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Bacteriostatic and bacteriocidal effects of extracts of foliage from various plant species on Bacillus thuringiensis var. thuringiensis Berliner
JOURNAL
OF
INVERTEBRATE
7, 273-280
PATHOLOGY
Bacteriostatic Foliage
and from
(1965)
Bacteriocidal Various
thuriugiensis
var.
Plant
Effects
of
Extracts
Species
on
Bacittus
thuringiensis
of
Berliner’
W. A. SMIRNOFF AND P. M. HUTCHISON~ Forest
Research
Laboratory, Accepted
Sillery, July
Quebec,
,Canada
17, 1964
The influence of foliage from each of 74 species of plants on the development of Bacillus thuringiensis var. thuringiensis Berliner was studied. The plant juices and their extracts produced either complete inhibition, a delay in normal growth, or showed no effect on the Bacillus. Leaf juices of conifers produced more inhibition than those of the other plants tested. Results with ethyl acetate, ethyl ether, ethyl alcohol, and water extracts varied considerably both within and between the different groups of plants, but on the whole, acetate extracts had more influence on the growth of B. thuringiensis. Parallel tests showed that several undetermined species of bacteria encountered on the surface of the foliage had no effect on normal growth of the Bacillus. Similarly, the different pH values of the juice extracts added on agar surface were not an influential factor. The results suggest possibilities for further development of the methods used, and for the extension of this work with phytocides to other microorganisms normally present in the phytocoenosis, as well as those which are introduced.
of these substanceshave antibacterial properties. Antibacterial substancespresent in phytoOnly a few studies on the influence of tides of higher plants are attracting more and phytocides on bacterial entomopathogensare more attention, not only in the field of medknown and these deal with the aspect of inicine but also in various specialized fields of hibitory action: Gukasian (1958) studied the biology. Tokin (1963) reported that an influence of coniferous foliage on Bacillus “hectare” of deciduous trees liberated 2 kg of dendrolimus Talalaev, and Serratia marcesvolatile substancesin 24 hours while a forest cenSBizio, while Kushner and Harvey (1962) of conifers liberated 5 kg of these substances basedtheir studies on the effects of 10 species during the sameperiod. It is easily imagined of conifers and 8 speciesof deciduous trees that the quantity of volatile substanceslibon B. cereus Frankland and Frankland, diferated greatly increaseswhen billions of manferent strains of B. thuringiensis Berliner, and dibles destroy the foliage during an insect S. marcescens Bizio. invasion, and it is quite possible that some In 1961, a study of the effect of B. thuringiensis and its commercial preparations on 1 Contribution No. 1105, Forest Entomology and Pathology Branch, Department of Forestry, Ottawa, the activity of Choristoneura fumiferana Canada. (Clemens) indicated that phytocides might be 2 Fourth-year student, Department of Biology, of some importance (Smirnoff, 1963). In the Queen’s University, Kingston, Ontario, Canada; summer of 1963, an investigation was started summer seasonal employment at the Quebec Forest in the Chicoutimi region of Quebec Province R;search Laboratory. 273
274
SMIRNOFF
EFFECT
ON THE DEVELOPMENT TUICES FRO&I VARIOUS
AND
HUTCHISON
TABLE 1 OF Bacillus thuringiensis VAR. thuringiensis OF FOLIAGE EXTRACTS AND PLANTS AS DETERMINED THROUGH MICROSCOPIC ANALYSES~ Culture
Plant
Juice
PH
development
Water
on the spot
Alcohol
Acetate
Ether
Conifers Abies balsatnea (Linnaeus) Miller La& laricina (Du Roi) K. Koch Picea glauca (Moench) Voss
3.6
0 , B3 , 3 Spores only
3.6
h-
Picea wariana (Miller) Britton, Sterns, Poggenburg
4.9
Pinus banksiana Lambert Thuja occidentalis Linnaeus
4.9
S, B3 Spores only
Spores only Bx
Bi
0
0, B”
0
N
0
N
Spores only
0
0
0
4.5
N, B”
0, B”
0
0, Bl
5.2
N, B”
N
N
0
N
0
0
N
N
N
0
N
Deciduous
Trees
Acer pennsylvanicum Linnaeus
4.6
Acer rubrum Linnaeus
4.6
Acer spicaturn Lamar&
4.7
N
N
0, Bs
0
Cells only
5.5
N
N
N
0
3
4.5
N
N
0
0
ix
N
Cells only
s
Amelanchiev Wiegand
N
laevis
Betzda papyvifera Marshall Fagus grandifolia Ehrhart Populus alba Linnaeus Populus balsamifera Linnacus
5.3
N
5.2
N
Tests
not made
5.1
N
ix
0, Bs
0, B”
Popztlus tremuloides Michaux
5.3
N
N
N
Cells only
Prz~us pennsylvanica Linnaeus
5.5
N, Bs
s
0,B”
0, Bx
QUCYCIIS rzlbra Linnaeus var. borealis (Michaux fils) Farwell
4.3
N, B”
N, B”
0, Bx
0, B’
Robinia acacia
5.9
0, Bi
N
is
Cells only B’
N
Tilia americana Linnaeus
6.0
N,B’
N
N
iY
N
Ulmzts americana Linnaeus
4.6
N
N
0, B”
0
0
pseudoLinnaeus
EFFECTS
OF EXTRACTS
OF FOLIAGE
TABLE
ON
B. thuringiensis
1 (Continued) Culture
Plant
275
development
on the spot
Alcohol
Acetate
PH
Juice
Water
5.7
0, B’
Cells only
ix
0, B”
N
Anaphalis margaritacea (Linnaeus) C. B. Clarke
5.5
N, Bl
N, BY
0
0
0
Apocy~m mifoliz~m
Cells only
0
0
Herbaceous Achilles millefolizrnt Linnaeus
androsaeLinnaeus
Ether
Plants
6.4
N, Bl
Tests
Aster macrophyllus Linnaeus
j.9
S, Bx
N, Bl
Bvassica kaber (DeCandelle) L. C. Wheeler
6.0
N, Bl.3
Tests
Capsella bursapastoris (Linnaeus) Medicus
6.0
N, Bl.3
N
Spores only BX
Spores only
N
Chrysanthemum leucanthemum Linnaeus
5.8
N
N
N, Bs
0, B1
s
Cirsium (Savi)
5.3
N, Bl
N
N, B”
0, B1
N
5.6
N, BX
N
N, B3
N
N
N
Cells only
Spores only
N
0, BY
N, Bx
0, B”
N
vulgare Tenor0
Cornus canadensis Linnaeus Diervilla Miller
tonicera
Elymus cf. virginiczrs Linnaeus Epilobium angustifolium Linnaeus Eupatorium Linnaeus Fragaria niana Geranium Britton
not
not
made
made
5.2
N, BX
6.0
Spores only
6.2
N, Bx
N, B3
N
0
N
5.5
N, B’
N, B”
N, Br
N
N
N
N
Bl
macutatum cf. virgiDuchesne
4.6
N, I31
N
Cells only
4.8
N, B’
PI;
0, BX
0
N
4.9
N. B’
N
0, B3
0
0
bicknellii
Hieracium aurantiuciuwz Linnaeus Hieracium cf. porantinum Allioni Oenotheva biennis Linnaeus
5.3
N, B3
N
N, BX
0, BX
0
l
5.7
0, B3
0
N
0
s
Phleum pratense Linnaeus
5.7
N, B’
N
N, BX
0, Br
Cells only
Plantago major Linnaeus
5.6
N, B1
N
N
N
N
Polygonum convolL vulus Linnaeus
4.5
N
N
N, B*
Cells only
N
276
SMIRNOFF
TABLE
AND
HUTCHISON
1 (Continued) Culture
Plant
PH
Juice Herbaceous
Ranunculus Linnaeus
development
Water
on the spot
Alcohol
Acetate
Ether
Plants
acris 5.7
N
0
N, Bx
0, BX
N
Rzlbus cf. idaeus Linnaeus
5.2
N, BX
N, Bx
0
0
0
Rumen pallidus Bigelow
5.1
N, BX
X, Bx
Bx
N
0
Solidago canadensis Linnaeus
6.1
0, B’
N, B”
BX
N
0
5.2
AT, B”
0
Cells only
Cells only
0
5.8
N, B”
N
N
N
N
6.9
Bl
Tests
6.2
N
N
N, Bs
N
0
5.7
N, B’
N
N, Bs
0, B’
N
4.7
N
Spores only
0
N
5.4
N, Bx
N
0
0
5.1
N, Bx
O,BX
N
4.6
N, B
0, B” Spores only
0
0
6.5
N
N
0
N
Solidago graminifolia Linnaeus var. nuttallii (Greene) Fernald Tararacum oficinale Weber Trifolium agrarium Linnaeus Trifolium hvbridum Linnaeus Vicia cracca Linnaeus
not
made
Shrubs Alnus rugosa (Du Roi) Sprengel var. americana (Rogel) Fernald Amelanchier bartramiana (Taush) Roomer Cornus stolonifera Michaux Corylus cornuta Marshall Fraxinzls nigra Marshall Lonicera villosa (Michaux) Roomer & Schultes var. calvescens (Fernald 8; Wiegend) Fernald My&a gale Linnaeus Philadelphus cororzarizl.5 Linnaeus Pyrus melanocarpa (Michaux) Willdenow (Syn. Aronia melanocarpa (Michaux) Britton)
5.3
N, B”
N
N
N
5.5
N, B”
N
0
0
6.0
S, B”
N, Br
N
N
0. R
Few cells only
48
N
N
EFFECTS
OF EXTRACTS
OF FOLIAGE
TABLE
ON
PH
Juice
Rktis tyfihina Linnaeus
5 .o
N
Ribes glandulosum Grauer
4.5
N
Rosa blanda Aiton
4.8
N
Rosa rugosa Thumberg
4.7
Salix bebbiana Sargent Salix cf. discolor Muhlenberg
277
1 (Continued) Culture
Plant
B. thuringiensis
development
Water
Cells only
on the spot
Alcohol
Acetate
Ether
0
0
0
N, B”
0, B”
N
N, BY
N, B
0
N
N, I31
N, B”
Cells cnly
0
N
4.9
N, BX
N
N
N
5.3
0, B”
N, BX
Cells only
0
N
N
N
B3
Cells
Salix kumilis Marshall
5.0
N, BX
N
Cells only
Sorbus cf. decora (Sargent) Schneider
5.0
N
N
0
0
N
Syringa vzdgaria Linnaeus
5.5
N
N
0
N
N
Vaccinium angustifolit&m Aiton
3.8
0
N
0, B”
0, B-’
Viburnum Linnaeus
cassinoides 5.3
Spores only
Spores only
0, BY
0
5’iburnum Marshall
trilobum 5.0
N
N
0
0
Cells
Mosses Lycopodium Linnaeus Pleurozium (Willder)
and Lycopods
clavatum schreberi Nitten
Pteridium aquilinum var. latiusculum (Desvaux) Underwood
4.8
N, B’
N
Cells only Ba
6.2
N
0, BX
Cells only
5.3
N, B”
N
N
a Control: Among the 354 tests performed only four deviations were noted from normal development. They N, BX; the juice of Eapatorium maculawere: the juice of Circiztm v&are, N, Br, and its alcohol extract, tzlm, N, Br ; and the water extract of Oenotkeva biennis, 0. B, presence of other bacteria encountered on the surface of the foliage; N, normal development of Bacillus thuvingiensis var. thuringiensis; this indicates ,normal multiplication, sporulation, crystal synthesis, and lysis ; 0, complete inhibition of B. thuringiensis, ‘that is no growth. Gram stain procedure showed: Br, oval bacteria (gram +) ; Ba, short rods (gram +) ; B3, clostridiumhke of above bacteria. Tests showed that these bacteria (gram +) ; B4, long fine rods (gram -) ; B X, mixture bacteria produced no effect on the normal metabolism of B. tkuringiensis.
278
SMIRNOFF
AND
which included 74 species of plants growing in the region. The purpose of this work was to add to the list of plants studied with respect to their possible effect on bacterial metabolism, and point the way to methods that could be used for future work in this field. MATERIALS
AND
METHODS
A list of the plants tested is shown in Table 1. Plant material was obtained between mid- June and mid-August and approximately 60 g of foliage extract from plant species was prepared the day of collection. The foliage was mascerated in a Waring Blendor and 10-g lots were extracted with 25 ml of the following solvents: ethyl ether, ethyl alcohol, ethyl acetate, and water. The foliage was left in contact with the various solvents for 1 hour and then strained through a single layer of nylon cloth. Resulting extracts were placed in small weighing bottles and left to evaporate until 1 ml remained. In the case of the water extraction, where normal evaporation would be a long procedure, a vacuum evaporation system at 40°C was used. The concentrated extracts were stored at 0°C. Leaf juices were extracted by squeezing some of the remaining mascerated material through a single layer of fine-mesh nylon cloth; in some cases a garlic press lined with the nylon cloth was used. A small amount of juice was immediately placed on a bacteriaagar surface, prepared as described below. The pH of all foliage juices was determined, using a Coleman Companion pH meter, Model 31, with the following Beckman microelectrode attachments: a sealed one drop, and a Beckman sealed fiber-type calomel electrode assembly. Sterilizations through bacteriological filters were not made in order to maintain conditions as natural as possible. Filtering the extracts would have eliminated bacteria normally present on the plants, and might have retained certain substances, or might have broken the structure of others, which could
HUTCHISON
have resulted in changed properties of the substances. The culture of B. thuringiensis var. thuringiensis” was obtained by inoculating 100 ml of nutrient broth medium with the microorganism and incubating at 28°C for 18 hours. Plates were prepared by mixing 2 ml of this culture medium containing the Bacillus with 100 ml of warm nutrient agar (40°C). Disposable plastic tri-plate petri dishes containing the agar-bacteria mixture were used to test the effect of ethyl acetate, ethyl alcohol, and water extracts on bacterial development, by placing a drop of the substance on the bacteria-agar surface. In all cases, the bacteria growing on the spot area and the colonies which developed approximately 1 cm from the spot were examined microscopically. The Buffalo black-carbol Fuchsin method of staining (Smirnoff, 1962) was used to examine the phases of development of the Bacillus. Each analysis consisted of one slide on which there were two smears: on the right end, bacteria taken directly from the spot area; on the left end, bacteria which developed 1 cm from the region where the substance had been placed on the agar. This second smear served as a control and was separated from the first one by a clear space. During analysis, the presence or absence, and state of development of B. thuringiensis were recorded. The presence of cells, spores, and crystals of this microorganism, and the existence of other species of bacteria normally encountered on the foliage surface, were also noted. These other bacteria were later characterized by the Gram procedure. RESULTS
AND
DISCUSSION
Results of the various analyses appear in Table 1, and can be grouped into four categories: 3 We used a culture of Bacillus thuringiensis var. thuringiensis Berliner, obtained from Dr. T. A. Angus, Insect Pathology Research Institute, Sault
Ste. Marie, Canada.
EFFECTS
OF EXTRACTS
OF FOLIAGE
1. The tested substances completely inhibited the growth of B. thuringiensis and other bacteria present on the plant foliage. 2. The tested substances completely inhibited B. thuringiensis but had no effect on other bacteria present. 3. The tested substances affected normal development of B. thuringiensis in the following two ways: (a) normal development of cells; complete absence of spores and of crystals; (b) normal development of cells and spores; complete absenceof crystals. 4. The tested substanceshad no effect on
ON
B. thuringiensis
279
Linnaeus, Acer rubrum Linnaeus, and .4cer spicatum Lamarck; Salix bebbiana Sargent and Salix cf. disrolor Muhlenberg; Solidago canadensis Linnaeus and Solidago graminifolia Linnaeus. There are several cases of “sporulation only” mentioned in the table but this phenomenon was not checked by repeated analysis. However, the extract of Viburnum cassinoides Linnaeus that acted as a crystal inhibitor has been studied in detail since 1962 (Smirnoff, 1965) and the stability of the new character was proven. When B. thuringiensis was mixed with each B. thuringiensis. of the speciesof bacteria found on the plant The percentage of complete inhibition of foliage, or with mixtures of two, three, or B. thuringiensis by the juices and the various four of these species,there was no effect on indiextracts was distributed as follows for juices normal development of B. thwingiensis, and for each extract: juices, 8.6 percent; cating that inhibition in the development of is mainly caused by subwater extract 10 percent; ether extract, 30 B. thuringiensis percent; alcohol extract, 34.3 percent; and stancesin the extract since these do not affect ethyl acetate, extract, 65.7 percent. It is clear the other bacteria or vice versa. that the ethyl acetate, alcohol, and ether exThe values for the measurementsof the tracts were more efficient inhibitors of growth. zones of inhibition surrounding the sensi Control tests showed that the solvents them- discs (plant juices absorbed on discs) are not selveshad no effect on the normal metabolism indicated in the table because of the inacof the microorganism. curacy of such a method of testing when using It appears that the pH of the plant juices unsterilized plant juices. An alteration or deand extracts had no influence on the develop- lay in the development of B. thuringiensis ment of B. thwingiensis; for instance, juice frequently occurred, but this could only be of LaGx Zakina (Du Roi) Ii. Koch (pH 3.6) detected through careful microscopic examination since bacterial flora normally present inhibited crystal synthesis, juice of Picea gZauca (Moench) Voss (pH 3.6) did not on plants could develop in the zones where was inhibited. affect the development of the Bacillus, while growth of B. thuringiensis juice of Vaccinium angustifolium Aiton (pH It is possible that the plant substancesnot 3.8) completely inhibited B. thuringiensis. soluble in water may be as important in Parallel tests made by adding a spot of liquid affecting B. thuringiensis as those that are buffer (pH 3.5 to 8.0) to nutrient agar soluble. It is also possiblethat foliage changes showed no noticeable effect on the develop- its properties when eaten by insects, as a ment of B. thuringicnsis. result of oxidation and attack by enzymes of ’ Some plants of the same genus but of dif- the salivary glands and intestinal juices. Further investigations are required to determine ferent species had a different effect on B. thuringiensis and on other bacteria. This is whether these complex factors are important in affecting the bacteriostatic and bacterioparticularly true for the following: Picea tidal effects of foliage on B. thuringiensis. glauca and Picea mariana (Miller) Britton, Sterns, Poggenburg; Acer pennsylvanicum The aim of this preliminary work will be
280
~MIRN~FFAND
reached if it serves as a basis for further studies on methods that should be used in future research on the influence of plant phytotides on insect pathogens. Knowledge of the bacteriostatic and bacteriocidal qualities of various plants will serve to provide a more accurate estimate of the possible efficiency of microorganisms pathogenic to insects. Knowledge of these qualities of various plants could help to determine the dosage, the survival, and the possible dissemination of the pathogen to be used. ACKNOWLEDGMENTS
The authors express their thanks to Dr. Lionel Daviault, Officer-in-Charge, Forest Entomology and Pathology Laboratory, Quebec, and to Dr. A. S. West, Professor of Zoology, Queen’s University, Kingston, Ontario, for their encouragement; to Dr. M. Grantner, Professor of Geobotany, Lava1 University, Quebec, for identification of the plants; and to Dr. J, R. Blais and Mr. J, G. Pilon of the Quebec Forest Research Laboratory for reviewing the manuscript.
HUTCHISON REFERENCES
A. B. 1958. Bacteriostatic and bacteriocid influence of the foliage and its chemical components on the diseases of Dendrolimus sibericus Tschtn. larvae. Rep. Siberian Branch Acad. Sci. USSR, 7, 85-101. KUSHNER, D. J., AND HARVEY, G. T. 1962. Antibacterial substances in leaves: Their possible role in insect resistance to disease. 1. Insect Pathol., 4, 155-184. SMIRNOFF, W. .4. 1962. A staining method for differentiating spores, crystals and cells of Bacillus thuringiensis (Berliner). J. Insect Pathol., 4, 384-386. SMIRNOFF, W. A. 1963. Tests of Bacillus thuringiensis var. thuringiensis Berliner and B. cereus Frankland and Frankland on larvae of Chovistoxeura jumijerana (Clemens). Can. Entomologist, 95, 127-133. SMIRNOFF, W. A. 1965. Inhibition of parasporal inclusions synthesis in crystalliferous sporeforming bacteria of the “cereus” group by an aqueous extract of Viburnum cassinoides L. (Caprifoliaceae) leaves. J. invertebrate Pathol., 7, 71-74. TOKIN, B. P. 1963. Results of the fourth conference on the problems of the phytoncides. J. Gen. Biol., 24, 230-235. (In Russian.) GUKASIAN,
OF
INVERTEBRATE
7, 273-280
PATHOLOGY
Bacteriostatic Foliage
and from
(1965)
Bacteriocidal Various
thuriugiensis
var.
Plant
Effects
of
Extracts
Species
on
Bacittus
thuringiensis
of
Berliner’
W. A. SMIRNOFF AND P. M. HUTCHISON~ Forest
Research
Laboratory, Accepted
Sillery, July
Quebec,
,Canada
17, 1964
The influence of foliage from each of 74 species of plants on the development of Bacillus thuringiensis var. thuringiensis Berliner was studied. The plant juices and their extracts produced either complete inhibition, a delay in normal growth, or showed no effect on the Bacillus. Leaf juices of conifers produced more inhibition than those of the other plants tested. Results with ethyl acetate, ethyl ether, ethyl alcohol, and water extracts varied considerably both within and between the different groups of plants, but on the whole, acetate extracts had more influence on the growth of B. thuringiensis. Parallel tests showed that several undetermined species of bacteria encountered on the surface of the foliage had no effect on normal growth of the Bacillus. Similarly, the different pH values of the juice extracts added on agar surface were not an influential factor. The results suggest possibilities for further development of the methods used, and for the extension of this work with phytocides to other microorganisms normally present in the phytocoenosis, as well as those which are introduced.
of these substanceshave antibacterial properties. Antibacterial substancespresent in phytoOnly a few studies on the influence of tides of higher plants are attracting more and phytocides on bacterial entomopathogensare more attention, not only in the field of medknown and these deal with the aspect of inicine but also in various specialized fields of hibitory action: Gukasian (1958) studied the biology. Tokin (1963) reported that an influence of coniferous foliage on Bacillus “hectare” of deciduous trees liberated 2 kg of dendrolimus Talalaev, and Serratia marcesvolatile substancesin 24 hours while a forest cenSBizio, while Kushner and Harvey (1962) of conifers liberated 5 kg of these substances basedtheir studies on the effects of 10 species during the sameperiod. It is easily imagined of conifers and 8 speciesof deciduous trees that the quantity of volatile substanceslibon B. cereus Frankland and Frankland, diferated greatly increaseswhen billions of manferent strains of B. thuringiensis Berliner, and dibles destroy the foliage during an insect S. marcescens Bizio. invasion, and it is quite possible that some In 1961, a study of the effect of B. thuringiensis and its commercial preparations on 1 Contribution No. 1105, Forest Entomology and Pathology Branch, Department of Forestry, Ottawa, the activity of Choristoneura fumiferana Canada. (Clemens) indicated that phytocides might be 2 Fourth-year student, Department of Biology, of some importance (Smirnoff, 1963). In the Queen’s University, Kingston, Ontario, Canada; summer of 1963, an investigation was started summer seasonal employment at the Quebec Forest in the Chicoutimi region of Quebec Province R;search Laboratory. 273
274
SMIRNOFF
EFFECT
ON THE DEVELOPMENT TUICES FRO&I VARIOUS
AND
HUTCHISON
TABLE 1 OF Bacillus thuringiensis VAR. thuringiensis OF FOLIAGE EXTRACTS AND PLANTS AS DETERMINED THROUGH MICROSCOPIC ANALYSES~ Culture
Plant
Juice
PH
development
Water
on the spot
Alcohol
Acetate
Ether
Conifers Abies balsatnea (Linnaeus) Miller La& laricina (Du Roi) K. Koch Picea glauca (Moench) Voss
3.6
0 , B3 , 3 Spores only
3.6
h-
Picea wariana (Miller) Britton, Sterns, Poggenburg
4.9
Pinus banksiana Lambert Thuja occidentalis Linnaeus
4.9
S, B3 Spores only
Spores only Bx
Bi
0
0, B”
0
N
0
N
Spores only
0
0
0
4.5
N, B”
0, B”
0
0, Bl
5.2
N, B”
N
N
0
N
0
0
N
N
N
0
N
Deciduous
Trees
Acer pennsylvanicum Linnaeus
4.6
Acer rubrum Linnaeus
4.6
Acer spicaturn Lamar&
4.7
N
N
0, Bs
0
Cells only
5.5
N
N
N
0
3
4.5
N
N
0
0
ix
N
Cells only
s
Amelanchiev Wiegand
N
laevis
Betzda papyvifera Marshall Fagus grandifolia Ehrhart Populus alba Linnaeus Populus balsamifera Linnacus
5.3
N
5.2
N
Tests
not made
5.1
N
ix
0, Bs
0, B”
Popztlus tremuloides Michaux
5.3
N
N
N
Cells only
Prz~us pennsylvanica Linnaeus
5.5
N, Bs
s
0,B”
0, Bx
QUCYCIIS rzlbra Linnaeus var. borealis (Michaux fils) Farwell
4.3
N, B”
N, B”
0, Bx
0, B’
Robinia acacia
5.9
0, Bi
N
is
Cells only B’
N
Tilia americana Linnaeus
6.0
N,B’
N
N
iY
N
Ulmzts americana Linnaeus
4.6
N
N
0, B”
0
0
pseudoLinnaeus
EFFECTS
OF EXTRACTS
OF FOLIAGE
TABLE
ON
B. thuringiensis
1 (Continued) Culture
Plant
275
development
on the spot
Alcohol
Acetate
PH
Juice
Water
5.7
0, B’
Cells only
ix
0, B”
N
Anaphalis margaritacea (Linnaeus) C. B. Clarke
5.5
N, Bl
N, BY
0
0
0
Apocy~m mifoliz~m
Cells only
0
0
Herbaceous Achilles millefolizrnt Linnaeus
androsaeLinnaeus
Ether
Plants
6.4
N, Bl
Tests
Aster macrophyllus Linnaeus
j.9
S, Bx
N, Bl
Bvassica kaber (DeCandelle) L. C. Wheeler
6.0
N, Bl.3
Tests
Capsella bursapastoris (Linnaeus) Medicus
6.0
N, Bl.3
N
Spores only BX
Spores only
N
Chrysanthemum leucanthemum Linnaeus
5.8
N
N
N, Bs
0, B1
s
Cirsium (Savi)
5.3
N, Bl
N
N, B”
0, B1
N
5.6
N, BX
N
N, B3
N
N
N
Cells only
Spores only
N
0, BY
N, Bx
0, B”
N
vulgare Tenor0
Cornus canadensis Linnaeus Diervilla Miller
tonicera
Elymus cf. virginiczrs Linnaeus Epilobium angustifolium Linnaeus Eupatorium Linnaeus Fragaria niana Geranium Britton
not
not
made
made
5.2
N, BX
6.0
Spores only
6.2
N, Bx
N, B3
N
0
N
5.5
N, B’
N, B”
N, Br
N
N
N
N
Bl
macutatum cf. virgiDuchesne
4.6
N, I31
N
Cells only
4.8
N, B’
PI;
0, BX
0
N
4.9
N. B’
N
0, B3
0
0
bicknellii
Hieracium aurantiuciuwz Linnaeus Hieracium cf. porantinum Allioni Oenotheva biennis Linnaeus
5.3
N, B3
N
N, BX
0, BX
0
l
5.7
0, B3
0
N
0
s
Phleum pratense Linnaeus
5.7
N, B’
N
N, BX
0, Br
Cells only
Plantago major Linnaeus
5.6
N, B1
N
N
N
N
Polygonum convolL vulus Linnaeus
4.5
N
N
N, B*
Cells only
N
276
SMIRNOFF
TABLE
AND
HUTCHISON
1 (Continued) Culture
Plant
PH
Juice Herbaceous
Ranunculus Linnaeus
development
Water
on the spot
Alcohol
Acetate
Ether
Plants
acris 5.7
N
0
N, Bx
0, BX
N
Rzlbus cf. idaeus Linnaeus
5.2
N, BX
N, Bx
0
0
0
Rumen pallidus Bigelow
5.1
N, BX
X, Bx
Bx
N
0
Solidago canadensis Linnaeus
6.1
0, B’
N, B”
BX
N
0
5.2
AT, B”
0
Cells only
Cells only
0
5.8
N, B”
N
N
N
N
6.9
Bl
Tests
6.2
N
N
N, Bs
N
0
5.7
N, B’
N
N, Bs
0, B’
N
4.7
N
Spores only
0
N
5.4
N, Bx
N
0
0
5.1
N, Bx
O,BX
N
4.6
N, B
0, B” Spores only
0
0
6.5
N
N
0
N
Solidago graminifolia Linnaeus var. nuttallii (Greene) Fernald Tararacum oficinale Weber Trifolium agrarium Linnaeus Trifolium hvbridum Linnaeus Vicia cracca Linnaeus
not
made
Shrubs Alnus rugosa (Du Roi) Sprengel var. americana (Rogel) Fernald Amelanchier bartramiana (Taush) Roomer Cornus stolonifera Michaux Corylus cornuta Marshall Fraxinzls nigra Marshall Lonicera villosa (Michaux) Roomer & Schultes var. calvescens (Fernald 8; Wiegend) Fernald My&a gale Linnaeus Philadelphus cororzarizl.5 Linnaeus Pyrus melanocarpa (Michaux) Willdenow (Syn. Aronia melanocarpa (Michaux) Britton)
5.3
N, B”
N
N
N
5.5
N, B”
N
0
0
6.0
S, B”
N, Br
N
N
0. R
Few cells only
48
N
N
EFFECTS
OF EXTRACTS
OF FOLIAGE
TABLE
ON
PH
Juice
Rktis tyfihina Linnaeus
5 .o
N
Ribes glandulosum Grauer
4.5
N
Rosa blanda Aiton
4.8
N
Rosa rugosa Thumberg
4.7
Salix bebbiana Sargent Salix cf. discolor Muhlenberg
277
1 (Continued) Culture
Plant
B. thuringiensis
development
Water
Cells only
on the spot
Alcohol
Acetate
Ether
0
0
0
N, B”
0, B”
N
N, BY
N, B
0
N
N, I31
N, B”
Cells cnly
0
N
4.9
N, BX
N
N
N
5.3
0, B”
N, BX
Cells only
0
N
N
N
B3
Cells
Salix kumilis Marshall
5.0
N, BX
N
Cells only
Sorbus cf. decora (Sargent) Schneider
5.0
N
N
0
0
N
Syringa vzdgaria Linnaeus
5.5
N
N
0
N
N
Vaccinium angustifolit&m Aiton
3.8
0
N
0, B”
0, B-’
Viburnum Linnaeus
cassinoides 5.3
Spores only
Spores only
0, BY
0
5’iburnum Marshall
trilobum 5.0
N
N
0
0
Cells
Mosses Lycopodium Linnaeus Pleurozium (Willder)
and Lycopods
clavatum schreberi Nitten
Pteridium aquilinum var. latiusculum (Desvaux) Underwood
4.8
N, B’
N
Cells only Ba
6.2
N
0, BX
Cells only
5.3
N, B”
N
N
a Control: Among the 354 tests performed only four deviations were noted from normal development. They N, BX; the juice of Eapatorium maculawere: the juice of Circiztm v&are, N, Br, and its alcohol extract, tzlm, N, Br ; and the water extract of Oenotkeva biennis, 0. B, presence of other bacteria encountered on the surface of the foliage; N, normal development of Bacillus thuvingiensis var. thuringiensis; this indicates ,normal multiplication, sporulation, crystal synthesis, and lysis ; 0, complete inhibition of B. thuringiensis, ‘that is no growth. Gram stain procedure showed: Br, oval bacteria (gram +) ; Ba, short rods (gram +) ; B3, clostridiumhke of above bacteria. Tests showed that these bacteria (gram +) ; B4, long fine rods (gram -) ; B X, mixture bacteria produced no effect on the normal metabolism of B. tkuringiensis.
278
SMIRNOFF
AND
which included 74 species of plants growing in the region. The purpose of this work was to add to the list of plants studied with respect to their possible effect on bacterial metabolism, and point the way to methods that could be used for future work in this field. MATERIALS
AND
METHODS
A list of the plants tested is shown in Table 1. Plant material was obtained between mid- June and mid-August and approximately 60 g of foliage extract from plant species was prepared the day of collection. The foliage was mascerated in a Waring Blendor and 10-g lots were extracted with 25 ml of the following solvents: ethyl ether, ethyl alcohol, ethyl acetate, and water. The foliage was left in contact with the various solvents for 1 hour and then strained through a single layer of nylon cloth. Resulting extracts were placed in small weighing bottles and left to evaporate until 1 ml remained. In the case of the water extraction, where normal evaporation would be a long procedure, a vacuum evaporation system at 40°C was used. The concentrated extracts were stored at 0°C. Leaf juices were extracted by squeezing some of the remaining mascerated material through a single layer of fine-mesh nylon cloth; in some cases a garlic press lined with the nylon cloth was used. A small amount of juice was immediately placed on a bacteriaagar surface, prepared as described below. The pH of all foliage juices was determined, using a Coleman Companion pH meter, Model 31, with the following Beckman microelectrode attachments: a sealed one drop, and a Beckman sealed fiber-type calomel electrode assembly. Sterilizations through bacteriological filters were not made in order to maintain conditions as natural as possible. Filtering the extracts would have eliminated bacteria normally present on the plants, and might have retained certain substances, or might have broken the structure of others, which could
HUTCHISON
have resulted in changed properties of the substances. The culture of B. thuringiensis var. thuringiensis” was obtained by inoculating 100 ml of nutrient broth medium with the microorganism and incubating at 28°C for 18 hours. Plates were prepared by mixing 2 ml of this culture medium containing the Bacillus with 100 ml of warm nutrient agar (40°C). Disposable plastic tri-plate petri dishes containing the agar-bacteria mixture were used to test the effect of ethyl acetate, ethyl alcohol, and water extracts on bacterial development, by placing a drop of the substance on the bacteria-agar surface. In all cases, the bacteria growing on the spot area and the colonies which developed approximately 1 cm from the spot were examined microscopically. The Buffalo black-carbol Fuchsin method of staining (Smirnoff, 1962) was used to examine the phases of development of the Bacillus. Each analysis consisted of one slide on which there were two smears: on the right end, bacteria taken directly from the spot area; on the left end, bacteria which developed 1 cm from the region where the substance had been placed on the agar. This second smear served as a control and was separated from the first one by a clear space. During analysis, the presence or absence, and state of development of B. thuringiensis were recorded. The presence of cells, spores, and crystals of this microorganism, and the existence of other species of bacteria normally encountered on the foliage surface, were also noted. These other bacteria were later characterized by the Gram procedure. RESULTS
AND
DISCUSSION
Results of the various analyses appear in Table 1, and can be grouped into four categories: 3 We used a culture of Bacillus thuringiensis var. thuringiensis Berliner, obtained from Dr. T. A. Angus, Insect Pathology Research Institute, Sault
Ste. Marie, Canada.
EFFECTS
OF EXTRACTS
OF FOLIAGE
1. The tested substances completely inhibited the growth of B. thuringiensis and other bacteria present on the plant foliage. 2. The tested substances completely inhibited B. thuringiensis but had no effect on other bacteria present. 3. The tested substances affected normal development of B. thuringiensis in the following two ways: (a) normal development of cells; complete absence of spores and of crystals; (b) normal development of cells and spores; complete absenceof crystals. 4. The tested substanceshad no effect on
ON
B. thuringiensis
279
Linnaeus, Acer rubrum Linnaeus, and .4cer spicatum Lamarck; Salix bebbiana Sargent and Salix cf. disrolor Muhlenberg; Solidago canadensis Linnaeus and Solidago graminifolia Linnaeus. There are several cases of “sporulation only” mentioned in the table but this phenomenon was not checked by repeated analysis. However, the extract of Viburnum cassinoides Linnaeus that acted as a crystal inhibitor has been studied in detail since 1962 (Smirnoff, 1965) and the stability of the new character was proven. When B. thuringiensis was mixed with each B. thuringiensis. of the speciesof bacteria found on the plant The percentage of complete inhibition of foliage, or with mixtures of two, three, or B. thuringiensis by the juices and the various four of these species,there was no effect on indiextracts was distributed as follows for juices normal development of B. thwingiensis, and for each extract: juices, 8.6 percent; cating that inhibition in the development of is mainly caused by subwater extract 10 percent; ether extract, 30 B. thuringiensis percent; alcohol extract, 34.3 percent; and stancesin the extract since these do not affect ethyl acetate, extract, 65.7 percent. It is clear the other bacteria or vice versa. that the ethyl acetate, alcohol, and ether exThe values for the measurementsof the tracts were more efficient inhibitors of growth. zones of inhibition surrounding the sensi Control tests showed that the solvents them- discs (plant juices absorbed on discs) are not selveshad no effect on the normal metabolism indicated in the table because of the inacof the microorganism. curacy of such a method of testing when using It appears that the pH of the plant juices unsterilized plant juices. An alteration or deand extracts had no influence on the develop- lay in the development of B. thuringiensis ment of B. thwingiensis; for instance, juice frequently occurred, but this could only be of LaGx Zakina (Du Roi) Ii. Koch (pH 3.6) detected through careful microscopic examination since bacterial flora normally present inhibited crystal synthesis, juice of Picea gZauca (Moench) Voss (pH 3.6) did not on plants could develop in the zones where was inhibited. affect the development of the Bacillus, while growth of B. thuringiensis juice of Vaccinium angustifolium Aiton (pH It is possible that the plant substancesnot 3.8) completely inhibited B. thuringiensis. soluble in water may be as important in Parallel tests made by adding a spot of liquid affecting B. thuringiensis as those that are buffer (pH 3.5 to 8.0) to nutrient agar soluble. It is also possiblethat foliage changes showed no noticeable effect on the develop- its properties when eaten by insects, as a ment of B. thuringicnsis. result of oxidation and attack by enzymes of ’ Some plants of the same genus but of dif- the salivary glands and intestinal juices. Further investigations are required to determine ferent species had a different effect on B. thuringiensis and on other bacteria. This is whether these complex factors are important in affecting the bacteriostatic and bacterioparticularly true for the following: Picea tidal effects of foliage on B. thuringiensis. glauca and Picea mariana (Miller) Britton, Sterns, Poggenburg; Acer pennsylvanicum The aim of this preliminary work will be
280
~MIRN~FFAND
reached if it serves as a basis for further studies on methods that should be used in future research on the influence of plant phytotides on insect pathogens. Knowledge of the bacteriostatic and bacteriocidal qualities of various plants will serve to provide a more accurate estimate of the possible efficiency of microorganisms pathogenic to insects. Knowledge of these qualities of various plants could help to determine the dosage, the survival, and the possible dissemination of the pathogen to be used. ACKNOWLEDGMENTS
The authors express their thanks to Dr. Lionel Daviault, Officer-in-Charge, Forest Entomology and Pathology Laboratory, Quebec, and to Dr. A. S. West, Professor of Zoology, Queen’s University, Kingston, Ontario, for their encouragement; to Dr. M. Grantner, Professor of Geobotany, Lava1 University, Quebec, for identification of the plants; and to Dr. J, R. Blais and Mr. J, G. Pilon of the Quebec Forest Research Laboratory for reviewing the manuscript.
HUTCHISON REFERENCES
A. B. 1958. Bacteriostatic and bacteriocid influence of the foliage and its chemical components on the diseases of Dendrolimus sibericus Tschtn. larvae. Rep. Siberian Branch Acad. Sci. USSR, 7, 85-101. KUSHNER, D. J., AND HARVEY, G. T. 1962. Antibacterial substances in leaves: Their possible role in insect resistance to disease. 1. Insect Pathol., 4, 155-184. SMIRNOFF, W. .4. 1962. A staining method for differentiating spores, crystals and cells of Bacillus thuringiensis (Berliner). J. Insect Pathol., 4, 384-386. SMIRNOFF, W. A. 1963. Tests of Bacillus thuringiensis var. thuringiensis Berliner and B. cereus Frankland and Frankland on larvae of Chovistoxeura jumijerana (Clemens). Can. Entomologist, 95, 127-133. SMIRNOFF, W. A. 1965. Inhibition of parasporal inclusions synthesis in crystalliferous sporeforming bacteria of the “cereus” group by an aqueous extract of Viburnum cassinoides L. (Caprifoliaceae) leaves. J. invertebrate Pathol., 7, 71-74. TOKIN, B. P. 1963. Results of the fourth conference on the problems of the phytoncides. J. Gen. Biol., 24, 230-235. (In Russian.) GUKASIAN,