race interactions of Medicago sativa and Colletotrichum trifolii

Physiological and Molecular Plant Pathology (1989) 35, 231-241

Accumulation of phenolic compounds in incompatible clone/race interactions of

Medicago sativa

and

Colletotr/chum trifolii* C . JACYN BAKERt§, NICHOLE R . O'NEILLt AND J . ROBERT TOMERLIN+ j' Microbiology and Plant Pathology Laboratory and Germplasm Quality and Enhancement Laboratory, respectively, Agricultural Research Service, U .S. Department of Agriculture, Beltsville, MD 20705, U .S .A . ++ Environmental Protection Agency, Washington, DC 20460, U .S.A .

(Accepted for publication March 1989)

Preliminary evidence had suggested that accumulation of phenolics, including phytoalexins, may play a role in natural and induced resistance interactions involving Colletotrichum trifolii and Medicago sativa . We examined specific race interactions with clones of resistant and susceptible genotypes . Stem sections of seedlings were excised, dipped in conidial suspensions of race 1 or 2 or water, incubated for 5 days, and analysed for phenolic compound accumulation by monitoring UV-absorbance and fluorescence . Phenolic compounds accumulated in resistant interactions of C . trifolii and alfalfa and therefore depended on both the phenotype of the clone and the race of the pathogen . The ability to quickly and quantitatively measure these compounds, as described in this report, will greatly facilitate investigations of the induction of resistance in alfalfa .

INTRODUCTION

Anthracnose of alfalfa (Medicago saliva L .), caused by Collelotrichum trifolii Bain, is an important disease which limits forage yield in warm areas of the southern and midatlantic United States [5, 9] . The primary defence against the disease is through the use of resistant cultivars such as Arc . During the late 1970's a second race of the pathogen, designated race 2, was found to be highly pathogenic on formerly resistant cultivars [30] . In the process of screening plants for resistance to both races of the pathogen, Ostazeski & Elgin [29] discovered that Arc or Arc-related populations had a higher survival rate when inoculated with a mixture of both pathogen races as compared to inoculation with race 2 alone . The authors suggested that prior inoculation of most Arc seedlings and selected clones with race 1 induced resistance against race 2 . We are interested in the specific biochemical mechanisms involved in this induced resistance . Histological studies [7] have shown that in both susceptible and resistant interactions the fungus germinates, develops appressoria, and penetrates the epidermis . However, only in the susceptible interaction does the fungus develop infection hyphae, penetrate internal cell walls and ramify throughout the host . It therefore appears that resistance * Mention of a trade name, proprietary product, or vendor does not imply its approval to the exclusion of other vendors that may also be suitable . § To whom correspondence should be addressed . 0885-5765/89/090231 + 11 $03 .00/0

© 1989 Academic Press Limited



232

C . J . Baker et al. is expressed prior to internal cell wall penetration and ramification of infection hyphae . Similar histological observations have been noted with other Colletotrichum species in which induced resistance has been demonstrated [11, 13, 19, 37] . Several mechanisms have been suggested to account for the unsuccessful penetration or reduced ramification of fungal development in resistant interactions . These include impenetrable papillae [37], containment of penetrating hyphae [11, 12], enhanced lignification [19], and phytoalexin production [2] . In most of these cases an increased production of phenolic compounds is apparent, many of which may be involved in phytoalexin accumulation and/or lignin-related materials which reinforce papillae and cell walls [1, 20, 24-26, 33, 42] . This increased accumulation of phenolic compounds contributes to the fluorescent characteristic often associated with the resistant interactions [1, 8, 20, 23, 27, 28, 33, 37, 38, 41] . The induced resistance observed in the alfalfa system requires the presence of genes for resistance to the inducer inoculum [29] . Thus the mechanism of genetic resistance of alfalfa to races of C. trifolii needs to be understood . In this study we are investigating the accumulation of phenolic compounds, including the phytoalexin medicarpin, which preliminary studies suggested correlated closely with resistant interactions [3, 4] . The present assay for induced resistance requires a 10 day incubation period after which the percentage of plants surviving is determined . Correlation of an extractable compound to resistance would allow a faster and more quantifiable means to assess various treatments on induced resistance in future studies . Detection of a fluorescent compound would allow increased sensitivity . The method developed in this study allows rapid extraction of compounds, removal of chlorophyll, and direct injection onto an HPLC column for quantification .

MATERIALS AND METHODS

Fungal inoculum Stock cultures of C. trifolii were stored as conidia or hyphae (suspended in a solution of 10 °, . nonfat dry milk) on silica gel at -20 °C [32] . Race 1 isolates used in this study include 271, WL 315, 3-5, 23R1, and Vertus Beck ; race 2 isolates include SB1, SB2, SB3, H4-2 and H5-3 . Isolates were grown on half-strength oatmeal agar at 22 ° C with 24 h fluorescent light [31] . Conidia were transferred to fresh agar plates bi-weekly . Inoculum was prepared from an equal number of plates of each isolate by suspending conidia from 7-day-old cultures in sterile distilled water containing two drops of Tween 20 per litre (approximately 0 .003 % w/v) . Subsequent spore germination was greatest if the distilled water was autoclaved after addition of the Tween 20 . Spore concentration was determined by haemocytometer, and the final concentration adjusted to 6 x 10 8 conidia ml - ' . Plants Clones of alfalfa were used because alfalfa is an autotetraploid and crosspollinated, making it genotypically heterogeneous within a cultivar . Resistance to each race of the pathogen is conditioned independently by a different, single dominant gene [10] . Alfalfa clones 1 RR, 4 SS, 15 RS, 16 RS and 20 SS were derived from the cultivar Arc ; clones 41 RS, 51 RR, 54 RS and 57 RR were derived from cultivar Saranac AR . The



Interactions of

M. sativa

and C.

trifolii

233

designation SS signifies susceptible to both race 1 and race 2 of C . trifolii ; RS, susceptible to race 2 and resistant to race 1 ; RR, resistant to both races . Clones susceptible to race I but resistant to race 2 (SR) have not yet been identified and therefore were not available for this study . Plants were maintained in the greenhouse at 26±4 ° C with 16 h light and were periodically cut back and the new growth used for experiments.

Injection inoculation of tissue Stems were injected with a 23 gauge hypodermic needle as described previously [31] .

Immersion inoculation of tissue Stem segments 5 cm in length were excised from about 2 cm below the growing tip, the leaves removed, and the segments immersed in inoculum . Control segments were immersed in water containing only Tween 20 . Five stem segments were placed on glass rods in sterile plastic petri plates lined on the bottom with Whatman No . 1 filter paper wetted by 3 ml sterile water . Plates were placed in a sealed plastic container and incubated at 22 °C for 5 days . The stems were then placed in plastic bags and frozen at -20 ° C until needed for extraction . Each 5 cm stem segment was treated as a replicate . Due to the large number of samples and the amount of time needed to assay each sample, only one replicate of each interaction was processed at one time . Due to the similarity of replicate results only three replicates were processed .

Sample preparation-liquid phase method Stem segments, about 1 g, were cut into 1-2 cm sections and ground in a mortar and pestle with 3 ml of 100 % ethanol . The supernatant was removed by pasteur pipette and pooled with subsequent ethanol washings . Five millilitres of ethanol were added to the tissue with continued grinding . After three grindings the stem residue lost most of its pigment. The ethanol extract was filtered through a 0 . 2 gm solvent resistant filter (alpha 200 Metricel, Gelman) and dried under air at 35-40 ° C . The residue was suspended in 9 ml chloroform and extracted three times with 5 ml 0 . 2 N sodium hydroxide . Each time the suspension was vortexed, centrifuged, and the upper layer removed and saved . The aqueous sodium hydroxide fraction was acidified with 1 N hydrochloric acid to a pH of about 3 and extracted three times with 5 ml ethyl acetate . The ethyl acetate layers were pooled, dried under air, and suspended in 3 ml 1000, ethanol .

Sample preparation

solid phase method

This extraction method was developed to reduce the chlorophyll content of samples, to extract fluorescent compounds as well as known phytoalexins, and to simplify and streamline the procedure for small samples . Each step was checked for efficiency and consistency using tissue from the 16RS/race 1 interaction ; 50 .tg of maackiain in 50 pl ethanol was added at appropriate steps as an internal standard and the recovered peak area compared to standards . Stem segments, about 0 . 1 g, were removed from the freezer, weighed and cut into approximately 0 . 5 cm long pieces which made subsequent grinding easier . Stem tissue was placed into 1 dram shell vials with 1 . 5 ml acetronitrile-water (4 :1 v/v) . The vial was placed in ice and the contents homogenized

234 C . J . Baker et al. with a Tekmar Tissumizer (Tekmar Co ., Cincinnati, Ohio, U .S .A .) for about 0 . 5 min at 80 % maximum speed . In preliminary tests the Tissumizer was found to give consistent results comparable to the mortar and pestle and was more practical for large numbers of samples . Extraction efficiency for compounds of interest was higher with aqueous acetonitrile than 100% ethanol . The homogenate was poured onto a 3 ml Baker-l0 SPE octadecyl (C18) disposable extraction column which had been preconditioned with methanol followed by acetronitrile-water (4 : 1 v/v) . Two 1 . 5 ml rinses of the homogenizer probe and vial were also run (approx . 1-2 ml min') through the column with a final 0 . 5 ml rinse of the column . The column retained over 95 % of the chlorophyll while compounds of interest were in the eluant (about 5 ml) . Aqueous acetic acid, 6 ml 0 . 1 %, and ethyl acetate, 1 . 25 ml, were added to the eluant and the mixture vortexed and centrifuged . The top phase of ethyl acetate was removed and saved . A second extraction of the mixture with 1 ml of ethyl acetate was added to the first . The samples were dried under nitrogen and suspended in 1 ml ethanol g- ' tissue . Chlorophyll content was approximated by measuring absorbance at 662 nm [43] . Analysis by TLC Thin-layer chromatography plates (0 . 25 mm, silica gel 60, Merck) were heated for 2 h at 85 °C Plates were spotted with 10 tl of sample and developed in chloroform : methanol, 25 : 1 . After air-drying the plates were examined under long and short wave UV light for fluorescent compounds and then sprayed with either 1 % ferric chloride [21] or 1 % vanillin in 50 % phosphoric acid followed by heating 15 min at 110°C [16] . Analysis by HPLC Samples, 20 µl, were analysed by reverse phase HPLC on an Alltech C18 column, 25 cm x 4. 6 mm diameter with 10 pm beads . The mobile solvent was 50 % acetonitrile in water with a flow rate of 1 ml min' . The effluent was monitored for absorbance at 210 nm with a UV monitor and with a fluorescence monitor set for 250 nm excitation and full range emission . Data was collected and peak area units (mV s - ') quantified by a Perkin Elmer Chrom II data system . Statistical analysis of data Relative peak area units were analysed using the Statistical Analysis System (SAS) . The experiment was first analysed as a split plot design with alfalfa clones as the main plot and fungus isolates as sub-plots . Because the main plot error term was not significantly different from the residual, subsequent analyses were considered to be simple randomized complete blocks . The clone by isolate interaction was significant for each fluorescent and UV-absorbing peak, as well as for the totals of each phenotype group (SS, RS, RR) ; consequently, the variation on peak area was further investigated using single degree of freedom contrasts . The experiment was designed to have an equal number of clones of each phenotype, however, it was found that 41 RS had been mistakenly included as an SS clone .



Interactions of M. sativa and C. trifolii

2 35

RESULTS Initial investigations of resistant and susceptible interactions of alfalfa tissue with C . trifolii involved TLC of stem tissue injected with conidial suspensions and were limited to two clones of each phenotype (4 SS, 20 SS ; 16 RS, 54 RS ; 1 RR, 57 RR) . Phenolic compounds could be detected in both susceptible and resistant interactions within 6 h, however, distinct differences between interactions were not clear by this technique

(a) F6

a,o Ca, O a> O tL F2

F5

F7

(b) U5

UI U7

0

5

10 Time (min)

15

20

FIG . 1 . HPLC elution profile of fluorescent (a) and UV-absorbing (b) compounds extracted from alfalfa stems inoculated with C . trifolii. The eluant was 50% aqueous acetonitrile at I ml min-' monitored for UV absorbance at 210 nm and for full range fluorescence excited at 250 nm .



C . J . Baker et al.

236 6000

m ô Y O U) D

2000



t;

54RS

16RS

15RS

41 RS

1 n o - T T T A

20SS

4SS

57RR

51RR

IRR

Genotype

Ftc . 2 . Accumulation of fluorescent compounds by specific race/clone interactions . Stems from alfalfa clones were inoculated with : 0, race l ; /, race 2 and ®, water. After incubation for 5 days the stems were extracted and analysed for fluorescent compounds . Bars represent the sum of peak areas of F2, 5, 6, and 7 . Error bars indicate the standard error of three replicate extractions .

until 12-24 h . These differences were maintained over the rest of the 14 day period . Three fluorescent spots, not found in water control inoculations, were commonly associated with the resistant interaction at RF 0. 31, 0 . 40, and 0 . 54 with yellow, purple, and yellow fluorescence respectively . SS clones generally showed very little accumulation of any of these three spots ; RR clones accumulated high levels in response to both race 1 and 2 ; RS clones accumulated high levels in response to race 1, but not race 2 . In an attempt to quantify these findings better, an improved extraction procedure and HPLC method was developed which allowed monitoring of fluorescent and UVabsorbing compounds, including known alfalfa phytoalexins . The liquid phase extraction procedure used above was laborious and time consuming, and while it removed chlorophyll which interfered with fluorescent compound detection, the extraction efficiency was low . The solid phase extraction procedure developed for this study consistently yielded greater than 94 per cent recovery of an internal standard, maackiain, which is chemically similar to medicarpin . More than 95 per cent of the chlorophyll which tended to interfere with chromatography was eliminated . This procedure allowed extraction and quantification of individual stems . During the development of the HPLC method it was found that, while elution profiles from stems extracted by different extraction techniques were similar, the profiles from stems inoculated by injection versus immersion were different . Instead of the three fluorescent TLC spots detected in needle inoculated tissue, as mentioned above, a single blue fluorescent TLC spot, R i, 0-4 1, [corresponds to peak F6, Fig . 1(a)] was observed with



Interactions of M. sativa and C . trifolii

237

1500

1250

Y O N

a

500

250

S 54RS

16RS

15RS

-+ -TA

41 RS

20SS

nT 4SS

7 57RR

ri~ 51RR

r IRR

Genotype FIG . 3 . Accumulation of total UV-absorbing compounds by specific race/clone interactions .

Stems from alfalfa clones were inoculated with : [3, race l ;/, race 2 and ®, water. After incubation for 5 days inoculated alfalfa stems were extracted and analysed for UV-absorbing compounds . Bars represent sum of peak areas of U 1, 5, and 7 . Error bars indicate standard error of three replicate extractions .

immersion inoculation . The latter inoculation procedure was used for all further experiments since it better represented the natural phenomenon . Stem segments of clones of each phenotype RS, RR, or SS, were inoculated with race 1, race 2 or water . Figure 1 shows the typical elution pattern from reverse phase HPLC of extracts from these stem segments . The profiles were monitored for fluorescence and UV absorbance . Seven fluorescent and five UV peaks were well resolved and quantified for each interaction . After initial examination of these peaks only F2, F5, F6, F7 and U1, U5, and U7 were found to correlate to clone/race interactions . Peak U5 was determined to be medicarpin based on co-chromatography and UVspectra comparison with authentic standard . In general the accumulation of fluorescent and UV-absorbing compounds in alfalfa stem tissue corresponded to both the phenotype of the clone and the race of the pathogen (Figs 2 and 3) . In interactions where resistance would be expected, the relative peak area of these compounds was always high . RS clones accumulated much greater levels of fluorescent and UV-absorbing compounds in response to race 1 compared to race 2 . RR clones accumulated high levels of compounds in response to both races . Interactions where susceptibility would be expected generally yielded relatively low levels of the compounds, with one exception, 20 SS/race 1 . Levels produced by this interaction were higher than other susceptible interactions . The stem pieces appeared uniformly green similar to other resistant interactions and unlike susceptible interactions which appeared dark and discoloured . Subsequent checks of the clone confirm that under greenhouse conditions it is susceptible to both race 1 and 2 .



238

C . J . Baker et al.

Peaks F6 and U5 were the most dominant peaks (Fig . 1), greater than 80 1, ,,, of the total in their respective groups . Nearly twice as much of the UV-absorbing compounds accumulated in RR clones compared to RS/race 1 interactions (Fig . 3), while the levels of fluorescent compounds were similar (Fig . 2) . When fluorescent and UV peaks appeared, the relative proportion to one another was similar in most interactions . However, in clones 15 RS and 20 SS peak F7 was four to five times greater than F5 ; in all other clones these two peaks were similar in area . DISCUSSION Limited TLC studies demonstrated a correlation between accumulation of phenolic compounds and resistance in alfalfa stems inoculated with C . trifolii . This initial work was corroborated by expanded and quantitative HPLC studies . Several distinct peaks were found to correlate with resistant interactions including one shown to be medicarpin, a known phytoalexin of alfalfa . In general the accumulation of fluorescent and UV-absorbing compounds in alfalfa stem tissue depended on both the phenotype of the clone and the race of the pathogen (Figs 2 and 3) . Clones of similar phenotypes responded in a similar manner ; RR clones accumulated high levels of these compounds in response to both races, RS clones accumulated high levels only in response to race 1, SS clones, except for the 20 SS/race 1 interaction, did not accumulate comparable levels of these compounds . The interaction of clone 20 SS with race 1 would normally be susceptible . However, since typical susceptible symptoms did not develop, it appears the bioassay conditions may have triggered the resistance mechanism in this clone . There is a strong correlation between the accumulation of these phenolic compounds and resistance . Comparison of numerous histological studies of Colletotrichum spp . on alfalfa and other hosts show obvious similarities in pathogen development . In both susceptible and resistant interactions, spore germination is followed by appressorial formation and direct penetration of the host epidermis . At this point there is either ramification of infection hyphae in susceptible interactions or containment of fungal development in resistant interactions [6, 7, 11, 13, 19, 34, 35, 37, 39] . The biochemical mechanisms responsible for containment of fungal pathogens in the resistant interactions are undoubtedly multifold . Indirect evidence such as presented in this report for alfalfa and elsewhere for other plants [1, 2, 19, 22, 28, 38, 40, 41, 45] suggests that phenolic metabolites play a role . Although not always shown to be directly responsible for resistance, phenolics are produced in association with resistance mechanisms, such as phytoalexin accumulation or production of lignin-like materials [17, 18, 42] . Interestingly, in the present study we found that different fluorescent compounds accumulated depending on the inoculation procedure, presumably due to placement of inoculum in contact with different tissue ; immersion inoculation involves epidermal tissue, while injection inoculation involves vascular tissue . Autofluorescence and phenolic compounds are commonly associated with hostpathogen interactions [44] and are often noted in greater intensity in resistant interactions [1, 8, 20, 23, 27, 28, 33, 38, 41] . In most of these cases the identities of the compounds responsible for the fluorescence are unknown, however, in some cases it has



Interactions of

M. sativa

and C.

trifolii

239

been at least partially attributed to phenolic compounds associated with phytoalexin accumulation [20, 24-26, 33] or with lignin, tannin, and melanin formation [1, 42] . Several plant-pathogen interactions, usually resistant interactions, including both fungal and bacterial pathogens have been found to have synchronous production of autofluorescence and phytoalexins . Mansfield et al . detected the blue fluorescence typical of wyerone acid and wyerone in broad bean leaf tissue resistant to Botrytis cinerea [26] . Also, the blue fluorescence characteristic of resveratrol, a potential phytoalexin of grape, was detected surrounding lesions caused by B . cinerea [24, 25] . Pierce and Essenberg have clearly demonstrated that brightly fluorescent cells from cotton cotyledons inoculated with Xanthomonas campestris pv . malvacearum contain much higher levels of phytoalexins, lacinilene, and dihydroxycadalene compared to less fluorescent cells [33] . In soybean, inoculated with Phytophthora megasperma f . sp . glycinea evidence by Holliday et al . also strongly suggests that autofluorescence observed in hypersensitive interactions is related to production of the phytoalexin, glyceollin [20] . Fluorescence has also been correlated with another class of phenolic compounds involved in structural barriers such as lignin [14, 15, 19, 36, 40, 42] . Increased fluorescence has been observed near or within papilla, localized wall appositions laid down in response to pathogen attack [1, 28] . Aist and Israel studied papillae induced by Erysiphe graminis f. sp . hordei using epifluorimetric and ultraviolet-microspectrophotometric methods [1] . They suggested that deposition of phenolics in oversize papillae prior to challenge by the pathogen and/or the presence of certain phenolics detected only in these oversize papillae could have rendered them resistant to pathogen penetration . Hammerschmidt and Ktic found that lignification in response to C. lagenarium occurred more rapidly and to a greater extent in cucumber plants which had been previously infected with the pathogen [19] . They also found that coniferyl alcohol, a lignin precursor, was toxic to the fungus suggesting that it might function as a phytoalexin . The results of this study demonstrate that UV-absorbing compounds and fluorescent compounds accumulate in resistant interactions of C . trifolii and alfalfa . Some of these products may be metabolites from defence mechanisms activated by recognition of the presence of the pathogen . The ability to quickly and quantitatively measure these compounds, as described in this report, will greatly facilitate our investigations of the induced resistance phenomenon in alfalfa, including studies of pathogen elicitors which trigger the host defence response . We wish to thank Dr Hans Van Etten for his gift of maackiain and Alice Churchill, Jim Hoffman and Susan Stanton for their excellent technical assistance and contributions throughout this project .

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