Hydroxamic acids in Aegilops species and effects on Rhopalosiphum padi behaviour and fecundity

Transcription

1 Bulletin of Insectology 66 (2): , 2013 ISSN Hydroxmic cids in Aegilops species nd effects on Rhoplosiphum pdi behviour nd fecundity Henriett ELEK 1, Lesley SMART 2, Jnet MARTIN 2, Shkoor AHMAD 2, Ruth GORDON-WEEKS 2, Angel ANDA 3, Suznne WELHAM 4, Peter WERNER 1, John PICKETT 2 1 KWS UK Limited, Thriplow, UK 2 Rothmsted Reserch, Hrpenden, UK 3 University of Pnnoni Georgikon Fculty, Keszthely, Hungry 4 VSN Interntionl Limited, Hemel Hempsted, UK Abstrct As n environmentlly comptible lterntive to the use of conventionl insecticides to control cerel phids, the possibility of exploiting nturl resistnce to insect pests in whet species ws investigted. Previous work, compring the ntibiotic nd ntixenotic effects of hexploid whet (Triticum estivum, AABBDD), tetrploid whet (Triticum durum, AABB) nd some A genome diploid species on the bird cherry-ot phid, Rhoplosiphum pdi L., found little difference between ccessions in the higher ploidy plnts, but the diploid species contined ttributes tht could be importnt in the breeding for resistnce ginst phids in the future. This study concentrted on wild ccessions of diploid Aegilops species to which the closest ncestor of the B genome donor of hexploid whet belongs. The phid R. pdi showed reduced ttrction nd n increse in the intrinsic rte of popultion growth on the B genome species tested by compred to the hexploid control. Investigtion of group of secondry metbolites, the hydroxmic cids or benzoxzinones showed tht lef tissue of one of these (Aegilops speltoides) contins high levels of DIMBOA-glucoside nd of the min glucone, 2,4-dihidroxy-7-methoxy-1,4-benzoxzin-3-one (DIMBOA) while no hydroxmic cids were found in the lef tissue of Ae. longissim, Ae. bicornis nd Ae. shronensis nd only trce levels in Ae. sersii. In those species, n unknown compound ws present, which my hve n effect on phid behviour. The effect of phid feeding on levels of hydroxmic cids in Ae. speltoides nd Ae. shronensis ws lso exmined. While loclised defence rection to phid feeding hd been identified in the hexploid nd tetrploid species, more systemic effect ws observed in the diploid Ae. speltoides. Key words: Aegilops species, Aegilops speltoides, phid behviour, Rhoplosiphum pdi, hydroxmic cids, plnt resistnce. Introduction Modern whet belongs to two species, the hexploid Triticum estivum (AABBDD, 2n = 42 chromosomes) nd the tetrploid T. durum (AABB, 2n=28 chromosomes). These species re grown round the world in ll temperte zones nd re economiclly the most importnt of the smll grined cerels. Under the influence of ncient nd modern humn cultivtion, whet hs developed through series of specition events driven by mphiploidy (hybridistion) between diploid Triticum species nd Aegilops species (Nevo et l., 2002). The mjor grss subfmilies rdited million yers go nd the Triticee tribe - where the ncestrl species of hexploid whet belongs - diverged within the Pocee bout 35 million yers go (Hung et l., 2002; Crver, 2009). Within the Triticee, the B genome diverged before the seprtion of the A nd D genomes (Crver, 2009). A number of potentil specition events hve been proposed, but it is most likely tht tetrploid whet (AABB) first rose from hybridistion of T. urrtu (AA) or T. monococcum ssp. boeoticum (AA) with Ae. speltoides (BB). Subsequent hybridistion of cultivted tetrploid with Ae. tuschii (DD) led to the bred nd spelt hexploid whets (AABBDD). Insect pests re ubiquitous nd diverse throughout the cultivtion zones for whet, but the most economiclly significnt phid pest of Europen cerel crops is the bird cherry-ot phid (Rhoplosiphum pdi L.). Fctors such s crowding, temperture, photoperiod nd poor host plnt qulity cn cuse n incresed production of lte (winged morphs) in phid popultions. Widespred dispersl of the lte my led to greter spred of trnsmitted diseses, prticulrly brley yellow dwrf virus (BYDV). Even low levels of ntibiosis cn be importnt in limiting infesttion levels of R. pdi (Hesler et l., 1999). Severl potentil sources of host plnt resistnce hve been identified ginst bird cherry-ot phid in Europe including lef phenolic content nd phenyllnine mmoni-lyse ctivity (Smith et l., 2004). In ddition to these, the importnce s n phid resistnce fctor of group of secondry metbolites, the hydroxmic cids (HAs), ws recognised in the 1990s. Experiments showed phids prefer to settle on plnts tht contin lower concentrtion of HAs (Givovich nd Niemeyer, 1991; Nicol et l., 1992) nd tht in rtificil diet n incresed level of HA concentrtion decresed the survivl rte of cerel phids (Brri et l., 1992). HAs were first discovered in the 1960s in rye, in reltion to fungl diseses, nd lter found in mize where they were ssocited with resistnce to the Europen corn borer, Ostrini nubillis (Hubner) (Klun nd Robinson, 1969; Niemeyer, 2009). HAs cn be found in the cultivted monocotyledons mize, whet (Nicol et l., 1992) nd rye. The HA 2,4-dihydroxy-1,4-benzoxzin- 3-one (DIBOA) cn be found in the wild Hordeum species (Brri et l., 1992) lthough no HAs present in the modern cultivted brley vrieties. The mximum recorded HA level in cultivted whet is between 1.4 to 10.9 mmol/kg fresh weight (FW) (Copj et l., 1991) nd nerly 40 mmol/kg FW ws found in wild rye (Nicol et l., 1992; Nicol et l., 1993).

2 2,4-Dihydroxy-7-methoxy-1,4-benzoxzin-3-one (DIMBOA), the min HA glucone to be found in whet extrcts, hs been shown to cuse ntibiosis, feeding deterrence, decresed performnce nd reduced reproduction in phids (Figuero et l., 2004). In ddition, it hs mutgenic effects nd ffects the level of genetic polymorphism in phid popultions (Figuero et l., 2004). As the concentrtion of HAs increses, phids need longer time to serch for suitble phloem vessels (Givovich nd Niemeyer, 1994; 1995) becuse they feed from single sieve tube nd the HA concentrtion cn be different between sieve tubes (Givovich et l., 1994). They lso spend longer period ingesting xylem fluid (Rmìrez nd Niemeyer, 1999), which does not contin HAs (Givovich nd Niemeyer, 1995). The HA level lso ffects virus trnsmission becuse phids tke longer time to ttch to the phloem nd re therefore less ble to trnsmit BYDV (Givovich nd Niemeyer, 1991; Nicol et l., 1992). In preliminry ntixenotic study we investigted the host plnt selection / settling behviour of R. pdi, on very diverse set of hexploid (T. estivum) nd tetrploid (T. durum) whets, few cultivrs showed moderte reduction in phid preference while severl were more preferred thn the hexploid control vriety, Solstice (Elek et l., 2009; Elek et l., 2013). Most of the diploid species tested, such s the A genome T. monococcum nd T. boeoticum, were less preferred by phids thn the hexploid control (Elek et l., 2012), but the gretest resistnce ws found in the B genome diploid species Ae. speltoides nd Ae. shronensis. This study investigted the ntibiotic nd ntixenotic effect of different B genome diploid species ginst R. pdi together with HPLC nlysis to evlute HA levels in the plnt tissues. Mterils nd methods Plnt mteril Accessions of Ae. shronensis ( , ), Ae. bicornis ( , ), Ae. longissim ( , ), Ae. speltoides ( ) nd Ae. sersii ( , ) were supplied by the John Innes Centre, nd the hexploid vriety Solstice by Rothmsted Reserch. Aphids R. pdi ws collected from volunteer whet plnts from the field in Thriplow, Herts, UK in September 2007 nd gin (refreshing the colony) in 2008 nd The colonies used in this study were estblished from one phid using the mildew resistnt spring whet vriety Tyblt s the culture plnt. Aphids were kept in glsshouse in temperture rnge of C nd 16:8 L:D, in cm insect cges. Colonies were checked regulrly to void contmintion with other phid species nd diseses. For the settling test, mixed ge lte phids were collected from the top of the rering cge with smll electric potter. For the fecundity test nd the phid feeding experiments, wingless phids (ptere) were gently shken off or removed from the culture plnts with pint brush. Settling test of phids on whet plnts This ws choice test in smll ventilted cylindricl chmber (12 cm dimeter by 20 cm high) between one 7 dy old seedling ech (growth stge 11, Tottmn nd Mkepece, 1979) of the test (Ae. speltoides or Ae. shronensis ) nd of the control hexploid plnt (Solstice). Plnts were growing in Levington F1 seedling compost in cm cells. To set up the experiment the cells were moved onto wet snd try to keep the humidity high during the testing period. Twenty rndomly llocted R. pdi lte were plced in ech cge nd there were 14 to 17 replictes per plnt species. Alte, which hd settled on ech of the two plnts, were counted nd recorded fter 2, 5 nd 24 hours. The test ws conducted in the glsshouse t 20 C, 16:8 L:D. For ech observtion the difference in the number of lte settling on Solstice compred to the test vriety ws clculted nd the full dtset ws nlysed using the liner mixed model routine with residul mximum likelihood estimtion (REML) in GENSTAT (13 th edition). Occsions nd cges were used s rndom terms, with test vriety, time (2, 5 or 24 hours) nd their interction used s fixed effects. No trnsformtion ws required. Aphid fecundity tests on whet plnts This ws no choice test used to determine the intrinsic rte of popultion increse (r m ) (Wytt nd White, 1977) of R. pdi on the test vrieties (Ae. speltoides , Ae. shronensis , nd Ae. longissim ) compred to the control plnt (Solstice). In seprte experiment, the r m ws determined on Ae. sersii ( ) compred to Solstice. Seven lte were put in cge with one plnt of ech test vriety to produce similrly ged neonte nymphs for the experiment. After 24 hours the lte were removed nd the nymphs were llowed to develop on those plnts for 3 dys until they hd moulted to 2 nd instr. At this ge the nymphs were lrge enough to trnsfer esily onto 7 dy old test seedlings (GS11) which were grown in Levington F1 seedling compost. One 3 dy old nymph ws plced on the middle prt of the first lef of ech of 8 to 10 replicte seedlings nd enclosed in 2 cm dimeter clip cge (McGillivry nd Anderson, 1957). The developing phids were monitored t the sme time ech dy until they produced their first nymph (time d). From the first dy of production, the new nymphs were removed nd their numbers recorded dily over period t lest equivlent to time d (equivlent to the dys from birth to the first nymph produced). The experiment ws crried out in glsshouse t 20 C nd 16:8 L:D. From the dt the intrinsic rte of popultion increse ws clculted using the formul [1] of Wytt nd White (1977). [1] Intrinsic rte of popultion increse: r m = c (log e Md) / d Where c is constnt = 0.74, d = pre-productive period in dys (from birth to the first nymph produced), Md = number of nymphs produced in the reproductive period equl to d. 214

3 The intrinsic rtes of popultion increse from the two experiments were nlysed using the liner mixed model routine (REML). Since no significnt differences were found between the experiments, the results of Ae. sersii re presented together with the rest of the test vrieties. The intrinsic rte of popultion increse, verge number of nymphs produced per dy nd the time tken from birth to produce the first nymph were nlysed using ANOVA. Vrition mong vrieties ws prtitioned into n overll comprison of diploid species ginst Solstice plus differences between the diploid species. Hydroxmic cid nlysis of different plnt tissues Levels of HAs were mesured in replicted 6 dy (GS 11), 9 dy (GS 11-12) nd 12 dy old (GS12-13) seedlings of Ae. shronensis ( , ), Ae. bicornis ( , ), Ae. longissim ( , ), Ae. speltoides ( ) nd Ae. sersii ( , ) grown in compost under controlled conditions (20 C nd 16:8 L:D), with two replictes per time point per vriety, ech replicte consisting of 3 plnts. Lef, coleoptile nd root tissue smples, frozen in liquid nitrogen, were ground in pestle nd mortr, ensuring the smple remined frozen during preprtion. The ground tissue ws trnsferred to n Eppendorf tube, which ws frozen to void the smple melting nd sticking to the side of the tube. 25 mg of frozen smple ws weighed into n Eppendorf tube contining 0.5 ml of 98% methnol + 2% cetic cid mixture. It ws sonicted for 10 minutes nd centrifuged for 10 minutes on 21.5 g t 4 C. Superntnt ws trnsferred into glss vil for nlysis by HPLC. A reversed phse HPLC procedure ws dpted from previously published methods (Bumeler et l., 2000). A therml hypersil C-18, (5µm, mm) column ws used, with mobile phse (A) HPLC grde wter nd (B) methnol/isopropnol (95/5) % cetic cid. The grdient profile of solvent A nd B ws: 0-2 min, 10% B; 2-11min, 10-50% B; 16-17min, 50 to 10% B. Injection volume ws 20µl, the flow rte ws 1 ml/min nd the run time 17 minutes. From the HPLC it ws possible to determine the levels of HAs found in the plnt tissues in mmol/kg fresh weight (FW) by using rnge of stndrd DIMBOA nd DIMBOA glucoside concentrtions. The synthetic stndrds were provided by Prof. Dieter Sicker, University of Leipzig. The HPLC method used could not seprte DIMBOA nd HDMBOA-glucoside (2-hydroxy-4,7-dimethoxy- 1,4-benzoxzin-3-one glucoside), nd therefore these compounds re presented s combined vlues throughout, lthough levels of the ltter re known to be low in whet (Oikw et l., 2002). HA levels were compred cross species, vrieties within species nd time points using two-wy ANOVA in GENSTAT. A squre-root trnsformtion ws required to stbilize the vrince. Aphid feeding experiment on plnts nd smpling for HA nlysis To determine the effects of R. pdi feeding on HA levels in lef tissues of Ae. speltoides ( ) nd Ae. shronensis ( ), btches of twenty R. pdi lte were put into replicted cges with one seedling (GS 11) for 24 hours to produce pre-conditioned nymphs for the experiment fter which the lte were removed. Nymphs were llowed to develop on those plnts for 3 dys until they moulted to 2 nd instr nd then they were trnsferred to 7 dy old test plnts (GS 11-12). Btches of twenty-five 3 dy old nymphs were put in clip cge on the middle prt of the first lef of ech of fifty 7 dy old (GS 11-12) test plnts, which were kept in controlled environment (20 C nd 16:8 L:D). Fifty equivlent plnts with clip cges, but no phids were lso kept under the sme conditions s controls. The first smpling ws done on pproximtely hlf of the replicte plnts fter 24 hours of phid feeding, hrvested in 3 replictes of 8 plnts ech. The phids were removed from the lef using smll pint brush, nd the bove ground prt of the plnt ws hrvested nd seprted into three prts; 1) the lef re immeditely under the clip cge where the phids hd been feeding, 2) the coleoptile nd 3) the reminder of the lef mteril. The tip of the first lef ws not included. These were wrpped in foil, lbelled nd frozen immeditely in liquid nitrogen. Control plnts were smpled in the sme wy s the test plnts. After 48 hours of phid feeding the smpling ws repeted on the remining plnts. Plnt smples were prepred for HPLC nlysis s described in the previous section. Dt were nlysed using three-wy ANOVA in GENSTAT. A squre-root trnsformtion ws required to stbilise the vrince. Results Settling test of phids on whet plnts There ws evidence of differentil response over time (P < 0.001), but no evidence of n interction (P = 0.994) or of differences between the test vrieties (P = 0.797). After 24 hours, significntly fewer phids (Solstice - test = 3.07, SE 0.71, P < 0.001) settled on Ae. speltoides thn on the hexploid control (figure 1). Similrly, when Ae. shronensis ws tested ginst Solstice significntly more phids settled on the control vriety (Solstice - test = 2.882, SE 0.60, P < 0.001). No differences were found t 2 or 5 hours, nd no difference ws found between the test vrieties in phid settling behviour. Aphid fecundity tests on whet plnts In ech of three spects of fecundity mesured the B genome diploids proved more chllenging to R. pdi thn the control hexploid Solstice (figure 2). R. pdi nymphs feeding on the diploid vrieties took 8-12 dys to strt to reproduce, while nymphs feeding on the hexploid vriety took 7-9 dys from birth (P < 0.001). The verge dily nymph production ws higher for Solstice, (5.2 nymphs/dy; SE 0.44), thn on the diploid vrieties, (3.3 nymphs/dy; SE 0.24) (P < 0.001). On the Aegilops species the verge totl nymph production ws only 39 while on Solstice it ws 63 during the test period (P < 0.001) (figure 2). This difference lso showed in the size of the dult phids, since phids 215

4 Averge number of settled lte b 2 hours 5 hours 24 hours Aegilops speltoides Solstice Figure 1. R. pdi lte settlement on Ae. speltoides nd the hexploid control (Solstice) fter 2, 5 nd 24 hours. No differences were found in the number of settled phids fter 2 nd 5 hours, but significntly more phids settled on Solstice fter 24 hours (P < 0.001). Different letters bove the columns indicte significnt difference t P = feeding on the diploid vrieties were noticebly much smller thn those on Solstice. There ws some vrition between the diploid vrieties (P < 0.001, figure 2), with the number of nymphs produced per dy on Ae. shronenesis nd Ae. sersii not significntly different to Solstice. The lowest nymph production ws noted on Ae. speltoides where phids took on verge 3 dys longer to produce the first nymph. The difference in intrinsic rte of popultion increse between the vrieties ws highly significnt (P < 0.001) the lowest r m ws clculted on Ae. speltoides (SE 0.009), which ws 33% lower thn the hexploid control Solstice (SE 0.009) (figure 2b). Hydroxmic cid in different plnt tissues When the coleoptiles nd the roots of Ae. shronensis, Ae. longissim, Ae. bicornis nd Ae. sersii were nlysed only low levels of HAs were detected compred to levels found previously in Ae. speltoides by Gordon- Weeks et l., (2010) (tble 1). Sum of verdge dily nymph production ) Solstice Ae. s hronensis Ae. sersii Ae. longissim Ae. speltoides r m b) b b b c Dys counted from the birth of the nymphs 0.2 Figure 2. ) Sum of the verge dily nymph production by R. pdi on the B genome Ae. speltoides, Ae. longissim, Ae. sersii, Ae. shronenesis nd on the hexploid Solstice. b) The intrinsic rte of popultion increse (r m ) of R. pdi on Aegilops sp. nd the hexploid control Solstice (P < 0.001). Different letters bove the columns indicte significnt difference t P =0.05. Tble 1. Averge levels of hydroxmic cids (mmol/kg fresh weight) in the root, coleoptile nd the lef tissues of different B genome species t 6 (GS 11), 9 (GS 11-12) nd 12 dys (GS 12-13). The levels of HAs (mmol/kg fresh weight) in Ae. speltoides s mesured by Gordon-Weeks et l. (2010) re presented in bold. Smpled re Root Coleoptile Lef 216 Species 6 dys 9 dys 12 dys DIMBOA (+HDMBOA-glu) DIMBOA (+HDMBOA-glu) DIMBOAglucoside DIMBOAglucoside DIMBOAglucoside mmol/kg fresh weight Ae. shronensis DIMBOA (+HDMBOA-glu) Ae. longissim SE 0.08 SE 0.09 SE 0.08 SE 0.09 SE 0.08 Ae. sersii SE 0.09 Ae. bicornis Ae. speltoides 5 SE SE SE SE SE SE 0.19 Ae. shronensis Ae. longissim Ae. sersii 2.97 SE SE SE SE SE SE 0.31 Ae. bicornis Ae. speltoides 26.5 SE SE SE SE SE SE 3.24 Ae. shronensis null null null null null null Ae. longissim null null null null null null Ae. sersii 4.29 E-05 null 3.14 E 1.92 E E E-05 Ae. bicornis null null null null null null Ae. speltoides 27 SE SE SE SE SE SE 1.21

5 mm/kg fresh weight mm/kg fresh weight phid +phid phid +phid phid +phid 0 phid +phid phid +phid phid +phid under the clip cge rest of the leves coleoptile under the clip cge rest of the leves coleoptile Figure 3. Chnges in DIMBOA-glucoside level in the lef tissue of Ae. speltoides fter 48 hours of R. pdi feeding. None of the prts of the plnt tested showed ny significnt difference in the DIMBOA-glucoside level fter phid feeding. The bove ground prts of the plnt were nlyzed seprtely; under the clip cge (the middle prt of the first lef where 25 phids were feeding), the coleoptile nd the rest of the leves not including the tip of the first lef. Figure 4. Chnges in DIMBOA (+ HDMBOA-glucoside) level in the lef tissue of Ae. speltoides fter 48 hours of R. pdi feeding. None of the prts of the plnt tested showed ny significnt difference in the DIMBOA (+ HDMBOA-glucoside) level fter phid feeding. The bove ground prts of the plnt were nlyzed seprtely; under the clip cge (the middle prt of the first lef where 25 phids were feeding), the coleoptile nd the rest of the leves not including the tip of the first lef. In the roots DIMBOA-glucoside levels were recorded between the diploid species (P = 0.021) nd there ws evidence of significnt chnges over time (P < 0.001) due to decrese with plnt ge. In the 6 dy old plnts the verge DIMBOA-glucoside level ws between 0.76 nd 1.35 mmol/kg FW, which decresed by dy 12 to mmol/kg FW. The DIMBOA (+HDMBOAglucoside) level vried between species (P = 0.027) but there ws no evidence of differences over time. The verge DIMBOA (+HDMBOA-glucoside) level ws lower t between 0.16 nd 0.48 mmol/kg FW in 6 dy old plnts nd mmol/kg FW in 12 dy old plnts. Higher levels of HAs were mesured in the coleoptiles nd gin there ws evidence of differences with plnt ge (P < 0.001) nd between the diploid species (P = 0.02) for DIMBOA-glucoside. In the coleoptiles of the 6 dy old seedlings the DIMBOA-glucoside rnged cross species from 1.3 to 2.97 mmol/kg FW, which decresed by dy 12 to mmol/kg FW (P < 0.001). The DIMBOA (+HDMBOA-glucoside) level showed some evidence of chnge over time (P = 0.045) due to generl decrese with plnt ge, to differences between the diploid species (P = 0.002) nd to n interction between vriety nd time point (P = 0.042), s one vriety showed n increse rther thn decrese with plnt ge. The DIMBOA (+HDMBOA-glucoside) level ws between 0.78 nd 3.22 mmol/kg FW in 6 dy old plnts nd between 0.33 nd 1.27 mmol/kg FW by dy 12. HPLC of the 6 dy old (GS 11) Ae. speltoides lef tissue extrcts detected high levels of DIMBOA-glucoside (27 mmol/kg FW) nd DIMBOA (+HDMBOAglucoside) (22.2 mmol/kg FW), but none of the known HAs could be detected in the lef tissue of the other B genome species Ae. shronensis, Ae. longissim nd Ae. bicornis (tble 1). In the lef tissue extrct of Ae. sersii, there were two peks, which coincided with DIM- BOA-glucoside nd DIMBOA (+HDMBOA-glucoside). The pek res for both retention times were too smll to be ble to confirm their identities nd further nlysis is required. The lef tissue of ll the B genome species contin n unidentified compound which eluted on the HPLC t minutes nd this my be the sme compound seen to elute t minutes in Ae. speltoides nd lso in T. estivum extrcts (unpublished dt). Effect of phid feeding on the HA levels of seedlings Across the experiment Ae. speltoides, showed nonsignificnt chnges in HA levels in the lef tissue smpled from immeditely under the clip cge in comprison with control plnts. For the DIMBOA-glucoside in Ae. speltoides, there ws evidence of difference between prts of the plnt (P < 0.001), with lower levels in coleoptiles compred to the leves, s found by Gordon-Weeks et l., (2010) in 9 dy old plnts (tble 1), but no other significnt differences. After 24 hours of phid feeding, the DIM- BOA-glucoside level ws similr in the presence nd bsence of phids, in the lef re where the phids were feeding we recorded 20.9 mmol/kg FW nd 23.5 mmol/kg FW in the control. In the coleoptile we mesured 8.7 mmol/kg FW in the bsence of phids nd 7.7 mmol/kg FW fter phid feeding (figure 3). After 48 hours we were still not ble to record sttisticl difference in the plnts in the presence nd bsence of the phids. Through the experiment for DIMBOA (+HDMBOAglucoside) in Ae. speltoides, there ws evidence of difference between prts of the plnt (P < 0.001), with incresed levels in the middle of the lef, compred to the coleoptiles nd the rest of the leves. There ws lso e- vidence of three-wy interction (P = 0.026), due to n increse in levels between 24 nd 48 hours in the middle 217

6 of the lef compred to slight decrese in the control. After 24 hours of phid feeding, the DIMBOA (+HDMBOA-glucoside) levels were similr in the test nd the control plnts. In the lef re where the phids were feeding we recorded 14.8 mmol/kg FW nd in the control 18.4 mmol/kg FW (figure 4), nd the difference ws still not significnt fter 48 hours of phid feeding. Ae. shronensis ws lso tested in this phid feeding ssy, but neither DIMBOA-glucoside nor DIMBOA (+HDMBOA-glucoside) were detected by HPLC in the control lef extrct, with no phid feeding, or in the lef in the immedite region of the feeding phids. Discussion nd conclusions These studies pull together series of observtions which provide more evidence bout the importnce of wild reltives of polyploid whet. The wild diploid species possess wide rnge of dptive diversity to diseses, pests nd ecologicl stress for this reson these species could be potentil germplsm source for further crop improvement. Preliminry studies of the hexploid (AABBDD, 2n = 42) nd tetrploid (AABB, 2n = 28) vrieties showed tht the hexploid whet does produce hydroxmic cids, but in moderte concentrtions of 1.2 to 7.8 mmol/kg FW (Elek et l., 2013). In the tetrploid T. durum there ws higher level of DIMBOA-glucoside leding to higher overll levels of 11.3 to 14.3 mmol/kg FW of totl HA. This reduction in the level of HA from the 4 to 6 species ws lso reported by Niemeyer nd Jerez (1997). When the A genome species Triticum boeoticum nd its cultivted form Triticum monococcum (2n = 14) ws investigted we could not detect ny hydroxmic cid in the lef tissue but we noticed negtive effect on phid settlement nd nymph production on certin ccessions (Elek et l., 2012). In our current studies we found similr effect during testing group of B genome species including the closest ncestrl of the B genome donor (Aegilops speltoides) of the hexploid whet. Most of the species tested hve only very low or non-detectble levels of the known HAs in the leves of seedlings. However, the ccession of Ae. speltoides (214008) tht ws tested contined over 20 mmol/kg FW DIMBOA in the 6 dy old seedling leves, which is considerbly higher level of HAs thn reported previously in either the 6 or 4 species. Niemeyer published similr observtions in 1988, he lso mesured high level of hydroxmic cids in the Ae. speltoides species, 14.3 mmol/kg FW, lthough he ws testing 10 dy old seedlings, lower level of HAs ws recorded in the tetrploid vrieties (4.2 mmol/kg FW) nd only 1.4 mmol/kg FW ws detected in the hexploid vrieties. Ae. shronensis, the other representtive of the B genome, is null hydroxmic cid expressor in the lef but it cn produce DIMBOAglucoside nd DIMBOA (+HDMBOA-glucoside) in the root nd coleoptile, therefore the metbolic pthwys re intct nd there is difference in the regultory control of expression compred to Ae. speltoides. It would be unwise for us to generlise from these limited results on single ccessions from ech species, but it is cler tht considerble scope exists mong the potentil diploid progenitors for vrition in the levels of HA. It is very possible tht the hybridistion event nd the development of the polyploid species hve limited the full rnge of llelic vrition tht could be vilble in the gene pool for breeding modern whet cultivrs. Ginoli nd Niemeyer (1997) studied the HAs level chnges in hexploid whet vriety Plet due to R. pdi phid feeding nd he reveled n infesttion of 25 phids for 48 hours is required to elicit n induced response; the ccumultion of HAs in the infested lefs. Studying the effect of R. pdi feeding, we estblished tht DIMBOA (+HDMBOA-glucoside) in the hexploid nd tetrploid vrieties ws higher fter 24 hours of phid feeding nd the pek ws mintined fter 48 h, while the DIMBOA-glucoside levels ws lower compred to plnts with no feeding dmge. We were ble to record significnt difference in the DIMBOA level between the control nd the infested plnts (hexploid vriety - Solstice) even fter 24 hours of phid feeding with further increse fter 48 hours. These effects within the host plnt were loclised nd did not spred to the bse of the lef or the rest of the plnt (Elek et l., 2013). The diploid Ae. speltoides behved differently to the other species, in tht there ws only smll nonsignificnt chnge in the HA level fter phid dmge. Gordon-Weeks et l. (2010) mde similr observtions, nd did not record significnt chnges in the levels of HAs produced by Ae. speltoides fter phid feeding. In Ae. shronensis there were no HAs in the lef tissue fter phid feeding. Since the HPLC method used could not seprte DIMBOA nd HDMBOA-glucoside, the possibility tht HDMBOA-glucoside increses in Ae. speltoides in response to phid feeding, s hs been reported for mize (Ahmd et l., 2011), will be investigted in future work. The settling test is choice test bsed on physicl nd chemicl differences between the test plnt nd the control plnt (the hexploid whet vriety Solstice). For the test, lte phids were chosen becuse their olfctory receptors re more developed thn in pterous phids nd lte re the primry colonising morph. Both B genome vrieties tested, whether they were HA producers or non-producers, were less ttrctive to R. pdi lte, nd significntly fewer phids settled on these plnts compred to the control. However, when the diploid species were tested ginst ech other there ws no difference in phid preference between them. The negtive effect on phids in the non-hydroxmic cid producing species could be cused by the unidentified compound, which ws detected by HPLC nd/or the chemicl nd physicl chrcteristics of the plnts, this will be investigted further. Nymph production on hexploid nd tetrploid vrieties ws not significntly different in previous studies (Elek et l., 2013), but settlement nd nymph production were significntly lower on the A nd B genome diploid species compred to the hexploid control (Elek et l., 2009; 2012). The extreme levels of HAs in Ae. speltoides clerly hd negtive effect on R. pdi host plnt selection nd nymph production, which my indicte tht this species 218

7 could be importnt in further crop improvement, by trditionl breeding, to use s resistnce source nd, combined with other defence mechnisms, to develop n effective phid resistnt vriety. The higher levels of HAs observed in Ae. speltoides correltes with the significntly lower intrinsic rte of popultion increse of R. pdi compred to tht on the hexploid control. Certinly the development nd production of the phids on this diploid species ws substntilly reduced to level which, if replicted in the field, would gretly fcilitte protection of the crop from phid dmge. The smll scle study reported here is unble to conclude tht the HAs re definitely responsible for the observed effects upon phid behviour nd growth. Indeed, the representtives of the species tested re extremely diverse nd must vry in mny spects of their defences ginst pests. 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8 NIEMEYER H. M., JEREZ J. M., Chromosoml loction of genes for hydroxmic cid ccumultion in Triticum estivum L. (whet) using whet neuploids nd whet substitution lines.- Heredity, 79: OIKAWA A., ISHIHURA A., IWAMURA H., Induction of HDMBOA-Glc ccumultion nd DIMBOA-Glc 4-Omethyltrnsferse by jsmonic cid in poceous plnts.- Phytochemistry, 61: RAMÌREZ C. C., NIEMEYER H. M., Slivtion into sieve elements in reltion to plnt chemistry: the cse of the phid Sitobion frgrie nd the whet, Triticum estivum.- Entomologi Experimentlis et Applict, 91: SMITH M. C., HAVLÌCKOVÁ H., STARKEY S., GILL S. B., HO- LUBEC V., Identifiction of Aegilops germplsm with multiple phid resistnce.- Euphytic, 135: TOTTMAN D. R., MAKEPEACE R. J., An explntion of the deciml code for the growth stge of cerels, with illustrtions.- Annls of Applied Biology, 93: WYATT J. I., WHITE F. P., Simple estimtion of intrinsic rtes for phids nd tetrnychid mites.- Journl of Applied Ecology, 14: Authors ddresses: Henriett ELEK (corresponding uthor: Peter WERNER, KWS UK LTD., 56 Church Street, Thriplow, Herts, SG8 7RE, UK; Lesley SMART, Jnet MARTIN, Shkoor AHMAD, Ruth GORDON- WEEKS, John PICKETT, Rothmsted Reserch, Hrpenden, Hertfordshire, AL5 2JQ, UK; Angel ANDA, University of Pnnoni Georgikon Fculty, Festetics u. 7, 8360 Keszthely, Hungry; Suznne WELHAM, VSN Interntionl Limited, The Wterhouse, Wterhouse Street, Hemel Hempsted, Hertfordshire HP1 1ES, UK. Received October 1, Accepted June 18,