Dale, J. L.

Jones, J. P.

English

UARK (University of Arkansas )

Journal of the Arkansas Academy of Science Volume 23 Article 24 1969 Effect of Culture Environment Upon Sporangium and Zoospore Production of Three Species of Phytophthora J. L. Dale University of Arkansas, Fayetteville J. P. Jones University of Arkansas, Fayetteville Follow this and additional works at: https://scholarworks.uark.edu/jaas  Part of the Plant Pathology Commons Recommended Citation Dale, J. L. and Jones, J. P. (1969) "Effect of Culture Environment Upon Sporangium and Zoospore Production of Three Species of Phytophthora," Journal of the Arkansas Academy of Science: Vol. 23 , Article 24. Available at: https://scholarworks.uark.edu/jaas/vol23/iss1/24 This article is available for use under the Creative Commons license: Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0). Users are able to read, download, copy, print, distribute, search, link to the full texts of these articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. This Article is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Journal of the Arkansas Academy of Science by an authorized editor of ScholarWorks@UARK. For more information, please contact scholar@uark.edu. 136Arkansas Academy of Science Proceedings, Vol. 23, 1969EFFECT OF CULTURE ENVIRONMENT UPONSPORANGIUM AND ZOOSPORE PRODUCTIONOF THREE SPECIES OF PHYTOPHTHORA 1J. L. Dale and J. P. Jones 2University of ArkansasSpecies of Phytophthora are cosmopolitan in nature and areresponsible for economically important diseases of potato, soybeans,tobacco, and tomato. The Phytophthora spp. are Phycomycetes andreproduce sexually by the formation of oospores, and asexually bythe production of sporangia and zoospores.Since species of Phytophthora are common and of economicimportance, conditions favoring their growth and reproduction havebeen studied rather extensively. These fungi have also been used forteaching purposes as examples to demonstrate asexual reproductionby zoospores.Various techniques have been used to promote oospore andsporangium production within the genus Phytophthora. For spor-angium and zoospore production Gooding and Lucas (2) grewPhytophthora parasitica (Dastur) var. nicotianae (Breda de Haan)Tucker on solid oatmeal agar and stripped aerial mycelium fromcultures after 6-20 days growth. The mycelium was then transferredto plates and moistened or floated on water at 24-26°C for 6-10 days.Fresh distilled water was then added to the cultures, they werechilled for 25 min at 8°C, and then returned to room temperature.Zoospores were released after about 15 min. This procedure re-quires from 16-32 days. Sporangia and zoospores can also be obtainedfrom oatmeal agar cultures by taking small squares of agar withthe organism and floating them on water in Petri dishes for 10-12days, during which time additional mycelium and sporangia develop.The cultures are then chilled for about 30 min and the mycelium andsporangia are then transferred to water at room temperature andzoospores are released. This latter method is also time consuming,and sporangium development and zoospore release are often erratic.Reproduction on semi-solid media has been studied and isused less extensively (3, 4). The authors have successfully used suchmedia for a number of years for both research and teaching pur-poses. For sporangium production by Phytophthora parasitica Dast.,the causal organism of a tomato disease known as buckeye rot, very'Published with the approval of the Director of the Arkansas Agri-cultural Experiment Station.'Professors of Plant Pathology, Department of Plant Pathology.136Journal of the Arkansas Academy of Science, Vol. 23 [1969], Art. 24Published by Arkansas Academy of Science, 1969I137Sporangium and Zoospore Productiongood results have been obtained by growing the organism on 1/10strength lima bean agar (LBA). After 7-10 days, when sporangiahave been produced, a small volume of mycelium and residual mediaare put on a microscope slide and an equal or slightly larger volumeof distilled water is added to the slide. The mycelium is teasedapart with needles to expose the sporangia to the distilled water,and the preparation is covered with a cover slip. Zoospores normallybegin to be released within a few minutes after the slide is prepared.If the film of water is maintained under the cover slip, the zoo-spores will usually stop swimming, encyst, and germinate by theformation of a germ tube within an hour.The authors have used P. parasitica for instructional purposesbecause it is readily available from diseased plants or professionalsources and it is easy to culture. The optimum conditions for repro-duction of this organism on semi-solid media have not been fullydetermined. The present study was undertaken to further delineateconditions favoring sporangium and zoospore production.MATERIALS AND METHODSThe cultures used in the study were: an isolate of P. parasiticaobtained locally from tomato, two isolates of P. parasitica var.nicotianae from tobacco (NC 1156 and NC 1030),' and an isolateof P. cactomm (Leb. & Cohn) Schroet. 2 Isolates were maintained onplates of solid LBA (Difco). A 4 mm disc of each organism on agarwas aseptically transferred to a 125 ml Erlenmeyer flask containing20 ml of sterile l/10strength LBA. Appropriate numbers of suchinoculated flasks were immediately incubated in the dark at tem-peratures of 20, 24, 28, and 32"C, and a series of flasks were alsoincubated at room temperature under normal light conditions. At3-day intervals, for a period of 18 days, individual flasks of eachorganism at each temperature were checked for sporangium forma-tion and zoospore release. Forty ml of sterile distilled water wasadded to each flask, the flasks were gently shaken on a shaker for20 min., and a 1 ml sample was scanned under low magnification tocheck for presence of zoospores. The zoospores in the flasks werethen inactivated by the addition of 1 ml of 1-1000 HgCl2 per flask.The numbers of zoospores in the diluted medium were determinedby the use of a hemacytometer, and the results were transferredinto number of zoospores per ml of original growth medium.'Cultures supplied by Dr. J. L. Apple, North Carolina State Uni-versity.fCulture supplied by Dr. Donald C. Erwin, University of California,Riverside. 137Journal of the Arkansas Academy of Science, Vol. 23 [1969], Art. 24https://scholarworks.uark.edu/jaas/vol23/iss1/24138Arkansas Academy of Science ProceedingsRESULTS AND DISCUSSIONThe results of tests are shown in Figures 1 and 2. Since fewzoospores were produced at 32°C, this temperature was omitted fromthe graphs. The results show that considerable variation occurredamong the isolates in number of zoospores released and that sporan-gium production and zoospore release was affected by time, tempera-ture, and light conditions. The isolate from tomato, P. parasitica,produced the largest number of zoospores, with 187,000/ml beingreleased from flasks incubated 15 days at room temperature in thelight (Fig. 1-A). Isolate NC 1030 of P. parasitica var. nicotianavproduced the second largest number of zoospores, with 176,000/mlreleased from cultures incubated in the dark at 20°C for 18 days(Fig. 2-A). Isolate NC 1156 from tobacco produced consistently lownumbers of zoospores at all temperatures (Fig. 2-B). Except forP. cactorum the cultures were somewhat cyclic in zoospore release,with periods of high zoospore release followed by periods of lowzoospore release. This undoubtedly resulted from the fact that zoo-spores were released in the flasks during incubation even thoughno distilled water was added, and a time interval was necessary forproduction of additional sporangia. Interestingly, P. cactorum re-leased a maximum number of zoospores after 9 days incubation atall but room temperature (Fig. 1-BK Except for P. parasitica, thelargest numbers of zoospores were generally produced by the culturesincubated in darkness, regardless of temperature.The results in the present study differ somewhat from thoseobtained by other workers. Dukes and Apple (1), using the techniqueof Gooding and Lucas (2), reported that tobacco isolate NC 1156 wasmore pathogenic and produced more sporangia than isolate NC 1030.This contrasts withthe low number of zoospores produced by NC 1156and the high number produced by NC 1030 in the present work.Harnish and Barnett (3) reported that P. cactorum grown on diluteLBA produced only a few sporangia when grown in darkness. Al-though grown on the same medium in the present work, a differentisolate of the organism produced more sporangia and zoospores indarkness than under conditions of fluctuating darkness and light.Results obtained in the present work thus indicate that culturalconditions have a pronounced effect on sporangium and zoospore pro-duction, and that the reaction of isolates is dependent upon the tech-nique used. The results also indicate that individual isolates mayvary even when grown under similar cultural conditions.For instructional purposes to demonstrate zoospore release andactivity, the utilization of 1/10 strength LBA as a growth mediums more simple, rapid, and reliable than use of other methods. WithP. parasitica, the cultures can be grown under normal conditionsof light and room temperature, and abundant zoospores for obser-138Journal of the Arkansas Academy of Science, Vol. 23 [1969], Art. 24Published by Arkansas Academy of Science, 1969139I 200r" ' "IA P. pgrositico /\ |flr.. D £ cfldotum180- / \ l8°x 20° C / \ x 20°Co 24°C / \ o 24°C160- A 28°C /» 160- A 28°CD Room temp. / u a Room temp</> 140- / / ui l40 '"D I ~O OC , / C /\O / / O I.ion / / 3 20-\o I-/ / o'A \£ i I ¦£ If\ \~inn I ~ 100- '7. \^£ 80- / / £ 80- '// \\\O° '' / ° en 7/ \ VV a__ ¦—— """"*" "*"^**¦ - A_ ___ _ —JA»-*^ |T-«,-=--=r'=jJt" i I 1^ —̂_ -J—I03*-—-f i =1 fe* °3 6 S &- «Incubation Time, Days Incubation Tine, DaysFigure 1. Effect of incubation time and temperature upon number of zoospores producedby (A) Phytophthoraparasitica, and (B) P. cactorum.139Journal of the Arkansas Academy of Science, Vol. 23 [1969], Art. 24https://scholarworks.uark.edu/jaas/vol23/iss1/24140Figure 2. Effect of incubation time and temperature upon number of zoospores produced by (A) Phytophthoraparasitica var. nicotianae NC 1030, and (B) P. parasitica var. nicotianae NC 1156.140Journal of the Arkansas Academy of Science, Vol. 23 [1969], Art. 24Published by Arkansas Academy of Science, 1969141Sporangium and Zoospore Productionvational purposes can be obtained as early as 6 days following incu-bation of a culture. The fungus will release even greater numbersof zoospores with longer incubation periods under the same con-ditions.LITERATURE CITED1. Dukes, P. D., and J. L. Apple. 1962. Relationship of zoosporeproduction potential and zoospores motility with virulence inPhytophthora parasitica var. nicotianae. Phytopathology 52:191-193.2. Gooding, G. V., and G. B. Lucas. 1959. Factors influencingsporangial formation and zoospore activity in Phytophthoraparasitica var. nicotianae. Phytopathology 49:277-281.3. Harnish, W. N., and H. L. Barnett. 1962. The effect of light onsexual and asexual sporulation of species of Phytophthora.Phytopathology 52:1218-1219. (Abstr.)4. Klein, H. Harvey. 1959. Factors affecting development andmorphology of reproductive structures of the soybean rootand stem rot Phytophthora. Phytopathology 49:376-379.141Journal of the Arkansas Academy of Science, Vol. 23 [1969], Art. 24https://scholarworks.uark.edu/jaas/vol23/iss1/24

Effect of Culture Environment Upon Sporangium and Zoospore Production of Three Species of Phytophthora

ScholarWorks@UARK

Journal of the Arkansas Academy of ScienceVolume 23 Article 241969Effect of Culture Environment Upon Sporangiumand Zoospore Production of Three Species ofPhytophthoraJ. L. DaleUniversity of Arkansas, FayettevilleJ. P. JonesUniversity of Arkansas, FayettevilleFollow this and additional works at: http://scholarworks.uark.edu/jaasPart of the Plant Pathology CommonsThis article is available for use under the Creative Commons license: Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0). Users are able toread, download, copy, print, distribute, search, link to the full texts of these articles, or use them for any other lawful purpose, without asking priorpermission from the publisher or the author.This Article is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Journal of the Arkansas Academyof Science by an authorized editor of ScholarWorks@UARK. For more information, please contact scholar@uark.edu, ccmiddle@uark.edu.Recommended CitationDale, J. L. and Jones, J. P. (1969) "Effect of Culture Environment Upon Sporangium and Zoospore Production of Three Species ofPhytophthora," Journal of the Arkansas Academy of Science: Vol. 23 , Article 24.Available at: http://scholarworks.uark.edu/jaas/vol23/iss1/24136Arkansas Academy of Science Proceedings, Vol. 23, 1969EFFECT OF CULTURE ENVIRONMENT UPONSPORANGIUM AND ZOOSPORE PRODUCTIONOF THREE SPECIES OF PHYTOPHTHORA 1J. L. Dale and J. P. Jones 2University of ArkansasSpecies of Phytophthora are cosmopolitan in nature and areresponsible for economically important diseases of potato, soybeans,tobacco, and tomato. The Phytophthora spp. are Phycomycetes andreproduce sexually by the formation of oospores, and asexually bythe production of sporangia and zoospores.Since species of Phytophthora are common and of economicimportance, conditions favoring their growth and reproduction havebeen studied rather extensively. These fungi have also been used forteaching purposes as examples to demonstrate asexual reproductionby zoospores.Various techniques have been used to promote oospore andsporangium production within the genus Phytophthora. For spor-angium and zoospore production Gooding and Lucas (2) grewPhytophthora parasitica (Dastur) var. nicotianae (Breda de Haan)Tucker on solid oatmeal agar and stripped aerial mycelium fromcultures after 6-20 days growth. The mycelium was then transferredto plates and moistened or floated on water at 24-26°C for 6-10 days.Fresh distilled water was then added to the cultures, they werechilled for 25 min at 8°C, and then returned to room temperature.Zoospores were released after about 15 min. This procedure re-quires from 16-32 days. Sporangia and zoospores can also be obtainedfrom oatmeal agar cultures by taking small squares of agar withthe organism and floating them on water in Petri dishes for 10-12days, during which time additional mycelium and sporangia develop.The cultures are then chilled for about 30 min and the mycelium andsporangia are then transferred to water at room temperature andzoospores are released. This latter method is also time consuming,and sporangium development and zoospore release are often erratic.Reproduction on semi-solid media has been studied and isused less extensively (3, 4). The authors have successfully used suchmedia for a number of years for both research and teaching pur-poses. For sporangium production by Phytophthora parasitica Dast.,the causal organism of a tomato disease known as buckeye rot, very'Published with the approval of the Director of the Arkansas Agri-cultural Experiment Station.'Professors of Plant Pathology, Department of Plant Pathology.136Journal of the Arkansas Academy of Science, Vol. 23 [1969], Art. 24Published by Arkansas Academy of Science, 1969I137Sporangium and Zoospore Productiongood results have been obtained by growing the organism on 1/10strength lima bean agar (LBA). After 7-10 days, when sporangiahave been produced, a small volume of mycelium and residual mediaare put on a microscope slide and an equal or slightly larger volumeof distilled water is added to the slide. The mycelium is teasedapart with needles to expose the sporangia to the distilled water,and the preparation is covered with a cover slip. Zoospores normallybegin to be released within a few minutes after the slide is prepared.If the film of water is maintained under the cover slip, the zoo-spores will usually stop swimming, encyst, and germinate by theformation of a germ tube within an hour.The authors have used P. parasitica for instructional purposesbecause it is readily available from diseased plants or professionalsources and it is easy to culture. The optimum conditions for repro-duction of this organism on semi-solid media have not been fullydetermined. The present study was undertaken to further delineateconditions favoring sporangium and zoospore production.MATERIALS AND METHODSThe cultures used in the study were: an isolate of P. parasiticaobtained locally from tomato, two isolates of P. parasitica var.nicotianae from tobacco (NC 1156 and NC 1030),' and an isolateof P. cactomm (Leb. & Cohn) Schroet. 2 Isolates were maintained onplates of solid LBA (Difco). A 4 mm disc of each organism on agarwas aseptically transferred to a 125 ml Erlenmeyer flask containing20 ml of sterile l/10strength LBA. Appropriate numbers of suchinoculated flasks were immediately incubated in the dark at tem-peratures of 20, 24, 28, and 32"C, and a series of flasks were alsoincubated at room temperature under normal light conditions. At3-day intervals, for a period of 18 days, individual flasks of eachorganism at each temperature were checked for sporangium forma-tion and zoospore release. Forty ml of sterile distilled water wasadded to each flask, the flasks were gently shaken on a shaker for20 min., and a 1 ml sample was scanned under low magnification tocheck for presence of zoospores. The zoospores in the flasks werethen inactivated by the addition of 1 ml of 1-1000 HgCl2 per flask.The numbers of zoospores in the diluted medium were determinedby the use of a hemacytometer, and the results were transferredinto number of zoospores per ml of original growth medium.'Cultures supplied by Dr. J. L. Apple, North Carolina State Uni-versity.fCulture supplied by Dr. Donald C. Erwin, University of California,Riverside. 137Journal of the Arkansas Academy of Science, Vol. 23 [1969], Art. 24http://scholarworks.uark.edu/jaas/vol23/iss1/24138Arkansas Academy of Science ProceedingsRESULTS AND DISCUSSIONThe results of tests are shown in Figures 1 and 2. Since fewzoospores were produced at 32°C, this temperature was omitted fromthe graphs. The results show that considerable variation occurredamong the isolates in number of zoospores released and that sporan-gium production and zoospore release was affected by time, tempera-ture, and light conditions. The isolate from tomato, P. parasitica,produced the largest number of zoospores, with 187,000/ml beingreleased from flasks incubated 15 days at room temperature in thelight (Fig. 1-A). Isolate NC 1030 of P. parasitica var. nicotianavproduced the second largest number of zoospores, with 176,000/mlreleased from cultures incubated in the dark at 20°C for 18 days(Fig. 2-A). Isolate NC 1156 from tobacco produced consistently lownumbers of zoospores at all temperatures (Fig. 2-B). Except forP. cactorum the cultures were somewhat cyclic in zoospore release,with periods of high zoospore release followed by periods of lowzoospore release. This undoubtedly resulted from the fact that zoo-spores were released in the flasks during incubation even thoughno distilled water was added, and a time interval was necessary forproduction of additional sporangia. Interestingly, P. cactorum re-leased a maximum number of zoospores after 9 days incubation atall but room temperature (Fig. 1-BK Except for P. parasitica, thelargest numbers of zoospores were generally produced by the culturesincubated in darkness, regardless of temperature.The results in the present study differ somewhat from thoseobtained by other workers. Dukes and Apple (1), using the techniqueof Gooding and Lucas (2), reported that tobacco isolate NC 1156 wasmore pathogenic and produced more sporangia than isolate NC 1030.This contrasts withthe low number of zoospores produced by NC 1156and the high number produced by NC 1030 in the present work.Harnish and Barnett (3) reported that P. cactorum grown on diluteLBA produced only a few sporangia when grown in darkness. Al-though grown on the same medium in the present work, a differentisolate of the organism produced more sporangia and zoospores indarkness than under conditions of fluctuating darkness and light.Results obtained in the present work thus indicate that culturalconditions have a pronounced effect on sporangium and zoospore pro-duction, and that the reaction of isolates is dependent upon the tech-nique used. The results also indicate that individual isolates mayvary even when grown under similar cultural conditions.For instructional purposes to demonstrate zoospore release andactivity, the utilization of 1/10 strength LBA as a growth mediums more simple, rapid, and reliable than use of other methods. WithP. parasitica, the cultures can be grown under normal conditionsof light and room temperature, and abundant zoospores for obser-138Journal of the Arkansas Academy of Science, Vol. 23 [1969], Art. 24Published by Arkansas Academy of Science, 1969139I 200r" '"IA P. pgrositico /\ |flr.. D £ cfldotum180- / \ l8°x 20° C / \ x 20°Co 24°C / \ o 24°C160- A 28°C / » 160- A 28°CD Room temp. / u a Room temp</> 140- / / ui l40 '"D I ~O OC , / C /\O / / O I.ion / / 3 20-\o I-/ / o 'A \£ i I ¦£ If\ \~inn I ~ 100- '7. \^£ 80- / / £ 80- '// \\\O° '' / ° en 7/ \ VV a__ ¦——""""*" "*"^**¦ - A_____—JA»-*^ |T-«,-=--=r'=jJt" i I 1^^—_ -J—I03*-—-f i =1 fe* °3 6 S &- «Incubation Time, Days Incubation Tine, DaysFigure 1. Effect of incubation time and temperature upon number of zoospores produced by(A) Phytophthoraparasitica, and (B) P. cactorum.139Journal of the Arkansas Academy of Science, Vol. 23 [1969], Art. 24http://scholarworks.uark.edu/jaas/vol23/iss1/24140Figure 2. Effect of incubation time and temperature upon number of zoospores produced by (A) Phytophthoraparasitica var. nicotianae NC 1030, and (B) P. parasitica var. nicotianae NC 1156.140Journal of the Arkansas Academy of Science, Vol. 23 [1969], Art. 24Published by Arkansas Academy of Science, 1969141Sporangium and Zoospore Productionvational purposes can be obtained as early as 6 days following incu-bation of a culture. The fungus will release even greater numbersof zoospores with longer incubation periods under the same con-ditions.LITERATURE CITED1. Dukes, P. D., and J. L. Apple. 1962. Relationship of zoosporeproduction potential and zoospores motility with virulence inPhytophthora parasitica var. nicotianae. Phytopathology 52:191-193.2. Gooding, G. V., and G. B. Lucas. 1959. Factors influencingsporangial formation and zoospore activity in Phytophthoraparasitica var. nicotianae. Phytopathology 49:277-281.3. Harnish, W. N., and H. L. Barnett. 1962. The effect of light onsexual and asexual sporulation of species of Phytophthora.Phytopathology 52:1218-1219. (Abstr.)4. Klein, H. Harvey. 1959. Factors affecting development andmorphology of reproductive structures of the soybean rootand stem rot Phytophthora. Phytopathology 49:376-379.141Journal of the Arkansas Academy of Science, Vol. 23 [1969], Art. 24http://scholarworks.uark.edu/jaas/vol23/iss1/24

https://scholarworks.uark.edu/cgi/viewcontent.cgi?article=3024&amp;context=jaas

Effect of Culture Environment Upon Sporangium and Zoospore Production of Three Species of Phytophthora

Abstract

Similar works

Full text

Available Versions

UARK (University of Arkansas )

ScholarWorks@UARK