J!Jf Phloem and Xylem Phenols

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SYMPOSIUM ON GENERAL WOOD CHEMISTRY Polyphenols in Ceratocystis minor Infected Pinus taeda: Fungal Metabolites, ,;J!Jf Phloem and Xylem Phenols

Richard W. Hemingway,. Gerald W. McGraw, and Stanley J. Barras

Since Ceratocystis minor is central to the death of pines infested by southern pine beetles, changesin polyphenolsof infected loblolly pine wereexamined with regard to accumulationof fungal metabolites and changesin concentrationsof fungitoxic and fungistatic phloem and xylem constitutents. C. minor grown in liquid culture produced 6,8-dihydroxy-3-hydroxymethyl-1H-2-benzopyran-l-one(I), 6,S-dihydroxy-3-methyl-1H-2-benzopyran-l-one(11), and 3,6,8-trihydroxy-3,4-dihydro-l-(2H)-naphthalenone (III). IsocoumarlnII appearedin both phloem and xylem of loblolly pine bolts within 7 to 10 days after inoculation with C. minor. Isocoumarin I was evident in both phloem and xylem after 14 to 17 days of incubation. The a-tetralone III wasnot detectedin either phloem or xylem. The condensedtannins, catechin,and flavanonolswere degradedby C. minor. The stilbenespinosylvin, pinosylvin monomethyl ether,and a compoundtentatively identified as resveratrolinitially accumulated,but their concentrations also diminished in later periods of incubation.

The blue-stainfungus Ceratocystis minor (Hedgc.)Hunt lyphenolsin southernpine phloem (Hergert, 1960;Porter, is generally introduced into the phloem and xylem of 1974; Hemingway and McGraw, 1976) and xylem southern pine trees during attack by the southern pine (Lindstedt and Misiorny, 1951; Erdtman, 1956) exhibit beetle Dendroctonus frontalis Zimmerman, and devel- fungitoxic or fungistaticproperties (Rennerfeltand Nacht, opment of C. minor in the xylem is consideredto be central 1955;Scheffer and Cowling, 1966). Additional phenols to the death of beetle-attacked trees (Nelson, 1934; might be produced as a host responseto infection by C. Basham, 1970). lt is commonly accepted that death of minor, as Shain has shown in Fornesannosus (Fr.) Karst. these trees is causedby a reduction in stem water con- infection of loblolly pine (Shain, 1967)and Norway spruce ductivity to such an extent that wilting occurs (Bramble (Shain and Hillis, 1971)and as Sbrimpton (1973) found and Holst, 1940;Basham, 1970). However, other mech- in lodgepolepine after attack by Dendroctonusponderosae anisms (e.g.,phytotoxins) may also be important. Hopkins. Accumulationof fungistatic or fungitoxic phloem The symptoms of southern pine beetle infestation are or xylem polyphenolsmight be a basisfor host resistance similar to thoseof Dutch elm disease,where bark beetles to the southern pine beetle. introduce Ceratocystisulmi (Buism.)C. Moreau (Banfield, EXPERIMENTALSECTION 1008)and translocatablephytotoxic metaboliteshave been Liquid Cultures. Isolates of C. minor and C. ulmi were suggestedby a number of studies (Zentmeyer, 1942;Di- grown in liquid shake culture on malt extract and on a mond, 1947;Feldman et al., 1950;Salemink et al., 1965). defined medium containing glucose,20 g; i-asparagine Increasesof respirationrate and electrolytemobility of leaf monohydrate, 2 g; KH2PO., 1.5 g; MgSO..7H2O,20 mg; cellsbefore the developmentof high water stresshave been FeCI3,10 mg; ZnSO..7H2O,20 mg; pyridoxin, 1 mg; and observedin Dutch elm disease(Landis and Hart, 1972). thiamin, 1 mg/L. Developmentof the funguswas followed Both high molecularweight carbohydrates(Feldman et al., by measuring pH and residual reducing sugar yields. 1950) and glycopeptides (Van Alfen and Turner, 1975) When the pH had increased from 3.5 to 5.5 and reducing reducestem water conductivity,but thesemetabolites have sugarconcentrations were comparatively low (normally 3 no effect on leaf physiology other than the development to 4 weeks on malt extract) (Claydon et al., 1974),the of high water stress. Alcohol-solublemetabolites of C. ulmi cultures were harvested;the filtrates were saturated with induce necroticlesions of leaf veins (Feldmanet al., 1950). NaCl and were extracted with chloroform, followed by Attention has recently been focusedon phenolic C-10 ethyl acetate. Ethyl acetate extracts were concentrated acid-dihydroisocoumarin tautomers which are produced and examined by TLC with diisopropyl ether-formic in highest yields by the most virulent strains of C. ulmi acid-water (90:7:3)or chloroform-acetone (9:1) and by PC (Claydon et al., 1974). Isocoumarinsare known for their with 1-butanol-aceticacid-water (6:1:2)and/or 6% acetic biological activity toward plant growth (Hardeggeret al., acid. Chromatogramswere viewed under UV light and 1966; Iwasaki et al., 1973; Kameda et aL, 1973). The sprayed with either FeCI3:K3Fe(CN)6or diazotized sulf- similarities in both the causeand symptomsof Dutch elm anilic acid, followed by K2CO3. Three major metabolites disease to destruction of pines by the southern pine obtained from C. minor were identified by their spectral beetle-microbial complex prompted us to examine the properties (McGraw and Hemingway, 1977). Phenolic phenolic metabolitesof C. minor and to determineif these metabolites of C. ulmi were identified by comparison of compoundsare formed in loblolly pine phloem and xylem TLC R, values,color reactions, and spectralproperties with at times when they could be translocated to the needles. published data (Claydon et al., 1974). Another objectiveof this study wasto examinechanges C. minor Inoculated Loblolly Pine. Loblolly pine in phloem and xylem polyphenol composition which were trees were felled on Aug 12 and 19, 1975,from the Ki- associated with infection by C. minor. Many of the po- satchieNational Forestin central Louisiana,and the stems were cut into bolts 1 m long (12 to 22 cm DIB). Six bolts - from tree A (Aug 12) were inoculated with C. minor at 18 Southern Forest Experiment Station, Forest Service, U.S. Department of Agriculture, Pineville, Louisiana sites-six sites around the circumference at each of three positions along the length of eachbolt. One control bolt 71360(R. W .H., S.J.B.) and the Department of Chemistry, Louisiana College,Pineville, Louisiana 71360(G. W .M.). was aseptically woundedwith a 1.5-cmpunch in a similar

~~rin.~~_f.r°m.AG.RICU~TURAJ:: ANI? F~ ~HEMIS!"Y. ~01. ~~, No. 4, ~~e 717~ ,July! AUf. ,1,977 HEMINGWAY

R,valu. Color reactioaa, on PC Paper Thin layer uv+ Ref- RAW 6HOAc DFW UV' NH.OH 8ulfanUie acid

6,8 -Dih ydroxy-3-metbyl. A 0.75 0.95 Ab yw "« after bue isocoumarin 6,8-Dih y droxy -3.hydroxymetbyl. A 0.78 0..7 0.18 0.70 Ab YW"Ol'after baR isocoumarin 3,6,8-Trihydroxy-a -tetralone A 0.77 0.63 0.57 yw yw or after base P. tGedGPolyphenola Catechin A 0.55 0.49 Ab Ab yw before baR Proanthocyanidin a Porter, 197. 0.20 0.55 Ab Ab yw before baR Proanthocyanidin b Porter, 197. 0.22 0.45 Ab Ab yw before hue Proanthocyanidin c Porter, 197. 0.88 0.38 Ab Ab yw before bue Dihydroquercetin glucoaide Heqel't.. 1960 0.45 0.56 Ab Ab yw-or after baR Dihydroquercetin A 0.71 0.37 Ab Ab wb-yw after bale Dihydromyricetin Hertert., 1960I 0.55 0.82 Ab Ab wb-yw after baR tranB-Ferulic acid A 0.76 0.31 bI bl pr after baR ciB-Ferulic acid A 0.76 0.61 bl bl pr after base Pinosylvin A 0.79 0.13 0.39 hi bl yw-or after bale Pinosylvin monomethyl ether A 0.85 0.10 0.80 hi wh yw-or after baR Resveratrol A 0.72 0.00 0.21 hi bl or-rd after hue G A, authentic compound geed for comparison. b Ab, ab8Orb8ultrafiolet lililt; bI, blue; yw, yellow; or, oraDte; wb, white; pr, purple; rd, red.

pattern. The secondtree B (Aug 19)was cut into 12 bolta; methanol extract was redissolved in methanol (1 mL/7 g six bolts wereinoculated with C. minor, and six bolts were of wood). aseptically wounded in the pattern described above. Wbatman No.1 papers were spotted with 40 JJ,Lof the The C. minor inoculated and control wound sites were methanol extracts from phloem and xylem, and the sampled periodically over a 24-dayincubation period by chromatograms were developed with I-butanol-acetic peeling the bark off at the xylem cambium in a zone3 to acid-water (6:1:2) and in the second dimension with 6% 5 cm wide and 6 to i8 cm long around the treatment site. acetic acid. The dried sheets were examined under UV The extent of different colored reaction zones of the light before and after fuming with ammonia and then were phloem (normal white phloem, a pale-yellow zone ahead sprayed with diazotized sulfanilic acid, followed by K~ of a dark-brown zone, and the black tissue where the Changes in concentrations of compounds were evaluated perithecia were abundant) was measured,and phloem of by visual comparision of spot intensity. Polyphenols of eachof these reaction zoneswas dissectedwith a scalpel. the phloem or xylem were identified by compariaon of their 'fo test the purity of the inoculation, phloem tissueswere chromatographic and spectral properties with either au- cultivated. Excellent growth of C. minor was obtained thentic compounds or p\lblished literature. The major from isolatesof both brown and black phloem. C. minor phenolic metabolite of C. minor, 6,8-dihydroxy-3- was obtained occasionallyfrom the yellow zone, but no hydroxymethylisocoumarin (I), was isolated from both other fungus waspresent. Sufficient treatment sites were phloem and xylem in sufficient quantities to crystallize. sampled to obtain 0.5 to 2.0 g dry weight of each color Melting points and spectral properties were identical with reaction zone. Care was taken to avoid inclusion of any those of I, obtained from liquid culture. outer bark in the phloem samples. The xylem directly below the treatment site wassampled by drilling a 2.5-cm RESUL1'8 AND DISCUSSION hole toward the pith; the shavingswere collected,and the Phenolic Metabolite. in Liquid Culture.. Culture depth of penetration of the blue stain wasmeasured. The filtrates from C. minor grown on both media contained control bolts were drilled to a comparabledepth. three major phenols. Chromatographic and UV spectral Phloem samples were dried under vacuum at room properties of these compounds are summarized in Tables temperature and ground with a mortar and pestle. The I and II, and detailed arguments for the proof of their ground phloem was extracted with 30 to 60 °C petroleum structure have been reported (McGraw and Hemingway, and then methanol by soaking for at least 24 h in 50 mL 1977). The most abundant compound was 6,S-di- of eachsolvent. Extractswere recovered by filtration, dried hydroxy-3-hydroxymethyl-lH -2- benzopyran-l-one (I) under vacuum,and weighed.The methanol-aolubleextract (Figure 1). This isocoumarin has not previously been wasredissolved in methanol(4 mL/g of phloem extracted), isolated as a fungal metabolite. A second isocoumarin was and 1 mL of this solution was applied to a O.4-gPoly- present in liquid cultures in much lower concentrations. amide-6 column to removethe condensedtannins. The It was identified primarily through its high-~lution ~ lower molecular weight polyphenolswere eluted with 10 spectrum as 6,8-dihydroxy-3-methyl-lH-2-benzopyran- mL of methanol;the eluatewas evaporated to drynessand I-one (II). Isocoumarin n was previously reported as a redissolved in 1 mL of methanol. metabolite of Ceratocystis fimbriata Ell. and Haist. Wood shavings (10 to 20 g) were dried under vacuum (Curtis, 1968), where it was found in small quantities along at room temperatureand ground with a Wiley mill to pa88 with larger proportions of the 6-methoxy analogue IV a I-mm screen. The ground wood was extracted with 30 (Stoessel, 1969). The clO8ely related dihydroisocoumarin to 60 °C petroleum and then methanol by soaking the V is a metabolite of C. ulmi (Claydon et aI., 1974). The wood for at least 24 h in ISOto 200 mL of solvent. The third compound isolated from liquid culture of C. minor petroleum-and methanol-solublematerials were recovered was the a-tetralone 3,6,8-trihydroxy-3,4-dihydro-l(2H)- by filtration, dried under vacuum, and weighed. The naphthalenone (ll1), which was previously identified 88 a

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R5 R4 blue-stained xylem did not differ, and the only change during the incubation period was a slight upward trend in the petroleum solubility of the blue-stained xylem of ~R3 tree A. Methanol-SolubleExtractives. The methanolsolubility O-H 0 of the normal white phloem decreasedduring the experiment, and, as with the petroleum solubility, this de- creasein concentTationof methanol-solublematerials was ~ ~ ~ ~ not dependent upon infection by C. minor (Table IV). I CHZOH H H OH The yellow and particularly the brown reaction zonesof II CH3 H H OH the aseptically wounded sites contained less methanol- IV CH3 H H solublematerial than did the normal white phloem. In the OCH3 XIII H OC OH early stagesof incubation of the C. minor inoculatedsites, CH3 CH3 the yellow reaction zone contained much less methanol- XVI CHZCHOtI:H3 H H OCH3 soluble material than did the normal white phloem. The difference between these two tissue types became less R4 pronounced later in the incubation period. Since the yellow reaction zone became more difficult to detect as a discrete zone,the convergencein the methanol solubility of these two tissue tyPes may reflect difficulties encountered in sampling. The brown and black reaction zonescontained far less O-H 0 methanol-soluble material than did the white or yellow R tissue. To determinehow much of this responsemay have ...1 R4 ~ resulted from fungal utilization of low molecular weight III OH.H HZ OH carbohydrates,the amounts of reducing sugarspresent in VI OH.H OH.H H the methanol extracts were determined after acid hy- drolysis. Results shown in Table V confirmed our sus- VII OH.H OH.H OH picion that someof this reduction in methanol solubility VIII ~ OH.H OH was related to the carbohydrates. A similar effect was reported by Barras and Hodges (1969). However, the R5 R4 major causeof the lower methanol solubility in the black and brown reaction zones than in normal white phloem R6ROOW R3 was a reductionin the amO\D1tsof tannins extractablewith methanol. 7 II The methanol solubility of the blue-stained wood was consistently lower than that of the normal wood, but no O-H0 substantial changeswere observedwith an increasein the ~ ~ ~ ~ ~ incubation period. V CH3,OH HZ H OH H C. minor Metabolites in Phloem and Xylem. The three major phenolic metabolites of C. minor grown in IX CH3,H HZ H OH H liquid culture were not detected in the white or yellow X CH3,OH OH,H H OH H reaction zone phloem at any stage of incubation. Iso- XI CH3,OH .0 H OH H coumarin II was detected in the brown reaction zone XII CH3,H HZ H H phloem 7 to 10 days after incubation, and isocoumarin I OCH3 was evident in the brown reaction zone after 14 days. XIV CH3,OH CH3,H CH3 OH CH3 Concentrationsof both of thesemetabolites increasedin XV CH3,OH HZ CH3 OH CH3 the black reaction zone with increasing length of incubation. However, the concentration of II remained at Figure 1. Phenolic metabolites of fungi. relatively low levels, but I reached very high concentrations. Mter 24 daysof incubation, when the peritheciahad amountsof petroleum-solublematerial in the normal white covered the entire reaction zone, I was the dominant phloem decreasedduring the incubationperiod in both the phenol in the methanol extracts. The a-tetralone III was C. minor inoculated and asepticallywounded bolts, 80 the not detectedin the phloem after any stagein development decreasewas not related to infection by C. minor (Table Ill). The petroleum solubility of the brown and black of the fungus. reactionzones of the C. minor infectedphloem was usually The xylem of control bolts and the normal white xylem higher than that of the white phloem. The more solid and of C. minor inoculated bolts contained none of the three tacky properties of the extract suggesteda higher pro- phenolic metabolites of C. minor. In the blue-stained portion of resin acids in these reaction zones than in xylem, II appearedafter 7 days of incubation, and I was normal phloem. In a concurrent study, Barras (1976) evident after 14 days. As was evident in the phloem found that the proportion of neutral lipids in the phloem samples,the concentration of n remainedat relatively low decreasedafter 4 weeks' growth of C. minor. Different levels,while the concentration of I continually increased inoculation sites varied considerably in the extent of re- until it becamethe dominant phenol of the blue-stained sinosus. The fungus appeared to develop well in sites xylem after 17 days. Because of interference by other where resinosuswas readily apparent,so resinosusdid not xylem constituentsat the R, valuesof ill, it wasimpossible occur sufficiently to retard the growth of C. minor. The to determine if small amounts of the a-tetralone were amounts of petroleum-soluble materials in normal and present in the blue-stained xylem.

720 J. AtI'w;.Food OleIn., Yd. 25, No.4, 1977 ~ ON GBNmW. WOODCHEMIS'l1ty

Incubation period. daJ8 Tigue 8 7 10 14 1'7 21 24 Phloem White (C. minor) 29 30 27 25 2& 22 23 (ateptic) 30 32 27 29 28 20 21 Yellow (C. minor) 20 21 20 22 20 22 20 (SEptic) 23 22 19 Brown (C. minor) 14 13 9.0 6.3 7.9 8.4 6.0 (ateptic) 17 16 12 Black (C. minor) 7.0 4.4 4.8 8.8 4.2 Xylem Normal 0.76 0.88 0.80 0.82 0.91 0.88 Blue stain 0.67 0.70 0.62 0.70 0.70 0.67

Table V. Changesin Carbohydrates and PoIypbenoia in phloem after 7 to 10 days of incubation, but their con- Methanol Extracta of Phloem Infated with C. minorA centrationsremained low. Resveratrol has not previously been reported in normal (Lindstedt and Misiomy,1951; Erdtman, 1956) or fungal-infected pines (Shain, 1967; Phloem H2O Shrimpton, 1973). Although PC and TLC R, valUM.color type insoluble - -- reactions,and UV spectrawere identical with th<.e of an White 166 31 authenticsample and similar to publishedliterature (Hillis Yellow 137 31 and Ishikura, 1968),identification of this compoundshould Brown 46.2 6.9 Black 63.6 3.8 be consideredtentative until sufficient amounts of crys- talline materials are obtained. 0 Samplesfrom tI'eeA after 14 daysof incubation. The concentrationsof catechin and proanthocyanidina were much lower in the brown reaction zone than in the Changes in Phloem and Xylem Polyphenols. Po- normal white phloem of both trees. In the later stagesof lyphenols in Aseptic Phloem. The major polyphenol incubation, only traces of catechin and one of the pro- present in the normal white phloem of both trees was anthocyanidinscould be detected. As suggestedby the low catechin (Table I). Large amounts of proanthocyanidins methanolsolubility and low proportion of methanol-soluble werealso p~t. The phloemof tzeeA differed from that tannins, these compounds were apparently polymerized of tree B becauseof the presenceof relatively large am- to suchan extent that they were no longerextractable witl1 ounts of a glucoside of dihydroquercetin in tree A. Fla- methanol. Dihydroquercetin and dibydromyricetin vanonol and flavonol aglyconeswere not detected in the concentrationswere relatively high in the brown reaction white phloem tissue of either tree. Only subtle changes zonethrough the first 10days of incubation,but by 24 days in the polyphenol composition of the white phloem were only tracesof dihydroquerretin remained. Ferulic acid was observed as incubation progressed. In tree A the con- also present in substantial proportions after 7 days of centration of the dihydroquercetin glucoside decreased, incubation, but its concentration decreasedthereafter. but no corresponding increasein dihYdroquercetin con- Neither quercetinnor myricetin wasobserved in the brown centration was detected. In the normal white phloem reaction zone of tree A or tree B. Small amounts of pi- tissues of both trees, the distribution of the proantho- n~ylvin, pin~ylvin monomethyl ether, and resveratrol cyanidins shifted to compoundsof lower R, valuesin the were present in the brown reaction zonephloem, but their 6% HOAc dimension and higher R, values in the 1- concentrations appearedto decreasewith increasing in- BuOH-HOAc-H~ dimension. In the yellow and brown cubation period. None of the polyphenols normally reaction zonesof the aseptically wounded sites, the am- p~nt in the phloemand none of the stilbeneswhich were ounts of proanthocyanidinsand catechinwere much lower found in infected phloem during early stages of fungal than in the normal white tissue. Small amountsof ferulic developmentcould be detected in the black reaction zone acid were also detected in the brown reaction zone. after 24 days of incubation. Polyphenols in C. minor Infected Phloem. Although Polypheool8in Normal White Xylem. In the first week catechin and proanthocyanidin concentrations in the of incubation, the wood of aseptically wounded bolts yellow reaction zones of C. minor infected tissues were contained only traces or undetectable quantities of fla- lower than those in the normal white phloem, these vonoids and stilbenes. However, the amounts of catechin compoundsremained the major constitutentsof the yellow and proanthocyanidins in the sapwood increased dra- phloemthroughout tl1e experiment. In tree A the gl~de matically in later incubation periods,and thMe OOIDpounds of dihydroquercetin remained a prominent constitutent were major phenolic constituents after 17 to 20 days of through 14 days of incubation, but thereafter its con- incubation. Proanthocyanidin b wasthe major compound centration decreasedmore rapidly than was observedin in the sapwood;proanthocyanidin a dominated this class the normal white phloem. Both dihydroquercetin and of compounds in the phloem (Table I). The stilbenes dihydromyricetin were detectable after 3 days of incu- pinQuercetin and myricetin were commonly found in sapwood of other conifers by Hergert not detected in the yellow reaction zonesof either tree. and Goldschmid (1958). Small amounts of pinosylvin, pinosylvin monomethyl Polypheools in C. minor Infected Xylem. As was ether, and ~trOI weredetected in yellow react.ionzone observedin the control bolts, tl1eamounts of catechinand

J. ~. Foc.sQwm.. v~ 25. No.4. 1977 721 proanthocyanidins initially increased in the blue-stained Banfield, W. M., Mall. Agric. Exp. Stn., Control Ser. Bull., 568 xylem of the C. minor inoculated bolts. The conrentrations (1968). of th~ compounds reached a maximum after 14 days and Barras,S. J., unpublishedresulta, SouthernForest Experimental thereafter decreased, as was ot.erved in the phl~m which Station, Pineville, La., 1978. was infected with C. minor. Concentrations ofpinoeylvin, Barras, S. J., Hoctce&, J. D.. Con. Entomol. 101, - (1~). Basham, H. G.. Phytopathology 60, 750 (1970). pinosylvin monomethyl ether, and particularly resveratrol Bramble, W. C., Holst, E. C., Phytopathology 30, 881 (1940). were much higher in the blue-stained wood than in the Claydon. N., Grove,J. F., H~ken. M., Phytochemi6try U, 2567 uninfected sapwood. In the later periods of incubation, (1974). the stilbenes were present in lower concentrations than in Condon. P., Ku~, J., Phytopathology SO,'JS1 (1~). the 7 to 14 days after inoculation. Condon, P., Kuc, J., Phytopathology 52, 182 (1982). CONCLUSIONS Curtis, R. F.. Ezpenentia 24, 1187 (1968). Dimond, A. E., Phytopathology 37, 7 (1947). Both iBOCOUIDarinsI and fi, which were produced by C. Erdtman. H., in "Penpectiva in Organic CbemiBtry", Tood. A minor in liquid culture, were also found in the phl~m and R., Ed., Interaclence,New York, N.Y., 1956,p 463. xylem of infected loblolly pine bolts. The a-tetralone m, Feldman.A. w., ~lli. N. E., Howard, F. L., Phytopathology which was found in liquid culture filtrates, was not found 40, 341 (1950). I in infected loblolly pine bolts. The concentration of n Findlay, J. A, Kwan, D., C4n. J. Chem. 11. 1617(1973a). I remained at relatively low levels, but I accumulated to high Findlay, J. A., Kwan, D., Can. J. Chem. 51, 3299 (1973b). I concentrations in both xylem and phl~m after 24 days of Hardener, E.. Rieder, W., Waller, A., KUBler,F., Hell/. Chim. Acta 49, 1283(1988). incubation. Since compounds of the iaocoumarin class are Hemingway, R. W., McGraw, G. W., Appl. Polym. Symp. 28, noted for their biological activity, I and fi may alter the 1349-1364 (1976). physiology of southern pine trees, other microorganisms Hergert, H. L., For. Prod. J. 10, 610 (1~). associated with the southern pine beetle, or certain life- Hergert, H. L., Goldschmid, 0., J. Or,. Chem. 23.1700 (1958). stages of the beetle itself. In addition, it should be em- Hillis. W. E., Ishikura, N.. Chromatography 32. 323 (1968). phasized that th~ changes occurred in bolts rather than Iwasaki. S., Muro, H., Sasaki.K., N~, s., Okuda,S., Seto, z., intact trees. Difficulties encountered in artificially ino- Tetrahedron Lett. 37, 3537 (1973). culating southern pine trees with C. minor suggest that Kameda,K, Aoki, H., Tanaka,H, Namiki, M., Agric. Bioi. Chem. analysis of southern pine beetle infested trees for th~ 37, 2137 (1973): changes in polyphenol composition should be attempted Landis, W. R., Hart. J. H.. Phytopathology 12. ~ (1972). Lindstedt. F., Miaiomy. A., Acta Chem. Scand. 5,121 (1951). next. These questions deserve further study. McGraw, G. W., Hemingway, R. W., Phytochemiltry, in preu C. minor was so effective in degrading flavonoids and (1977). stilbenes, which are generally known for their fungistatic Nelson, R M., Phytopathol. Z. 7, 327 (1934). or fungitoxic properties, that apparently these compounds Porter, L. J.. N. Z. J. Sci. 7, 327 (1974). would not contribute to host resistance to C. minor. Rennerfelt, E., Nacht, G., Sl/en: &t. Ti4.kr. 49, 419 (1955). ACKNOWLEDGMENT Salemink.C. A., Rebel,H., Kerli~, L. C. P., Tcbernoft',V ., Sc~e I 149, 202 (1965). The authors are indebted to J. H. Hart, Department of s..a. T., Aoki. H.. Namiki, M.. M\makata.K. Agric. Bioi. Chem. Plant Pathology, Michigan State University, East Lansing, 32, 1432(1968). Mich., for an isolate of C. ulmi and authentic resveratrol; Scheffer,T. C., Cowling, E. B., Annu. ReI/.Phytopathol. 4,147 L. Shain, Department of Plant Pathology, University of (1988). Kentucky, Lexington, Ky., for pin~ylvin and pinosylvin Shain. L., Phytopathology 57, 1034(1987). monomethyl ether; W. E. Hillis, Division of Building Shain, L., Hillis, W. E., Phytopathology II, 841 (1971). Research, Commonwealth Scientific and Industrial Re- Shrimpton, D. M., Can. J. &t. 51, 527 (1973). Stoessel,A., Biochem. Biophya. Rea.Commun. 35, 188 (1989). search Organization, Highett, Australia, for several fla- Van Alfen, N. K., Turner, N. C., Plant PhYlioI. 51, 312 (1975). vonoids; J. W. Rowe, Forest Products Laboratory, U.S. Zentmeyer, G. A, Science H, 512 (1942). Forest Service, Madison, Wis., for 1H NMR spectra; and the High Resolution Mass Spectroscopy Laboratory, Florida State University, Ta11ahassee,Fla., for spectra. J. Receivedfor reviewSeptember 23, 1976. ~pted December28, D. Hodges, School of Forestry, Mississippi State Univ- 1976. Supported in part by the U.S. Department of Agriculture ersity, State College, Miss., provided valuable di&CUSSions SouthernPine BeetleResearch and DevelopmentProgram. This of the physiology of this disease. paper was presented at the 172nd National Meeting of the American Chemical Society, San Franciaco, Calif., Aug 1976. I LITERATURE CITED Mention of trade names is solely to identify equipment and Aldridge, D. C., Grove,J. F., Thrner, W. B., J. Chem.Soc. C, 1~ materials used and does not imply endorsementby the U.S. (1966). Department of Agriculture.

722 J. Awk:. F(xxt Q8n., Vol. 25. ~. 4, 1977