Inhibition of Blumeria graminis germination and germling development within colonies of oat mildew

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1 Physiologicl nd Moleculr Plnt Pthology (1) 58, 9±228 doi:1.16/pmpp.1.33, ville online t on Inhiition of Blumeri grminis germintion nd germling development within colonies of ot mildew T. L.W. ARVER 1 *, P.. ROBERTS 1,B.J.THOMAS 1 nd M. F. LYNGKáR 2 1 Institute of Grsslnd nd Environmentl Reserch, Aerystwyth, eredigion SY23 3EB, U.K. nd 2 Risù Ntionl Lortory, DK-4 Roskilde, Denmrk (Accepted for puliction April 1) Germintion y Blumeri grminis D Speer. spp. vene, hordei nd tritici, ws gretly suppressed when conidi fell within colonies of. spp. vene or hordei estlished on susceptile ot or rley, respectively. On helthy ot or rley, nd when distnt from powdery mildew colonies, ll. spp. formed norml ppressori. This ws lso true when conidi germinted within estlished rley mildew colonies. Within rley mildew colonies, ppressori of f. sp. hordei penetrted epiderml cells ( formed hustori) more frequently thn ppressori distnt from colonies. Similrly,. spp. vene nd tritici, normlly unle to infect rley, frequently penetrted epiderml cells sutending estlished rley mildew colonies. Thus, colony estlishment induced rley epiderml cell ccessiility, even to non-pthogenic. spp. In contrst, when ll three. spp. germinted within estlished ot mildew colonies, most formed norml, hyph-like germ tues. Since they did not form ppressori, nd were thus unle to ttempt penetrtion, it ws impossile to determine whether ot mildew colonies induced ccessiility of underlying ot epiderml cells. However, when super cil structures of estlished colonies were removed, germlings of ll. spp. formed ppressori freely on cells contining ot mildew colony hustori. Furthermore, these cells showed high level induced ccessiility not only to f. sp. vene ut lso to the normlly non-pthogenic. spp. This indicted tht fctors disrupting germintion nd further development y conidi lying within ot mildew colonies were produced from the super cil colony structures nd not y hustoriumcontining plnt cells. The fctors pper to hve limited moility. *c 1 Acdemic Press Keywords: Blumeri grminis; Erysiphe grminis; ot mildew; rley mildew; germintion; germ tues; iocontrol. INTRODUTION Blumeri grminis D Speer (syn. Erysiphe grminis D) cuses powdery mildew of cerels nd other grsses. The disese is spred minly y wind-lown sexul conidi. Following deposition on host, conidi germinte nd germling morphogenesis proceeds through series of distinct steps. Emergence of the primry germ tue () provides the rst visile sign of germintion (within 3±1 min). The remins short (pprox. 5±1 mm) nd septte, nd is thought to hve t lest three functions, nmely, ttchment to the host surfce [5±7, 11, 26], gining ccess to host wter [2], nd recognising host surfce fetures [4, 6, 7, 38]. The fetures recognized proly include hydrophoicity nd cutin nd cellulose rekdown products relesed y fungl enzymes [5, 6, 1, 14, 32, 35, 36]. This recognition engges processes tht led to elongtion of second germ tue [4, 5, 38] tht emerges shortly fter the [5, 6, 26, 38]. Elongtion of the second germ tue is pre-requisite to it eventully di erentiting specilized infection structure, * To whom ll correspondence should e ddressed. the ppressorium. As it grows epiphyticlly, the elongting ppressoril germ tue forms single septum nd itself recognizes host surfce fetures [4±6, 17]. This recognition leds to swelling of the distl cell nd eventully to di erentition of hooked ppressoril loe t the tue pex (Fig. 1). At 8, ppressori re formed y 1± 12 h fter inocultion. Occsionlly, rst- or lter-formed germ tues fil to contct, or recognize contct with the lef, in which cse they remin s short, non-functionl ``susidiry germ tues'' [7, 26, 38]. In this cse, reserves permitting, susequent germ tue will tke on the functions nd the next-formed germ tue will elongte. Similrly, elongted germ tues sometimes fil to contct, or respond to, the host surfce, in which cse they remin s undi erentited, hyph-like structures incple of infection [4, 5, 38]. When functionl ppressorium is formed, n infection peg emerges from eneth the ppressoril loe nd ttempts to penetrte the underlying epiderml cell (12± 16 h fter inocultion) [1]. The ttcked cell responds nd ppill deposition is initited. However, in comptile reltionship, the infection peg often penetrtes the /1/59+ $35./ *c 1 Acdemic Press

2 21 T. L. W. rver et l. () () 1 mm 1 mm response site, enters the cell nd n immture primry hustorium is formed (15±24 h). The hustorium then develops digitte processes from ech end of the ellipsoidl hustoril ody nd sors nutrients from the infected cell [1, 3, 18]. These re exported to the ppressorium, hyphe emerge from the ppressoril germ tue or from the mother conidium, nd n epiphytic hyphl weft grows t ccelerting rte over the lef surfce [3, 18]. After 2±3 dys, nutrients supplied y the growing primry hustorium ecome insu cient to support the young colony, nd second genertion of hyphl ppressori is initited in response to drkness during the dily cycle [3, 18]. These hyphl ppressori re usully sited either on the cell contining the primry hustorium or within one or two cells distnce of it. Penetrtions from these ppressori form secondry hustori (usully etween two nd six) nd hyphl growth ccelertes further. Successive genertions of S S App App FIG. 1. ( nd ) Light nd LTSEM microgrphs, respectively, of ``norml'' B. grminis f. sp. vene germlings 24 h fter inocultion. Ech conidium (c) hs formed short, septte primry germ tue () nd second ppressoril germ tue (App) tht hs elongted (to pprox. 4 mm), swollen nd hs distinct, hooked picl loe (H). A single septum (S) is evident within the ppressoril germ tue (pprox. 1±15 mm from the conidium). H H hustori (with incresing numers in ech genertion) re formed nightly. By 4±5 dys the rst conidiophores develop nd sporultion commences shortly fterwrds. Even in comptile host±b. grminis interctions where some primry penetrtion ttempts succeed, mny fil to penetrte the ppill defence. It is now well estlished tht the ility of B. grminis to penetrte cerel epiderml cells is drmticlly ected y the outcome of prior ttcks y the fungus [8,, 21, 23, 24, 27±29, 33, 34]. This hs implictions for the eld sitution where individul cells my e confronted y sequentil ttcks s conidi re deposited continully from the eril pool. Thus, if n initil ttck is successful, the cell shows induced ccessiility (sensu Ouchi et l. [34]) so tht lter ttcks on tht cell will lmost invrile succeed. By contrst, filed initil ttcks induce ``inccessiility'' (sensu Kunoh et l.[21]) within the cell so tht susequent ttcks lmost invrily fil. While these induced chnges re essentilly loclized phenomen, eing expressed most strongly in the single cell directly sujected to erlier ttck, there is some trnsmission of e ects to immeditely djcent epiderml cells lthough the e ects re often undetectle t two cells distnce [8, 21, 23, 27±29, 33]. Lyngkjñr nd rver's previous studies [8, 27±29] involved doule inocultion procedures wherey the rst, ``inducer'' inoculum ws llowed to develop nd then its super cil fungl structures (conidi, germ tues, hyphe) were removed efore the second, ``chllenge'' inoculum ws pplied. The inducer inoculum ws given mximum of 48 h incution efore removl of super cil structures i.e. where primry penetrtion hd succeeded, primry hustorium ws formed ut no hyphl ppressori or second genertion hustori were initited. The success (or filure) of chllenge ttck ws then determined in reltion to the presence of inducer primry hustori or ppille remining s intrcellulr evidence of successful or filed inducer ttck, respectively. We undertook the present work to extend Lyngkjñr nd rver's erlier studies [8, 27±29]. Here, however, we imed to llow colonies from inducer inoculum to develop second genertion hustori efore pplying chllenge inoculum. We intended tht germling ppressori of the chllenge inoculum would ttck host cells t the time tht inducer colonies were inititing their third genertion hustori. Thus, our intention ws to determine whether the presence of multiple hustori (second nd third genertion) produced y developing colony enhnced the expression of induced ccessiility in nery, unttcked cells. We executed our rst experiments using ot (Aven stiv L.) inoculted with comptile isolte of B. grminis f. sp. vene Mrchl. However, we found it impossile to gther su cient dt to ful l our ojective. Where chllenge conidi were deposited within the oundries of n estlished f.sp. vene colony derived

3 Inhiition of B. grminis development 211 from inducer inocultion, very few of these conidi seemed to germinte. Furthermore, very few of those tht did germinte formed n ppressorium. The mjority of germlings produced highly norml hyph-like germ tues nd did not pper to ttempt penetrtion of the host cells. The work reported here ws therefore redirected towrds exploring the e ects tht estlished colonies might hve on germintion nd germling development y B. grminis conidi deposited within estlished B. grminis colonies. We investigted these e ects with respect to f. sp. vene colonies on susceptile ots nd f. sp. hordei colonies on susceptile rley. Pthogens nd plnts MATERIALS AND METHODS An isolte of B. grminis f. sp. vene rce 5, ws multiplied on plnts of ot cv. Selm grown in spore-proof glsshouse under nturl lighting conditions nd minimum temperture of 128. Similrly, isolte 1 of B. grminis f. sp. hordei nd n isolte of B. grminis f. sp. tritici (originlly supplied y A. Dniels, Aventis, U.K.), were multiplied on rley cv. Plls nd whet cv. Rind, respectively. One dy efore conidi were required for experimenttion, infected leves were shken to remove older conidi nd ensure supply of vigorous young conidi for inoculum. For ll experiments, fully expnded second-formed leves of cerel plnts were used. Studies of ot (A. stiv L.) used cvs Selm nd Mldwyn. Neither hs known mjor gene resistnce to B. grminis f. sp. vene rce 5. Studies of rley (Hordeum vulgre L.) used cv. Plls, which is susceptile to B. grminis f. sp. hordei isolte 1. Since the second leves of these cvs re susceptile to virulent fungl isoltes, penetrtion ttempts from mny ppressori re successful nd reltively few ttcks fil in ssocition with ppill deposition y living epiderml cells, nd few result in deth of ttcked cells ([8], Lyngkjñr nd rver, unpulished). Neither ot cv. nor Plls rley is susceptile to B. grminis f. sp. tritici, Plls is not susceptile to B. grminis f. sp. vene, nd the ot cvs re not susceptile to B. grminis f. sp. hordei. As expected from previous studies [13], preliminry oservtions suggested tht filure of infection y inpproprite. spp. ws explined y comintion of two fctors: either ppill responses prevented penetrtion or ttcked cells died rpidly in response to ttck. Importntly, even on inpproprite cerel spp., conidi of ll fungl. spp. ppered to germinte nd develop normlly up to the stge of ppressoril di erentition. To produce disese-free experimentl plnts, seeds were sown singly in pper tues (15 1 mm) contining soil : pet : snd (6 : 3 : 1; v : v). Plnts were grown to full expnsion of the second-formed lef (pprox. 18 dys) in spore-proof glsshouse under nturl lighting conditions nd minimum temperture of 128. Experimentl designs nd procedures Doule inocultion experiments: estlished colonies in plce. All of these experiments involved using rst low density inocultion to estlish young colonies on test leves nd then, 72 h lter, pplying second, high density inocultion. In this wy, conidi of the second inocultion were deposited oth within estlished colonies where they ly touching or etween the super cil hyphe, or distnt from colonies when they ly on cells tht hd no contct with hyphe of estlished colonies. For the rst inocultion, in ll cses nine leves of Selm or Mldwyn ot, or of Plls rley were inoculted with conidi of the pproprite f. sp. (vene or hordei, respectively). Pper tues contining the plnts were lid on ench, nd their second-formed leves were rrnged dxil surfce up (nd held t using smll weights t lef pex nd se), efore plcing spore settling tower over them. onidi were lown from infected leves into the tower nd slide plced mong the leves ws used to trp conidi nd monitor spore density which ws djusted to pprox. 2±5 conidi mm 2. The inoculted plnts nd four dditionl helthy plnts were then trnsferred to growth cinet t 8 nd under 12 h light/12 h drk cycle, with mmol m 2 s 1 photon ux density during the light period. The rst light period commenced immeditely fter inocultion. Under these conditions, previous studies [3, 9, 12] llowed us to nticipte the pttern of colony development. Following estlishment of functionl primry hustorium t round h fter inocultion, secondry hyphl elongtion nd rnching ws expected to ccelerte up to the drk period eginning 6 h fter inocultion, when, in the 6±72 h drk period, hyphl ppressori nd then second genertion hustori would e initited. One of the nine inoculted leves ws cut, xed, clered nd microscopiclly exmined 71 h fter inocultion to con rm tht colonies were developing s expected. When this ws con rmed, four of the inoculted leves were xed 72 h fter inocultion for reference. At 72 h, second inocultion ws pplied to the remining four inoculted leves, nd the second-formed leves of the four helthy plnts were inoculted simultneously to provide control. The second inocultion ws djusted to pprox. 5 conidi mm 2. According to requirement, conidi for the second inocultion were

4 212 T. L. W. rver et l. either of the f. sp. pproprite to the host (i.e. the sme isolte s used for estlishing colonies vi the rst inocultion) or of n inpproprite f. sp. normlly unle to infect the test leves. Following the second inocultion, leves were returned to the growth cinet nd incuted for further 24 h efore ll were xed. Doule inocultion experiments: estlished colonies removed. Thirteen second-formed leves of Selm ot were inoculted with pprox. 2 conidid mm 2 of B. grminis f. sp. vene rce 5. These, nd 12 helthy (uninoculted) leves, were incuted s descried ove, ut for 96 h, lthough one inoculted lef ws xed, clered nd microscopiclly exmined 95 h fter inocultion. Microscopy of this lef con rmed the nticiption [3] tht the third genertion of hustori hd een initited y this time. At 96 h fter the rst inocultion, the dxil surfce of ll leves (including the helthy ones) ws pinted with cellulose cette/cetone solution which ws llowed to dry (pprox. 2±5 min) efore the dried cette lm ws peeled wy. This removed ll super cil fungl structures due to the rst inocultion (colony hyphe, filed germlings nd conidi) nd the epicuticulr lef wxes from inoculted nd helthy leves [1, 27]. As expected [27], epiderml cells contining hustori formed y colonies on leves treted with the rst inoculum remined live (s judged y lck of cytoplsmic grnultion nd whole-cell uto uorescence). Four leves tht hd received the rst inoculum were then inoculted for second time using 5 conidi mm 2 of either f. sp. vene, f. sp. hordei or f. sp. tritici, nd di erent sets of four helthy leves were lso inoculted simultneously with the di erent. spp. All were incuted for further 36 h efore xtion. Preprtion for light microscopy As descried previously (e.g. [13]) leves were xed nd clered y procedure tht voids displcing ungerminted conidi nd loosely ttched germlings. Brie y, centrl 3 mm segments of leves were excised, nd lid inoculted surfce up on tissue pper pds moistened with 3 : 1 ethnol : glcil cetic cid (v : v) to x nd lech the leves. They were then trnsferred to pds moistened with wter nd susequently to pds moistened with lctoglycerol (lctic cid : glycerol : wter, 1 : 1 : 1, v : v) to cler. For studies of fungl development where leves re xed efore lrge colonies re llowed to develop, it is possile to ttin good resolution of pthogen nd host using unstined leves mounted without coverslips, nd viewed with no-coverslip ojectives. However, ecuse of di rction round hyphe of colonies estlished y the rst inoculum, it ws impossile to use this pproch to resolve detils of second inoculum development within estlished colonies. We needed, therefore, to stin specimens. The rst experiment involved doule inoculting Selm ot with B. grminis f. sp. vene, leving estlished colonies from the rst inocultion in plce. Here, stining ws chieved y plcing smer of Trypn lue in lctoglycerol (.1 g per 1 ml) on coverslip, lowering the dxil (inoculted) surfce of the lef segment onto the coverslip, nd then inverting the mount onto microscope slide smered with lctoglycerol. This procedure ws devised to llow stining without displcing loosely ttched germlings. We used this to gther dt on the developmentl chrcteristics of germlings formed y conidi of the second inoculum in reltion to their proximity to estlished colonies. However, we found tht even with cre some ungerminted conidi were displced; they could e seen moving over reltively long distnces if specimens were viewed immeditely fter mounting when the liquid medi were owing over segments. Therefore, for this experiment we hd limited con dence in dt for germintion frequency y conidi of the second inoculum in reltion to their proximity to estlished colonies. To llow ssessment of germintion frequency s well s the morphologicl chrcteristics of germlings formed y the second inoculum, we devised stining technique tht cused no detectle displcement of ungerminted conidi. This ws used for ll further experiments. As efore, leves were xed, leched, nd clered, ut dishes contining the segments supported on tissue pper were held on slnt to void ccumultion of free liquids round segments nd the possiility tht free liquid would move onto the segment surfce. To stin, segments supported on slnted dishes were spryed very lightly using hnd-pumped tomizer (Schott, Germny) from distnce of pprox. 4 cm with lcoholic methyl lue stin (.2 g methyl lue in 1 ml 95 % ethnol). A smll mount of spry (six pumps) ws pplied nd then sprying ws suspended for 1 min to llow the ethnol to evporte. In this wy, no visile droplets of liquid ccumulted. A further spry ws then pplied. This process ws repeted until microscope inspection showed colonies, germlings nd ungerminted conidi to e stined su ciently (usully three cycles). Repeted oservtion (efore nd fter spry stining, without coverslip) of spores locted distntly from estlished colonies, showed tht the procedure did not displce ungerminted conidi. Following stining, specimens were mounted without coverslip nd viewed y trnsmitted light nd di erentil interference contrst microscopy using or 4 ojectives. Oservtions were mde immeditely ecuse lthough stining remined good for pprox. 24 h, it fded therefter.

5 Dt collection Doule inocultion experiments: estlished colonies in plce. Our im in these experiments ws to determine the e ect colonies estlished from the rst inocultion might hve on conidil germintion nd the development of germlings from the second inocultion. For the rst experiment where coverslips were tted, on ech control lef (inoculted once, 24 h efore xtion) 5 rndomly selected, germinted conidi were exmined. onidi were clssi ed s germinted if t lest one short germ tue hd een produced. For ech of these germlings, note ws tken of whether they hd developed normlly or hd developed normlly to di erentite n ppressoril germ tue (illustrted in Fig. 1). Appressoril germ tues were recognized y: the presence of single septum within the tue, swelling towrds the tue pex, nd the presence of hooked Mx. lterl distnce Lo1 () olony 72 h fter inocultion Mx. longitudinl distnce () olony 96 h fter inocultion L2 L1 (c) Inner nd outer s of 96 h colony Inner Lo2 Outer 1 µm 1 µm FIG. 2. Drwings of B. grminis f. sp. vene colonies xed 72 h () nd 96 h () nd (c) fter inoculting ot cv. Selm. () Positioning of co-ordintes for mesurement of mximum longitudinl nd lterl mesurements. ˆ conidium. () Positioning of co-ordintes mrking the lst rnches on leder hyphe. Mesurement of Lo1 to Lo2 gives the longitudinl distnce etween lst rnches; L1 to L2 gives lterl distnce etween lst rnches. (c) The inner nd outer s of 96 h colony [sme s shown in ()]. The inner lies within n imginry oundry line linking the points t which the lst hyphl rnch occurred on the colony's mjor runner hyphe. The outer (shded) ly etween the inner oundry nd the outer oundry which ws de ned y n imginry line joining the tips of the colony's mjor hyphl rnches. Inhiition of B. grminis development 213 picl loe. Note ws lso mde of whether ppressori were ssocited with successful penetrtion s evidenced y the presence of hustorium. Anorml germlings were those which hd either filed to form n elongted germ tue (t lest 15 mm), or where the elongted germ tue hd not di erentited into n ppressoril germ tue. On doule-inoculted lef segments xed 24 h fter ppliction of the second inoculum, oservtions were mde for 5 germinted conidi tht ly on lef epiderml cells locted ``distnt'' from the nerest colony, nd germlings were clssi ed in the sme wy s on controls. Distnt cells were de ned s eing t lest four epiderml cells wy from the nerest cell in contct with hyphe elonging to colonies formed y the rst inoculum. Similr clssi ctions were mde for 5 germlings rising from conidi locted within the outer ``oundries'' of colonies estlished y the rst inoculum. The colony oundry ws de ned y n imginry line joining the tips of colony's leder hyphe (Fig. 2). Of course, on doule inoculted leves, some germlings deriving from the rst inocultion filed to penetrte nd estlish colonies, nd we could not distinguish etween these nd germlings from the second inocultion. Some of these filed germlings would inevitly e included in our smples. However, since the density of the second inocultion (ppox. 5 conidi mm 2 ) ws 1±25 times greter thn the rst (pprox. 2±5 conidi mm 2 ), nd since mny germlings from the rst inocultion formed colonies, the gret mjority of dt would necessrily relte to germlings derived from the second inocultion. Therefore, we introduced only smll errors due to inclusion of oservtions of germlings from the rst inocultion. We wished to guge the growth mde y colonies in the period 72±96 h fter inocultion i.e. from the time of pplying second inoculum to the time of xtion for doule inoculted leves. Drwings of two ``typicl'' B. grminis f. sp. vene colonies re shown in Fig. 2() nd () ( for 72 h nd 96 h colonies, respectively). As mesures of colony size, the mximum longitudinl nd lterl distnces from tip to tip of the longest hyphe [Fig. 2() nd ()] were mesured for 1 colonies on ech lef segment xed 72 h fter inocultion, nd for 1 colonies xed 96 h fter inocultion. For the 96 h segments, mesurements were lso mde of the mximum longitudinl nd lterl distnces etween the lst rnches on leder hyphe [Fig. 2()]. From these mesurements, it ws deduced tht the lengths nd redths of 72 h colonies were pproximtely equivlent to the lst-rnch length nd redth distnces seen in 96 h colonies. Thus, when oserving 96 h colonies, the position of the lst rnch on leder hyphe o ered n pproximte, ut resonle, indiction of the loction of leder hyphl tips t 72 h when the second inoculum ws

6 214 T. L. W. rver et l. pplied. This informtion llowed us to modify dt collection criteri for susequent experiments. In remining experiments where the fungus ws sprystined, dditionl dt were collected. As descried ove, on controls (inoculted once, 24 h efore xtion) 5 germlings were clssi ed for normlity of development, ut in ddition, 1 rndomly selected conidi were exmined to determine whether they hd germinted. Similrly, on doule-inoculted leves xed 24 h fter the second inocultion, 5 germlings lying on epiderml cells distnt from colonies were clssi ed for normlity of development, nd 1 conidi on such cells were exmined for germintion. In reltion to estlished colonies, dt were collected from two di erent s within colonies. The inner ws de ned y n imginry oundry line linking the points t which the lst hyphl rnch occurred on the colony's mjor runner hyphe [Fig. 2(c)]. The outer ly etween the inner oundry nd the outer oundry which ws, s efore, de ned y n imginry line joining the tips of colony's mjor hyphl rnches [Fig. 2(c)]. On ech lef segment, mny rndomly selected colonies were exmined nd dt were collected from within ech until we hd ccumulted oservtions from 1 conidi to determine whether they hd germinted, nd from 5 germinted conidi for normlity of germling development. As considered ove, on doule inoculted leves, only smll errors would hve een introduced due to inclusion of dt deriving from the rst inoculum in the dt sets. For ll lef segments xed 24 h fter the second inocultion nd in ll experiments, men percentges were clculted, s pproprite, for spore germintion, norml germling development, nd hustorium formtion. Dt sets were gthered from ech loction on doule inoculted leves (distnt from colonies, nd, s pproprite, within colony outer nd inner s) nd for controls inoculted once 24 h efore xtion. Percentges were trnsformed to rcsine squre roots (trnsformed vlue ˆ 18/p rcsine [Z/(1)]) to normlize dt nd stilize the vrince throughout the dt rnge, efore eing sujected to nlysis of vrince (using Genstt [15]). Doule inocultion experiments: estlished colonies removed. Our im here ws to determine whether ny e ects of the rst inocultion on susequent performnce of conidi nd germlings rising from the second inocultion, depended: (1) upon the presence of colony hyphe derived from the rst inocultion; or (2) whether the presence of hustori within epiderml cells ws su cient to medite these e ects. On ech control lef segment, dt to ssess germintion nd normlity of germling development were gthered s descried ove. On leves inoculted twice, germintion ws determined for 5 conidi locted on epiderml cells t lest four cells wy from the nerest cell contining hustorium formed y colonies rising from the rst inoculum (distnt cells), nd 25 germlings on distnt cells were scored for their developmentl chrcteristics. Similrly where conidi of the second inocultion ly on epiderml cells contining hustori formed y colonies of the rst inocultion, germintion ws determined for 5 conidi nd germling development ws ssessed for 25 germinted conidi. As descried ove, men dt from ech lef segment were clculted s percentges tht were trnsformed prior to nlysis of vrince. Low temperture scnning electron microscopy (LTSEM) To complement light microscopy nd id imge interprettion, su-smples of doule inoculted leves were exmined y LTSEM. Thus, when the centrl 3 mm lef segments were excised nd xed for light microscopy, the djcent sipetl 1 mm lef lde segment from some smples ws tken for LTSEM. Following estlished procedure [11], these segments were ttched to copper SEM stu with colloidl grphite nd frozen rpidly y introducing the stu to pre-cooled stu holder (pprox. 198) under n rgon ush, nd trnsferring the ssemly to the precooled ( 198) stge of n Emscope SPA sputtercryo system. ryo- xtion in this wy voided ice crystl formtion on specimens. The specimen ws then sputter coted with gold efore detiled oservtion on the cold stge (pprox. 168) of JEOL JSM 84A SEM, with 3. or 5. kv electron ccelerting voltge. SEM imges were recorded using digitizer ord nd softwre (SemAfore 2, JEOL) running under Windows 2 operting system. The gures presented here re from digitized imges enhnced using Adoe Photoshop 2 to mximize detils of res of interest. RESULTS Selm ot doule-inoculted with f. sp. vene, coverslips pplied Dt for spore germintion will not e descried ecuse pplying coverslips to specimens ws seen to displce mny ungerminted spores. Fig. 3 shows tht deposition within the oundry of n estlished colony hd drmtic e ect on germling development. On control leves (inoculted only once, 24 h efore xtion) nd when germlings were distnt from estlished colonies on doule-inoculted leves, the gret mjority of germlings developed norml ppressori. By contrst, when conidi were locted within the oundry of n estlished colony, more thn 7 % of germlings were norml

7 Inhiition of B. grminis development 215 (%) () () pex. When germlings were within the oundry of estlished colonies, they frequently mde direct contct with hyphe of the estlished colony [Fig. 4(), (), (d) nd (e)]. ontct could e vi the conidium, the germ tues, or oth. However, norml germlings were formed even where no direct contct ws mde [Fig. 4(c) nd ( f)]. On controls nd when germlings were distnt from estlished colonies on doule inoculted leves, pprox. 15±3 % of germlings successfully penetrted lef epiderml cells to form hustorium [Fig. 3()]. By contrst, when germlings were locted within the oundry of n estlished colony, less thn 5 % of germlings formed hustori. (%) 3 1 Experiments where specimens were viewed without coverslips, nd hyphe of estlished colonies remined in plce Ots inoculted rst with f. sp. vene nd secondly with. spp. vene, hordei or tritici ontrol [Fig. 3()]. Sometimes more thn one short germ tue ws formed, nd none elongted, ut this ws reltively rre. Most often, conidi tht germinted formed short nd second germ tue tht grew to length fr greter thn norml for n ppressoril germ tue (Fig. 4). These long tues filed to form hooked ppressoril loe t their pex. Sometimes they ppered to e slightly swollen t the point where n ppressoril loe would e expected (3±5 mm from the conidium), ut the germ tue then nrrowed nd extended s hyph-like tue with prllel wlls nd simple, rounded Distnt Within onidium loction reltive to colony FIG. 3. Development of B. grminis f. sp. vene germlings on ot cv. Selm second-formed leves 24 h fter inocultion onto helthy control leves or onto leves ering B. grminis f. sp. vene colonies derived from rst inocultion 72 h erlier. All mteril ws xed 24 h fter the second inocultion. () Percentge of morphologiclly norml germlings tht filed to develop n ppressorium. () Percentge of germlings with pprently norml ppressori tht penetrted plnt epiderml cells successfully to form hustorium. On controls ( ), dt re sed on counts of 5 rndomly selected germlings, while on leves ering colonies, counts were mde for 5 germlings locted distnt from colonies ( ) nd further 5 germlings were locted within outer colony oundries (h), on ech of four replicte leves in ll cses. Percentge dt were trnsformed to ngles for nlysis of vrince. Where columns re heded y di erent letters, LSD tests pplied to ngletrnsformed dt indicted signi cnt di erence (P 5.5) etween mens within dt set. Germintion. When spry stined specimens were mounted without coverslips to void displcing ungerminted conidi we felt con dent tht dt gthered to descrie spore germintion were vlid. Here, therefore, we considered percentge germintion of conidi locted on control leves, nd, where leves ore colonies estlished from the rst inocultion, we considered the germintion of conidi deposited distnt from colonies nd lying within the outer nd inner s of colonies [Fig. 2(c)]. Fig. 5() nd () show tht when f. sp. vene conidi were inoculted onto Mldwyn or Selm ot leves ering estlished f. sp. vene colonies, germintion rtes were signi cntly depressed if conidi ly within the inner of n estlished colony. Here, germintion percentges were pproximtely hlved compred with loctions distnt from colonies or on controls inoculted only once. This ws true for experiments involving oth ot cvs. There ws no di erence etween controls nd loctions distnt from colonies on doule-inoculted leves. In oth experiments, there ws tendency for conidi within the outer of colonies to show slightly lower germintion thn t distnt loctions or on controls, ut this e ect ws smll nd signi cnt only for the experiment with cv. Mldwyn. When f. sp. tritici conidi were inoculted onto Mldwyn ot leves ering f. sp. vene colonies, e ects of loction on germintion y f. sp. tritici conidi were very similr [Fig. 5(c)] to those seen in Fig. 5() nd (). Germintion frequency y conidi locted within the inner of f. sp. vene colonies ws out hlf tht seen for conidi distnt from colonies or on controls, ut germintion ws not depressed if conidi were within the outer of colonies.

8 216 T. L. W. rver et l. () u A (d) A SGT A 5 µm 3 µm () u (e) A A 5 µm ' 3 µm (c) (f) A A 5 µm 3 µm FIG. 4. Light ()±(c) nd LTSEM (d)±( f) microgrphs showing morphologiclly norml germlings of B. grminis f. sp. vene growing within the outer oundry of B. grminis f. sp. vene colonies on Selm ot leves. olonies were estlished from rst inocultion pplied 72 h erlier thn the second inocultion from which the germlings derive. All mteril ws xed 24 h fter the second inocultion. Often, germlings derived from the second inoculum hd contct with hyphe of estlished colonies (), (), (d) nd (e) vi either the conidium, germ tues or oth, lthough in some cses no contct ws mde (c) nd ( f). Sometimes, conidi were ungerminted () nd () or hd formed only one [ottom spore, ()] or more short germ tues. Where long germ tue ws produced, short germ tue with the ppernce of typicl primry germ tue ()±(d) nd ( f) ws lmost invrily visile. [In (e) it is prole tht primry germ tue is hidden eneth the conidium]. Occsionlly germlings with long germ tue hd more thn one short germ tue (d) in which cse one of these mde contct with the host surfce nd hd the chrcteristics of primry germ tue, while the other(s) ws typicl of redundnt susidiry germ tue. Frequently, norml long germ tues ecme distinctly swollen towrds region pprox. 3±5 mm from the conidium efore the tue nrrowed nd continued growth s hyph-like tue with prllel wlls nd simple rounded pex [germling on right in (), centrl germling in () nd (e)], lthough in some cses no sign of swelling ws evident ( f). A ˆ norml long germ tue; ˆ conidium; u ˆ ungerminted conidium; ˆ conidium with short germ tue only; ˆ hyph of estlished colony; ˆ primry germ tue; SGT ˆ susidiry germ tue. For f. sp. hordei, the suppression of germintion ssocited with deposition within f. sp. vene colonies ppered even greter thn for conidi of other. spp. [Fig. 5(d)]. When f. sp. hordei conidi were deposited within the inner of colonies, germintion ws reduced y nerly 7 % (to pprox. 17 %) compred with loctions distnt from the colonies (ppox. 53 % germintion). Agin, suppression of germintion ws less mrked for conidi deposited within the outer of f. sp. vene colonies, lthough even here signi cntly lower percentge germinted thn if locted distnt from colonies. Interestingly, there ws lso smll ut signi cnt reduction in germintion for f. sp. hordei conidi locted distnt from f. sp. vene colonies on doule inoculted leves compred with controls. This e ect ws not seen in ny other comintion.

9 1 8 () Inhiition of B. grminis development 217 () 6 onidi tht germinted (%) (c) c (d) c d ontrol Distnt Outer Inner ontrol Distnt Outer Inner onidium loction reltive to colony onidium loction reltive to colony FIG. 5. Percentges of B. grminis. spp. vene, tritici nd hordei conidi tht hd germinted 24 h fter inocultion onto secondformed ot leves of helthy controls, or onto leves ering B. grminis f. sp. vene colonies derived from rst inocultion 72 h erlier. All mteril ws xed 24 h fter the second inocultion. Dt re for f. sp. vene conidi on ot cvs Mldwyn () nd Selm (), nd for. spp. tritici (c) nd hordei (d) on cv. Mldwyn. On controls ( ) dt re sed on counts of 1 rndomly selected conidi, while on leves ering colonies, counts were mde for 1 conidi locted distnt from colonies ( ), 1 within the outer of colonies ( ), nd 1 locted within the inner of colonies ( ), on ech of four replicte leves in ll cses. Percentge dt were trnsformed to ngles for nlysis of vrince. Where columns re heded y di erent letters, LSD tests pplied to ngletrnsformed dt indicted signi cnt di erence (P 5.5) etween mens within dt set. Germling development. Fig. 6() nd () show tht when f. sp. vene conidi germinted on Mldwyn or Selm ot leves ering estlished f. sp. vene colonies, very high percentge (474 %) developed normlly if conidi ly within the inner of n estlished colony. As descried for the rst experiment (Fig. 4), norml germlings were those tht filed to form long germ tue, or, where long germ tue ws formed, it did not di erentite n ppressorium. In generl, normlities were infrequent (ppox. 4±12 %) for germlings on controls or locted distnt from colonies. They were lso reltively infrequent when germinted conidi ly within the outer of estlished colonies (518 %). When f. sp. tritici germlings developed on Mldwyn ot leves ering f. sp. vene colonies, e ects of loction on germling development y f. sp. tritici [Fig. 6(c)] were very similr to those seen in Fig. 6() nd (). On controls nd in loctions distnt from colonies, very few germlings developed normlly (57 %). Within the outer of f. sp. vene colonies, the normlity frequency ws slightly ut signi cntly higher (pprox. 16 %). However, when f. sp. tritici conidi were within the inner colony, the mjority of germlings developed normlly (77 %). The normlities were similr to those shown in Fig. 4. As with e ects on germintion, the e ects of germling loction on the frequency of norml germling development ppered greter for f. sp. hordei thn other. spp. As in other cses, when f. sp. hordei germlings were on control leves or distnt from f. sp. vene colonies, very low frequency of normlity ws seen [59%, Fig. 6(d)], nd when locted within the inner of colonies normlities were extremely frequent [82 %, Fig. 6(d)]. However, in contrst to other comintions, reltively high normlity frequency [ppox. 49 %, Fig. 6(d)] ws seen when germinted f.sp hordei conidi were locted in the outer of f. sp. vene colonies. These dt indicted tht f. sp. hordei ws more sensitive

10 218 T. L. W. rver et l. 1 8 () c () c Germinted conidi developing normlly (%) (c) c (d) c ontrol Distnt Outer Inner ontrol Distnt Outer Inner onidium loction reltive to colony onidium loction reltive to colony FIG. 6. Percentges of B. grminis. spp. vene, tritici nd hordei conidi tht germinted ut formed norml germ tue structures ( filed to form ppressori) 24 h fter inocultion onto second-formed ot leves of helthy controls, or onto leves ering B. grminis f. sp. vene colonies derived from rst inocultion 72 h erlier. All mteril ws xed 24 h fter second inocultion. Dt re for f. sp. vene conidi on ot cvs Mldwyn () nd Selm (), nd for. spp. tritici (c) nd hordei (d) on cv. Mldwyn. On controls ( ), dt re sed on counts of 5 rndomly selected germlings, while on leves ering colonies, counts were mde for 5 germlings locted distnt from colonies ( ), 5 within the outer of colonies ( ),nd 5 within the inner of colonies ( ), on ech of four replicte leves in ll cses. Percentge dt were trnsformed to ngles for nlysis of vrince. Where columns re heded y di erent letters, LSD tests pplied to ngle-trnsformed dt indicted signi cnt di erence (P 5.5) etween mens within dt set. to the in uences of f. sp. vene colonies thn either f. sp. vene itself or f. sp. tritici. In ddition to eing more sensitive to the in uence of f. sp. vene colonies, f. sp. hordei germlings ppered to show greter rnge of morphologicl normlities (Fig. 7) thn germlings of the other. spp. Hustorium formtion. Germlings of f. sp. hordei never formed hustori in ot cv. Mldwyn irrespective of whether they ttcked control leves or leves previously infected with f. sp. vene. Hustorium formtion y f. sp. tritici ws lso extremely rre; no germlings formed hustori in control leves or on cells distnt from estlished f. sp. vene colonies. Within the outer of f. sp. vene colonies, only 1 % of f. sp. tritici germlings formed hustorium nd within the inner, only 1.5 % did so. There were no di erences in the percentges of f. sp. vene germlings forming hustori in epiderml cells of control leves nd cells distnt from estlished colonies of f. sp. vene (Fig. 8). On Mldwyn [Fig. 8()], there ws no di erence etween germlings distnt from colonies nd those within the outer of colonies, lthough within the inner of colonies signi cntly fewer germlings formed hustori thn in ny other loction. In the experiment using Selm [Fig. 8()], similr percentges of germlings formed hustori in controls, in cells distnt from colonies nd in the inner of colonies. However, when germlings were locted within the outer of colonies, signi cntly higher percentge formed hustori thn in ny other loction. Brley cv. Plls inoculted rst with f. sp. hordei nd secondly with. spp. hordei, vene or tritici.

11 Inhiition of B. grminis development 219 () (c) u 3 µm 3 µm u () (d) 3 µm 3 µm FIG. 7. Light microgrphs showing exmples of norml germling structures formed y B. grminis f. sp. hordei when conidi germinted within the oundry of f. sp. vene colonies growing on Mldwyn ot leves. olonies were estlished from rst inocultion pplied 72 h erlier thn the second inocultion from which the germlings derive. All mteril ws xed 24 h fter the second inocultion. () The elongted germ tue is simple, hyph-like structure showing little sign of swelling or picl hooking, nd two sept re evident. The rst is close to the spore, t the position where the single septum forms in morphologiclly norml ppressoril germ tues, nd the second is pproximtely 35 mm from the conidium i.e. pproximtely where the picl ppressoril hook is formed y norml ppressoril germ tue. Note: three ungerminted conidi re locted close to hyphe (top right of microgrph). () As in (), two sept re present in the elongted germ tue. The centrl cell of this tue is swollen, ut further hyph-like cell hs een formed. Smll protruernces re evident on the centrl cell, the most distl of which (directed downwrds) is reminiscent of poorly developed ppressoril loe. (c) Agin two sept re present in the elongted germ tue. The centrl cell is swollen nd shows distinct protruernces, ut these re clerly too smll to e mistken for ppressoril loes. The distl cell of the germ tue is slightly swollen with clu-like pex. (d) When rst seen, this germling ws mistkenly thought to hve formed norml ppressorium with hooked picl loe (doule rrowhed). The rnch cell, seprted from the hooked cell y septum nd growing left from the centrl portion of the hooked cell, ws thought to e secondry hyphl growth. However, creful exmintion showed tht no hustorium ws present eneth the hooked structure. Therefore the cell growing left represented norml rnching of the elongted germ tue nd the hooked cell cnnot e interpreted s norml ppressoril structure. Severl exmples of similr germlings were seen. u ˆ ungerminted conidium; ˆ hyph of estlished colony; ˆ primry germ tue; rrowheds indicte sept in elongted germ tue; doule rrowheds indicte ppressorium-like structure. Germintion. Dt from rley showed trends very similr to those from ot (Fig. 9). onidi of ll. spp. germinted well on Plls rley control leves, nd eqully well on cells distnt from estlished colonies of f. sp. hordei. There ws slight tendency for germintion to e reduced when conidi were locted within the outer of f. sp. hordei colonies, ut the reduction ws smll nd signi cnt only in the cse of f. sp. vene. In ll cses, germintion ws signi cntly reduced where conidi were locted within the inner of colonies. However, the degree of reduction in germintion ws reltively smller thn tht seen within the inner of f. sp. vene colonies on ot (where germintion rtes were t lest hlved; Fig. 5). The mximum reduction ws seen with f. sp. vene where on distnt cells pprox. 69 % germinted compred to pprox. 39 % within the inner of f. sp. hordei colonies (i.e. reduction of 43 %) while there were only 32 nd 31 % reductions in germintion of f. sp. hordei nd tritici, respectively. Germling development. Extremely low frequencies of germling normlity were seen when conidi of ny. spp. germinted within f. sp. hordei colonies growing on rley (Fig. 1). This ws in complete contrst to the sitution seen when conidi germinted within colonies of f. sp. vene growing on ot. Even when the germlings

12 2 T. L. W. rver et l. 5 () () Germinted conidi forming hustori (%) c c ontrol Distnt Outer Inner ontrol Distnt Outer Inner onidium loction reltive to colony onidium loction reltive to colony FIG. 8. Percentges of B. grminis f. sp. vene conidi tht hd germinted on second-formed leves of ot cvs Mldwyn () nd Selm (), nd tht formed morphologiclly norml ppressori from which plnt epiderml cells were penetrted successfully, resulting in hustorium formtion, 24 h fter inocultion onto helthy control leves or onto leves ering B. grminis f. sp. vene colonies derived from rst inocultion 72 h erlier. All mteril ws xed 24 h fter the second inocultion. On controls ( ), dt re sed on counts of 5 rndomly selected germlings, while on leves ering colonies, counts were mde for 5 germlings locted distnt from colonies ( ), 5 within the outer of colonies ( ) nd 5 within the inner of colonies ( ), on ech of four replicte leves in ll cses. Percentge dt were trnsformed to ngles for nlysis of vrince. Where columns re heded y di erent letters, LSD tests pplied to ngle-trnsformed dt indicted signi cnt di erence (P 5.5) etween mens within dt set. were intimtely ssocited with hyphe of n estlished colony they lmost lwys formed norml ppressori (Fig. 11). For f. sp. hordei conidi, there ws no di erence in normlity frequency etween germlings locted within the inner of colonies compred with controls, germlings locted distnt from colonies or those locted within the colony outer. For. spp. vene nd tritici, there ws signi cnt increse in the frequency of normlity within the inner of f. sp. hordei colonies. However, even here only pprox. 1 % of germlings ppered norml (s compred with 7±8 % normlity for ny. spp. germinting within the inner of f. sp. vene colonies on ot). Hustorium formtion. When f. sp. hordei germlings ttcked control rley leves, epiderml cells distnt from estlished colonies, or were locted within the outer of colonies, 13±14 % of ppressori were ssocited with successful penetrtion ttempts s evidenced y the presence of hustorium eneth their ppressori (Fig. 12). However, when germlings were locted within the inner of colonies, the percentge of ppressori with hustori ws signi cntly incresed nd lmost douled in frequency (22.5 %). On controls nd on cells distnt from f. sp. hordei colonies, germlings of. spp. vene nd tritici never formed hustori (Fig. 12). By contrst, when germlings of these inpproprite. spp. were locted within the inner of f. sp. hordei colonies, they frequently formed hustorium (17.5 nd 14.5 % for vene nd tritici, respectively, Figs 12 nd 13), nd even if they were locted within the outer of colonies, few germlings formed hustori (4 nd 2 %, respectively). From csul oservtion, it ws pprent tht when germlings of the inpproprite. spp. locted within f. sp. hordei colony formed hustorium, this hustorium ws most frequently formed in n epiderml cell tht lso contined hustorium of the estlished colony (illustrted in Fig. 13). However, dt to con rm this oservtion were not collected. Doule inocultion of Selm ot: hyphe of estlished colonies removed Removing the super cil hyphe of B. grminis f. sp. vene colonies tht hd een previously llowed to develop for 96 h efore re-inocultion, llowed us to determine whether e ects on germintion nd germling development depended upon the presence of hyphe or whether the presence of hustori within epiderml cells ws su cient to medite these e ects. Where colony hyphe were removed, hustori formed y the colony were clerly visile, nd epiderml cells contining those hustori ppered to remin live s evidenced y lck of whole-cell uto uorescence nd sence of cytoplsmic grnultion or disorgniztion. This ws s expected from erlier studies [8, 27±29] where hustoriumcontining cells lso survived removl of super cil structures. ells contining colony hustori oviously ly within the outer oundry of colonies s de ned erlier y n imginry line linking tips of leder hyphe. Furthermore, from our oservtions nd Hirt's [18]

13 Inhiition of B. grminis development () () onidi tht germinted (%) () (c) c ontrol Distnt Outer Inner Germinted conidi developing normlly (%) () (c) ontrol Distnt Outer Inner onidium loction reltive to colony FIG. 9. Percentges of B. grminis. spp. hordei (), vene () nd tritici (c) conidi tht hd germinted 24 h fter inocultion onto second-formed cv. Plls rley leves of helthy controls, or onto leves ering B. grminis f. sp. hordei colonies derived from rst inocultion 72 h erlier. All mteril ws xed 24 h fter the second inocultion. On controls ( ), dt re sed on counts of 1 rndomly selected conidi, while on leves ering colonies, counts were mde for 1 conidi locted distnt from colonies ( ), 1 within in the outer of colonies ( ), nd 1 within the inner of colonies ( ), on ech of four replicte leves in ll cses. Percentge dt were trnsformed to ngles for nlysis of vrince. Where columns re heded y di erent letters, LSD tests pplied to ngle-trnsformed dt indicted signi cnt di erence (P 5.5) etween mens within dt set. onidium loction reltive to colony FIG. 1. Percentges of B. grminis. spp. hordei (), vene () nd tritici (c) conidi tht germinted ut formed norml germ tue structures ( filed to form ppressori) 24 h fter inocultion onto second-formed cv. Plls rley leves of helthy controls, or onto leves ering B. grminis f. sp. hordei colonies derived from rst inocultion 72 h erlier. All mteril ws xed 24 h fter the second inocultion. On controls ( ), dt re sed on counts of 5 rndomly selected germlings, while on leves ering colonies, counts were mde for 5 germlings locted distnt from colonies ( ), 5 within the outer of colonies ( ), nd 5 within the inner of colonies ( ), on ech of four replicte leves in ll cses. Percentge dt were trnsformed to ngles for nlysis of vrince. Where columns re heded y di erent letters, LSD tests pplied to ngle-trnsformed dt indicted signi cnt di erence (P 5.5) etween mens within dt set.

14 222 T. L. W. rver et l. () () H S H App App µm µm App H FIG. 11. LTSEM microgrphs of norml B. grminis f. sp. hordei germlings tht developed when in intimte contct with hyphe of B. grminis f. sp. hordei growing on rley lef. The germlings grew from conidi inoculted 24 h erlier, while the colonies derived from inocultion mde 96 h erlier. In () the conidium on the left hs formed two susidiry germ tues, proly ecuse it ws deposited upon the hyphe of the colony nd filed to perceive the underlying host lef surfce [38]. The primry germ tue of this conidium is presumly hidden from view. App ˆ ppressoril germ tue; ˆ conidium; H ˆ hooked picl loe of ppressorium; ˆ hyphe of colony; ˆ primry germ tue; S ˆ susidiry germ tue. detiled studies, it is most likely tht hustoriumcontining cells ly within the inner of colonies whose hyphe hd een removed. On the other hnd, epiderml cells four cells distnt from hustoriumcontining cells proly ly outside ( for cropetl nd sipetl cells), or only just within ( for lterl cells), the oundries of colonies tht hd een removed. Thus, these epiderml cells were regrded s comprle to cells previously de ned s distnt from colonies. Germintion nd germling development. Overll, slightly ut signi cntly (P 5.5) fewer conidi germinted on control leves (pprox. 63 %) thn on doule inoculted leves. However, unlike experiments where colony hyphe were left in plce, there ws no di erence in germintion etween conidi distnt from cells contining colony hustori (germintion ˆ pprox. 68 %), nd conidi overlying epiderml cells contining colony hustori (germintion ˆ pprox. 69 %). There ws signi cnt (P 5.1) di erence in germintion etween the three fungl. spp., with f. sp. hordei conidi germinting slightly less well (pprox. 62 %) thn either. spp. vene (pprox. 7 %) or tritici (pprox. 68 %). These smll di erences presumly re ect uncontrolled vrition in inoculum qulity etween. spp. However, there ws no interction etween conidium loction nd. spp. indicting consistency of these smll e ects within the experiment. For germinted conidi, there ws only very low frequency of germling normlity in ll loctions nd most germlings formed pprently norml ppressori. For ll. spp. nd ll loctions, normlity ws never seen for more thn 6 % of germlings even if the germlings were in contct with epiderml cells contining hustori formed y f. sp. vene colonies tht hd een removed. Thus, in complete contrst to experiments where colonies were undistured, the loction of conidi on epiderml cells tht hd een within the oundries of colonies estlished y the rst inoculum hd no e ect on either their germintion or on susequent germling development. Hustorium formtion. Although loction hd no e ect on ppressorium formtion, it hd drmtic e ect on the percentge of germlings tht penetrted epiderml cells successfully to produce hustori (Fig. 14). When f. sp. vene germlings ttcked control leves or epiderml cells distnt from cells contining hustorium formed erlier y n f. sp. vene colony, pprox. 42 % formed hustori. However, when ttck ws on cell contining hustorium formed erlier, 92 % did so. Thus, cells tht contined colony hustori were conditioned to high level of ``ccessiility'' to susequent ttck y f. sp. vene. The presence of f. sp. vene colony hustori in epiderml cells of ot lso conditioned them to high level of ccessiility to susequent ttck y germlings of. spp. tritici nd hordei. Thus, 9 % of f. sp. tritici nd 58 % of f. sp. hordei germlings penetrted successfully nd formed hustori when ttcked cells contined f. sp. vene colony hustori. By contrst, neither of these inpproprite. spp. formed hustori in control ot leves or in cells distnt from cells contining f. sp. vene colony hustori. DISUSSION We were very surprised y our initil nding from Selm ot tht germintion nd germling development were mrkedly nd dversely ected when B. grminis conidi

15 Inhiition of B. grminis development () t 4 Ht Appt Appt t 3 Ht 1 Hh Germinted conidi forming hustori (%) () (c) NA NA Apph FIG. 13. Light microgrph of two B. grminis f. sp. tritici germlings locted within the inner of B. grminis f. sp. hordei colony. The colony ws estlished from f. sp. hordei conidium deposited 72 h erlier thn the f. sp. tritici conidi. The specimen ws xed 24 h fter deposition of the f. sp. tritici conidi. Both f. sp. tritici germlings produced norml ppressori from which hustori were formed. Apph ˆ primry ppressorium of f. sp. hordei colony; Appt ˆ ppressorium of f. sp. tritici germling; h ˆ conidium of f. sp. hordei,t ˆ conidium of f. sp. tritici. Hh ˆ primry hustorium of f. sp. hordei colony; Ht ˆ primry hustorium of f. sp. tritici germling. h NA ontrol NA Distnt Outer Inner onidium loction reltive to colony FIG. 12. Percentges of B. grminis. spp. hordei (), vene () nd tritici (c) conidi tht hd germinted on second-formed cv. Plls rley leves, nd tht formed morphologiclly norml ppressori from which plnt epiderml cells were penetrted successfully, resulting in hustorium formtion, 24 h fter inocultion onto helthy control leves or onto leves ering B. grminis f. sp. hordei colonies derived from rst inocultion 72 h erlier. All mteril ws xed 24 h fter the second inocultion. On controls ( ), dt re sed on counts of 5 rndomly selected germlings, while on leves ering colonies, counts were mde for 5 germlings locted distnt from colonies ( ), 5 within the outer of colonies ( ) nd 5 within the inner of colonies ( ), on ech of four replicte leves in ll cses. Percentge dt were trnsformed to ngles for nlysis of vrince. Where columns re heded y di erent letters, LSD tests pplied to ngle-trnsformed dt indicted signi cnt di erence (P 5.5) etween mens within dt set. NA: dt ˆ zero, therefore excluded from nlyses. were deposited within estlished ot mildew colonies. This ws ecuse considerle ody of evidence indictes tht deposition of B. grminis conidi, nd conidi of other powdery mildew fungi, close to n estlished B. grminis colony cn gretly enhnce the chnces of these conidi producing successful infections. This evidence comes from investigtions of whet nd rley infected with their pproprite f. sp. nd susequently inoculted with n inpproprite f. sp. of B. grminis or with less closely relted Erysiphcee. Thus Mosemn nd his co-workers [3, 31] found tht whet could e ttcked y B. grminis f. sp. hordei if leves were lredy infected with virulent isolte of B. grminis f. sp. tritici or if the two fungi were inoculted simultneously. Tsuchiy nd Hirt [37] inoculted B. grminis f. sp. hordei-infected rley with f. sp. tritici, B. grminis isolte from Agropyron, or with powdery mildew fungi from 49 di erent dicotyledonous species; the fungi included species from the gener Erysiphe, Spherothec, Podospher, Microspher nd Uncinul. Of the 51 fungi tested, 45 were le to infect the rley nd 3 formed sporulting colonies. Tsuchiy nd Hirt [37] noted tht these infections lmost invrily occurred close to estlished f. sp. hordei colonies nd detiled histologicl studies of the B. grminis isolte from Agropyron nd of Spherothec fuligine showed tht successful infections y these fungi were lmost invrily estlished in rley epiderml cells contining B. grminis f. sp. hordei hustori or in neighouring cells. Similrly, Ouchi et l. [33] concluded tht induced `ccessiility' of rley to ttck y whet