Ice Nucleation Activity in Lichens - Applied and Environmental ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUIY 1988, p. 1678-1681 0099-2240/88/071678-04$02.00/0 Copyright ©3 1988, American Society for Microbiology

Vol. 54, No. 7

Ice Nucleation Activity in Lichens THOMAS L. KIEFT

Department of Biology, New Mexico Instituite of Mining and Technology, Socorro, New Mexico 87801 Received 18 February 1988/Accepted 30 March 1988

A newly discovered form of biological ice nucleus associated with lichens is described. Ice nucleation spectra of a variety of lichens from the southwestern United States were measured by the drop-freezing method. Several epilithic lichen samples of the genera Rhizoplaca, Xanthoparmelia, and Xanthoria had nuclei active at temperatures as warm as -2.3°C and had densities of 2.3 x 106 to more than 1 x 108 nuclei g-1 at -5sC (2 to 4 orders of magnitude higher than any plants infected with ice nucleation-active bacteria). Most lichens tested had nucleation activity above -8°C. Lichen substrates (rocks, plants, and soil) showed negligible activity above -8°C. Ice nucleation-active bacteria were not isolated from the lichens, and activity was not destroyed by heat (70°C) or sonication, indicating that lichen-associated ice nuclei are nonbacterial in origin and differ chemically from previously described biological ice nuclei. An axenic culture of the lichen fungus Rhizoplaca chrysoleuca showed detectable ice nucleation activity at - 1.9°C and an ice nucleation density of 4.5 x 106 nuclei g- 'at -5°C. It is hypothesized that these lichens, which are both frost tolerant and dependent on atmospheric moisture, derive benefit in the form of increased moisture deposition as a result of ice nucleation.

weight) of lichen thallus liter-'. A minimum of 10 drops per dilution was tested. The concentrations of ice nuclei were calculated from the formula of Vali (16): N() = (-ln f)/V, where N(7) is the nucleation frequency at temperature T, f is the proportion of droplets unfrozen, and V is the volume of individual droplets. The number of nuclei per gram of lichen was calculated by dividing the concentration of nuclei per liter by the density of the lichen suspension (in grams per liter). Drops of distilled water used as controls in each nucleation spectrometer analysis always supercooled to -10 to -15°C. Other controls consisted of suspensions of INA bacteria (P. syringae Cit7, provided by Steven Lindow, University of California, Berkeley) and non-INA bacteria (Escherichia coli ATCC 8739). Suspensions of lichen substrates (rocks, tree bark, and soil) were also tested for ice nucleation activity. Intact lichen thalli were tested for ice nucleation activity by suspending 0.1 g of lichen thallus in 3.0 ml of distilled water and chilling the suspension to -1, -2, -3, -4, -5, -6, or -7°C. Lichens were considered ice nucleation active at a particular temperature if the water froze within 10 min. Heating and sonication. The heat stability of ice nuclei from the lichen Rhizoplaca chrysoleuca was tested by heating samples of a homogenized lichen suspension (0.1 g/liter in distilled water) for 10 min at 40, 50, 60, 70, 80, 85, and 95°C. These and an unheated sample were tested for ice nucleation activity by the drop-freezing assay. The effect of sonication on R. chrysoleuca nuclei was tested by sonicating a lichen homogenate suspension (1.0 g/liter) for 3 min (Branson model 250, 95-W output) at 0°C. Sonicated and nonsonicated suspensions were tested by the drop-freezing assay. Attempts to culture INA bacteria. Attempts to isolate INA bacteria from lichens showing ice nucleation activity at -5°C and above were made by streaking lichen suspensions on nutrient agar and agar media selective for P. syringae and E. herbicola. The selective medium for E. herbicola was nutrient agar with 5% NaCl. The P. syringae medium (devised by Myron Sasser, University of Delaware) contained 12.0 g of sorbitol, 0.8 g of K2HPO4 3H20, 0.13 g of MgSO4. 7H20, 0.2 g of L-histidine, 128 mg of Cetrimide (Sigma), 100 ,ug of cycloheximide, 50 ,ug of Benomyl (Chem Service, West

In the absence of heterogeneous ice nuclei, water supercools to temperatures well below 0°C. The majority of ice nuclei active at relatively warm temperatures (above -5°C) have been found to be biological in origin. These are primarily ice nucleation-active (INA) bacteria which occur on crop and wild plants, where they can contribute to frost damage (8-13). These INA bacteria (e.g., INA strains of Pseudomonas syringae and Erwinia herbicola) have received greater attention as a result of the recent environmental release of genetically engineered "ice-minus" mutants for frost protection of crop plants (3, 4). The study reported here was undertaken to assess ice nucleation activity in lichens. These frequently overlooked associations of fungi and algae have not been included in surveys of plants for ice nucleation activity. As a basis for this investigation, it was hypothesized that lichens, many of which are both frost tolerant and dependent on the atmosphere for moisture (7), might benefit from ice nucleation through enhanced moisture uptake. Lichens were therefore considered good candidates for ice nucleation activity. MATERIALS AND METHODS

Samples. Lichens with a variety of growth forms (crustose, foliose, fruticose, gelatinous, and umbelliferous), sampled from a variety of substrates (rocks, trees, and soil) and from habitats with widely differing moisture contents (semiarid deserts to alpine forests) were collected from sites in the southwestern United States (Table 1). Samples were collected during July through October 1987 (except Letharia spp., which were collected in June 1986) and refrigerated (5°C) prior to testing. Ice nucleation assays. Ice nucleation activities of lichens were determined with an ice nucleation spectrometer. Suspensions of cell material were placed in 10-lI droplets on the surface of a paraffin-coated, temperature-controlled aluminum cold plate. As the temperature was slowly lowered (approximately 0.3°C min-'), the number of drops frozen was scored. Lichen suspensions were prepared by grinding lichens in a mortar and pestle, suspending them in distilled water, and homogenizing them in a ground-glass handoperated homogenizer. Serial 10-fold dilutions were used to make suspensions ranging in density from 0.01 to 10 g (dry 1678

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TABLE 1. Sources of lichens tested for ice nucleation activity Lichen

Sampling site

Elevation

Habitat

Substrate

Rhizoplaca chrysoleuca Rhizoplaca chrysoleuca Xanthoparmelia sp. Xanthoria elegans Platismatia sp. Acarospora sp. Acarospora sp. Acarospora sp. Leptogium sp. Psora decipiens Xanthoparmelia sp. Xanthoparmelia sp. Letharia sp.

Magdalena Mountains, N.Mex. Near Jemez Springs, N.Mex. Near Jemez Springs, N.Mex. Near Jemez Springs, N.Mex. Magdalena Mountains, N.Mex. Platoro, Colo. Near Jemez Springs, N.Mex. Near Socorro, N.Mex. Near Socorro, N.Mex. Near Los Alamos, N.Mex. Magdalena Mountains, N.Mex. Near Socorro, N.Mex. Sierra Nevada Mountains, Calif. Magdalena Mountains, N.Mex. Jemez Mountains, N.Mex.

3,060 2,290 2,290 2,290 3,050 3,200 2,290 1,570 1,490 1,980 3,050 1,570 1,870

Alpine meadow Semiarid desert Semiarid desert Semiarid desert Alpine forest Alpine forest Semiarid desert Semiarid desert Semiarid desert Pinyon-juniper woodland Alpine meadow Semiarid desert Alpine forest

Rock-rhyolitic volcanic tuff Rock-rhyolitic volcanic tuff Rock-rhyolitic volcanic tuff Rock-rhyolitic volcanic tuff Trees-Pseudotsuga taxifolia (Douglas fir) Rock-andesite Rock-rhyolitic volcanic tuff Rock-sedimentary conglomerate Rock-silicified sedimentary breccia Soil Rock-rhyolitic volcanic tuff Rock-sedimentary conglomerate Trees-Pseudotsuga taxifolia (Douglas fir)

3,050 2,440

Alpine forest Alpine forest

Soil Trees-Abies concolor (white fir)

Peltigera sp. Usnea sp.

(m)

Chester, Pa.), 20 ,ug of Botran (Aldrich), 100 ,ug of rifampin, and 15 g of agar per liter of H20. Lichen fungal culture. An axenic culture of the lichen fungus R. chrysoleuca, isolated previously (2), was kindly supplied by Vernon Ahmadjian, Clark University. The culture was grown in malt-yeast extract medium (1), suspended in distilled water, and tested for ice nucleation activity by the drop-freezing assay. RESULTS Ice nucleation activities of lichens. Nearly all the lichens tested showed ice nucleation activity at temperatures warmer than -8°C, with several of the samples showing nucleation activity at temperatures warmer than -5°C (Fig. 1). Of particular note were R. chrysoleuca, Xanthoria elegans, and Xanthoparmelia sp. samples, which had detectable ice nucleation activity at -3°C or warmer and which showed nucleus densities of at least 2.3 x 106 nuclei g-' at -5°C. All lichens showed greater ice nucleation activity than the distilled water controls, which always supercooled to -10°C or colder. Suspensions of lichen substrates (rocks, tree bark, and soil) tested by the same method showed little activity: less than 10% of the drops of these materials held in suspension froze at temperatures above -8°C. Lichen-associated nuclei were active at the same warm temperatures in whole lichens as in suspensions of homogenized lichen fragments, indicating that ice nucleation activity is not an artifact of thallus disruption. Effects of heating and sonication. The ice nuclei of the lichen R. chrysoleuca were relatively heat stable; temperatures above 70°C were required to greatly reduce ice nucleation activity (Table 2). Sonication at the intensity tested did not diminish the ice nucleation activity of R. chrysoleuca. Attempts to culture INA bacteria. The selective medium for P. syringae yielded no isolates. Isolates obtained on nutrient agar amended with 5% NaCl were not of the genus Erwinia. None of the isolates from either nutrient agar or nutrient agar with 5% NaCl showed any ice nucleation activity at -5°C when tested by the drop-freezing assay. Ice nucleation in lichen fungal culture. The axenic culture of the mycobiont R. chrysoleuca showed detectable ice nucleation activity at -1.9°C and a density of 4.5 x 106 nuclei g-1 at -5°C (Fig. 2).

DISCUSSION The relatively warm temperatures at which some of the lichen samples nucleated ice indicate a biological source of heterogeneous nuclei. The very high densities of nuclei (106 to 108 nuclei g-1) active at -5°C in the most active of the lichens are higher than the maximum 103 nuclei g-1 observed in plant leaf tissue (9) and the approximately 102 to 104 nuclei g-1 of decaying leaf litter (15) active at the same temperature. In plants showing ice nucleation activity at -5°C and warmer temperatures, 95 to 100% of the ice nuclei have been attributed to INA bacteria (9). However, the failure to culture INA bacteria from lichens suggests a nonbacterial source of lichen nuclei. The very high densities of ice nuclei associated with the most active lichens also suggest a nonbacterial source, since INA bacterial populations usually have fewer than 1 nucleus per cell (8, 9) and the lichens typically harbor fewer than 105 total bacteria g-1 (unpublished data). Furthermore, unlike bacterial ice nucleation, which is destroyed by mild heat or sonication (13), the activity of lichen-associated ice nuclei resisted 70°C heat and sonication. The evidence therefore points to a nonbacterial source of ice nuclei. Organic and inorganic dust particles, including clay minerals, all of which only show freezing nucleation at temperatures below -8°C (8, 9), are not likely candidates as lichen-associated ice nuclei. Moreover, nuclei of this nature would not be found concentrated only on lichens. Thus, having eliminated bacterial and dust-derived nuclei, one must conclude that the ice nucleation activity of these lichens is the result of a substance produced by the lichens themselves. This substance could be a product of the phycobiont, the mycobiont, or a combination of the two. The presence of ice nuclei active at relatively warm temperatures in the axenic fungus R. chrysoleuca points to the mycobiont as the source of ice nuclei and further underscores the nonbacterial source of these nuclei. This is the first finding of biological ice nucleation activity in any fungus. Axenic phycobiont cultures have not yet been tested for ice nucleation activity and therefore cannot be ruled out as possible contributors to ice nucleation activity in lichens. Lichen-associated ice nuclei may be membrane-bound proteins analogous to those found in INA bacteria (14, 17). Alternatively, one or more of the secondary metabolites

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TABLE 2. Effects of 10-min heat treatment on ice nucleation activity in the lichen R. chrysoleuca Temp of heat treatment (°C) T50 (oC)a -2.3 Control (no heat treatment) .................... 40 .................... -2.3 50 .................... -2.3 60 .................... -3.3 70 .................... -2.7 80 .................... -14.6 85 .................... -14.6 95 .................... -14.6 a T50, Temperature at which 50% of drops froze in drop-freezing assay.

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occurring as precipitates on the surfaces of lichen hyphae (5, 6) may be the source of the observed ice nuclei. Lichen-associated ice nuclei active at temperatures warmer than -5°C appear not to be universal among lichens. This could be due to phenotypic variations in response to environmental conditions as well as genotypic differences among lichens. The most active lichen samples came from volcanic tuff surfaces at two sites. However, other lichens from the same rocks (e.g., Acarospora sp.) had only moderate ice nucleation activity. Three Acarospora samples from widely separated sites showed nearly identical ice nucleation spectra (Fig. 1B), while Xanthoparmelia samples from different sites had widely different activities (Fig. 1A and C). There was no correlation between elevation and ice nucleation activity. Epiphytic and soil lichens exhibited the least ice nucleation activity. Ice nucleation activity at temperatures warmer than -5°C may enhance the uptake of atmospheric moisture by enhancing condensation and/or causing deposition of ice from water vapor to occur earlier as the temperature drops below 0°C. Once an ice lattice is formed, further deposition occurs more readily. When the temperature rises, the lichen thallus may then absorb moisture from the melting ice. Also, lichens may derive a secondary benefit in that gradual ice crystal formation at relatively warm temperatures may be less stressful than the sudden formation of large crystals at lower temperatures. This has been proposed as a benefit of INA bacteria to frost-tolerant plants (8). Lichen-associated ice nuclei might even contribute to atmospheric ice nuclei, as has been suggested for other biological ice nuclei (8, 9, 13). All the lichens tested were sampled from environments in which winter daily temperatures typically range above and below 0°C. For these lichens in winter, the hypothesized deposition would be of greatest advantage. Lichens have not

-2

Temperature (degrees C) FIG. 1. Ice nucleation spectra of lichens. (A) Lichens showing ice nucleation activity at temperatures above -3°C: R. chrysoleuca (O), Xanthoparmelia sp. (-), Xanthoria elegans (A), all collected near Jemez Springs, and R. chrysoleuca (0) from the Magdalena Mountains (see Table 1). (B) Lichens with intermediate ice nucleation activity: Platismatia sp. from the Magdalena Mountains (0), Acarospora sp. from near Jemez Springs (-), Acarospora sp., from Platoro (A), Acarospora sp. collected near Socorro (0), and Leptogium sp. collected near Socorro (L). (C) Lichens with least nucleation activity: Psora decipiens collected near Los Alamos (0), Xanthoparmelia sp. from the Magdalena Mountains (O), Xanthoparmelia sp. collected near Socorro (A), Letharia sp. from the Sierra Nevada Mountains (0), Peltigera sp. from the Magdalena Mountains (L), and Usnea sp. from the Jemez Mountains (A).

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ICE NUCLEATION ACTIVITY IN LICHENS

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been tested for seasonal patterns of ice nucleation, nor have lichens from nonfreezing environments been tested. Further work is required to determine the chemical nature and possible selective advantage of ice nucleation activity in lichens. ACKNOWLEDGMENTS

I thank Steven Lindow, Alan Blyth, J. Alvin Smoake, and Gerardo Gross for helpful discussions; Ellen DeBruin of the Herbarium, Museum of Southwestern Biology, University of New Mexico, for help with lichen identifications; Andrew Campbell for mineral identifications; and Sandra Kieft, Feyzan Okmen, and Tracy Ruscetti for technical assistance. LITERATURE CITED 1. Ahmadjian, V. 1973. Methods of isolating and culturing lichen symbionts and thalli, p. 653-659. In V. Ahmadjian and M. E. Hale (ed.), The lichens. Academic Press, Inc., New York. 2. Ahmadjian, V., L. A. Russell, and K. C. Hildreth. 1980. Artificial reestablishment of lichens. I. Morphological interactions between the phycobionts of different lichens and the mycobionts Cladonia cristatella and Lecanora chrysoleuca. Mycologia 78:73-89. 3. Crawford, M. 1987. California field test goes forward. Science 236:511. 4. Crawford, M. 1987. Vandals hit Lindow plot. Science 236:1181. 5. Hale, M. E. 1983. The biology of lichens, 3rd ed. Edward Arnold, Melbourne, Australia. 6. Hawksworth, D. L., and D. J. Hill. 1984. The lichen-forming fungi. Blackie, Glasgow, Scotland. 7. Kershaw, K. A. 1985. Physiological ecology of lichens. Cam-

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bridge University Press, Cambridge, England. 8. Lindow, S. E. 1982. Epiphytic ice nucleation-active bacteria, p. 335-362. In G. Lacy and M. Mount (ed.), Phytopathogenic prokaryotes, vol. 1. Academic Press, Inc., New York. 9. Lindow, S. E. 1983. The role of bacterial ice nucleation in frost injury to plants. Annu. Rev. Phytopathol. 21:363-384. 10. Lindow, S. E., D. C. Arny, and C. D. Upper. 1978. Distribution of ice nucleation-active bacteria on plants in nature. Appl. Environ. Microbiol. 36:831-838. 11. Lindow, S. E., D. C. Arny, and C. D. Upper. 1978. Erwinia herbicola: a bacterial ice nucleus active in increasing frost injury to corn. Phytopathology 68:523-527. 12. Lindow, S. E., D. C. Arny, C. D. Upper, and W. R. Barchet. 1978. The role of bacterial ice nuclei in frost injury to sensitive plants, p. 249-263. In P. Li and A. Sakai (ed.), Plant cold hardiness and freezing stress-mechanisms and crop implications. Academic Press, Inc., New York. 13. Maki, L. R., E. L. Galyon, M. Chang-Chien, and R. Caldwell. 1974. Ice nucleation induced by Pseudomonas syringae. Appl. Microbiol. 28:456-459. 14. Phelps, P., T. H. Giddings, M. Prochoda, and R. Fall. 1986. Release of cell-free ice nuclei by Erwinia herbicola. J. Bacteriol. 167:496-502. 15. Schnell, R. C., and G. Vali. 1973. World-wide source of leafderived freezing nuclei. Nature (London) 246:212-213. 16. Vali, G. 1971. Quantitative evaluation of experimental results on the heterogeneous freezing nucleation of supercooled liquids. J. Atmos. Sci. 28:402-409. 17. Wolber, P. K., C. A. Deininger, M. W. Southworth, J. Vandekerckhove, M. van Montagu, and G. J. Warren. 1986. Identification and purification of a bacterial ice nucleation protein. Proc. Natl. Acad. Sci. USA 83:7256-7260.