Black Shank Disease Fungus - ACS Symposium Series (ACS

Chapter 27

Black Shank Disease Fungus Inhibition of Growth by Tobacco Root Constituents and Related Compounds 1

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Downloaded by PENNSYLVANIA STATE UNIV on August 1, 2012 | http://pubs.acs.org Publication Date: January 9, 1991 | doi: 10.1021/bk-1991-0449.ch027

Maurice E . Snook , Orestes T . Chortyk , and Alex S. Csinos 1

Russell

Research Center, Agricultural Research Service, U.S. Department of Agriculture, P.O. Box 5677, Athens, G A 30613 Department of Plant Pathology, University of Georgia, Coastal Plain Experiment Station, Tifton, G A 31793

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Tobacco root phenolics were investigated for their possible role in resistance of tobacco toPhytophthoraparasitica var. nicotianae (black shank). Chlorogenic acid (CA), scopolin, and scopoletin were found to be significantly increased in apparently healthy root tissue, adjacent to infected tissue. In contrast, C A and scopolin concentrations remained relatively constant throughout the length of control roots, while scopoletin increased slightly from the proximal to the distal end. Roots of resistant and susceptible varieties showed similar trends in concentrations of phenolics. Chlorogenic acid, scopolin, scopoletin, and structurally related compounds were evaluated for inhibition of growth of black shank fungus in a laboratory bioassay. At a 4000 ppm dosage level, C A produced 25% inhibition of fungal growth, while scopoletin gave 39% inhibition at 1000 ppm dosage. The activity of C A was shown to reside in the caffeic acid portion of the molecule. Free phenolic acids were also investigated and were found to be very active against black shank growth, with mono-hydroxycinnamic acids being more active than the 3,4-di-hydroxy acid and the o-hydroxy acid being the most active. Dihydro-cinnamic acids were slightly more active than the corresponding cinnamic acids. While scopoletin and esculetin were active in the laboratory bioassay, their glucose-derivatives were found to be completely inactive.

Phytophthora parasitica var. nicotianae (black shank) is a fungus that only attacks tobacco and also causes extensive physical damage, resulting in large economic losses for farmers. It affects both flue-cured and burley types of tobaccos. The fungus attacks the roots of the plant and eventually blocks water and nutrient transport, resulting in the death of the plant. Several varieties of tobacco are known to be highly resistant to the black shank fungus. Several reports have shown that phenolic compounds of tobacco increase in tissues that have been exposed to pathogens, such as tobacco mosaic virus (1), blue mold (Peronospera tabacina) (2), and black root rot (Thielaviopsis This chapter not subject to U.S. copyright Published 1991 American Chemical Society In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

27. SNOOK E T A L .

Black Shank Disease Fungus

389


basjcoja) (3). Recently, Gasser, et al. (3) reported that chlorogenic acid (3-O-caffeoylquinic acid, C A ) and scopolin (6-methoxy-7- glucosylcoumarin) increased in callus tissue cultures of black root rot-resistant tobacco varieties. They also reported that C A and scopoletin (6-methoxy-7-hydroxycoumarin), phenolics present in tobacco root tissue, were toxic to the black root rot fungus in laboratory bioassays. We, therefore, have investigated the levels of the major tobacco phenolics in roots of susceptible and resistant varieties grown in black shank infected and disease free fields. Also, a number of tobacco root phenolics and related compounds were tested for inhibition of growth of the black shank fungus in a laboratory bioassay. MATERIALS AND METHODS Tobacco was grown in 1989 at the University of Georgia Coastal Plain Experiment Station, Tifton, G A , under standard cultural practices. Varieties ( N C 2326, N C 82, McNair 944, Coker 371, and V A 509) were grown in both a black shank infected field and in a disease free field. Susceptible varieties were sampled in July, while resistant varieties were sampled in August. The soil was gently washed from the roots. Single roots, attached to the main tap root and possessing visible signs of disease incidence (as indicated by brown discoloration), were detached. Root hairs were removed and the entire root length was cut into 1-cm segments and each segment was immediately frozen in dry-ice. Roots ranged from 4-7 mm in diameter at their point of attachment to 2-3 mm at their tip end (distal end). The segments were freeze-dried and chopped with a sharpened spatula. Each segment was extracted with 2.5 m L M e O H (containing 0.14 mg 5,7-dimethoxycoumarin as ISTD) by ultrasonication for 10 min. Then, 2.5 m L water were added and the solution ultrasonicated for an additional 10 min. The samples were filtered and analyzed by high performance liquid chromatography, as described before (4). Briefly, a Waters uBondapak C18 column was used with a concave gradient solvent program from 13% M e O H / H O (containing 0.08 M K H P 0 buffer adjusted to p H 4.45) to 50% M e O H / H 0 . A Hewlett-Packard 1040 Diode Array Detector, set at 340 nm, was employed. A typical chromatogram of a tobacco root extract is shown in Figure 1. The laboratory bioassay of Phvtophthora parasitica var. nicotianae was similar to that already described (5). Stock P. parasitica var. nicotianae was grown on V - 8 juice agar. Additional V-8 juice agar was prepared, autoclaved, cooled to 45°C, and the test compounds were added in quantities to prepare 250 m L solutions of each concentration of test compound. The agar and chemicals were mixed well, dispensed into 60X15 mm plastic petri plates, and allowed to solidify. Each concentration of test material was replicated ten times. Plugs of the fungus culture, cut out with a 5mm #2 cork borer, were placed with fungal culture side down on the edge of the test plates. Plates were placed in Ziploc bags and incubated at 27°C for 14 days or until fungal growth had reached the opposite edge of the agar control plates. Radial mycelial growth was measured and the percent inhibition of growth determined. Scopolin was isolated from freeze-dried, flue-cured tobacco roots. The ground roots were extracted with M e O H and the extract was separated by silicic acid column chromatography, eluted first with ethyl acetate and then with acetone. The acetone eluent contained the scopolin, which was purified by preparative reverse-phase C18 chromatography on a Waters PrepPak 500 C18 column, using a 35% M e O H / H 0 solvent. 2

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In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


390

NATURALLY OCCURRING PEST BIOREGUIATORS

Q U J o Q. 0

U

01

10 T 1 me

Figure 1.

20 (min,

30

High Performance Liquid Chromatogram of Tobacco Root Phenolics.


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27. SNOOK ET AL.

Black Shank Disease Fungus

R E S U L T S A N D DISCUSSION


Tobacco Root Chemistry Versus Fungal Attack The major phenolic compound in tobacco roots (Figure 1) is chlorogenic acid ( C A ) , correctly termed 3-O-caffeoylquinic acid (3-O-CQA). Only minor to trace amounts of the 4- and 5 - O - C Q A isomers are found in the roots, but occur in larger quantities in the leaves. Two coumarin derivatives are found in major amounts in root tissue: scopoletin (6-methoxy-7-hydroxycoumarin) and its glucoside, scopolin. Both C A and scopolin can reach levels of 1% of the dry weight of the root. Very little scopolin or scopoletin is found in the leaves. Roots from resistant and susceptible plants, grown in disease free and infected fields were analyzed for their phenolic contents. Whole roots were removed from the tap root, cut into 1-cm segments and analyzed for C A , scopolin, and scopoletin (Figures 2 and 3). It was determined that C A and scopolin remained relatively constant throughout the entire length of a root, from point of attachment at the tap root to the distal end (Figures 2a and 2b). Scopoletin however, tended to increase towards the distal end of the root. Both resistant and susceptible tobacco roots gave these same trends. The fungus attacks the roots of the plant by first entering the root hairs and then advancing towards the tap root. The disease will eventually engulf the tap root and lower stalk, becoming visible as a brown to black coloration on the stalk surface; hence, the name "black shank". Frequently, one observes apparently healthy roots attached to an infected tap root with infection advancing from the tap root outward to the root tip. Roots, with varying degrees of fungal attack (as indicated in Figures 2c and 3a-3d), were analyzed for their phenolics. Roots, under pressure of fungal attack, snowed different patterns of phenolics in their tissues, than roots grown in disease-free fields. Chlorogenic acid, scopolin, and scopoletin all increased significantly in tissue adjacent to visibly-infected tissue. This trend was consistent whether the infection was progressing from the tip end (Figure 2c), from the tap root outward (Figures 3a,3b,3c), or had entered a root hair at the midsection of the root (Figure 3d). Both susceptible (Figures 2c,3a) and resistant (Figures 3b-3d) varieties showed similar increases in these phenolics in root tissues adjacent to infection. Also, there was no difference in flue-cured (Figures 3b,3c) versus burley (Figure 3d) resistant varieties. Gasser et al. (3) found that callus cultures of resistant tobaccos gave an increase in phenolics, while susceptible tobaccos gave a decrease, when challenged by black root rot. In contrast, both resistant and susceptible whole root tissue showed increases in phenolics, when challenged by black shank. Perhaps, callus cultures infected with black shank would show the same effect observed by Gasser for black root rot. Laboratory Bioassays of Tobacco Root Compounds The reported activity of C A and scopoletin in laboratory bioassays of black root rot (3) prompted us to examine these and related compounds for activity towards black shank. C A and scopoletin were tested for inhibition of growth of black shank in our laboratory bioassay (Table I). Chlorogenic acid produced a 25% inhibition of growth at the highest level tested (4000 ppm), while scopoletin gave 39% inhibition of growth at a 1000 ppm dosage rate. The constituent parts of chlorogenic acid (caffeic and quinic acids) were also tested (Table I) and showed that the activity of chlorogenic acid lies in the caffeoyl moiety of the molecule.


391


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NATURALLY

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OCCURRING PEST BIOREGULATORS

10

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ROOT SECTION # (HIGHEST # DISTAL END) - G A (Y1) •SCOPOLIN (Y1)

McNair 944 Root Healthy Disease Free Field

•SCOPOLETIN (Y2)

% DRY WGT. (Y2)

% DRY WGT. (Y1)

0.07

0.4

2b

0.06

0.3

0.06 0.04

0.20.03 0.02

0.1 -

0.01 0 1

2

3

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6

6

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10

ROOT SECTION # (HIGHEST # DISTAL END) NC 2326 Root A Black Shank Nursery % DRY WGT. (Y1)

% DRY WGT. (Y2)

CA (Y1) S C O P O L E T I N (Y2) SCOPOLIN (Y1) DISEASE AREA

0.6 -

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ROOT SECTION # (HIGHEST # DISTAL END) Figure 2.

Root Levels of Phenolics in Healthy Resistant Varieties (2a-2b) and a Black Shank Infected Susceptible Tobacco Variety (2c).



CA (Y1)

Figure 3.

-e-

SCOPOLIN (Y1)

S C O P O L E T I N (Y2)

-*-

DISEASE A R E A

ROOT SECTION # (HIGHEST # DISTAL END)

Coker 371 Black Shank Nursery

Levels of Phenolics in Black Shank Infected Tobacco Roots of Different Varieties.

ROOT SECTION # (HIGHEST # DISTAL END)

N C 2326 Root B Black Shank Nursery


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NATURALLY OCCURRING PEST BIOREGULATORS

TABLE I


Dosage (ppm) 31.25 62.5 125 250 500 1000 2000 4000 a

% I N H I B I T I O N O F GROWTH O F B L A C K SHANK FUNGUS ( R a c e 0 ) BY T O B A C C O ROOT P O L Y P H E N O L S

Chlorogenic Acid

Quinic Acid

1 1 2 2 6 11 14 25

1 2 0 4 11 4 11 NT

Caffeic Acid 0 0 0 25 59 71 74 NT

Scopoletin

0 0 0 17 38 39 NT NT

a

NT - n o t t e s t e d

Laboratory Bioassavs of Phenolic Acids The high activity observed for caffeic acid was further investigated by studying the activity of a number of related compounds, in order to determine structure-activity relationships. Table II compares the activities of mono-hydroxycinnamic acids to caffeic acid (3,4-dihydroxy-cinnamic acid). A l l of the mono-hydroxy acids were more active than caffeic acid. o-Hydroxycinnamic (o-coumaric) acid was the most active and gave 91% inhibition of fungal growth at only 62.5 ppm. As p-hydroxy-cinnamic acid can exist in either the cis or trans configuration, both were tested. The data (Table III) showed that the cis-isomer was more active than the trans-isomer. Saturation of the double bond in the aliphatic portion of the molecule was investigated by testing the dihydro-derivatives of p-hydroxycinnamic and 3,4-dihydroxycinnamic acids. The dihydro-acids were slightly more active than the corresponding cinnamic compounds. Laboratory Bioassavs of Coumarins The activities of scopoletin (6-methoxy-7-hydroxycoumarin) and esculetin (6,7-dihydroxycoumarin) were compared to each other and to those of their respective glucosides, scopolin and esculin (Table IV). The dihydroxy-coumarin was found to be just as active as the methoxy-hydroxy-compound. Thus, the presence of a free hydroxyl group at position-6 is not required for activity. Surprisingly, substitution of a glucose on the hydroxyls of both compounds resulted in the complete loss of activity. Gasser (3) found similar results with the activity of scopolin towards black root rot. SUMMARY We have found compounds (chlorogenic acid and scopoletin) in tobacco roots that inhibit the growth of the black shank fungus in a laboratory bioassay.



37 57 75 100 100

14 39 87 100 100

62.5

125

250

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1000

R= - C H = C H - C 0 0 H

17

m-Coumaric

0

p-Coumaric

100

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96

91

65

o-Coumaric

% I N H I B I T I O N O F GROWTH O F B L A C K SHANK FUNGUS BY H Y D R O X Y - C I N N A M I C A C I D S

31.25

Dosage (ppm)

TABLE I I .

78

75

22

2

0

0

Caffeic



R-

% I N H I B I T I O N O F GROWTH OF B L A C K SHANK FUNGUS BY H Y D R O X Y - C I N N A M I C A C I D S

-CH-CH-COOH

TABLE I I I .



T A B L E I V . % I N H I B I T I O N O F GROWTH O F B L A C K SHANK FUNGUS BY COUMARINS


NATURALLY OCCURRING PEST BIOREGULATORS

398


However, levels of these compounds in roots of disease-free, resistant, and susceptible varieties were comparable. Although levels of these compounds have been shown to increase in roots in response to infection, the levels of increase were similar in resistant and susceptible roots. Free phenolic acids were shown to be very active in inhibiting the growth of black shank. Free phenolic acids do not occur in tobacco, but could play a role in resistance. Perhaps, they are produced at the narrow point of attack of the fungus. Because of their high activity, only small amounts would be needed to block the advance of the fungus through the root. Further research is needed to determine if this is a viable mode of resistance and to search for other chemicals that may be responsible for the observed black shank resistance of certain tobacco varieties. L I T E R A T U R E CITED 1. 2. 3. 4. 5.

Fritig, B.; Hirth, L. ActaPhytopathol.Acad. Sci. Hung. 1971, 6, 21-29. Cohen, Y.; Kuc, J. J. Phytopathology 1981, 71, 209. Gasser, R.; Kern, H.; Defago, G. J. Phytopathology 1988, 123, 115-123. Snook, M . E.; Mason, P. F.; Sisson, V. A. Tob. Sci. 1986, 30, 43-49. Csinos, A. S.; Fortnum, B. A.; Gayed, S. K.; Reilly, J. J.; Shew, H . D. In Methods for Evaluating Pesticides for Control of Plant Pathogens; Hicky, K. D., Ed.; APS Press: St. Paul, MN; pp 231-236.

RECEIVED July 18, 1990


Black Shank Disease Fungus - ACS Symposium Series (ACS

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