Inoculation strategies to assess biological interactions between Fusarium and Alternaria species infecting sorghum

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1 University of Nersk - Linoln DigitlCommons@University of Nersk - Linoln Ppers in Plnt Pthology Plnt Pthology Deprtment 2008 Inoultion strtegies to ssess iologil intertions etween Fusrium nd Alternri speies infeting sorghum Denn L. Funnell-Hrris USDA-ARS, Denn.Funnell-Hrris@rs.usd.gov Jeffrey F. Pedersen USDA-ARS, jpedersen1@unl.edu Follow this nd dditionl works t: Prt of the Other Plnt Sienes Commons, Plnt Biology Commons, nd the Plnt Pthology Commons Funnell-Hrris, Denn L. nd Pedersen, Jeffrey F., "Inoultion strtegies to ssess iologil intertions etween Fusrium nd Alternri speies infeting sorghum" (2008). Ppers in Plnt Pthology This Artile is rought to you for free nd open ess y the Plnt Pthology Deprtment t DigitlCommons@University of Nersk - Linoln. It hs een epted for inlusion in Ppers in Plnt Pthology y n uthorized dministrtor of DigitlCommons@University of Nersk - Linoln.

2 404 Epidemiology / Épidémiologie Inoultion strtegies to ssess iologil intertions etween Fusrium nd Alternri speies infeting sorghum Denn L. Funnell-Hrris nd Jeffrey F. Pedersen Astrt: New ultivrs of sorghum re eing developed for inresed food use, s livestok feed, nd for the ioenergy industry y mnipultion of seondry metoli pthwys through reeding. Previous work hs suggested tht suh modifitions my hve n impt on ultivr response to fungl pthogens. We investigted four inoultion strtegies tht my e used in studies involving pthogens of sorghum. Plnts of elite sorghum genotypes Whetlnd nd RTx430, ommonly used in reeding progrms, were inoulted with isoltes of Alternri lternt, Fusrium equiseti, Fusrium prolifertum, Fusrium solni, Fusrium thpsinum, nd Fusrium vertiillioides reovered from field-grown sorghum. Wound inoultion of the pedunles of heds t nthesis demonstrted tht isoltes of F. vertiillioides nd F. thpsinum produed men lesion lengths tht were signifintly greter thn ontrol inoultions. The length of this ssy ws deresed y inoulting fungi onto plnts of very erly mturing sorghum line (N247). Sprying heds t nthesis with onidil suspensions over three onseutive dys in the greenhouse ws onduted to ttempt to mimi onditions in the field. Results indited tht plnts treted with onidi of F. thpsinum my develop mture grin with redued germintion. Seed nd seedling inoultions produed responses over reltively short time period. Seed inoultions were onduted in mnner similr to orn iossy. Seedling inoultions, onduted in Mgent vessels, llowed for mesurement of effets of inoulum on roots, stlks, nd leves. Following inoultion of RTx430 nd Whetlnd plnts grown in Mgent vessels with F. thpsinum, nerly ll mesurements were signifintly different from those of the wter ontrols exept for perent root infetion of RTx430. Key words: sorghum pthogens, Alternri lternt, Fusrium equiseti, Fusrium solni, Fusrium thpsinum, Fusrium vertiillioides, Sorghum iolor, inoultion methods. Funnell-Hrris nd Pedersen: sorghum / hed light / inoultrésumé : De nouveux ultivrs de sorgho plus produtifs sont à l heure tuelle développés pr mnipultion des / Fusrium speies 413 voies métoliques seondires durnt le proessus de séletion. Ces nouveux ultivrs sont destinés à l limenttion des humins, à elle du étil insi qu à l industrie des ioénergies. Des trvux préédents ont suggéré que de telles modifitions peuvent influener l réponse des ultivrs qunt à leur résistne ux gents pthogènes fongiques. Nous vons étudié qutre strtégies d inoultion qui peuvent être utilisées dns les études reltives ux gents pthogènes qui s ttquent u sorgho. Des plnts de génotypes élites de sorgho Whetlnd et RTx430, ommunément utilisés dns les progrmmes de séletion, ont été inoulés ve des isolts d Alternri lternt, de Fusrium equiseti, de Fusrium prolifertum, de Fusrium solni, de Fusrium thpsinum et de Fusrium vertiillioides otenus de plnts de sorgho ultivés en hmps. L inoultion de lessures usées ux pédonules des épis rrivés u stde de l nthèse démontré que les isolts de F. vertiillioides et de F. thpsinum produisient des lésions dont les longueurs moyennes étient psslement plus grndes que elles produites pr les inoultions témoins. L durée de e iotest été rourie en inoulnt des plnts d une lignée très hâtive de sorgho (N247) ve des hmpignons. Afin d essyer de reproduire les onditions de plein hmp, les épis, u stde de l nthèse, ont été vporisés, en serre, ve une suspension onidile, et e, sur une période de trois jours onséutifs. Les résultts ont montré que les plnts trités ve les onidies de F. thpsinum peuvent produire des grins mtures, mis que leur tux de germintion ser réduit. Aepted 10 June D.L. Funnell-Hrris.1 USDA-ARS, Grin, Nersk, 314 Biohemistry Hll, Linoln, J.F. Pedersen. USDA-ARS, Grin, Forge Nersk, 314 Biohemistry Hll, Linoln, Forge nd Bioenergy Reserh, Deprtment of Plnt Pthology, University of NE , USA. nd Bioenergy Reserh, Deprtment of Agronomy nd Hortiulture, University of NE , USA. 1 Corresponding uthor (e-mil: Denn.Funnell-Hrris@rs.usd.gov). Cn. J. Plnt Pthol. 30: (2008)

3 Funnell-Hrris nd Pedersen: sorghum / hed light / inoultion methods / Fusrium speies 405 Les inoultions de grines et de plntules induisent des réponses à rève éhéne. Les inoultions de grines furent effetuées selon une méthode semlle à elle utilisée pour les iotests effetués sur le mïs. Les inoultions des plntules, fites dns des oîtes Mgent, ont permis de mesurer les effets de l inoulum sur les rines, les tiges et les feuilles. À l suite de l inoultion ve F. thpsinum des plnts de RTx430 et de Whetlnd ultivés dns les oîtes Mgent, presque toutes les mesures effetuées vriient psslement de elles provennt des solutions témoins queuses, suf pour e qui est du tux d infetion des rines de RTx430. Mots-lés : gents pthogènes du sorgho, Alternri lternt, Fusrium equiseti, Fusrium solni, Fusrium thpsinum, Fusrium vertiillioides, Sorghum iolor, méthodes d inoultion. Introdution Sorghum ultivrs re eing developed for inresed use for food, livestok feed, nd for the ioenergy industry (Tylor et l. 2006). Chrteristis modified inlude deresed lignin (Funnell nd Pedersen 2006) nd, in grin, redued pigments (Pedersen nd Toy 2001), inresed lysine (Tesso et l. 2006), nd deresed mylose ( wxy ) strh (Pedersen et l. 2005). Suh modifitions in the si struture nd iohemistry of the plnt my ffet intertions with fungi, pthogeni or non-pthogeni (Funnell nd Pedersen 2006). Speies of Fusrium (Fusrium vertiillioides (S.) Nirenerg (previously known s Fusrium moniliforme Sheldon), Fusrium prolifertum (Mtsushim) Nirenerg, Fusrium thpsinum Klittih, Leslie, Nelson et Mrss sp. nov., Fusrium solni (Mrt.) Appel & Wollenw. emend. Snyd. & Hns., nd Fusrium equiseti (Cord) S. sensu Gordon) nd memers of the Alternri lternt (Fr.:Fr.) Keissl. omplex were used in this study. These speies re redily isolted from sorghum plnts nd from sorghum field soil (Leslie et l. 1990, 2005; Funnell nd Pedersen 2006). Some of these speies, e.g., F. vertiillioides nd F. thpsinum, my dmge sorghum grin or forge mteril prior to hrvest (Menkir et l. 1996). All of these speies n olonize sorghum ut not produe ovious symptoms (Funnell nd Pedersen 2006). Sine ll n produe toxins (Leslie et l. 2005), infetions y these speies pose prolems for the yield nd qulity of the hrvested rop nd possile prolems during storge (Cstor nd Frederiksen 1980). Four different iossys were exmined for use to sreen sorghum germplsm. One stndrd sorghum ssy involves quntittively ompring lesions resulting from wound inoultion of pedunles of developing heds (Jrdine nd Leslie 1992). This ssy hs een used to hrterize pthogeniity or virulene of the F. moniliforme omplex ssoited with sorghum (Jrdine nd Leslie 1992) nd to ompre the response of sorghum lines to inoultion (Funnell nd Pedersen 2006). The seond ssy ws sed on sorghum field ssy to ssess grin mold responses (Fores et l. 1989) with modifitions to the protool for inoulum delivery nd onditions of infetion. The third ssy ws sed on orn ssy (Bon et l. 1994) in whih sorghum grin ws inuted with fungl onidi. The lst ssy ws modified from tht of sorghum (Leslie et l. 2005), the mjor modifition eing tht onidi were inoulted onto pprently sterile plnts grown in plnt growth medium in Mgent vessels. Mterils nd methods Fungl isoltes Fungl isoltes were otined from sorghum or sorghum fields. FRC-M-0900, isolted from sorghum field soil in Texs, represented F. solni. Stlk isoltes, FRC-M-1141 from Cliforni nd FRC-M-3790 from Missouri, represented F. vertiillioides nd F. thpsinum, respetively (oth formerly known s F. moniliforme). These three isoltes were otined from the Fusrium Reserh Center, Pennsylvni Stte University, USA. Isolte H02-765S ws isolted from sorghum grin grown in Nersk nd represented F. equiseti. Two representtives of the A. lternt omplex, isoltes H02-755S nd H02-781S-3, originted from sorghum grin grown in Nersk. Isolte H02-811L-2, reovered from lef tissues from sorghum plnts grown in Nersk, represented F. prolifertum. For omprison of plnt responses, the F. thpsinum isolte hd een known from previous work to e highly virulent (Funnell nd Pedersen 2006). The F. solni isolte served s the wekly virulent isolte sine F. solni is ommonly isolted from sorghum soil nd deris (Leslie et l. 1990). Isoltes of F. vertiillioides, F. equiseti, F. prolifertum, nd A. lternt were used in some ssys to represent different fungl speies ommonly isolted from sorghum nd hd differing responses in preliminry ssys. Fusrium nd Alternri spp. isoltes were identified y morphologil hrteristis, nd identifition ws onfirmed y sequene nlysis of onserved DNA regions (GenBnk. Nos. EF152426, EF , EF152428, EF152430, EF152432, EF152433, EU016680, nd EU595566). Plnt mterils Eh iossy ws onduted using Whetlnd nd RTx430, sine these elite sorghum genotypes hve een ommonly used in reeding progrms nd in other reserh on sorghum nd grsses in generl (Rodriguez-Herrer et l. 2006). RTx430 hs een shown to e highly suseptile to grin mold diseses (Rodriguez-Herrer et l. 2006) ut no pulished dt of response of Whetlnd to grin mold disese hs een found. Other genotypes used in different iossys inluded very erly mturing lines N246 nd N247, BTx631, whih lks the purple wound response, nd Tx2911 nd Tx2927, reported to hve resistne to grin mold diseses (Ntionl Plnt Germplsm System, USDA-ARS;

4 406 Cn. J. Plnt Pthol. Vol. 30, 2008 Reltively len, true-reeding seed ws generted in the greenhouse y gging heds t nthesis. No fungl growth or teril ontmintion ws deteted when seeds were plted onto wter gr (0.6 %) or germinted in sterile wter. Biossys were onduted in greenhouses or growth hmers. For iossys onduted in greenhouses, plnts were grown in 25.4 m dimeter pots, 2 plnts per pot. Soil mix used ws 83% soil (Generl Exvting, Linoln, Ner.), 6% #3 vermiulite, hortiulturl grde perlite (Therm-O-Rok Est In., New Egle, P.), 4% pet moss (Conrd Ffrd, In., Agwm, Mss.), nd 3% snd (Generl Exvting). Biossys Wound-inoultion iossy The wound-inoultion ssy ws essentilly s desried y Jrdine nd Leslie (1992). Briefly, roth ultures were prepred y inoulting 50 ml sterile tues ontining 5 ml of full-strength potto dextrose roth (PDB; Beton, Dikinson nd Co., Sprks, Md.) nd 10 sterile toothpiks with n gr plug from 1-week-old one-hlf strength potto dextrose gr (PDA) ulture of eh fungl isolte, then inuting t 20 C for 10 dys. The dy t whih plnts rehed nthesis, defined s pproximtely hlf of the plnts hving pniles with 50% of the nthers exerted, ws noted, nd two weeks following this dte, pedunles were wound inoulted with fungl-infested toothpiks. Toothpiks inuted in sterile roth served s the negtive ontrol. Inoultions were rndomized so tht eh plnt within pot ws inoulted with different isoltes or with fungl isolte nd sterile roth. Eighteen dys following inoultion, pedunles were split longitudinlly nd the totl length of the disolortion, whih my inlude regions with disontinuous disolortion, ws mesured. Two ssys were onduted with RTx430 nd Whetlnd. Plnts were inoulted with F. solni, F. vertiillioides, F. thpsinum, F. equiseti, nd A. lternt (H02-781S-3) or with sterile toothpiks inuted in sterile roth. Levene homogeneity of vrine (HoV) tests (SAS 2000) were onduted on dt from iossys with the sme plnt genotype. Bsed on results from these nlyses, the two iossys onduted on Whetlnd plnts were omined nd nlyzed s rndomized omplete loks with individul ssys serving s loks, wheres those from RTx430 plnts were nlyzed s seprte experiments. Wound inoultions were onduted on very erly mturing genotypes N246 nd N247 (Pedersen nd Toy 1999). Ten plnts eh of N247 nd N246 were inoulted with F. solni, F. thpsinum, nd A. lternt or with sterile toothpiks inuted in sterile roth. This ssy ws onduted twie. HoV tests were onduted on dt from iossys with the sme plnt genotype. Bsed on these nlyses, the two iossys onduted on N247 plnts were omined nd nlyzed s rndomized omplete loks with individul ssys serving s loks, wheres those from N246 plnts were nlyzed seprtely. All nlyses of vrine were ompleted using Pro GLM (SAS 2000), nd men lesion lengths nd lest signifint differenes (LSDs) mong isoltes were reported. Spry inoultion iossy The protool for the spry inoultion iossy ws developed sed on sorghum grin mold field ssy (Fores et l. 1989) with refinements for inoulum delivery nd onditions for infetion. Two ssys were onduted, eh with genotypes RTx430 nd Whetlnd. For the first ssy, plnts were inoulted with F. thpsinum, F. vertiillioides, F. solni, F. prolifertum, nd A. lternt (H02-755S); Whetlnd plnts were inoulted two weeks prior to plnts of RTx430 euse of differenes in loom dte. Individul plnts were spryed with 10 ml of fungl onidil suspension (1 105 to onidi ml 1), wter, or left untreted. Spryed plnts were immeditely overed with plsti gs (Minigrip relosle gs, 12 in 12 in, 4 Mil; Minigrip/Zip-pk, Sequin, Tex.) tht were losed y folding the open end nd stpling ner eh pedunle. Untreted plnts were neither spryed nor gged. For the seond ssy, Whetlnd nd RTx430 seeds were sown nd individul plnts were oserved until eh rehed nthesis (>50% of nthers exerted) nd inoulted over 3 onseutive dys. Tretments inluded onidil suspensions of F. equiseti, A. lternt (H02-781S-3), F. thpsinum, or sterile purified wter. Inoultions of RTx430 plnts were onduted 84 to 104 dys fter plnting (dp), wheres inoultions of Whetlnd were onduted 90 to 139 dp. Individul plnts were spryed with freshly prepred onidil suspension or sterile wter then immeditely overed with plsti gs. Untreted plnts were neither spryed nor gged. The tretment ws repeted over three onseutive dys, nd on the fourth dy, the gs were removed. Inoulum ws prepred in the following mnner. For Fusrium isoltes, gr loks from PDA ultures of eh isolte were pled in the entre of 60 mm Petri dishes filled with 1.5% gr ontining 80 mmol/l KCl. For A. lternt isoltes, 2% wter gr plte ws point-inoulted from PDA ulture followed y plement of -1 m2 filter pper (Whtmn #1, Whtmn Interntionl Ltd., Midstone, Englnd) over the inoultion site. Fungi were inuted t room temperture or 28 C for t lest 5 dys. Conidil suspensions were prepred in sterile filter-purified wter nd diluted to the onentrtion to onidi ml 1. Sporultion on miniml medi ws undnt ut myelil growth ws redued; therefore, no filtrtion step ws needed nd the onidil suspension ould e pplied y sprying with 473 ml plsti ottles (Ntionl Industries for the Blind, Alexndri, V.) without logging the spry pprtus. Mture grin ws hrvested nd seed threshed nd ulked y tretment. The following three mesurements were mde on eh seed smple: (i) men mss of seed, (ii) perent of seeds ppering dmged, nd (iii) perent germintion. Men seed mss ws determined y susmpling nd otining the totl mss of 100 seeds (in three replites). To determine perent of dmged seed, three individuls exmined susmples of 100 seeds (for totl of 300 seeds sreened) from eh tretment nd seprted them into the following two lsses: (i) helthy ppering or (ii) those tht ppered disolored, moldy, smll, or deformed. Greenhouse-grown uninfeted seed were used for omprison. To determine perent germintion, pproximtely 100 seeds from eh tretment were distriuted into four Petri dishes etween sterile, wet pper towels. Petri dishes ontining

5 Funnell-Hrris nd Pedersen: sorghum / hed light / inoultion methods / Fusrium speies 407 seeds were inuted t 25 C, exmined dily, nd, if dishes ppered dry, sterile purified wter ws dded. Seeds were sored fter 72 h s germinted if the rdile nd shoot hd grown pst the length of the seed. Spry inoultion iossys were nlyzed onsidering dt reorded y eh of the three individuls s repeted mesurements using Pro GLM. Min effets for tretments were presented where genotype tretment effets were not signifint. Where genotype tretment effets were signifint, tretment effets were lulted for eh sorghum line used. Correltions mong tretments were mde using Pro CORR Pro GLM (SAS 2000). Seed inoultion iossy The mize ssy of Bon et l. (1994) served s the sis for the ssy in the present work. Approximtely 100 surfe-sterilized, greenhouse-produed seed of either RTx430 or Whetlnd were inoulted with suspension of onidi of either F. thpsinum or F. solni, or with sterile purified wter. Conidi were produed on KCl gr inuted t room temperture for 7 to 10 dys. For the fourth repetition, vile mroonidi of the F. solni isolte from n older KCl ulture were used. Condil suspensions were prepred in sterile purified wter t onentrtion of onidi ml 1 sine preliminry ssys indited tht this onentrtion of F. thpsinum onidi inuted with grin of RTx430 redued germintion of seed y pproximtley one-hlf when ompred with seed inuted in sterile purified wter. Seeds were inuted for two dys in the drk t 25 C then two dys in the drk t 4 C nd the numer of germinted seeds per Petri dish were reorded. The ssy ws onduted four times. The dt were nlyzed using Pro GLM (SAS 2000) s two genotypes three tretments design with eh experiment onsidered replition. Seedling inoultion iossy This ssy is modifition of tht of Leslie et l. (2005) for sorghum. Seedlings were grown in Mgent vessels on Murshige nd Skoog (MS) plnt growth medium tht ws modified to enhne sorghum growth (Medium 11; Elkonin nd Pkhomov 2000). Plnts were inuted with fungl isolte nd were infeted without wounding. Infetion of roots ws ompred with uninoulted ontrols. The ssy ws onduted within four to six weeks, depending on plnt genotype. Greenhouse-grown seeds were surfe sterilized nd trnsferred septilly to 0.6% wter gr nd inuted for 4 7 dys in growth inutor t 25 C with ontinuous light. Germinted seeds were trnsferred to sterile Mgent vessels prepred with 50 ml MS sl medium with Gmorg s vitmins nd 87.6 mmol/l surose, mended with KH2PO4 to inrese the onentrtion to 8.8 mmol/l (Sigm), nd solidified with 0.7% phytolend gr (Cisson Lortories, In., Rexerg, Ind.). Seedlings ontinued to e inuted t 25 C with ontinuous light. Eight to fifteen dys following trnsfer of seedlings to Mgent vessels, plnts were inoulted with 0.5 ml of spore suspension or sterile purified wter pplied t the se of stlk on the plnt growth medium nd inuted in growth hmer t 22 C, 12 h light. Fungi used for inoultions were F. solni, F. thpsinum, nd A. lternt (H02-755S). Conidi of Fusrium spp. were grown on KCl gr while onidi of A. lternt were grown on 2% wter gr, nd suspensions were prepred in sterile purified wter t the onentrtions indited in Results. Sixteen to twenty-one dys fter inoultion, eh plnt ws removed from the plnt growth medium. The myelil mt ws refully removed so s not to dmge the roots. Plnts were pled in wter-dmpened pper towels while the following mesurements were mde: (i) perent of disolored stlk nd leves; (ii) perent of disolored roots; (iii) fresh mss of stlk nd leves; nd (iv) length of disolortion from se of stlk following removl of loose, dmged leves. To determine the perent of root piees with fungl growth, pproximtely 1 m long non-djent root piees (no root tips) were exised from eh plnt, surfe-sterilized in 1% sodium hypohlorite, nd plted onto dihlorn (Ultr Sientifi, North Kingstown, R.I.) nd hlormpheniol (Sigm) medium (DCPA), whih ws semi-seletive for Fusrium nd Alternri (Funnell nd Pedersen 2006). Four to seven dys following plting, the perent of root piees with fungl growth ws reorded. Whetlnd seeds were plted onto wter gr nd four dys lter, seedlings were trnsferred to Mgent vessels. Thirteen dys fter plting of seed, rndomly hosen plnts were inoulted with onidi of F. thpsinum, F. solni, or with sterile purified wter; plnts were sored 31 dys fter plting of seed. Seventeen dys fter plting of seed, seedlings were inoulted with A. lternt onidi or sterile purified wter nd responses were sored 33 dys fter seed plting. Four dys following plting of RTx430 seeds, seedlings were trnsferred to prepred Mgent vessels. Two weeks fter seeds were plted, seedlings were inoulted with onidi of F. thpsinum, F. solni, A. lternt, or with wter. Plnts were sored 31 dys fter plting of seed. Root piees were plted onto DCPA. To exmine the response of genotypes red for grin mold resistne to isoltes of F. thpsinum, F. solni, nd A. lternt, seedings of Tx2911 nd Tx2927 were inoulted. Seedlings of Tx2911 were trnsplnted into prepred Mgent vessels seven dys fter sowing onto wter gr, then inoulted fter 15 dys, nd sored 29 dys fter plting of seed. Seedlings of Tx2927 were trnsplnted into prepred Mgent vessels six dys following plting of seeds onto wter gr, inoulted t 14 dys, nd sored 35 dys fter plting of seed. Root piees were plted onto DCPA. Whetlnd, RTx430, nd Tx2911 hve the purple wound response, wheres Tx2927 lks this response ( phenotype lled tn plnt olor). To ddress the possiility tht disolortion in roots nd other plnt prts ws due to the umultion of pigments or other phenolis in plnts with the purple phenotype (Hung nd Bkhouse 2005), nother tn genotype, BTx631, ws inoulted with onidi from the sme three fungi used in the seedling iossy. BTx631 seedlings were trnsplnted into prepred Mgent vessels 5 dys following plting of seed. Inoultion with onidi of F. thpsinum nd F. solni or with wter ourred 13 dys following plting of seed, nd plnts were

6 408 Cn. J. Plnt Pthol. Vol. 30, 2008 sored 32 dys fter plting of seed. Seedlings were inoulted with onidi of A. lternt or with wter 20 dys following seed plting, nd plnts were sored 39 dys fter plting of seed. Root piees were plted onto DCPA. Seedling iossys were nlyzed s ompletely rndom designs using Pro GLM. Men perent root disolortion, perent shoot disolortion, shoot mss, lesion lengths, perent infeted root piees, nd LSDs mong isoltes were reported. Correltion oeffiients were derived nd tested for signifine using Pro CORR. Results Wound inoultion iossys Inoultion of Whetlnd nd RTx430 HoV tests reveled tht the ssys with Whetlnd were not signifintly different (P = 0.58); therefore, the results were omined for nlysis. Inoultion with F. vertiillioides resulted in men lesion length tht ws signifintly different from tht of the sterile roth ontrol nd following inoultion with ll fungl isoltes exept F. thpsinum (Tle 1). Inoultion with F. thpsinum yielded men lesion length tht ws signifintly greter thn tht resulting from inoultion with F. equiseti, F. solni, or the sterile roth ontrol (Tle 1). Men lesion lengths resulting from inoultion of RTx430 plnts were signifintly different for tretment in oth iossys (P < 0.01). When HoV tests were onduted, the experiments were signifintly different (P < 0.01); therefore, the iossys were nlyzed seprtely. In spite of this sttistil differene, oth ssys demonstrted tht inoultion of RTx430 plnts with either F. vertiillioides or F. thpsinum yielded men lesion lengths tht were signifintly different from ll other tretments (Tle 1). Inoultion of erly mturing lines N246 nd N247 These ssys were onduted to determine whether very erly mturing lines N246 nd N247 ould shorten the durtion of the wound inoultion iossy nd distinguish etween highly virulent nd wekly virulent fungi. Using the HoV test, it ws determined tht results from the two ssys onduted on N247 plnts were not signifintly different (P = 0.16). Men lengths of lesions resulting from inoultion of N247 were signifintly different (P < 0.01; Tle 2). Following inoultion with F. thpsinum, men lesion lengths were signifintly greter thn those resulting from inoultion with F. solni nd A. lternt (Tle 2). The HoV test of iossys onduted on N246 plnts reveled tht the results were signifintly different (P < 0.01). Therefore, the two experiments were nlyzed seprtely. In the first ssy, plnts inoulted with F. thpsinum hd signifintly greter men lesion length ompred with other tretments, s expeted (Tle 2). However, in the seond ssy, there were no signifint differenes etween tretments (Tle 2). Spry inoultion iossy Heds of RTx430 nd Whetlnd plnts were spryinoulted with onidi of F. thpsinum, F. solni, F. vertiillioides, F. prolifertum, or A. lternt (H02- Tle 1. Men lesion lengths* resulting from wound inoultion of Whetlnd nd RTx430 y Fusrium vertiillioides, Fusrium thpsinum, Fusrium equiseti, Fusrium solni, nd Alternri lternt. Tretment F. vertiillioides F. thpsinum F. solni F. equiseti A. lternt Sterile roth Men lesion length (mm) Sorghum genotype Whetlnd Sorghum genotype RTx430 Assy d d d *Totl length of disolortion, whih my inlude regions with disontinuous disolortion, ws mesured. Comined results of two ssys. Mens with the sme letter re not sttistilly different. Assys re reported seprtely euse of signifint differenes in HoV. Mens with the sme letter re not sttistilly different within eh ssy. Assy Tle 2. Men lesion lengths* resulting from wound inoultion of very erly mturing genotypes, N247 nd N246, with Fusrium thpsinum, Fusrium solni, nd Alternri lternt. Tretment F. thpsinum F. solni A. lternt Sterile roth Men lesion length (mm) Sorghum genotype N Sorghum genotype N246 Assy *Totl length of the disolortion, whih my inlude regions with disontinuous disolortion, ws mesured. Comined results of two ssys. Mens with the sme letter re not sttistilly different. Assys re reported seprtely euse of signifint differenes in HoV. Mens with the sme letter re not sttistilly different within eh ssy. 755S), then inuted for one dy in plsti gs. Both wter-treted nd untreted ontrols were lso inluded. There were no signifint effets due to tretment for mss per 100 seeds, perent of helthy seed y visul ssessment, nd perent germintion (P = 0.10, P = 0.31, nd P = 0.44, respetively). Sorer effets were signifint (P < 0.01) for visul ssessment of seed. There were no signifint orreltions etween the three mesurements. Whetlnd nd RTx430 plnts were inoulted y sprying onidil suspension of either F. thpsinum, F. equiseti, or A. lternt (H02-781S-3) onto heds s they rehed nthesis for three onseutive dys. Wter-treted nd untreted ontrols were lso inluded. The effets of tretments were signifint for perent of helthy ppering seed (P = 0.02; Tle 3). Untreted seed hd the highest men perent helthy ppering seed, nd this perentge ws signifintly greter thn tht following tretment with Assy

7 Funnell-Hrris nd Pedersen: sorghum / hed light / inoultion methods / Fusrium speies 409 Tle 3. Men mss, perent helthy seed, nd perent germintion of mture grin resulting from spry inoultion of heds t nthesis of sorghum genotypes RTx430 nd Whetlnd with onidi of Fusrium thpsinum, Fusrium solni, nd Alternri lternt s ompred with wter-spryed heds or untreted heds.* Comined tretments of lines Whetlnd nd RTx430 Men mss (g) per 100 mture seeds otined from fungus-spryed, wterspryed, or untreted heds Spry tretment Men perent helthy seed Men perent germintion RTx430 Whetlnd None F. equiseti A. lternt F. thpsinum Wter *Arithmeti mens of three mesurements for eh tretment on eh line. Comprisons were mde on eh mesurement for eh sorghum genotype. Mens with the sme letter re not sttistilly different. F. thpsinum or wter (Tle 3). There my hve een n effet of tretment on perent germintion sine grin from untreted nd wter-treted plnts germinted t higher rte thn grin from plnts treted with F. thpsinum (P = 0.06; Tle 3). Plnt genotype tretment effets for seed mss were signifint (P = 0.01), nd effets of tretment were signifint for oth RTx430 nd Whetlnd (P < 0.01 nd P = 0.03, respetively). Tretment of RTx430 plnts with sterile wter or F. thpsinum onidi yielded seed with signifintly higher men msses (g) per 100 seeds thn other tretments, while sprying F. thpsinum onidi onto Whetlnd plnts yielded grin with signifintly lower men msses per 100 seeds thn other tretments (Tle 3). To onfirm tht visul inspetion ould e used s n indition of grin helth, orreltions were onduted. The perent of helthy ppering seed ws positively orrelted with perent germintion (R = 0.61; P < 0.01). Seed inoultion iossys Effets of tretments on germintion were not signifint (P = 0.06) following the inution of seed of sorghum genotypes RTx430 nd Whetlnd in either sterile wter (75.1%), sterile wter ontining onidi of F. thpsinum (71.3%), or F. solni (70.5%) (LSD0.05 = 5.2). Effets of genotype were signifint (P < 0.01) with men germintion of 62.9% for RTx430 seed ross ll tretments nd men germintion of 81.7% for Whetlnd seed ross ll tretments (LSD0.05 = 4.2). those otined from wter ontrol plnts (Tle 4). Following inoultion with F. solni onidi, only men perent root disolortion nd shoot mss were signifintly different from those found in the wter ontrol (Tle 4). Seedlings grown in Mgent vessels lso were inoulted with A. lternt isolte H02-755S, whih in preliminry wound inoultion iossys, resulted in men lesion lengths similr in length to those of H02-781S-3 (dt not shown). Perent root disolortion, shoot mss, nd lesion length were signifintly different mong tretments (P < 0.01; P < 0.01 nd P = 0.047, respetively) ut not for perent shoot disolortion (P = 0.054) (Tle 4). When plnts of genotype RTx430 were inoulted with onidi of F. thpsinum, F. solni, or A. lternt, the mesurements of men perent root disolortion, shoot disolortion nd infeted root piees, nd mss nd lesion length were signifint mong tretments (P < 0.01; Tle 4). Nerly ll mesurements resulting from inoultion with onidi from the three fungl speies were signifintly different from those otined following pplition of wter to plnts. Exeptions were the men lesion length resulting from inoultion with F. solni onidi nd the perent infeted root piees following inoultion with onidi of F. thpsinum s ompred with wter ontrols (Tle 4). Additionlly, plnts treted with F. solni onidi hd signifintly higher men mss when ompred with tht of plnts treted with onidi of either F. thpsinum or A. lternt (Tle 4). Seedling inoultion iossys Inoultion of Whetlnd nd RTx430 When the genotype Whetlnd ws inoulted with onidi of F. thpsinum or F. solni, or with sterile wter, the mesurements of perent root disolortion nd shoot disolortion, shoot mss, nd men lesion length were signifintly different mong tretments (P < 0.01, P < 0.01, P < 0.01, nd P = 0.02, respetively) (Tle 4). For ll mesurements, inoultion with F. thpsinum onidi resulted in men mesurements tht were signifintly different from Inoultions of genotypes red for resistne to grin mold pthogens nd tn genotypes When plnts of genotype Tx2911 were inoulted with onidi of F. thpsinum, F. solni, or A. lternt, s ompred with sterile wter ontrols, mesurements of funglinoulted plnts were signifintly different exept for perent shoot disolortion following inoultion with F. solni onidi (Tle 4). The mens of perent root disolortion following inoultion with onidi of either A. lternt or F. solni were signifintly higher thn the

8 410 Cn. J. Plnt Pthol. Vol. 30, 2008 Tle 4. Men perent root disolortion, perent infeted root piees, shoot mss (g), stlk lesion length (mm), nd perent shoot disolortion following inoultion of ultured sorghum seedlings with Fusrium thpsinum, Fusrium solni, nd Alternri lternt. Roots Shoots Genotype Whetlnd Tretment* FRC-M-3790 FRC-S-0900 Wter Perent disolored Perent infeted nt nt nt Mss (g) Lesion length (mm) Perent disolored Whetlnd H02-755S Wter nt nt RTx430 FRC-M FRC-S H02-755S Wter 2.8 d d Tx2911 FRC-M-3790 FRC-S-0900 H02-755S Wter Tx2927 FRC-M-3790 FRC-S-0900 H02-755S Wter BTx631 FRC-M-3790 FRC-S-0900 Wter BTx631 H02-755S Wter *The pproximte numer of onidi pplied to eh plnt ws s follows: Whetlnd (first ssy), ; Whetlnd (seond ssy), ; RTx430, ; Tx2911, ; Tx2927, ; BTx631 (first ssy), ; nd BTx631 (seond ssy), The length of nerosis or disolortion rising from the se of stlk is defined s the lesion. Comprisons were mde on eh mesurement for eh sorghum genotype. Mens with the sme letter re not sttistilly different. nt, not tested. sme mesurement fter inoultion with F. thpsinum onidi (Tle 4). Plnts of genotype Tx2927 were inoulted with onidi from eh of the three fungl isoltes, nd the mesurements of perent of root nd shoot disolortion nd shoot mss (P < 0.01) s well s men lesion length (P < 0.01) nd perent of infeted root piees (P = 0.01) were signifintly different mong tretments (Tle 4). Only plnts treted with F. thpsinum onidi hd stlk lesions tht were signifintly higher thn tht of the wter ontrol (Tle 4). Plnts treted with onidi of either of the two Fusrium spp. hd men perent root infetions tht were signifintly higher thn those of the wter-treted plnts or plnts inoulted with A. lternri onidi (Tle 4). For mens of perent root or shoot disolortion, tretment with onidi of ll three fungi yielded results tht were signifintly higher thn those otined with wter lone (Tle 4). Applition of A. lternt onidi resulted in signifintly higher men perent root disolortion thn ll other tretments, while tretment with F. solni onidi resulted in signifintly higher men perent shoot disolortion s ompred with ll other tretments (Tle 4).

9 Funnell-Hrris nd Pedersen: sorghum / hed light / inoultion methods / Fusrium speies 411 Sorghum genotypes Whetlnd, RTx430, nd Tx2911 produe drk pigments in response to pthogens, insets, or injury, wheres Tx2927 hs the tn response. To inlude n dditionl tn genotype in this nlysis, plnts of BTx631 were inoulted with onidi of the two Fusrium speies or with wter. Following inoultion with F. thpsinum onidi, mesurements for shoots (mss, lesion length, nd perent shoot disolortion) were signifintly higher thn those for wter, while mesurements for roots (perent disolortion nd perent infeted root piees) were not (Tle 4). Applition of F. solni onidi to BTx631 plnts resulted in signifintly higher men perent shoot disolortion nd perent root disolortion, with signifintly redued shoot msses s ompred with wter-treted plnts (Tle 4). When plnts of genotype BTx631 were inoulted with onidi of A. lternt, ll mesurements were signifintly different from those resulting from pplition of sterile wter (Tle 4). In generl, when onsidered ross ll ssys, ll mesurements were orrelted in iologilly relevnt mnner. For exmple, perent root disolortion nd perent infetion of root piees were diretly orrelted (R = 0.23, P < 0.01 when onsidering only fungl inoultions; R = 0.44, P < 0.01 when wter ontrols re inluded). Disussion A set of isoltes of Fusrium nd Alternri spp. ssoited with sorghum were used in four different inoultion strtegies. Eh of these strtegies ould e used to ssess iologil intertions etween fungl speies nd sorghum, or to ompre responses of different genotypes of sorghum, under onditions of the prtiulr ssy. Tle 5 summrizes the four ssys, inluding the length of time to otin nlyzle mteril, the plnt prt nlyzed, nd the extent of nlysis required. The wound inoultion ssy of Jrdine nd Leslie (1992) is vlule for omprison of fungl isoltes s well s omprison of sorghum genotypes (Funnell nd Pedersen 2006) with regrd to fungl oloniztion of the pedunle of mturing heds following wound inoultion. By using very erly mturing lines, suh s N247, the durtion of this ssy ould e redued y nerly eight weeks (Tle 5), strtegy tht might e used for omprison of oloniztion y different fungi. An unexpeted onsequene of this study ws the oservtion tht wound inoultion of genotype N246 resulted in men lesion lengths tht were not signifintly different y tretment in one ssy, while tretment ws signifint for genotype N247. We used wound inoultion on plnts from severl geneti kgrounds (Funnell nd Pedersen 2006) nd only genotype N246 responded in this mnner. It hs een oserved tht erly physiologil mturtion in sorghum my e diretly ssoited with senesene (Crst et l. 1999). In ryegrss, it hs een shown tht wounding ould eliit moleulr responses lso involved in senesene (Le Deunff et l. 2004). Perhps, in the present work, wounding of N246 resulted in senesene, even in the roth-only ontrol. The response of the N246 genotype to wound inoultion of pedunles indites tht reful seletion of plnt genotypes for rtifiil inoultions is ritil efore extensive use in studies for iologil hrteriztion of intertions etween fungi nd plnts (Miedner et l. 2003). Diret pplition of fungl onidi to sorghum flowers nd developing grin would pper to mimi nturl infetion (Nvi et l. 2005). Sorghum grin mold is likely used y speies omplex tht my inlude Curvulri lunt, memers of the F. moniliforme omplex, Alternri spp., Bipolris spp., Cldosporium spp., or Phom spp. (Nvi et l. 2005). Trekegn et l. (2006) reported tht moist nd wrm onditions post-flowering, inluding 3 to 19 dys of preipittion, were highly fvorle for grin mold infetions. Attempts hve een mde to reprodue onditions tht were fvorle for grin mold infetions in the field, following ontrolled inoultions with known fungl speies, y the use of overhed sprinkler systems nd gging (Bndyopdhyy nd Mughogho 1988; Fores et l. 1989). However, reports of greenhouse spry inoultions, in whih onidi hd een pplied to heds nd mture grin ssessed, re not ville. In the present study, we ttempted to provide onditions tht would llow for suessful infetion of flowering heds under greenhouse onditions. When lrge numers of onidi were pplied to eh hed t nthesis, over three-dy period, during whih time heds remined gged in plsti, iologilly relevnt results were hieved. However, it lso ws oserved tht plnts spryed with wter lone hd redued qulity of the pperne of the seed, similr to tht of plnts spryed with onidi of F. thpsinum (Tle 3). In mize, it hs een suggested tht fungi, prtiulrly F. moniliforme, ould provide protetion to kernels from other invding fungi (Keder et l. 1994). Previously, it ws reported tht symptomti grin nd lef tissue of field-grown sorghum plnts ould hror Fusrium nd Alternri spp., inluding pthogeni speies (Funnell nd Pedersen 2006). Perhps, during the ssy onduted in the present study, onditions provided y gging heds over three dys llowed for infetion of the developing grin y pre-existing fungi in wter ontrols. Sprying heds with high onentrtions of onidi of wekly virulent speies my hve somehow prevented these internl infetions, perhps y ompetition or y indution of defense responses (Little nd Mgill 2003). Seed inoultions were onduted to determine whether F. thpsinum nd F. solni isoltes ould infet nd inhiit germintion of sorghum grin. In four ssys, there were no signifint differenes due to tretment (P = 0.06). However, there ppered to e trend for lower germintion rtes in fungl-inoulted grin thn wter-treted grin. During preliminry ssys in whih RTx430 seeds were inuted with onidil suspensions of the two fungl speies or with wter, germinted seeds were trnsplnted nd llowed to grow to mturity. When mture grin olleted from eh hed were plted onto gr medium ontining the fungiide penthloronitroenzene, there were no signifint differenes in fungl growth from seeds otined from plnts grown from inoulted grin (dt not shown). This suggests tht if there ws systemi growth in the plnt, it did not infet grin or it infeted grin t low level. This is onsistent with results from mize, in whih field-grown plnts emerging from F. moniliforme-treted seed did not hve signifintly higher levels of F. moniliforme-infeted kernels s ompred with wter-treted seed (Munkvold et l. 1997).

10 412 Cn. J. Plnt Pthol. Vol. 30, 2008 Tle 5. Comprison of four strtegies for inoultion of sorghum with Fusrium or Alternri spp. Experimentl method Durtion (dys)* Plnt prt nlyzed Anlysis required Wound inoultion- Whetlnd, RTx430 Wound inoultion-n247 Spry inoultion Seed inoultion Seedling inoultion Pedunle Pedunle Mture grin Germinted seeds Roots, shoot Split pedunle, mesure lesion length Split pedunle, mesure lesion length Weigh, exmine grin; germintion tests Count germinted seeds Exmine roots nd shoots; mesure shoot lesion length; plte nd sore root piees *Length of time to otin nlyzle plnt mteril during the ssys. Previous work showed tht Fusrium spp. isolted from vrious soures, inluding grin of sorghum, perl millet or mize, or sorghum deris, ould use in vitro infetion of sorghum seedlings (Gourley et l. 1977; Leslie et l. 2005). In the present study, n ssy ws developed to infet sorghum seedlings in highly ontrolled nd rpid mnner using seedlings trnsplnted nd grown on MS medium. This medium hd een previously modified to enhne growth of sorghum nd inluded reltively low onentrtion of surose (-90 mmol/l) (Elkonin nd Pkhomov 2000; Tomes 1985). All of the sorghum genotypes tested ppered to e suseptile to F. thpsinum following pplition of lrge numer of spores ner the stlk, on the MS medium, s indited y stlk lesion lengths. In ontrst, F. solni produed stlk lesions tht were not signifintly different from the wter ontrol on nerly ll genotypes exept Tx2927 (Tle 4). However, t lest some genotypes lso ppered to e infeted y F. solni nd A. lternt isoltes tht were wekly virulent in other ssys. In prtiulr, roots were highly disolored, whih ould e due to n umultion of phenoli ompounds in response to hllenge y these fungi (Hung nd Bkhouse 2005). Additionlly, ll of the genotypes tested hd roots infeted s result of pplition of t lest one of these fungi (Tle 4). Fusrium solni is ommonly isolted from sorghum field deris (Leslie et l. 1990), wheres sorghum grin nd lef tissue re olonized y Alternri spp. (Funnell nd Pedersen 2006). In the present work, whether the high levels of onidi, the surose levels in the MS medium, or other onditions in the Mgent vessels resulted in root infetions y these fungi is not known. Fusrium onidi n e indued to germinte t quite low levels of surose (-15 mmol/l; Beyer et l. 2004). On the other hnd, Gómez-Ariz et l. (2007) hve shown tht when 300 mmol/l surose ws dded to liquid MS medium in whih rie ws eing grown, plnt defense responses were primed prior to infetion y pthogens, inluding F. vertiillioides. It is not known whether the ddition of surose to the plnt growth medium ffeted the intertion of fungi with sorghum. In summry, eh of the four iossys were le to provide informtion out individul fungl isoltes ssoited with sorghum or plnt mterils for further study of fungl intertions with sorghum. The use of n erly mturing line, suh s N247, ppered to onsiderly shorten the durtion of the wound inoultion iossy (Tle 5), whih ws useful to ompre different fungl speies ssoited with sorghum. The spry, seed, nd seedling inoultion s- sys my lso identify plnt mterils for investigtions of gene expression, enzyme tivities, or produts of seondry metolism during sorghum fungl intertions (Muthukrishnn et l. 2001; Slzmn et l. 2005). Aknowledgements We thnk J. Toy for growing nd mintining len seed nd P. O Neill for overseeing lortory work nd for ssistne with sttistis. We thnk T. Eisenhuer nd M. Eeling for tehnil ssistne. We lso thnk T. Clemente for dvie on plnt ulture. Mention of trde nmes or ommeril produts in this rtile is solely for the purpose of providing speifi informtion nd does not imply reommendtion or endorsement y the U.S. Deprtment of Agriulture. This rtile is in the puli domin nd not opyrightle. It my e freely reprinted with ustomry rediting of soure. Referenes Bon, C.W., Hinton, D.M., nd Rihrdson, M.D A orn seedling ssy for resistne to Fusrium moniliforme. Plnt Dis. 78: Bndyopdhyy, R., nd Mughogho, L.K Evlution of field sreening tehniques for resistne to sorghum grin molds. Plnt Dis. 72: Beyer, M., Röding, S., Ludewig, A., nd Verreet, J.-A Germintion nd survivl of Fusrium grminerum mroonidi s ffeted y environmentl ftors. J. Phytopthol. 152: Cstor, L.L., nd Frederiksen, R.A Fusrium hed light ourrene nd effets on sorghum yield nd grin hrteristis. Plnt Dis. 64: Crst, O.R., Xu, W.W., Rosenow, D.T., Mullet, J., nd Nguyen, H.T Mpping post flowering drought resistne trits in grin sorghum: ssoition etween QTLs influening premture senesene nd mturity. Mol. Gen. Genet. 262: Elkonin, L.A., nd Pkhomov, N.V Influene of nitrogen nd phosphorous on indution of emryoni llus of sorghum. Plnt Cell Tissue Orgn Cult. 61: Fores, G.A., Frederiksen, R.A., nd Seitz, L.M Assessment of sorghum grin mould: disese severity nd rop loss. Seed Si. Tehnol. 17: Funnell, D.L., nd Pedersen, J.F Retion of sorghum lines genetilly modified for redued lignin ontent to infetion y Fusrium nd Alternri spp. Plnt Dis. 90: Gómez-Ariz, J., Cmpo, S., Ruft, M., Estopà, M., Messeguer, J., Sn Segundo, B., nd Co, M Surosemedited priming of plnt defense responses nd rod-

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