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.