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Effects of volatile substances released by foliage of Abies balsamea
JOURNAL
OF
INVERTEBRATE
Effects
PATHOLOQY
of Volatile
19,
32-35
(1972)
Substances Abies
Released
by Foliage
of
balsamea
W . A. SMIRNOFF Laurentian
Forest
Research
CentTe,
Department of the Environment, Quebec, Canada
P.O. Box 3800, Ste-Foy,
Quebec IO,
Received May 3, 197i
The principal substances released by the foliage of Abies balaamea (pinene a! and 8, limonene, phellandrene, fenchone, and thujone) were tested on a few entomopathogenie species of Bacillus “cere-us group” and on several common bacteria such as Escherichia coli, Pseudomonas sp., and Staphylococcus sp. E. wli proved to be the most resistant of all the bacteria tested, and B. cereus was the most resistant entomopathogenic species. The results of these experiments conducted under laboratory conditions brought light on the causes responsible for the B. thuringienais inactivation observed in balsam fir stands during the course of treatments against insect pests feeding in these stands. The strong bactericidal effect of the substances tested suggests the possibility of using them for sanitation.
INTRODUCTION
Thus, the effect of each of the main composing substances released by the foliage of A. balsamea was determined for certain species of the entomopathogenic bacilli of the “cereus group” and some bacteria commonly encountered in our environment.
The specific action of certain substances produced by the foliage of trees on living organisms has aroused interest in recent years. These substances, mostly ethers, aldehydes, and terpenes, can have bactericidal, bacteriostatic, mutagenic, or stimulating properties. Such phenomena are extremely important in the eflicacy of entomopathogenie microorganisms, some of which, such as Badus thuringienti, are now used extensively in insect control programs in the field. Several authors have observed that foliage extracts have a bactericidal effect on B. thuringiensis (Gukasian, 1958; Kushner and Harvey, 1962; Smirnoff and Hutchison, 1965; Maksymiuk, 1970). Also, it was demonstrated that the volatile substances released by 34 species of plants have a specific spectrum of inhibition on 6 varieties of B. thuringiensis and B. cereus (Smirnoff, 1967). In addition to evaluating the resistance of the entomopathogens to these volatile substances, we are also attempting to determine their activity against microorganisms in general and their possible use in disinfection of the environment. Copyright 0
MATERIAL AND METHODS Concentrates of the following substances naturally released by the foliage of A. balsamea in a gaseous form were tested: pinene a and 8, limonene, phellandren, fenchone, and thujone. The substances were tested on entomopathogenic spore-forming B. cereus, on the spore and crystalliferous forms of B. thuringiensis and B. ckndrolimus, and on the following common bacteria: Staphylococcus aureus, Streptococcus faecalis, Sarcina lutea, Bacillus subti1i.s.~ The enterobacteria Escherichia wli, Proteus vulgaris, and Serratia marcescens and the pseudomonad P. aeruginosa were also tested. The bacteria concentration in nutrient 1 These bacteria were kindly supplied by Dr. L. Gauvreau, Director, Department of Bacteriology, Faculty of Medicine, Lava1 University, Quebec, P.Q. 32
1972 by Aosdemic
Press,
Inc..
SUBSTANCES
broth was determined by counting the viable cells on agar and by nephelometry. First, the effect of each substance at. concentrations of l/250, l/500, l/1000, and l/2000 was determined on the growth of each bacterium
FROM
iYtbif?S
33
in nutrient broth containing 1 X lo4 cells/ ml. Eecacse the tested substances were lighter than water and have specific degrees of suspension in nutrient broth, they always tend to remain on top of the medium. Thus:
FIG. 1. Degree of inhibition of the four bacteria after 1,2,3, and 4 hr of exposure to the substances in nutrient broth. B. c. = Bacillus cereus; B. th. = Bacillus thuringiensis; E. c. = Escherichia c&i; S. a. = Staphylococcus aureus. Period of exposure: top left quadrant of each dish, 1 hr; top right, 2 hr; lower left, 4 hr; and lower right, 3 hr.
34
SMIRNOFF
shaking during incubation was made to ensure a uniform spreading of the substance in the medium, which in turn favors a permanent contact of the bacteria and the substance and prevents any loss from volatilization. Turbidity was measured at the beginning of the tests and at 24-hr periods for a week. The total absence of growth after seeding on nutrient agar was the criterion for inhibition. Also, the effects of the same concentrations of the substances were studied on B. cereus, B. thuringiensis, E. coli, and 8. aureuS over a definite period of time. The bacteria were incubated in nutrient agar with the same concentrations of the substances used above and reseeded hourly during an S-hr period on nutrient agar (Fig. 1). The count of colonies served to determine the inhibiting effect of the substances in the shorter period of time. Smeared preparations of the entomopathogenie bacteria were examined frequently to determine whether the tested substances had effects other than bactericidal. Modifications in spore germination, cell development, spore and crystal formation, and appearance of sporoblasts were some of the features observed. Eight replicates were made for each test, representing some 720 tests altogether. continuous
TABLE
RENJLTS
AND
DISCUSSION
The tests in nutrient broth revealed that each species of bacteria had a different response to the action of each substance tested. The spore-forming, entomopathogenic species of Bacillus and B. subk%s were more susceptible to the substances than the other bacteria studied (Fig. 1). B. cereus was more resistant than B. thwingieti and B. dendrolimus to the substances except for phellandrene and thujone. These two substances had a strong inhibiting effect on the bacilli tested and on P. vubati and S. jaeca& at concentrations of l/4000 and l/8000 (Table 1). Also, it was observed that E. coli and P. aeruginosa were more resistant than 8. marcescens, P. vulgar& S. aureus, S. lutea, and S. faecuZi.s (Table 1). Arbitrary indices of resistance were determined for each substance by determining the average minimal concentration required to inhibit bacteria. Thujone proved to be the most powerful and was given a value of 1.0. The substances were listed as follows: thujone, 1.0; phellandrene, 0.64; fenchone, 0.47; limonene, 0.36; /3-pinene, 0.24; rw-pinene, 0.19. Indices of resistance were also calculated for the nonentomopathogenic and the entomopathogenic bacteria tested. E. coli, the 1
THE CONCENTRATIONS OF VARIOUS SUBSTANCES IN FOILAGE OF Abies bakamea INACTIVATION OF THE TESTED BACTERIA Substsnces from A&s
WHICH PBOVOKED TOTAL
balsamea foliage
Bacteria tested
Serratia marcescens Sarcina lulea Staphylococcus aweus Streptococcus faecalis Proteus vulgaris Pseudomonas aeruginosa Escherichia coli Bacillus subtilis Bacillus cereus Bacillus dendrolimus Bacillus thuringiensis
Limonene
a-Pinene
p-F’inene
Phellandrene
1:500 1:2ooo 1:2cClo 1:2ooo 1:lcQo Nil 1:2ooo 1:4000 1:looo 1:4oLIo 1:4CKIo
1:1ocJo 1:500 1:2ooo 1:looo Nil Nil 1:500 1:2oocl 1:1ooo 1:2Doo 1:2uoo
Nil 1:2cQo 1:2000 Nil Nil Nil 1:loLIo 1:2ooo 1:2GQo 1:4ooo 1:2uoo
1:looo 1:looo 1:4ooo 1:lOoo 1:4ooo 1:500 1:500 1:4ooo 1:8ooo 1:2ooo 1:2000
Fe&one 1:2Cloo 1:2000 1:1000 1:mo 1:2000 1:500 Nil 1:8ooo 1:moo 1:4000 1:4mo
Thujone 1:4oocl 1:8000 1:4oGu 1:8cOo 1:2ooo 1:4ooo 1:500 1:8ooo 1:8oMl 1:4000 1:4000
SUBSTANCES
most resistant species, was given a value of 1.0. The bacteria were rated as follows: E. coli, 1.0; P. aeruginosa, 0.90; S. marcescens, 0.53; P. vulgaris, 0.50; S. aweus, 0.30; 8. lutea, 0.29; S. faecdis, 0.28; B. cereus, 0.22; B. thuringknsis, 0.16; B. subtilis, 0.16; B. dendrolimus, 0.15. Indices of resistance were also determined for the entomopathogenic bacteria alone. The most resistant species, B. cereus, was given the value 1.0, followed by B. thuringiensis 0.80, and B. dendrolimus 0.73. Examination with the microscope revealed that cell development and spore and crystal formation remained normal where no inhibition was observed. The above results were confirmed by tests on the effect of the studied substances for definite periods of bime on B. cereus, B. thuringiensis, E. coli, and S. aureus growing on nutrient agar. It was observed that the two bacilli were more sensitive to the action of the substances than S. aureus and E. coli and that B. thuringiensis was more rapidly inhibited by the substances t.han B. cereus (Fig. 1). This has already been demonstrated (Smirnoff, 1967). Also, cell growth and spore and crystal formation remained normal. COBCLUSIONS
The results obtained revealed the effect and the role that. the substances studied might have on the survival of entomopathogenie bacilli under laboratory conditions. These results, along with those of other studies conducted on the survival of B. thuringietis in six forest types of the Province of Quebec (unpublished), permits the statement that the efficacy of large-scale sprayings with commercial preparations of B. thuringiensis depends upon the survival of this bacillus in the field. Thus, in the light of these findings it will be necessary to modify the efficiency standard and the dosage of the bacillus with regard to the inhibiting effect of the plant substances for
FROM
Abies
35
specific phytocoenosis. The degree of survival of microorganisms will not be the same in a stand of balsam fir, in an apple orchard, and in a field of carrots or onions. The following recommendations would ensure greater success in commercial applications of B. thuringiensis: the nominal dosage of B. thuringienzis in stands of A. balsamea should be increased and a protect,ive coating on the spores of the bacillus should be used. Geon latex proved to be a good sticking agent, causing the spores to stay on the foliage, but its action as a protective coating agent. against t,he inhibiting action of the plant. foliage should be investigated further.
The author wishes to express his sincere thanks to Dr. F. Bender, Forest Products Laboratory, Department of Fisheries and Forestry, Ottawa, for supplying concentrated extracts of the substances and for his criticism of the manuscript, and to Mr. J. ValBro, Forest Research Laboratory, Quebec, for his technical assistance. REFEREXCES
W. G. 1959. Les phytoncides en medecine. Acad&mie des Sciences 02 l’llkraine, Kiev, pp. Z-39. (In Russian.) GUKASIAN, A. B. 1958. Met bactCriostatique et bactericide des feuillages et de leurs composants chiiques sur les maladies de Dendrolimus sibiricus. Rep. Siberian Branch of Academy of Sciences of USSR, 7, 85-101. (In Russian.) G. T. 1962. KUSHNER, D. J., AND H‘IRVEY, Antibact’erial substances in leaves; their possible role in insect resistance to disease. J.
DROBOT’KO,
Insecl
Pathol.,
4, 155-184.
B. antibacterial
MAKSYMICK,
Bacillus
1970. Occurrence and nature of substances in plants affecting thuringiensis and other entomogenous
bacteria. J. Inverkbr. Pathol., 15, SMIRNOFF, W. A. 1967. Effet des volatiles Bmises par le feuillage sur la survivance de 6 vari&& Bacillus SMIRROPF,
cereus. Phytoprotection, W. A. AND HUTCHISOX,
356-371.
substances des plantes du group0
48, 119-127. P.
M.
1965.
Bacteriostatic and bactericidal effects of extracts of foliage from various plant species on Bacillus thuringiensis var. thuringiensis Berliner. J. Insect Pathol., 7, 2X3-280.
OF
INVERTEBRATE
Effects
PATHOLOQY
of Volatile
19,
32-35
(1972)
Substances Abies
Released
by Foliage
of
balsamea
W . A. SMIRNOFF Laurentian
Forest
Research
CentTe,
Department of the Environment, Quebec, Canada
P.O. Box 3800, Ste-Foy,
Quebec IO,
Received May 3, 197i
The principal substances released by the foliage of Abies balaamea (pinene a! and 8, limonene, phellandrene, fenchone, and thujone) were tested on a few entomopathogenie species of Bacillus “cere-us group” and on several common bacteria such as Escherichia coli, Pseudomonas sp., and Staphylococcus sp. E. wli proved to be the most resistant of all the bacteria tested, and B. cereus was the most resistant entomopathogenic species. The results of these experiments conducted under laboratory conditions brought light on the causes responsible for the B. thuringienais inactivation observed in balsam fir stands during the course of treatments against insect pests feeding in these stands. The strong bactericidal effect of the substances tested suggests the possibility of using them for sanitation.
INTRODUCTION
Thus, the effect of each of the main composing substances released by the foliage of A. balsamea was determined for certain species of the entomopathogenic bacilli of the “cereus group” and some bacteria commonly encountered in our environment.
The specific action of certain substances produced by the foliage of trees on living organisms has aroused interest in recent years. These substances, mostly ethers, aldehydes, and terpenes, can have bactericidal, bacteriostatic, mutagenic, or stimulating properties. Such phenomena are extremely important in the eflicacy of entomopathogenie microorganisms, some of which, such as Badus thuringienti, are now used extensively in insect control programs in the field. Several authors have observed that foliage extracts have a bactericidal effect on B. thuringiensis (Gukasian, 1958; Kushner and Harvey, 1962; Smirnoff and Hutchison, 1965; Maksymiuk, 1970). Also, it was demonstrated that the volatile substances released by 34 species of plants have a specific spectrum of inhibition on 6 varieties of B. thuringiensis and B. cereus (Smirnoff, 1967). In addition to evaluating the resistance of the entomopathogens to these volatile substances, we are also attempting to determine their activity against microorganisms in general and their possible use in disinfection of the environment. Copyright 0
MATERIAL AND METHODS Concentrates of the following substances naturally released by the foliage of A. balsamea in a gaseous form were tested: pinene a and 8, limonene, phellandren, fenchone, and thujone. The substances were tested on entomopathogenic spore-forming B. cereus, on the spore and crystalliferous forms of B. thuringiensis and B. ckndrolimus, and on the following common bacteria: Staphylococcus aureus, Streptococcus faecalis, Sarcina lutea, Bacillus subti1i.s.~ The enterobacteria Escherichia wli, Proteus vulgaris, and Serratia marcescens and the pseudomonad P. aeruginosa were also tested. The bacteria concentration in nutrient 1 These bacteria were kindly supplied by Dr. L. Gauvreau, Director, Department of Bacteriology, Faculty of Medicine, Lava1 University, Quebec, P.Q. 32
1972 by Aosdemic
Press,
Inc..
SUBSTANCES
broth was determined by counting the viable cells on agar and by nephelometry. First, the effect of each substance at. concentrations of l/250, l/500, l/1000, and l/2000 was determined on the growth of each bacterium
FROM
iYtbif?S
33
in nutrient broth containing 1 X lo4 cells/ ml. Eecacse the tested substances were lighter than water and have specific degrees of suspension in nutrient broth, they always tend to remain on top of the medium. Thus:
FIG. 1. Degree of inhibition of the four bacteria after 1,2,3, and 4 hr of exposure to the substances in nutrient broth. B. c. = Bacillus cereus; B. th. = Bacillus thuringiensis; E. c. = Escherichia c&i; S. a. = Staphylococcus aureus. Period of exposure: top left quadrant of each dish, 1 hr; top right, 2 hr; lower left, 4 hr; and lower right, 3 hr.
34
SMIRNOFF
shaking during incubation was made to ensure a uniform spreading of the substance in the medium, which in turn favors a permanent contact of the bacteria and the substance and prevents any loss from volatilization. Turbidity was measured at the beginning of the tests and at 24-hr periods for a week. The total absence of growth after seeding on nutrient agar was the criterion for inhibition. Also, the effects of the same concentrations of the substances were studied on B. cereus, B. thuringiensis, E. coli, and 8. aureuS over a definite period of time. The bacteria were incubated in nutrient agar with the same concentrations of the substances used above and reseeded hourly during an S-hr period on nutrient agar (Fig. 1). The count of colonies served to determine the inhibiting effect of the substances in the shorter period of time. Smeared preparations of the entomopathogenie bacteria were examined frequently to determine whether the tested substances had effects other than bactericidal. Modifications in spore germination, cell development, spore and crystal formation, and appearance of sporoblasts were some of the features observed. Eight replicates were made for each test, representing some 720 tests altogether. continuous
TABLE
RENJLTS
AND
DISCUSSION
The tests in nutrient broth revealed that each species of bacteria had a different response to the action of each substance tested. The spore-forming, entomopathogenic species of Bacillus and B. subk%s were more susceptible to the substances than the other bacteria studied (Fig. 1). B. cereus was more resistant than B. thwingieti and B. dendrolimus to the substances except for phellandrene and thujone. These two substances had a strong inhibiting effect on the bacilli tested and on P. vubati and S. jaeca& at concentrations of l/4000 and l/8000 (Table 1). Also, it was observed that E. coli and P. aeruginosa were more resistant than 8. marcescens, P. vulgar& S. aureus, S. lutea, and S. faecuZi.s (Table 1). Arbitrary indices of resistance were determined for each substance by determining the average minimal concentration required to inhibit bacteria. Thujone proved to be the most powerful and was given a value of 1.0. The substances were listed as follows: thujone, 1.0; phellandrene, 0.64; fenchone, 0.47; limonene, 0.36; /3-pinene, 0.24; rw-pinene, 0.19. Indices of resistance were also calculated for the nonentomopathogenic and the entomopathogenic bacteria tested. E. coli, the 1
THE CONCENTRATIONS OF VARIOUS SUBSTANCES IN FOILAGE OF Abies bakamea INACTIVATION OF THE TESTED BACTERIA Substsnces from A&s
WHICH PBOVOKED TOTAL
balsamea foliage
Bacteria tested
Serratia marcescens Sarcina lulea Staphylococcus aweus Streptococcus faecalis Proteus vulgaris Pseudomonas aeruginosa Escherichia coli Bacillus subtilis Bacillus cereus Bacillus dendrolimus Bacillus thuringiensis
Limonene
a-Pinene
p-F’inene
Phellandrene
1:500 1:2ooo 1:2cClo 1:2ooo 1:lcQo Nil 1:2ooo 1:4000 1:looo 1:4oLIo 1:4CKIo
1:1ocJo 1:500 1:2ooo 1:looo Nil Nil 1:500 1:2oocl 1:1ooo 1:2Doo 1:2uoo
Nil 1:2cQo 1:2000 Nil Nil Nil 1:loLIo 1:2ooo 1:2GQo 1:4ooo 1:2uoo
1:looo 1:looo 1:4ooo 1:lOoo 1:4ooo 1:500 1:500 1:4ooo 1:8ooo 1:2ooo 1:2000
Fe&one 1:2Cloo 1:2000 1:1000 1:mo 1:2000 1:500 Nil 1:8ooo 1:moo 1:4000 1:4mo
Thujone 1:4oocl 1:8000 1:4oGu 1:8cOo 1:2ooo 1:4ooo 1:500 1:8ooo 1:8oMl 1:4000 1:4000
SUBSTANCES
most resistant species, was given a value of 1.0. The bacteria were rated as follows: E. coli, 1.0; P. aeruginosa, 0.90; S. marcescens, 0.53; P. vulgaris, 0.50; S. aweus, 0.30; 8. lutea, 0.29; S. faecdis, 0.28; B. cereus, 0.22; B. thuringknsis, 0.16; B. subtilis, 0.16; B. dendrolimus, 0.15. Indices of resistance were also determined for the entomopathogenic bacteria alone. The most resistant species, B. cereus, was given the value 1.0, followed by B. thuringiensis 0.80, and B. dendrolimus 0.73. Examination with the microscope revealed that cell development and spore and crystal formation remained normal where no inhibition was observed. The above results were confirmed by tests on the effect of the studied substances for definite periods of bime on B. cereus, B. thuringiensis, E. coli, and S. aureus growing on nutrient agar. It was observed that the two bacilli were more sensitive to the action of the substances than S. aureus and E. coli and that B. thuringiensis was more rapidly inhibited by the substances t.han B. cereus (Fig. 1). This has already been demonstrated (Smirnoff, 1967). Also, cell growth and spore and crystal formation remained normal. COBCLUSIONS
The results obtained revealed the effect and the role that. the substances studied might have on the survival of entomopathogenie bacilli under laboratory conditions. These results, along with those of other studies conducted on the survival of B. thuringietis in six forest types of the Province of Quebec (unpublished), permits the statement that the efficacy of large-scale sprayings with commercial preparations of B. thuringiensis depends upon the survival of this bacillus in the field. Thus, in the light of these findings it will be necessary to modify the efficiency standard and the dosage of the bacillus with regard to the inhibiting effect of the plant substances for
FROM
Abies
35
specific phytocoenosis. The degree of survival of microorganisms will not be the same in a stand of balsam fir, in an apple orchard, and in a field of carrots or onions. The following recommendations would ensure greater success in commercial applications of B. thuringiensis: the nominal dosage of B. thuringienzis in stands of A. balsamea should be increased and a protect,ive coating on the spores of the bacillus should be used. Geon latex proved to be a good sticking agent, causing the spores to stay on the foliage, but its action as a protective coating agent. against t,he inhibiting action of the plant. foliage should be investigated further.
The author wishes to express his sincere thanks to Dr. F. Bender, Forest Products Laboratory, Department of Fisheries and Forestry, Ottawa, for supplying concentrated extracts of the substances and for his criticism of the manuscript, and to Mr. J. ValBro, Forest Research Laboratory, Quebec, for his technical assistance. REFEREXCES
W. G. 1959. Les phytoncides en medecine. Acad&mie des Sciences 02 l’llkraine, Kiev, pp. Z-39. (In Russian.) GUKASIAN, A. B. 1958. Met bactCriostatique et bactericide des feuillages et de leurs composants chiiques sur les maladies de Dendrolimus sibiricus. Rep. Siberian Branch of Academy of Sciences of USSR, 7, 85-101. (In Russian.) G. T. 1962. KUSHNER, D. J., AND H‘IRVEY, Antibact’erial substances in leaves; their possible role in insect resistance to disease. J.
DROBOT’KO,
Insecl
Pathol.,
4, 155-184.
B. antibacterial
MAKSYMICK,
Bacillus
1970. Occurrence and nature of substances in plants affecting thuringiensis and other entomogenous
bacteria. J. Inverkbr. Pathol., 15, SMIRNOFF, W. A. 1967. Effet des volatiles Bmises par le feuillage sur la survivance de 6 vari&& Bacillus SMIRROPF,
cereus. Phytoprotection, W. A. AND HUTCHISOX,
356-371.
substances des plantes du group0
48, 119-127. P.
M.
1965.
Bacteriostatic and bactericidal effects of extracts of foliage from various plant species on Bacillus thuringiensis var. thuringiensis Berliner. J. Insect Pathol., 7, 2X3-280.