PHYSIOLOGICAL EFFECTS OF MUCUS FROM THE WOOD WASP, [Manuscript received 2 November 1972]

Transcription

1 PHYSOLOGCAL EFFECTS OF MUCUS FROM THE WOOD WASP SREX NOCTLO F. ON THE FOLAGE OF PNUS RADATA D. DON. By L. K. FONG* and R. K. CROWDENt [Manuscript received 2 Nvember 1972] The effects f mucus frm S. nctili n the physilgy and bichemistry f needles f P. radiata are described. Sme dramatic changes in respiratry rate and mde are nted as well as effects n the activity f perxidase and amylase enzymes. These changes are crrelated with the appearance f the characteristic symptms assciated with altered water balance within the needle tissues. The similarity between changes caused by the mucus and natural and induced leaf senescence is discussed. 1. NTRODUCTON When the wd wasp Sirex nctili F. (Hymenptera: Siricidae) attacks radiata pine trees Pinus radiata D. Dn. it elicits a cmplex syndrme f respnses within the tree. These are described in detail by Cutts (l969a 1969b). A series f rapid physilgical changes becmes manifest within 1-14 days f attack; these have been shwn by Cutts (1 969b) t be caused slely by the wasp's mucus secretin. They can be induced by artificial injectin f the mucus int the tree i.e. withut the direct participatin f the wasp and withut the simultaneus intrductin f the fungal symbint Amylstereum are/atum (Fr.) Bidin (Gaut 1969) int the vipsitin hle. Sme f the systemic respnses e.g. increases in dry weight f fliage and general retardatin f grwth may ultimately be reversed in healthy trees but thse mre susceptible t S. nctili attack may lse chlrphyll fllwed by premature and cmplete abscissin f the fliage. The mucus alne des nt cause tree death but rather death fllws frm the cmbined effect f "mucus cnditining" and eventual invasin f the sapwd by the symbitic fungus. n these experiments we have attempted t characterize and quantitate sme f the immediate physilgical and bichemical changes in P. radiata needles which result frm mucus treatment. * Frest Research nstitute Frestry and Timber Bureau Department f Natinal Develpment Hbart; present address: Btany Department University f Tasmania Bx 252C G.P.O. Hbart Tas. 71. t Btany Department University f Tasmania Bx 252C G.P.O. Hbart Tas. 71. Aust. J. bi. Sci

2 366 L. K. FONG AND R. K. CROWDEN. METHODS (a) Experimental The effects f artificial applicatin f mucus were investigated by (i) injectin f mucus int whle trees; and (ii) uptake f mucus slutins by excised twigs. (i) Whle Tree Experiments These were cnducted n vigrus (5-6 yr ld) P. radiata rammets chsen frm clned material grwn at the Frest Research nstitute Field Statin at Hbart. Fur trees were each injected with 1 ml f mucus slutin (5 % in deinized water) using the methd f Cutts (1969b). Cntrl trees were injected with 1 ml f deinized water. The effects f this treatment were investigated in l-yr-ld needles taken frm branches f the third and furth whrl behind the sht apex. Randm samples f needles were taken at peridic intervals extending frm 3 weeks befre t 7 weeks after the mucus injectin. Because the appearance f effects was rather irregular alng the length f the needles with a brad tendency t a prgressin frm apex t base ur investigatins were cncentrated n a standard l-cm segment frm the base f the fascicle. (ii) Experiments n Excised Twigs Branch tips (abut 5 cm in length) were selected frm trees which had previusly been shwn t have high susceptibility t the mucus. Each f six twigs in every trial was allwed t take up 1 ml f 1 % mucus slutin thrugh the ends f the cut stem. They were then std in deinized water in glass cntainers fr the duratin f the experiment the water being replaced regularly. Cntrl twigs were std in deinized water thrughut. n sme experiments mucus was autclaved at 15 b/in 2 fr 15 min befre use. (b) Analysis At harvest duplicate lts f l-cm segments frm 5 needles remved frm trees (r twigs) f like treatments were weighed and their respiratin rate in darkness was determined in a Warburg apparatus at 25 C. The same needle segments were then used t prepare extracts fr further examinatins. (i) Cld-water Extracts The 5 needle segments were grund with sand in 1 ml deinized water and centrifuged at 2 g fr 1 min. The supernatant after cncentratin t O' 5 ml (by dialysis using plyethylene glycl ml. wt. 4) was used in the assay f varius enzyme activities and fr electrphretic investigatin. n excised twig experiments the extracts used were nt cncentrated. The cld-water extracts were used in the fllwing assays: (1) Sluble prtein cntent was measured by the methd f Lwry et al. (1951) and by the biuret reactin (Layne 1957). (~) Perxidase was assayed spectrphtmetrically by the prcedure f. Maehly and Chance (1955) but with -dianisidine as substrate (1'25xl- 3 M in O'M acetate buffer ph 4 5). (3) B-amylase was assayed by the methd f Bernfield (1955). (4) Electrphresis n acrylamide gels was carried ut accrding t the prcedures f Mills and Crwden (1968). (5) The residue frm the cld-water extractin was assayed fr chlrphyll by the methd f Arnn (1949) but using 8 % ethanl. (ii) Ht-water Extracts A secnd batch f 5 needle segments was hmgenized with 1 ml f deinized water and biled fr 2 hr. The slurry was cled and centrifuged at 1 g fr 1 min and the super-

3 EFFECT OF S. NOCTlLO MUCUS ON P. RADATA 367 natant stirred fr 1 hr with 1 g each f Zekarb-225 acetate and Dwex-l H+. The ht-water extracts were used fr the fllwing assays: () Sluble sugar cntent was determined using the anthrne methd f Trevelyan and Harrisn (1952). (2) The cmplement f sugars in the supernatant was examined by thin-layer chrmatgraphy n cellulse (Whatman CC 41) using the slvent systems n-butanl-acetic acid-water (4 : 1 : 5 v/v)(upper layer) and n-butanl-benzene-pyridine-water (5 : 1 : 3 : 3 v/v). Authentic sugar samples were used as references. Visualizatin was achieved by u~ing aniline hydrgen phthalate and anisaldehyde-sulphuric acid sprays (Hugh 1954). (iii) Anatmical Studies Transverse sectins 22 flm thick were cut n a. freezing micrtme frm the same needles used fr cld-water extracts. These sectins were remved frm the regin 5 em belw the standard sample segments. Examinatin was made f tissues particularly the phlem after the sectins had been stained in tannic acid-ferric chlride-lacmid stain (Cheadle et al. 1953). A cunt f starch grains was made n the stellar and enddermal tissues f the' needle sectins after staining in idine-ptassium idide. Fr this purpse an average cunt was btained in fur separate fields per sectin at 6 magnificatins. Twenty needles per sample taken at randm were examined fr bth mucus-treated and cntrl trees. (iv) Statistical Analysis Cmparisns made between sets f data btained ver specified time intervals befre and after mucus treatment have been analysed by the t-test. Values f P are shwn in the legends t the apprpriate figures.. RESULTS Several preliminary investigatins f the mucus effect have been.cnducted since These trials shwed that trees varied appreciably in their respnse t mucus treatment just as trees in the field display a varying susceptibility t attack and injury by S. nctili. The results reprted in this paper fr whle trees derive essentially frm an experiment cmmenced in the early spring f 1969 in which trees f high susceptibility were used. The twig experiments were cnducted during autumn During the first week after mucus treatment sme differences were apparent between the results btained fr twigs and thse fr intact trees. t is thught that these differences are due t physical damage caused by the remval f twigs frm the trees since bth cntrl and experimental twigs shwed discrepancies f the same rder. Within a week r s after twig excisin the levels f mst measured parameters in the cntrl twigs tended twards thse f cntrl trees. The treated trees and twigs develped yellw dry needles which abscissed easily (cf. Cutts 1969b). n whle trees prgressive defliatin ccurred abve the injected regin branches belw this regin being apparently unaffected. The ld needles n affected branches abscissed earlier than yung nes and apical buds ften wilted within abut 3 mnths f treatment. During the perid f the experiments cntrl trees and twigs remained green and withut defliatin. Prgressive changes in the measured characteristics are illustrated in the figures. Figure 1 shws changes in the weights f needle segments fllwing mucus treatment. t is difficult t accunt fr the persistent decline in the weight f cntrl needle

4 368 L. K. FONG AND R. K. CROWDEN segments (tree experiments). We have assumed that it is related t the cntinued pruning f the trees during the curse f the experiment r that it reflects sme seasnal change. Despite this uncertainty the effect f mucus treatment n needle weight is clear. A crrespnding decline in weight f cntrl needle segments was nt apparent in the twig experiments. Hwever these experiments were cnducted under mre unifrm envirnmental cnditins in a glasshuse. n whle-tree experiments the dry weight f needle segments increased [Fig. (a)]. Hwever with the excised twigs treatment with mucus appeared t have n effect n needle dry weight [Fig. 1 (b)]. nstead twig needles tended t lse water mre rapidly and t a greater extent than did needles n intact trees [Figs. l(e) and led)]. Thus althugh the dry weight/wet weight rati f needle segments was increased by mucus in bth tree and twig experiments [Figs. lee) and 1 (!)] the reasns fr the increases are different. Changes in the weight f needle segments were assciated with anatmical disturbances particularly in the phlem where severe physical distrtin and cllapse f cells became evident within a few days f mucus treatment. Figures 5-9 shw prgressive stages in the desiccatin and cllapse f phlem and ther nn-lignified elements f the needles e.g. ray parenchyma mesphyll enddermis and secretry cells lining the resin canals. Cutts (l969b) reprted that starch accumulated in the enddermal and mesphyll tissues f the needles during the first 1 days after mucus treatment. Our results have nt cnfirmed this bservatin and shw instead that the starch cntent f needles is reduced by mucus (Fig. 2). Thus in experiments with whle trees the starch cntent f leaf cells was shwn t fall-slwly during the first week but rapidly thereafter. n the experiments with twigs the act f excisin alne caused a substantial fall in the starch cntent f bth treated and cntrl needles alike. Hwever after sme 3 weeks cntrl needles began t increase their starch cntent but the level in needles f mucus-treated twigs remained lw. That mucus affects the chlrphyll cntent f needles is evident frm the characteristic yellwing f the fliage f affected trees. Figures 3(a) and 3(b) shw the decline in chlrphyll cntent in bth tree and twig experiments. The amunt f sluble prtein in needles was increased after mucus treatment [Figs. 3(e) and 3(d)] and in additin higher levels f activity f tw enzymes perxidase and amylase were nted. Perxidase activity increased prgressively frm the secnd day after treatment right thrugh t needle abscissin [Figs. 3(e) and 3(f)]. Electrphresis using plyacrylamide gels shwed that nine ande-migrating and tw very weak cathde-migrating perxidases were present in the extracts (Fig. 1). Mucus treatment resulted in enhanced activities f all the regular perxidases (except bands 1 and 2) and the appearance f tw new perxidases bands 7 and 8. Amylase activity in needles f treated trees was increased (after a sharp initial fall) [Fig. 2(e)]. This increase in amylase activity (after day 7) was crrelated with the decline in starch cntent (r = '75) and was als reflected in an increased level f sluble sugars [Fig. 2(e)] appearing in the needle extracts. n experiments with twigs n the ther hand any effect f mucus n amylase activity was very slight cmpared t that f twig remval and n distinct crrelatin with starch r sluble sugar cntent was apparent.

5 18 -- n S 16.:c n. iii ~» 14 (a) EFFECT OF S. NOCTLO MUCUS ON P. RADATA Tree needles.' - - -! - ~-.- '" -....' "... ~ er- 6 -_ Twig needles 15 (b) ----'l--- n -& _.- &- -.-&- - _. -& n E '-'.:c n. iii ~..<: '" <11 ~ 5 4 (c) ! - _. i ~ '. '. -v--~o.2.. Q (d) 4 - '. 3 2 Q.' 't --- )t & ~ 1... & ' '& 3.:c n.~..<: '4 ] -.:c '35. n. ~ i!' (e) '.... e.. t '.'... '. ---~-~~~----- '55 '45 35 ().'. '& &.~-~&-'~-n o--4-- '3 ~_~2::----L--~--.L.--+~2~:--~ '25~----71~----~2~----~3 Time (days) Fig..-Effects f S. nctili mucus n dry weight (a and b) fresh weight (c and d) and dry weight/fresh weight rati (e and f) f P. radiata needle segments frm intact trees (a c e) and islated twigs (b d ). Cntrl. Treated with raw mucus.... Treated with autclaved mucus. P values are as fllws: Days Raw Autclaved Cntrl mucus mucus Dry weight Trees < 1 Twigs Fresh weight Trees < 1 Twigs < 1 1

6 37 L. K. FONG AND R. K. CROWDEN C 15.;; en -5 1 E '" 5 " ci Z (a) T Tee needles -e...!.... ' ----A._'. ~~~ ~ ~ ~~~ (b) Twig needles (c) ~ ~ l' (d) l\.t.~\ " ' '..'. t". ~...t./~. :1.... / ""/-... _.' :'_.. : :'?/ ~~~i E 5 "" (e) E '-"... 4 "" '" ::> -.- -~-.--- 'n... - <U 3 - :: ::> -.- " <fl 7 ( /:...-. _ : /' : ~..j:...: 5 \ " ~\ -_ ~.\ / :._...--e 2 \'~&'. ;'.' e- _.' : ~1~------~2~~------~3 Time (days) Fig. 2

7 EFFECT OF S. NOCTLO MUCUS ON P. RADATA 371 Chrmatgraphic examinatin f "free" sugars present in needle extracts shwe-i n qualitative differences due t mucus treatment. The fllwing sugars (with their relative amunts) were identified: glucse +++ 'galactse ++ arabinse + manrtse + ribsetrace amunt nly. Anther majr effect f mucus treatment was a significant stimulatin f respiratry activity. Madden (1968) has previusly reprted that attack by S. nctili results in an enhanced rate f respiratin f phlem tissue in the tree trunk. n the present experiments we have shwn that the rate f respiratin f tree needles had risen t 2 5 times the cntrl rate by day 9 (Fig. 4). The respiratry mde was als altered; the higher values fr the respiratry qutient indicate the adptin f a mre anaerbic mde f respiratin (R.Q.--= 1 37 fr treated needles at day 28 whereas fr cntrl needles it remained at apprximately 1 thrughut the experiment). Excisin ftwigs resulted inan initial sharp fall in respiratry activity. Hwever in the cntrl twigs respiratin rate subsequently recvered t near nrmal and by this time the effect f mucus n the respiratin f treated twigs was apparent. Excisin alne caused a drastic change in respitatry qutient f twig needles and the change was exaggerated when cupled with mucus treatment. The altered respiratry rate and the assciated change in' respiratry qutient almst certainly bear n ther parameters which were measured cncurrently e.g. starch and sluble sugar levels. Due t this interactin between the respiratry rate and the levels f ptential substrates it is difficult t interpret the significance f changes in these substances. Cutts ( 969b r has reprted n the heat stability f the active principle in the mucus. Our data suggest that autclaved mucus may be mre effective than raw mucus in prmting the physilgical respnses. Thus cmpared with raw mucus autclavedmucus prmtes higher levels' f amylase acfivity and sluble sugars [Figs. 2(d) and 2(/)] advances the nset f chlrphyll disappearance by almst a week [Fig. 3(b )] and causes a greater departure f respiratry qutient frm the cntrl level [Fig. 4(d)].Raw mucus caused a greater effect with perxidase levels alne [Fig. 3(j)]; Cutts (1969b) als reprted that the mucus was highly specificfr P. radiata and was withut effect n Ppulus seedlings and Eucalyptus saplings. Hwever we fund that when disks f radish leaf were flated n a slutin f mucus ( 5 mg/ml) in the dark they shwed lss f chlrphyll and develpment f paque areas within Fig 2.~Effects f S. nctili mucus n starch (a and b) amylase activity (c and d) and sluble sugar (e and f) in P. radiata needle segments frm intact trees (a c e) and islated twigs (b dl). One unit f amylase activity = 2 85 mg maltse per millilitre extract in 5 min at 25 C. Sluble sugar estimated as milligrams glucse per millilitre extract. Cntrl. Treated with raw mucus.... Treated with autc1aved mucus. P values are as fllws: Days Cntrl Raw Autclaved mucus mucus Starch Trees Twigs Amylase Trees Trees '2 1 Twigs < 1 '~)1 < 1 Sluble Trees sugar Twigs '2 < 1 '1

8 372 L. K. FONG AND R. K. CROWDEN 3 days (cntrl disks shwed lss f chlrphyll after 1 days) suggesting that S. nctili mucus culd accelerate senescence in this tissue. A similar result was btained using needle segments f P. radiata. Additin f kinetin ( 1 mg/ml) inhibited the effect in bth radish disks and P. radiata needle segments. V. DSCUSSON Mucus frm dissected glands f S. nctili has been shwn t induce a prgressive series f changes in l-yr-ld needles f P. radiata which may lead eventually t their death and abscissin frm the tree. The rapidity with which sme f the symptms appear (e.g. increase in respiratry activity within 24 hr see Figure 4) shws that the active principle in the mucus is transprted rapidly and efficiently frm the site f applicatin t the needles. The immediate effect f mucus appears t invlve sme impairment f the nrmal water relatinships f the needles which causes tissue desiccatin distrtin and eventual cllapse f the translcatry cells f the phlem. Cncurrent with these effects are sme dramatic changes in the rates f certain metablic activities e.g. respiratin as well as changes in the levels f perxidases and amylases. t is nt pssible at this stage t determine which f the ttal bserved effects are primary and which are f secndary imprtance. Hwever there is clearly a cnsiderable degree f interactin f the effects bth in respect t the time f their appearance and in their manifestatin within the needle tissues. n general ur results supprt the bservatins reprted earlier by Cutts (1969b). n ne respect hwever there is an apparent anmaly which requires explanatin. Cutts claimed that the increase- in needle dry weight (trees) culd be largely attributed t starch accumulatin in the mesphyll cells. He further suggested n the basis f shading experiments that this starch was due t current phtsynthesis either by an increased rate f phtsynthesis r an impeded rate f translcatin f phtsynthate frm the needles r bth. We did nt bserve starch accumulatin in ur experiments (Fig. 2). Mrever the rapid lss f chlrphyll frm mucusaffected needles (Fig. 3) and the failure f these needles (in experiments with twigs) t restre starch t the cntrl level [Fig. 2(b)] are taken as evidence f lwered phtsynthetic activity. Fig. 3.-Effects f S. nctili mucus n chlrphyll (a and b) sluble prtein (c and d) and perxidase (e and ) h:vels in P. radiata needle segments frm intact trees (a c e) and islated twigs (b d ). Chlrphyll cntent is expressed as milligrams per millilitre extract per gram dry tissue sluble prtein as milligrams equivalent t 1 ml bvine serum albumin and perxidase activity as change in ptical density at 43 nm per minute per gram dry tissue. Cntrl. Treated with raw mucus.... Treated with autclaved mucus. P values are as fllws: Days Chlrphyll a Trees Chlrphyll b Trees Ttal chlrphyll Twigs Prtein Trees Twigs Perxidase Trees Twigs Cntrl 3 ' Raw mucus < 1 < 1 < 1 < 1 5 < 1 1 Autclaved mucus < 1 < 1 1

9 EFFECT OF S. NOCTLO MUCUS ON P. RADATA 373 2:5 ;;' bo ' E bo E '-"' 1 5 'E '" 'E <.J >...c: C. ::a 5 U (a) !--"O _..;. _ Tree needles \ \. \ ' Q'S2 3 ~ ~... ' (b) '6. \ Twig needles -.. \. \ j...".' \ 6 D 1 2 ". 6 ". ' (c) g -! '.' ' (d)./. -.f- _& _ _..._ - _e O.X-.-'----~----B ~ (e) ' ~ () ~..6 '. 2 Time (days) Fig

10 374 L. K. FONG AND R. K. CROWDEN Since cnducting the present experiments we have learned (persnal cmmunicatin frm T. E. Bird) that Cutts did nt bserve starch increase in all trees injected with mucus. n general nly thse trees that had sme degree f resistance t S. nctili attack shwed psitive accumulatin f starch. Hwever ur experiments Tree needles.-.. (a) (b) 1... bo Twig needles ~ 5 t : i 1.' 1 "i. 1 ill..:<.s ::l ---:-- 1.:: ill.~ bo» '-A:...- >< DO \ :)~ (c) "E.. ~ ~ i. " ::l. c:r. :. 3.' e: l-l.. ~ '5' ill '" :: 7 5 (d) \ - 8 'i ~...-a../o~._ e ~ ~_ Time (days) Fig. 4.-Effects f S. nctili mucus n xygen uptake (a and b) and respiratry qutient (c and d) f P. radiata needle segments frm intact trees (a and c) and islated twigs (b and d). Cntrl. Treated with raw mucus... Treated with autclaved mucus. P values are as fllws: Days Cntrl Raw Autclaved mucus mucus Oxygen Trees < 1 uptake Twigs Respiratry Trees qutient Twigs were carried ut n trees deliberately chsen fr their high susceptibility t S. nctili in rder t maximize the effects. t is clear that sme additinal wrk is necessary t determine the spectrum f physilgical respnses t mucus in trees f varying susceptibility. t is pssible that such a study culd prvide infrmatin f ptential use fr field diagnsis f tree susceptibility and may have applicatin in future silvicultural practice. As an alternative explanatin t accunt fr the increase in needle dry weight we prpse that mucus injectin int the tree trunk may well stimulate a shrt-term

11 EFFECT OF S. NOCTLO MUCUS ON P. RADATA 375 translcatin f substances int the needles frm ther parts f the tree. This translcate may well be carbhydrate in nature and may in fact serve as a precursr Figs Phtmicrgraphs f traverse sectins f P. radiata needle segments. 5 cntrl. 6 frm mucus-treated tree 7 days after injectin. 7 tissues f vascular regin in untreated needle. 8 tissues f vascular regin 4 days after mucus treatment shwing distrtin f phlem. 9 vascular tissues 7 days after treatment shwing extensive necrsis f phlem and ray parenchyma. c resin canal; m mesphyll; p phlem; r ray; t transfusin tissue; x xylem. Scale n Figure 6 applies t bth Figures 5 and 6 scale n Figure 9 t Figures 7 8 and 9. fr starch synthesis in resistant trees. The pssibility f a similar translcatin int needles n excised twigs is f curse eliminated and therefre n cmparable dry

12 376 L. K. FONG AND R. K. CROWDEN weight increase f twig needles culd be expected [Fig. 1 (b)]. The perid during which inwards translcatin may ccur must necessarily be brief because f the rapid deteriratin f the phlem cells after mucus treatment. 8 ~1l ~1 +- Origin +-9 M c M c +- Marker dye Fig. O.-Phtgraphs and interpretative drawings f acrylamide gel electrphretgram stained t detect perxidase activity f extracts frm needle segments 7 days after injectin f trees with mucus (M) r water (C). n many respects the bichemical changes which S. nctili mucus causes in P. radiata needles are similar t thse which accmpany the nset f senescence either natural (Wlhuse 1967) r disease-induced in the leaves f ther plants. Thus Shaw and Sambrski (1957) Cllins and Scheffer (1958) Sctt and Smillie (1966) and Nemeth and Klement (1967) have all reprted stimulated respiratry activity in diseased plants whilst Peth6 (1966) nted higher levels f reducing sugars as well as increased respiratin in maize leaves infected with crwn gall. Dke and Hirai (1969) have reprted increased amylase activity which was crrelated with a stimulated rate under dark cnditins f starch degradatin in tbacc leaf disks infected with tbacc msaic virus. Shaw and Cltel (1961) bserved an increase in the dry weight f susceptible rust-infected wheat leaves accmpanied by increase in the nitrgen cntent and level ffree amin acids. Finally Weber et al. (1967) Lvrekvich et al. (1968) Jennings et al. (1969) and Curtis (1971) have all recrded increases in perxidase activities in variusly diseased plant tissues.

13 EFFECT OF S: NOCTlLO MUCUS ON P. RADATA 377 Variatins frm the cntrl levels in each f these parameters are induced in the fliage f P. radiata by S. nctili mucus. The affected needles are eventually abscissed and shed. Thus it is pssible t interpret these effects f mucus in terms f induced premature senescence. The reactin f the mucus in the standard senescence test using radish disks and its reversal by kinetin lend a measure f supprt t this ntin. There is little knwledge yet abut the chemical nature f the active principle in mucus but the fact that it is quite stable at high temperatures suggests that it may be a substance f relatively lw mlecular weight. Cutts (1969b) suggested that mucus may interact in sme unknwn way with natural grwth hrmnes within the tree. On his interpretatin mucus acts essentially as a "tree-cnditining" agent which brings abut a reductin in the effectiveness f the tree's natural resistance mechanisms. By this means the successful establishment f the fungus is prmted and survival f the wasp's eggs and larvae is enhanced. The syndrme f respnses in the affected needles described in this paper may express a type f hypersensitivity in which the tree by premature senescence and shedding f its needles attempts t remve the mucus txin thus increasing its chances f recvery. n this cntext hypersensitivity is synnymus with resistance. Field bservatins n trees attacked by S. nctili indicate that thse trees which shed their ld needles sn after a mderate degree f attack ften recver but death usually results when the yung needles are als affected. Yung needles are mre likely t be affected in the highly-susceptible trees. V. ACKNOWLEDGMENTS We shuld like t thank the Frest Research nstitute Frestry and Timber Bureau Department f Natinal Develpment Hbart fr making facilities available t us during the curse f this wrk. One f us (L.K.F.) gratefully acknwledges receipt f apstgra<;luate schlarship frm the Frest Research nstitute. V. REFERENCES ARNON D.. (1949).-Cpper enzymes in islated chlrplasts. Plyphenl xidase in Beta vulgaris. P. Physil. Lancaster BERNFELD P. (1955).-Amylases c and {3. n "Methds in Enzymlgy". (Eds. S. P. Clwick and N. O. Kaplan.) Vl. 1. pp (Academic Press nc.: New Yrk.) CHEADLE V.. GFFORD E. M. JR. and ESAU K. (1953).-A staining cmbinatin fr phlem and cntiguus tissues. Stain Technl COLLNS R. P. and SCHEFFER R. P. (1958).-Respiratry respnses and systemic effects in Fusarium-infected tmat plants. Phytpathlgy COUTTS M. P. (1969a).-The mechanism f pathgenicity f Sirex nctili n Pinus radiata.. Effects f the symbitic fungus Amylstereum sp. (Thelphraceae). Aust. J. bi. Sci COUTTS M. P. (1969b).-The mechanism f pathgenicity f Sirex nctili n Pinus radiata.. Effects f Sirex nctili mucus. Aust. J. bi. Sci CURTS C. R. (1971).-Disc electrphretic cmparisns f prteins and perxidases frm Phaselus vulgaris leaves infected with Agrbacterium tumefasciens. Can. J. Bt DOKE N. and HRA T. (1969).-Starch metablism in tbacc leaves infected with tbacc msaic virus. Phytpath. Z GAUT. P. C. (1969).-dentity f the fungal symbint f Sirex nctili. Aust. J. bi. Sci

14 378 L. K. FONG AND R. K. CROWDEN HOUGH L. (1954).-Analysis f mixtures f sugars by paper and cellulse clumn chrmatgraphy. n "Methds in Bichemical Analysis". (Ed. D. Glick.) Vl. 1. pp (nterscience: New Yrk.) JENNNGS P. H. BRANNAMAN B. L. and ZSCHELE F. P. JR. (1969).-Perxidase and plyphenl xidase activity with Helminthsprium leaf spt f maize. Phytpathlgy LAYNE E. (1957).-Spectrphtmetric and turbidimetric methds fr measuring prteins.. Biuret methd. n "Methds in Enzymlgy". (Eds. S. P. Clwick and N. O. Kaplan.) Vl. 3. pp (Academic Press nc.: New Yrk.) LOVREKOVCH L. LOVREKOVCH H. and STAHMANN M. A. (1968).-The imprtanct; f perxidases in the wildfire disease. Phytypathlgy LOWRY O. H. ROSEBROUGH N. J. FARR A. L. and RANDALL R. J. (1951).-Prtein measurement with the Flin phenl reagent. J. bii. Chem MADDEN J. L. (1 968).-Physilgical aspects f hst tree favurability fr the wdwasp Sirex nctili F. Prc. ecl. Sc. Aust MAEHLY A. C. and CHANCE B. (1955).-The assay f catalases and perxidases. n "Methds f Bichemical Analysis". (Ed. D. Glick.) Vl. 1. pp nterscience: New Yrk.) MLLS A. K. and CROWDEN R. K. (1968).-Distributin f sluble prteins and enzymes during early develpment f Pisum sativum. Aust. J. bii. Sci NEMETH J. and KLEMENT Z. (1967).-Changes in respiratin rate f tbacc leaves infected with bacteria in relatin t the hypersensitive reactin. Acta phytpathl. Acad. Sci. Hung PETHO M. (1966).-Effect f crwn gall infectin n the respiratin and n the cntent f reducing sugars and ket-acids in maize. Acta phytpathl. Acad; Sci. Hung SCOTT K. J. and SMLLE R. M. (1966).-Metablic regulatin in diseased leaves.. The respiratry rise in barley leaves infected with pwdery mildew. P. Physil. Lancaster SHAW M. and COLOTELO N. (1961).-The physilgy ~ hst-parasite relatins. V. The effect f stem rust n the nitrgen and aminacids in wheat leaves. Can. J. Bt SHAW M. and SAMBORSK D. J. (l957).-the physilgy f hst-parasite relatins.. The pattern f respiratin in rusted and mildewed cereal leaves. Can. J. Bt TREVELYAN W. E. and HARRSON J. S. (l952).-studies n yeast metablism.. Fractinatin and micrdeterminatin f cell carbhydrates. Bichem. J WEBER D. J. CLARE B. and STAHMANN M. A. (l967).-enzymic changes assciated with induced and natural resistance f sweet ptat t Ceratcystis fimbriata. Phytpathlgy WOOLHOUSE H. W. (ED.) (l967).-"aspects f the Bilgy f Ageing." [Symp. Sc. exp Bi (Cambridge University Press: Lndn.)