Growth of noninfected and PIasmodiopkova bvassicae infected cabbage callus in culture1 P. H. WILLIAMS,M. N. REDDY,AND J. 0. STRAND BERG^ Can. J. Bot. Downloaded from www.nrcresearchpress.com by Laurentian University on 10/26/17 For personal use only. Departrtret~tof Plant Pathology, Utrivet~sityof Wisconsin, Mndison, Wisconsitr Received February 17, 1969 , 1969. Growth of noninfected and PlastrroWILLIAMS,P. H., REDDY,M. N., and S ~ A N D B E RJ.G0. diophorn brnssicae infected cabbage callus in culture. Can. J. Bot. 47: 1217-1221. Noninfected cabbage callus and Plast~~odiophorabrassiccre infected callus isolated fro111 clubroot galls were maintained for over 40 successive transfers o n Murashige-Skoog's medium modified to contain 0.5 mg/l of a-naphthalene acetic acid. Infected callus if transferred at 7- to 12-day intervals grew rapidly, doubling its dry weight about every 3 days, whereas noninfected callus doubled its dry weight about every 5 days. Approximately one-third of the cells in infected callus contained the parasite in various stages of its life cycle, ranging from small vegetative plasmodia to mature resting sporangia. Sporangia isolated from callus were viable and produced clubroot inoculated on cabbage seedlings. When a high percentage of the plasmodia in infected cells in any portion of a callus underwent sporogenesis, the callus growth slowed and the tissues became brown. By transferring only actively proliferating callus a high percentage of plasmodia could be maintained in the vegetative condition. Infected callus resembles closely gall tissue from natural clubroot both cytologically and chemically and thus should be a useful material for studying parasitism and the processes of hypertrophy and hyperplasia in a contaminant-free system. Introduction A lack of success in culturing obligate fungal parasites on artificial media has prompted the development of tissue culture techniques for the study of these organisms. Callus tissue infected with a single parasite provides a means for examining the physiology of parasitism without the influence of contaminating organisms. A comparison of the nutritional requirements of infected and noninfected callus tissues could make it possible to ascertain the nutritional factors required to grow the obligate parasite in vitro. Although there have been a number of reports of obligate fungi being grown in monoxenic culture with their host tissues (1, 1S), few of these have led to productive programs on the nature of obligate parasitism or to the culture of the obligate parasite itself. AS in the work of Nozzolillo and Craigie (18) with Puccinia helianthi Schw. on Helianthus annuus L. or in the work of Heim and Gries (7) with Erysiphe cichoracearum D.C. on H . annuus, the fungus killed the callus and subsequently died out, before many successive transfers of the culture lThis work was supported in part by the National Kraut Packers Association, in part by the American Cancer Society, Grant No. IN-35G-13, and in part by Public Health Service Grant A1 4149. Published with the approval of the Director, Wisconsin Agricultural Experiment Station Project No. 2007. 2Present address: Central Florida Experiment Station, Sanford 32771. could be made. Milholland (14) was unable to maintain Puccinia granzinis var. tritici infected wheat callus in culture beyond a single transfer. The most successful cultivation of an obligate parasite on host callus has been that of Cutter (3) for Gymnosporangium juniperi-virginianae Schw. on Juniperus virginiarza L. Infected calluses were maintained up to 8 years by successive transfers and eventually led to the isolation of the fungus on artificial medium. Turel and Ledingham (22) succeeded in producing abundant sterile mycelium and spores of Melampsora lini (Pers.) Lev. on the cotyledons of flax in organ culture and Nakamura (17) has maintained contaminant-free Peronospora parasitica on turnip root explants in culture. With the exception of the systemically infected juniper callus grown by Cutter, most of the fungi in tissue culture grew superficially on the callus and eventually the noninfected tissues outgrew the invaded cells. Unlike many of the filamentous obligate fungal parasites, Plasmodiophora brassicae Wor. remains entirelv within the host cells where it stimulates a callus-like gall on the roots and hypocotyls of various crucifer species (2). Previous cytochemical observations indicated that the Dresence of the ~arasitein crucifer cells was an important factor in the hyperplastic and hypertrophic response leading to gall formation (23). Because of the superficial similarity between clubroot gall tissue and tissue culture Can. J. Bot. Downloaded from www.nrcresearchpress.com by Laurentian University on 10/26/17 For personal use only. 1218 CANADIAN JOURNAL OF BOTANY. callus, it appeared that a study of P. brassicae infected callus in monoxenic culture would enable cytochemical and physiological studies on the fundamental processes underlying gall formation and parasite growth. Comparisons of various parameters for growth and metabolism of infected and noninfected cabbage callus with those already reported in the literature for natural clubroot galls would determine the validity of using tissue culture callus for study of the clubroot disease. In addition to providing a material for physiological work, infected callus would be a contaminant-free source of P. brassicae plasmodia (11) suitable for attempted in vitro culture of the parasite. This study is a more detailed account of the previously reported work on the monoxenic culture of P. brassicae in cabbage callus (21). Recently Ingram (9) also has reported on the growth of P. brassicae infected crucifer callus in tissue culture. Materials and Methods Infected cabbage callus was isolated from young actively growing clubroot galls and maintained on a synthetic medium. Clubroot galls were produced on cabbage plants, Brassica oleracea var. capitata L., Jersey Queen, grown in P. brassicae infested soil using the methods described previously (25). Infected roots were removed from the soil 25-30 days after the plants were transplanted into infested soil and then washed thoroughly in running tap water. Young white clubs between 2 and 3 mm in diameter were selected and further washed by shaking them for several minutes in sterile water in a stoppered flask. Clubs were transferred to a 57, chlorox solution in a petri dish for 5 rnin, cut into 4- to 5-rnm sections, and then transferred to 207, chlorox. After 5 rnin in 20% chlorox the sections were rinsed for 10 min in 10 changes of sterile water and placed individually into test tubes containing 5 ml of the tissue culture medium. The chemically defined medium of Murashige and Skoog (MS) (16) as modified by Linsmeier and Skoog (12) was further modified by using a-naphthalene acetic acid (NAA) (0.5 mg/l) instead of indole-3-acetic acid (IAA) and by adding kinetin (6-furfural aminopurine) at 1.0 mg/l (21). The medium was adjusted to pH 5.8 with NaOH before it was autoclaved and its pH was 5.6 after autoclaving. The tissue explants were placed in the dark at 22-24 OC. Tissues contaminated with bacteria or fungi were discarded. After 7-10 days, tissue pieces showing cell proliferation were transferred to fresh medium in test tubes and as callus developed it was excised, examined for the presence of the parasite, numbered, and transferred to flasks of the culture medium. Actively growing callus was divided and explants transferred to fresh MS medium every 7-12 days. Only cream or light-colored callus was transferred. Cultures were maintained in the dark at 22-24'C in 125-1111 Erlenmeyer flasks or 6-oz VOL. 47, 1969 prescription bottles containing 25 ml of the MS isolation medium. T o each bottle, four or five explants were transferred each weighing 3&45 mg (dry weight). Noninfected cabbage callus was obtained from hypocotyls of noninfected plants in the same way as described above for infected galls, or from hypocotyls of germinating Jersey Queen seed as follows. Cabbage seed was surface-sterilized for 10 min in 107, chlorox, then washed thoroughly in five changes of sterile water. Seeds were then transferred to flasks or bottles (four per bottle) containing 25 ml of Hildebrandt's D tissue culture medium (8). After 10-15 days on this medium the small seedlings began to develop callus from their roots, hypocotyls, and cotyledons. After 25-30 days callus originating from the hypocotyl and root regions of the seedling was excised, numbered, and transferred to fresh medium. Rapidly growing clones were transferred every 7-12 days on D medium. After 2 4 weeks the rapidly growing clones were transferred to MS medium supplemented with 500 mg/l of casamino acids (Difco) and 2.0 mg/l of 2,4-dichlorophenoxyacetic acid (2,4-D) in place of the NAA. This enriched medium was better for the initial transfer of noninfected callus from D medium because callus which was transferred directly to MS medium became brown and died. To avoid this transfer shock callus was brought to the MS medium over a period of two or three transfers by eliminating the casamino acid in the first transfer and replacing the 2,4-D with NAA in the second or third transfers. After the noninfected tissues had become adapted to MS medium they were grown and maintained in the same way as infected callus. Growth rates of infected and noninfected callus on MS medium were established by determining the fresh and dry weights of 10 tissue pieces from two bottles at specified intervals. For comparison, callus derived from pith of Nicotiarza tabacst~tzL. var. Wisconsin No. 38 was grown and sampled in the same way as cabbage callus. These experiments were repeated at least 4 times. lnfected and noninfected tissue culture callus was examined cytologically at different times between 2 and 12 days after explants were transferred to fresh medium. Pieces were fixed in formalin - acetic acid - alcohol (FAA) and dehydrated in a tertiary-butanol series. After standard paraffin embedding and sectioning techniques sections were stained in safranin and fast green (10). Ten sections from each of three different callus pieces taken at different times were observed for parasite development and host cell response. The number and volume of cell nuclei and nucleoli from infected cells and from noninfected cells in normal callus were estimated from diameter measurements, assuming that the nuclei and nucleoli were spherical. The volume of infected and noninfected cells was estimated from length and width measurements, assuming the cells were cylindrical. The number of cells per milligram of infected or noninfected callus was estimated by hydrolyzing 20-30 mg of tissue in 2 ml of 107, chromic acid for 8-12 h and then counting the separated cells with a hemocytometer. Infected and noninfected callus, 8 days after transfer to fresh medium, were analyzed for a number of metabolites by the identical procedure outlined previously for the separation of metabolites in clubroot galls (24). Callus tissue was washed in distilled water to remove Can. J. Bot. Downloaded from www.nrcresearchpress.com by Laurentian University on 10/26/17 For personal use only. WILLIAMS ET AL.:GROWTH excess medium and agar, then frozen in liquid nitrogen. Frozen pieces were powdered, lyophilized, dried at 80 OC for 24 h, and then stored at room temperature over CaC12. Samples of dried tissue (10 mg to 20 mg) were analyzed for total N by a Kjeldahl procedure (24), for total lipids by the quantitative sulfuric acid charring method (20), and for total sugars with the anthrone reagent (4). The quantity of starch was determined by the iodine method of Hassid (6), protein with the Lowry reagent (13), and free amino acids by the method of Yemm and Cocking (27). Four classes of phosphorous compounds, inorganic, acid labile, lipid phosphate, and nucleotide phosphates, were separated and extracted by the procedure of Smillie and Krotkov (19) and analyzed for phosphate by the Fiske and Subbarow method (5). T o determine the viability and infectivity of P. brassicae, resting sporangia produced in tissue culture 25- to 30-day-old callus pieces which had turned brown were minced in a petri dish under aseptic conditions with a sterile razor blade. The released sporangia were suspended (105 spores/ml) in a dilute salt solution (20) in 5-ml vials. Three-day-old Jersey Queen seedlings grown on water agar from surface-sterilized seed were placed in the vials containing sporangia. After 1-week root hairs were examined for infection and seedlings were transferred t o sterilized quartz sand and watered with a balanced nutrient solution. Thirty days later the seedlings were washed free from sand and observed for clubroot. 1219 OF CABBAGE CALLUS doubled its weight every 5 days. Tobacco pith callus grew at a similar rate as noninfected cabbage callus. Infected callus had a loose, irregular shape, and white to cream-colored growth (Fig. 2B) whereas noninfected callus was yellow and had a firm consistency (Fig. 2A). Infected callus averaged (4.5 rt 0.8)103 cellslmg fresh weight and contained about 8.670 dry matter. Noninfected callus averaged (16.2 5)103 cellslmg fresh weight and contained 12.1y0 dry matter (Table 11). Frequently, 7-12 days after transfer, small sectors of an infected callus piece began to darken and slowed in growth whereas other sections remained light-colored and continued to proliferate rapidly. Tissues which were not transferred to fresh media at regular intervals usually completely darkened and became necrotic in 3-4 weeks. Occasionally a tissue piece darkened within a few days of transfer without any apparent reason. Frequently necrosis of a whole infected tissue piece was preceded by the production of many small rootlets and profuse root hairs. Results Between 1 and 5% of the P. brassicae infected cabbage root explants produced continually growing callus on tissue culture. Most of the explants died as bacteria within the tissues multiplied. Individual clones of tissue isolated from the same root explant had different growth rates. The gross morphological characters of certain clones changed with repeated passage on the culture medium. Some clones continued as undifferentiated callus, whereas others produced many thickened structures resembling poorly differentiated roots. By selecting only undifferentiated tissue for transfers, the differentiating property of some clones could be eliminated. Root-hair-like structures were also observed on some infected and occasionally on noninfected callus. These root-hair-like structures could also be eliminated by selective transfer of callus tissue. By transferring fast-growing pieces of callus to fresh media at 7- to 12-day intervals, several rapidly growing clones have been maintained for more than 40 transfers. Figure 1 depicts the growth rate of infected callus clone, JQ-2, compared with a rapidly growing noninfected clone. Infected callus doubled its dry weight every 3 days whereas noninfected tissue -- P. B R A S S I C A E I N F E C T E D CABBAGE T I S S U E CULTURE /I > TISSUE CULTURE 0 2 4 GROWTH 6 8 10 12 PERIOD IN DAYS FIG. 1. Increase in dry weights of noninfected and Plasmodiophora brassicae infected cabbage callus grown o n a chemically defined medium. Can. J. Bot. Downloaded from www.nrcresearchpress.com by Laurentian University on 10/26/17 For personal use only. 1220 CANADIAN JOURNAL OF The various stages of parasite growth and host cell response found in natural clubroot galls (23) were also observed in tissue culture. Infected callus had numerous sites of meristematic activity with many cells containing small plasmodia. Characteristic "Krankheitsherde" were common and when the parasite occupied many cells in a localized area, host cell division slowed while plasmodia enlarged and eventually sporulated. An average of 34% of the callus cells contained the parasite in various stages of development. Darkening and necrosis of the tissue were usually accompanied by sporulation of the parasite. As was found in clubroot galls, host cell responses such as nuclear and nucleolar enlargement (23) and increased starch content (24) were restricted to the parasitized cells. Nuclear and nucleolar volumes increased an average of 4.5 and 23.8 times, respectively, compared to cells from noninfected callus (Table I). As was found previously with clubroot galls (24) total nitrogen, free amino acids, protein, total lipids, and starch were all higher in infected than in noninfected callus tissues (Table 11). Of the various phosphate compounds, lipid and nucleotide phosphates were slightly higher in TABLE I A comparison of size of cell, nucleus, and nucleolus in noninfected and Plosmodiophora brassicae-infected cabbage callus tissue in culturea Factor measured Noninfected Cell size, p 3 7260 Nucleus size, p 3 195.5 Nucleolus size, p 3 1 .6 a Infected Increase in infected 61879 878 38.8 8.5 4.5 23.8 Data are the averagc of 30 randomly sampled cclls. BOTANY. VOL. 47, 1969 infected tissues than in noninfected callus. Unlike in clubroot gall tissue, sugar levels were lower in the infected callus than in noninfected callus (Table 11). Resting sporangia collected from monoxenic cultures of P. brnssicne were viable as numerous plants became infected and produced clubroot galls 30 days after having their roots dipped in spore suspensions. Discussion The comparative ease with which P. brassicae infected cabbage callus was isolated and maintained in tissue culture on a chemically defined medium suggests its potential usefulness for the contaminant-free study of obligate host-parasite relations. Cytologically and ultrastructurally the parasite and the parasitized tissue culture cells are similar to those in natural clubroot galls (24, 26). Metabolites shown to increase in infected tissue culture callus also resemble those known to be high in natural clubroot galls (24). Although the changing amounts of metabolites in growing callus were not followed, it is unlikely that callus would show the sharp increases in starch, sugars, and nucleic acids that are known to occur when cabbage seedlings become infected with P. brassicae, mainly because the callus is a heterogenous mass of cells at different stages of infection. Some cells contain mature resting sporangia whereas others contain young vegetative plasmodia. In the case of young cabbages (24) the rapid buildup of starch and sugars in the gall reflected a response of the host to invasion by the parasite. Unlike in the cabbage plant TABLE I1 A comparison of various metabolites in noninfected and Plasmodiophora brassicae infected cabbage callus tissue cultures at 8 days after transfer to fresh medium Metabolite Dry wt. (mg/g fresh wt.) Total nitrogen Protein Free amino acids Total sugars Starch Total lipids Total phosphate compounds (a) Inorganic P (b) Acid-labile P (c) Lipid phosphates (d) Nucleotide P Noninfected, mg/g dry wt. Infected, mg/g dry wt. Increase in infected, mg Can. J. Bot. Downloaded from www.nrcresearchpress.com by Laurentian University on 10/26/17 For personal use only. FIG. 2. T h e appearance of noninfected cabbage callus 16 days after transfer (A) and (B) Plaa~~oriiopl~ora brasricne infected cabbage callus 8 days after transfer. Can. J. Bot. Downloaded from www.nrcresearchpress.com by Laurentian University on 10/26/17 For personal use only. Can. J. Bot. Downloaded from www.nrcresearchpress.com by Laurentian University on 10/26/17 For personal use only. WILLIAMS ET 1 I I AL.: GROWTH about one-third of all the cells in tissue culture callus are continuously infected. The localization of large amounts of starch in the infected cells in callus is similar to that in the cabbage root h ~ p o c o t ~galls. l As it does in the clubroot galls, the starch in infected callus cells disappeared upon sporogenesis of the parasite (24). Unlike natural galls the infected callus had a lower sugar content than noninfected callus. Noilinfected callus had 2 times as much sugar per gram dry weight as noninfected h ~ ~ o c o t (24) ~ l s Or as infected The tively low levels of sugars in the infected callus may reflect a rapid metabolizing of sugars since the respiration of infected callus tissue doubles in the presence of excess exogenous glucose or sucrose (unpublished data). Conversely the high Sugar contellt of noninfected tissue c0~1ldresult from an accumulation of sugars in tissues which are unresponsive metabolically to exogenous~y supplied sugars (unpublished data). Infected callus can be maintained in an active log phase Of growth through many transfers by frequent transfer and selection of actively growing sectors of tissue. The rapid growth of infected to nOninfected suggests that the parasite is providing some form of growth in addition to that in the medium. Although it is unknown whether the parasite is directly promoting growth by providing growth factors, or whether it is stirnulating- the host to do so, the tissue culture system seems to be feasible for investigating the problem. Acknowledgments The authors acknowledge the assistance of Dr. N. T. Keen with some chemical analyses and of Eugene Herrling and Steve Vicen with the figures. 1. BRIAN,P. W. 1967. Obligate parasitism in fungi. Proc. Roy. Soc. B. 168: 101-118. 2. COLHOUN,J. 1958. Clubroot disease of crucifers caused by Plasmodiophora brassicae Woron. Commonw. Mycol. Inst. Phytopathol. Pap. 3. 3. CUTTER,V. M. 1959. Studies on the isolation and growth of plant rusts in host tissue cultures and upon synthetic media. I. Gymnosporangium. Mycologia, 51: 248-295. 4. FAIRBURN, N. J. 1953. A modified anthrone reagent. Chem. Ind. 1953: 86. 5. FISKE,C. H. and S u n n ~ ~ o w Y., 1925. The colorimetric determination of phosphorus. J. Biol. Chem. 66: 375-400. 6. HASSID,W. Z. and NEUFELD,E. F. 1964. Quantitative determination of starch in plant tissues. pp. 33-36. I n R. J. Smith, J. N. BeMiller, and M. L. 1221 OF CABBAGE CALLUS Wolfrom, (Editors) Methods in carbohydrate chemistry. IV. Academic Press, New York. 7. HEIM,J. M. and GRIES,G. A. 1953. The ci~ltureof Erysiplre cickorncearro~r on sunflower tumor tissue. ~ ~ ~ y ~ ~ and single cell cultures of higher plants as a basic experimental method. pp. 383-421. It2 H. F. Linskens and M. V. Trace'. (Editors) Modern methods of plant analysis. Springer-Verlag, Berlin. 9. INGRAM,D. S. 1969. Growth of Plnsr~~odiophora brnssicae in host callus J. Gen. Microbial. 55: 9-18. 10. JENSEN, W. A. 1962. Botanical histochemistry. W. H. F,eman and co., sari F~~~~~~~~~ 11. KEEN,N. T., REDDY,M. N., and WILLIAMS, P. H. 1969. Isolation and properties of Plnsrnorli~~hora brassicne plasmodia from infected crucifer tissues and fmm tissue culture callus. Phytopathology, 59: 637-644. 12. LINSMEIR, E. M. and SKOOG,F. 1965. Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant. 18: 100-129. 13. LOWRY,0. H., ROSEBROUGH, N. J., FARR,A. L., and RANDALL, R. J. 1951. Protein measurement with the Folin phenol reagent, J. Biol, Chem. 193: 265-275. 14. MILHOLLAND, R. D. 1962. Callus tissue from wheat for infection studies of Plrccinin grnrninis var. tritici. Phytopathology (Abstr.), 52: 21. 15. MOREL.G. 1948. Recherches sur la culture associhe de parasites obligatoires et de tissus vegitaux. Ann. Epiphyt. (Paris), 14: 123-234. 16. MURASHIGE,T. and SKOOG,F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 14: 473-497. 17. NAKAMURA, H. 1965. The use of tissue cultures in the culture of obligate parasites, pp. 535-540. In P. R. White and A. R. Grove, (Edrtors) Proc. Int. Conf. Plant Tissue Culture. University Park, Penn. 18. NOZZOLILLO, C. and CRAIGIE,J. H. 1960. Growth of the rust fungus Plrccinia ~ ~ e ~ i aont ~tissue t ~ ~ iCUItures of its host. Can. J. Bot. 38: 227-233. 19. SMILLIE, R. M. and KROTKOV,G. 1960. The estimation of nucleic acids in some algae and higher ~ l a n t s .Can. J. Bot. 38: 31-49. 20. STRANDBERG, J. 0. 1968. Lipid metabolism in clubroot of cabbage. Ph.D. Thesis. University of Wisconsin, Madison. 21. STRANDBERG, J. O., WILLIAMS, P. H., and YUKAWA, Y. 1967. Monoxenic culture of Plasmodiouhora brassicae with cabbage tissue. ~ h y t o ~ a t h o l o g y(Abstr.) , 57: 903. 22. TUREL,F. L. M. and LEDINGHAM, G. A. 1957. Production of aerial mycelium and uredospores of Melnnlpsora lini (Pers.) LCv. on flax leaves in tissue culture. Can. J. Microbiol. 3: 813-819. 23. WILLIAMS,P. H. 1966. A cytochemical study of hypertrophy in clubroot of cabbage. Phytopathology, 56: 521-524. 24. WILLIAMS,P. H., KEEN,N. T., STRANDBERG, J. O., and MCNABOLA, S. S. 1968. Metabole synthesis and degradation during clubroot development in cabbage hypocotyls. Phytopathology, 58: 921-928. 25. WILLIAMS, P. H. and WALKER,J. C. 1963. Races of clubroot in North America. Plant Dis. Rep. 47: 608-61 1. 26. WILLIAMS, P. H. and YUKAWA, Y. B. 1967. Ultrastructural studies on the host-parasite relations of Plnsttlodiophora brnssicae. Phytopathology, 57: 682hR7 27. YEMM,E. W. and COCKING,E. C. 1955. The determination of amino acids with ninhydrin. Analyst, 80: 209-21 3. , "