PHYTOMELANIN: DEVELOPMENT AND ROLE IN ITYBRID RESISTANCE TO ELE CTELL{III LARVAE (\:PYRALLIDAE).

C.E. ROGERST, R.E STAFFORDT, and G.L. KREITNER2 1 USDA Conservaton and Production Research Laboratory, Agricultural Research Service, P.O. Drawer 10, Bushland, Texas, U.S.4.79012. 2 Deparhnent of Agronomy, Kansas State University, Manhattan, Kansas, U.S.A. 66506.

ABSTRACT phytomelanin was studied by collectng achenes from Thcre was a highly signilicant negative correlation (r = aqjacent heads within a line segregating for the characteristic. - 0.93) between achene iqiury by lawae of Homoeosorna Three achenes were collected from the heads dailv from dav 0 electellam (Hulst) and the pnesence of phytomelanin in though 18 post-fertilization, and at 3-day intervâls thereafter pericarps during 4 years of field tests. Ph5rtomelanin through day 29 post-fertilization. As achenes were collected. becomes evident microscopically as early as 3 days after they were cut into quarters and placed in either 907o ethanol achene fertilization in genotypes expressing the charac- or glutaraldehyde, wtere they remained until further pro- teristic. The phytomelanin layer develops as a metabolic cessing. Fixed pericarps of each age grouping were precipitaæ from the catabolism of hypodermal cells, and subsequently processed, embedded in plaitic, secùoned, and has a syneqgistic effect on early lignilication and hardening examined microscopically for phytomelanin development and of sclerenchyma cell walls. Young larvae of H. elecællum other morphological differences. showed a signilicantly greater feeding prefetence for RHA The resistance of pericarps containing phytomelanin to 26,6 pericarps lacking phSomelanin than for RHA 265 penetraton was determined in the greenhouse-by a penetre pericarps having phytomelanin in both no-choice and free- meter. Immature achenes were collècted 0, 5, 10, 15, and 20 choice whole-achene feeding trials in the laboratory. days afær fertilization, and the force required to penetrate the Young larvae feeding on ground pericarps of RHS 265 and pericarp in the crown and equator arèas was- determined. RHA 266 incorporated in a wheat-germ diet experienced a Penetrometer readings were obtained for frve achenes from significantly higher mortality when feeding on the RHA five different plan-ts êt e€ch age for about l(X) lines segregating 265 diet Among rcleased hybrid variet5r parental lines, tle for the characteristic. Data were subjected to an anâly-sis oT pericarp of 39Vo of the RHA's and 97Vo of the cmsHA's variance a$ s,igryficpurtly different means were separa-ted by have phytomelanin. Duncan's Multiple Ranee TesL . .Iaboratory liioassayi The effect of phytomelanin on injury to immature achenes- by larvae of H, electellun was INTRODUCTION studied by Yo bioassay techniques. In the first study, A phytomelanin (armored) layer develops between the immature achenes were collected from RHA 265 (a hypodermis and sclerenchyrna in the pericarp of some phytomelanin line) and RHA 266 (a line lackingphytomelaniù) genotypes of cultivated sunflower, L. at 2 to 3 day inærvals through 9 days posrtertiiization ané (E.D. PutÇ 1944). Although the layer has been described as stored in a freezer for subsequent feeding trials. Feeding trials carboniferons, resinous, and amorphous (Sarkany, 1947; wele^accomplished by placing whole achenes of desiréd age Kiewnick, 19641' and Putt, 1944), its chemical nature has not in 28 ml cups containing five newly hatched H. electellum been defined nor h4ve the physiological processes contributing larvae. In free-choice feeding trials, three achenes each of to its development been clarified. RHA 265 and RHA 266 were placed in each of five cups. In The phytomelanin layer is pliable and soft in the pericarp of 4qcholce feeding tials, only achenes of either RHA 2-65 or immature achenes, but becomes rigid, dense, and hard in the RHA 266 were placed in a respective cup. On each of five pericarp of mature achenes (Kiewnick, 1964). The usefulness consecutive days the achenes were removed from the cups of the phytomelanin layer in protecting sunflower achenes and e-valuatgd fo-r pericarp injury and larval growth (-or from injury by larvae of Homoeosoma nebulella (Hubner) in mortality). Three fresh achenes of each RHA line were put in Europe is well documented (Shapiro, 1975). However, the the cups each day as the old ones were removed. Each impact of phytomelanin in protecting achenes of hybrid combinaton of achene genotype versus age was replicated varieties from injury by larvae of Homoeosoma electellum five times. (Hulst) in North America is negligible, and its a role as _ The,second bioassay consisted offeeding newly hatchedl{ useful resistance mechanism is largely discounted (Kinman, e-le,c,tellum on different-aged pericarps of RIIA 265 and RHA 1964). Recent investigations have indicated that we need to 266 ground to a 6Gmeih powdei incorporated in a wheat re-examine the potential role of phytomelanin as a useful tool geryr dief Pericarp mash of a desired agè and concentration reducing economic losses caused larvae in by of 1{ in the wheat germ diet ',ras poured ca. 5 ml per each of 25 electellum in North America (Rogers, 1980). one-dram vials. Each vial was infested with two newlv hatched larvae, the weaker of which was removed thê following day. Detailed records were kept on larval MAf,ERIALS AND METHODS development and mortality, and on pupal weight and Field Trials - Several sunllower lines were planted in mo4aqty. Data from both bioassays were subjecæd to an ryplicated, randomized blocks at Bushland, Texas, each year analysis of variance test" and significantly diffêrent means from 1976 to 1980. Plantings were made in laæ April to were separated by the Duncan's Multiple Ranse Test assure-that flowering closely coincided with the seasonal peak Parental Lines Achenes of male and femâe parental of sunllower flight and ovi-positional activities. Fifûeen lines for-currently -used hybrid varieties were analfzed for plants from each of the 7.5 m rows were tagged and numbered phytomelanin. The occurrence of phytomelanin wàs deter- to facilitate subsequent collection and summation of data. mined by visual examination of' immature achenes, by Detailed records were kept on flowering date, anthocyanin scraping away the epidermis of mature achenes, and by pigmentation ofthe pericarp and inflorescence, frassiness ofthe soaking mature achenes in a solution of potassium dichromate- inflorescence, the presence of phytomelanin, and sclerenchyma sulfuric acid (Carlson et al., 1972). Five achenes from each and thickness. Data from each ofthe parameters were subjected line were tested by the various methods, and recorded as to regression analyses to determine their correlation with having no, partial, or complete phytomelanization of the achene injury by H. electellum. The correlation of larval pencarp. injury with the parameters was accomplished by pairing arhenes of plants within entries having coincident flowering dates across years. RESUTTS AND DISCUSSION Phytomelanin Development - The development of Field Trials - Parameters possibly affecting injury by larvae

138 H. electellum were assessed ftorn 274 paired samples of tion, resulting in cells that are impacted and disarranged. of -cells pericarps phytomelanin are à"t.n"t from heads with coincident flowering dates within Hvriodermal in lacking Achenes having lorv injury ratings were paired with reidtively larger, thin-walte{ fod neaty arranged in defined ènri.t. pericarps of àôh.net having a highér injury ràting There^w^as a highly columns. ruiq-ttre sclerenchymal cell walls in much earlier,. and ( l6level) negative correlation (r : 0.9 3.)-between achenes having phytomelanh ligrrify significant pericarps lacking phyte oeircent iniired achénes and percent pericarps with phyto- become thickei tliari cell walls in ilelanin. fhe correlation of aôhene injury by H. electellum melanin. layers. of iarvae with other parameters studied was not statistically Microscopic examinations suggest that inner cell contents which at the 5% level. For example, percent frassiness of the hvpoderinis disintegrate and discharge cell iisniflt"-t hypo' ttie inflo.escence, anthocyanin pigmentation, and sclerer preci-pitaæ into the inter-cellular spaces between the cÈv-J tttictness-had r vâues ôf-0.55' -o.47, and 0.40, âermÎs and sclerenchyma in the pericarps of genotypes reipectivelv. Hence, it appears that genotypes having formins the phvomeÉnin laver. Sarkany (1947) proposed ohvtomelaiin in the perièarp suffered significantly less ttrat cdfootrydraiæs of hypoderinal cell walls become lVdraæg à"Éene iniurv bv H. èlectellum during the 4 years than bv increaseô cellular mètabolism ofthe "cell layer underneath Phytomelanin genotypes laikin! phytomelanin in ttre peqca!?' The presence tlie epidermis" to form the -ofphytomelanin layer. its layer 6f ptryiometaninln the pericarp had no.sigrificant effects on has no ohvsical structure its owru and assumes eitheioercent oil in the àchenes or on sclerenchyma thickness momhol'oiy as it fills intercellular spaces between the In many cases, achenes having phytomelanin hvooderni-s and sclerenchyma and hardens. in the'pericam. -îhe the the'pericaip had a higher per.cgnt oil-than achenes iuxtaposition of cômpacted hypodermal cells, in fagking walls result ohvtomelanin in the peiicarp. Also, sclerenchyma thickness ptrvtométaniri-sienificantlv layer, and denser sclerench5rma cell pericarps when phyto- àvéraeed 239.43tt fôr periéarps having phytomelanin and in harder than occurs iSg.fgp in peribarps iacking phytomelanin These data mehriin is hêking. In fact, there is evidence thet p,hyto- iricorporatiôn-oi phytomelanin into the melanin and the larly ligrification of associated sclererr indicatb that'the pericarP o"ricum of hvbrid varieties as a resistanèe mechanism would chvmal cell walls interâct iynergistically to increase showed irave n'o deléterious effects on oil production. haidness in immature acheies. Penetrometer studies Phvtomelanin Development Microscopically' deposi- that the crown and equatorial areas ofRHA 266 achenes are tion ôf phvtomelanin between the- hypodermis and scleren- much less resistant-to penetration by the 5th day after phytomelanin (Thble In chvma ôf s'unflower pericarps becomerevident by the 3rd day fertilization than are acheires with l). achene fertilization- (Rogers and Kreiher, l98l). all achenes tested, the crown was much more resistant to aflËr given Deposition of phvtomelanin continues at a rapid rate from the penetration than the equatorial area on any day during Peredovik achenes 3rd ttroush ttrè t3ttr dav afær achene fertilizaton. Thereafter, àchene maturation. Although the crown of phyomelânin depositioir seems to wane, and hardening of the was equally resistant to penetration as the crown of other iaier becomes more pronounced as the pericarp matures. entries'witli phytomelaniri the equatorial area of Peredovik 'The phytomelanin in the pericarp of was no more-plistant to penetration as the crown of fæqetra- expression oif phyto sunflower is thought to be-controlled by a-single dominant tion than wai the equatorial areas of entries lacking achenes ex- sene (Pml) (John-son and Beard, 1977). However, distinct melanin. Perhaps the weak sides of Peredovik inomÈolosicàl differences that occur in the pericarps with and plains why this variety appears to be injured as readily by without àhvtomelanin, and differences in quantity and Îarvae ofÉL electellum in the United Staæs as are the achenes lines (e.g ., enty 2522, earliness bfphytomelanin deposition among .pericarps with of hvbrid varieties. Some experimental phytomelanin duggest that additonal modifying genes. may Tabie 1) offer high potentials as parental lines fo1 developin-g àe6rmine the phytomelanin characteristics of a perigarp. hvbrid varieties ltrdt are resistant to injury by larvae of IL Hvoodermal ceils-in the pericarp of achenes having phyto- eiectellum" where both crowns and equatorial areas ofachenes méianin undergo early, accelerated cell division and prolifera- would be more resistant to peneta-tion (Rogers, l98l).

Ibble l. Force required to penetrate tùe pericarp of achenes at 5-day intervals after fertilization.

Mean grams force required to penetrate pericarp on dayv % I l0 achenes Entry w/Pmld Crown Side Crown Side Crown Side Hybrid 894 20 49.8bcd 2l.8abc l49.4bc 73.6ab l99.0bcd l32.lab RHA 266 0 57.5a-d 27.9ab 96.8d 18.8d I 10.3d 5 l.3c RHA 265 100 44.lcd l6.4bcd l4l.4bc 91.5a l59.0bcd l243ab Peredovik 100 70.6a 31.8a l22.3cd 52.5b l95.3bcd 51.8c 2522 #8 100 55.8a-d 7.2de I l4.0cd 45.5bcd 260.lab 160.0a

a/ Zygosity of Pml for entries were not known. b/ Means followed by different letters are significantly different at the 596 level.

Iaboratory Bioassays European literature (Kiewnick, greater as achene age increased (Thble 2). Pericarp hardness 1964 and Sarkany, 1947) -states that the phytomelanin layer éould have inlluenced results in the 5 to 9 day age category, forms a physical barrier that prevents pericarp penetration by but hardness should have had little influence on feeding larvae ofl{ nebulella Does the phyûomelanin function solely preference in the soft, pliable pericarps of the 2 19 4 day- to day-age groupings. Physical evidence of as a physical barrier to larvae of H. electellum? Field ihrough the 4 7 -total observations indicate that earlier hardening of pericarps feedirig usually involvéd ihe peîcarp-in RHA 266, but having phytomelanin plays a significant role in protecting often bnly involved consumption of the epidermis and mahrring achenes from injury by H. 'electellum larvae. hypodermis of RHA 265 pericarps as larvae burrowed along However, it appears that something other than hardness thé longihrdinal axis of achenes. There was no significant functions to reduce larvae injury to pericarps having difference in mortality of larvae feeding on whole-achenes phytomelanin during early stages of achene development of RHA 265 and RHA 266. Hence, acute toxicity of phyto (Table 2). melanin to early-instar larvae was not evident in the results of In whole-achene feeding trials in the laboratory, early- these trials. However, chronic toxicity or gustatory inhibition instar larvae of H. electellun showed a significantly (5% by phytomelanin could have influenced the apparent feeding level) greater preference for RHA 266 achenes than for RHA preference of H. elecællum larvae for RHA 266 achenes. feeding pericarp 265 achenes- following the 2nd day post-fertilization of Responses- of H. electellum larvae on achenes. The differential feeding preference for RHA 266 mash incorporated in a wheat germ diet at a l0%o achenes was greater in freechoice trials, and appeared to be concentration are summarized in Thble 3. The incorporation

139 of dry pe.ncarp mash in the wheat germ diet at l}o/o g" Rlê 265 pericarp mash from co-ncenfration made the diet -the 2 to 4 day_old and 3 to 5 drier than controlled diei oay-orq acnenes than trom other treatments. biased Also, total larval yliî! T.1v,l*"e the bioassay resutts. N;;".th"Ë;.; mortality was sigrrificanly hieher srgrurlc-ant cltlerences (5olo) for larvae feeding on RHA in larval feeding responses on zo) pencarp mash of 2 to.4 day_old RHA 265 and RHA pericarp and 3 to-5 day_old 266 mash suglgésted a chronic achenes than for larvae feeding due on some of the other en'tries. ro in.'immaturel".i"urpr. First_ Pupal mortality was greatest l9f:i?rnstar larvae .phyromgtùin frôm larvae feeaing on the 3 to 5 had a significantly higher mortality whdn feeding day-old achenes.

k!I"-1-lr.if ro immarure R HA 265 (w/phytometanin) and RH^A__26Q-"y- @19 pfyromelanin) y"ung laruae of H. electellutà iir whote-acÉe;"è'f,.'nËi-ty l;;àid rriats in the laboratory. x o/o peicarp destroyed/rep./dayd Achene age RHA Free-Choice No-Choice (days) no. feeding feeding 5-9 265 1.88a 4.58a 266 8.24b 41.42b 4-7 265 6.48a l6.l4a 266 I1.02b 34.06b J-) 265 6.22a 2O.l0a 266 r6.20b 27.90b 2-4 265 23.88a 20.00b 266 34.r2b 28.60b t-2 265 15.80a 25.02a 266 l6.7Ùa 19.80a

a/ Means followed by_different letters within an ..ase_qrouo,, are sigrifrcanty diferent atthe 50Â level (thi ,ôi"rËæril.

Growth of survivine larvae was severely affecûed by the containing.ground pericarps. diet containing 2 a {day-otd;d a t" ?'aîv:orâ;Ëd;; Excessive drying of the diet or (Table 3 )' Larvae were usuallv smaller *tten feeaing àn trte uii#JËirrÉ."rËllËtirfry"fthËËîffiir:,iËr,"u.beenresponsiblefor diet containing ground f"li; ;l;iî;;ilrs using ground pericarrrs RHA 265 pericarps tha, ;h;;f;àid i"- th;;ËJ;i;;"ff.âi.t on the diet containine eround RiIA 266 pericams.._tffi AËËrilà"0 a tyo concentration. ilù,:i:$t_irdË"Ë;'b:îiï "t diet, and a hisher size' developmental p;È'od, ana lupJ mair fiË' ruruiuing mortalitv îîuritv tarvae Iginrr-qnù, î;-* i;â:ing on the diet contaiiing were usuallv sienincglt1 ere.ater for specimens_.ea.eà c-*d-frÈÀ)"0ï'ffi"'frJ'vvw* r\ '""* on the standard diet than for specimens reared d tË;ie;

Table 3' Results of laboratory bioassays incorporating ground pericarp in the lanal diet at a l0olo concentration.d o/o Mortahty x Larval Length (mm) at Achene age x Larval pupal and First Total ti.Ti,î x RHA line instars larva Pupar 4 days 7 days l-2day ïËi' 265 4b 3qd 18.7b 2.Oabc 3.9b 266 8b 23.5cd 21.8b 24de 0.0c 2.rab ,.ib 23.td 24.Ob 2-4day 265 48a 80a 0.0c t.1c 2.4cd 28.0b 20.6b 266 t2b 44cd I l.lbc 2.2a 3.5b 25.7bcd 3-5day 19.8b 265 64a 88a 33.3a l.8bc 2.6cd 27.7bc tg.1b 266 zOb 68abc 25.0b r.1c 2.8bcd 26.8bcd i8.lb 4-7 day 265 ?99 64a-d 2o.0b t.jc 2.3d 266 20b 28.9ab 20.5b 56cd 0.0c 2.2; i.:u. 32.3a 21.7b Checkb/ 0b 8e 0.0c 6.6a 16.2e 3l.l a

a/ values followed bv different letûers are significantly different at the 5zo level (Duncan's Multiple b/ Standard wheat gérm aiet wim no t dàï;;"rË, Range Test).

t40 Tabte 4. Phytomelanin rating in the pericarp o.f commonly We feel that hybrid varietes developed from parental lines used RHA and cmsHA parental sunllower lines. homozygous dominant for phytomelanin would result in considerable reduction in yield loss due to achene injury by Assay method indicating phytomelanin'/ feeding larvae of H. electellum. Parental Potassium dichromate - ' line Scraping sulfuric acid LITERATURE CITED RHA 265 + CARLSON. E.C.. P.F. KNOWLES, and J.E. DILLE. 266 1 1972. Sunflower varietal resistance to sunflower moth larvae. 269 Califontia Agiculture 26(6):ll - 13. 270 + + JÔlil.rsoN, A.L. and'B:H. BEARD. 1977. Sunflower 272 + T moth damage and inheritance of the phytomelanin layer in 274 + + sunllower achenes. Crop Science 17:369 372. KIEWNICK, V.L. 1964. The phytomelanin- layer in the cmsHA 60 + + pericarp of Helianthus annuus as a barrier against Homoeo- 89+ + soma nebulellaHb. Z. Pflanzenkr. Pflanzenchutz 7l;294 99+ + 301. - ll3 + KINMAN, M.L. 1964. Comments, rounétable discussion. 224 T Proceedings Firct Intemational Sunflower Conference June 300 + incomplete 17 18. Texas A&M University, College Station. l0 pages. PUTT,- E.D. 1944. Histological observations on the location of pigments in the akene wall of sunflower a/ * indicates positive test for phytomelanin; - indicates (Helianthus annuus L). Scienffic Agriculture 25:185 nesative test for phytomelanin;lnèomplete indicates part of 190. - peiicarp "+" arid irart of pericarp ROGERS, C.E. 1980. Biology and breeding for and disease resistance in oilseed crops, pages 359 389. In. M.K. Harris (Ed.), Biology and breeding for resistance- to Parçntal Lines - We have examined most of the public- and pathogens in agicultural plants. Proceedings released hybrid variety parental lines for phytomelanin. The International Shon Course in Host Plant Resistance, Tëxas pericam cit lgV" of thi-RHA lines and 97Vo of the cmsHA Agicultural Expeiment Station Miscellaneous Publication iines hâve phytomelanin.-selected American sunflower breeders have (MP) 1451.605 pages. not activefu for phytomelanin, and its zygosity ROGERS, C.E. 198 1. Breeding sunflower for resistance to among hybhd varieties is undetermined Usualty, the !!A and diseases in the United States. Proceedings fine hàs fhytomelanin in its pericarp when the sister cmsHA EUCARPIA Symposium Prague, Czechoslovakia OcL26 line has ihyromelaniru but thêre are-exceptions. Also,-the HA 30.1981. - line may la-ck phytomelanin when the respectiv,e cmsHA line ROGERS, C.E. and G.L. KREITNER 1981. Aphysical has it, aid sometimes the HA line has phytomelanin while the basis for pericarp resistance to larvae of the sunflower moth, cmsHA counterpart lacks it. The pericarp is most often pages 17 18. Prcceedings Sunflower Forum and Research oositive or nesative for phytomelanin, but sometimes only Workshop.- Sunflower Association of Ameica 27 pages. bartial' or incoriplete development of phytomelanin is evidenl SARKAI.IY, S. 1947. Sunllower breeding in connection Phytomelanin expression in commonly used hybrid variety with the phyûomelanin queston. Agrartudomangi Szemele parental lines is summarized in Table 4. Scraping away the 2O:97 103. èpidermis and hvpodermis of mature achenes gave a positive SHARPIO,- I.D. 1975. Achievements in plant breeding in rêsponse for phytomelanin in ca. 75Vo of the samples where plant resistance to pests, pages 162 167. Proceedings 8th soaking mature- achenes in a potassium dichromatesulfuric International Plant Prctection Congress,- Moscow, USSR acid solution had indicaæd a positive response.