- Email: [email protected]
Phytophthora cinnamomi in Hawaiian forest soils: technique for enumeration and types of propagules recovered
Soil Biol. Biochem. Vol. 12, pp. 89 to 91 © Pergamon Press Ltd 1980. Printed in Great Britain
0038-0717/80/0101-0089502.00/0
SHORT COMMUNICATION Phytophthora cinnamomi in Hawaiian forest soils: technique for enumeration and types of propagules recovered J. T. KLIEJUNAS* and J. T. NAGATA Department of Plant Pathology, University of Hawaii, Beaumont Agricultural Research Center, Hilo, CA 96720, U.S.A.
(Accepted 7 July 1979)
Phytophthora cinnamomi Rands has been isolated from soils on soil dilution plates and by placing soil on an agar surface (Hendrix and Kuhlman, 1965; Tsao and Ocana, 1969). The main disadvantage of these methods is the small amount of soil sampled. McCain et al. (1967) sieved soil to concentrate chlamydospores of the fungus from large quantities of soil. Although McCain et al. (1967) and Hwang and Ko (1978) did not recover P. cinnamomi from field soils passed through 38/am mesh, Cother and Griffin (1973) and Reeves (1975) reported chlamydospores of the fungus < 38#m. Little research has been done to determine the types and dimensions of P. cinnamomi propagules present in naturally-infested soils. In our studies on P. cinnamomi in Hawaiian forest soils, we needed an efficient technique to isolate the fungus. We report experiments to compare various plating techniques and media, and describe the types of P. cinnamomi propagules recovered from field soils. Organic muck histosols associated with ohia [Metrosideros collina (Forst.) Gray polymorpha (Gaud.) Rock] forests o n the island of Hawaii were used. Moisture contents ranged from 57 to 87% by volume, organic matter content from 24 to 62% and bulk densities from 0.13 to 0.24. Unless otherwise stated, soils were collected, air-dried overnight in the laboratory, sieved < 6 mm, and then immediately used. Colonies of P. cinnamomi were located by scanning plates at x 100 for characteristic mycelium. When identity of suspected P. cinnamomi colonies was uncertain the colony was transferred to 20% V-8 juice agar and identified when characteristic mycelium with chlamydospores had formed.
Modified Kerr's medium was not used in subsequent experiments since AM was as effective and was easier to prepare. When AM was compared with PloVP medium (Tsao and Ocana, 1969) no significant differences were observed in numbers of P. cinnamomi colonies (an average of 14 g- 1 on each medium), but AM was easier to handle and examine. Washing disturbed the soft surface of PIoVP and soil organic particles present interfered with examination. Fewer and more restricted colonies of Pythium spp developed on AM. AM, with two modifications, was used in subsequent experiments. Reducing the agar concentration from 4 to 3% increased the growth rate of P. cinnamomi and made colonies easier to distinguish. V-8 juice was centrifuged at 15,000 rev. min- t for 10 min to remove suspended particles that interfered with microscopic examination. The modifications had no apparent effect on recovery of P. cinnamomi. Incubation at 20° vs 24°C, in light vs dark, and washing and examining plates after 16, 24 and 36 h were compared. A temperature of 24°C increased growth of P. cinnamomi in both light and dark treatments throughout and colonies were easier to detect. Incubation in the dark reduced development of Pythium spp regardless of temperature. At 16 h colonies had not developed sufficiently for positive identification and at 36 h Pythium spp masked P. cinnamomi. Incubation at 24°C in the dark for 24 h permitted maximum recognition of P. cinnamomi and was used in subsequent experiments. Two subsamples of soil were collected from each of two forest sites and used the same day. One 50g moist subsample was placed on 38/am-mesh, washed under running tapwater for 5 min, and the residue on the sieve removed by backwashing. A second 50 g subsample was not washed. Water was added to each subsample to bring the volume to 150 ml. The mixture was then mixed in an Omni mixer for 30s at 1500rev. min-1, placed on a magnetic stirrer, and a 3 ml subsample dispersed on each of 10 plates of modified AM. An additional five, 3 ml subsamples were placed in weighed aluminum dishes and dried at t05°C overnight to determine average oven-dry weight of soil per plate. The number of P. cinnamomi colonies recovered from washed subsamples (2.4 and 4.8 colonies g-1) was significantly less than from nonwashed subsamples (13.4 and 27.6 g- 1), indicating that propagules < 38/am dia were present. When organic muck soil was autoclaved, infested with chlamydospores of P. cinnamomi (Hemmes and Wong, 1975), and treated in the same manner, 352 colonies g-1 were recovered from the nonsieved sample and 60 colonies g- ~ from the sieved. Soils from six forest sites were washed on 38/am-mesh and filtrates baited with lupine (Lupinus angustifolius L.) radicles (Chee and Newhook, 1965). P. cinnamomi was recovered from the filtrates of four of the six soils. Sieving was inefficient and eliminated recovery of propagules <38/am. In subsequent experiments, washing
Development of isolation technique Soil dilutions (Hendrix and Kuhlman, 1965) of 1:10, 1:20, and 1:100 of three soils were placed on the antibiotic medium (AM) of McCain et al. (1967) and on modified Kerr's (Hendrix and Kuhlman, 1965) medium. After 72 h at 20°C, colonies of Pythium spp on both media and at all dilutions had overgrown the plates. P. cinnamomi could be distinguished only by tediously scanning entire plates for the presence of its characteristic mycelium. The numbers of P. cinnamomi colonies ranged from 0 to 3.1 g - L When 1 g soil per plate was placed on the two media, dispersed with 3 ml water and incubated under the same conditions, similar results were obtained. When the sieving technique was tested using the same three soils and two media, the 149/am-mesh plugged up immediately because of the high soil organic matter content. Propagules of P. cinnamomi ranged from 1,1 to 7.8g -1.
*Present address: Insect & Disease Management, Region 5, U.S. Forest Service, 630 Sansome Street, San Francisco, CA 94111, U.S.A. 89
90
Short communications
Fig. 1. Propagules of Phytophthora cinnamomi recovered from Ohia forest soils on the island of Hawaii. (A) Cluster of encysted zoospores. Ten germinating cysts are present. (B) Sporangium with germinating cysts retained inside. (C) A chlamydospore, 57.9 #m dia. (D) A hyphal swelling, 19.8 by 23.2 #m.
of soils on 38/am-mesh was omitted and subsamples from a 50 g sample were plated. Propagules of P. cinnamomi in forest soils The types and dimensions of propagules recovered from three forest sites sampled every 2 weeks for a year were recorded. Ten plates per site were examined at each sampiing. Of 4744 colonies of P. cinnamomi observed, 55~o originated from zoospores, 18~o from chlamydospores or hyphal swellings, 15~o from organic matter, 11~o from pieces of hyphae, and 1% from directly-germinating sporangia. Oospores were not observed. Encysted zoospores were sometimes seen in clusters on plates (Fig. 1A), often with an empty sporangium nearby, indicating that they may have been released during the 24 h incubation. In some instances, zoospores had encysted and germinated while still in the sporangium (Fig. 1B). Numerous individual zoospores were also observed on plates during some periods of the year but not others. Their presence was -elated to environmental conditions present at time of field ~ r - , l i n g (Kliejunas and Nagata, unpublished). Cysts ra,.sed from 10.5 to 11.8 #m dia, Directly-germinating sporangia were only occasionally seen. Sporangia were normal in size (Waterhouse, 1970) and no microsporangia similar to those found in vitro (Ho and Zentmyer, 1977) were seen. Chlamydospores ranged from 15.4 to 82.0/*m dia (mean, 44.2 #m) (Fig. IC). Of 175 chlamydospores measured, 12~ were <38 #m dia. Oblong thin-walled structures which were apparently hyphal swellings (Fig. 1D) were recorded
as chlamydospores. Blackwell (1949) distinguished between hyphal swellings which are not cut off by septa and chlamydospores which are. The hyphal swellings ranged from 14.5 by 23.2#m to 34.4 by 37.6#m and all were small enough to pass through 38 #m mesh. They lacked the inner wall-layer of typical chlamydospores. Colonies also originated from pieces of organic matter which when teased apart contained no discernable propagule, and from pieces of hyphae with no discernable propagule present. On nine occasions when zoospores were observed on plates, a second 50 g sample of the same soil was mixed with 200 ml water, placed on 38 #m mesh, and washed 10 times with 200 ml water. The filtrates were collected separately, passed through 20 #m mesh, and baited with lupine. P. cinnamomi was recovered from the 20 #m filtrates on five of the nine occasions. When 0.5 ml samples of the filtrates were examined microscopically, propagules similar to P. cinnamomi cysts were observed but none had germinated to enable identification. Sporangia, chlamydospores, or hyphal swellings were not seen in the filtrates. On five other occasions when zoospores were observed on plates, a second soil sample from the same site was collected and tested as above the same day. P. cinnamomi was recovered by baiting from < 20 #m filtrates on three of the five occasions, indicating propagules < 20/zm were present in field soils. To determine if the plating technique induced indirect sporangial germination, mycelium with mature sporangia of P. cinnamomi (6 mm dia mycelial discs incubated in the dark for 3 days at 24°C in soil extract) were added to
Short communications plates with 3 ml of soil mixture and incubated as usual. The experiment was performed twice with soil from each of the three sites but indirect germination of the added sporangia was not observed. Zoospores were absent from plates where the mycclial discs had been. These results strongly suggested that free zoospores were present in field soils. We conclude that ohia forest organic muck soils are at times a favorable environment for in situ sporangial production and subsequent indirect germination, and a high potential for population increase of P. cinnamomi exists in these soils.
Acknowledgements--Journal Series Paper 2313 of the Hawaii Agricultural Experiment Station. Supported in part by funds from the U.S. Forest Service. REFERENCES
BLACKWELL E. M. (1949) Terminology in Phytophthora. Mycolooical Papers 30, 1-23. CHEE E. J. and NEWHOOKF. J. (1965) Improved methods for use in studies on Phytophthora cinnamomi Rands and other Phytophthora species. New Zealand Journal of Agricultural Research 8, 88-95. COTH~RE. J. and GRn~FIND. M. (1973) Formation of chla-
91
mydospores by Phytophthora drechsleri. Transactions of the British Mycological Society 61, 379--402. HEMM~ D. E. and WOSG L. D. S. (1975) Ultrastructure of chlamydospores of Phytophthora cinnamomi during development and germination. Canadian Journal of Botany 53, 2945-2957. HENDRIX F. F. JR and KUHLMAN G. H. (1965) Factors affecting direct recovery of Phytophthora cinnamomi from soil. Phytopathology 55, 1183-1187. Ho H. H. and ZErCrMYERG. A. (1977) Morphology of Phytophthora cinnamomi. Mycologia 69, 701-713. HWANG S. C. and Ko W. H. (1978) Biology of chlamydospores, sporangia, and zoospores of Phytophthora cinnamomi in soil. Phytopathology 68, 726-731. MCCAIN A. H., HOLTZMANNO. V. and TRUJtLLO E. E. (1967) Concentration of Phytophthora cinnamomi chlamydospores by soil sieving. Phytopatholooy 57, 1134-1135. REEVESR. J. (1975) Behaviour of Phytophthora cinnamomi Rands in different soils and water regimes. Soil Biology & Biochemistry 7, 19-24. TSAO P. H. and OCANA G. (1969) Selective isolation of species of Phytophthora from natural soils on an improved antibiotic medium. Nature 223, 636-638. WATEgHOUS~ G. M. (1970) The genus Phytophthora de Bary. 2nd (revised) edn. Mycological Papers 122.
0038-0717/80/0101-0089502.00/0
SHORT COMMUNICATION Phytophthora cinnamomi in Hawaiian forest soils: technique for enumeration and types of propagules recovered J. T. KLIEJUNAS* and J. T. NAGATA Department of Plant Pathology, University of Hawaii, Beaumont Agricultural Research Center, Hilo, CA 96720, U.S.A.
(Accepted 7 July 1979)
Phytophthora cinnamomi Rands has been isolated from soils on soil dilution plates and by placing soil on an agar surface (Hendrix and Kuhlman, 1965; Tsao and Ocana, 1969). The main disadvantage of these methods is the small amount of soil sampled. McCain et al. (1967) sieved soil to concentrate chlamydospores of the fungus from large quantities of soil. Although McCain et al. (1967) and Hwang and Ko (1978) did not recover P. cinnamomi from field soils passed through 38/am mesh, Cother and Griffin (1973) and Reeves (1975) reported chlamydospores of the fungus < 38#m. Little research has been done to determine the types and dimensions of P. cinnamomi propagules present in naturally-infested soils. In our studies on P. cinnamomi in Hawaiian forest soils, we needed an efficient technique to isolate the fungus. We report experiments to compare various plating techniques and media, and describe the types of P. cinnamomi propagules recovered from field soils. Organic muck histosols associated with ohia [Metrosideros collina (Forst.) Gray polymorpha (Gaud.) Rock] forests o n the island of Hawaii were used. Moisture contents ranged from 57 to 87% by volume, organic matter content from 24 to 62% and bulk densities from 0.13 to 0.24. Unless otherwise stated, soils were collected, air-dried overnight in the laboratory, sieved < 6 mm, and then immediately used. Colonies of P. cinnamomi were located by scanning plates at x 100 for characteristic mycelium. When identity of suspected P. cinnamomi colonies was uncertain the colony was transferred to 20% V-8 juice agar and identified when characteristic mycelium with chlamydospores had formed.
Modified Kerr's medium was not used in subsequent experiments since AM was as effective and was easier to prepare. When AM was compared with PloVP medium (Tsao and Ocana, 1969) no significant differences were observed in numbers of P. cinnamomi colonies (an average of 14 g- 1 on each medium), but AM was easier to handle and examine. Washing disturbed the soft surface of PIoVP and soil organic particles present interfered with examination. Fewer and more restricted colonies of Pythium spp developed on AM. AM, with two modifications, was used in subsequent experiments. Reducing the agar concentration from 4 to 3% increased the growth rate of P. cinnamomi and made colonies easier to distinguish. V-8 juice was centrifuged at 15,000 rev. min- t for 10 min to remove suspended particles that interfered with microscopic examination. The modifications had no apparent effect on recovery of P. cinnamomi. Incubation at 20° vs 24°C, in light vs dark, and washing and examining plates after 16, 24 and 36 h were compared. A temperature of 24°C increased growth of P. cinnamomi in both light and dark treatments throughout and colonies were easier to detect. Incubation in the dark reduced development of Pythium spp regardless of temperature. At 16 h colonies had not developed sufficiently for positive identification and at 36 h Pythium spp masked P. cinnamomi. Incubation at 24°C in the dark for 24 h permitted maximum recognition of P. cinnamomi and was used in subsequent experiments. Two subsamples of soil were collected from each of two forest sites and used the same day. One 50g moist subsample was placed on 38/am-mesh, washed under running tapwater for 5 min, and the residue on the sieve removed by backwashing. A second 50 g subsample was not washed. Water was added to each subsample to bring the volume to 150 ml. The mixture was then mixed in an Omni mixer for 30s at 1500rev. min-1, placed on a magnetic stirrer, and a 3 ml subsample dispersed on each of 10 plates of modified AM. An additional five, 3 ml subsamples were placed in weighed aluminum dishes and dried at t05°C overnight to determine average oven-dry weight of soil per plate. The number of P. cinnamomi colonies recovered from washed subsamples (2.4 and 4.8 colonies g-1) was significantly less than from nonwashed subsamples (13.4 and 27.6 g- 1), indicating that propagules < 38/am dia were present. When organic muck soil was autoclaved, infested with chlamydospores of P. cinnamomi (Hemmes and Wong, 1975), and treated in the same manner, 352 colonies g-1 were recovered from the nonsieved sample and 60 colonies g- ~ from the sieved. Soils from six forest sites were washed on 38/am-mesh and filtrates baited with lupine (Lupinus angustifolius L.) radicles (Chee and Newhook, 1965). P. cinnamomi was recovered from the filtrates of four of the six soils. Sieving was inefficient and eliminated recovery of propagules <38/am. In subsequent experiments, washing
Development of isolation technique Soil dilutions (Hendrix and Kuhlman, 1965) of 1:10, 1:20, and 1:100 of three soils were placed on the antibiotic medium (AM) of McCain et al. (1967) and on modified Kerr's (Hendrix and Kuhlman, 1965) medium. After 72 h at 20°C, colonies of Pythium spp on both media and at all dilutions had overgrown the plates. P. cinnamomi could be distinguished only by tediously scanning entire plates for the presence of its characteristic mycelium. The numbers of P. cinnamomi colonies ranged from 0 to 3.1 g - L When 1 g soil per plate was placed on the two media, dispersed with 3 ml water and incubated under the same conditions, similar results were obtained. When the sieving technique was tested using the same three soils and two media, the 149/am-mesh plugged up immediately because of the high soil organic matter content. Propagules of P. cinnamomi ranged from 1,1 to 7.8g -1.
*Present address: Insect & Disease Management, Region 5, U.S. Forest Service, 630 Sansome Street, San Francisco, CA 94111, U.S.A. 89
90
Short communications
Fig. 1. Propagules of Phytophthora cinnamomi recovered from Ohia forest soils on the island of Hawaii. (A) Cluster of encysted zoospores. Ten germinating cysts are present. (B) Sporangium with germinating cysts retained inside. (C) A chlamydospore, 57.9 #m dia. (D) A hyphal swelling, 19.8 by 23.2 #m.
of soils on 38/am-mesh was omitted and subsamples from a 50 g sample were plated. Propagules of P. cinnamomi in forest soils The types and dimensions of propagules recovered from three forest sites sampled every 2 weeks for a year were recorded. Ten plates per site were examined at each sampiing. Of 4744 colonies of P. cinnamomi observed, 55~o originated from zoospores, 18~o from chlamydospores or hyphal swellings, 15~o from organic matter, 11~o from pieces of hyphae, and 1% from directly-germinating sporangia. Oospores were not observed. Encysted zoospores were sometimes seen in clusters on plates (Fig. 1A), often with an empty sporangium nearby, indicating that they may have been released during the 24 h incubation. In some instances, zoospores had encysted and germinated while still in the sporangium (Fig. 1B). Numerous individual zoospores were also observed on plates during some periods of the year but not others. Their presence was -elated to environmental conditions present at time of field ~ r - , l i n g (Kliejunas and Nagata, unpublished). Cysts ra,.sed from 10.5 to 11.8 #m dia, Directly-germinating sporangia were only occasionally seen. Sporangia were normal in size (Waterhouse, 1970) and no microsporangia similar to those found in vitro (Ho and Zentmyer, 1977) were seen. Chlamydospores ranged from 15.4 to 82.0/*m dia (mean, 44.2 #m) (Fig. IC). Of 175 chlamydospores measured, 12~ were <38 #m dia. Oblong thin-walled structures which were apparently hyphal swellings (Fig. 1D) were recorded
as chlamydospores. Blackwell (1949) distinguished between hyphal swellings which are not cut off by septa and chlamydospores which are. The hyphal swellings ranged from 14.5 by 23.2#m to 34.4 by 37.6#m and all were small enough to pass through 38 #m mesh. They lacked the inner wall-layer of typical chlamydospores. Colonies also originated from pieces of organic matter which when teased apart contained no discernable propagule, and from pieces of hyphae with no discernable propagule present. On nine occasions when zoospores were observed on plates, a second 50 g sample of the same soil was mixed with 200 ml water, placed on 38 #m mesh, and washed 10 times with 200 ml water. The filtrates were collected separately, passed through 20 #m mesh, and baited with lupine. P. cinnamomi was recovered from the 20 #m filtrates on five of the nine occasions. When 0.5 ml samples of the filtrates were examined microscopically, propagules similar to P. cinnamomi cysts were observed but none had germinated to enable identification. Sporangia, chlamydospores, or hyphal swellings were not seen in the filtrates. On five other occasions when zoospores were observed on plates, a second soil sample from the same site was collected and tested as above the same day. P. cinnamomi was recovered by baiting from < 20 #m filtrates on three of the five occasions, indicating propagules < 20/zm were present in field soils. To determine if the plating technique induced indirect sporangial germination, mycelium with mature sporangia of P. cinnamomi (6 mm dia mycelial discs incubated in the dark for 3 days at 24°C in soil extract) were added to
Short communications plates with 3 ml of soil mixture and incubated as usual. The experiment was performed twice with soil from each of the three sites but indirect germination of the added sporangia was not observed. Zoospores were absent from plates where the mycclial discs had been. These results strongly suggested that free zoospores were present in field soils. We conclude that ohia forest organic muck soils are at times a favorable environment for in situ sporangial production and subsequent indirect germination, and a high potential for population increase of P. cinnamomi exists in these soils.
Acknowledgements--Journal Series Paper 2313 of the Hawaii Agricultural Experiment Station. Supported in part by funds from the U.S. Forest Service. REFERENCES
BLACKWELL E. M. (1949) Terminology in Phytophthora. Mycolooical Papers 30, 1-23. CHEE E. J. and NEWHOOKF. J. (1965) Improved methods for use in studies on Phytophthora cinnamomi Rands and other Phytophthora species. New Zealand Journal of Agricultural Research 8, 88-95. COTH~RE. J. and GRn~FIND. M. (1973) Formation of chla-
91
mydospores by Phytophthora drechsleri. Transactions of the British Mycological Society 61, 379--402. HEMM~ D. E. and WOSG L. D. S. (1975) Ultrastructure of chlamydospores of Phytophthora cinnamomi during development and germination. Canadian Journal of Botany 53, 2945-2957. HENDRIX F. F. JR and KUHLMAN G. H. (1965) Factors affecting direct recovery of Phytophthora cinnamomi from soil. Phytopathology 55, 1183-1187. Ho H. H. and ZErCrMYERG. A. (1977) Morphology of Phytophthora cinnamomi. Mycologia 69, 701-713. HWANG S. C. and Ko W. H. (1978) Biology of chlamydospores, sporangia, and zoospores of Phytophthora cinnamomi in soil. Phytopathology 68, 726-731. MCCAIN A. H., HOLTZMANNO. V. and TRUJtLLO E. E. (1967) Concentration of Phytophthora cinnamomi chlamydospores by soil sieving. Phytopatholooy 57, 1134-1135. REEVESR. J. (1975) Behaviour of Phytophthora cinnamomi Rands in different soils and water regimes. Soil Biology & Biochemistry 7, 19-24. TSAO P. H. and OCANA G. (1969) Selective isolation of species of Phytophthora from natural soils on an improved antibiotic medium. Nature 223, 636-638. WATEgHOUS~ G. M. (1970) The genus Phytophthora de Bary. 2nd (revised) edn. Mycological Papers 122.