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A morphological change in the fungal symbiont Neotyphodium lolii induces dwarfing in its host plant Lolium perenne

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A morphological change in the fungal symbiont Neotyphodium lolii induces dwarfing in its host plant Lolium perenne
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  A morphological change in the fungal symbiont   Neotyphodiumlolii  induces dwarfing in its host plant   Lolium perenne W. R. SIMPSON a, *, J. SCHMID b , J. SINGH b , M. J. FAVILLE a , R. D. JOHNSON a a AgResearch Grasslands Research Centre, Private Bag 11008, Palmerston North 4442, New Zealand b Institute of Molecular Biosciences (IMBS), Massey University, Palmerston North, New Zealand a r t i c l e i n f o Article history: Received 28 September 2011Received in revised form10 November 2011Accepted 14 November 2011Available online 22 November 2011 Corresponding Editor :Paola Bonfante Keywords: Colony morphologyDwarfing  Lolium perenneNeotyphodium a b s t r a c t The endophytic fungus  Neotyphodium lolii  forms symbiotic associations with perennial rye-grass( Loliumperenne )andinfectionistypicallydescribedasasymptomatic.Herewedescribea naturally occurring New Zealand  N. lolii  isolate that can induce dwarfing of   L. perenne  andsuppressfloralmeristemdevelopmentinthedwarfedplants.Furthertothiswedemonstratethattheobservedhostdwarfingcorrelateswithareversiblemorphologicalchangeintheen-dophytethatappearsassociatedwithcolonyage.Myceliumisolatedfromnormallygrowing plants had a typical cottony appearance in culture whereas mycelium from dwarfed plantsappearedmucoid.Cottonycoloniescouldbeinducedtoturnmucoidafterprolongedincuba-tion and seedlings inoculated with this mucoid mycelium formed dwarfed plants. Mucoidcoloniesontheotherhandcouldbeinducedtoformcottonycoloniesthroughadditionalfur-ther incubation and these did not induce dwarfing. The reversibility of colony morphologyindicates that the mucoid dwarfing phenotype is not the result of mutation. Ten isolatesfrom other locations in New Zealand could also undergo the reversible morphologicalchangesinculture,inducedwarfingandhadthesamemicrosatellitegenotypeasthesrcinalisolate, indicating that a  N. lolii  genotype with the ability to dwarf host plants is common inNew Zealand. ª  2011 British Mycological Society. Published by Elsevier Ltd. All rights reserved. Introduction ForagegrassesbelongingtothePoaceaesubfamilyPooideae,in-cludingseveralimportantforageandturfspecies,oftenharbourendophyticfungibelongingtothegenera Neotyphodium and Epi-chlo € e  (Clavicipitaceae, Ascomycota).  Neotyphodium  endophyteslive entirely within the intercellular spaces of their grass hostswith the endophyte relying entirely on the host plant for dis-semination  via  the seed or through vegetative structures(Philipson & Christey 1986; Schardl  et al.  2004). The fungus canthereforebeconsideredtobeamaternallytransmittedcompo-nent of the plant and infection is typically symptomless.The association is mutually beneficial in agricultural set-tings, since the endophyte confers a number of biotic and abi-otic advantages to the host,including enhanced plant growth,protection from some mammalian and insect herbivores, en-hanced resistance to nematodes, resistance to some fungalpathogens and in some associations, enhanced drought toler-ance (Arachevaleta  et al.  1989; Kimmons  et al.  1990; Gwinn &Gavin 1992; Schardl & Phillips 1997; Schardl 2001; Scott 2001; Johnson  et al.  2003; Spiering   et al.  2006). Endophyte infectionhas also been implicated in modification of root morphology,osmotic adjustment, and mineral uptake (Malinowski &Belesky 1999; Malinowski  et al.  1999). *  Corresponding author . AgResearch Grasslands Research Centre, Private Bag 11008, Palmerston North 4442, New Zealand. Tel.: þ 64 6 3518127; fax:  þ 64 6 351 8032.E-mail address: wayne.simpson@agresearch.co.nz journal homepage: www.elsevier.com/locate/funbio fungal biology 116 (2012) 234 e 240 1878-6146/$  e  see front matter  ª  2011 British Mycological Society. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.funbio.2011.11.006  Neotyphodium lolii  can be grown axenically in culture anddemonstratepronouncedcolonymorphologyvariation.Myce-liumcanbesparseorabundantandappearfelted,cottony,ag-gregated or waxy (Christensen  et al.  1991).This paper describes the identification and characterisa-tion of a  N. lolii  genotype common within New Zealand thatis able to undergo reversible morphological changes andwhichalsohasasignificantimpactonhostplantarchitecture,leading to dwarfism. This is the first report of a naturally oc-curring endophyte causing dwarfing of its host plant. Materials and methods Fungal isolation and culture Fungus was isolated from endophyte-infected plants follow-ing surface sterilisation of plant tissue, as described byChris-tensen  et al.  (2002), and grown on Potato Dextrose Agar (PDA)(Difco   Becton, Dickinson and Co., USA) supplemented withtetracycline at a final concentration of 5  m gml  1 . After 2weeks, fungal mycelium was either transferred directly tofresh PDA or first macerated and plated as follows. A 6 mmmyceliaplugwas placed in anEppendorftube with 1 ml of Po-tato Dextrose Broth (PDB) and ground by hand, using a plasticpestle. The resulting macerate was transferred to a PDA plateand spread evenly. Cultures were incubated at 22   C in thedark for up to 8 weeks with observation. Seedling inoculation Seed was surface-sterilised and inoculated as described byLatch & Christensen (1985). Plants were grown for  ca.  6 weeksbefore indentifying infected individuals using an immunoblotassay as described below. Immuno-detection of endophyte Plants were grown to at least the 3 e 4 tiller stage before detec-tion of endophyte was undertaken. Tillers were cut basally  ca. 5 mmfromsoillevel.Wherenecroticsheathtissuewaspresentit was carefully peeled off the tiller before making a transversecut. The freshlycut end ofthe tiller was placed onto a nitrocel-lulose membrane (NCM) (0.45  m m) leaving a circular outline of the moist cut end. A positive and a negative control tiller wereblottedtothemembraneusingplantsofknownendophytesta-tus. Surfaces on blotted sheets with no bound protein wereblocked by immersion in a milk protein blocking solution (BS)(Tris(hydroxymethyl) methylamine 2.42 g, NaCl 2.92 g, Non-fatmilkpowder5 g,1 MHCl10mlmadeupto1 lwithROwaterad- justedtopH7.5).Membraneswereshakenonanorbitalshakerfor at least 2 h at room temperature. BS was decanted off themembrane and it was rinsed 2   with fresh BS before adding 25  m l primary antibody (rabbit anti-endophyte produced atAgResearchinconjunctionwithMasseyUniversity’sSmallAn-imal Production Unit) in 25 ml BS (1:1000 dilution). Following 15 min shaking at room temperature the membrane was incu-bated overnight at 4   C. Excess primary antibody was removedby decanting and rinsing twice in fresh BS. The secondaryantibody (goat anti-rabbit IgG-AP, sc-2034, Santa CruzBiotechnology,USA)wasadded,6.25  m lin25 mlBS(1:4000dilu-tion) and shaken for 15 min at room temperature before incu-bating at 4   C for 5 h. Excess secondary antibody was removedby decanting and rinsing twice in BS. Chromogens were pre-pared by dissolving separately 20mg Fast Red TR (SigmaF-2768) in 12.5 ml Tris buffer (Tris(hydroxymethyl) methyl-amine 24.2 g in 1 l RO water adjusted to pH 8.2) and 12.5 mg of naphtholAS-MXphosphate(SigmaN4875)in12.5 mlTrisbufferper 10 cm 2 of NCM. Chromogen solutions were combined andthe NCM immersed, shakenat roomtemperaturefor  ca. 15 minuntilredcolourdevelopsoncontrolpositiveblot.Developmentwas stopped by rinsing three times in RO water. Preparation and inoculation of clonal plantlets Clonal material was prepared as described by Simpson (2009)using Murashige and Skoog agar culture media, plants wereallowed to develop to the 3 e 5 tiller stage and individual tillersremoved and inoculated by making an incision at the base of the tillers as described. After 4 weeks, inoculated plantletswere planted in potting soil then grown in a greenhouse for 6weeks. The infection statusof inoculated plantlets was exam-inedusingimmunoblotandconfirmedusinglightmicroscopy. Microscopic examination Tillers were selected from mature plants for endophyte detec-tion. Any necrotic sheath tissue was peeled back off the pseu-dostem exposing clean, live sheath tissue. The outermost of the remaining sheaths was removed and manipulated undera dissecting microscope at 16  magnification. The sheath waslaid on a cutting surface adaxial epidermis facing up, a shallowtransversecutwasmadewithascalpelandtheepidermisgentlylifted, separated and pulled off the sheath. The epidermaltissue was mounted on a drop of aniline blue stain (glycerol50%, lactic acid 25%, water 24.95 %, aniline blue 0.05%) ona 25  75  1 mm microscope slide and covered witha 22  22mm cover slip, heated over a naked flame, allowed tocoolandexaminedat100  and400  usingacompoundmicro-scope. To examine excised floral meristems clonal plants werevernalised and then exposed to long days to induce flowering.Representativetillersweredissected,following0,5,and14dex-posuretolongdays,usingascalpeltoexposethemeristemandexamined under a dissecting microscope at 20  magnification. Endophyte elimination Endophyte was eliminated from infected plants by fungicidetreatment as described by Latch & Christensen (1982). Plantswere checked for negative tillers by immunoblot andendophyte-free daughter tillers developing from these werethen potted individually. Simple sequence repeat (SSR) genotyping of   Neotyphodiumlolii  isolates Total plant or plant and endophyte DNA was extracted from100 mg fresh weight of endophyte-free or endophyte-infectedplant tissue, respectively, using the QIAgen DNA extractionkit (DNeasy  Plant Mini Kit) as per the manufacturer’sA morphological change in  N. lolii  induces dwarfing in  L. perenne  235  instructions and quantified using a Hoefer Scientific TKO 100Fluorometer. PCR primers designed to SSRs used for genotyp-ing included the B10 and B11 primers (Moon  et al.  1999) aswell as those with an ‘ans’ prefix (Kirkby  et al.  2011) whichwere mined from a  N. lolii  expressed sequence tag (EST) re-source (Bassett  et al.  2009). The forward primers for both B10and B11 were fluorescently labelled with 5 0 -fluorescein phos-phoramidite (6-FAM) (Invitrogen), whereas the forward ‘ans’primers were synthesised (Integrated DNA Technologies,Inc.) with a 21 nucleotide M13 tail sequence at the 5 0 -terminus(5 0 -TGTAAAACGACGGCCAGT-3 0 ), to facilitate universal label-lingofPCRproductsbyanM13primer(Schuelke2000)labelled withFAM.Reverse‘ans’primersweresynthesisedwiththese-quence5 0 -GTTTCTT-3 0 atthe5 0 -terminusendtopromotenon-templated adenylation at the 3 0 -terminus end of PCR product(Brownstein  et al.  1996). Genotyping reactions with the B10and B11 primers were performed as described by Moon  et al. (1999), and for the ‘ans’ primers as described by Faville  et al. (2004),exceptthatafinalconcentrationof2.5 mMmagnesiumchlorideand0.75unitsofPlatinumTaqDNApolymerase(Invi-trogen, Carlsbad, CA, USA) were used. Genotypic analysisfollowedFaville etal. (2004),exceptthatcapillaryelectrophore-sis on an ABI 3100 Genetic Analyser (Applied Biosystems,FosterCity,California,USA)usedPOP-7  polymer.Electrophe-rograms were analysed using ABI Prism GeneScan v3.7(Applied Biosystems) and fragments were sized using GeneMarker v1.75 (SoftGenetics LLC, PA, USA). Results Observation of dwarfed plants in a perennial ryegrass population infected with  Neotyphodium lolii During routine growth of seventy endophyte-infected peren-nial ryegrass seedlings seventeen were observed to bedwarfed with fine-leaved tillers and dark green pigmentation(Fig 1A) compared to the remaining plants which retaineda normal phenotype (Fig 1B). Colony morphology of endophyte isolated from dwarfed andnormal plants differs To investigate further the nature of the observed host plantdwarfing the infecting fungus was isolated from both normaland dwarfed plants and grown axenically. Dwarfed plantswere found to host an endophyte that had an in-culture mor-phology that differed from the endophyte isolated from nor-mal-phenotype host plants. The fungus that emerged fromnormal-phenotype plants was filamentous with aerial whiteand cottony hyphae (Fig 2A), whereas the fungus isolatedfrom the dwarf plants was mucoid (Fig 2B). We will subse-quently refer to these two colony morphologies as ‘whiteand cottony’ or ‘mucoid’, respectively. Fig 1  e  Dwarfed (A) and normal- (B) phenotype diploid perennial ryegrass (  Lolium perenne  ) plants observed in a populationconsisting predominantly of normal-phenotype plants. A fungicide-treated dwarfed plant (C) displays the normal pheno-type. Vernalised and induced plants (D) infected with mucoid (left) and white cottony (right)  N. lolii . Note inflorescence for-mation on the white and cottony-infected plant but not the mucoid endophyte-infected plant. Excised tiller meristems (E) of white and cottony-infected (left) and mucoid-infected (right) perennial ryegrass tillers following vernalisation and 0, 5, and14 d exposure to long days. 236 W. R. Simpson  et al.  The mucoid endophyte is responsible for host plant dwarfing To determine if the mucoid fungus isolated from the dwarfedplantswasresponsiblefortheobservedhostphenotype,singletillers were treated with fungicide to cure the plants of fungalinfection.Tillerscuredofendophyteinfectionwereconfirmedasendophyte-freebyanimmunoblotassayanddevelopedintoplants that displayed a normal phenotype (Fig 1C). Having shown that dwarfed plants cured of endophyte infectionreturn to a normal phenotype we then determined if artificialinoculation of endophyte-free seedlings with the mucoid fun-gus results in dwarfing of the infected plants. Of 341 seedlingsinoculated 81 died and of the surviving 260 plants 20 becameinfected (7.7 % infection). This infection frequency was lowcomparedtothatofthewhiteandcottonyfunguswherethein-fection frequency was 27.4 %. All mucoid-infected plants de-veloped a dwarf phenotype (Fig 1A). Endophyte colony morphology can undergo spontaneouschange  in vitro  that directly predicts the fate of the host plantafter inoculation We observed that mycelium from a freshly isolated whiteand cottony colony from a normal-phenotype plant, whenmacerated and plated at low density onto PDA initially de-veloped into white and cottony colonies as expected(Fig 2C). However, after prolonged incubation (4 e 5 weeks)100 % of the colonies displayed a mucoid phenotype(Fig 2D). When these mucoid colonies were incubated fora further 3 e 4 weeks we observed reversion at low frequencyto white and cottony colonies (Fig 2E and F). To determinethe effect of endophyte colony morphology on host pheno-type, mycelium from freshly isolated cottony and mucoidcolonies and mycelium from secondary cottony colonies,i.e. colonies that had developed from mucoid colonies,were used to inoculate endophyte-free perennial ryegrassseedlings. Of 73 plants inoculated with white and cottonyfungus, 27.4 % (20/73) became infected and in all casesgave rise to normal-phenotype plants. This was also trueof white and cottony colonies that had developed from mu-coid colonies with 29.4 % (30/102) of inoculated plants be-coming infected and showing a normal phenotype. Thiscontrasts sharply with the inoculations with mucoid fungus,in which 7.9 % (6/76) of plants became infected, all of whichwere dwarfed. These experiments clearly demonstrate thatthe change from white and cottony to mucoid is reversibleand that only the mucoid fungus induces dwarfing in itshost grass. Fig 2  e  White and cottony (A) and mucoid (B) mycelium of   Neotyphodium lolii  emerging from surface-sterilised tissue piecesincubated on PDA at 22   C. Colony transformation over time: white and cottony colonies (C) become mucoid (D), and finallyrevert back to white and cottony (E). The reversion back to white and cottony is sporadic throughout the culture plate (F). A morphological change in  N. lolii  induces dwarfing in  L. perenne  237  Clonal plants infected with mucoid  Neotyphodium lolii  arealso dwarfed To eliminate any possibility of host plant genotype, which isunique for each seedling due to the out-crossing nature of  Loliumperenne ,havinganimpactontheobserveddwarfingphe-notype, further inoculation experiments were performed onclonallyderivedryegrassplants.Asingleperennialryegrassge-notype was grown axenically and divided into a number of cloned plantlets which were maintained as endophyte-free orwereinoculatedwitheitherwhiteandcottonyormucoidendo-phyte. Only mucoid-infected clones displayed a dwarf pheno-type whereas the white and cottony-infected clones orendophyte-free clones displayed a normal phenotype. Theseplantsweresubsequentlyusedinfurtherexperimentstoexam-ine the effect of endophyte status on host flowering. Dwarfed plants infected with the mucoid endophyte do not flower under long day conditions following vernalisation In the srcinal population of dwarfed plants floral emergencewasneverobserveddespiteplantsbeingexposedtovernalisa-tionconditions.Tostudythisinmoredetailandtoremovepos-sible plant genotype affects, infected clonal plants weretreated to induce flowering. Dwarfed plants did not flower,while normal-phenotype white and cottony endophyte-infected plants did (Fig 1D). Tillers of both normal-phenotypeand dwarfed plants were dissected to directly examine floralmeristems following 0, 5, and 14 d exposure to long days. Thedevelopment of floral meristems of dwarfed plants infectedwith mucoid endophyte was suppressed compared to nor-mal-phenotypeplantsinfectedwiththewhiteandcottonyen-dophyte. Although some rudimentary development of thefloral meristems occurred in the mucoid endophyte-infectedplant by day 14, floral emergence did not occur (Fig 1E). Mucoid and white and cottony endophytes areindistinguishable from each other and ten additionalwild-type isolates with eight microsatellite markers Todetermine thegenetic background of the mucoid and whiteandcottonyendophytesweperformedSSRanalysisusingeightmicrosatellite markers that have been shown to discriminate Neotyphodium lolii  strains. SSR analysis was also performed onten additional  N. lolii  isolates sourced from diverse locationsin New Zealand (Table 1). Results showed that the mucoid andwhite and cottony endophytes are indistinguishable fromeach other as well as from the additional  N. lolii  isolates, withproduct sizes (  0.5 bp) of 177 bp, for markers B10 and B11,303 bp for ans015, 389 bp for ans017, 311 bp for ans025, 325 bpfor ans030, 265 bp for ans036, and 264 bp for ans056, and pro-vides evidence that the fungal strains srcinally isolated inthis study are typical of New Zealand wild-type  N. lolii . The ability to form mucoid isolates that dwarf their hosts iswide-spread among New Zealand ecotype wild-type Neotyphodium lolii To determine whether the additional  N. lolii  isolates could alsoundergo morphological change, further observations weremade. Isolates were incubated on PDA plates and observedover time. All of the isolates srcinally grew with a white andcottony phenotype but formed mucoid colonies at a frequencyof100 %after4 e 5weeksofgrowth.Afterfurtherincubationfor2 e 3 weeks a low frequency switch back to the white and cot-tony phenotype was observed. We also tested white and cot-tony and mucoid mycelium from five of the isolates for theirability to induce dwarfing by inoculating endophyte-free rye-grassseedlings.Comparabletoresultsobtainedforthesrcinalwhite and cottony and mucoid endophytes isolated in thisstudy, none of the plants infected with white and cottony en-dophyte were dwarfed, whereas all plants infected with themucoid endophyte were dwarfed. Re-isolation of endophytefromdwarfedandnormal-phenotypeplantsconfirmedawhiteand cottony or mucoid phenotype, respectively. Discussion In this study an observation was made of a number of dwarf seedling plants amongst an otherwise normal-phenotypepopulation of seedlings. The endophyte from normal-pheno-typeplantswaswhiteandcottonyandtypicalof  Neotyphodiumlolii  colonies whereas the endophyte from dwarf plants wasmucoid. We further demonstrated that elimination of endo-phyte from dwarf phenotype plants restored normal growthand that infection of endophyte-free perennial ryegrass withthe mucoid endophytecaused dwarfing. In subsequent exper-iments we discovered that the wild-type endemic white andcottony endophyte could be induced to switch to a mucoidphenotype in culture at a high frequency and that this was re-versible.Akeyfindingwasthatmucoidorcottonycoloniesob-tained in this way behaved in a manner similar to the srcinalmucoid and cottony colonies, causing dwarfed or normalplants, respectively, when inoculated into the host.Although the mechanism underling the observed morpho-logical change in the fungus is unknown, colony age appearsto be a factor. Aging-induced changes in the appearance of colonies are common in many fungi but in most cases arereadily reversible when transferred to fresh growth medium(Griffiths 1992; Maheshwari & Navaraj 2008). This was not ob-served in the present study whereby colony morphology wasstable between subcultures. On this basis the morphologicalchanges observed with  N. lolii  are likely to be due to fungal Table 1 e  N. lolii  strains used in this study. Designation Origin A11754 Hawkes Bay a A11293 Auckland a A11602 Auckland a A11787 Gore a (South Island)A12440 Europe a A10351 Auckland a ID 256 KaikoheID 374 Waiau (South Island)ID 379 Waiau (South Island)ID 418 Palmerston Northa Seed sourced from the Margot Forde Forage Germplasm Centre(MFFGC). 238 W. R. Simpson  et al.
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