Growth response to and morphology of mycorrhizas of Thysanotus (Anthericaceae: Monocotyledonae)

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1 New Phytol. (1988), 19, Growth response to and morphology of mycorrhizas of Thysanotus (Anthericaceae: Monocotyledonae) BY P. A. MCGEE Department of Agricultural Biochemistry, Waite Agricultural Research Institute, Uni-versity oj Adelaide, Glen Osmond, S.A., Australia, 564 {Received 15 July 1987; accepted 29 February 1988) SUM MARY Seedlings of Thysanotus were inoculated in pot culture with fungi that form vesicular-arbuscular or ectomycorrhizas on other hosts. Mycorrhizas formed by both fungi were similar in morphology to each other and to mycorrhizas observed in field-collected plants. Hyphae of mycorrhizal fungi penetrated between epidermal cells and ramified between the cortex and epidermis. Spread of mycorrhizas in pot-cultured T. tenellus Endl. occurred only in the presence of a companion plant. In soils with low levels of nutrients, mycorrhizal plants of T. tenetlus grew faster than non-mycorrhizal plants, with roots of infected plants growing relatively faster than shoots. Key words: Mycorrhiza, Thysanotus, morphology, growth response, mycorrhizal spread. I N T R O D tj C T I O N Mycorrhizas are symbiotic associations between soil-.borne fungi and tbe roots of plants. Commonly accepted types of mycorrhizas include vesiculararbuscular (VA) mycorrhizas, ectomycorrbizas, ericoid, arbutoid and orchid mycorrhizas (Harley & Smith, 1983). Description of root-fungus associations from field-collected materials as mycorrbizas is problematic as there is no indication of mutual benefit and consistancy of association. Variation in morpbology owing to host, fungus (Kope & Warcup, 1986) and environment (Mosse, 1973) and the presence of saprophytic fungi in roots (Williams, 1 985) necessitates pot studies to confirm that associations are mycorrbizal. Further, while hyphal continuity between different hosts may link similar types of mycorrhiza (Heap & Newman, 198), this is not always the case. For instance, members of the Gentianaceae have endomycorrhizas tbat differ from tbe VA mycorrhizas of their companion plants (McGee, 1985) and the fungus that forms orchid mycorrhizas in Rhizatithella gardneri also forms ectomycorrbizas on Melaleuea uneinata (Warcup, 1985). Hyphal links between mycorrhizas may be important for the movement of nutrients, particularly carbon, between plants (Whittingham & Read, 1982), and in some cases in the spread of mycorrhizas (McGee, 1985). Thysanotus is a genus of about 49 species of perennial monocotyledons found mostly across southern Australia (Brittan, 1987). Some species have rhizomes or tubers, some have adventitious root systems; some have perennial leaves while the leaves of others die back during periods of stress. On the roots of several field collections of Thysanotus (Anthericacease: Dablgren, CliflFord & Yeo, 1985), McGee (1986a) observed a fungal 'mantle' on the surface of the cortex after the epidermis had sloughed. In these associations, there was no indication of plant growth response. There were a number of different fungi present including septate and nonseptate forms, hyphae of different diameter and degree of melanization. As only flowering plants were examined, tbere was no indication of tbe development of the association. To investigate these problems, seedlings of Thysanotus were inoculated under controlled conditions. Tbe fungi were chosen trom a group of fungi that were first inoculated on to seedlings of Thysanotus in pot culture to determine if they formed mycorrhizas. MATERIALS AND METHODS Growth of plants and synthesis of 7Tiycorrhizas The mycorrhizas of Thysanotus funeifolius (Salisb.) J. H. Willis & Court, T. multifiorus R. Br., T. patersonii R. Br. and T. tenellus Endl. were examined. Seed of T. multifiorus and T. patersonii was obtained

2 46 P. A. McGee Companion plant Thysanotus seedling roots and placed in 3 " glutaraldehyde in -2 M potassium phosphate buffer at ph 6-8. Fixation was overnight at 4 C. Segments were rinsed and stored in phosphate buffer. Dehydration in an ethanol series of 1 steps was followed by replacement with LR White in three steps. Roots were then agitated for d at 4 C. Polymerization was under nitrogen gas and u.v. light at room temperature for 1-12 h. Sections were cut with a glass knife, stained with either Toluidine Blue at ph 44 or Amido Black. Roots of T. juncifolius, T. multiflorus, T. patersonii and T. tenellus were examined using a light microscope. 1 cm Figure 1. Pot system used to inoculate and mycorrhizal seedlings of Thysanothus. from commercial sources. Further seed of T. patersonii and seed ot T. juncifolius and T. lenelhts was collected from plants grown in the glasshouse after transplantation from the field. Seed of Thysanotus was sown on the surface of moist, autoclaved sand. Seedlings were transplanted to tubes in pots (Fig. 1) or to pots of soil mix. Soil used was a mix of autoclaved sand (9%) with autoclaved forest soil (1% ; Warcup & McGee, 1983). Plants were grown in a growth room with a 12 h day, a day temperature of 2-22 C and a night temperature of C and a PAR of 24 /imol m" s ' at plant level from a bank of Philips TLF 65/8OW33RS white tubes. Mycorrhizal fungi used were Glotnus clarum Nicol & Schenck (McGee, 1986 ft), a fungus with a mucilaginous outer layer on all structures, and Peziza whilei (Gilkey) l>appe, a fungus without a mucilaginous, outer layer. Infection of seedlings occured from either pads of -5 g mycorrhizal roots of Melaleuca uncinata placed at the base of the tube or from the mycorrhizas of companion plants of M. uncinata grown in the pots. When a pad of inoculum was placed beneath seedlings in pot culture, most roots spread laterally and only those roots that grew down through the pad became infected. The tubes forced roots of Thysanotus to grow through the pad of inoculum after the contractile root had 'pulled' the crown of the seedling into the soil. Growth respotise of Thysanotus to mycorrhizal infection To determine the growth response of Thysanotus to mycorrhizal infection, seedlings of T. tenellus were inoculated with either P. whitei or G. clarum in tbe presence or absence of a companion plant or left uninoculated after transplanting to pots of 85 g soil mix. Six replicates of each treatment were established. After eight weeks, plants were harvested and the roots of M. uncinata carefully removed from those of T. tenellus. Roots of T. tenellus were placed in water and examined for mycorrhizas under a dissecting microscope. Degree of infection was scored according to the following scale:, no infection; 1, infection only within the area of the inoculum; 2, infection beyond the inoculum but not to the bottom of the pot; 3, infection extensive and to the bottom of the pot. Presence of tubers was noted and the number of leaves longer than 1 cm as well as the total length of the leaves determined. Shoots and roots were then oven dried and weighted. RES LILTS Morphology of mycorrhizas of Thysanotus In uninfected roots of Thysanotus (Fig. 2) there was a single layer of irregular epidermal cells surrounding the cortex. Large air spaces between the epidermis and cortex were common. In the outer laver of the Morphology of mycorrhizas of Thysanotus Roots of Thysanotus are flexible and tear when sectioned by hand. Roots were Hxed, embedded in London Resin (LR) White as follows and then sectioned. Roots were washed from moist soil and placed immediately in deionized water. Segments of root about 2--4 cm long were cut from selected Figure 2. Transverse section of an uninfected root of Thysniiotluis lenclhis Bar, 5 /mi.

3 Mycorrhizas of Thysanotus 461 Figure 3. Longitudinal section of a root tip of Thysanotus ju?7cifolius infected with Glomus clarum. Short cells (open arrow) of the cortex are still attached to the epidermis. Hyphae are found between the epidermis and cortex (closed arrow) in the zone of root elongation. Bar, 5/(m. ff Figure 4. Transverse section of a young root of Thysanotus patersonii collected from the park with mycorrhizal fungi (closed arrow) between the epidermis and cortex. Bar, 28 ^ Figure 5. Transverse section of a root of Thysanotus juncifoliiis infected with Glomus clarum, showing blue stained exudate between hyphae (arrowed) of the mycorrhiza. Bar, 5 //m. A single hypha of G. clarum was found in one cell in this instance. No arbuscules were observed. Hyphae penetrated into the zone of elongation of the root tip between the cortex and the epidermis (Fig. 3). Away from the tip of the root, turgid and collapsed fungal cells were present in the mycorrhizas, suggesting death of the older hyphae and continual reinfection. The staining reaction of infected roots was similar to that of uninfected roots. Hyphae of both VA mycorrhical and ectomycorrhizal fungi stained blue with Toluidine Blue and remained unstained with Amido Black. Use of Toluidine Blue also showed a blue deposit surrounding the hyphae of all fungi (Fig. 5) between the tissues, suggesting exudation of phenolic substances by the fungi, or by the host only in the presence of the fungi. cortex there were long and short cells. The short cells were in close contact with the epidermal layer (Fig. 3) when it was present. The long cells separated from the epidermis soon after differentiation. Staining reactions of the epidermis and cortex differed. Epidermal cells stained with Toluidine Blue were blue in colour suggesting lignification of the cell walls; walls of cortical cells were purple, suggesting absence of phenolic substances. Sections treated with Amido Black stained heavily between the cells of the outer layer of the cortex. Only those roots of Thysanotus growing in or near the inoculum or the mycorrhizas of M. iincinata were found with fungal infections. Away from mycorrhizas of the inoculum or the companion plant, roots of Thysanotus were not mycorrhizal. Morphology of roots infected with VA mycorrhizal and ectomycorrhizal fungi were similar except for the presence of septae in the hyphae of P. whitei. In infected roots (Figs 3-5), hyphae penetrated between epidermal cells and proliferated within the air spaces between the epidermis and the cortex. In only one case was penetration of the cortex observed. Growth response of Thysanotus to tnycorrhizal infection After 8 weeks, two plants from the control and one inoculated with Glomus clarum had died. One further plant inoculated with G. clarum was uninfected. Growth of Thysanotus tenellus was variable (Table 1). Root and total dry weight of mycorrhizal plants were consistently greater than those of non-mycorrhizal plants. Differences occurred in the growth of shoots. The number of leaves was clearly different in mycorrhizal and non-mycorrhizal plants, though no clear trends were obtained for total length of leaves and shoot dry weight. There was greater spread of infection in roots of T. tenellus in the presence of a companion plant. Only in the presence of a companion plant did infection of roots spread to the bottom of the pot. The diflference in the spread of mycorrhizas between G. clarum and Peziza zvhitei may reflect differences in the growth of hyphae in soil and on roots, with P. whitei more likely to grow along the surface of a root than G. clarum. Even with better spread of mycorrhizas in the presence of a companion plant, total dry weight

4 462 P. A. McGee Table 1. Growth and mycorrhizal infection of Thysanotus tenellus either uninocidated or inocutated ivtth chopped roots or hv a companion ptant (Melaleuca uncinata) infected with Pcziza vvhitei {P.w.) or Glomus clarum {G.e.) {mean + SE) Inoculated M. uncinata Treatment Nil* P.wt G.c.t P.W.: G.c.t Mean degree of infection Mean number of tubers Mean number of leaves Mean total leaf lengtb cm Root dry wt (mg) Sboot dry wt (mg) Total dry wt (mg) Root/sboot ratio ± * Mean of four relicates. f Mean of four replicates, non-mycorrbizal plants excluded. X Mean of six replicates., No infection; 1, infection limited to tbe area of tbe pad of inoculum; 2, lnfectu but not to tbe bottom of tbe pot; 3, infection to tbe bottom of tbe pot. beyond tbe pad of inoculum, of mycorrhizal T. tenettus was not necessarily greater in the presence of a companion plant. T. tenetlus infected with G. clarum was heavier in the presence than in the absence of a companion plant, hut not when infected with P. whitei, where growth was similar. DISCUSSION The fungi that form mycorrhizas in 'Thysanotus in the field are unknown, though they appeared be connected to VA mycorrbizas or ectomycorrhizas in adjacent hosts (McGee, 1986a). The morphology of pot cultured associations were similar to tbose observed in the field. Clearly, associations between Glomus clarum or Peziza whitei and Thysanotus were mycorrhizas. There was botb a consistent association and morpbology, and there was a positive growth response of Thysanotus to infection by both fungi in soil with low concentrations of phosphate. However, the mycorrbizas of Thysanotus differ from otber commonly studied mycorrhizas. Fungi that from mycorrbizas witb a similar morphology in Thysanotus may form either VA mycorrbizas or ectomycorrbizas in otber plants. Spread of mycorrbizas is enhanced by tbe presence of a companion plant in some circumstances. Also, growtb response to infection is greater in tbe roots tban tbe sboots. Tbe similarity of mycorrbizas in Thysanotus formed by G. clarum and P. whitei suggests tbat tbe pbysiology of infection is markedly different from tbat of commonly studied mycorrbizas. Tbat most bypbae are contained in tbe zone between tbe cortex and epidermis suggests tbat presence of bypbae in tbe cortex is not critical for transfer of nutrients from tbe fungus to tbe bost, and tbat VAM and ectomycorrbizal fungi can function similarly. Tbe results also suggest tbat tbe bost restricts tbe growtb of byphae such that constant re-infection from fungal sources external to the root is necessary for spread of mycorrhizas. Whether poor spread occurs because of restricted supply of carbobydrate, low ligbt or some otber factor needs to be investigated. Tbe observation of an increase in root/sboot ratio in mycorrbizal Thysatiotus tenellus is contrary to tbe commonly observed decrease in root/sboot ratio upon infection (Smitb, 198). Tbe increase suggests tbat in Thysanotus, roots are a strong sink for carb;)bydrate. As many species of Thvsanotiis are found in babitats witb sbort seasons of growtb (Brittan, 1987) and as tbe sboot tissue dies back in periods of stress, usually summer, rapid development of underground storage organs would improve the chances of regeneration of seedlings tbe following season. Tbe increase in root/sboot ratio was due mostly to tbe increase in root dry weigbt upon infection. Transfer of nutrients from tbe companion plant \\a mycorrbizal fungi is one possibility, particularly as spread of mycorrbizas in Thysanotus relies on tbe presence of a companion plant. However, growtb of tubers and tbe companion plant suggest tbat tbere is a limited 'pool' of nutrients available in tbe system of pot culture used. In experiments, not reported bere, tbe size of tbe companion plant at tbe time of transplanting of tbe seedling of Thvsanotus appeared to be critical to tbe development of Thvsanotus and its mycorrbizas. Wbere growtb of tbe companion plant bad ceased prior to transplanting tbe seedling

5 Mycorrhizas of Thysanotus 463 of Thysanotus, the Thysanotus would only develop three short leaves. On the other hand, seedlings transplanted with very small companion plants did not rapidly become mycorrhizal, owing to the lack oi contact between the roots of Thysa7wtus and the mycorrhizas of the companion plant. A successful experiment relied upon selection of companion plants of appropriate size and degree of myeorrhizal development and the rapid germination of seeds of Thysanotus, a process that rec]uired a degree of luck. While lack of shoot growth may be related to poor mineral nutrition, other nutritional factors may be important. Lack of increased shoot growth in the presence of mycorrhizas, and the carbon source for root growth, need to be investigated further. While the mycorrhizas formed by G. clarum and T. zvhitei had a similar morphology, the morphology of the association between Thysanotus and its different mycorrhizal fungi needs to be examined in more detail. Some questions that need to be examined include; are there differences between the morphology of the associations with fungi that form VAIVl and ectomycorrhizas on other plants? Is the exudation of phenolics associated with conditions of the pot culture or a common phenomenon of the mycorrhizas of Thysanottts} Does the extent of the spread of mycorrhizas relate to the supply of carbohydrate from the companion plant and if so, does any carbohydrate move into the hyphae external to the root of Thysanotus, from Thysanotus} Further work is also required to elucidate the nutritional physiology of the mycorrhizas of Thysanotus. While it is clear that these associations differ markedly from commonly studied VA mycorrhizas and ectomycorrhizas, the mycorrhizas of Thysanotus offer insights into the process of nutrient transfer and the interaction between svmbionts. A C K N O W L! : D G H M E N T S I wish to thank i^rs J. H. Warcup and S. E. Smith for advice and encouragement. Professor A. E. Asiiford for suggesting the method of embedding and sectioning and her comments on the work, Waite Agricultural Research Institute Research Committee for the opportunity to work with Professor Ashford. The work was completed while on a L'niversity of Adelaide Postgraduate Research Award. R V. F li R E N C K S BRITTAN, N. II. (1987). Thysanotus. Flora of Australia 45, DAHI.GREN, R. M. T., Ci.iri-oiio, H. T. & Yi-o, V. V. (1985). The Families of the.monocotyledons : Structure, Taxonomy. Springer-Verlag, Berlin. HARI.EY, J. L. & SMITH, S. M. (1983). Mycorrhizal Evolution and Symbiosis, Academic Press, London. HEAP, A.J. & NEWMA.N, E. I. (198). Links between roots i^y hyphae of vesicular-arbuscular mycorrhizas. New Plivtologist 85, KopE, H. H. & WARCUP, J. H. (1986). Synthesised ectomycorrhizal associations of some Australian herbs and shrubs. New Phytotogist 14, MCGEE, P.A. (1985). Lack of spread of endomycorrhizas of Centaurium (Gentianaceae). New Phvtologist 11, MCGEE, P. A. 986(1). Mycorrhizal associations of plant api-cies in a semi-arid community..australian Journal of Botanv 34, MCGEE, P. A. (1986/;). Further sporocarpic species of Glomus (lindogonaceae) from South Australia. Transactions of tlie British Mycological Society 87, MossE, 13. (1973). Advances in the study of vesicular-arbuscular mycorrhiza. Annual Review of Phytopathology 11, SMITH, S. li. (198). Mycorrhizas of higher autotrophic plants. Biological Reviews 55, WAHC^IP, J. II. (1985). Rhizanthella gardneri (Orchidaceae), its Rhizoctonia endophyte and close association with Melaleuca uncinata (Myrtaceae) in Western Australia. New Phvtologist 99, WARCUP, J. H. & MCGEE, P. A. (1983). The mycorrhizal assocations of some Australian Asteraceae. New Phvtologist 95, WHITTINGHAM, J. & READ, O. J. (1982) Vesicular arbuseular mycorrhiza in natural vegetation systems Nutrient transfer between plants with mycorrhizal interconnections. New Phytologist 9, WILLIAMS, P. G. (1985) Orchidaceous rhizoctonias in pot cultures of vesicular-arbuscular mycorrhizal fun^i. Canadian Journal of Botany 63,

COMPONENTS OF VA MYCORRHIZAL INOCULUM AND THEIR EFFECTS ON GROWTH OF ONION

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