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Contribution of eicosapolyenoic fatty acids to the sesquiterpenoid phytoalexin elicitor activities of Phytophthora infestans spores
Physiologicaland MolecularPlant Pathology(1988) 32, 49-59
Contribution of eicosapolyenoic fatty acids to the sesquiterpenoid phytoalexin elicitor activities of Phytophthora infestans spores J.
R. CREAMER a n d R . M . BOSTOCKi-
Departmentof Plant Patholog)',Universityof California, Davis, California95616, U.S.A. (Acceptedfor publieationMay 1987)
The concentrations of arachidonic (AA) and eicosapentaenoic (EPA) acids in lyophilized sporangia from isolates representing four races ofPh)'tophthorainfestansand in lyophilized zoospores and newly-formed cystospores of an isolate of race 0 were determined. The predominant eicosapolyenoic acid was EPA, and the levels of EPA and AA were similar in all four isolates. In the race 0 isolate, the total concentration of EPA and AA was greater in sporangial tissue than in tissues from zoospores or cystospores. Treatment of spores with bromine eliminated AA, EPA and other unsaturated fatty acids and aboIished phytoalexin elicitor activity. Addition of a mixture ofllplds from P. infestans containing free and esterified EPA and AA or of pure eicosapolyenoic acids to brominated sporangia restored 76 and 55%, respectively, of the elicitor activity present in untreated sporangia. The elicitor activities of these mixtures were significantly greater than the activities of the lipids or free fatty acids. Bromine treatment also abolished the elicitor activities of zoospore and cystospore tissues, and the treated tissues retained the capability to significantly enhance Ark elicitor activity. The results demonstrate a requirement of unsaturated lipid in spore tissues ofP. infestansfor elicitor activity and provide evidence that the levels ofelcosapolyenolc acids in lyophilized sporangia ofP. infestansare sufficient to account for the major portion oftheir elicitor activity, provided that the components which enhance fatty acid elicitor activity are also present.
INTRODUCTION T h e p o l y u n s a t u r a t e d fatty acids, a r a c h i d o n i c (AA) a n d e i c o s a p e n t a e n o i c (EPA) acids, are a b u n d a n t c o m p o n e n t s in tissue lipids o f Pto'tophthora infestans a n d b o t h acids are efficient elicitors o f s e s q u i t e r p e n o i d p h y t o a l e x i n s a n d o t h e r reactions associated with the h y p e r s e n s i t i v e response ( H R ) in p o t a t o t u b e r [1, 4]. Previous studies clearly d e m o n s t r a t e d a r e q u i r e m e n t for these a n d related eicosapolyenoie or d o c o s a p o l y e n o i c acids in lipids for tile elicitation o f t h e response in potato, a n d the s t r u c t u r a l features in fatty acids necessary for activity h a v e b e e n identified [1-3, 18]. T i l e elicitor activities o f these fatty acids a n d acyl lipids c o n t a i n i n g t h e m are e n h a n c e d b y b r a n c h e d fl-glucans, also f o u n d in extracts o f f u n g a l tissue. H o w e v e r , these g l u c a n s a l o n e a r e i n a c t i v e as elicitors o f s e s q u i t e r p e n o i d p h y t o a l e x i n s in p o t a t o [4, 17, 18]. T h e physiological significance o f t h e fatty acids as i m p o r t a n t elicitors o f t h e H R has b e e n q u e s t i o n e d [10] b e c a u s e the threshold c o n c e n t r a t i o n o f A A or E P A for sesquiterAbbreviations used in text: AA, arachldonie acid; EPA, eicosapentaenoic acid; FAME, fatty acid methyl esters; HR, hypersensitive response. "['Towhom correspondence should be addressed. 0885-5765/88]010049+ 11 S03.00/0 © 1988 Academic Press Limited
50
J.R. Creamer and R. M. Bostock
penoid phytoalexin accumulation in potato tuber (cv. Kennebec) is approximately 16 nmol when applied to tile upper surface of a disc and maximum sesquiterpenoid accumulations are achieved with approximately 330 nmol or more. The latter corresponds to the concentration of these acids (free and esterified) in approximately 5 mg of lyophilized mycelium [1, 3]. It therefore seems unlikely that comparable levels ofelicitor acids would be available in the incompatible spore-plant tissue combinations typically used by investigators, although the concentrations of these elicitors in spores have not been reported. Tile contribution of the fl-glucans appear to be important in this regard for their marked enhancement of fatty acid elicitor activity [17, 18]. Furthermore, the glucans reveal elicitor activity with concentrations of eicosapolyenoic acids that are below tile tbreshold of induction ofdetectable levels ofsesquiterpenes. The role ofthe fatty acids as elicitors is complicated by the diversity of fractions from P. infestans mycelium capable of eliciting tile response [1-3, 6, 9]. Extraction with a mixture of chloroform and methanol removes a significant portion of elicitor activity from fungal tissue but leaves a small amount of elicitor acids and a considerable amount of elicitor actMty in the residual material. Nevertheless, AA and EPA were found free or bound in all fractions with elicitor activity, although they were present at low or trace levels in some complex fractions, including insoluble cell wall preparations [3]. Another report suggests that there may be elicitors ofsesquiterpenoid phytoalexins devoid of AA and EPA in filtrates of senescent cultures of P. bfestans [14]. However, the activity of these components relative to other fractions from mycelia is very low. Evidence for alternative elicitors ofthe response in potato has been presented by Zook & Kuc [26] and may also be inferred from the elicitor activity associated with living but not autoclaved or etbanol-treated tissues ofBipolaris carbonumand other fungi nonpathogenic to potato [16]. In general, members of the higher fungi (Ascomycetes, Basidiomycetes and Fungi Imperfecti) do not contain AA or EPA, although AA was detected in minor amounts in the fatty acids from two Penicillium spp. [25]. In the light of these observations, we believed it important to re-examine the relationship of tbe eicosapolyenoic acids to the elicitor activity in spores ofP. infestans and cultures of certain eliciting fungi nonpathogenic on potato. In this study we provide precise information on the levels of EPA and AA in P. infestans spores and present further evidence for the requirement of eicosapolyenoic acid lipids in the elicitor activity oflyophilized sporangia, cystospores and zoospores. We describe a method for the inactivation of the eicosapolyenoic acids in tissues ofP. infestans with bromine which circumvents tile limitations encountered when attempting their complete removal by solvent extraction. We also determined the fatty acid composition ofmycelia of Ceratocystisfimbriata and Bipolaris carbonum, which elicit a strong HR, and examined the effect ofseveral methods of attenuation of these fungi on the elicitor actMty of their tissues. MATERIALS AND METHODS
Fungal culture Phytophthora infestans (Mont.) de Bary races 0, 4, 1.4 and 1.2.3.4 were maintained on amended lima bean agar [5] at 20 °C for 14 days. An isolate of Ceratocystisfimbriata El 1. and Halst. pathogenic oll almond (Prunus dulcis (Mill.) Webb) was obtained by hyphal tip culture from a bark canker. A culture ofBipolaris carbonum (Ullstrup) Shoemaker was
Phytoalexin elicitor activities of P. infestans spores
51
a gift of E. E. Butler of our Department. These isolates were maintained on potato dextrose agar (PDA, Difco) at room temperature. Sporangia, zoospores and cystospores ofP. infestans were obtained from cultures as described previously [12]. Sporangial suspensions in sterile deionized water were collected from cultures, passed through two to four layers of sterile cheesecloth and washed with sterile deionized water over filter paper (Whatman No. 1). Zoospores were obtained from sporangia incubated in sterile distilled water for 2-3 h at 10 °C. They were collected by filtering ttle suspension through a nylon mesh filter (15 [am mesh) or by carefully decanting the supernatant and allowing the sporangia to sediment for 20-30 min, centrifuging at 3000 r min -1 for 1 min, and removing the supernatant. Cystospores were formed by agitating a suspension ofzoospores in a test tube on a Vortex mixer for 2 min [12]. The suspensions ofzoospores and cystospores were evaluated for concentration and purity with a light microscope. For fatty acid analyses, spore suspensions were immediately frozen in a dry ice/acetone bath, lyophilized and weighed. In the experiments testing the elicitor activities ofzoospores and cystospores, the spore suspensions were frozen immediately in liquid nitrogen and lyophilized. For race 0, 5.4 x 10 s lyophilized sporangia weighed approximately 1 mg (mean of nine determinations from three preparations). Non-viability of spores was confirmed by absence of growth on amended lima bean agar. Mycelia ofB. carbonum and C.fimbriata were collected from 10-day-old shake cultures in 500-ml flasks of potato dextrose broth (PDB, Difco). The mycelia were collected on a colander through four layers of cheesecloth, rinsed with deionized water, lyophilized, ground to fine fragments using a mortar and pestle, and weighed. Mycelial fragments were transferred onto PDA (C. fimbriata) or cornmeal agar (B. carbonum) after the different treatments to determine viability.
Attenuation offi~ngal tissue The following treatments were examined to determine their effect on the viability and elicitor activity of C.fimbriata and B. carbonum tissues. Lyophilized mycelium and conidia were (1) sonicated in water for 5 min at maximum intensity (treatment A); (2) autoclaved for I0 min (treatment B); (3) exposed to 2 ml of diethyl ether, the solvent evaporated and sterile water added (treatment C); (4) exposed to propylene oxide, the gas allowed to dissipate and sterile water added (treatment D); or (5) frozen in water in a dry ice/acetone bath and thawed (three cycles, treatment E). Combinations of these treatments were also tested (see Table 4).
Fatty acid analysis Lyophilized tissues were treated with 1 N methanolic-HC1 for 2 h at 100 °C, cooled and the fatty acid methyl esters (FAME) extracted three times with hexane [2]. The FAME were analysed by GLC using a 1.8 m x 2 mm i.d. glass column packed with 10% SP-2330 on Chromosorb W AW (Supelco) programmed at 150-250 °C at 10 °C m i n - l [2]. Methyl esters ofstandard fatty acids were used for identification and quantitation, with methyl arachidate or methyl heptadecanoate as an internal standard. Authentic lipids were purchased from Sigma.
52
J.R. Creamer and R. M. Bostock
Treatment 0fPhytophthora infestans spores with bromine Since bromine is a powerful nucleophile which readily adds to double bonds of unsaturated fatty acids [23], we used it to eliminate EPA and AA in killed spores. Samples containing 3-24 mg of lyophilized sporangia, zoospores or cystospores were mixed with 2 ml ofcarbon tetrachloride and 50-100 lal of bromine (Sigma) in a capped test tube and incubated for 24-48 h at 45 °C. The solvent was removed under nitrogen and the tubes were left uncapped for 48 h to allow residual bromine to dissipate. Analysis of FAME from the brominated sporangia confirmed the elimination of all unsaturated fatty acids. The brominated spores were suspended in the same test tube by sonication in sterile water with or without additional elicitor lipids. T o determine the effect ofbromine on the enhancing activity offungal glucans, a glucan preparation from mycelium ofrace 0 ofP. infestans was obtained as described previously [4] and treated with bromine. T h e concentrations of glucan and AA applied to each potato disc were 100lag (glucose equivalents) and 50 lag, respectively.
Elicitor activity assay Elicitor activities of samples were determined with cv. Kennebec potato tuber discs as described in previous publications [2, 3, 13]. A 50-100 lal sample of a treatment suspension was applied uniformly to the upper surface o f a 2 cm diameter tuber disc. T u b e r discs were visually rated for hypersensitive browning 96 h after treatment and the accumulations of the sesquiterpenoid phytoalexins, rishitin and lubimin, were determined by T L C [22] or by G L C [13]. Ph.ytophthora infestans lipids were obtained by extracting mycelia with a mixture of chloroform and methanol (2:1, by volume) and nonlipid contaminants were removed with 0.75% aqueous KCI [2]. Tile class distribution, fatty acid composition and elicitor activity ofP. infestans lipids have been reported elsewhere [8, 15]. In one treatment, sporangial tissue equivalent to approximately 5.4 x 10 s sporangia (I mg) was added to each tuber disc. Another treatment contained tile brominated samples to which were added lipids with an eicosapolyenoic acid content equal to that found in the untreated sporangia. In a third treatment, pure AA and EPA were added to the brominated samples at concentrations equivalent to those present in total sporangial lipids. Brominated cystospore and zoospore preparations were assayed for elicitor activity with and without exogenous AA. One hundred micrograms of treated spore tissue were added to each disc. T h e AA concentration was I0 lag per disc. In experiments with the fungi non-pathogenic on potato, 100 lal containing 1 mg of a suspension oftreated or untreated mycelium was applied to each potato disc.
RESULTS
Concentration of EPA and AA in P. infestans spores Similar to their abundance in mycelium [1-3], EPA and AA were abundant fatty acids in sporangia from all races ofP. infestans examined (Tables 1 and 2). The predominant ~icosapolyenoic acid was EPA. There was little variation among three races in the concentration ofelicitor fatty acids (Table 1). Race 0 zoospore and cystospore tissues had
Phytoalexin elicitor activities of P. infestans spores
53
TABLE 1
Total concentrations(Itg rag- t dry weight) of arachidonicand e#osapentaenoicacids in sporangia of three rates ofPhytophthora infestans" Race Race4 Racel.4 Racel.2.3.4
AA 7.84-0-6 7"54-0.2 7.04-0.2
EPA 29-44-2-0 24.44-0.8 27-24-1"1
AA+EPA 37.2±2.4 31-9±1-0 34.2±1.3
aEach value is the mean and standard error obtained from at least three determinations.
TABLE2 Arachidonic and eicosapentaenoicacid concentrations (fig rag-1 dry weight) in different spore types of Phytophthora infestans raceO" Spore type
AA
EPA
AA+ EPAb
Sporangia Zoospores Cystospores
2.74-0.4 2"04-0.4 1"44-0.4
31.4+8.4 !8-5 4-5.9 14"44-4"2
34-1 y 20"5z 15-8 z
"Values are the means and standard errors obtained from at least three experiments. bValues followedby the same letter are not significantlydifferent, Duncan's multiple range test (P= 0-05).
similar concentrations of E P A and AA, whereas sporangia had significantly higher concentrations of the elicitor fatty acids (expressed on a per milligram basis). Effect of bromine on the elicitor activi{y of sporangia, zoospores and cystospores Bromine completely eliminated the unsaturated fatty acids in lyophilized sporangia of race 0 o f P . infestans (Fig. 1) and abolished their sesquiterpenoid phytoalexin elicitor activity (Table 3). Addition ofmycelial lipids from race 0 or pure A A and EPA restored 76 and 55%, respectively, o f the elicitor activity of the sporangia treated with bromine. W h e n applied alone to the discs, ttle lipid extract and elicitor fatty acids contained 38% and 22%, respectively, of the elicitor activity present in the lyophilized sporangia. Bromine also abolished the elicitor activity in zoospore and cystospore tissue (Table 4). Addition o f b r o m i n a t e d spore tissue to pure AA significantly enhanced rishitin and lubimin levels. Although cystospores elicited somewhat higher levels of the phytoalexins than zoospores, this effect was not apparent after treatment with bromine and addition of AA. T r e a t m e n t of the partially purified glucan with bromine did not significantly reduce its enhancement o f AA elicitor activity (t-test, P = 0 - 0 5 ) . Rishitin and lubimin accumulations were 3324_37 lagg -1 fresh weight in discs treated with the g l u c a n + A A and 265 4-45 lag g - x fresh weight in discs treated with the brominated glucan + AA.
54
J. R. Creamer and R. M. Bostock (a)
!
(b)
EPA
1
I g
~ i
0
1
i
2
4
:
(3
,
i
8
DO 12 0 2 Retention time (min}
4
6
8
I0
12
Flo. 1. Gas-liquid chromatograms showing fatty acid methyl ester profiles from (a) Ph)'lophlhora infestans race 0 sporangia; (b) P. infestans sporangia treated with bromine; (c) Ceratocystisfimbriata mycelium; and (d) Bipolaris carbonum mycelium. Peaks corresponding to arachidonic (AA) and eicosapentaenoic (EPA) acids are indicated.
Effect of different methods of sterilization on the elicitor activity of tissues o f t . fimbriata and B. carbonum Spores of C.fimbriata and B. carbonum elicited rishitin accumulation in tuber discs. Only viable tissues of C.fimbriata and B. carbonum elicited the response (Table 5). All three methods of inactivation--heat, treatment with ether or treatment with propylene oxide---abolished the elicitor activity oftheir tissues. Fatty acid composition ofC. fimbriata and B. carbonum Mycelia of C. fimbriata and B. carbonum did not contain AA or EPA (Fig. 1). The predominant fatty acids in these fungi were palmitic, oleic and linoleic acids. DISCUSSION
Our results provide additional support for the hypothesis that eicosapolyenoic acid lipids play an important role in elicitation ofthe HR by tissues ofP. infestans. Our results further
Phytoalexin elicitor activities of P. infestans spores
55
TABLE 3
Sesquiterpenoid phytoalexin elicitor actidties ol'killed sporangia of Phytophthora infestans race 0 before and after treatment u'ith bromine Elicitor activity (% ofuntreated sporangia) b
Treatment* Sporangia Sporangia + bromine Sporangia + bromine + P. infestans lipids Sporangia + bromine + EPA + AA P. infestans lipids EPA+AA Water
100 v 0z 76 w 55 x 38 y 22 y 0
*Sporangia were killed by freezing and lyophilizing. T h e y were then exposed to bromine as described in materials and methods. P. infestans lipids containing EPA and AA or pure EPA and AA were added to sporangial tissues equivalent to their concentrations in untreated sporangia. This corresponded to approximately 34.1 lag EPA + AA rag- 1 dry weight sporanglal tissue (Table 2). Sample containing ! mg dry weight sporangia with or without additional components as indicated was applied to each potato disc. bValues were derived from rishitin and lubimin accumulations 96 h after treatment and are expressed as the percentage of the accumulations induced by killed sporangia not treated with bromine. This corresponds to 520__. 28 lag g - l fresh weight. Values for sesquiterpene accumulations were derived from nine determinations from three experiments. Values followed by the same letter indicate the treatments did not differ significantly, Duncan's multiple range test (P=0.05). Values for the water control were not included in the analysis since sesquiterpcne phytoalexins were not detected in any sample.
TABLE 4
Sesqulterpenoidph)'toalexin elicitor activities of killed zoospores and c.)'stospores ofPhytophthora infestans race 0 before and after treatment with bromine
Treatment* Cystospores Zoosporcs Cystospores + bromine Zoospores + bromine AA Cystospores + bromine + AA Zoospores + bromine + AA Water
Rishitin + Lubimin b (lag g - 1 fresh weight) 258 x 188 y 0 0 57z 161 y 141 y 0
~Spore preparations were frozen and lyophilized. Portions were treated with bromine as described in materials and methods. Approximately 100 lag of brominated or untreated spores in water were applied to tbe upper surface ofeach potato disc. Approximately 10 lag AA were applied to each disc in treatments using AA. bEach value is ttle mean of rishitin+lubimin accumulations 96 h after treatment from at least nine determinations from two experiments. Values followed by tbe same letter are not significantly different, Duncan's multiple range test (P=0.05). Values for the brominated spores and water control were not included in the analysis since sesquiterpenes were not detected in any ofthese samples.
J. R. Creamer and R. M. Bostock
56 TABLE5
Effects of various treatments on the dability and elicitor activity of tissues of Ceratocystis fimbriata and Bipolaris carbonum"
Fungus C.fimbriata
B. carbonum
Treatments b
tiypersensitive response and sesquiterpene accumulation
Growth in culture
A
+
+
AB AC AD E
--+
---+
A
+
+
AB AC AD
----
----
aResults were obtained from at least three experiments of each treatment. +indicates response observed in all samples in all experiments; --indicates response not observed. Vl'reatment codes: A=sonicated in water; B=autoclaved for 15 min; C=treated with diethyl ether; D=treated with propylene oxide; E=repeated freezing and thawing. In all treatments, 100 ttl containing 1 mg of a suspension of treated myeelia and conidia were applied to each potato disc. See Materials and *lethods for further details.
indicate that tile levels of eicosapolyenoic acid lipids in sporangia are sufficient to account for the major portion of their elicitor activity, provided that the enhancing activity of the other components is included. Bromine abolished the elicitor activity of spores and provided a relatively simple method for completely eliminating detectable levels o f E P A , AA and the other unsaturated fatty acids, avoiding losses that occur in the residual material during solvent extraction and subsequent transfers. Addition o f the lipid mixture to brominated sporangia restored approximately 76% of their elicitor activity. Addition o f p u r e EPA and AA restored 55% o f their activity, a result suggesting that the lipid mixture contains factors, in addition to E P A and AA, that contribute to elicitor activity. O n e possible factor is homo-y-linolenic acid (cis-8,11,14-eicosatrienoic acid), which is present in small amounts in lipids ofP. infestans and is a fatty acid which has significant elicitor activity in ttle presence ofmycelial glucans [6, 8, 15, 18]. Another possibility is the formation ofliposomes by phospholipids during sonication, structures which could a u g m e n t the dispersion o f s o m e elicitor molecules (e.g. neutral lipid containing AA and EPA). Liposomes might also present the elicitors in a form more readily taken up by potato cells or could protect them from degradation by extracellular enzymes [7]. T h e reconstitution experiments indicate that the bromine treatment abolished a portion (approximately 24%) of the elicitor activity in sporangia that was not recovered upon addition of lipid. Bromine could oxidize glucan oligomers forming a gluconic acid residue at the reducing end, or modify other nonlipid components, some o f w h i c h m a y coutribute to H R induction. Nevertheless, this treatment did not significantly alter the enhancing activity of the partially purified glucan preparation. Further research is necessary to determine the precise structural features in glucan oligomers
Phytoalexin elicitor activities of P. infestans spores
57
required for enhancing eicosapolyenoic acid-elicitor activity. Our experiments also confirm that cystospores and zoospores contain components which significantly enhance AA elicitor activity with properties distinct from unsaturated lipid. It is likely that these components are fl-glucans which occur in all spore stages and in mycelium of Phytophthora spp. [24]. The relatively constant levels of EPA and AA during encystment ofzoospores appear to rule out the possibility that changes in the levels of these acids account for the increased elicitor activity ofcystospores relative to zoospores, reported by Henfling et al. [12] and also observed to some extent in our study. Such differences might be attributed to the rapid synthesis ofglucan enhancers or induction 0fspecific conformational changes in the mycolaminarans during encystment [20, 21]. Our results also support those of Lisker & Ku~ [16] and Zook & Ku6 [26] which suggested that the induction of sesquiterpenoid accumulation and the H R in potato tuber can occur via different mechanisms, although the specific nature ofsuch mechanisms remains elusive. The absence ofeicosapolyenoic acids in tissues of C.fimbriata and B. carbonum and the fact that their tissues must be viable for elicitation support the case for multiple mechanisms. These investigators observed that autoclaving or ethanol abolished the elicitor activity ofB. carbonum. In this paper, we have shown that two other methods of inactivation--treatment with ether or propylene oxide---also eliminate elicitor activity in both B. carbonum and C. fimbriata. This is in marked contrast to P. ilfestans and other Oomycctcs reported to contain eicosapolyenoic acids [25] in which killing does not eliminate elicitor activity [16]. We conclude that the evidence presented here and elsewhere [1-4, 8, 17, 18] weighs strongly in favor of a significant role for eicosapolyenoic acids in the elicitor activity of P. infestans tissues. Although inoculation with living spores from an incompatible race elicits a stronger reaction in tuber discs than treatment with killed spores [12], the higher elicitor activity of living spores may arise from an increase in fungal biomass during ingress and formation ofspecialized infection structures that efficiently deliver elicitors to plant cell receptors. It is also possible that elicitors different than the eicosapolyenoic acids are produced during the interaction between living spores and potato tissue similar to the specific elicitors described in the Cladosporiumfidvum-tomato interaction [11]. Biochemical mutants ofP. infestans deficient in EPA and AA would provide an additional critical test of the role of these compounds in elicitation, but it is unlikely that such mutants could be obtained given the gross disruption of membrane structure expected after removal ofsuch abundant fatty acids [3, 8]. Perhaps a more useful approach will be to determine ifa common reactive intermediate is formed by potato cells in response to different elicitors. Nevertheless, the eicosapolyenoic acids provide a significant opportunity for obtaining information about the participation of specific microbial lipids in signal transduction mechanisms in plant-pathogen interaction, and are useful in studies of regulation of isoprcnoid metabolism during hypersensitivity expression [19]. This work was supported in part by National Science Foundation Grant PCM8308563, a faculty research grant from the University of California, Davis, and a crop science fellowship to J.R.C. from Chevron Chemical Company, Richmond, California.
58
J.R. Creamer and R. M. Bostock
REFERENCES 1. BOSTOCK,R. M. (1981). The identification of polyunsaturated fatty acids from Phytophthora infeslans as elicitors ofhypersensitivity in potato tuber and studies ofrelated terpenoid metabolism. Ph.D. Thesis, University of Kentucky. Lexington, Kentucky, U.S.A. 2. Bos-rocK, R. M., Kud, J. A. & LAt.~E, R. A. (1981). Eicosapentaenoie and arachidonic acids from Ph.)'tophthora infestans elicit fungitoxic sesquiterpenes in the potato. Sdence 212, 67-69. 3. BosTocK, R. M., LAL~E, R. A. & Ku+,J. A. (1982). Factors affecting the elicitation ofsesquiterpenold phytoalexin accumulation by eicosapentaenoic and arachidonic acids.in potato. Plant Physiology 70, 1417-1424. 4. BOSTOCK,R. M., SCIIAEFFER,D. A. & HAMMERSCHMIDT,R. (1986). Comparison of elicitor activities of arachidonic acid, fatty acids and glucans from Ph)tophthora infestans in hypersensitivity expression in potato tuber. Physiological and ~ lolecular Plant Pathology 29~ 349-360. 5. BaucI<, R. I., FRY, W. E. & APet.E, A. E. (1980). Use of metalaxyl, an acylalanlne fungicide, on developmental stages ofPhytophthora infestans. Phytopathology 70, 597-601. 6. BRYAN, I. B., RA'rn.~tELt.,W. G. & Fm~.'~D,J. (1985). The role ofllpid and non-lipid components of Ph)'tophthora infestans in the elicitation of the hypersensitive response in potato tuber tissue. Physiological Plant Pathology 26, 331-355. 7. CttA.~cE, M. L. & NEw, R. R. C. (1984). Enhancement ofefScacy of antifungal agents by entrapment inside liposomes. In Mode of action ofantifungal agents, Ed. by A. P.J. Trinci & J. F. Ryley, pp. 377-385. Cambridge University Press, Cambridge. 8. CRE,~,.~mR,J.R. & BOSTOCK,R. M. (1986). Characterization and biological activity ofphospholipids from Ph.)'tophthora infestans in the hypersensitive response of potato tuber. Physiological and Molecular Plant Pathology 28, 215-225. 9. CURR:ER, W. W. (1974). Characterization ofthe induction and suppression of terpenoid accumulation in the potato-Ph.)tophthora infestans interaction. Ph.D. Thesis, Purdue University, Lafayette, IN, U.S.A. 10. DARVttL, A. G. & ALBERSHE:.~X,P. (1984). Phytoalexins and their elicitors--a defense against microbial infection in plants. Annual Review of Plant Physiology 35, 243-275. !1. DE WIT, P . J . G . M . & SrlK.~:A.~,G. (1982). Evidence for the occurrence of race and cuhivar-specifie elicitors of necrosis in intercellular fluids of compatible interactions of Cladosporiumfulvum and tomato. Physiological Plant Pathology 21, 1-I 1. 12. HE.~rH.~o,J. W. D. M., BOSTOCK,R. M. & Kud,J. (1980). Cell walls ofPhytophthom infestans contain an elicitor ofterpene accumulation in potato tubers. Phytopathology 70, 772-776. 13. HE.~rLIyC,J. W. D. M. & Ku(:,J. (1979). A semi-micro method for the quantitation ofsesquiterpenoid stress metabolites in potato tuber tissue. Phytopathology 69, 609-612. 14. KEL'~AY,P., BRYAN,I. B. & FRIL'~D,J. (1985). The elicitation ofthe hypersensitive response ofpotato tuber tissue by a component of the culture filtrate of Phytophthora infestans. Physiological Plant Pathologv 26, 343-355. 15. KURA.~TZ,M.J. & OSMAY,S. F. (1983). Class distribution, fatty acid composition, and elicitor activity of Phytophthora infestans mycelial lipids. Physiological Plant Pathology 22s 363-370. 16. L:SKER,N. & Ku~:,J. (1977). Elicitors ofterpenoid accumulation in potato tuber slices. Phytopathology 67, 1356-1359. 17. MANIARA, G., LAI.~E, R. & Ku6, J. (1984). Ollgosaccharides from Phytophthora infestans enhance the elicitation ofsesquiterpeuold stress metabolites by arachidonie acid in potato. PhysiologicalPlant Pathology 2'1, 177-186. 18. PREIStG,C. L. & Kcd, J. A. (1985). Arachidonic acid-related elicitors of the hypersensitive response in potato and enhancement of their activities by glucans from Phytophthom infestans (Mont.) de Bary. Archives of Biochemistry and Biophysics 236, 379-389. 19. STER.~tER, B. A. & BOSTOCK, R. M. (1987). Involvement of 3-hydroxy-3-methylglutaryl coenzyme A reductase in the regulation of sesquiterpenoid phytoalexin synthesis in potato. Plant Physiology, B4, 404-408. 20. TOKU.~AOA,j . & BARTmCKt-GARcXA,S. (1971). Cyst wall formation and endogenous carbohydrate utilization during synchronous encystment of Ph.ytophthora dnnamomi zoospores. Archives of Microbiology 79, 283-292. 21. TOKU.~AG^, J. & BAWr,'qCKt-GARctA, S. (1971). Structure and differentiation of the cell waU of Phytophthorapalmivora cysts, hyphae, and sporangia. Archives of Microbiology 79, 293-310. 22. VAR.~S,J. L., Ku~,J. & WtLLtA.~tS,E. B. (1971). Terpenoid accumulation as a biochemical response ofthe potato tuber to Phytophthora infestans. Phytopathology 61, 174-177. 23. WALK~R, B. L. (1975). Structural determination of liplds by chemical means. In Analysis of Lipids and Lipoproteins, Ed. by E. G. Perkins, pp. 108-122. American Oil Chemists' Society, Champaign, USA.
Phytoalexin elicitor activities of P. infestans spores
59
24. WA.~O,M. C. & BARTNICKI-GARCIA,S. (1980). Distribution ofmycolaminarans and cell wallfl-glucans in the life cycle of Ph.ytophthora. ExperimentalMycology4, 269-280. 25. WAssEF, M. K. (1977). Fungal liplds. In Advances in Lipid Research, Volume 15, Ed. by R. Paoletti & D. Kritchevsky, pp. 159-232. Academic Press, New York. 26. ZOOK, M., & Kc6, J. (1984). Etlcitatlon ofsesqulterpenoid stress metabolites in potato tuber slices by Hdminthosporium earbonum. Phytopathology74, 849 (Abstr.).
Contribution of eicosapolyenoic fatty acids to the sesquiterpenoid phytoalexin elicitor activities of Phytophthora infestans spores J.
R. CREAMER a n d R . M . BOSTOCKi-
Departmentof Plant Patholog)',Universityof California, Davis, California95616, U.S.A. (Acceptedfor publieationMay 1987)
The concentrations of arachidonic (AA) and eicosapentaenoic (EPA) acids in lyophilized sporangia from isolates representing four races ofPh)'tophthorainfestansand in lyophilized zoospores and newly-formed cystospores of an isolate of race 0 were determined. The predominant eicosapolyenoic acid was EPA, and the levels of EPA and AA were similar in all four isolates. In the race 0 isolate, the total concentration of EPA and AA was greater in sporangial tissue than in tissues from zoospores or cystospores. Treatment of spores with bromine eliminated AA, EPA and other unsaturated fatty acids and aboIished phytoalexin elicitor activity. Addition of a mixture ofllplds from P. infestans containing free and esterified EPA and AA or of pure eicosapolyenoic acids to brominated sporangia restored 76 and 55%, respectively, of the elicitor activity present in untreated sporangia. The elicitor activities of these mixtures were significantly greater than the activities of the lipids or free fatty acids. Bromine treatment also abolished the elicitor activities of zoospore and cystospore tissues, and the treated tissues retained the capability to significantly enhance Ark elicitor activity. The results demonstrate a requirement of unsaturated lipid in spore tissues ofP. infestansfor elicitor activity and provide evidence that the levels ofelcosapolyenolc acids in lyophilized sporangia ofP. infestansare sufficient to account for the major portion oftheir elicitor activity, provided that the components which enhance fatty acid elicitor activity are also present.
INTRODUCTION T h e p o l y u n s a t u r a t e d fatty acids, a r a c h i d o n i c (AA) a n d e i c o s a p e n t a e n o i c (EPA) acids, are a b u n d a n t c o m p o n e n t s in tissue lipids o f Pto'tophthora infestans a n d b o t h acids are efficient elicitors o f s e s q u i t e r p e n o i d p h y t o a l e x i n s a n d o t h e r reactions associated with the h y p e r s e n s i t i v e response ( H R ) in p o t a t o t u b e r [1, 4]. Previous studies clearly d e m o n s t r a t e d a r e q u i r e m e n t for these a n d related eicosapolyenoie or d o c o s a p o l y e n o i c acids in lipids for tile elicitation o f t h e response in potato, a n d the s t r u c t u r a l features in fatty acids necessary for activity h a v e b e e n identified [1-3, 18]. T i l e elicitor activities o f these fatty acids a n d acyl lipids c o n t a i n i n g t h e m are e n h a n c e d b y b r a n c h e d fl-glucans, also f o u n d in extracts o f f u n g a l tissue. H o w e v e r , these g l u c a n s a l o n e a r e i n a c t i v e as elicitors o f s e s q u i t e r p e n o i d p h y t o a l e x i n s in p o t a t o [4, 17, 18]. T h e physiological significance o f t h e fatty acids as i m p o r t a n t elicitors o f t h e H R has b e e n q u e s t i o n e d [10] b e c a u s e the threshold c o n c e n t r a t i o n o f A A or E P A for sesquiterAbbreviations used in text: AA, arachldonie acid; EPA, eicosapentaenoic acid; FAME, fatty acid methyl esters; HR, hypersensitive response. "['Towhom correspondence should be addressed. 0885-5765/88]010049+ 11 S03.00/0 © 1988 Academic Press Limited
50
J.R. Creamer and R. M. Bostock
penoid phytoalexin accumulation in potato tuber (cv. Kennebec) is approximately 16 nmol when applied to tile upper surface of a disc and maximum sesquiterpenoid accumulations are achieved with approximately 330 nmol or more. The latter corresponds to the concentration of these acids (free and esterified) in approximately 5 mg of lyophilized mycelium [1, 3]. It therefore seems unlikely that comparable levels ofelicitor acids would be available in the incompatible spore-plant tissue combinations typically used by investigators, although the concentrations of these elicitors in spores have not been reported. Tile contribution of the fl-glucans appear to be important in this regard for their marked enhancement of fatty acid elicitor activity [17, 18]. Furthermore, the glucans reveal elicitor activity with concentrations of eicosapolyenoic acids that are below tile tbreshold of induction ofdetectable levels ofsesquiterpenes. The role ofthe fatty acids as elicitors is complicated by the diversity of fractions from P. infestans mycelium capable of eliciting tile response [1-3, 6, 9]. Extraction with a mixture of chloroform and methanol removes a significant portion of elicitor activity from fungal tissue but leaves a small amount of elicitor acids and a considerable amount of elicitor actMty in the residual material. Nevertheless, AA and EPA were found free or bound in all fractions with elicitor activity, although they were present at low or trace levels in some complex fractions, including insoluble cell wall preparations [3]. Another report suggests that there may be elicitors ofsesquiterpenoid phytoalexins devoid of AA and EPA in filtrates of senescent cultures of P. bfestans [14]. However, the activity of these components relative to other fractions from mycelia is very low. Evidence for alternative elicitors ofthe response in potato has been presented by Zook & Kuc [26] and may also be inferred from the elicitor activity associated with living but not autoclaved or etbanol-treated tissues ofBipolaris carbonumand other fungi nonpathogenic to potato [16]. In general, members of the higher fungi (Ascomycetes, Basidiomycetes and Fungi Imperfecti) do not contain AA or EPA, although AA was detected in minor amounts in the fatty acids from two Penicillium spp. [25]. In the light of these observations, we believed it important to re-examine the relationship of tbe eicosapolyenoic acids to the elicitor activity in spores ofP. infestans and cultures of certain eliciting fungi nonpathogenic on potato. In this study we provide precise information on the levels of EPA and AA in P. infestans spores and present further evidence for the requirement of eicosapolyenoic acid lipids in the elicitor activity oflyophilized sporangia, cystospores and zoospores. We describe a method for the inactivation of the eicosapolyenoic acids in tissues ofP. infestans with bromine which circumvents tile limitations encountered when attempting their complete removal by solvent extraction. We also determined the fatty acid composition ofmycelia of Ceratocystisfimbriata and Bipolaris carbonum, which elicit a strong HR, and examined the effect ofseveral methods of attenuation of these fungi on the elicitor actMty of their tissues. MATERIALS AND METHODS
Fungal culture Phytophthora infestans (Mont.) de Bary races 0, 4, 1.4 and 1.2.3.4 were maintained on amended lima bean agar [5] at 20 °C for 14 days. An isolate of Ceratocystisfimbriata El 1. and Halst. pathogenic oll almond (Prunus dulcis (Mill.) Webb) was obtained by hyphal tip culture from a bark canker. A culture ofBipolaris carbonum (Ullstrup) Shoemaker was
Phytoalexin elicitor activities of P. infestans spores
51
a gift of E. E. Butler of our Department. These isolates were maintained on potato dextrose agar (PDA, Difco) at room temperature. Sporangia, zoospores and cystospores ofP. infestans were obtained from cultures as described previously [12]. Sporangial suspensions in sterile deionized water were collected from cultures, passed through two to four layers of sterile cheesecloth and washed with sterile deionized water over filter paper (Whatman No. 1). Zoospores were obtained from sporangia incubated in sterile distilled water for 2-3 h at 10 °C. They were collected by filtering ttle suspension through a nylon mesh filter (15 [am mesh) or by carefully decanting the supernatant and allowing the sporangia to sediment for 20-30 min, centrifuging at 3000 r min -1 for 1 min, and removing the supernatant. Cystospores were formed by agitating a suspension ofzoospores in a test tube on a Vortex mixer for 2 min [12]. The suspensions ofzoospores and cystospores were evaluated for concentration and purity with a light microscope. For fatty acid analyses, spore suspensions were immediately frozen in a dry ice/acetone bath, lyophilized and weighed. In the experiments testing the elicitor activities ofzoospores and cystospores, the spore suspensions were frozen immediately in liquid nitrogen and lyophilized. For race 0, 5.4 x 10 s lyophilized sporangia weighed approximately 1 mg (mean of nine determinations from three preparations). Non-viability of spores was confirmed by absence of growth on amended lima bean agar. Mycelia ofB. carbonum and C.fimbriata were collected from 10-day-old shake cultures in 500-ml flasks of potato dextrose broth (PDB, Difco). The mycelia were collected on a colander through four layers of cheesecloth, rinsed with deionized water, lyophilized, ground to fine fragments using a mortar and pestle, and weighed. Mycelial fragments were transferred onto PDA (C. fimbriata) or cornmeal agar (B. carbonum) after the different treatments to determine viability.
Attenuation offi~ngal tissue The following treatments were examined to determine their effect on the viability and elicitor activity of C.fimbriata and B. carbonum tissues. Lyophilized mycelium and conidia were (1) sonicated in water for 5 min at maximum intensity (treatment A); (2) autoclaved for I0 min (treatment B); (3) exposed to 2 ml of diethyl ether, the solvent evaporated and sterile water added (treatment C); (4) exposed to propylene oxide, the gas allowed to dissipate and sterile water added (treatment D); or (5) frozen in water in a dry ice/acetone bath and thawed (three cycles, treatment E). Combinations of these treatments were also tested (see Table 4).
Fatty acid analysis Lyophilized tissues were treated with 1 N methanolic-HC1 for 2 h at 100 °C, cooled and the fatty acid methyl esters (FAME) extracted three times with hexane [2]. The FAME were analysed by GLC using a 1.8 m x 2 mm i.d. glass column packed with 10% SP-2330 on Chromosorb W AW (Supelco) programmed at 150-250 °C at 10 °C m i n - l [2]. Methyl esters ofstandard fatty acids were used for identification and quantitation, with methyl arachidate or methyl heptadecanoate as an internal standard. Authentic lipids were purchased from Sigma.
52
J.R. Creamer and R. M. Bostock
Treatment 0fPhytophthora infestans spores with bromine Since bromine is a powerful nucleophile which readily adds to double bonds of unsaturated fatty acids [23], we used it to eliminate EPA and AA in killed spores. Samples containing 3-24 mg of lyophilized sporangia, zoospores or cystospores were mixed with 2 ml ofcarbon tetrachloride and 50-100 lal of bromine (Sigma) in a capped test tube and incubated for 24-48 h at 45 °C. The solvent was removed under nitrogen and the tubes were left uncapped for 48 h to allow residual bromine to dissipate. Analysis of FAME from the brominated sporangia confirmed the elimination of all unsaturated fatty acids. The brominated spores were suspended in the same test tube by sonication in sterile water with or without additional elicitor lipids. T o determine the effect ofbromine on the enhancing activity offungal glucans, a glucan preparation from mycelium ofrace 0 ofP. infestans was obtained as described previously [4] and treated with bromine. T h e concentrations of glucan and AA applied to each potato disc were 100lag (glucose equivalents) and 50 lag, respectively.
Elicitor activity assay Elicitor activities of samples were determined with cv. Kennebec potato tuber discs as described in previous publications [2, 3, 13]. A 50-100 lal sample of a treatment suspension was applied uniformly to the upper surface o f a 2 cm diameter tuber disc. T u b e r discs were visually rated for hypersensitive browning 96 h after treatment and the accumulations of the sesquiterpenoid phytoalexins, rishitin and lubimin, were determined by T L C [22] or by G L C [13]. Ph.ytophthora infestans lipids were obtained by extracting mycelia with a mixture of chloroform and methanol (2:1, by volume) and nonlipid contaminants were removed with 0.75% aqueous KCI [2]. Tile class distribution, fatty acid composition and elicitor activity ofP. infestans lipids have been reported elsewhere [8, 15]. In one treatment, sporangial tissue equivalent to approximately 5.4 x 10 s sporangia (I mg) was added to each tuber disc. Another treatment contained tile brominated samples to which were added lipids with an eicosapolyenoic acid content equal to that found in the untreated sporangia. In a third treatment, pure AA and EPA were added to the brominated samples at concentrations equivalent to those present in total sporangial lipids. Brominated cystospore and zoospore preparations were assayed for elicitor activity with and without exogenous AA. One hundred micrograms of treated spore tissue were added to each disc. T h e AA concentration was I0 lag per disc. In experiments with the fungi non-pathogenic on potato, 100 lal containing 1 mg of a suspension oftreated or untreated mycelium was applied to each potato disc.
RESULTS
Concentration of EPA and AA in P. infestans spores Similar to their abundance in mycelium [1-3], EPA and AA were abundant fatty acids in sporangia from all races ofP. infestans examined (Tables 1 and 2). The predominant ~icosapolyenoic acid was EPA. There was little variation among three races in the concentration ofelicitor fatty acids (Table 1). Race 0 zoospore and cystospore tissues had
Phytoalexin elicitor activities of P. infestans spores
53
TABLE 1
Total concentrations(Itg rag- t dry weight) of arachidonicand e#osapentaenoicacids in sporangia of three rates ofPhytophthora infestans" Race Race4 Racel.4 Racel.2.3.4
AA 7.84-0-6 7"54-0.2 7.04-0.2
EPA 29-44-2-0 24.44-0.8 27-24-1"1
AA+EPA 37.2±2.4 31-9±1-0 34.2±1.3
aEach value is the mean and standard error obtained from at least three determinations.
TABLE2 Arachidonic and eicosapentaenoicacid concentrations (fig rag-1 dry weight) in different spore types of Phytophthora infestans raceO" Spore type
AA
EPA
AA+ EPAb
Sporangia Zoospores Cystospores
2.74-0.4 2"04-0.4 1"44-0.4
31.4+8.4 !8-5 4-5.9 14"44-4"2
34-1 y 20"5z 15-8 z
"Values are the means and standard errors obtained from at least three experiments. bValues followedby the same letter are not significantlydifferent, Duncan's multiple range test (P= 0-05).
similar concentrations of E P A and AA, whereas sporangia had significantly higher concentrations of the elicitor fatty acids (expressed on a per milligram basis). Effect of bromine on the elicitor activi{y of sporangia, zoospores and cystospores Bromine completely eliminated the unsaturated fatty acids in lyophilized sporangia of race 0 o f P . infestans (Fig. 1) and abolished their sesquiterpenoid phytoalexin elicitor activity (Table 3). Addition ofmycelial lipids from race 0 or pure A A and EPA restored 76 and 55%, respectively, o f the elicitor activity of the sporangia treated with bromine. W h e n applied alone to the discs, ttle lipid extract and elicitor fatty acids contained 38% and 22%, respectively, of the elicitor activity present in the lyophilized sporangia. Bromine also abolished the elicitor activity in zoospore and cystospore tissue (Table 4). Addition o f b r o m i n a t e d spore tissue to pure AA significantly enhanced rishitin and lubimin levels. Although cystospores elicited somewhat higher levels of the phytoalexins than zoospores, this effect was not apparent after treatment with bromine and addition of AA. T r e a t m e n t of the partially purified glucan with bromine did not significantly reduce its enhancement o f AA elicitor activity (t-test, P = 0 - 0 5 ) . Rishitin and lubimin accumulations were 3324_37 lagg -1 fresh weight in discs treated with the g l u c a n + A A and 265 4-45 lag g - x fresh weight in discs treated with the brominated glucan + AA.
54
J. R. Creamer and R. M. Bostock (a)
!
(b)
EPA
1
I g
~ i
0
1
i
2
4
:
(3
,
i
8
DO 12 0 2 Retention time (min}
4
6
8
I0
12
Flo. 1. Gas-liquid chromatograms showing fatty acid methyl ester profiles from (a) Ph)'lophlhora infestans race 0 sporangia; (b) P. infestans sporangia treated with bromine; (c) Ceratocystisfimbriata mycelium; and (d) Bipolaris carbonum mycelium. Peaks corresponding to arachidonic (AA) and eicosapentaenoic (EPA) acids are indicated.
Effect of different methods of sterilization on the elicitor activity of tissues o f t . fimbriata and B. carbonum Spores of C.fimbriata and B. carbonum elicited rishitin accumulation in tuber discs. Only viable tissues of C.fimbriata and B. carbonum elicited the response (Table 5). All three methods of inactivation--heat, treatment with ether or treatment with propylene oxide---abolished the elicitor activity oftheir tissues. Fatty acid composition ofC. fimbriata and B. carbonum Mycelia of C. fimbriata and B. carbonum did not contain AA or EPA (Fig. 1). The predominant fatty acids in these fungi were palmitic, oleic and linoleic acids. DISCUSSION
Our results provide additional support for the hypothesis that eicosapolyenoic acid lipids play an important role in elicitation ofthe HR by tissues ofP. infestans. Our results further
Phytoalexin elicitor activities of P. infestans spores
55
TABLE 3
Sesquiterpenoid phytoalexin elicitor actidties ol'killed sporangia of Phytophthora infestans race 0 before and after treatment u'ith bromine Elicitor activity (% ofuntreated sporangia) b
Treatment* Sporangia Sporangia + bromine Sporangia + bromine + P. infestans lipids Sporangia + bromine + EPA + AA P. infestans lipids EPA+AA Water
100 v 0z 76 w 55 x 38 y 22 y 0
*Sporangia were killed by freezing and lyophilizing. T h e y were then exposed to bromine as described in materials and methods. P. infestans lipids containing EPA and AA or pure EPA and AA were added to sporangial tissues equivalent to their concentrations in untreated sporangia. This corresponded to approximately 34.1 lag EPA + AA rag- 1 dry weight sporanglal tissue (Table 2). Sample containing ! mg dry weight sporangia with or without additional components as indicated was applied to each potato disc. bValues were derived from rishitin and lubimin accumulations 96 h after treatment and are expressed as the percentage of the accumulations induced by killed sporangia not treated with bromine. This corresponds to 520__. 28 lag g - l fresh weight. Values for sesquiterpene accumulations were derived from nine determinations from three experiments. Values followed by the same letter indicate the treatments did not differ significantly, Duncan's multiple range test (P=0.05). Values for the water control were not included in the analysis since sesquiterpcne phytoalexins were not detected in any sample.
TABLE 4
Sesqulterpenoidph)'toalexin elicitor activities of killed zoospores and c.)'stospores ofPhytophthora infestans race 0 before and after treatment with bromine
Treatment* Cystospores Zoosporcs Cystospores + bromine Zoospores + bromine AA Cystospores + bromine + AA Zoospores + bromine + AA Water
Rishitin + Lubimin b (lag g - 1 fresh weight) 258 x 188 y 0 0 57z 161 y 141 y 0
~Spore preparations were frozen and lyophilized. Portions were treated with bromine as described in materials and methods. Approximately 100 lag of brominated or untreated spores in water were applied to tbe upper surface ofeach potato disc. Approximately 10 lag AA were applied to each disc in treatments using AA. bEach value is ttle mean of rishitin+lubimin accumulations 96 h after treatment from at least nine determinations from two experiments. Values followed by tbe same letter are not significantly different, Duncan's multiple range test (P=0.05). Values for the brominated spores and water control were not included in the analysis since sesquiterpenes were not detected in any ofthese samples.
J. R. Creamer and R. M. Bostock
56 TABLE5
Effects of various treatments on the dability and elicitor activity of tissues of Ceratocystis fimbriata and Bipolaris carbonum"
Fungus C.fimbriata
B. carbonum
Treatments b
tiypersensitive response and sesquiterpene accumulation
Growth in culture
A
+
+
AB AC AD E
--+
---+
A
+
+
AB AC AD
----
----
aResults were obtained from at least three experiments of each treatment. +indicates response observed in all samples in all experiments; --indicates response not observed. Vl'reatment codes: A=sonicated in water; B=autoclaved for 15 min; C=treated with diethyl ether; D=treated with propylene oxide; E=repeated freezing and thawing. In all treatments, 100 ttl containing 1 mg of a suspension of treated myeelia and conidia were applied to each potato disc. See Materials and *lethods for further details.
indicate that tile levels of eicosapolyenoic acid lipids in sporangia are sufficient to account for the major portion of their elicitor activity, provided that the enhancing activity of the other components is included. Bromine abolished the elicitor activity of spores and provided a relatively simple method for completely eliminating detectable levels o f E P A , AA and the other unsaturated fatty acids, avoiding losses that occur in the residual material during solvent extraction and subsequent transfers. Addition o f the lipid mixture to brominated sporangia restored approximately 76% of their elicitor activity. Addition o f p u r e EPA and AA restored 55% o f their activity, a result suggesting that the lipid mixture contains factors, in addition to E P A and AA, that contribute to elicitor activity. O n e possible factor is homo-y-linolenic acid (cis-8,11,14-eicosatrienoic acid), which is present in small amounts in lipids ofP. infestans and is a fatty acid which has significant elicitor activity in ttle presence ofmycelial glucans [6, 8, 15, 18]. Another possibility is the formation ofliposomes by phospholipids during sonication, structures which could a u g m e n t the dispersion o f s o m e elicitor molecules (e.g. neutral lipid containing AA and EPA). Liposomes might also present the elicitors in a form more readily taken up by potato cells or could protect them from degradation by extracellular enzymes [7]. T h e reconstitution experiments indicate that the bromine treatment abolished a portion (approximately 24%) of the elicitor activity in sporangia that was not recovered upon addition of lipid. Bromine could oxidize glucan oligomers forming a gluconic acid residue at the reducing end, or modify other nonlipid components, some o f w h i c h m a y coutribute to H R induction. Nevertheless, this treatment did not significantly alter the enhancing activity of the partially purified glucan preparation. Further research is necessary to determine the precise structural features in glucan oligomers
Phytoalexin elicitor activities of P. infestans spores
57
required for enhancing eicosapolyenoic acid-elicitor activity. Our experiments also confirm that cystospores and zoospores contain components which significantly enhance AA elicitor activity with properties distinct from unsaturated lipid. It is likely that these components are fl-glucans which occur in all spore stages and in mycelium of Phytophthora spp. [24]. The relatively constant levels of EPA and AA during encystment ofzoospores appear to rule out the possibility that changes in the levels of these acids account for the increased elicitor activity ofcystospores relative to zoospores, reported by Henfling et al. [12] and also observed to some extent in our study. Such differences might be attributed to the rapid synthesis ofglucan enhancers or induction 0fspecific conformational changes in the mycolaminarans during encystment [20, 21]. Our results also support those of Lisker & Ku~ [16] and Zook & Ku6 [26] which suggested that the induction of sesquiterpenoid accumulation and the H R in potato tuber can occur via different mechanisms, although the specific nature ofsuch mechanisms remains elusive. The absence ofeicosapolyenoic acids in tissues of C.fimbriata and B. carbonum and the fact that their tissues must be viable for elicitation support the case for multiple mechanisms. These investigators observed that autoclaving or ethanol abolished the elicitor activity ofB. carbonum. In this paper, we have shown that two other methods of inactivation--treatment with ether or propylene oxide---also eliminate elicitor activity in both B. carbonum and C. fimbriata. This is in marked contrast to P. ilfestans and other Oomycctcs reported to contain eicosapolyenoic acids [25] in which killing does not eliminate elicitor activity [16]. We conclude that the evidence presented here and elsewhere [1-4, 8, 17, 18] weighs strongly in favor of a significant role for eicosapolyenoic acids in the elicitor activity of P. infestans tissues. Although inoculation with living spores from an incompatible race elicits a stronger reaction in tuber discs than treatment with killed spores [12], the higher elicitor activity of living spores may arise from an increase in fungal biomass during ingress and formation ofspecialized infection structures that efficiently deliver elicitors to plant cell receptors. It is also possible that elicitors different than the eicosapolyenoic acids are produced during the interaction between living spores and potato tissue similar to the specific elicitors described in the Cladosporiumfidvum-tomato interaction [11]. Biochemical mutants ofP. infestans deficient in EPA and AA would provide an additional critical test of the role of these compounds in elicitation, but it is unlikely that such mutants could be obtained given the gross disruption of membrane structure expected after removal ofsuch abundant fatty acids [3, 8]. Perhaps a more useful approach will be to determine ifa common reactive intermediate is formed by potato cells in response to different elicitors. Nevertheless, the eicosapolyenoic acids provide a significant opportunity for obtaining information about the participation of specific microbial lipids in signal transduction mechanisms in plant-pathogen interaction, and are useful in studies of regulation of isoprcnoid metabolism during hypersensitivity expression [19]. This work was supported in part by National Science Foundation Grant PCM8308563, a faculty research grant from the University of California, Davis, and a crop science fellowship to J.R.C. from Chevron Chemical Company, Richmond, California.
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J.R. Creamer and R. M. Bostock
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