MICROSCOPIC AND HISTOCHEAIICAL CHANGES IN IIOUGLAS-FIR BARK ACCOMPANYING FUNGAL INVASION'
I'lrillia~rz D. Ross' aid Malcolnz E. Corden Ilepa~t~ncnt\of Fo~cstProducts and Botany and Plant P<~thology,Oregon State Univcrdy Corv,1lli5, Oregon 97331 (Received 9 J~lly1973)
ABSTRACT Dorlglas-fir bark in the proccbss of I~iological clcterioration contained t\vo types of tissr~r "1)leaching." Sclcreid walls \tTcre "bleached" as \\,all lignin was removed in tissues infested \vith Bispora betzllinu, and parc.nchynla and sclcreids were "l~leachedas a rcs~iltof thc selective rcrllovnl of condensed tannins from \\-all surfaces and lnmina in tissues con- sistcntly i~lfestcd\vith an nnidentified fungus resembling Isariu. Rle;lchcd tissne symptol~~s \\-c.rc>not p~)ducedin hark tissncs inoculated with B. betrllil~u or the fungus resellll~ling Isczria, I~litI)oth fungi significantly altered specific bark coniponents. Atl(litiotrc11 l;c,r~rc;orcl.r: Pscrrrlotsrigu nlel~ziesii, lignin, condensed tannin, Bispora hettrlincl, tlecay.
INTHODUCTIOS tion of colored materials, and to identify AIxroscopic examination of cuts through the a'socinted the ],ark of Douglas-fir frecluently reveals I'REVIOUS WORK 1)iologically degraded tissues of two dis- tinct types. In one case, tan-colored scle- General descriptions of outer hark and rcids with normally lignified walls stand of ontoge~lyand structure of the i1lner bark out against a backgro~mdof "bleached," and periden-n layers of Douglas-fir are available (<:hang 1954; Grillos 1956; white parenchyma, and in the seco~ldcase, Grillos and Smith 1959; Ross and Krahmer "l)lcached," white sclereids appear on a 1971) . 1)nckground of dark 11ro\v11 pi~renchyn~a. The recldisll-brown appearance of the It was suspected fro111 these ol~servations cut surface of Douglas-fir outer bark is due that two 11iajor classes of phenolic coin- largely to polymeric polypheilols in the polrents in Douglas-fir bark, lignin and phloem tissne ( IIergert 1960). Specific condensed tanni~~s,mere differentially de- fractions of these polyphenols h:lvc, been graded. named ( e.g. phenolic acids, phlol npl~enes, The present study was initiated to con- and tannins) depending primarily on their firm the location of 1igni11 and condensed solubility in various solve~lts(Kurt11 et al. tannins in bark tissues, to describe "l~leach- 1949). All these fractions are chemically ing" of specific cell types through degrada- similar and are polymers of leucocya~lidins and catechins (Fujii and Kurth 1966). I11 the present study, all reddish-brow11 ma- ' This research was s~ipporteclin part 1,y hlc- 111tirt.-Stennis frunds. Appreciation is extended to terials encrusting cell walls and residual Dr. C. J. K. Wang for ~rrifyingthe identification protoplasts of bark cells are called con- of Uisl~oruhctrtlitra, and to Dr. Hol)ert L. Krahmer densed tan~lins. for hrlpf~~l;tdvicc and criticism. This paper is part of the 1'11.U. dissertation of the, senior aiithor, and Although lignin fractions froin Douglas- is I'aper No. 907 of the Forcst Rrsearch Labora- fir bark have bee11 described (Kurt11 and to1.1, School of I'orc.stry and Technical Paprr 3622 Smith 1954; Kiefer aild Kurth 1953; Fiolmes of the, Oregon Agricultul.al Experiment Station. and Kl~rth1961; Fujii and Kurth 1966), a 'Presc~~tatldrcss: Box 3354, ITniversity Station, l'la~lt Science Division, trniversity of Wyoming, sul~stancethat fits the criteria for lignin 1,an-:u~~ic~,\l'yo~r~ing 82070. suggested 1)y Aranns and Brauns (1960) 130 \VJI,I,IA~\C I). ~~OSSAND ;\IAI,COI,~SI E. COHDEN
IS. 3 1) Cr~ss-s~~cti~~iof Doi~glas-fir I)ar!i fl.on~a 235-y~ar-oldtree, \\~iih(A) vasc~ilarcan- I)iunr, (H) perider~rilayer, (C) innel.-l~arkphloem, (D) a dotted line illustrating a possible location for friture pt~ridrrnldifferelltiatio~i in the inner I~ark,(E) outer l)ar!i \vitli, (F) the ends of sclereids \ isil)lc., and ( C: ) sclcricds dc~ca~.c~d. I\.IODIYICATI~N oP' DOUGLAS-FIRBARK BY FUNGI 131 and Kratzl ( 1965) has not beell isolated. this residue was considered the third Thus, the presence in sclereicl walls of "lignin" fraction. ligni1-1 meeting several criteria of the latter ;illthors was sought in the present study. MATEHIALS AND hlETHOUS Early lignin preparations from Douglas- Isolation and selection of fungi associated fir bark failed to give a positive red to with decay of bark violet reaction when treated with Wiesner reagent (phloroglucinol-HC1) (Kurth and To isolate fungi associated with decay Smith 1954); however, Fujii and Kurth symptonis in bark, individual bark cells or ( 1966) later reported that a Ilouglas-fir small groups of cells with discrete evidence bark preparation "which contained some of fungal attack were plated in a range of I~astfibers and cork fragments" did give a solid media ( Ross 1971) . Followiilg in- positive TViesner test. Not all cell types in cubation at 20-24 C, individual fungi were tree I~arksgive a uniform \Viesner reaction, transferred to potato-dextrose agar slants Ilut fibers and sclereids of coniferous I~arks for storage. generally do ( Srivastava 1966). Two of these media were particularly Tlie chemical composition of isolated successful for culturing bark fungi. A11 sclereid fractions from Douglas-fir bark has inner-bark extract medium was prepared by becn studied, and three "lignin" fractions leaching 20 g of dry inner-bark fines (see have been described (Kiefer and Kurth below) in 500 ml of water for $4 hr. hlineral 1953). The total lignin content of the salts (iron reduced to ?/lo strength) and sclereids was 44.8% as deterinilled by the vitamins of Bf-2 medium (Fihraeus and Klason procedure: however, one fraction, Tulla~lder1956) were added with 90 g/liter constituting 49% of the Klason lignin, was of sucrose. The medium was sterilized by isolated 1)y extracting the sclereids with filtration through a Millipore HA filter, 1%h'aOI3. This fraction, termed "phenolic and agar (15 g/liter) was added when a acids," contained a relatively low methoxyl solid medium was desired. I11 addition, value (4.3%), a high carboxyl content modifications of the Bf-2 medium were (4.9-5.3%), gave a relatively low yield of used in which NI14N03(1 g/liter) was sub- vanillin ( 1.639 ) by nitrobenzene oxidation, stituted for s spa rag in, phenylalanine was hut failed to give a positive T17icsner reac- omitted, the iron content was reduced to lAo tion (Kiefer and Kmth 1953). strength, anil various carbon sources re- A second lignin preparation was obtained placed sucrose. I)y extraction with a dioxane-HC1 reagent Fungi repeatedly isolated from bark following the removal of "phenolic acids" were incubated in a lirluid inner-bark ex- with 1% NaOH. This fraction was prob- tract medium to determine their ability to ably most similar to lignins fotmd in the modify the bark polyphenols. The nledium wood of gyrnnospen~lsandhad ;L methoxyl (100 ml) in 250-ml Erle~lmeyerflasks was content of 13.5-14.3%. The results of a inoculated with mycelial fragments and the IViesner test and vanillin yield followi~lg cultures were incubated 24 weeks in the ~iitrol~enzeneoxidation were not given for dark at 20-24 C, either still or 011 a rotary this preparation ( Kiefer and Kurth 1953). shaker. Fungi causing precipitation of About one-follrth of the Klason lignin re- reddish phenolic materials in the inedium mained after the first two extractions and were retained for further study.
2) Cross-section of i11nc.r bark with (A) a sclereid with conderised tannins in the lumen, (B) an axial l~arenchq~i~acell containing starch grains, (C) ray parenchyma, and (11) sieve cells. :3) Cross-section of outer-hark phloe~ilwith (A) an ol~ter bark sclereid with condensed tannin cncr~lstingthe outer cell-wall layer, (R) expanded phloem parenc*hynla cell and (C) crl~shrtlsieve c,c.lla. 132 WILLIA~I n. ~ossANI) A~ALCOLAIE. CORDEN
F~c:s.4-7. 3) A split pit~ceof infested outer-hark ph1oe111 containing white ("bleached") sclereids a~ldtliukeued parnichq-111atissr~e. 5 ) Longitndinal section of tx11.k containing "1)leached" sclereids, with (A) parenchyma cells con- tiii~~ing,rlarL~,~~c.d residi~nl protoplast5 ;1nc1 fungal hyphacl, a~~d(B) degraded sclereid walls. \IODIFICATI~N OF DOUGLAS-FIIIIIAIIK I~YFUNGI 133
Incul~ationof fungi on burk while lying on the silica gel. Sterile Bf-2 Bark samples and isolated sclereids were nledium was added to allow thorough incubated with fungal isolates in an at- \vetting of the silica gel via the paper teinpt to reproduce specific decay symp- wicks. The slide cultures were incubated t0111~. in the dark ;at 20-24 C. Vial c~lltures. Outer bark was cut into Preparation of isolated sclereid frclctions 1-cm cubes and a 2-mm hole was bored through the center of each cube. Two bark \\'hole bark call be ground and then frac- cnbes were enclosed in each vial (21 x tionated by screening into homogeneous 70 mm) 1)y attaching a glass rod (2 x 60 batches of cork, sclereids, and parenchyma mm) to the vial stoppers and passing the cells (Hergert and Kurth 1952; Kiefer and rod through the holes in the two bark cubes. Kurth 1953). Methods used in this study Rloisture content of the bark was main- and descriptio~lsof the resulting bark frac- tained by submerging the lowest bark tions are given by Ross and Krahmer sainple halfway in water in the bottom of ( 1971 ) . Washed sclereids that passed the vial. The upper bark sample was above through a 100-mesh Tyler screen were con- the water in a saturated atmosphere. These sidered free of other cell types. Material hark sample assemblies were sterilized with passing through a 170-mesh screen (bark ethylene oxide, the bark cubes inoculated fines) was rich in pare~lchymatousmaterial mith mycelial fragments and the vial cul- and was used in making bark extract tures incrtbated in the dark at 20-24 C. medium. Jar cultures. Hark samples of various I
6) C;ross-section of bark containing "l,leached" sclcrcids, \\.it11 (A) bore holes oriented longit~~dinall~ in thick sclel.eid \valls, and (B) darkened condensed tannins in parcncllyma tissue. 7 ) h "l~leached" selercid, \vith ( A ') dar!ienc:d tanuins in sclerrid lrlmcn, and ( R ) residual "1,lcached \\all n~utcrial. 134 \VII,I,IAXI I). ROSS AND XIALCOLXI E. COHDEN
FIGS.8-9. 8) Outer-bark tissne contxining (A) "bleached" parenchyma cells that appear to ha7,e had condensed tannins removed by fungal activity, (B) "nn1,leached but infested tissue slightly darkcwed, and ( C ) a light-colored sclcrcid with tannins relno\ cd fro111 the o~~ter\vall layer. 9) Phlorn~tissue containing (.-\) scle~.eidwith tannins in its lulnen, I: H) sclereids with nulnerouv I,orc holes it1 their n~allsand cl~~tpyI~lmina, and (C) "blcachcd" parenchynla cells. iodide was used to stain starch, and Sudan periderm layers periodically form ill the I\' and ferric chloridc were used for outer part of the inner bark (Fig. 1-D) and sr11)erized tissues aid polyphenols, respec- living inner-bark cells are transfonnc:d to tively ( Johansen 1940). dead outer bark phloem. In cross-section, old growth Douglas-fir The thick-lvalled sclereids (Figs. 2-A and ])ark consists largely of reddish-brown 3-A) are the only cells in the inner and phloem traversed 1)y many buff-colored, outer bark that stain reddish-violet wit11 the anastomosillg peridern1 (cork) layers (Fig. \T7iesner reagent, and the color is very simi- 1). All tissue outside the innermost peri- lar to that given I)y lignified woodl ele- clerm layer is dead and is here termed outer ments. Scattered periderm and parei~chyma bark ( Fig. 1-E ) . The light tat\-colored cells may stuiil also but ~~suallyonly when tisstle l~etweenthe v:iscular canil)iunl and they are near ail injury. At maturity, the innermost periderrn layer is iilner bark sclereitls are filled with reddish-l~rowncon- (Fig. 1-C). h4any cells of the inner bark densed tannins (Figs. 2-A and 3-A) th,.~tare are living. Outer-bark plilocin is encrusted coi~vei-tedto phlobaphene-like 1nateri:~lfol- with condeiised tanniiis and appears darker lowii~gtreatment with 72% H2S04,ai~d are than inner bark phloem where heavy tannin released as intact units when the seconclary deposits have not yet occurred ( Fig. 1). walls are hydrolyzed by the acid. The vasc~~larcaml~il~m adds cells to the Most parenchyma cells of the inner bark inner bark, but the inner bark fails to in- are living and contain starch, tannins, :und crease greatly ill tliick~iess l~ecal~senew other storage products (Fig. 2-B&C).1bVhen \IOI)IPlC;A'l'ION OF DOUCLAS-FIR HARK 13Y FUNGI 135 ii new periderm forms \vitliin tlie inrier hark thick secoi~darywalls and condensed tan- (Fig. 1-11), tlie storage products disappear nins in their lumin,~,change from light tan from cells that are to 11ecome either phello- to reddish-brown a\ their outer wall layer gcn or outer bark phloem. Cells destined to becomes ililpregnated with condelised tan- I~econreouter bark phloem l~ecomegreatly nin, as do the surrounding parencllyma dilated (Fig. 2-13 vs. Fig. 3-B), mld then walls. Thus, tlie sclereid is the only bark light reddish-l~rown nlaterials encrust tlie cell that contains both lignin and condensed espandcd walls and residual protoplasts. ta~lni~l. The outer layer of outer-bark sc:lereid cell walls (i.e. the primary cell walls of the parcnchylna cells from which the sclereids "Bleaching" of sclereid walls is common dvvelopetl) also l~ecolnesi~npregr~ated with in the outer ])ark of Douglas-fir, especially reddish-l,ro\v~~m:lterials ( Fig. 3-A). A thin at the ]lase of trees a hundred years or more zone of pcriclinal divisions forins along an in age. Generally, the most advanced de- al)r~~ptI~o~uiidal-y I,etwee~) expanded par- gradation is in the outer bark initlway be- e~~cllymucells and tllc smaller, undilated, tween the outer slurface and the vascular light-colored parenchy~naof the inner bark. cam1)iuln. As the decay progresses, it moves This zo~lc.of cell divisious stains pink with inward to~vardthe cambium more rapidly Suldan I\<', suggesting the l~eginning of than it spreads outward. Infested outer sl~l~erizationof the periderni. bark phloem is darkened, and the periderm l'hesc observations arc consistent with layers l~ecorne dull gray rather t1i;ul the prcvions cllemical descriptions of Donglas- normal light I~uffcolor. When the \)ark is fir hark. For exalnple, brown materia1 in broken longitudinally, the normall?. rcltldish- the o~lterwall layer of outer-bark sclereids bro\vn sclereicls appear as white streaks was descril~edby Kiefer and Kurth (1953) and are easily distinguished from tl~esur- as "phenolic acids" soluble in dilute alkali. rounding phloem tissue (Fig. 4). Rlicro- Ilergert ( 1960) found high concentrations scopic examination reveals heavy acchumu- of monomeric catechins and leucocyanidins lations of fungal liyphae and dark reddish- ill the c:unbial zone of thc bark and their 1,rown materials in the parenchyma tissue coilcentratio11 decreased centrifugally from ( Fig. 5-A) . Lightly-"bleaclled" sclereids tlic c:wil)i~lnl where the polymeric poly- have bore holes ruilning longit~~diiially pller~ols increitsed. This irnplies that the \vitliin their thick walls (Fig. 6-A); as do colorless precltrsors for tlie reddish-brown, cells of wood attacked by soft-rot fungi high-molecular-weight condensed tannins (Fig. 3C in Duncan 1960). As decay occur ill parenchyma cells of the inner hark progresses, the sclereid walls 1)t.come ancl polymerize 01-I dilated parenchy~~iacell "bleached," fail to stain with \\'iesi~er \valIs and residual protoplasts at the time reagent, and are easily crushed u11dc.r pres- of peridern1 for~u:~tion.As condensed tan- sure as t11oup;l) the lignin con1ponc:nt of the nins increase in molecnlar weight, they \valls has been degraded. I11 adva~iced S~OW21 concoillitant increase ill their affin- stages of decay, the secondary \valls of ity for cellulose (13our and Evelyn 1958). sclerrids are completely decomposed. Thus, PossilIly tlie disappearance of starch and decayed sclereids (Figs. 4 and 7) more other storage products at tlie time of closely resem1)le elenlents of w1ritc.-rotted parenchyma expansion nlay contribute to wood tl~anthose of soft-rotted wood (Figs. synthesis of condensed tanilins. 5 and 7 in Di.111can 1960), which ill c>uternal 'To slimmai-ize, at periderni formation, appearance generally is siiliilar to 11rown- small nonlignified, living parenchyma cells rot decay. of tlir i~inerbark lose their storage ma- Condensecl tainiiils in the lumina of terials, increase greatly il) size, and become "bleached" sclereids and 011 parenchylila impregnated with conclensed tailnins. Inner- cell wiills and protoplasts are darker in [)ark sclcrcids, which contain Iiguin in their decaying bark than in normal tissuc,s (Fig. 136 \+'ILLIAI\L 11. ROSS AND XIALCO1,hI E. CC)RDEN
1-(; vs. F; Figs. 5 and 6 vs. 3). Although cells occur imnlediately under the outer elarkelled, these condensed tannins are not cortical tissues of the bark, and when su1,stantially remo\led from the tissl~es. "l~leaching"is severe, the sclereids become Thick, greenish-lllack hyphae often pro- detached from the surrounding decayed liferate along the periderm layers st~rromnd- tissues. ing decayed phloem, suggesting that peri- Degradation of condensed tannins in derm layers slow the advance of bark bark presents n picture that is the in~~erse fungi. \C'lien heavily infested, the peri- of that given by tissues co~ltaiiiing derms change from a light tau to a dull "bleached sclereids. In the former case, gray color. \\'here periderm tissue was the parenchyma cell walls are white and I~rokeiialong the edges of fissures in the the sclereids are tan, while in the latter bark ( Fig. 1) , severe sclereid degradatioi~ case, the sclereids are "hleached" white and occurred up to the next intact periderm. parenchyma rerllains a dark reddish-11rown 111 an attenipt to identify microorganisms (Fig. S vs. 4). Xlicroscopic exan~ination capn1)le of attacking bark, "bleached of bark with "bleached parenchyma re- sclercicls werc isolated horn the outer bark veals that not only are tannins removed of each of eight trees and plantecl in nine from parenchyma cells and the outer \valls different media (Ross 1971). Of 177 fungal of sclereids, bi~tbore holes penetrate the colonies ol~tained,110 were Aispora betu- thick sclereid \valls and the tannins are re- lintr (cda.) Hughes. This fui~gns has inoved from the sclereid lumina as well greenish-11lack hyphae similar to hyphae (Fig. 9). oftr11 ol~served\vithin walls of "1)leached \Vhen condensed tannins are removed scle~ids. from the outer cell wall layers of sclereids Hi"?x)r tlires short, thick 1)rown sy~rnemataformed. BHAUNS,F. E., AKU D. A. BRAUNS. 1960. The Xo conidia were produced. The next most chemistry of lignin-supp1ernent;II vol~nne. Academic Press, New York. 804 pp. prevalent flil~g~iswas isolated only sis CHAN~:, Y. 1954. Bark structure on North times. A~nericail conifers. (U.S. Dep. Agric. Tech. The I.scrric~-likefungus caused a lienvy Bull. 1095). 86 pp. precipitation of phelklic compollents of Ilo~rsca,K. II., AND hl. E. CORIIEX. 1970. En- l~arkextract medium witl~in24 11r. When \ironmental infl~~enceson the sensitivity of F~tsn~ir~ii~oxys~~orr~in f. sp. lycol~crsici to ii~clil)ateclwith \vl~oleor outer bark sarn- ~ncthylisothiocyanate. I'hytopathology 60: ples ilr slide cu1tm.e for six nionths, the 1347-1350. fmigal hyphae colonjzcd the ])ark uniformly DUNCAS, C. G. 1960. II7ood-attacking ca- and penetrated some sclereid walls, hut pacities and physiology of soft-rot fungi. (U.S. failed to "l~leacli" parenchyma or "empty" For. Prod. Lab. Rep. 2173) 28 pp. F~HKAEUS,G., AKD V. TULI~AYDEH.1956. Effect sclcrcid Inmina. The fungus did not notice- of indole-3-acetic acid on the formation of :~l~lyalter isolated sclereids in slide c11l- oxidascs in f~mgi. Physiol. Plant. 9:41)4-501. t~ires. FUJII, hl., ANn E. R. KURTII. 1966. The cherni- cal nature of conifer phenolic acids. Ethanoly- sis prodl~cts fro111 Douglas-fir. Tappi 49: CONCLUSIONS 92-96. From ~nicroscopicand I~istocllemicalex- (;~ c.on~pcnlents of I>ouglah-fir 1)ark 11y frmgi. Roux, D. G., AVII S. R. EVELYS. 1958. (:on- 1'h.D. Thesis, Ort,gon State trniv. 84 pp. densed tannins. 1. Study of complex-antho- Ross, W. I)., ANT) R. L. KHAII~IEH.1971. Some cyanins present in condensed tannins. Bio- sonrces of variation in strr~ctl~ralcharacter- chew. J. 69:530-538. ictics of Donglas-fir lurk. Wood Fiber 3: SHIVASTAVA,L. hf. 1966. IIistochc~nical str~dies ,'3,54(i. on lignin. Tappi 49: 173-1 83.