Folia Microbiol.39 (5), 409-414 (1994) Accumulation of Antifungal Compounds in Tea Leaf Tissue Infected with Bipolaris carbonum B.N. CHAKRABORTYand A. SAttA Plant PathologyLaboratory, Departmentof Botany, Universityof North Bengal P.O. - N.B.U. 734 430, Dr. - Darjeelin~ WestBenga~India ReceivedMay 13, 1994 ABSTRACT. Varietal resistance of tea towardsBipolaris carbonum was tested followingdetached leaf inoculationtechnique. Among the fourteenvarieties tested, three were found to be highlysusceptible,while other three were resistant. Leaf exudates and diffusatescollected from the resistantvarietieswere more fungitoxicthan those from the susceptibleones. Two antifungal compounds isolated from healthy and B. carbonum-infected tea leaves exhibited clear inhibition zones at RF 0.8 and 0.65, respectively,in a chromatographicbioassay.On the basis of their color reaction on TLC and UV-spectra these were identified to be catechinand pyrocatechol.Resistantand susceptiblevarietiesaccumulated439-510 and 187-212 lag/g fresh mass tissue of pyrocatechol,respectively,2 d after inoculationwithB. carbonum, whilea low concentration(45 -58 lag/g) of this compound was detectedin healthyleaf tissue. Tea (Camellia sinensis (L.) O. KUNTZE) is one of the most important plantation crops of India. It forms the backbone of the economy of North-East India. Foliar diseases (Sarmah 1960; Chakraborty 1987) drastically reduce the quality and quantity of tea production. Besides being perennial, it is likely that the tea plant in the course of its adjustment to varying environments, has also evolved a very effective defense mechanism which successfully wards off most of the fungal pathogens (Chakraborty et al. 1994). The biochemical mechanisms responsible for containment of fungal pathogens in the resistant interactions are undoubtedly multifold. Oligophenols are the major constituents of tea leaves (Wang 1991) and their antifungal activity against Pestalotiopsis theae has been reported (Chakraborty et al. 1989). In this communication, the involvement of antifungal compounds in determining resistance of tea to Bipolaris carbonum is discussed. M A T E R I A L S A N D METHODS Plant material. Fourteen varieties of tea plants (TV-1, TV-9, TV-14, TV-16, TV-17, TV-18, TV-19, TV-20, TV-23, TV-25, TV-26, Teenali-17/1/54, TS-449, CP-1) produced at the Tocklai Experimental Station, Jorhat (Assam) were collected from the clone house of Mohurgong and Gulma Tea Estate, Darjeeling (West Bengal) and grown in the Phytopathological Experimental Garden of the department as described by Chakraborty and Saha (1994). Fungal cultures. A virulent strain of Bipolaris carbonum (syn. Helminthosporium carbonum), anamorph of Cochliobolus carbonum NELSON (IMI 298762), was obtained from the departmental stock culture collection. It was originally isolated from the naturally infected leaves of tea plants grown in Gayabari Tea Estate, Darjeeling (Chakraborty 1987). Glomerella cingulata and Pestalotiopsis theae were obtained from the Tocklai Experimental Station, Jorhat. Helminthosporium oryzae, H. Maydis and H. sativum were obtained from the Indian Agricultural Research Institute, New Delhi. Inoculation technique and disease assessment. Detached leaf inoculation technique of Dickens and Cook (1989) was followed with modification for artificial inoculation of tea leaves. Fully expanded young tea leaves were detached from plants, washed with sterile distilled water, blotted and then placed in plastic trays lined with moist blotting paper. Conidial suspension of B. carbonum was prepared from 10-d-old culture and the conidia concentration was adjusted to 1/nL (i.e. 106/mL) by serial dilution and hemocytometer count. Twenty ttL droplets ( 2 - 4 per leaf) of conidial suspension of the fungus were placed on the adaxial surface of each leaf with a hypodermic syringe. In control sets, drops of sterile distilled water were placed on leaves. Each tray was covered with a glass lid and sealed with petroleum jelly in order to minimize the drying of drops during incubation. Assessment of inoculum infectivity and symptom development were done on the basis of percent drops that resulted in lesion production after 3 d of inoculation (Chakraborty and Saha 1989a). Observations were based on 50 inoculated leaves for each treatment and average of three separate experiments. 410 B.N.CHAKRABORTY and A. SAHA Vol. 39 Collection and bioassay of leaf diffusates. Drop diffusate technique of Muller (1958) was followed for collection of leaf exudates and leaf diffusates from six varieties of tea (TV-9, TV-16, TV-17, TV-18, TV-25, TV-26). Thirty young tea leaves of each variety were placed on moist blotting paper kept in plastic trays. Drops of spore suspension of B. carbonum and sterile distilled water drops were placed separately on the adaxial surface of leaves, covered with glass lid, sealed with petroleum jelly and incubated for 2 d at 28 ~ in the dark. Spore suspension as well as water drops were collected separately and centrifuged. Finally the diffusates (spore-free supernatant) and exudates (water drops) were passed through sintered glass fdter (G5) and their biological activities were evaluated on spore germination and germ-tube growth of B. carbonum following the method of Chakraborty and Saha (1989b). Extraction of antifungal compounds. Young tea leaves (50 g) of four varieties (TV-9, TV-18, TV-25, TV-26) were collected from the experimental garden and kept in plastic trays. Half of the leaves of each variety were inoculated with conidial suspension of B. carbonum as described earlier, while the other half was maintained as control in water. Both healthy and inoculated leaves of the varieties were harvested separately 2 d after inoculation, kept in Erlenmeyer flasks and vacuum-infiltrated with 40 % aqueous ethanol (15 mL/g fresh mass tissue) following the facilitated diffusion technique of Keen (1978). The flasks containing the plant tissue immersed in the ethanol solution were stoppered and placed on a rotary shaker (1.7 Hz, 25 ~ Shaking for 12 h removed most of the extractable antifungal compounds from tea leaves; these were separated by filtration and the filtrates were concentrated in vacuo to approximately one-half volume at 45 ~ The concentrated solution was extracted three times with ethyl acetate and the organic layers pooled and dehydrated with MgSO4. Both the aqueous and the ethyl acetate fractions were then concentrated in vacuo separately to dryness, dissolved in methanol (50 lag/g) and analyzed by TLC. Fungitoxicity assay. Ethyl acetate and water fractions of healthy and inoculated tea-leaf extracts were spotted on TLC plates (silica gel G), developed in chloroform-methanol (9:1, V/V) and the chromatogram inhibition assay (Hofmans and Fuchs 1970) was performed using B. carbonum as the test organism. Fungitoxicity was ascertained by the presence of inhibition zones, which appeared as white spots surrounded by blackish background of mycelia. Diameter of the inhibition zone(s) and RF values were noted. The color reaction with vanillin-H2SO4, FeCI3-K3Fe(CN)6, diazotized 4-nitroaniline and Folin- Ciocalteau reagent (Harborne 1973) on TLC plates at this RF were noted for comparison. Finally, silica gel from TLC plates corresponding to the inhibitory zones was scraped off, eluted with methanol and rechromatographed, and once again eluted with methanol. These eluates were used for determination of ED50 values of the antifungal compounds against B. carbonum. Relative antifungal activity of compound II was also tested on spore germination of P. theae, G. cingulata, H. oryzae, H. maydis and 11. sativum as described by Werder and Kern (1985). Spectral anatysis and quantification of antifungal compound 11. Antifungal compound II was examined by UV-spectrophotometer (Shimadzu 160) and the maximum absorption was determined. Quantification of this compound was done from UV-spectrophotometric curve by considering the molar absorption coefficient of 6 000 at 214 nm (Williams and Hemming 1988) and expressed in ~tg/g fresh mass tissue. RESULTS Pathogenicity test of B. carbonum. Pathogenicity of this organism was tested on fourteen tea varieties; B. carbonum was most virulent on TV-18, TV-9 and TV-17 but least on TV-26, TV-25 and TV-16 (Table I). Biological activities of exudates and diffusates of tea leaves. Diffusates collected 2 d after inoculation with B. carbonum from the resistant tea varieties (TV-26, TV-25, TV-16) were more fungitoxic than those from the susceptible varieties (TV-18, TV-9, TV-17). Leaf exudates also contained some fungitoxic substances (Table II). Characteristics of the antifungal compounds of tea leaves. Crude extracts of both the water fraction and the ethyl acetate fraction obtained from four varieties (TV-9, TV-18, TV-25, TV-26) were bioassayed for the detection of antifungal compound(s). The ethyl acetate fraction of healthy as well as B. carbonum inoculated leaves exhibited two inhibition zones I and II on the TLC plates (Fig. 1) bioassay. Ethyl acetate fraction from healthy extracts of all four varieties exhibited a prominent inhibition zone (I) at RF 0.80. In the case of inoculated leaf extract of TV-9 and TV-18, there was no evidence of such an inhibition zone (I); however, traces were evident in the case of inoculated leaves of resistant varieties (TV-25 and TV-26). On the other hand, the ethyl acetate fraction from infected leaf extracts 1994 A N T I F U N G A L C O M P O U N D S IN T E A L E A F T I S S U E 411 of all four varieties showed an inhibition zone (II) at RF 0.65. Appearance of these two antifungal zones were confirmed by three repeated experiments. The average diameter of these zones exhibited on the chromatogram inhibition assay are presented in Table III. Positive color reactions of the antifungal compounds I and II were evident on TLC plates after spraying with various chromogenic reagents (TabT a b k I. Pathogenicity test (lesion production, %) of B. carbonum on le IV). Compound I showed brown detached leaves of tea varieties (3 d after inoculation) color reaction when sprayed with vaniUin- H2SO4 which corresponded Variety % Vadety % with the RF value and color reaction of authentic catechin. Compound II Teenali-17/1/54 31.9 -+ 1.4 TV-18 58 +- 4.6 showed brick-red and deep-blue color TV-20 25.2 _+ 3.1 TV-9 56 -+ 3.1 when sprayed with diazotized 4-nitroCP-1 24.7 _+ 1.3 TV-17 52 -+ 1.5 aniline and Folin-Ciocalteau reagent, TV-23 21.6 +- 3.1 TV-19 38.6 +- 3.3 respectively. UV absorption spectrum TV-16 5.7 -+ 1.2 TV-1 38.6 -+ 1.0 of partially purified (by preparative TV-25 2.8 -+ 0.9 TV-14 37.0 _+ 4.1 TLC) compound II revealed 2max at TV-26 1.9 +-- 0.8 TS-449 34.5 _+ 2.4 214 and 276 nm which was identical with authentic pyrocatechol (Fig. 2). Fungitoxicity assay. With healthy extracts, catechin showed the highest fungitoxic activity in the spore germination assay (Table V). Pyrocatechol showed the highest fungitoxic activity in resistant varieties (TV-25, TV-26) 2 d after inoculation in comparison with the susceptible varieties (TV-9, TV-18). Relative antifungal activity of isolated pyrocatechol against six fungal species are presented in Table VI. Table II. Effect of leaf exudate and diffusate of resistant and susceptible tea varieties on germination and germ tube growth ofB. carbonum Varieties Germination b,d percentage Mean germ tube c,d length, lain E D E O E O 57 24.0 ( - 5 8 ) 66 29.0 ( - 56) 76 35.5 ( - 54) 109 57 ( - 4 7 . 6 ) 130 71 ( - 45.2) 122 79 ( - 35.2) E D E D E D 98 75 96 76 97 79 266 138 ( - 4 8 . 2 ) 249 176 ( - 2 9 . 3 ) 263 177 ( - 32.7) Resistant TV-26 TV-25 TV-16 Accumulation of pyrocatechol in tea leaves. Two days after inoculation maximum accumulation of pyrocatechol was detected in B. carbonum-infected tea leaf tissue. In the resistant varieties (TV-25, TV-26) pyrocatechol accumulated in greater amounts (439510 ~tg/g fresh mass) in comparison to that in the susceptible varieties (187212 ~tg/g; Table VII). Healthy leaf tissue contained a very low amount (45-88 ~tg/g) of this compound. Treatment a Susceptible TV-18 TV-9 TV-17 Distilled water (control) 98 (-22.6) (-20.5) ( - 18.5) 316 aE - exudate, D - diffusate; both collected after 2 d of incubation. bAveragc of 500 spores. CAverage of 50 germ tubes. dValues in parentheses indicate percentage reduction ( - ) in relation to exudate. 412 B.N. C H A K R A B O R T Y and A. SAHA Vol. 39 TV-18 TV-26 A B C D Fig. L T L C - plate bioassay of ethyl acetate fraction from healthy and B. carbonum infected leaf extracts of Camellia sinensis. A, C: healthy leaf extracts; B, D: infected leaf extracts. Table IIL T L C - plate bioassaya of the ethyl acetate fraction from healthy and B. carbonum infected leaf extracts of Camellia sinensis Varieties Treatment b Compound I (R F = 0.8) c Compound II (R F = 0.65) c Resistant TV-26 TV-25 Susceptible TV-18 TV'-9 Table IV. Color reaction of the antifungal c o m p o u n d s 1 and II in visible light after spraying with chromogenic reagents Chromogenic spray Vanillin - H2SO4 H I 22.5 5.5 4.2 11.2 H I 22.0 6.3 4.2 9.8 H I 18.2 0 0 4.0 H I 18.6 0 0 5.1 aAverage (of three replicates) diameter of the inhibition zone (ram); organism tested: B. carbonum; incubation period: 3 d. bH - healthy, I - infected (2 d after inoculation). CSolvent system: c h l o r o f o r m - m e t h a n o l (9:1, V/V). I II brown - F o l i n - Ciocalteau reagent - deep blue FeCI3 - K3Fe(CN)6 Diazotized 4-nitroaniline - blue brick red ANTIFUNGAL C O M P O U N D S IN T E A LEAF TISSUE 1994 I 1.5- I /, A I A i I ! 413 Table V. Fungitoxic effect (EDs0 a) of catechin and pyrocatechol isolated from different tea varieties in the spore germination test of B. carbonum Variety Treatment b Catechin Pyrocatechoi 1 I I 1.0 -- TV-26 I H 4.2 10.8 15.2 5.6 5.0 11.6 16.1 6.3 H 7.2 I _ c 34.6 18.6 H 8.4 I _ c I I TV-25 H I / TV-18 ! 0.5 t l ! I u TV-9 I ,A', / 0 I I '-'1 200 ~ 250 300 38.9 16.8 alnhibitory concentration equivalent to mg fresh mass of tea leaves causing 50 % reduction in spore germination. bH - healthy, I - infected (2 d after inoculation). CNot detected. nm VI. Relative antifungal activity of isolated pyrocatechol (50 mg/L) on spore germination of different fungi (inhibition a, %) Table Fig. 2. U V spectra of authentic pyrocatechol (A) and antifungal compound II (B) extracted from tea leaves (variety TV-26) inoculated with B. carbonum. Fungal species Table VII. Quantitative estimation of pyrocatechola in resistant and susceptible tea varieties inoculated with B. carbonura Varieties Healthy Infected Resistant TV-26 TV-25 88 62 510 439 Pestalotiopsis theae Glomerella cingulata Helminthosporium oryzae H. maydis H. sativum DISCUSSION Susceptible TV-18 TV-9 % b- 92 89 94 80 73 a o f spore germination (in relation to distilled water control). In the present investigation the drop diffusate method has proved outstandingly successful in evaluating biological activities of exudates and diffusates collected from a!ag fresh mass tissue; extraction 2 d after inthe leaves of resistant and susceptible tea varieties. Difoculation with B. carbonum. fusates obtained from resistant varieties after 2 d of inoculation with B. carbonum were more fungitoxic than the susceptible ones. Leaf exudates also contained some fungitoxic substances. Fungitoxicity of leaf diffusates has been implicated in the natural defense mechanism of plants against attack by a fungal pathogen in several instances (Purkayastha and Roy 1975; Sinha and Hait 1982; Chakraborty and Saha 1989b). The presence of antifungal compounds in the leaf extracts of Camellia japonica has also been established (Hamaya et al. 1984) and these were characterized as camellidin I and II (Nagata et al. 1985). However, leaves of both C. sinensis and C. sasanqua did not show the inhibitory activity. The drop diffusate method has often been criticized due to the low solubility of many antifungal compounds in pure water. Secondly, diffusates give no indication of the concentration of antifungal compounds within the inoculated tissues. Hence, the facilitated diffusion technique suggested by Keen (1978) was followed to detect the antifungal substance in tea leaves infected with B. carbonum. On the chromatogram inhibition assay catechin and pyrocatechol were found to be fungitoxic against 45 187 58 212 B. carbonum. Catechin (compound I) could be detected mainly from the healthy leaf extracts of TV-9, TV-18, TV-25 and TV-26 which exhibited a prominent inhibition zone at RF 0.80 on TLC plate and showed the highest fungitoxic activity in the spore germination assay. No such activity was evident with B. carbonum infected leaf extracts of susceptible varieties (TV-9, TV-18) but traces were evident in the 414 B.N. C H A K R A B O R T Y and A. SAHA Vol. 39 resistant varieties (TV-25, TV-26). Catechins are flavon-3-ols with two hydroxyl groups in the side ring. These include gallic acid esters with the acid moiety attached to the hydroxyl groups. The presence of four forms of catechins, epicatechin, epicatechin gallate, epigallocatechin, and epigallocatechin gallate have been reported from tea (Wang 1991). Antimicrobial activity of tea catechin to Streptococcus mutans was also demonstrated by Kawamura and Takeo (1989). Pyrocatechol (compound II) showed a positive color reaction of phenols with the chromogenic sprays and also exhibited an inhibition zone on TLC plate at RF 0.65 as well as markedly inhibited spore germination. This compound was identical to authentic pyrocatechol as determined by TLC and UV-spectrophotometry. It is known that catechin is oxidatively cleaved to simpler phenols and phenolic acids, such as pyrocatechol, phloroglucinol and protocatechuic acid (Sambandam 1982). The breakdown of the antifungal catechin was almost complete in the susceptible varieties but traces of catechin were evident in the resistant varieties after inoculation with B. carbonum. Accumulation of pyrocatechol in resistant varieties increased markedly in comparison with the susceptible varieties after 2 d of inoculation with B. carbonum. However, a low concentration of this compound was detected in healthy leaf tissues. In the susceptible varieties, although catechin is broken down completely, accumulation of the antifungal pyrocatechol is not greater than in the resistant varieties. It seems highly probable that the increased level of pyrocatechol is associated with differential responses of tea varieties against infection by B. carbonum. The authors wish to thank Professor M. 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