGGE method for detecting arbuscular mycorrhizal fungi in cultivated soils. Soil Biol Biochem. 2005; 37:1589–1597. 10.1016/j.soilbio.2005.01.020. DOI

del Pilar Martínez-Diz M, Andrés-Sodupe M, Berbegal M, Bujanda R, Díaz-Losada E, Gramaje D. Droplet Digital PCR Technology for Detection of Ilyonectria liriodendri from Grapevine Environmental Samples. Plant Dis. 2020; 104:1144–1150. 10.1094/PDIS-03-19-0529-RE. PubMed DOI

Dinkins RD, Barnes A, Waters W. Microarray analysis of endophyte-infected and endophyte-free tall fescue. J Plant Physiol. 2010; 167:1197–1203. doi: 10.1016/j.jplph.2010.04.002 PubMed DOI

Asaf S, Khan AL, Khan MA, Al-Harrasi A, Lee IJ. Complete genome sequencing and analysis of endophytic Sphingomonas sp. LK11 and its potential in plant growth. 3 Biotech. 2018; 8:389. 10.1007/s13205-018-1403-z. PubMed DOI PMC

Koh S, Vicari M, Ball JP, Rakocevic T, Zaheer S, Hik DS, et al.. Rapid Detection of Fungal Endophytes in Grasses for Large-Scale Studies. Functional Ecology. 2006; 20:736–742. https://doi.org/3806624.

Kaul S, Sharma T, Dhar MK. “Omics” Tools for Better Understanding the Plant–Endophyte Interactions. Frontiers in Plant Science. 2016; 7. doi: 10.3389/fpls.2016.00955 PubMed DOI PMC

Reinecke P, Fokkema NJ. An evaluation of methods of screening fungi from the Haulm base of cereals for antagonism to Pseudocercosporella herpotrichoides in wheat. Trans Br Mycol Soc. 1981; 77:343–350. 10.1016/s0007-1536(81)80036-8. DOI

Murray DIL, Gadd GM. Preliminary studies on Microdochium bolleyi with special reference to colonization of barley. Trans Br Mycol Soc. 1981; 76:397–403. 10.1016/S0007-1536(81)80065-4. DOI

Fatemi L. Shadmani S, Jamali A. Biocontrol activity of endophytic fungus of barley, Microdochium bolleyi, against Gaeumannomyces graminis var. tritici. Mycologia Iranica. 2018; 5:7–14. 10.22043/mi.2019.118205. DOI

Matušinsky P, Sedláková B, Bleša D. Compatible interaction of Brachypodium distachyon and endophytic fungus Microdochium bolleyi. PLoS ONE. 2022; 17:e0265357. 10.1371/journal.pone.0265357. PubMed DOI PMC

Hong SK, Kim WG, Choi HW, Lee SY. Identification of Microdochium bolleyi Associated with Basal Rot of Creeping Bent Grass in Korea. Mycobiology. 2008; 36:77–80. doi: 10.4489/MYCO.2008.36.2.077 . PubMed DOI PMC

Hodges CF, Campbell DA. Infection of adventitious roots of Agrostis palustris by Idriella bolleyi. J. Phytopathol. 1996; 144:265–271. 10.1111/j.1439-0434.1996.tb01527.x. DOI

Damm U, Brune A, Mendgen K. In vivo observation of conidial germination at the oxic–anoxic interface and infection of submerged reed roots by Microdochium bolleyi. FEMS Microbiol Ecol. 2003; 45:293–299. 10.1016/S0168-6496(03)00161-2. PubMed DOI

Kirk JJ, Deacon JW. Control of the take–all fungus by Microdochium bolleyi, and interactions involving M. bolleyi, Phialophora graminicola and Periconia macrospinosa on cereal roots. Plant Soil. 1987; 98:231–237. 10.1007/BF02374826. DOI

Sieber T, Riesen T, Müller E, Fried P. Endophytic fungi in four winter wheat cultivars (Triticum aestivum L.) differing in resistance against Stagonospora nodorum (Berk.) Cast. & Germ. = Septoria nodorum (Berk.) Berk. J. Phytopathol. 1988; 122:289–306. 10.1111/j.1439-0434.1988.tb01021.x. DOI

Duczek LJ. Biological control of common root rot in barley by Idriella bolleyi. Can. J. Plant Pathol. 1997; 19:402–405. 10.1080/07060669709501067. DOI

Comby M, Gacoin M, Robineau M, Rabenoelina F, Ptas S, Dupont J, et al.. Screening of wheat endophytes as biological control agents against Fusarium head blight using two different in vitro tests. Microbiological Research. 2017; 202: 11–20. doi: 10.1016/j.micres.2017.04.014 . PubMed DOI

Trouvelot A, Kough JL, Gianinazzi-Pearson V. Estimation of VA mycorrhizal infection levels. Research for method having a functional significance. In: Physiological and Genetical Aspects of Mycorrhizae (Gianinazzi-Pearson V and Gianinazzi S, Eds.). INRA Press, Paris. 1986; 217–221. ISBN 2-85340-774-8.

Hernández-Restrepo M, Groenewald JZ, Crous PW. Taxonomic and phylogenetic re-evaluation of Microdochium, Monographella, and Idriella. Persoonia. 2016; 36:57–82. 10.3767/003158516X688676. PubMed DOI PMC

Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, Leunissen JA. Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res. 2007; 35:71–74. doi: 10.1093/nar/gkm306 PubMed DOI PMC

Walsh K, Korimbocus J, Boonham N, Jennings P, Hims M. Using Real-time PCR to Discriminate and Quantify the Closely Related Wheat Pathogens Oculimacula yallundae and Oculimacula acuformis. J Phytopathol. 2005; 153:715–721. 10.1111/j.1439-0434.2005.01045.x. DOI

Zhu H, Wen F, Li P, Liu X, Cao J, Jiang M, et al.. Validation of a Reference Gene (BdFIM) for Quantifying Transgene Copy Numbers in Brachypodium distachyon by Real-Time PCR. Appl Biochem Biotechnol. 2014; 172:3163–3175. doi: 10.1007/s12010-014-0742-4 . PubMed DOI

Higuchi R, Fockler C, Dollinger G, Watson R. Kinetic PCR Analysis: Real-time Monitoring of DNA Amplification Reactions. Nat Biotechnol. 1993; 11:1026–1030. doi: 10.1038/nbt0993-1026 PubMed DOI

He C, Wang W, Hou J. Characterization of Dark Septate Endophytic Fungi and Improve the Performance of Liquorice Under Organic Residue Treatment. Front Microbiol. 2019; 10:1364. doi: 10.3389/fmicb.2019.01364 PubMed DOI PMC

Tellenbach C, Grünig CR, Sieber TN. Suitability of quantitative real-time PCR to estimate the biomass of fungal root endophytes. Appl Environ Microbiol. 2010; 76:5764–5772. doi: 10.1128/AEM.00907-10 . PubMed DOI PMC

Sprague R. Gloeosporium decay in Gramineae. Phytopathology. 1948; 38:131–136.

Hoes JA. Dynamics of the microflora of subterranean parts of winter wheat in the dryland area of Washington. Phytopathology. 1962; 52:736.

Reinecke P. Microdochium bolleyi at the stem base of cereals. Journal of Plant Diseases and Protection. 1978; 85:679–685.

Chow YY, Rahman S, Ting ASY. Interaction dynamics between endophytic biocontrol agents and pathogen in the host plant studied via quantitative real-time polymerase chain reaction (qPCR) approach. Biol Control. 2018; 125:44–49. 10.1016/j.biocontrol.2018.06.010. DOI

Latz MAC, Kerrn MH, Sørensen H, Collinge DB, Jensen B, Brown JKM, et al.. Succession of the fungal endophytic microbiome of wheat is dependent on tissue-specific interactions between host genotype and environment. Sci Total Environ. 2021; 759:143804. doi: 10.1016/j.scitotenv.2020.143804 PubMed DOI

Tao G, Liu ZY, Hyde KD, Lui XZ, Yu ZN. Whole rDNA analysis reveals novel and endophytic fungi in Bletilla ochracea (Orchidaceae). Fungal Diversity. 2008; 33:101–122. http://cmuir.cmu.ac.th/jspui/handle/6653943832/60035.

Bayman P, Lebron LL, Tremblay RL, Lodge DJ. Variation in Endophytic Fungi from Roots and Leaves of Lepanthes (Orchidaceae). The New Phytologist. 1997; 135:143–149. 10.1046/j.1469-8137.1997.00618.x. PubMed DOI

Cook D, Gardner DR, Ralphs MH, Pfister JA. Swainsoninine Concentrations and Endophyte Amounts of Undifilum oxytropis in Different Plant Parts of Oxytropis sericea. J Chem Ecol. 2009; 35:1272–1278. doi: 10.1007/s10886-009-9710-9 . PubMed DOI

Liu J, Nagabhyru P, Schardl CL. Epichloë festucae endophytic growth in florets, seeds, and seedlings of perennial ryegrass (Lolium perenne). Mycologia. 2017; 109:691–700. doi: 10.1080/00275514.2017.1400305 . PubMed DOI

Abdelfattah A, Tack AJM, Lobato C, Wassermann B, Berg G. From seed to seed: the role of microbial inheritance in the assembly of the plant microbiome. Trends Microbiol. 2023; 31:346–355. doi: 10.1016/j.tim.2022.10.009 . PubMed DOI

Hodgson S, de Cates C, Hodgson J, Morley NJ, Sutton BC, Gange AC. Vertical transmission of fungal endophytes is widespread in forbs. Ecol Evol. 2014; 4:1199–1208. doi: 10.1002/ece3.953 PubMed DOI PMC

Kouzai Y, Kimura M, Yamanaka Y, Watanabe M, Matsui H, Yamamoto M, et al.. Expression profiling of marker genes responsive to the defence-associated phytohormones salicylic acid, jasmonic acid and ethylene in Brachypodium distachyon. BMC Plant Biol. 2016; 16:1–11. 10.1186/s12870-016-0749-9. PubMed DOI PMC

Sandoya GV, Buanafina de Oliveira MM. Differential responses of Brachypodium distachyon genotypes to insect and fungal pathogens. Physiol Mol Plant Pathol. 2014; 85:53–64. 10.1016/j.pmpp.2014.01.001. DOI