Isolation of a Fungus from Mycorrhizas of Nothofagus cliffortioides (Hook. f.) Oerst.

Transactions and Proceedings of the Royal Society of New Zealand, Volume 87, 1959, Page 235

Isolation of a Fungus from Mycorrhizas of Nothofagus cliffortioides (Hook. f.) Oerst. By B. C. Arnold Botany Department, University of Canterbury [Received by the Editor, February 21, 1958.] Summary A fungus resembling Mycelium radicis Fagi (Chan) has been isolated from the mycorrhizas of Nothofagus cliffortioides. Secretions of the fungus in liquid culture show strong auxin activity. It is suggested that secretions of auxin by fungi of ectotrophic mycorrhizas tend to inhibit growth of the host roots, and thus enable the mantle to envelop them. Introduction The ectotrophic mycorrhizas of Nothofagus cliffortioides bear a close resemblance to those of Fagus both in anatomy and in external morphology (Clowes, 1951; Harley, 1937). The question arises whether the same, or similar, species of fungi are responsible for the production of mycorrhizas both in Fagus and in Nothofagus. In the present investigation the fungus isolated from Nothofagus exhibits many features in common with Mycelium radicis Fagi (Chan) which is suspected of causing mycorrhizas of Fagus. No proof of mycorrhizal capacity by synthesis from seedlings and fungus has been attempted. Nevertheless, the very presence of the isolated fungus, and its possible effects on the structure and physiology of the host roots are matters that require evaluation. Methods and Materials Isolations were made from clean, white mycorrhizas of the simple or pyramidal type (Harley, 1937). Excised portions, 2–5 mm in length, were surface sterilized in a commercial antiseptic “Nados”, containing 2.25% Sodium hypochlorite, and 15% Sodium chloride. After immersion for three seconds in Nados, the mycorrhizas were transferred under a hood by sterilized tweezers to agar media in Petrie dishes. If a substantial drop of Nados was carried over with the portion of mycorrhiza, and deposited with it on the agar, sterilization was severe so that in 90% of the dishes no growth at all ensued. By reducing the amount of Nados carried over to the agar the intensity of sterilization could be controlled. This was effected by dabbing the mycorrhiza on sterile glass until there remained as little of the Nados as was desired. Media used in isolations were:— Prune agar—(Rawlins, 1933). Gelatin agar—(Kligman, 1943). Malt agar—30 gms Maltexo and 20 gms agar to 1,000 ml of tap water. Plain agar—20 gms agar to 1,000 ml of tap water. “Difco” Potato dextrose agar. For comparisons of linear growth rates of mycelium on agar, the basal medium of Harley, was employed (Harley, 1939).

Variant components were:— Peptone 10 gms per litre Asparagine 2 gms per litre (NH4)2SO4 2 gms per litre KNO3 2 gms per litre The liquid medium employed was a modification of Melin and Nilsson's formula (Melin & Nilsson, 1950) as follows:— Dextrose 20.0 gm K2HPO4 0.5 gm CaCl2 0.05 gm NaCl 0.025 gm MgSO4.7H2O 0.15 gm (NH4)2HPO4 0.25 gm CuSO4.SH2O 0.0000002 gm MoO3 0.0000005 gm ZnSO4.H2O 0.0000005 gm MnCl2 0.000005 gm H3BO3 0.000005 gm Distilled water 1000 ml Iron citrate 1.2 ml of a 1% solution per litre of medium. A stock solution of micronutrients was made up to 1,000 times required strength, and added in appropriate dilution to the macronutrients. Estimation of auxin-activity of secretions of the fungus in the above liquid medium, were made using the split-pea test according to the method of Went and Thimann (Went & Thimann, 1937), with the modification that the seedlings were exposed to a red photographic safe light for four hours continuous illumination, thirty-two hours before the stems were to be excised (Kent and Gortner, 1951). Since, as was found later, the incidence of red light was less than 0.5 foot candles, this pre-treatment proved ineffective, but was not, however, essential for the test since the potency of the test products was demonstrable without pre-treatment. The curvatures induced in the pea stems were of the order of those obtained by earlier workers. (Shackell, 1937). A pure line No. 2790 of Alaska peas supplied by the Crop Research Division of the D.S.I.R. was used, eight split sections of stems being employed with each test solution. As a control, an uninoculated sample of the liquid medium was tested. A 50 P.P.M. solution of Indole Acetic Acid in distilled water was used as a standard. The contents of liquid cultures were filtered through cotton wool, and 40ml of each sample tested in a Petrie dish. Characteristics of the Fungus in Pure Culture When excised mycorrhizas of N. cliffortioides were subjected to mild surface sterilization, a variety of common soil organisms were isolated on Petrie dishes of agar, among them being species of Aspergillus, Penicillium, Mucor, Verticullium, Trichoderma, various bacteria, and the fungus which is described below. To prevent the growth of the ubiquitous members of the rhizosphere, severe sterilization was applied to the mycorrhizas in the manner already outlined so that no growth ensued in about 90% of the Petrie dishes. This necessitated inoculation

of at least 60 Petrie dishes at each isolation experiment in order to obtain sufficient samples. The fungus which emerged after severe sterilization was considered likely to be the cause of mycorrhizas since the innermost hyphae of the Hartig net would be less exposed to the sterilizing fluid than would be non-mycorrhizal fungi located on the surface of the mycorrhizas. The mycorrhizal suspect grew fairly rapidly on agar (Table 1). When isolated under mild sterilization, it competed successfully at times with Penicillium and Verticillium, but occasionally was overgrown by Penicillium. Bacteria frequently emerged with the suspected mycotroph after light sterilization, but in the majority of cases the fungus was dominant. Young colonies are near-white at first. “Leading” hyphae at the colony margin, growing on the agar surface are widely separate (Fig. 1), but hyphae penetrating the medium grow close together, making slower progress than the surface hyphae. Aerial hyphae do not generally rise more than 2 mm above the substratum. On Malt and Peptone agar aerial mycelium is relatively abundant, rapidly turning grey on the latter medium. Apical segments of hyphae on agar are broad (about 14μ), smooth, and rounded at the tips. Under high magnification, reticulate thickenings are apparent within the outwardly smooth walls. The protoplast absorbs many stains deeply and evenly. The first septa are usually no closer than 600μ to the tip. Branch hyphae are smaller than the “leaders” (as fine as 2μ) and emerge roughly 450μ behind the tip at right angles. Branch hyphae may enlarge to the size of the “leaders”. Whereas the tips of surface hyphae on agar are rounded, the extremities of aerial hyphae often taper to a point. The paths of the main surface hyphae on agar are sinuous and over-lapping (Fig. 1). Most remarkable is the frequent, close twining of fine hyphae around larger hyphae (Fig. 2). This tendril-like behaviour might be well suited to the ensnaring of roots by a mycorrhizal fungus. Coils of hyphae aggregated into a mass about 500μ in diameter, are found in older parts of the colony both on aerial and on submerged mycelium. In colonies which have long overgrown the agar, superficially white circular growths about 2–3 mm in diameter appear on the surface. Their bulk constituent is tightly woven pseudoparanchyma, darkly pigmented in the interior. Septation of most hyphae increases with age, and dark pigments appear in the walls. Oil globules are abundant. On some media submerged hyphae become black quite rapidly; but the aerial mycelium remains nearly white for a long period in most cases. On Prune and plain agar the aging submerged hyphae become brownish pink and eventually black. On Peptone a distinct green colouration of submerged hyphae precedes the eventual blackening. Old mycelium both aerial and submerged, gives rise to irregular conidia, either by intercalary septation or from hyphal extremities (Fig. 3). Conidia vary from round to rounded oblong. Anastomosis of hyphae is common (Fig. 4), and is most abundant on hyphae growing over a glass surface. Apart from the possession of larger, round tipped hyphae, and more rapid growth, the isolate from Nothofagus shows significant resemblance to Mycelium

radicis Fagi (Chan) which has been isolated from mycorrhizas of Fagus sylvatica (Harley, 1939). Comparisons between the two are listed in Table I. As far as possible the tabulation has been arranged to follow the form used by Harley (1939). Table. I Character Isolate of Nothofagus Mycelium radicis Fagi (Chan) Colour (a) Brownish pink, Brown on Prune and Plain agar. Green, Grey green, Black green Black. (b) Grey, Grey green, Black on Peptone and Asparagine. Septation Frequent Frequent Clamps None None Anastomosis Common on all media. Abundant on old media and glass surfaces. Especially on old and poorly nourished cultures. Conidia Formed by constriction. Formed by constriction. Hyphal diameter (a) Main hyphae 14μ (9μ in variant form) (a) 4–5μ (b) Branch hyphae 2μ+ (b) 2μ Smaller in poor culture. Smaller in poor culture Aerial Hyphae Most abundant on Malt and Peptone agar Especially on Malt and Prune agar Coils Common on all media Especially on poor media Hyphal growth Sinuous on normal agar; corkscrew like on glass surface or agar film Corkscrew-like Hyphal tips (a) Rounded on surface of agar Tapering (b) Tapering on aerial mycelium Pigmentation Dark sclerotium-like regions arise erratically, though predominantly in older regions Black sclerotium-like growth in oldest (central) regions of colonies A comparison was made of the growth rates of the Nothofagus isolate with those of Mycelium radicis Fagi (based on radial spread) on different agar media (Tables II (a) and (b)). Each measurement for the respective media was averaged from 10 Petrie dishes, except for KNO3, in which case 2 dishes were discounted owing to contamination. The original source of test material was from an isolation made on Prune agar. Subsequently the fungus was twice sub-cultured on gelatin-potato-dextrose agar (Kligman, 1943). Inocula of about 8 cu mm were taken at random from the periphery of 3 dishes of similar origin in which hyphae had slightly overgrown the edge of agar. Growth of the Nothofagus isolate was faster on all media than was that of Mycelium radicis Fagi (Chan) (Harley, 1939). In both fungi, growth was greatest on Peptone. Ammonia produced greater growth than Nitrate with the Nothofagus isolate. The reverse case held for Mycelium radicis Fagi (Chan). Table. II (a). Mean Lateral Extension of Mycelium radicis Fagi (Chan) on Agar (mm). From Harley (1939). Days from Inoculation Source of Nitrogen 5 6 11 26 Nitrate 7.0 8.4 10.3 20.0 Ammonia 7.0 8.0 9.2 12.0 Peptone 8.5 9.4 12.1 22.0 Asparagine 5.4 6.4 9.8 18.6

Fig. 1.—Hyphae in situ on surface of agar at margin of colony showing branching. Fig. 2.—Spiralling of minor hyphae around major hyphae. Fig. 3.—Conidia on submerged hyphae in liquid peptone medium. Fig. 4.—Anastomosis of hyphae. Fig. 5.—Coils of spontaneous variant.

Table. II (b). Average Growth Rates in Radial Spread on Agar Media of Isolate of Nothofagus Measured in mm. Days from Inoculation Agar Media 3 5 6 8 10 KNO3 3.68 17.06 25.0 40.03 Overgrown (NH4)2SO4 15.5 31.85 3 plates Overgrown Overgrown overgrown Peptone 24.45 41.15 Most plates Overgrown Overgrown overgrown Asparagine 5.6 21.8 29.2 39.20 Overgrown Plain agar 1.0 5.55 9.8 17.7 26.6 Malt agar 3.95 12.45 16.0 27.03 3 plates overgrown Auxin Activity of Exudates of the Fungus Filtrates of cultures aged ten weeks, which had been grown at room temperature on liquid media containing dextrose, were tested by the split-pea test. Strong positive curvatures were obtained with filtrates of cultures which were darkly pigmented. The weakest curvatures were elicited by exudates from the cultures showing the least amount of pigmentation. Only negative curvatures were obtained with the control solutions. Drawings of the curvatures of the split-pea stems (made on tracing paper supported by a glass plate) indicated that the exudates having greatest activity were approximately equal in strength to a 50 P.P.M. solution of Indole acetic acid. A concentration of auxin-like substances of equivalent strength, if liberated by mycorrhizal fungi would have important repercussions on the morphogenesis and physiology of infected roots. Emergence of A Spontaneous Variant In the course of growth-measurements of cultures on Petrie dishes, an unusual density of hyphae was noted at the margins of one colony growing on malt agar. The main body of the colony had a greyish brown colour characteristically found on malt, but the new sector was of light red hue and extended further radially. (The parent colony had originally been isolated on Prune agar and sub-cultured twice on Gelatin-potato-dextrose agar before being transferred to malt agar.) Repeated sub-cultures of the new growth were made. Despite an erratic inheritance of colour, there was a regular transmission of morphological characters through a variety of media. The feature by which the colonies were first distinguished was a red colouration which generally occurred in submerged hyphae, though small discrete patches of the aerial mycelium were often red or pink. With age the red colouration was masked, giving place to black or olive-black. Colour proved, eventually, to be a variable characteristic. There was, however, a constant difference in the form of marginal surface hyphae from the parental type. “Leading” hyphae were narrower (9μ), septa and branch hyphae occurred nearer the tip, the first branches being at a distance of about 180μ, and the arrangement of the hyphae at the margin of the colony was much more compact. But in other microscopic features, the relation to the parent type was obvious. Coils were frequent (Fig. 5), minor hyphae were entwined round larger hyphae though usually only on ageing mycelium, anastomoses were common and conidia on aerial or submerged hyphae were similar to the parental type.

Discussion It is not known whether mycorrhizas of Nothofagus are produced by a single species of fungus, or whether several unrelated species or genera are capable of forming the association. Some writers have suggested that Omphalia, Russula, and Boletus cause mycorrhizas of Nothofagus, chiefly on the basis of the frequent proximity of these fungi to the roots of the tree (Birch, 1937; Rawlings, 1951). Such observational evidence is not, however, very cogent; nor is the isolation of a particular species of fungus from mycorrhizal roots a sufficient proof that it is a regular, or the sole cause of mycorrhizas. Complete proof can be obtained only when aseptic cultures of both host and fungus are brought together with the formation of mycorrhizas in the absence of any other fungi. Nevertheless, when a fungus is regularly isolated from mycorrhizas under proper conditions of sterility it is reasonable to assume that its presence is important to the association. The fungus isolated repeatedly from Nothofagus mycorrhizas after severe sterilization has been shown to secrete substances possessing strong auxin activity. In this connection it is noteworthy that histological changes which accompany the transformation of roots of Fagus into mycorrhizas (Clowes, 1950) are symptomatic of excess auxin, though almost certainly other factors are involved Similar histological effects are present in mycorrhizas of N. cliffortioides (work unpublished). These observations are consistent with the findings of Slankis, who showed that secretions of mycorrhizal fungi may be instrumental in the transformation of roots into mycorrhizas (Slankis, 1948). Working with excised roots of Pine in water culture, he showed further, that the effects produced by fungal exudates were similar to those obtained by exposing the roots to suitable concentrations of auxins (Slankis, 1949, 1950). In both cases, there resulted a profuse branching and stunting of roots which simulated mycorrhizas in a remarkable manner. A possible conclusion is that secretions of auxin by fungi of ectotrophic mycorrhizas tend to inhibit growth of the host roots, and thus enable the mantle to envelop them, and spread throughout the system. Acknowledgments I am indebted to the Research Committee of the University of New Zealand for a grant in support of this work. References Birch, T. T. C., 1937. A synopsis of Forest Fungi of Significance in New Zealand. The N.Z. Journal of Forestry, Vol. 4, p. 109. Clowes, F. A. L., 1951. The Structure of Mycorrhizal Roots of Fagus sylvatica. The New Phytol. Vol. 50, p. 1. Harley, J. L., 1937. Ecological observations on the mycorrhiza of beech (Preliminary note), J. Ecol. Vol. 25, p. 421. — 1939. Beech mycorrhiza: re isolation and the effects of root extracts upon Mycelium radicis Fagi (Chan). The New Phytol. Vol. 38, p. 352. Kent, M.. and Gortner, W. A., 1951. Effects of Pre-illumination on the response of split pea stems to Growth Substances. Bot. Gaz. Vol. 112, p. 307. Kligman, A. M., 1943. Some cultural and genetic problems in the cultivation of the mushroom. Agaricus campestris. Fr. Amer. Journ. Bot. Vol. 30, p. 745. Melin, E. and Nilsson, H., 1950. Transfer of Radio-active Phosphorus to Pine Seedlings by Means of Mycorrhizal Hyphae. Physiol Plantarum. Vol. 3, p. 88. Rawlings, G. B., 1951. The Mycorrhizas of Trees in New Zealand Forests. Forest Products Research Notes, Vol. 1, No 3.

Rawlins, T. E., 1933. Phytopathological and Botanical Research Methods. John Wiley & Sons Inc., New York. Shackell, E., 1937. Investigations on the specificity of the action of auxins for the Avena and Pea Tests. The Austral. Jour. Exptl. Biol. and Med. Sci. Vol. 15, p. 33. Slankis, V., 1948. Einfluss von Exudaten von Boletus variegatus auf die dichotomische Versweigung isolierter Kiefernwurzeln. Physiol. Plantarum. Vol. 1, p. 390. — 1949. Wirkung von β Indolylessigsaure auf die dichotomische Verzweigung isolierter Wurzeln von Pinus silvestris. Svensk Bot. Tidskr Vol. 43, p. 603. — 1950. Effect of α Napthaleneacetic Acid on Dichotomous Branching of Isolated Roots of Pinus silvestris. Physiol. Plantarum. Vol. 3, p. 40. Went, F. W. and Thimann, K. V., 1937. Phytohormones. The Macmillan Coy., New York. B. C. Arnold, Botany Department, University of Canterbury, Christchurch.