JOURNAL
OF INVERTEBRATE
PATHOLOGY
25, 267-268
(1975)
NOTES Photoprotection
Against Spores
Inactivation by Ultraviolet
It is well known that nonionizing rays which have a high photon energy show a remarkable inactivating effect on microorganisms, including insect pathogens. It is known that ultraviolet (UV) rays with a wavelength of 253.7 nm induces a striking photoinactivation on spores of Bacillus thuringiensis var. alesti (= Bacillus cereus var. alesti) so that they are unable to germinate and grow out (C. Vago and M. C. Busnel. Antonie van Leeuwenhoek 18, 125-127, 1955). Because nucleic acids show a maximum of extinction near 260 nm, it is suggested that the UV-induced death at 253.7 nm may be correlated to a photoreaction of the genetic material, especially DNA. The natural resistance of spores, as compared with vegetative cells, against UV may correspond to their content of dipicolinic acid (N. Crecz, T. Tang, and H. A. Frank. J. Bacterial. 113, 1058-1060, 1973), the Ca salt oi’ which shows spikes of UV extinction at 263, 270, and 279 nm. But this natural protection is evidently not sufficient for survival of B. thuringiensis in the field (E. S. Raun, G. R. Sutter, and M. A. Revelo. J. Invertebr. Pathol. 8, 365-375, 1966; R. D. Frye, C. G. Scholl, E. W. Scholz, and B. R. Funke. J. Invertebr. Pathol. 22,50 54, 1973). We found that a dose of 18 mW/sec/cm” of 253.7 nm-UV inactivates 99.9% of the var. darmspores of B. thuringiensis stadiensis.
In order to test their protective quality against inactivation of B. thuringiensis spores by UV, some substances with different optical features were selected. These were: (1) a black material which would absorb light over a broad area of the spectrum, hence, fine carbon particles dis-
of Baci//us Rays
persed in water in form of India ink; (2) a material which would absorb UV comparable to DNA, hence RNA, which has a maximum of extinction near 260 nm; (3) dipicolinic acid, which is supposed to act as natural UV-protectant of Bacillus spores (see above); (4) material which would absorb UV over a broad band and emit the absorbed ray-energy as light of longer wavelengths, so-tailed fluorochromes, hence we a physiological cell used riboflavine, component which is photosensitive, fluorescein-Na, a relatively photostable artificial fluorescent dye, and two fluorochromes of plant origin, aesculine and berberine-SO,. For our investigations B. thuringiensis var. darmstadiensis was propagated in submerged cultures. After sporulation, the biomasses were sedimented and washed three times with water. The spores were resuspended and the experimental protective substances were added at a concentration of 0.1 or 0.01%. Then each spore suspension was spread evenly on opened sterile Petri dishes by indirect spraying. The spore deposits (see Table 1 for densities) in the open Petri dishes (50 cm2) were exposed to the UV beam emitted by a low pressure Hg burner (Sterisol NN 30/82, Original Hanau). The emission of this lamp is nearly monochromatic at the Hg line of 253.7 nm. At a distance of 130 cm, there was an UV intensity of about 60 pW/cm”. After exposure for 3.5 and 7 min, respectively, the spore deposits in the Petri dishes were overlayed with nutrient agar and incubated at 37°C for 24 hr. Subsequently, the number of colonies per dish was counted and numerically compared with that in nonirradiated Petri dishes. The results of a representative experiment is shown in Table 1.
267 Copyright 0 1975 by Academic Printed in the United States.
thuringiensis
Press, Inc. All rights of reproduction
in any form
reserved.
268
NOTES
Effect
of UV against
Irradiation (253.7 nm) doses = 60pW/cm* untreated 5 5 5 5 5 5 5 5
control min min min min min min min min
unprotected
TABLE 1 and protected spores of Bacillus
Colonies Additives without protection India ink RNA dipicolinic acid riboflavine berberine-SO4 aesculine fluorescein-Na
‘Calculated from dilution plates. bCalculated from the foregoing column. ‘Against survival % of unprotected spores
0.1% 0.1% 0.1% 0.1% 0.1% 0.1% 0.1%
w/v w/v w/v w/v w/v w/v w/v
X lo*/50 (mean) 680U 0.9 0.9 2.7 5.5 3.3 7.2 7.9 10.0
thuringiensis
cm*
var. darmstadiensis
Survival %b (mean) = 100.00 0.13 0.13 0.38 0.80 0.48 1.06 1.16 1.47
Significance of means at 0.05 level of probabihtyC
+ + + + + +
(=0.13).
These results indicate that some fluorochromes may act as useful photoprotectants against UV. Of all the tested materials fluorescein-Na showed maximal protection followed by aesculine and berberine-SO,. Dipicolinic acid, riboflavine, and RNA were also better protectants than India ink, which has been occasionally recommended as a protectant. Because the UV of sunlight in the field may inactivate 50% of B. thuringiensis spores within 30 min or 80% within 60 min,
respectively (G. E. Cantwell and B. A. Franklin. J. Invertebr. Pathol. 8, 256-258, 1966), it would appear possible to extend this survival time significantly by using such UVprotectants as berberine-SO,, aesculine, or fluorescein-Na. ALOYSIUS KRIEG Biologische Bundesans talt ftir Land- und Forstwirtschaft Darmstadt, Germany Received June 7.1974
OF INVERTEBRATE
PATHOLOGY
25, 267-268
(1975)
NOTES Photoprotection
Against Spores
Inactivation by Ultraviolet
It is well known that nonionizing rays which have a high photon energy show a remarkable inactivating effect on microorganisms, including insect pathogens. It is known that ultraviolet (UV) rays with a wavelength of 253.7 nm induces a striking photoinactivation on spores of Bacillus thuringiensis var. alesti (= Bacillus cereus var. alesti) so that they are unable to germinate and grow out (C. Vago and M. C. Busnel. Antonie van Leeuwenhoek 18, 125-127, 1955). Because nucleic acids show a maximum of extinction near 260 nm, it is suggested that the UV-induced death at 253.7 nm may be correlated to a photoreaction of the genetic material, especially DNA. The natural resistance of spores, as compared with vegetative cells, against UV may correspond to their content of dipicolinic acid (N. Crecz, T. Tang, and H. A. Frank. J. Bacterial. 113, 1058-1060, 1973), the Ca salt oi’ which shows spikes of UV extinction at 263, 270, and 279 nm. But this natural protection is evidently not sufficient for survival of B. thuringiensis in the field (E. S. Raun, G. R. Sutter, and M. A. Revelo. J. Invertebr. Pathol. 8, 365-375, 1966; R. D. Frye, C. G. Scholl, E. W. Scholz, and B. R. Funke. J. Invertebr. Pathol. 22,50 54, 1973). We found that a dose of 18 mW/sec/cm” of 253.7 nm-UV inactivates 99.9% of the var. darmspores of B. thuringiensis stadiensis.
In order to test their protective quality against inactivation of B. thuringiensis spores by UV, some substances with different optical features were selected. These were: (1) a black material which would absorb light over a broad area of the spectrum, hence, fine carbon particles dis-
of Baci//us Rays
persed in water in form of India ink; (2) a material which would absorb UV comparable to DNA, hence RNA, which has a maximum of extinction near 260 nm; (3) dipicolinic acid, which is supposed to act as natural UV-protectant of Bacillus spores (see above); (4) material which would absorb UV over a broad band and emit the absorbed ray-energy as light of longer wavelengths, so-tailed fluorochromes, hence we a physiological cell used riboflavine, component which is photosensitive, fluorescein-Na, a relatively photostable artificial fluorescent dye, and two fluorochromes of plant origin, aesculine and berberine-SO,. For our investigations B. thuringiensis var. darmstadiensis was propagated in submerged cultures. After sporulation, the biomasses were sedimented and washed three times with water. The spores were resuspended and the experimental protective substances were added at a concentration of 0.1 or 0.01%. Then each spore suspension was spread evenly on opened sterile Petri dishes by indirect spraying. The spore deposits (see Table 1 for densities) in the open Petri dishes (50 cm2) were exposed to the UV beam emitted by a low pressure Hg burner (Sterisol NN 30/82, Original Hanau). The emission of this lamp is nearly monochromatic at the Hg line of 253.7 nm. At a distance of 130 cm, there was an UV intensity of about 60 pW/cm”. After exposure for 3.5 and 7 min, respectively, the spore deposits in the Petri dishes were overlayed with nutrient agar and incubated at 37°C for 24 hr. Subsequently, the number of colonies per dish was counted and numerically compared with that in nonirradiated Petri dishes. The results of a representative experiment is shown in Table 1.
267 Copyright 0 1975 by Academic Printed in the United States.
thuringiensis
Press, Inc. All rights of reproduction
in any form
reserved.
268
NOTES
Effect
of UV against
Irradiation (253.7 nm) doses = 60pW/cm* untreated 5 5 5 5 5 5 5 5
control min min min min min min min min
unprotected
TABLE 1 and protected spores of Bacillus
Colonies Additives without protection India ink RNA dipicolinic acid riboflavine berberine-SO4 aesculine fluorescein-Na
‘Calculated from dilution plates. bCalculated from the foregoing column. ‘Against survival % of unprotected spores
0.1% 0.1% 0.1% 0.1% 0.1% 0.1% 0.1%
w/v w/v w/v w/v w/v w/v w/v
X lo*/50 (mean) 680U 0.9 0.9 2.7 5.5 3.3 7.2 7.9 10.0
thuringiensis
cm*
var. darmstadiensis
Survival %b (mean) = 100.00 0.13 0.13 0.38 0.80 0.48 1.06 1.16 1.47
Significance of means at 0.05 level of probabihtyC
+ + + + + +
(=0.13).
These results indicate that some fluorochromes may act as useful photoprotectants against UV. Of all the tested materials fluorescein-Na showed maximal protection followed by aesculine and berberine-SO,. Dipicolinic acid, riboflavine, and RNA were also better protectants than India ink, which has been occasionally recommended as a protectant. Because the UV of sunlight in the field may inactivate 50% of B. thuringiensis spores within 30 min or 80% within 60 min,
respectively (G. E. Cantwell and B. A. Franklin. J. Invertebr. Pathol. 8, 256-258, 1966), it would appear possible to extend this survival time significantly by using such UVprotectants as berberine-SO,, aesculine, or fluorescein-Na. ALOYSIUS KRIEG Biologische Bundesans talt ftir Land- und Forstwirtschaft Darmstadt, Germany Received June 7.1974
