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Control of anthracnose disease of grape vines by garlic oil
N otes and brief articles
35°
observed to be the most effective agains t all the fungal species. Inhibitory effects were more pronounced against Rhiz octonia solani followed by F usarium chlamydosporum and Curvu laria luna ta. The growth inhi bition of th e test fungi by the volatile factors was stat istically significant (P = 0 '01 ) in relation to treatments. Microscopic observation showed tha t the volatile sub stances caused morphological alteration s such as stun ted mycelial growth, granulation, swelling and lysis of the fungal hypha e. In some cases hypha 1 tip s were highly branched compared with the cont rols. Sporulation of C. lunata and F . chlamydosporum was also ret ard ed considerably. The inhibition or stimulation of th e growth of the te st fungi is pre sumably caused by the produ ction of various volatile substances (Afifi, 1975, 1976 ; Cat ska et al., 1975), but it may be significant to note that the seeds of each plant do not act in a uniform manner, and each fungus shows variable sensitivities to the products from the different seeds. The authors are grateful to Dr R. Y. Roy and Dr B. Rai for suggestions and critic ism. Thanks are also due to the Head of the Botany Department, Banaras Hindu Uni versity for laboratory facilit ies and to the C.S.I.R. and U .G .C. Government of India for financial assistan ce.
REFERENCES AFIFI, A. F. (1975). Effect of volatile sub stances from species of Labiatae on rhizospheric and phyl losphe ric fungi on Phaseolus vulgaris. Phy topathologische Z eitschrift 83, 296-302. AFIFI, A. F. (1976). Volatile and gaseous exudates of eith er germinating seeds or roots of Okra plant in relation to Okra wilt disease caused by some Fusarium spp . Ze ntralblatt f ar Bakteriologie 131MFI, A. F. & EL-GINDY, A. A. (1978). Effect of volatile and gaseous exudates of both germi nating seeds and root s of some crops on spore germi nation of some soil fungi. Journal oj Indian Botanical Society 57, 266-271. CATSKA, V., AFIFl, A. F. & VANCURA, V. (1975). The effect of volatil e and gaseous metabolites of swelling seeds on germinatio n of fungal spores. Folia Microbiologica 20, 152-15 6. DENNIS, C . & WEBSTER, J. (1971). Antagonistic properties of species groups of Trichoderma. II. Production of volatile antibiotics. Transactions oj the British My cological Society 57, 41-48. HOLM, R. E. (1972). Volatile metabolites controlling germination in buried weed seeds . Plant Physiology, Lancaster 50, 293. VANCURA, V. & STOTZKY, G . (1971). Excr etio n of germinat ing plant seed. Folia Microbiologica 16, 512.
CONTROL OF ANTHRACNOSE DISEASE OF GRAPE VI NES BY GARLIC OIL S. V . AMONKAR AND L. VI J AY AL A K S H MI
Biology and Agriculture Div ision, Bhabha A tomic R esearch Cent re, T rombay , B ombay 400 085, Maharashtra, India
Antibacterial and medicinal properti es of garlic have been reviewed extensively by Cavallito & Bailey (1944) and Stoll & Seebeck (1951). The use of garlic oil as a mosquito larv icidal agent was shown by Amonkar & Reeves (19 70), and Amonkar & Banerji ( 1971). Murthy & Amonkar (1974), reported its antifungal property using 11 species of plant-pathogenic fungi. Tansey & Appleton ( 197 5) and Appleton & Tansey (197 5) conducted deta iled studies on the inhibitory property of garlic extract using several species of fungi . Agrawal ( 197 8) has used Allium spp. extr acts to demon strate similar effects on some fungal speci es. The present report deals with the feasibility of using garlic oil against Sphaceloma ampelinum de Bary, the causative agent of anthracnose disease in vineyards . Trans. Br. mycol. Soc. 73 (2), (1979). 0007-1536/79/2828-5560 $00.35
The pathogen was isolated from infected grapeleaves from Hyderabad. Throughout the investigations, glucose-phosphate-agar medum (glucose 1 %; Bacto-peptone 0'2 %; KH zP04 0'05 %; MgS04 .7H20 0'05 %; Bacto agar 1'5 %; distilled water 100 ml GPA) was used. The pathological material was isolated on GP A medium in Petri dishes and incubated at a room temperature of 27 ± 1 °C for 7 days. The ident ity of the causative agent, Sphaceloma ampelinum, was confirmed, after which the fungus was streaked on GPA slope s. The slopes were incubated at room temperature and the se served as the stock cultures of the pathogen for further tests. The mycelial growth inhibition te sts were done using 3-da y-old fungal cultures and GPA agar medium. Emulsion of garlic oil was obtained at
Printed in Great Britain
© 1979 The Brit ish Mycological Society
Notes and brief articles 20,uIJml concentration level with sterile Tween-Be as an emulsifying agent. This was prepared in sterile distilled water and added to 20 ml presterilized melted PGA agar in tubes kept at 45°, in order to give concentrations of 100, 200, 300, 400, 500 and rooo ppm levels. The plates were poured in triplicate and allowed to dry for some time in the incubator before inoculation. Central spot inoculations were made on non-treated as well as on treated GPA medium plates. These were then incubated at room temperature for 7 days. This experiment was repeated three times in order to confirm the findings. The spore germination inhibition tests were done similarly. In order to obtain spore material, the test pathogen was streaked in duplicate on two slopes and allowed to sporulate at room temperature. The spores were harvested using Tween-So as a surfactant. The spore suspensions were pooled and centrifuged at 3000 rpm for 15 min. The spore pellet thus obtained was washed three times in sterile distilled water and resuspended in 5 ml distilled water. This served as an inoculum for the inhibition tests. The same garlic oil concentrations were again prepared in melted GPA. Each concentration was used in triplicate and 0'2 ml of the spore suspension was added to each of the tubes. The contents of the tubes were thoroughly mixed and Petri dishes were poured, inoculated and incubated at room temperature. The results on spore germination were recorded after the microscopic examination of the dishes. This experiment was also repeated thrice. The results of the mycelial and spore germination inhibition tests are given in Table 1.
Table.
1.
351
These results clearly indicated that garlic oil at 200 ppm and above inhibited mycelial growth and spore germination of S. ampelinum. These data suggest that garlic oil can be used at desired dose levels above 200 ppm dose in spray programmes in vineyards in order to combat anthracnose disease caused by S. ampelinum. REFERENCES
AMONKAR, S. V. & REEVES, E. L. (1970). Mosquito control with active principle of garlic 'Allium sativum L.' Journal of Economic Entomology 63, 1172-1175. AMONKAR, S. V. & BANERJI, A. (1971). Isolation and characterization of larvicidal principle of garlic. Science 174, 1343. AGRAWAL, P. (1978). Effect of root and bulb extracts of Allium spp. on fungal growth. Transactions of the British Mycological Society 70, 439-441. ApPLETON, J. A. & TANSEY, M. R. (1975). Inhibition of growth of zoopathogenic fungi by garlic extract. Mycologia 67, 882-885. CAVALLITO, C. J. & BAILEY, J. H. (1944). Allicin, the antibacterial principle of Allium sativum. I. Isolation, physical properties and antibacterial action. Journal of American Chemical Society 66, 1950-1951. MURTHY, N. B. K. & AMONKAR, S. V. (1974). Effect of a natural insecticide from garlic (Allium sativum L.) and its synthetic form (Diallyl-disulphide) on plant pathogenic fungi. Indian Journal of Experimental Biology 12, 208-209. STOLL, A. & SEEBECK, E. (1951). Chemical investigations on allicin, the specific principle of garlic. Advances in Enzymology 11, 377-400. TANSEY, M. R. & ApPLETON, J. A. (1975). Inhibition of fungal growth by garlic extract. Mycologia 67, 40 9- 4 13.
Inhibitory doses of garlic oil for the mycelial development and spore germination of Sphaceloma ampelinum* Dose of garlic oil used (ppm) A.....
,-
°
100
Mycelium
+
+
Spores
+
200
3°0
400
_
500
1000
-, Growth or germination; -, no growth or no germination. * Each set had three replicates per dose; the experiments were repeated three times.
Trans. Br. mycol. Soc. 73 (2), (1979). 0007-1536/79/2828-5560 $00.35
Printed in Great Britain
© 1979 The British Mycological Society
35°
observed to be the most effective agains t all the fungal species. Inhibitory effects were more pronounced against Rhiz octonia solani followed by F usarium chlamydosporum and Curvu laria luna ta. The growth inhi bition of th e test fungi by the volatile factors was stat istically significant (P = 0 '01 ) in relation to treatments. Microscopic observation showed tha t the volatile sub stances caused morphological alteration s such as stun ted mycelial growth, granulation, swelling and lysis of the fungal hypha e. In some cases hypha 1 tip s were highly branched compared with the cont rols. Sporulation of C. lunata and F . chlamydosporum was also ret ard ed considerably. The inhibition or stimulation of th e growth of the te st fungi is pre sumably caused by the produ ction of various volatile substances (Afifi, 1975, 1976 ; Cat ska et al., 1975), but it may be significant to note that the seeds of each plant do not act in a uniform manner, and each fungus shows variable sensitivities to the products from the different seeds. The authors are grateful to Dr R. Y. Roy and Dr B. Rai for suggestions and critic ism. Thanks are also due to the Head of the Botany Department, Banaras Hindu Uni versity for laboratory facilit ies and to the C.S.I.R. and U .G .C. Government of India for financial assistan ce.
REFERENCES AFIFI, A. F. (1975). Effect of volatile sub stances from species of Labiatae on rhizospheric and phyl losphe ric fungi on Phaseolus vulgaris. Phy topathologische Z eitschrift 83, 296-302. AFIFI, A. F. (1976). Volatile and gaseous exudates of eith er germinating seeds or roots of Okra plant in relation to Okra wilt disease caused by some Fusarium spp . Ze ntralblatt f ar Bakteriologie 131MFI, A. F. & EL-GINDY, A. A. (1978). Effect of volatile and gaseous exudates of both germi nating seeds and root s of some crops on spore germi nation of some soil fungi. Journal oj Indian Botanical Society 57, 266-271. CATSKA, V., AFIFl, A. F. & VANCURA, V. (1975). The effect of volatil e and gaseous metabolites of swelling seeds on germinatio n of fungal spores. Folia Microbiologica 20, 152-15 6. DENNIS, C . & WEBSTER, J. (1971). Antagonistic properties of species groups of Trichoderma. II. Production of volatile antibiotics. Transactions oj the British My cological Society 57, 41-48. HOLM, R. E. (1972). Volatile metabolites controlling germination in buried weed seeds . Plant Physiology, Lancaster 50, 293. VANCURA, V. & STOTZKY, G . (1971). Excr etio n of germinat ing plant seed. Folia Microbiologica 16, 512.
CONTROL OF ANTHRACNOSE DISEASE OF GRAPE VI NES BY GARLIC OIL S. V . AMONKAR AND L. VI J AY AL A K S H MI
Biology and Agriculture Div ision, Bhabha A tomic R esearch Cent re, T rombay , B ombay 400 085, Maharashtra, India
Antibacterial and medicinal properti es of garlic have been reviewed extensively by Cavallito & Bailey (1944) and Stoll & Seebeck (1951). The use of garlic oil as a mosquito larv icidal agent was shown by Amonkar & Reeves (19 70), and Amonkar & Banerji ( 1971). Murthy & Amonkar (1974), reported its antifungal property using 11 species of plant-pathogenic fungi. Tansey & Appleton ( 197 5) and Appleton & Tansey (197 5) conducted deta iled studies on the inhibitory property of garlic extract using several species of fungi . Agrawal ( 197 8) has used Allium spp. extr acts to demon strate similar effects on some fungal speci es. The present report deals with the feasibility of using garlic oil against Sphaceloma ampelinum de Bary, the causative agent of anthracnose disease in vineyards . Trans. Br. mycol. Soc. 73 (2), (1979). 0007-1536/79/2828-5560 $00.35
The pathogen was isolated from infected grapeleaves from Hyderabad. Throughout the investigations, glucose-phosphate-agar medum (glucose 1 %; Bacto-peptone 0'2 %; KH zP04 0'05 %; MgS04 .7H20 0'05 %; Bacto agar 1'5 %; distilled water 100 ml GPA) was used. The pathological material was isolated on GP A medium in Petri dishes and incubated at a room temperature of 27 ± 1 °C for 7 days. The ident ity of the causative agent, Sphaceloma ampelinum, was confirmed, after which the fungus was streaked on GPA slope s. The slopes were incubated at room temperature and the se served as the stock cultures of the pathogen for further tests. The mycelial growth inhibition te sts were done using 3-da y-old fungal cultures and GPA agar medium. Emulsion of garlic oil was obtained at
Printed in Great Britain
© 1979 The Brit ish Mycological Society
Notes and brief articles 20,uIJml concentration level with sterile Tween-Be as an emulsifying agent. This was prepared in sterile distilled water and added to 20 ml presterilized melted PGA agar in tubes kept at 45°, in order to give concentrations of 100, 200, 300, 400, 500 and rooo ppm levels. The plates were poured in triplicate and allowed to dry for some time in the incubator before inoculation. Central spot inoculations were made on non-treated as well as on treated GPA medium plates. These were then incubated at room temperature for 7 days. This experiment was repeated three times in order to confirm the findings. The spore germination inhibition tests were done similarly. In order to obtain spore material, the test pathogen was streaked in duplicate on two slopes and allowed to sporulate at room temperature. The spores were harvested using Tween-So as a surfactant. The spore suspensions were pooled and centrifuged at 3000 rpm for 15 min. The spore pellet thus obtained was washed three times in sterile distilled water and resuspended in 5 ml distilled water. This served as an inoculum for the inhibition tests. The same garlic oil concentrations were again prepared in melted GPA. Each concentration was used in triplicate and 0'2 ml of the spore suspension was added to each of the tubes. The contents of the tubes were thoroughly mixed and Petri dishes were poured, inoculated and incubated at room temperature. The results on spore germination were recorded after the microscopic examination of the dishes. This experiment was also repeated thrice. The results of the mycelial and spore germination inhibition tests are given in Table 1.
Table.
1.
351
These results clearly indicated that garlic oil at 200 ppm and above inhibited mycelial growth and spore germination of S. ampelinum. These data suggest that garlic oil can be used at desired dose levels above 200 ppm dose in spray programmes in vineyards in order to combat anthracnose disease caused by S. ampelinum. REFERENCES
AMONKAR, S. V. & REEVES, E. L. (1970). Mosquito control with active principle of garlic 'Allium sativum L.' Journal of Economic Entomology 63, 1172-1175. AMONKAR, S. V. & BANERJI, A. (1971). Isolation and characterization of larvicidal principle of garlic. Science 174, 1343. AGRAWAL, P. (1978). Effect of root and bulb extracts of Allium spp. on fungal growth. Transactions of the British Mycological Society 70, 439-441. ApPLETON, J. A. & TANSEY, M. R. (1975). Inhibition of growth of zoopathogenic fungi by garlic extract. Mycologia 67, 882-885. CAVALLITO, C. J. & BAILEY, J. H. (1944). Allicin, the antibacterial principle of Allium sativum. I. Isolation, physical properties and antibacterial action. Journal of American Chemical Society 66, 1950-1951. MURTHY, N. B. K. & AMONKAR, S. V. (1974). Effect of a natural insecticide from garlic (Allium sativum L.) and its synthetic form (Diallyl-disulphide) on plant pathogenic fungi. Indian Journal of Experimental Biology 12, 208-209. STOLL, A. & SEEBECK, E. (1951). Chemical investigations on allicin, the specific principle of garlic. Advances in Enzymology 11, 377-400. TANSEY, M. R. & ApPLETON, J. A. (1975). Inhibition of fungal growth by garlic extract. Mycologia 67, 40 9- 4 13.
Inhibitory doses of garlic oil for the mycelial development and spore germination of Sphaceloma ampelinum* Dose of garlic oil used (ppm) A.....
,-
°
100
Mycelium
+
+
Spores
+
200
3°0
400
_
500
1000
-, Growth or germination; -, no growth or no germination. * Each set had three replicates per dose; the experiments were repeated three times.
Trans. Br. mycol. Soc. 73 (2), (1979). 0007-1536/79/2828-5560 $00.35
Printed in Great Britain
© 1979 The British Mycological Society