response of three root rot fungi to strawberry phenolics and the relation of phenolics to disease...

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Mycopathologia vol. 59, 1, pp. 37-40, 1976 RESPONSE OF THREE ROOT ROT FUNGI TO STRAWBERRY PHENOLICS AND THE RELATION OF PHE- NOLICS TO DISEASE RESISTANCE* S. NEMEC 1 Agricultural Research Service, U.S. Department of Agriculture, Orlando, Florida 32803, U.S.A. Abstract Pythium irregulare, Rhizoctonia solani, and Alternaria alternata, usually associated with strawberry root rot diseases, were sensitive in vitro to several phenolics present in strawberry roots, fruits, and leaves, P. irregulare was the most sensitive. Eighteen strawberry cultivars were divided into two types, based on qualitative phenolic content. Five contained an unidentified xanthone and generally less kaempferol-7-glucoside than the remaining thirteen. Al- though these differences were not correlated with resistance to three strawberry diseases, quantitative difference of certain phenolics may be important in seasonal resistance to root rot pathogens. Introduction Tissue necrosis is a common reaction of plants to infection by various pathogens. Associated with this necrosis is an enhanced polyphenol oxidase activity, an increased pro- duction of polyphenols, and increase 0 2 consumption. Ac- cumulation of oxidized phenols at tile infection site is responsible for a necrotic reaction and is antimicrobial (6). Phenolics are generally less fungitoxic than their quinones, and it is unlikely that phenolics remain unchanged after an infected cell has been damaged (22). In many studies, attempts to relate resistance in plants to phenolic content have achieved varying results. Little information on this subject in regard to strawberry diseases has been published. Jarvis (10) grew seven isolates of * Cooperative Investigations, Agricultural Research Ser- vice, United States Department of Agriculture and Plant and Soil Science Department, School of Agriculture, Southern Illinois University,. Carbondale, Illinois. 1 Research Plant Pathologist, formerly located at Carbon- dale, Illinois. Phytophthorafi'agariae Hickman on liquid and solid media containing some phenolics identified from strawberry roots. In general, those polyphenols depressed growth on solid media, but a number of them enhanced growth on liquid media. Pyrocatechol, cinnamic acid, and p-cresol, in general, inhibited growth on both media. Mussell & Staples (15) detected phytoalexin-like compounds from two inoculated strawberry cultivars resistant to P. fraga- riae. Only one of these substances was detected in the susceptible cultivar Blakemore, and both were partially inhibitory to mycelial growth of P. fragariae 'on two media. Recently, Nemec (18) investigated the phenolic composition in healthy roots of four strawberry cultivars. The effect of some of these phenolics on the growth of Pythium irregulare Buisman and Rhizoctonia solani Ktihn, two pathogens of strawberry root rot diseases, and Alternaria alternata (Fr.) Kessler (= A. tenuis Nees), a suspected weak root pathogen, was examined in this study. In addition, the phenolics in a collection of cultivars were surveyed in an attempt to establish a relationship of phenolic content with resistance to three diseases. Materials and methods The phenolics chosen for testing against R. solani, P. irregulare, and A. alternata are listed in table 1. Of these phenolics, gallic acid, chlorogenic acid, quercetin, d- catechin, ellagic acid, an unidentified compound related to arbutin (18), and salicylic acid (19), occur in straw- berries. Protocatechuic acid was included because it is widespread in angiosperms (7), may form under conditions of plant stress, and because it has been proven toxic to certain plant pathogens (13). D-quinic acid was included because it is a constituent of chlorogenic acid. Rutin (quercetin 3-/3-rutinoside) was used to provide quercetin in a glycoside form ; and tannin, because it is a derivative of 37

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Mycopathologia vol. 59, 1, pp. 37-40, 1976

RESPONSE OF THREE ROOT ROT FUNGI TO STRAWBERRY PHENOLICS AND THE RELATION OF PHE- NOLICS TO DISEASE RESISTANCE*

S. NEMEC 1

Agricultural Research Service, U.S. Department of Agriculture, Orlando, Florida 32803, U.S.A.

Abstract

Pythium irregulare, Rhizoctonia solani, and Alternaria alternata, usually associated with strawberry root rot diseases, were sensitive in vitro to several phenolics present in strawberry roots, fruits, and leaves, P. irregulare was the most sensitive. Eighteen strawberry cultivars were divided into two types, based on qualitative phenolic content. Five contained an unidentified xanthone and generally less kaempferol-7-glucoside than the remaining thirteen. Al- though these differences were not correlated with resistance to three strawberry diseases, quantitative difference of certain phenolics may be important in seasonal resistance to root rot pathogens.

Introduction

Tissue necrosis is a common reaction of plants to infection by various pathogens. Associated with this necrosis is an enhanced polyphenol oxidase activity, an increased pro- duction of polyphenols, and increase 0 2 consumption. Ac- cumulation of oxidized phenols at tile infection site is responsible for a necrotic reaction and is antimicrobial (6). Phenolics are generally less fungitoxic than their quinones, and it is unlikely that phenolics remain unchanged after an infected cell has been damaged (22).

In many studies, attempts to relate resistance in plants to phenolic content have achieved varying results. Little information on this subject in regard to strawberry diseases has been published. Jarvis (10) grew seven isolates of

* Cooperative Investigations, Agricultural Research Ser- vice, United States Department of Agriculture and Plant and Soil Science Department, School of Agriculture, Southern Illinois University,. Carbondale, Illinois. 1 Research Plant Pathologist, formerly located at Carbon- dale, Illinois.

Phytophthorafi'agariae Hickman on liquid and solid media containing some phenolics identified from strawberry roots. In general, those polyphenols depressed growth on solid media, but a number of them enhanced growth on liquid media. Pyrocatechol, cinnamic acid, and p-cresol, in general, inhibited growth on both media. Mussell & Staples (15) detected phytoalexin-like compounds from two inoculated strawberry cultivars resistant to P. fraga- riae. Only one of these substances was detected in the susceptible cultivar Blakemore, and both were partially inhibitory to mycelial growth of P. fragariae 'on two

media. Recently, Nemec (18) investigated the phenolic composition in healthy roots of four strawberry cultivars. The effect of some of these phenolics on the growth of Pythium irregulare Buisman and Rhizoctonia solani Ktihn, two pathogens of strawberry root rot diseases, and Alternaria alternata (Fr.) Kessler (= A. tenuis Nees), a suspected weak root pathogen, was examined in this study. In addition, the phenolics in a collection of cultivars were surveyed in an attempt to establish a relationship of phenolic content with resistance to three diseases.

Materials and methods

The phenolics chosen for testing against R. solani, P. irregulare, and A. alternata are listed in table 1. Of these phenolics, gallic acid, chlorogenic acid, quercetin, d- catechin, ellagic acid, an unidentified compound related to arbutin (18), and salicylic acid (19), occur in straw- berries. Protocatechuic acid was included because it is widespread in angiosperms (7), may form under conditions of plant stress, and because it has been proven toxic to certain plant pathogens (13). D-quinic acid was included because it is a constituent of chlorogenic acid. Rutin (quercetin 3-/3-rutinoside) was used to provide quercetin in a glycoside form ; and tannin, because it is a derivative of

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gallic and catechin nuclei. Other important phenolics of strawberry, such as kaempferol, scopoletin, and certain condensed tannins, were not used because of their pro- hibitive cost or unavailability. Cyanidin is present in strawberry roots as cyanidin-3-monoglucoside (18), and was also included in limited tests with P. irregulare.

Phenolics reported in table 1 were dissolved in 1- to 2-ml quantities of appropriate solvents, unless they were readily soluble in phosphate buffer. After addition to sterile 0.02 M phosphate buffer at pH 6 and 8, the buffer was diluted to 0.01 M with cooled potato-dextrose-agar, mixed, and poured into 15-ram petri dishes. Final concentrations of the phenolics were 50 and 500 ppm. Fungi were added to these dishes by inverting on the agar surface a 5-mm plug from a petri dish culture. As many as three plugs per dish were used for the slower growing A. alternata.

Mean colony diameter in mm was determined from eight plugs for each concentration of phenolic at each pH. These measurements were made after 1 to 5 days of in- cubation at 21~2 ~'C.

Phenolic composition of roots of 14 cultivars was de- termined by paper chromatography, as previously re-

ported (18).

Results

Fungal response to phenolies P. irregulare was the most sensitive of the three fungi grown on agar containing the phenolics (table 1). It was completely inhibited by 500 ppm protocatechuic acid, salicylic acid, and quercetin at both pH's for at least 5 days. Some reduction in growth occurred in the presence of 500 ppm of tannin at both pH's, and in the presence of 500 ppm D-quinic acid and D-catechin, pH 6. P. irregulare was able to utilize gallic acid, increasing growth by as much

Table I. Growth of three fungi associated with strawberry root rot On petato- dextrose-agar containing phenol ics present in strawberry roots at t~o pH values and two concentratlonsa,b.

Pythian irregulare Alternaria alternata Rhizoctonla solani

pH 6 pH 8 pH 6 pH 8 pH 6 pH 8 ppm

Phenolics 50 500 50 500 50 500 50 500 50 500 50 500

G a l l i c ac id 148 131 97 116 94 91 96 106 102 91 106 100 Protocatechuic acid 38 0 25 0 98 93 84 108 52 35 59 45 Salicylic a c i d 2B 0 86 0 92 17 92 87 64 0 58 49 Chlorogenic acid 96 94 log I00 iO0 102 I00 102 98 92 104 104 D-qulnlc acid 94 66 92 104 96 95 102 104 102 94 103 Ill Quercetin 54 0 50 0 95 74 I00 33 53 0 45 0 Arbutin 103 105 95 75 104 102 95 95 102 I02 102 97 D-catechin 78 67 IO5 89 I02 lO0 I02 I02 I06 97 97 97 Rutln 122 89 94 109 98 95 100 I00 87 87 105 95 Tannin 109 60 89 51 89 89 102 102 90 86 96 B5 s ac ld I00 98 98 95 99 99 IO0 94 98 97 IOl 104

a Protocatechuic ac id , D-quinic ac id , r u t i n , and t a n n i n were not de tec ted on paper chromatograms o f roo t e x t r a c t s .

b Data presented as percentage o f r a d i a l growth o f c o n t r o l s . Colony d iameter measured in mm I -5 days a f t e r i n c u b a t i o n a t 20-21~

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as 31 ~o, whereas A. alternata and R. solani did not. Growth ofP. irregulare was unaffected by 50 and 500 ppm of cyanidin hydrochloride.

Growth ofA. alternata was unaffected by protocatechuic acid, which at 500 ppm reduced growth of R. solani by almost two-thirds at pH 6 and nearly one-half at pH 8. Both A. alternata and R. solani were more sensitive to salicylic acid at pH 6 than at pH 8; however, R. solani was more sensitive to quercetin at both pH's than A. alternata. The growth of all three fungi was essentially un- affected by chlorogenic acid, ellagic acid, rutin, and ar- butin.

Phenolics in roots o f strawberry cultivars

In a previous report (18), four strawberry cultivars were assayed for root phenolics during the summer. Of these, only Howard 17 (Premier) differed qualitatively from Sunrise, Surecrop, and Blakemore. Howard 17 contained a xanthone, a component not present in the other cultivars, but less kaempferol-7-glucoside than generally present in the other cultivars. Subsequently, for this study, 14 ad- ditional cultivars were assayed for phenolics during the same time of the year and were found to be similar in con- tent to either Howard 17 or the other three cultivars.

The 18 cultivars comprising both types of phenolic com- position appear in table 2. Each is given a disease rating based on its reaction to three diseases: leaf spot, leaf scorch, and red stele. Resistant and susceptible reactions to each disease are found among both phenolic types, except for the red stele disease with the Howard 17 type of phenolic composition. There the cultivars give either a susceptible or unknown reaction. Although there appears to be a higher proportion of susceptible reactions among the cultivars with the Howard 17 type of phenolic composition, distinct reactions of resistance and susceptibility in both phenolic types indicate that no relation exists between phenolic composition and disease resistance.

Although the major distinction between cultivars was the presence or absence of the xanthone component, seasonal differences were noted in the composition of root phenolics. Winter-grown roots of all cultivars contained fewer phenolics than summer-grown roots did. Chlorogenic acid, scopoletin glycoside, and kaempferol-7-glucoside were not usually detected in roots from winter-grown plants, and when detected, most appeared to be in low concentration. Both naphthoquinone and cyanidin-3-

monoglucoside were absent in root extracts during the winter. Apparently, more ellagic acid was synthesized during the winter, because this band became more intense

Table 2. Disease resistance ratings of five straw- berry cultivars with the Howard 17 type of root phenolic composition and 13 cultivars with the Surecrop type of phenolic composition for three diseases

Diseases and Ratings a,b

Cultivars Leaf Spot Leaf Scorch Red Stele

Howard 17 type Jerseybelle VS S S Raritan S S S Klondike VS 1 S 1 U Armore S S S Howard 17 R R S

Surecrop type Redchief R R R Redglow S I R Fairfax R R S Florida 90 VS VS S Earlibelle VR VR S Midland R R S Redcoat S 2 U U Redstar S R S Cyclone R U S Tenn. Beauty R R S Sunrise VS R R Blakemore S VR S Surecrop R R R

a S = susceptible; VS = very susceptible; I = intermediate; R = resistant; VR = very resistant, and U = unknown.

b All ratings taken from Scott, D.H., G.M. Darrow & Lawrence (20), except that those indicated l are from McGrew, J.R. (14), and those indicated 2 are from Nemec, S. (17).

then, and one new phenolic appeared that was not detected during the summer. This phenolic was blue in uv light, and blue-violet in uv light plus NH3-fuming. It has an Rf of 0.33 in BAW (butanol : acetic acid : H20 ) 4 : 1 : 5 and an Rf of 0.23 in 10 ~o acetic acid. This phenolic was not identified.

Discussion

Phenolics have a wide range of toxicity to microorganisms (4). In this study, chlorogenic acid, arbutin, D-catechin, rutin, and ellagic acid were essentially noninhibitory to the three fungi tested. Similar findings have been cited for other fungi and chlorogenic acid (1, 2), catechin (1, 9), arbutin (21), and ellagic acid (8). Each of the remaining phenolics was inhibitory to one or all of the fungi, with quercetin and salicyclic acid the most inhibitory. Salicyclic acid derivati- ves in the bark ofPopulus candicans have also been reported

to have fungistatic activity (11), but quercetin has been found nontoxic to Gloeosporium perennans Zeller and Childs (9).

Quantitative amounts of phenolics in plants vary, depending on tissue, varieties, season, and other factors examined (12, 5). Although growth of many plant patho- gens is generally inhibited by 10 3M and higher con- centrations of phenolics (4), these concentrations may not occur in the infected tissue. The quantities used in this study are within the range of concentrations to which many other plant pathogens are sensitive, and 50 ppm may exceed the normal levels of certain phenolics measured in leaf disks ofFragaria vesca cv. Alpine (3).

No correlation was apparent between the qualitative phenolic composition of 18 strawberry cultivars and their resistance to three diseases. Resistance to strawberry root rots was not evaluated, because disease ratings for this group of diseases are incomplete. Seasonal quantitative differences of phenolics in strawberry roots may be signi- ficant in resistance to root diseases if levels decrease below the point at which they are antimicrobial. Chlorogenic acid and scopoletin glycoside decline in strawberry roots during the winter, particularly at a time when roots are susceptible to attack by Pythium spp. (16).

Summary

Three strawberry root rot fungi Pythium irregulare, Rhi- zoctonia solnai, and Alternaria alternata were sensitive in vitro to several phenolics present in the strawberry plant. P. irregulare was the most sensitive of the three fungi grown on agar containing the phenolics. Of the phenolics detected in the plant, salicyclic acid and quercetin were the most inhibitory. The growth of all three fungi was essentially unaffected by chlorogenic acid, ellagic acid, rutin and arbutin. Eighteen strawberry cultivars were divided into two types, based on qualitative phenolic content. Five contained an unidentified xanthone and generally less kaempferol-7-glucoside than the remaining thirteen. No correlation was apparent between the quali- tative phenolic composition of 18 strawberry cultivars and their resistance to three diseases.

References

1. Byrde, R. J. W., A. H. Fielding & A. H. Williams. 1960. The role of oxidized polyphenols in the varietal resis- tance of apples to brown rot. In: Phenolics in Plants in

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Health and Disease (J. B. Pridham, Ed.): 95-99. Pergamon Press, Oxford.

2. Condon, P. & J. Kud. 1960. Isolation of a fungitoxic compound from carrot root tissue inoculated with Ceratocystis fimbriata. Phytopathology 50: 267-270.

3. Creasy, L. L. 1971. Role of phenylalanine in the bio- synthesis of flavonoids and cinnamic acid in straw- berry leaf disks. Phytochemistry 10:2705~711.

4. Cruichshank, I. A. M. & D. R. Perrin. 1964. Patho- logical function of phenolic compounds in plants. In : Biochemistry of Phenolic Compounds (J. B. Harborne, Ed.): 511 544. Academic Press, London.

5. Dawson, R. F. & E. Wada. 1957. Flavonoids and depsides of the green tobacco leaf. I. Rutin and chloro- genic acid. Tobacco Sci. 1 : 47 50.

6. Goodman, R. M., Z. Kirfily & M. Zaitlin. 1967. The Biochemistry and Physiology of Infectious Plant Disease. Van Nostrand, Princeton.

7. Harborne, J. B. & N. W. Simmonds. 1964. The natural distribution of the phenolic aglycones. In : Biochemistry of Phenolic Compounds (J. B. Harborne, Ed.): 77-127. Academic Press, London.

8. Hart, J. H. & W. E. Hillis. 1972. Inhibition of wood- rotting fungi by ellagitannins in the heartwood of Quercus alba. Phytopathology 62 :620 626.

9. Hulme, A. C. & K. L. Edney. 1960. Phenolic substan- ces in the peel of Cox's Orange Pippin apples with reference to infection by G. perennans. In: Phenolics iv. Plants in Health and Disease (J. B. Pridham, Ed.): 87 94. Pergamon Press, Oxford.

10. Jarvis, W. R. 1961. Growth of isolates of Phytoph- thora fragariae Hickman in the presence of various polyphenols. Trans. Brit. Mycol. Soc. 44: 357-364.

11. Klopping, H. L. & G. J. M. van der Kerk. 1951. Antifungal agents from the bark of Populus candicans. Nature, Lond. 167 : 996-997.

12. Lee, S. & D. J. Le Tourneau. 1958. Chlorogenic acid content and verticillium wilt resistance of potatoes. Phytopathology 48:268 274.

13. Link, K. P., H. R. Angell & J. C. Walker. 1929. The isolation of protocatechuic acid from pigmented onion scales and its significance in relation to disease resistan- ce in onion. Jour. Biol. Chem. 81 : 369 375.

14. McGrew, J. R. 1970. Strawberry Diseases. U.S.D.A. Farmers Bull. 2140. 27 pp.

15. Mussell, H. W. & R. C. Staples. 1971. Phytoalexinlike compounds apparently involved in strawberry resistan- ce to Phytophthora fragariae. Phytopathology 61: 515-517.

16. Nemec, S. 1970. Bimonthly changes in fungi associated with apparently healthy and root rot-diseased straw- berry roots. Phytopathology 60:586 (Abstr.).

17. Nemec, S. 1971. Studies on resistance of strawberry varieties and selections to Mycosphaerella fragariae in southern Illinois. Plant Dis. Reptr. 55: 573-576.

18. Nemec, S. 1973. Phenolics in the strawberry root. Ann. Bot. 37: 935441.

19. Portes, L. & A. Desmouli6res. 1901. Normal occur- rence of salicylic acid in strawberries; resulting ana- lytical errors. Ann. de Chim. Analyst. 6: 401-407.

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20. Scott, D. H., G. M. Darrow & F. J. Lawrence. 1972. Strawberry varieties in the United States. U.S.D.A. Farmer's Bull. 1043. 22 pp.

21. Sieb, E. 1964. Tlae behavior in vitro of scab of pome fruit trees in relation to the glucosides in the leaves of the host plant. Phytopath. Z. 51 : 479-490.

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