t lphnlxd Atvt nd nl Oxlt rdtn n th trt Intrtn Ctn tvCrphntr prt Andr nnn, . Mr nd I rtnl prtnt d rtzn dll nt, Unvrt dl Std dll , 000 trb, IAY ASAC. lphnlxd (O tvt f Er MAEIAS A MEOS (Castanea sativa) pn htnt br t nltd ll trl. C. parasitica d n th td Cryphonectria th th hpvrlnt trn f nldd th dAntnn Itln hpvrlnt trn parasitica nd t nndl vrlnt lt M8, n nd t nndl dAfr lt M8. lzd lttvl nd prd th tvt n hlth h trn nd th lt r rn nd ntnd n C. sativa ntrl br. O tvt f hhr drn A (f t 2 C th 4h phtprd. r xpr prn nd fll n thn drn r. An nvr nt r prfrd, n M, n, Sptbr nd O rltnhp fnd btn O tvt nd z f tbr. th nr prdd b th fn. A ltv nhbtn lnt trl tlzd fr th xprnt r n f n nz b th vrlnt trn fnd n rld r lltd fr tp f C. sativa n ltrfn (IE xprnt. Oxlt ltn ntrl tnd n ntrl Itl. h r r ntnd fnd n nrt htnt br t nltd th th th t nd n tt tb ntnn dnzd tr th hpvrlnt trn nd th th vrlnt lt. nd nbtd n rth hbr th 2h pht nfnt dffrn r fnd n xlt ltn prd t 2 C nd 80% rltv hdt. btn th hpvrlnt trn nd th vrlnt lt. dA ltn. h fn rn n plt nhbtn f O fr hlth htnt br fnd t ld d fr C. parasitica th th n f l dffrnt nntrtn f xl d. (. Extrtn f dA prfrd b th lll ln thd drbd b Mrr nd dd ( nd dfd b lbrht t l. (0. h vrlnt trn f th htnt blht pthn Inltn. h r r nltd th trn Cryphonectria parasitica (Mrr. rr prd n ltr, nd lt M8 b rvn d f br th lrr nt f xl d (OA thn th hpvrlnt r brr nd b pln pl f f l n (6, 2, 20. nnn nd lbrht (26 rprtd n tn fr th dvnn d f dld ltr rn ltn f OA n t f ppl nftd th hp n A. h nl r tpd th prfl t vd vrlnt nd vrlnt trn f C. parasitica. vr, n dtn nd th r nbtd n th bv nd dt r vlbl bt prdtn f OA n htnt br tn fr , 0, 20 nd 0 d. nnltd nd ndd t nftd b vrlnt nd hpvrlnt trn f th r r d ntrl. Aftr , 0, 20 nd 0 d, fn. th z f th nr rd. Chtnt tr r OA rprtd t pl rl n th pthn f l nltd n xprntl plt th nd M8 vrl htpthn bntn (, , , 22, 2 b fr th vrln . h vrt f pt t pt t hlt dvlnt tn. St (2 hd d ftr r. n nhbtr fft b xl d n plphnlxd O xtrtn nd . Inftd t r l (EC4..8.:O tvt n pnh lv. On f th ltd t , 0, 20 nd 0 d fr nltn. Old pbl rl f O n dd plnt t xdz nrt t nd th dvnn d f nr r phnl, prdn pnd, h nn, tht r lltd prtl. h t fr bt 20 nr tx t th nvdn rrn (, 4, , 24. r tlzd fr h pl. lth nd ndd br h f th r t nvtt th rltn t r lltd fr th ntrl. On r f btn O tvt nd OA ltn n Erpn t, rnd t fn pdr n rtr th ld 2, htnt (Castanea saliva Mll. br t nftd th xtrtd n 4 l f 0. M trt bffr p .0, 0.2% hpvrlnt trn nd n n vrlnt lt f C. tn, t 4 C fr h th ntl ttn. h pn parasitica. n ntrfd t 40,000 fr 20 n t 4 C. Aftr ntrftn th prntnt lltd nd dlzd xhtvl nt vrl hn f dtlld tr t 4 C. O tvt rd th dblb ptr phttr (d , rn Elr, O r, Ill.. h rtn xtr l f 0. M trt bffr, p .0, 0 M p nd 200 f rd xtrt. h rtn flld fr 0 n t 0 C nd th nr f brbn t 40 n rrdd. O tvt x prd nr f brbn t 40 nn frh ht t 0 C. h rfrn vtt ntnd th rtn xtr bt th bld rd xtrt. Isoelectrofocusing. IEF prfrd n 0% rld l n vrtl lb nt th phln p 2.0 (S, St. , M.. n rrd t t 4 C nd t 20 ntl th rrnt drppd t .0 A. p rnt r d b ttn tn fr th l, n th n dtlld tr, nd rn th p. h l bjtd t tnn b n t n 0. M trt bffr p .0 th 0 M p b trt. Extraction and determination of soluble and insoluble oxalic acid from the infected tissues. Inftd nd hlth br t r drd t 60 C fr 2 h nd rnd t pdr th ptl n rtr. Soluble oxalic acid extraction. h htnt pdr (bt xtrtd t t 20 C fr 0 n th 0 l f 0.0 M rCI bffr, p .8, nd ntrfd t 40,000 fr 20 n. h prntnt lltd, d jtd t p .0 th 0. C, nd trtd th rbt xd ptl (hrnr Mnnh t lnt rb d (. h prntnt thn d fr lbl xl d dtrntn. Insoluble oxalic acid extraction. h pllt fr th bv ntrftn xtrtd t t 60 C fr 0 n th 0 l f C. Aftr ntrftn bv, th prntnt lltd nd d fr th nlbl xl d dtrntn. Oxl d ntnt f th xtrt dtrnd b th xlt xd nzt t (S dfd b hnn th t f nbtn f th nzt rtn t 0 n t C. Abrbn f th ndn d rd t 0 n th dbl b ptrpht tr (rn Elr d . h dt r xprd frh ht. Effect of oxalic acid on PPO activity. 0.1 l f hlth br rd xtrt nbtd fr n t 2 C n l f 0. M trt bffr p .0, t dffrnt nntr tn f xl d (S, 0, 0, 20, 40, 60 nd 80 M p vl f th rtn xtr r vntll djtd th KO. h rtn trtd b ddn 0M p nd rd bv. ESUS PPO activity. O tvt n ll th pld t dsRNA isolation. h dA pttrn f trn hhr n th nth f M nd Otbr thn n n nd nd lt M8 r nfrd (r (26. Sptbr (r , b. O tvt t th dvnn Inoculations. h z f th nr prdd n h d f th nr d b M8 lhtl dlnd t 0 xprnt b trn nd lt M8 ftr 0 d d prd th th hlth ntrl. O tvt n th fr nltn rprtd n r 2. h prrn nrt t vr l r bnt. h plt f th z f th d ftr drn n nd Sptbr thn f th nr vr O tvt f th ntrl t r drn M nd Otbr. Ilt M8 prdd lrr rprtd n r 4. nr thn trn . r th xprntl plt Isoelectrofocusing. h nz pttrn f O r nltn, trn M8 jdd vrlnt nd trn rprtd n r . h hlth ntrl t hd hpvrlnt. fr nz n M nd n nd fv nz n Sptbr nd Otbr n nz fd t p 4., while the remaining ones focused at alkaline pH values. 6b); about one tenth of total oxalic acid was accumulated The advancing edge of the necrotic tissues inoculated with in soluble form. No differences were recorded in accumu- strain TR57/a always showed the pattern of the control, lation of oxalic acid between the hypovirulent strain and while the isoenzyme at pI 8.5 and the one at pI 7.8 were the virulent isolate. lacking in the advancing edge of the necrotic tissues inocu- Effect of oxalic acid on PPO activity. No inhibition of lated with the isolate M18 in the May and June experi- PPO activity was found at all the concentration of oxalic ments, and in the fall experiments. acid used. In the necrotic tissues from infection with strain TR57/a and isolate M18, PPO isoenzymes were weaker and fewer than in the control healthy tissue. However, in the necrosis ISCUSSIO caused by the virulent isolate M18, had fewer PPO isoen- Studies of herbaceous tissues of several plant species, in zymes than necrosis caused by the hypovirulent strain short-term experiments (4-7 days), showed an increase of TR57/a. PPO activity following infections by virus, bacteria, fungi Extraction and determination of soluble and insoluble or mechanical injury (1, 2, 4, 8, 16, 18, 21). Our results on oxalic acid from the infected tissues. Oxalic acid accumu- chestnut bark tissues and in long-term experiments indi- lated in the necrotic tissues only (Figure 6c); no accumula- cated that in the advancing edge of lesions caused by both tion of oxalic acid was recorded in the advancing edge of the hypovirulent strain (TR57/a) and the virulent isolate the infections produced by either the hypovirulent strain or (M18) of C. prt, the PPO activity of C. tv is not the virulent isolate, compared with the control. Most of the increased compared with the healthy tissue. The kind of oxalic acid was accumulated as insoluble oxalate (Figure tissues examined and the period of time investigated from th nltn ld nt fr th dffrnt bhvr f nd ntttv hn f O tvt rtrd t C.