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Axenic culture of Peridermium pini
108
Mycol. Res. 9 5 (1): 108-115 (1990) Printed in Great Britain
Axenic culture of Peridermium pini
M. H. PEI* A N D R. G . P A W S E Y t Department of Forestry, University of Aberdeen, Aberdeen AB9 2UD, U.K.
Axenic cultures of Peridermium pini have been established on modified Shenk & Hildebrandt's and Harvey & Grasham's media following inoculation with naturally infected cortex tissue and artificially inoculated callus tissue of Pinus sylvestris, and with aeciospores. A small proportion of the fungal colonies obtained formed immature, mostly binucleate spores with smooth, irregularly thickened walls. Hyphae produced in culture, except those in the infrequently produced spore-proliferating colonies, were mononucleate. Actively growing cultures of P. pini were able to infect host callus tissues in vitro but no infection occurred in the inoculation tests in vivo.
Attempts to culture rust fungi on artificial media were first proved successful by Hotson & Cutter (1951) and since then more than 30 species of Puccinia, Gymnosporangium, Mel-
ampsora, Phragmidium, Cronartium, Uromyces, Melampsoridium and Pucciniastrum have been grown axenically. Of the more
than twenty species of pine blister rusts (Peterson, 1973), Cronarfium fusiforme (Hedgcock & Hunt) Cummins (Hollis, Schmidt & Kimbrough, 1972), C. ribicola 0. C. Fisch.) Rabenh. (Harvey & Grasham, 1974). C. quercuum (Miyabe) Shirai (Yamazaki & Katsuya, 1987) and Endocronartium harknessii (Moore) Hiratsuka (syn. Peridermium harknessii Moore) (Allen, Blenis & Hiratsuka, 1988) have now been grown separate from their hosts. Axenic cultures of C. fusiforrne, C. ribicola and E. harknessii were first established by inducing growth of mycelium into yeast extract-peptone medium from infected tissues of their pine hosts. Axenic cultures of C. fusiforrne were also obtained from basidiospores, aeciospores and urediniospores (Hare, 1978) and of C. ribicola from basidiospores (Diner & Mott, 1982). Axenic cultures of these biotrophic fungi, which had long been considered to be unculturable, provide suitable material f o r the studies of physiology, genetics and host-parasite interactions. Accounts of a number of intensive studies on many aspects of Puccinia graminis in axenic culture have been reviewed by Maclean (1982a, b). This paper reports, for the first time, the establishment of axenic cultures of Peridermium pini (Pers.) LCV.and some of its characteristics in culture. Present address: AFRC Arable Crops Research Institute, Long Ashton Research Station, Bristol BS18 9AF. U.K. t Present address: Llain Brongwyn, Cwm-Cou, Newcastle Emylyn, Dyfed, Wales SA38 9PR. U.K.
MATERIALS A N D M E T H O D S Culture media The media used for the establishment of the axenic culture of P. pini were modifications of Shenk & Hildebrandt's medium (Shenk & Hildebrandt, 1972). The basal medium (mg I-') consisted of KNO, (625), CaCI, .2 H 2 0 (50), MgSO,. 7H,O (loo), NH,H,PO, (75), KI (0.25), Na2Mo0,.2H,0 (0.025), CuSO,. 5 H 2 0 (0.05) and CoC1,. 6H,O (0.025). Other components added to the basal medium are shown in Table 1. To each litre of medium 8 g bacto agar was added and the pH was adjusted to 5.7-5.8 with 1 N-HCI and I N-NaOH before autoclaving at 121 OC for 15 min. A modified Harvey & Grasham's medium (Harvey & Grasham, 1974) containing the same strength of the six basal mineral salts, 4 g I-' 'Lab-Lemco' broth (Oxoid Ltd), 30 g 1-' sucrose and 8 g 1-' bacto agar was also used. Petri dishes (9 cm diam) were used in all studies and were sealed with Nescofilm (Bando Chemical Industries Ltd) after inoculation.
Cultures of P . pini From infected cortex. In spring 1987 naturally infected, 2- to 10-yr-old Scots pine branches, which were bearing aecia, were collected from Altyre, Morayshire in north-east Scotland. The following isolation procedure was adopted. The dead outer layers of the bark surrounding aecia were scraped off with a scalpel. A block of tissue approx. 2.0 x 1.0 cm and 0.4-0.6 cm deep, with a small portion of phloem beneath, was then excised From the area of the stem exposed by bark removal. This mycelium-carrying tissue was surface-sterilized by washing in running tap water for 10 min, followed by
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M. H. Pei and R. G. Pawsey Table 1. Media for establishment of axenic culture of Periderrnium prnr Compounds
KDGY
KDSY
KDGM
KDSM
BNGY
BNSY
BNGM
BNSM
KDS
BNS
'L-L' broth (g I-') Yeast extract (g I-') Malt extract (g I-') Sucrose (g I-') Glucose (g I-') Kinetin (mg I-') 2.4-D* (mg I-') BAP* (mg I-') NAA' (mg I-') 2.4-D, 2,4-dichlorophenoxyaceticacid; BAP, 6-benzylaminopurine; NAA, I-naphthaleneacetic acid
immersion in 70% ethanol for 2 min, rinsing three times in distilled water, soaking in 20 % Domestos (commercial bleach, Lever Brothers Ltd) for 25 min, and finally rinsed three times in sterilized water. The surface-sterilized blocks were then cut aseptically into small segments approximately 5 x 5 mm in size. Six such segments were laced on the surface of 30 ml solidified KDSM medium in each Petri dish and the inoculated dishes were sealed and incubated at room temperature (77-24') in a glass cabinet. From aeciospores. Three- to 5-yr-old Scots pine branches with aecium-bearing lesions were collected in May-June, and stored at -12'. Spores, labelled M87-1, M87-2, M87-3 (according to the year of collection and individual aeciumbearing trees) were collected from Moray forest in 1987, and T87-1, T88-1 spores were collected from infected pine trees in Thetford forest in East Anglia, England in 1987 and 1988. Dead layers of bark surrounding aecia were scraped off, and intact aecia were removed with sterile forceps. Spores were then shaken out of these detached aecia into a sterile Petri dish in a laminar flow cabinet. Aeciospores were dusted onto the surface of the different agar media (Table 1) in Petri dishes using a sterilized hair brush. Inoculated plates were sealed and incubated at room temperature. During the first week of incubation, the inoculated Petri dishes were examined daily and any contaminants were removed with a sterilized scalpel. Two plates of each of the 10 media in Table 1 were inoculated with M87-1 spores. M87-2, M87-3, T87-1 and T88-1 spores were inoculated directly only on KDSM medium. Spores used for initiating the fungal cultures were taken from aecia which had been stored at - 12' for a period from a few days to I yr. From callus tissues inoculated with aeciospores. Scots pine callus tissue cultures were developed from embryos on Murashige & Skoog's medium (Murashige & Skoog, 1962) supplemented with 30 g I-' sucrose, 2.0 mg I-' kinetin, 0.5 mg I-' 2,443 and 8 g 1-I bacto agar. Aeciospores collected as described previously were inoculated into calluses 8-13 mm diam by using a sterilized hair brush. The Petri dishes containing the inoculated callus cultures were sealed and incubated in the dark at 25'.
Cytology Nuclei. Small segments (about 1mm3) of hyphae and immature spore masses were removed from the surface and periphery of colonies growing on agar. These were soaked in 1 N-HCI for 30 rnin at 20°, washed in distilled water several times, and stained with Giemsa solution (stock:buffer as 1:7) for 20 rnin (Colotelo & Grinchenko, 1962). They were then mounted in the same staining solution and squashed gently before examination under a light microscope. Spore wall and septa1 pore. Samples of naturally developed aecia, spore-proliferating (from M88-4 spore source) and vegetative cultures were prepared as described by Jones (1973) for transmission electron microscope (TEM) examination. Mycelial fragments approx. 2 x 2 mm were removed from colony margins and from the upper surface of fungal colonies and fixed in 1% formaldehyde/3 % glutaraldehyde in 0.2 M sodium cacodylate (pH 6.5) for 1.5 h, washed in 0.2 M sodium cacodylate buffer for 30 min, and soaked in 1% (v/v) osmium tetroxide in 0-1 M phosphate buffer (pH 6.1) for 30 min. Finally they were rinsed in distilled water, stained for 5 rnin in 2% aqueous uranyl acetate, dehydrated through an ethanol series and infiltrated with LR White resin (London Resin Company Ltd, England). Capsules containing resin and samples were incubated at 60° for 2 d. Sections were cut on an ultramicrotome using glass knives, stained in 5 % saturated aqueous uranyl acetate for 10 min, rinsed in distilled water and stained in lead citrate solution for 5 rnin (Reynolds, 1963). Sections were examined and photographed using a Philips EM301 transmission electron microscope. Samples of naturally developed aecia were prepared following the same procedure except the material was infiltrated with resin for 5 d. Pathogenicity of culture In vitro. Scots pine callus tissues were cultured from embryos on Murashige & Skoog's medium and transferred to fresh medium every 4 wk. Calluses 6-9 mm diam were used for inoculation. The fungal colonies used for callus inoculation were grown on modified Harvey & Grasham's medium. Inoculum from each of four selected fungal cultures was used to inoculate 20 calluses per culture. Two of the cultures were derived from M87-2 spores; one culture being 7 months old,
Culture of Peridermium pini 10-12 mm diam, subcultured once and showing slow growth, and the other 6-wk-old, 3-4 mm diam and growing actively at the time of inoculation. The two other cultures were 8 months old and derived from T87-1 spores; one 4 - 6 mm diam, with a smooth surface, the other about 12 mm diam and with a white, fluffy surface. Both of the T87-1 cultures had been subcultured twice and were growing actively at the time of inoculation. The colonies were removed from the fungal culture medium and those larger than 6 mm diam were subdivided into 2-4 pieces. Calluses were inoculated by placing the mycelial side of the fungal inoculum against the callus surface. Inoculated calluses were kept at 25O in the dark. Eight weeks after inoculation, calluses were fixed in formalinacetic acid-alcohol (FAA) and embedded in paraffin wax. Microtome sections, 12-15 um thick, were stained with safranin-fast green (1.5 g safranin and 0.5 g fast green in 100 ml 60% ethanol) for 3-5 min, then destained in 100% ethanol for 1-2 min.
In vivo. Current or 1-yr-old Scots pine shoots were inoculated with fungal inoculum derived from axenic cultures in three ways. (1) Seven-month-old M87-2 cultures and 8-wk-old T88-2 cultures were both inoculated into current and 1-yr-old shoots. The inoculum was placed under the bark flap incised at an angle to a depth of 1-1.5 mm with a scalpel. The inoculated area was sealed with Nescofilm. (2) Colonies of the same type as used in (I) were cut into small pieces and macerated in distilled water. Using a disposable syringe with a large diameter needle, the mycelial suspension was injected into current Scots pine shoots 4-5 mm diam. (3) Eight-monthold T87-1 colonies (a) with a smooth surface and (b) with a white, fluffy surface were inoculated separately into 1-yr-old Scots pine shoots after wounding as described in (I). Thirty to forty current or I-yr-old shoots were used for each treatment.
RESULTS Culturing experiments Isolation from infected cortex. The main experiment was conducted in May-July 1987 with material taken from ten branches from four trees. Out of 78 cortex inocula, 14 became contaminated. The contaminants were distinguished from P. pini by their diverse morphology and rapid growth. After 2 wk, slow-growing fungal hyphae started to develop from the surfaces of the uncontaminated inocula which were showing some degree of callus development. Six weeks after inoculation, all the cortex discs showed evidence of the
110 development of long straight aerial hyphae. However, only in five cases was there also penetration of the medium during 8 wk of incubation (Fig. I). These medium-penetrating hyphae formed twisted, short, downward-developing branches in the agar medium. This fungal mycelium established in the medium was divided into 3-5 parts, and transferred onto fresh KDSM medium. By this means, 20 colonies were isolated from the 5 pieces of infected cortex tissue. The cortex tissues from which medium-penetrating hyphae had been removed often produced new hyphae which grew into the medium. Proliferation of aerial hyphae ceased on most of the cortex segments after 4 months except on those forming medium-penetrating hyphae.
Isolation from aeciospores. Work was concentrated on spores collected from Moray in 1987 (M87-1, -2, -3) and on those from Thetford collected in 1987 and 1988 (T87-1, T88-I). Spores germinated on all the media soon after inoculation. Spores which had been stored at - 12' for several months usually took longer to germinate. After germination, twisted, corkscrew-like hyphae started to develop from germ-tubes (Figs 2, 3). Irregular swellings (Fig. 3), which were strongly stained by HC1-Giemsa solution and morphologically different from those forming on Moray spore germ-tubes described by Gibbs et al. (1988) soon after germination, were produced frequently at the hyphal tips in both Moray and Thetford isolates. Three weeks after inoculation, colonies 1.52 mm diam began to appear on the surface of KDSM, KDGM, BNGM, and BNSM media. Growth of the colonies from M872 aeciospores on the tested media 5 wk after spore-seeding is indicated in Table 2.
Isolation from inoculated calluses. Callus infection was identified by the formation of aerial hyphae on the callus surface. Twelve weeks after inoculation, hyphae from 8 out of a total of 63 rust-infected calluses penetrated the medium (Fig. 4). Nineteen colonies were then obtained from these inoculated calluses following the same technique described under 'isolation from cortex'.
Colony development. Two types of hypha were formed in axenic culture of the fungus. One (type A) was long, straight, relatively unbranched, often aerial, growing mostly on the surface of the colony and spreading outwards (Fig. 5). These were up to 6 mm in length, with branches which often grew into the medium forming the second type of hypha (type B) which were twisted, usually corkscrew-like and with many branches (Figs 6, 7). These type B hyphae were intertwined to
Fig. 1. Aerial hyphae of P. pini From naturally infected cortex of P. sylvestris colonizing medium 8 wk after cortex isolation (bar = 1 mm). Fig. 2. Initiation of vegetative hyphae from an M87-1 spore on KDSM medium 2 wk after inoculation (bar = 40 WM). Fig. 3.
Hyphal development of a T87-1 spore on KDSM medium 8 d after inoculation. Irregularly shaped, strongly stained swellings (arrow) frequently formed at hyphal tips (bar = 20 vm). Fig. 4. Hyphae from the infected callus of P. sylvestris colonizing the medium 12 wk after callus inoculation with aeciospores (bar = 1.5 mm). Fig. 5. Long, straight hyphae (type A), mostly superficial. Fig. 6. Twisted, cork-screw like hyphae (type B), the main hyphal structure constituting stromata in culture. Fig. 7. Twisted hyphae starting from long, straight hyphae. Hyphae in Figs 3, 5-7 were stained with HCI-Giemsa (Figs 5-7, bar = 10 urn). Fig. 8. A colony derived From M87-2 spores, with a white f l u 6 surface and irregular margin. Fig. 9. Stromatal colony from T87-1 spores, with smooth surface and forming loose, rapidly extending hyphae (Figs 8, 9, bar = 2 mm).
M. H. Pei and R. G. Pawsey
Fig. 1-9. For caption see facing page
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Culture of Peridermium pini Table 2. Axenic growth of Peridermium pini 5 wk after inoculation with M87-2 aeciospores
Media KDGY KDGM KDSM KDSY KDS
Colony Growth
+
+++ ++++ ++
-
Media BNGY BNGM BNSM BNSY BNS
Colony Growth
+ +++ ++++ ++
-
+ Some branched hyphae developed from germ-tubes, with aggregated hyphae visible as scanty white spots. + + Hyphae grew more extensively, some formed colonies 0.5 mm diam, no stromata developed. Colonies composed of central stromata, 2-25 mm diam. ++ Central stromata well-developed, 3-4 mm diam. - No colonies established.
+++ ++
constitute a stroma in the centre of the developing colony. Cells of both hyphal types were mononucleate. Though many small hyphal branches similar to haustoria were seen in axenic culture, no structures identical to those formed in the natural host tissue were observed. The colonies of P. pini derived from aeciospores on KDSM medium varied in morphology and structure. Some colonies appeared fluffy on the surface (Fig. 8) while others had a smooth, often mucilaginous surface (Fig. 9). Thin, fastgrowing hyphal networks were also formed by both of these colonies usually 2 months after inoculation (Fig. 10). The size of the colonies appeared to vary depending on the distribution density of the colonies on the medium, smaller colonies occurring in areas of more dense distribution. Changes in the colony morphology occurred frequently during the period (several months) of incubation. -
Cytology Nuclei. Two hundred and sixty spores from the T88-1 sporederived colonies were examined by HC1-Ciemsa staining. As a result, 9.6 % were one-; 73.8 %, two-; 10.4 %, three-; 5.8 %, four- and 0 4 % six-nucleate. Of 420 spores from the M88-4 spore-derived colony, 7.6 % were one-; 86.9 %, two-; 4.8 %, three-; and 0.7%, four-nucleate. In general, the same number of nuclei were present in adjacent spores. Spore-proliferating hyphae were usually binucleate (Fig. 15). The cells of all vegetative hyphae examined were found to be mononucleate. Spore wall. Naturally formed aeciospores had thick electron dense walls and were ornamented on the surface with annular knobs resembling a stack of 6-7 discs, with a constriction between each disc (Fig. 16). By contrast, walls of immature spores formed in culture varied in thickness and were smooth on the surface without any ornamentation (Fig. 17). Septa1 pore. In hyphae, the septal pore was located in the centra area of the septum. The septal wall, which was tapered to form the pore margin, was equally separated by an electron-lucent region which extended from the margin of the central pore to the inner limit of the outer layers of the longitudinal wall (Figs 18, 19). Both sides of the septal pore were covered by a dark electron-dense diaphragm which often appeared hollow towards the pore centre (Fig. 18).
-
Sporulation of culture. Spores were formed in culture on only three occasions. O n the first occasion, one sporulating colony formed among hundreds of the colonies derived from M87-3 spores on KDSM 10 wk after inoculation. O n the second occasion, six out of 700 colonies from T88-1 spores on Harvey & Crasham's medium became orange/pinkish 6 wk after inoculation. They were concentrated in one area of the plate 2.5 cm diam. After 9 wk, orange-coloured spore masses formed in the centre of the colonies. O n the third occasion, one colony developed from M88-4 spores 10 wk after inoculation and produced immature spores similar to those formed in the colonies derived from T88-I spores. The spores produced in culture were frequently attached directly together (Fig. XI), some in clusters on single hyphae (Fig. 12), or sometimes linked by short hyphae forming spore-chains (Fig. 13), which resembled an early stage of aeciospore development (van der Kamp, 1969). The spores were always irregularly shaped and apparently immature (Fig. 14). Orange-coloured areas occasionally developed in some colonies derived from both Moray and Thetford spores, and waterdrop-like fluids were present on some of these pigmented and non-pigmented colonies but none contained structures comparable to spermatia. ~
-
Pathogenici& 1n vivo. Approximately half of the axenic inocula regained growth and extended newly formed hyphae into the inoculated calluses within 4 wk of inoculation. However, although a proportion of the 6-wk-old M87-2 inocula regained growth, none of the 7-month-old M87-2 inocula showed any sign of new growth. Aerial hyphae usually arose from the area of callus surface surrounding the site of inoculation (Fig. 20). Sections of calluses fixed 8 wk after inoculation with inocula from the two cultures of 8-month-old T87-1 colonies and the culture of 6-wk-old M87-2 colonies revealed the formation of intercellular hyphae and haustoria in the area near the inoculation site (Fig. 21). Infection appeared to be confined to a limited area of callus tissue and mainly involved the development of intercellular hyphae. Haustoria formed in host cells frequently showed a resemblance to undifferentiated hyphae (Figs 22, 23). In vivo. Calluses usually formed around the wounds made with a scalpel or injection needle. Though colonization of bacteria or other fungi occasionally occurred on inoculation sites, examination of hundreds of free-hand-sections revealed no evidence of the characteristic haustoria and intercellular hyphae of P. pini.
DISCUSSION Axenic cultures of P. pini have been established by isolating mycelium from naturally infected Scots pine cortex and artificially inoculated calluses, and by seeding aeciospores
Fig. 10. A colony from T87-1 spores on KDSM medium 3 months after spore-seeding (transferred after 6 wk). Fig. 11. Immature spores forming closely attached together in culture. Fig. 12. A cluster of immature spores developing from a hyphal end (Figs 11, 12, bar = 10 pm). Fig. 13. An immature spore forming at the end of a linking hypha. Fig. 14. Immature spores produced on a T88-1 colony. Note several 3-nucleate spores (arrow) forming in the same area. In Figs 11-14, nuclei were stained with HC1-Giemsa. Fig. 15. Binucleate hyphae from a spore-forming colony derived from M88-4 spores (Figs 13-15, bar = 20 pm). Fig. 16. Section of a naturally formed spore, showing the stacks of discs comprising the vermcae on spore surface (bar = 1 pm). Fig. 17. Section of immature spores with smooth, irregularly thickened walls (bar = 2 pm). Figs 18, 19. Septa1 pore with a pair of electron-dense diaphragms, which often appear hollow towards centre, an electron lucent layer equally separating septa1 layer (arrow) (bar = 0.5 pm).
Culture of Peridermium pini
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Fig. 20. Aerial hyphae arising from inoculum-attached callus surface of P. sylvestris 4 wk after inoculation with mycelium hom M87-3 colonies (bar = 1 mm). Fig. 21. Haustoria (arrow) in callus tissue of P. sylvestris 8 wk after inoculation with a 7-month-old T87-1 colony. Figs 22, 23. Haustoria (arrow) developed following callus inoculation, resembling undifferentiated hyphae (Figs 21-23, bar = 20
pm).
directly on agar-nutrient media. The technique of isolating axenic cultures of biotrophic fungi from dual cultures was successfully applied by Cutter and co-workers in the early days of rust fungus culture (Hotson & Cutter, 1951; Cutter, 1959, 1960). In the case of P. pini preliminary attempts failed either because of a high percentage of contamination or excessive sterilization which led to death of the external layers of host tissue. Successful isolation was not achieved until larger portions of cortex material were prepared and subdivided into small pieces after sterilization. Urediniospores have been used almost exclusively to establish axenic cultures of rust fungi. Contaminant-free urediniospores have been obtained either by setting up aseptic leaf cultures or by collection from freshly opened pustules on intact, rust-infected plants (Maclean, 1 9 8 2 ~ ) .Establishing axenic cultures of P. pini from aeciospores proved much easier since no previous isolation treatments were needed and fungal colonies could be obtained in profusion. The well-developed, relatively large aecial peridium undoubtedly prevents the great majority of the contained aeciospores from becoming contaminated, especially in the early stages of sporulation. Moreover, aeciospores of P. pini inside unbroken aecia stored at low temperature ( - 12O) can be used even up to 1 yr after collection. Inoculation density, at least for spores from some sources used in the experiments, appeared to be an important factor for the successful initiation of saprotrophic growth of P. pini. In preliminary experiments, no axenic colonies were developed in dishes in which the medium was sparsely seeded with M87-2, M87-3 spores, although colonies were consistently formed when spores were inoculated at a higher density. The success of axenic growth in relation to race and inoculum
density in several forma speciales of Puccinia graminis has been described previously by Hartly & Williams (1971a, b), Kuhl ef al. (1971), Green (1976), and Maclean (1982~). In previous studies of axenic culture of pine blister rusts, Hollis ef al. (1972) described one mycelial culture of C. fusiforrne (isolated from pine gall tissue) that produced mature aeciospores capable of infecting its alternative host. Harvey & Grasham (1974) described the formation of immature spores in some cortex-derived cultures of C. ribicola. No records of sporulation of spore-derived cultures of C. fusiforrne or C. ribicola have been found in the available literature (Hare, 1978; Amerson & Mott, 1978; Diner & Mott, 1982). In axenic cultures derived from aeciospores of P. pini, immature spores were formed infrequently and were concentrated in a limited area. The concentration of spore-forming colonies in a small area may indicate that a stimulus, most likely chemical, occurred in localized areas of the medium. The reluctance of a number of pine blister rusts to sporulate on artificial medium seems to be a phenomenon common to this group of rusts, unlike Melampsora species a range of which have been reported as sporulating in culture (Turel, 1969; Yamaoka & Katsuya, 1985). In the work on axenic culture of Cronarfium ribicola, cultured mycelium infected both tissue cultures and germ-free pine seedlings (Harvey & Grasham, 1974). Hare (1978) recorded that artificially grown cultures of C. fusiforme formed typical infection structures in P. ellioffii Engelm. var. ellioffii when injected into succulent shoots. However in his work, no haustoria were observed in inoculated callus tissues which appeared resistant to basidiospore inoculation (Hare, 1978; Jacobi, Amerson & Mott, 1982). Compared with callus infection established by inoculation with aecios~ores(Pei.
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M. H. Pei and R. G. Pawsey 1989), infection of host callus tissues by axenically grown mycelium of P. pini tended to be slower and the haustoria formed in cells frequently resembled undifferentiated hyphae. Nevertheless, the results showing that the mycelia from actively growing colonies of P. pini formed haustoria and intercellular hyphae in callus tissues suggest that the failure of in viuo infection was not simply due to loss of pathogenicity of the cultures. The method of inoculating spores of P. pini after wounding living pine shoots has been used by van der Kamp (1970), Klingstrom (1972), Olembo (1971), and Gibbs et a l . (1988). Following the inoculation of shoots with axenically grown mycelium, the sudden environmental change, slowness in regaining mycelial growth and the defensive reaction stimulated by the wounding of tissue may reduce infectivity, whereas aeciospores are well-adapted to germinsting under variable conditions and capable of establishing infection in a short period of time.
REFERENCES Allen, E. A,, Blenis, P. V. & Hiratsuka, Y. (1988). Axenic cultures of Endocronartium harknessii. Mycologia 80, 12G123. Amerson, H. V. & Mott, R. L. (1978). Technique for axenic production and application of Cronartium fusiforme basidiospores. Phytopathology 68, 673675. Colotelo, N. & Grinchenko, A. A. (1962). Growth of Kabatiella on different media. Canadian Journal of Botany 40, 439-446. Cutter, V. M., Jr (1959). Studies on the isolation and growth of plant rusts on host tissue cultures and upon synthetic media. I. Gymnosporangium. Mycologia 51, 248-295. Cutter, V. M., Jr (1960). Studies on the isolation and growth of plant rusts on host tissue cultures and upon synthetic media. 11. Uromyces atri-triphylli. Mycologia 52, 726-742. Diner, A. M. & Mott, R. L. (1982). Axenic cultures from basidiospores of Cronartium ribicola. Canadian Journal of Botany 60, 1950-1955. Gibbs, J. N., England, N. & Wolstenholme, R. (1988). Variation in the pine stem rust fungus Peridermium pini in the United Kingdom. Plant Pathology 37, 45-53. Green, G. J. (1976). Axenic culture of Puccinia species collected in Canada. Canadian Journal of Botany 54, 1198-1205. Hare, R. G. (1978). Axenic culture of Cronartium fusiforme from three spore forms. Canadian Journal of Botany 56, 2641-2647. Hartley, M. J. & Williams, P. G. (1971~).Interactions between strains of Puccinia graminis f. sp. tritici in axenic culture. Transactions of the British Mycological Society 57, 129-136. Hartley, M. J. & Williams, P. G. (1971 b). Morphological and cultural differences between races of hrrcinia graminis f. sp. tritid in axenic culture. Transactions of the British Mycological Society 57, 137-144.
Harvey, A. E., Grasham, J. L. (1974).Axenic ~ l t u r of e the mononucleate stage of Cronartium ribicola. Phytopathology 64, 1028-1033. Hollis, C. A., Schmidt, R. A. & Kimbrough, 1. W. (1972). Axenic culture of Cronartium fusifonne. Phytopathology 62, 1417-1419. Hotson, H. H. & Cutter, V. N., Jr (1951). The isolation and culture of Gymnosporangium juniperi-virginianae Schw. upon artificial media. Proceedings of the National Academy of Sciences, U S A 37, 400-403. Jacobi, W. R.. Arnerson, H. V. & Mott, R. L. (1982). Microscopy of cultured loblolly pine seedlings and callus inoculated with Cronartium fusiforme. Phytopathology 72, 138-143. Jones, D. R. (1973). Ultrastructure of septal pore in Uromyces dimlthi. Transactions of the British Mycological Society 61, 227-235. Klingstrom, A. (1972). Melampsora pinitorqua (Braun) Rostr. and Pm'dermium pini (Willd.) Kleb. Inoculation problems and techniques. In Biology of Rust Resistance in Forest Trees (Proceedings of a NATO-IUFRO Advanced Study Institute, Aug. 1969), pp. 313-323. Washington, D.C., USA: Forest Service, USDA. Kuhl, J. L., Maclean, D. J., Scott, K. J. & Williams, P. G. (1971). The axenic culture of Pucdnia species from uredospores, experiments on nutrition and variation. Canadian Journal of Botany 49, 201-209. Maclean, D. J. (1982~).Axenic culture and metabolism. Part A: Isolation and characteristics of axenic culture. In The Rust Fungi (ed. R. J. Scott & A. K. Chakravarti), pp. 37-84. London, U.K.: Academic Press. Maclean, D. J. (1982 b). Axenic culture and metabolism. Part B: Metabolism of axenic cultures. In The Rust Fungi (ed. R. J . Scott & A. K. Chakravarti), .pp.. 85-120. London, U.K.: Academic Press. Murashige, T. & Skoog, F. (1962).A revised medium for the rapid growth and bioassay with tobacco tissue cultures. Physiologia Plantarum 15, 473-497. Olembo, T. W. (1971). A study of the mode of infection of Pinus sylvestris by Peridermium pini (Pers.) LCV. Forestry 44, 67-79. Pei, M. H. (1989). Inoculation of callus tissues of Scots pine, Corsican pine and three varieties of mountain pine with Pm.dermium pini. Mycological Rtsearch 93, 503-507. Peterson, R. S. (1973). Studies of Cronartium (Uredinales). Reports of the Totton' Mycological Institute 10, 203-233. Reynolds, E. S. (1963). The use of lead citrate at high pH as an electron opaque strain in electron microscopy. Journal of Cell Biology 17, 208-212. Shenk, R. U. & Hildebrandt, A. C. (1972). Medium and technique for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Canadian Journal of Botany 50, 199-204. Turel, F. L. M. (1969). Saprophytic development of the flax rust Melampsora lini, race No. 3. Canadian Journal of Botany 47, 821-823. Van Der Kamp, B. J. (1969). Peridmiurn pini (Pers.) LPv. and the resin-top disease of Scots pine. 11. Lesion anatomy. Forestry 42, 185-201. Van Der Kamp, B. J. (1970). Peridermium pini (Pers.) Lev. and the resin-top disease of Scots pine. 111. Infection and lesion development. Forestry 43, 73-88. Yamaoka, Y. & Katsuya, K. (1985). Germinability and mfective ability of urediniospores produced in axenic cultures of five Melampsora species, as afFected by the artificial media on which the culture grew. Annals of the Phytopathological Society of Japan 51, 435-442. Yamazaki, S. & Katsuya, K. (1987). Axenic cultures of Cronartium qwrcuum and their pathogenicity. Annals of the Phytopathological Society of Japan 53, 643-646.
(Received for publication 2 8 November 1989 and in revised form 17 M a r c h 1990)
Mycol. Res. 9 5 (1): 108-115 (1990) Printed in Great Britain
Axenic culture of Peridermium pini
M. H. PEI* A N D R. G . P A W S E Y t Department of Forestry, University of Aberdeen, Aberdeen AB9 2UD, U.K.
Axenic cultures of Peridermium pini have been established on modified Shenk & Hildebrandt's and Harvey & Grasham's media following inoculation with naturally infected cortex tissue and artificially inoculated callus tissue of Pinus sylvestris, and with aeciospores. A small proportion of the fungal colonies obtained formed immature, mostly binucleate spores with smooth, irregularly thickened walls. Hyphae produced in culture, except those in the infrequently produced spore-proliferating colonies, were mononucleate. Actively growing cultures of P. pini were able to infect host callus tissues in vitro but no infection occurred in the inoculation tests in vivo.
Attempts to culture rust fungi on artificial media were first proved successful by Hotson & Cutter (1951) and since then more than 30 species of Puccinia, Gymnosporangium, Mel-
ampsora, Phragmidium, Cronartium, Uromyces, Melampsoridium and Pucciniastrum have been grown axenically. Of the more
than twenty species of pine blister rusts (Peterson, 1973), Cronarfium fusiforme (Hedgcock & Hunt) Cummins (Hollis, Schmidt & Kimbrough, 1972), C. ribicola 0. C. Fisch.) Rabenh. (Harvey & Grasham, 1974). C. quercuum (Miyabe) Shirai (Yamazaki & Katsuya, 1987) and Endocronartium harknessii (Moore) Hiratsuka (syn. Peridermium harknessii Moore) (Allen, Blenis & Hiratsuka, 1988) have now been grown separate from their hosts. Axenic cultures of C. fusiforrne, C. ribicola and E. harknessii were first established by inducing growth of mycelium into yeast extract-peptone medium from infected tissues of their pine hosts. Axenic cultures of C. fusiforrne were also obtained from basidiospores, aeciospores and urediniospores (Hare, 1978) and of C. ribicola from basidiospores (Diner & Mott, 1982). Axenic cultures of these biotrophic fungi, which had long been considered to be unculturable, provide suitable material f o r the studies of physiology, genetics and host-parasite interactions. Accounts of a number of intensive studies on many aspects of Puccinia graminis in axenic culture have been reviewed by Maclean (1982a, b). This paper reports, for the first time, the establishment of axenic cultures of Peridermium pini (Pers.) LCV.and some of its characteristics in culture. Present address: AFRC Arable Crops Research Institute, Long Ashton Research Station, Bristol BS18 9AF. U.K. t Present address: Llain Brongwyn, Cwm-Cou, Newcastle Emylyn, Dyfed, Wales SA38 9PR. U.K.
MATERIALS A N D M E T H O D S Culture media The media used for the establishment of the axenic culture of P. pini were modifications of Shenk & Hildebrandt's medium (Shenk & Hildebrandt, 1972). The basal medium (mg I-') consisted of KNO, (625), CaCI, .2 H 2 0 (50), MgSO,. 7H,O (loo), NH,H,PO, (75), KI (0.25), Na2Mo0,.2H,0 (0.025), CuSO,. 5 H 2 0 (0.05) and CoC1,. 6H,O (0.025). Other components added to the basal medium are shown in Table 1. To each litre of medium 8 g bacto agar was added and the pH was adjusted to 5.7-5.8 with 1 N-HCI and I N-NaOH before autoclaving at 121 OC for 15 min. A modified Harvey & Grasham's medium (Harvey & Grasham, 1974) containing the same strength of the six basal mineral salts, 4 g I-' 'Lab-Lemco' broth (Oxoid Ltd), 30 g 1-' sucrose and 8 g 1-' bacto agar was also used. Petri dishes (9 cm diam) were used in all studies and were sealed with Nescofilm (Bando Chemical Industries Ltd) after inoculation.
Cultures of P . pini From infected cortex. In spring 1987 naturally infected, 2- to 10-yr-old Scots pine branches, which were bearing aecia, were collected from Altyre, Morayshire in north-east Scotland. The following isolation procedure was adopted. The dead outer layers of the bark surrounding aecia were scraped off with a scalpel. A block of tissue approx. 2.0 x 1.0 cm and 0.4-0.6 cm deep, with a small portion of phloem beneath, was then excised From the area of the stem exposed by bark removal. This mycelium-carrying tissue was surface-sterilized by washing in running tap water for 10 min, followed by
109
M. H. Pei and R. G. Pawsey Table 1. Media for establishment of axenic culture of Periderrnium prnr Compounds
KDGY
KDSY
KDGM
KDSM
BNGY
BNSY
BNGM
BNSM
KDS
BNS
'L-L' broth (g I-') Yeast extract (g I-') Malt extract (g I-') Sucrose (g I-') Glucose (g I-') Kinetin (mg I-') 2.4-D* (mg I-') BAP* (mg I-') NAA' (mg I-') 2.4-D, 2,4-dichlorophenoxyaceticacid; BAP, 6-benzylaminopurine; NAA, I-naphthaleneacetic acid
immersion in 70% ethanol for 2 min, rinsing three times in distilled water, soaking in 20 % Domestos (commercial bleach, Lever Brothers Ltd) for 25 min, and finally rinsed three times in sterilized water. The surface-sterilized blocks were then cut aseptically into small segments approximately 5 x 5 mm in size. Six such segments were laced on the surface of 30 ml solidified KDSM medium in each Petri dish and the inoculated dishes were sealed and incubated at room temperature (77-24') in a glass cabinet. From aeciospores. Three- to 5-yr-old Scots pine branches with aecium-bearing lesions were collected in May-June, and stored at -12'. Spores, labelled M87-1, M87-2, M87-3 (according to the year of collection and individual aeciumbearing trees) were collected from Moray forest in 1987, and T87-1, T88-1 spores were collected from infected pine trees in Thetford forest in East Anglia, England in 1987 and 1988. Dead layers of bark surrounding aecia were scraped off, and intact aecia were removed with sterile forceps. Spores were then shaken out of these detached aecia into a sterile Petri dish in a laminar flow cabinet. Aeciospores were dusted onto the surface of the different agar media (Table 1) in Petri dishes using a sterilized hair brush. Inoculated plates were sealed and incubated at room temperature. During the first week of incubation, the inoculated Petri dishes were examined daily and any contaminants were removed with a sterilized scalpel. Two plates of each of the 10 media in Table 1 were inoculated with M87-1 spores. M87-2, M87-3, T87-1 and T88-1 spores were inoculated directly only on KDSM medium. Spores used for initiating the fungal cultures were taken from aecia which had been stored at - 12' for a period from a few days to I yr. From callus tissues inoculated with aeciospores. Scots pine callus tissue cultures were developed from embryos on Murashige & Skoog's medium (Murashige & Skoog, 1962) supplemented with 30 g I-' sucrose, 2.0 mg I-' kinetin, 0.5 mg I-' 2,443 and 8 g 1-I bacto agar. Aeciospores collected as described previously were inoculated into calluses 8-13 mm diam by using a sterilized hair brush. The Petri dishes containing the inoculated callus cultures were sealed and incubated in the dark at 25'.
Cytology Nuclei. Small segments (about 1mm3) of hyphae and immature spore masses were removed from the surface and periphery of colonies growing on agar. These were soaked in 1 N-HCI for 30 rnin at 20°, washed in distilled water several times, and stained with Giemsa solution (stock:buffer as 1:7) for 20 rnin (Colotelo & Grinchenko, 1962). They were then mounted in the same staining solution and squashed gently before examination under a light microscope. Spore wall and septa1 pore. Samples of naturally developed aecia, spore-proliferating (from M88-4 spore source) and vegetative cultures were prepared as described by Jones (1973) for transmission electron microscope (TEM) examination. Mycelial fragments approx. 2 x 2 mm were removed from colony margins and from the upper surface of fungal colonies and fixed in 1% formaldehyde/3 % glutaraldehyde in 0.2 M sodium cacodylate (pH 6.5) for 1.5 h, washed in 0.2 M sodium cacodylate buffer for 30 min, and soaked in 1% (v/v) osmium tetroxide in 0-1 M phosphate buffer (pH 6.1) for 30 min. Finally they were rinsed in distilled water, stained for 5 rnin in 2% aqueous uranyl acetate, dehydrated through an ethanol series and infiltrated with LR White resin (London Resin Company Ltd, England). Capsules containing resin and samples were incubated at 60° for 2 d. Sections were cut on an ultramicrotome using glass knives, stained in 5 % saturated aqueous uranyl acetate for 10 min, rinsed in distilled water and stained in lead citrate solution for 5 rnin (Reynolds, 1963). Sections were examined and photographed using a Philips EM301 transmission electron microscope. Samples of naturally developed aecia were prepared following the same procedure except the material was infiltrated with resin for 5 d. Pathogenicity of culture In vitro. Scots pine callus tissues were cultured from embryos on Murashige & Skoog's medium and transferred to fresh medium every 4 wk. Calluses 6-9 mm diam were used for inoculation. The fungal colonies used for callus inoculation were grown on modified Harvey & Grasham's medium. Inoculum from each of four selected fungal cultures was used to inoculate 20 calluses per culture. Two of the cultures were derived from M87-2 spores; one culture being 7 months old,
Culture of Peridermium pini 10-12 mm diam, subcultured once and showing slow growth, and the other 6-wk-old, 3-4 mm diam and growing actively at the time of inoculation. The two other cultures were 8 months old and derived from T87-1 spores; one 4 - 6 mm diam, with a smooth surface, the other about 12 mm diam and with a white, fluffy surface. Both of the T87-1 cultures had been subcultured twice and were growing actively at the time of inoculation. The colonies were removed from the fungal culture medium and those larger than 6 mm diam were subdivided into 2-4 pieces. Calluses were inoculated by placing the mycelial side of the fungal inoculum against the callus surface. Inoculated calluses were kept at 25O in the dark. Eight weeks after inoculation, calluses were fixed in formalinacetic acid-alcohol (FAA) and embedded in paraffin wax. Microtome sections, 12-15 um thick, were stained with safranin-fast green (1.5 g safranin and 0.5 g fast green in 100 ml 60% ethanol) for 3-5 min, then destained in 100% ethanol for 1-2 min.
In vivo. Current or 1-yr-old Scots pine shoots were inoculated with fungal inoculum derived from axenic cultures in three ways. (1) Seven-month-old M87-2 cultures and 8-wk-old T88-2 cultures were both inoculated into current and 1-yr-old shoots. The inoculum was placed under the bark flap incised at an angle to a depth of 1-1.5 mm with a scalpel. The inoculated area was sealed with Nescofilm. (2) Colonies of the same type as used in (I) were cut into small pieces and macerated in distilled water. Using a disposable syringe with a large diameter needle, the mycelial suspension was injected into current Scots pine shoots 4-5 mm diam. (3) Eight-monthold T87-1 colonies (a) with a smooth surface and (b) with a white, fluffy surface were inoculated separately into 1-yr-old Scots pine shoots after wounding as described in (I). Thirty to forty current or I-yr-old shoots were used for each treatment.
RESULTS Culturing experiments Isolation from infected cortex. The main experiment was conducted in May-July 1987 with material taken from ten branches from four trees. Out of 78 cortex inocula, 14 became contaminated. The contaminants were distinguished from P. pini by their diverse morphology and rapid growth. After 2 wk, slow-growing fungal hyphae started to develop from the surfaces of the uncontaminated inocula which were showing some degree of callus development. Six weeks after inoculation, all the cortex discs showed evidence of the
110 development of long straight aerial hyphae. However, only in five cases was there also penetration of the medium during 8 wk of incubation (Fig. I). These medium-penetrating hyphae formed twisted, short, downward-developing branches in the agar medium. This fungal mycelium established in the medium was divided into 3-5 parts, and transferred onto fresh KDSM medium. By this means, 20 colonies were isolated from the 5 pieces of infected cortex tissue. The cortex tissues from which medium-penetrating hyphae had been removed often produced new hyphae which grew into the medium. Proliferation of aerial hyphae ceased on most of the cortex segments after 4 months except on those forming medium-penetrating hyphae.
Isolation from aeciospores. Work was concentrated on spores collected from Moray in 1987 (M87-1, -2, -3) and on those from Thetford collected in 1987 and 1988 (T87-1, T88-I). Spores germinated on all the media soon after inoculation. Spores which had been stored at - 12' for several months usually took longer to germinate. After germination, twisted, corkscrew-like hyphae started to develop from germ-tubes (Figs 2, 3). Irregular swellings (Fig. 3), which were strongly stained by HC1-Giemsa solution and morphologically different from those forming on Moray spore germ-tubes described by Gibbs et al. (1988) soon after germination, were produced frequently at the hyphal tips in both Moray and Thetford isolates. Three weeks after inoculation, colonies 1.52 mm diam began to appear on the surface of KDSM, KDGM, BNGM, and BNSM media. Growth of the colonies from M872 aeciospores on the tested media 5 wk after spore-seeding is indicated in Table 2.
Isolation from inoculated calluses. Callus infection was identified by the formation of aerial hyphae on the callus surface. Twelve weeks after inoculation, hyphae from 8 out of a total of 63 rust-infected calluses penetrated the medium (Fig. 4). Nineteen colonies were then obtained from these inoculated calluses following the same technique described under 'isolation from cortex'.
Colony development. Two types of hypha were formed in axenic culture of the fungus. One (type A) was long, straight, relatively unbranched, often aerial, growing mostly on the surface of the colony and spreading outwards (Fig. 5). These were up to 6 mm in length, with branches which often grew into the medium forming the second type of hypha (type B) which were twisted, usually corkscrew-like and with many branches (Figs 6, 7). These type B hyphae were intertwined to
Fig. 1. Aerial hyphae of P. pini From naturally infected cortex of P. sylvestris colonizing medium 8 wk after cortex isolation (bar = 1 mm). Fig. 2. Initiation of vegetative hyphae from an M87-1 spore on KDSM medium 2 wk after inoculation (bar = 40 WM). Fig. 3.
Hyphal development of a T87-1 spore on KDSM medium 8 d after inoculation. Irregularly shaped, strongly stained swellings (arrow) frequently formed at hyphal tips (bar = 20 vm). Fig. 4. Hyphae from the infected callus of P. sylvestris colonizing the medium 12 wk after callus inoculation with aeciospores (bar = 1.5 mm). Fig. 5. Long, straight hyphae (type A), mostly superficial. Fig. 6. Twisted, cork-screw like hyphae (type B), the main hyphal structure constituting stromata in culture. Fig. 7. Twisted hyphae starting from long, straight hyphae. Hyphae in Figs 3, 5-7 were stained with HCI-Giemsa (Figs 5-7, bar = 10 urn). Fig. 8. A colony derived From M87-2 spores, with a white f l u 6 surface and irregular margin. Fig. 9. Stromatal colony from T87-1 spores, with smooth surface and forming loose, rapidly extending hyphae (Figs 8, 9, bar = 2 mm).
M. H. Pei and R. G. Pawsey
Fig. 1-9. For caption see facing page
111
Culture of Peridermium pini Table 2. Axenic growth of Peridermium pini 5 wk after inoculation with M87-2 aeciospores
Media KDGY KDGM KDSM KDSY KDS
Colony Growth
+
+++ ++++ ++
-
Media BNGY BNGM BNSM BNSY BNS
Colony Growth
+ +++ ++++ ++
-
+ Some branched hyphae developed from germ-tubes, with aggregated hyphae visible as scanty white spots. + + Hyphae grew more extensively, some formed colonies 0.5 mm diam, no stromata developed. Colonies composed of central stromata, 2-25 mm diam. ++ Central stromata well-developed, 3-4 mm diam. - No colonies established.
+++ ++
constitute a stroma in the centre of the developing colony. Cells of both hyphal types were mononucleate. Though many small hyphal branches similar to haustoria were seen in axenic culture, no structures identical to those formed in the natural host tissue were observed. The colonies of P. pini derived from aeciospores on KDSM medium varied in morphology and structure. Some colonies appeared fluffy on the surface (Fig. 8) while others had a smooth, often mucilaginous surface (Fig. 9). Thin, fastgrowing hyphal networks were also formed by both of these colonies usually 2 months after inoculation (Fig. 10). The size of the colonies appeared to vary depending on the distribution density of the colonies on the medium, smaller colonies occurring in areas of more dense distribution. Changes in the colony morphology occurred frequently during the period (several months) of incubation. -
Cytology Nuclei. Two hundred and sixty spores from the T88-1 sporederived colonies were examined by HC1-Ciemsa staining. As a result, 9.6 % were one-; 73.8 %, two-; 10.4 %, three-; 5.8 %, four- and 0 4 % six-nucleate. Of 420 spores from the M88-4 spore-derived colony, 7.6 % were one-; 86.9 %, two-; 4.8 %, three-; and 0.7%, four-nucleate. In general, the same number of nuclei were present in adjacent spores. Spore-proliferating hyphae were usually binucleate (Fig. 15). The cells of all vegetative hyphae examined were found to be mononucleate. Spore wall. Naturally formed aeciospores had thick electron dense walls and were ornamented on the surface with annular knobs resembling a stack of 6-7 discs, with a constriction between each disc (Fig. 16). By contrast, walls of immature spores formed in culture varied in thickness and were smooth on the surface without any ornamentation (Fig. 17). Septa1 pore. In hyphae, the septal pore was located in the centra area of the septum. The septal wall, which was tapered to form the pore margin, was equally separated by an electron-lucent region which extended from the margin of the central pore to the inner limit of the outer layers of the longitudinal wall (Figs 18, 19). Both sides of the septal pore were covered by a dark electron-dense diaphragm which often appeared hollow towards the pore centre (Fig. 18).
-
Sporulation of culture. Spores were formed in culture on only three occasions. O n the first occasion, one sporulating colony formed among hundreds of the colonies derived from M87-3 spores on KDSM 10 wk after inoculation. O n the second occasion, six out of 700 colonies from T88-1 spores on Harvey & Crasham's medium became orange/pinkish 6 wk after inoculation. They were concentrated in one area of the plate 2.5 cm diam. After 9 wk, orange-coloured spore masses formed in the centre of the colonies. O n the third occasion, one colony developed from M88-4 spores 10 wk after inoculation and produced immature spores similar to those formed in the colonies derived from T88-I spores. The spores produced in culture were frequently attached directly together (Fig. XI), some in clusters on single hyphae (Fig. 12), or sometimes linked by short hyphae forming spore-chains (Fig. 13), which resembled an early stage of aeciospore development (van der Kamp, 1969). The spores were always irregularly shaped and apparently immature (Fig. 14). Orange-coloured areas occasionally developed in some colonies derived from both Moray and Thetford spores, and waterdrop-like fluids were present on some of these pigmented and non-pigmented colonies but none contained structures comparable to spermatia. ~
-
Pathogenici& 1n vivo. Approximately half of the axenic inocula regained growth and extended newly formed hyphae into the inoculated calluses within 4 wk of inoculation. However, although a proportion of the 6-wk-old M87-2 inocula regained growth, none of the 7-month-old M87-2 inocula showed any sign of new growth. Aerial hyphae usually arose from the area of callus surface surrounding the site of inoculation (Fig. 20). Sections of calluses fixed 8 wk after inoculation with inocula from the two cultures of 8-month-old T87-1 colonies and the culture of 6-wk-old M87-2 colonies revealed the formation of intercellular hyphae and haustoria in the area near the inoculation site (Fig. 21). Infection appeared to be confined to a limited area of callus tissue and mainly involved the development of intercellular hyphae. Haustoria formed in host cells frequently showed a resemblance to undifferentiated hyphae (Figs 22, 23). In vivo. Calluses usually formed around the wounds made with a scalpel or injection needle. Though colonization of bacteria or other fungi occasionally occurred on inoculation sites, examination of hundreds of free-hand-sections revealed no evidence of the characteristic haustoria and intercellular hyphae of P. pini.
DISCUSSION Axenic cultures of P. pini have been established by isolating mycelium from naturally infected Scots pine cortex and artificially inoculated calluses, and by seeding aeciospores
Fig. 10. A colony from T87-1 spores on KDSM medium 3 months after spore-seeding (transferred after 6 wk). Fig. 11. Immature spores forming closely attached together in culture. Fig. 12. A cluster of immature spores developing from a hyphal end (Figs 11, 12, bar = 10 pm). Fig. 13. An immature spore forming at the end of a linking hypha. Fig. 14. Immature spores produced on a T88-1 colony. Note several 3-nucleate spores (arrow) forming in the same area. In Figs 11-14, nuclei were stained with HC1-Giemsa. Fig. 15. Binucleate hyphae from a spore-forming colony derived from M88-4 spores (Figs 13-15, bar = 20 pm). Fig. 16. Section of a naturally formed spore, showing the stacks of discs comprising the vermcae on spore surface (bar = 1 pm). Fig. 17. Section of immature spores with smooth, irregularly thickened walls (bar = 2 pm). Figs 18, 19. Septa1 pore with a pair of electron-dense diaphragms, which often appear hollow towards centre, an electron lucent layer equally separating septa1 layer (arrow) (bar = 0.5 pm).
Culture of Peridermium pini
114
Fig. 20. Aerial hyphae arising from inoculum-attached callus surface of P. sylvestris 4 wk after inoculation with mycelium hom M87-3 colonies (bar = 1 mm). Fig. 21. Haustoria (arrow) in callus tissue of P. sylvestris 8 wk after inoculation with a 7-month-old T87-1 colony. Figs 22, 23. Haustoria (arrow) developed following callus inoculation, resembling undifferentiated hyphae (Figs 21-23, bar = 20
pm).
directly on agar-nutrient media. The technique of isolating axenic cultures of biotrophic fungi from dual cultures was successfully applied by Cutter and co-workers in the early days of rust fungus culture (Hotson & Cutter, 1951; Cutter, 1959, 1960). In the case of P. pini preliminary attempts failed either because of a high percentage of contamination or excessive sterilization which led to death of the external layers of host tissue. Successful isolation was not achieved until larger portions of cortex material were prepared and subdivided into small pieces after sterilization. Urediniospores have been used almost exclusively to establish axenic cultures of rust fungi. Contaminant-free urediniospores have been obtained either by setting up aseptic leaf cultures or by collection from freshly opened pustules on intact, rust-infected plants (Maclean, 1 9 8 2 ~ ) .Establishing axenic cultures of P. pini from aeciospores proved much easier since no previous isolation treatments were needed and fungal colonies could be obtained in profusion. The well-developed, relatively large aecial peridium undoubtedly prevents the great majority of the contained aeciospores from becoming contaminated, especially in the early stages of sporulation. Moreover, aeciospores of P. pini inside unbroken aecia stored at low temperature ( - 12O) can be used even up to 1 yr after collection. Inoculation density, at least for spores from some sources used in the experiments, appeared to be an important factor for the successful initiation of saprotrophic growth of P. pini. In preliminary experiments, no axenic colonies were developed in dishes in which the medium was sparsely seeded with M87-2, M87-3 spores, although colonies were consistently formed when spores were inoculated at a higher density. The success of axenic growth in relation to race and inoculum
density in several forma speciales of Puccinia graminis has been described previously by Hartly & Williams (1971a, b), Kuhl ef al. (1971), Green (1976), and Maclean (1982~). In previous studies of axenic culture of pine blister rusts, Hollis ef al. (1972) described one mycelial culture of C. fusiforrne (isolated from pine gall tissue) that produced mature aeciospores capable of infecting its alternative host. Harvey & Grasham (1974) described the formation of immature spores in some cortex-derived cultures of C. ribicola. No records of sporulation of spore-derived cultures of C. fusiforrne or C. ribicola have been found in the available literature (Hare, 1978; Amerson & Mott, 1978; Diner & Mott, 1982). In axenic cultures derived from aeciospores of P. pini, immature spores were formed infrequently and were concentrated in a limited area. The concentration of spore-forming colonies in a small area may indicate that a stimulus, most likely chemical, occurred in localized areas of the medium. The reluctance of a number of pine blister rusts to sporulate on artificial medium seems to be a phenomenon common to this group of rusts, unlike Melampsora species a range of which have been reported as sporulating in culture (Turel, 1969; Yamaoka & Katsuya, 1985). In the work on axenic culture of Cronarfium ribicola, cultured mycelium infected both tissue cultures and germ-free pine seedlings (Harvey & Grasham, 1974). Hare (1978) recorded that artificially grown cultures of C. fusiforme formed typical infection structures in P. ellioffii Engelm. var. ellioffii when injected into succulent shoots. However in his work, no haustoria were observed in inoculated callus tissues which appeared resistant to basidiospore inoculation (Hare, 1978; Jacobi, Amerson & Mott, 1982). Compared with callus infection established by inoculation with aecios~ores(Pei.
115
M. H. Pei and R. G. Pawsey 1989), infection of host callus tissues by axenically grown mycelium of P. pini tended to be slower and the haustoria formed in cells frequently resembled undifferentiated hyphae. Nevertheless, the results showing that the mycelia from actively growing colonies of P. pini formed haustoria and intercellular hyphae in callus tissues suggest that the failure of in viuo infection was not simply due to loss of pathogenicity of the cultures. The method of inoculating spores of P. pini after wounding living pine shoots has been used by van der Kamp (1970), Klingstrom (1972), Olembo (1971), and Gibbs et a l . (1988). Following the inoculation of shoots with axenically grown mycelium, the sudden environmental change, slowness in regaining mycelial growth and the defensive reaction stimulated by the wounding of tissue may reduce infectivity, whereas aeciospores are well-adapted to germinsting under variable conditions and capable of establishing infection in a short period of time.
REFERENCES Allen, E. A,, Blenis, P. V. & Hiratsuka, Y. (1988). Axenic cultures of Endocronartium harknessii. Mycologia 80, 12G123. Amerson, H. V. & Mott, R. L. (1978). Technique for axenic production and application of Cronartium fusiforme basidiospores. Phytopathology 68, 673675. Colotelo, N. & Grinchenko, A. A. (1962). Growth of Kabatiella on different media. Canadian Journal of Botany 40, 439-446. Cutter, V. M., Jr (1959). Studies on the isolation and growth of plant rusts on host tissue cultures and upon synthetic media. I. Gymnosporangium. Mycologia 51, 248-295. Cutter, V. M., Jr (1960). Studies on the isolation and growth of plant rusts on host tissue cultures and upon synthetic media. 11. Uromyces atri-triphylli. Mycologia 52, 726-742. Diner, A. M. & Mott, R. L. (1982). Axenic cultures from basidiospores of Cronartium ribicola. Canadian Journal of Botany 60, 1950-1955. Gibbs, J. N., England, N. & Wolstenholme, R. (1988). Variation in the pine stem rust fungus Peridermium pini in the United Kingdom. Plant Pathology 37, 45-53. Green, G. J. (1976). Axenic culture of Puccinia species collected in Canada. Canadian Journal of Botany 54, 1198-1205. Hare, R. G. (1978). Axenic culture of Cronartium fusiforme from three spore forms. Canadian Journal of Botany 56, 2641-2647. Hartley, M. J. & Williams, P. G. (1971~).Interactions between strains of Puccinia graminis f. sp. tritici in axenic culture. Transactions of the British Mycological Society 57, 129-136. Hartley, M. J. & Williams, P. G. (1971 b). Morphological and cultural differences between races of hrrcinia graminis f. sp. tritid in axenic culture. Transactions of the British Mycological Society 57, 137-144.
Harvey, A. E., Grasham, J. L. (1974).Axenic ~ l t u r of e the mononucleate stage of Cronartium ribicola. Phytopathology 64, 1028-1033. Hollis, C. A., Schmidt, R. A. & Kimbrough, 1. W. (1972). Axenic culture of Cronartium fusifonne. Phytopathology 62, 1417-1419. Hotson, H. H. & Cutter, V. N., Jr (1951). The isolation and culture of Gymnosporangium juniperi-virginianae Schw. upon artificial media. Proceedings of the National Academy of Sciences, U S A 37, 400-403. Jacobi, W. R.. Arnerson, H. V. & Mott, R. L. (1982). Microscopy of cultured loblolly pine seedlings and callus inoculated with Cronartium fusiforme. Phytopathology 72, 138-143. Jones, D. R. (1973). Ultrastructure of septal pore in Uromyces dimlthi. Transactions of the British Mycological Society 61, 227-235. Klingstrom, A. (1972). Melampsora pinitorqua (Braun) Rostr. and Pm'dermium pini (Willd.) Kleb. Inoculation problems and techniques. In Biology of Rust Resistance in Forest Trees (Proceedings of a NATO-IUFRO Advanced Study Institute, Aug. 1969), pp. 313-323. Washington, D.C., USA: Forest Service, USDA. Kuhl, J. L., Maclean, D. J., Scott, K. J. & Williams, P. G. (1971). The axenic culture of Pucdnia species from uredospores, experiments on nutrition and variation. Canadian Journal of Botany 49, 201-209. Maclean, D. J. (1982~).Axenic culture and metabolism. Part A: Isolation and characteristics of axenic culture. In The Rust Fungi (ed. R. J. Scott & A. K. Chakravarti), pp. 37-84. London, U.K.: Academic Press. Maclean, D. J. (1982 b). Axenic culture and metabolism. Part B: Metabolism of axenic cultures. In The Rust Fungi (ed. R. J . Scott & A. K. Chakravarti), .pp.. 85-120. London, U.K.: Academic Press. Murashige, T. & Skoog, F. (1962).A revised medium for the rapid growth and bioassay with tobacco tissue cultures. Physiologia Plantarum 15, 473-497. Olembo, T. W. (1971). A study of the mode of infection of Pinus sylvestris by Peridermium pini (Pers.) LCV. Forestry 44, 67-79. Pei, M. H. (1989). Inoculation of callus tissues of Scots pine, Corsican pine and three varieties of mountain pine with Pm.dermium pini. Mycological Rtsearch 93, 503-507. Peterson, R. S. (1973). Studies of Cronartium (Uredinales). Reports of the Totton' Mycological Institute 10, 203-233. Reynolds, E. S. (1963). The use of lead citrate at high pH as an electron opaque strain in electron microscopy. Journal of Cell Biology 17, 208-212. Shenk, R. U. & Hildebrandt, A. C. (1972). Medium and technique for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Canadian Journal of Botany 50, 199-204. Turel, F. L. M. (1969). Saprophytic development of the flax rust Melampsora lini, race No. 3. Canadian Journal of Botany 47, 821-823. Van Der Kamp, B. J. (1969). Peridmiurn pini (Pers.) LPv. and the resin-top disease of Scots pine. 11. Lesion anatomy. Forestry 42, 185-201. Van Der Kamp, B. J. (1970). Peridermium pini (Pers.) Lev. and the resin-top disease of Scots pine. 111. Infection and lesion development. Forestry 43, 73-88. Yamaoka, Y. & Katsuya, K. (1985). Germinability and mfective ability of urediniospores produced in axenic cultures of five Melampsora species, as afFected by the artificial media on which the culture grew. Annals of the Phytopathological Society of Japan 51, 435-442. Yamazaki, S. & Katsuya, K. (1987). Axenic cultures of Cronartium qwrcuum and their pathogenicity. Annals of the Phytopathological Society of Japan 53, 643-646.
(Received for publication 2 8 November 1989 and in revised form 17 M a r c h 1990)