Six media were compared for sporangial production and radial growth of Phytophthora cactorum (Lebert & Cohn) Schroeter. The media included: frozen lima bean agar (FLB), clarified V-8 juice agar (V-8 juice), fresh potato dextrose agar (PDA), Difco corn meal agar (DCM), Difco lima bean agar (DLB), and a semi-synthetic medium (GN) containing salts, thiamine, glucose and nitrate. Sporangial production was greatest on FLB, followed by V-8 juice; all isolates consistently produced more sporangia on these two media than on the other media tested. Sporangial production was substantially less on DCM, while DLB, PDA, and GN gave very poor sporangial production. The best radial growth occurred on DLB, followed by V-8 juice, FLB, DCM, PDA, and GN, respectively. There was no significant difference in radial growth on V-8 juice and DLB, nor on V-8 juice and FLB; however, growth was significantly less (P=0.05) on all other media.Seis meios de cultura foram avaliados em sua eficiência para produzir esporângios de Phytophthora cactorum (Lebert & Cohn) Schroeter. Os meios incluíram: Feijão-lima-ágar (FLB) gelado; suco V-8-ágar clarificado (suco V-8); batata-dextrose-ágar (PDA) fresco; Farinha de milho-ágar, da Difco (DCM); Feijão-lima-ágar, da Difco (DLB); e um meio semi-sintético (GN) contendo sais, tiamina, glucose e nitrato. A produção de esporângios foi maior em FLB, seguido por suco V-8; todos os isolados de Phytophthora cactorum produziram consistentemente mais esporângios nestes dois meios. A produção de esporângios foi substancialmente menor em DCM, enquanto que em DLB, PDA e GN foi mínima. O maior crescimento radial do fungo ocorreu em DLB, seguido por suco V-8, FLB, DCM, PDA e GN, respectivamente. Não houve diferença significativa (P=0,05) no crescimento radial em suco V-8 e DLB, nem em suco V-8, e FLB; entretanto, o crescimento foi significativamente menor (P=0,05) em todos os outros meios de cultura

Fortes, Joel Figueiredo

Pecknold, Paul C.

Embrapa: Sistema Eletrônico de Editoração de Revistas

EVALUATION OF MEDIA FOR SPORANGIAL PRODUCTION AND RADIAL GROWTH OF PHVTOPHTHORA CACTORUM' JOEL F1GUEIREDO FORTES 2 e PAUL C. PECKNOLD 3 ABSTRACT - Six media were compaied for sporangial production and radial growth of PaytophtJara cactorum (Lebert & Cohn) Schroe ter. The media included: frozen lima bean agar (FLB), clarificd V-8 juice agar (V-8 juice), fresh potato dextrose agax (PDA), DLfco com meal agar (DCM), Difco lima bean agax (DLB), and a scmi-tynthetic medium (GN) containing salts, thiamine, glucose and nitrate. Sporan-gial production was greatest on FLB, followed by V-8 juice; ali isolates consistentiy produced mor sporangia on these two media than on the other media tested. Sporangial production was substantially less on DCM, while DLB, PDA, and GN gave very poor sporangial production. The best radial growth occurred on DLI), foliowed by V-8 juice, FLB, DCM, PDA, and Gr'4, respectively. There was no signi-ficant difference in radial growth on V-8 juice and •DLB, nor on V-8juice and FLB; however, growth was significantly less (P- 0.05) on ali other media. Index terms: fungus, asexual reproduction, natural media. AVALIAÇÃO DE MEIO PARA PRODUÇÃO DE ESPOROS E CRESCIMENTO RADIAL DE PHYTOPHTI4ORA CACTORUM RESUMO Seis meios de cultura foram avaliados em sua eficiência para produzir esporângios de I4zytophthora cactorum (Lebert & Cohn) Schroeter. Os meios incluíram: Feijêo-lima-ágar (FLB) ge-lado; suco V.8-ágar clarificado (suco V-8); batata-dextrose-égar (PDA) fresco; Farinha de milho-ágar, da Difco CDCM 1; Feijio-lima-ágar, da 0 ifco (D LB); e um meio semi-sintético (GN) contendo sais, tiamina, glucose e nitrato. A produçáb de esporângiosfoi maior em F LB, seguido por suco V-8; todos os isolados de Phytophthora cactorum produziram consistentemente mais esporângios nestes dois meios. A produço de esporângios foi substancialmente menor em 0CM, enquanto que em DLB. PDA e GN foi mínima. O maior crescimento radial do fungo ocorreu em DLB, seguido por suco V-B, FLB, 0CM, PDA e GN, res-pectivamente. Ngo houve diferença significativa (P - 0,05) no crescimento radial em suco V-8 e DLB, nem em sucoV-8, e FLB; entretanto, o crescimento foi significativamente menor (P- 0,05) em todos OS Outros meios de cultura. Termos para indexaço: fungo, reproduço assexuada, meios naturais. INTRODUCTION To promote aporangial production, Phy toph-thora cactorum and other Pliytophthora species were. grown on a variety of defined (Hendrix 1964) and undefined media (Chee & Newhook 1965, Harnisli 1965, Hyre & Cox 1931, Mclntosh 1969, Mifler 1955, Schiffrnann-Nadel & Cohen 1966, Tucker 1931 e Waterhouse 1931). Wilson (1914) found media made with grain or leguminous sceds. (e.g. peas, beans, and oat) gave the most satisfactory growth for Phytophthora spp. Waterhouse (1931) found tliat many of these sarne Aceito para pub1icaço em 16 de julho de 1980. Parte da tese de M.S., apresentada pelo primeiro autor na Universidade de Purdue, Ind., USA. Eng? Agr9, Ph.D., Unidade de Execução de Pesquisa de Âmbito Estadual (LYEPAE-Cascata) - EMBRAPA, Cai-xa Postal 403, CEP 96.100- Pelotas, RS. Associate Professor, Ph.D., Department of Botany and Piant Pathology, Purdue University, West Lafayette, IN 47907, USA. media provided good sporangial production and concluded that the moisture content of the media probably regulated the production of sporangia. Tucker (1931), in his studies of the genus Phyto-phthora found oatmeal agar to be satisfactory for early and abundant production of sporangia; liowever, he concluded that no single medium was most favorable for sporangium formation, and that different isolates of the sarne species showed decided variation in sporangial production on the sarne medium. Harnish (1965), studying the effect of light on production of oospores and aporangia by four isolates ofP. cactorusn, alo found consid-erable variabiity between isolates when grown on clarified lima bean agar. Difco lima bean and V-8 juice agars are twa of the rnosc cornmonly used media for production of sporangia by P. cactorum; however, an assortmnent of other media are routinely employed to maximize sporangial production. Whule sporangial production on various media lias been documented by several Pesq. agropec. bras., Brasília, 16(2)213-218, mar. 1981. 214 	 J.F. FORTES e P.C. PECKNOLD authors (Chee & Newh.00k 1965, Harnish 1965, Hyre & Cox 1931, Miller 1955, Schiffmann-Nadel & Cohen 1966, Tucker 1931, Waterhouse 1931), there are no studies in which sporangial produc-tion iii P. cactorum has been quantitatively evaluat-cd on various media. lndeed, what quantitative studies have been done ib evaluate media for sporangial production by Phytoph thora spp have usualty entailed such a variety of methods for growing the organism and counting sporangia that quantitative comparisons between species and media are impossible to make. Also, many siudies have only included one isolate of the respective Phytophthora species, thus not allowing for differences between isolates iii their production of sporangia. Considering the importance ofP. cactorum as a plant pathogen and the frequent need to consis-tently produce large numbera of sporangia for taxonomic, physiological, etiological, and epide-miological purposes, a detailed, quantitative study of those media commonly used to prõmote spo-rangial production appeared warranted. MATERIAL AND METHODS Isolates of P. cactorum were furnished by tlie follow-ing personnel from their respective geographical locations (isolate designations fóllow each souxce):Dr. C.N. Clayton, Department of Plant Pathology, North Carolina State University at Raleigh (North Carolina); Dr. D.L. Mc-lntsh, Research Station, Summerland, B.C. Canada (Canada); Dr. Herb Aldwinclde, Geneva Experiinent Station, New York (Geneva); Dr. J. Sewell, East Malling Research Station, Maidstone, Kent, England (England); Dr. S.M. Mircetich, Plani Pathology Depaxtment, Univer-sity of California, Davis, California (California); Dr. Zofia Borecki, Institute of Pomology 96400 Skierniewice, V.I., Pomologidzna 18, Poland (Poland). Stock cultures were maintained on PDA slants ai room temperature (20 0 25°C). Pathogenicity tests were conducted both on six-day-old safflower seediings (cv. Nebraska-10) and on foiix to flve-month-old Gumes Golden apple seedlings in the coty-ledon stage. Seedlings were placed in paper cups contam-ing 225 mI of sterde distilled water with ca 1,200 zoosporeslmL After four days, the seedlings were removed, surface sterilized with 5% sodium hypochioride for 1 mi-nute, rinsed twice in sterile distiled water, blotted dry, and plaled to PDA medium containing 10 ppm of puna-dciii, 200 ppm of vancomycin, and 180,000 units o! polymyxin B sulfate. Pesq. agropec. bras., Brasília, 16(2)213-218, mar. 1981. The semi-synthetic medium (GN) was that used by Hendrix (1964) to study sterol unduction of reproduction and stimulation of growth of species of Pythb..m and Ptiytophthora. Frozen lima bean agar (FLB) was prepared by steanaing 142 g o! frozen Fordhook lima beans ia 400 mI of distilled water for 30 minutes, filtering through cheese cloth, then bringing the volume up to 1 liter. Af ter adding 10 g of agar, the solution was autoclaved for 30 minutes. Clarified V-8 juice agai (V-8 juice) was pre-pared by mixing 4.5 g o! CaCO 3 with 300 mlofV-Bjuicc (Campbell Co.), centrifuging at. 3,000 RPM for 5 minutes and diluting 200 ml of the supernatant with 800 ml of deionized water. Agar (15 g) was added and the mediuin then autoclaved for 30 minutes. Fresh potato dextrose agar (PDA) was prepared by steaming 200 g of peeled, sliced potatoes in 400 miof distffled water for 30 minutes, filtering through chedse cloth, and bringing the volume up to 1 liter. Dextrose (10 g)  and agar (12 g) were added before autoclaving for 30 minutes. Difco corn meal agar (DCM) and Difco lima bean agax (DLB) were prepared according to manufacturers Instruction. tnoculum disks (6 mm diameter), taken from the mar-gin of seven-day-old cultures o! P. cactorum grown on IDCM, were placed in the center of petri dishes with the different media. Cultures were grown at 250.  0.5 Cunder continuous iliumination. Light was provided by two Champion f90 t 171w, white fluorescent tubes suspended at a height of 45 cm. Each experinient was airanged iii a comptetcly randomized design with thiee replicates per isolate per media. Media were evaluated for sporangial production and radial growth atter seven days of incubation. Evaluation of radial growth was determined by measuring the colony diameter. Sporangial production was evaluated as follows: three plugs (6 mm diameter) were taken in a triangular fashion 5 mm in from the margin of seven-day-old cultures. Plugs were placed ia viais containing 1% lactophenol to kill mycelia and to prevent contamination. From the mid-die of each plug. a 1 mm wide strip was taken and cut into four 1 sq mm blocks. Each biock was then placed on a slide, covered with a cover slip, and gently squashed to avoid disruption ol' sporangia. The total number ef spo-rangia per block were then counted using X400 magnifi-cation. Thus a total of 12 sq mm per petri plate were counted. The average nwnber of sporangia per sq mm of each culture was then calculated. RESULTS Resulta of sporangial production on different media by T4iytophthora cactorum are shown ia Fig. 1. There was significant difference (P - 0.05) among the media tested ia supporting sporangial production, and also in sporangial production by differentisolates (Table 1). Of the six media tested, EVALUATION OF MEDIA FOR SPORANGIAL PRODUCTION 	 215 FLB supported maximum sporangial production, poorest production of sporangia; however, the followed by V-8 juice then DCM, with significant difference was not significant at P - 0.05 among difference at P- 0.05. DLB, GN and PDA gave the tbe media in this 1at group (Table 1). 50 45 	DFLB V-8 JUICE 40 	 PDA DCN 35 	 moLa •cM 30 25 20 z 15 o 10 5 o • NORTH CAROLINA 1 	 CENEVA 	 CALIFORNIA 	 POLAND 	 ENCLAND 	 CANADA P. Cactorum 150 LATES FIO. 1. Sporangial production of Phytophthora cactorutn lsolates after one week growth on frozen lima bean aar (FLB), clarified V-8 juce agar (V.8 luice), potato dextrose aqar (PDA). Difco Uma bean agar (OLB), Difco corn meal agar (0CM), and semi-synthetic medium (GN). TABLE 1. Sporangial production by I-liytophthora c.actorum isolates after 7 days growth on various media. Phytophthora cactorum isolates Culture Media England 	 California 	 Poland 	 Geneva 	 Canada 	 North Carolina Media Meansb Sporarigia per square millimeter FLB 48.2 	 55.4 	 36.7 	 29.8 	 23.3 	 16.7 3S0A V-8 Juice 17.9 	 16.3 	 13.1 	 20.1 	 9.5 	 6.9 14.58 DCM 5.3 	 0.7 	 2.8 	 4.8 	 9.0 	 3.1 4.3C GN 3.0 	 0.3 	 0.3 	 0.8 	 6.7 	 0.6 2.00D DLB 3.3 	 0.2 	 2.2 	 0.7 	 1.0 	 0.5 1.3D PDA 0.7 	 0.3 	 1.3 	 0.3 	 0.8 	 0.3 0.6D Isolate Meansb 	 13.1A 	 12.2A13 	 9.413C 	 9.46C 	 9.00 	 4.7C a FLB= frozen Uma bean agar; V-8juice=clarified V-8juice agar; 0CM- Oifco com meal agem; GN semi-synthetic me-dium; DLB. Difco lima bean agar; and PDA-fresh potatodextroseagar b Values foi Iowed by the sarne letter do not differ significantly at P 0.05 Pesq. agropec. bras., Brasflia, 16(2):213-218, mar. 1981. 9 8 7 6 ce 1- w o >- Z2 o -J o 216 	 J.F. FORTES e P.C. PECKNOLD Radial growth of P. cactorum on the different less supportive of radial growth than DLB, V-8 media are shown in Fig. 2. Radial growth was juice, and FLB (Table 2), greatest on DL, followedby V-8juice, FLB, DCM, PD.A, and GN agar, respectively (Table 2). There 	 DISCUSSION AND CONCLIJSIONS was no significant difference between V-8 juice and DLB, nor between V-8juice and FLB; however, 	 Studies with P. cactorum frequently require DCM, PDA, and GN were significantly (F = 0.05) large quantities of sporangia to be readily availa- A 	 ri 	 zo 9. PIO lii 	 Iii 	 I!1I 	 III 	 liii 	 iii NORTH CAROLINA 	 CENEVA 	 CALIFORNIA 	 POLAND 	 - 	 ENCLAND 	 CANADA P. Cactorum ISOLATES FIG. 2. Colon,y diameter of .l4zytophtliora cactcrum Isolates af ter seven days growth on frozen lima bean agar (FLB). clarified V-8 juice agar (V-8 juice), potato dextrose agar (PDA), and semi-synthetic medium (GN). TABLE 2. A comparison oC colony diameter of T4ytophthora cactorurn isolates alter 7 days growth õn various media. Phytophthora cactorum isolates Culture Media Medi - North.Caroluna Poland Canada 	 California Geneva England Meansb Colony dumeter (mm) •DL13 87.6 87.5 81.5 	 86.3 81.6 85.1 85.1A V-8 87.0 85.5 83.0 	 83.8 86.6 81.0 84.5AB FLB 85.5 86.1 84.6 	 84.3 84.3 81.0 83.913 0CM 81.6 80.4 80.4 	 80.0 80.1 81.0 80.7C PDA 73.0 71.3 70.0 	 67.8 64.6 69.5 69.4D GN 53.6 45.6 50.0 	 43.0 41.6 42.5 46.1E Isolate Means 781E 76.2D 74.9C 	 74.1B 73.5A8 73.QA a F LB = frozen lima bean agar; V-8 juice. clarified V-8 juice agar; DCM.Difcocorn meal agar: GN - semi-synthetic me-dium; DLB. Difco lima bean agar; and PDA= fresh potato dextrose agar. b Values fo!lowed by the sarne letter do not differ significantly at P. 0.05. Pesq. agropec. bras., Brasília, 16(2):213-218, mar. 1981. EVALUATION OF MEDIA FOR SPOR.ANGIAL PRODUCTION 	 217 Me. Whlle several media are routinely used for production of sporangia, consistently, large quantities of sporangia are not obtained. This is in part due to the varied effects that Iight, moisture, oxygen, temperature, and nutrients have on sporangial production and, ia part, due to the capricious nature of the organism. Hyre & Cox (1931), working with ten different media, found oat agar and lima bean agar to be best for maxirnum sporulation of P. p1aseoU. Of the six media evaluated in our study, the most conslstent and highest yields of sporangia were obtained on FLB and V-8 juice, with FLB the highest yields (Fig. 2 and 3). These fmdings are in agreement with Miier (1955), who reported sporulation of P. infestans on V-8 juice agar to be almost equal to that on fresh lima bean agar. Likewise, the poor sporangial production on PDA is in agreement with results by Chee & Newhook (1965) for P. cinflamomi, in which they observed that sporangia were produced sparingly and inonsistentIy on both fresh and Difco PDA. Of interest was the difference in sporangiat production between FLB and DLB, with FLB giving almost a 30-fold increase iii sporangia as compared to DLB. This is especially noteworthy since DLB is fre-quently used for sporangial production with many Phytop!thora spp. A possible reason for this large difference is that in the processing ofcommercially preparcd lima bean agar, sporangial promoting substances are removed and/or destroyed. Schiff-mann-Nadel & Cohen (1966) alio found poor sporangial production on eleven comercially prepared (Difco) agar culture media. Only five of tise eleven media supported sporangial production of F. citrophthora, and of these five, only Bacto--agar and lima bean agar gave appreciable numbers of sporangia. Tucker (1931) concluded that no single media was best for sporangial production among species of Phytophthora. However, from previous studies (Hyre & Cox 1931, Miller 1955, Schiffmann-Nadel & Cohen 1966) and our results, it does appear that certain media, e.g. FLB and V-8 juice, are superior for sporangial production, with possibly a universal similarity among species of Phytophthora as to whic1 media best support sporangial production. Also noteworthy is the apparent lack of correia- tion between sporangial production and radial growth (Fig. 3). However, this sarne lack of corre-lation was reported by Schfffmann-Nadel & Cohen (1966), with P. citrophthora, iii which eleven media were tested and no correlation between sporangiat prodction and radial growth was found. Apparen-tly, separate and unique substances are required for vegetative vs. reproductive growth of Phytoph-thora spp. e, JtI 	 DCII 	 CII 	 DLI 	 104 CULTIJE MEDIA FIG. 3. A comparison of growth end sporangl& production with six Isolates of Phytopthora cactorum on frozen lima bean agar (FLB), clarified V-8 juice agar (V-8 juice), potato dextrose agar (PDA). Difco lima bean agar (DLB). Difcocorn meal agar (DCM), and semi-synthetic medium (GN). A difference ia sporangial production among the isolares of P. cactorum used was also apparent. For example, the isolate from England showed an almost three-fold increase ia sporangia when com-paxed to the North Carolina isolate. A similar difference ia sporangial production among Phyto-phthora isolates also was noted by Tucker (1931), who found that different isolates of the sarne spe eles showed detided variation ia that a part icu-lar isolate would sometirnes produce sporangia Pesq. agropec. bras., Brasília, 16(2):213-218, mar. 1981. 218 	 J.F. FORTES e P.C. PECKNOLD sparsely on a meduni which was favorable for sporulation with other conspecific isolates. ACKNOWLEDGEMENTS • The authors extend their appreciation to E.B. Williams for assistance with certan aspccts of the work and• to R.J. Green for a critical review of the manuscript. REFERENCES CHEE K. & NEWHOOK, F.J. Improved methods foi use in studies on I4zytophthora cinnanvrni Rands and other I4iytophthora species. N.Z.J. Agric. Res., 8: 88-95, 1965. HÀRNISH, W.N. Effect of light on production of oospo-res and sporangia in species of t4iytophthora. Myco-logia, 57:85-90. 1965. I-IENDRIX, J.W. Influence of sterols ongrowth and reproduction of Pythiuni and I4sytoph 1thora spp. Phytopathology, 55:790-7, 1964. HYRE, R.A. & COX, R.S. Studies in the physiology of Paytophthora ph~oli. Phytopathology, 41: 562, 1931. MCINTOSH, D.L. Resistance to fliytophthora cactorum among open-poUinated seedlings of apple varieties and species. Proc. Am. Soe. Hort. Sci, 93:71-6, 1969. MILLER, P.M. V-8 juice as a general-purpose medium for fungi and bacteria Phytopathology, 45:46 1-2, 1955. SCHIFFMANr4-NADEL, M. & COHEN, E. lhe effect of agar culture and light on myceial growth and sporangium formation P. cifrophtIora (R.E.Sm. and E.H.Sm.) Leonlan. Israel J. Jiot,, 15:203-6, 1966. TIJCKER, C.M. Thc taxonomy of dia genusI4ytopht/wra DeBary. Coimbra, Missouri Agricultural Experiment Station, 1931. (Research BuUetin, 153). WATERHOUSE, G.M. fie production of conidia m the genus Pnytophtlwra. Trans. GuI. Mycol. Soe., 15: 311-21, 1931. WILSON, G.W. Studies in North American Peronosporales V. Review of thegenusI4ytophthora. Mycologia,6: 54-63, 1914. Peaq, agropee. bras., Brasília, 16(2):213-28, mar. 1981. 

Avaliação de meio para produção de esporos e crescimento radial de Phytophthora cactorum

http://seer.sct.embrapa.br/index.php/pab/article/download/16853/11182

Avaliação de meio para produção de esporos e crescimento radial de Phytophthora cactorum

Abstract

Similar works

Full text

Available Versions

Embrapa: Sistema Eletrônico de Editoração de Revistas