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Development of Haustorium in Taphrina maculans Butler

M. S. Pavgi and Rajendra Upadhyay1

College of Agriculture, Banaras Hindu University, India

Received August 17, 1966

The species of Taphrina have been known to grow subcuticularly on the host surface or intercellularly within the host tissues. Taphrina maculans Butler inciting leaf spot of turmeric (Curcuma longa L.) grows both subcuticularly and intercellularly. This pathogen being a semiobligate foliicolous parasite, produces haustoria penetrating the leaf cells of this host. A haustorium is known to be the principal food absorbing organ of highly adopted parasitic fungi obligate parasites, obtaining nourishment from the host cells without killing them. Elaboration in its form is an evidence of high degree of adoptation by the haustorial parasite in establishing maximum contact with the host protoplasm. Variations in size and shape of the haustorium from the button-like type of Albugo candida (Pers.) Kuntze (de Bary 1863) to a digitately branched structure of Erysiphe graminis DC. (Smith 1900) have been reported in literature. The haustoria are too nonspecific in their developmental pattern to form a basis for classification or a distinction between allied genera (Fraymouth 1956). Development of a membraneous sheath around the haustorium has been reported earlier by several workers, but the nature of its origin and function are not yet precisely known. Fraymouth (1956) suggested that the sheath was laid down by the host cell and composed of a modified cellulose-callose. Ehrlich and Ehrlich (1963) observed presence of a sheath of naked protoplasm outside the haustorial membrane in Puccinia gvaminis tritici Erikss. and E. Henn. and believed it to have formed through contact of oozed out protoplasm of the host cell. The nature of selective or nonspecific permeability of this membrane is also not known . Appressed contact of the haustorium with host nucleus has been frequently reported in the rust fungi. Sappin-Trouffy (1892, 1896) first pointed out the association of rust haustoria with nuclei of the penetrated host cells. Colley (1918) frequently noticed the host cell nuclei dented by the haustoria of Cronartium ribicola J. C. Fisch. Allen (1928) observing as many as 82% haustorium: host nucleus contacts in Puccinia striiformis West . suggested a definite affinity between the 2 organelles, although Rice (1935) believed it a variable phenomenon . The question as to which of these members initiated such a contact has remained controversial . Allen (1928) regarded it possible only through movement of the host nucleus towards the haustorium , while Cunningham (1931) reported evidence on the growth of the haustorium progressing towards the host cell nucleus. The order Taphrinales includes parasitic species which were considered earlier as obligate in their parasitism, but success to grow them on artificial media has now established their facultative nature. Development of hasutoria in this group of fungi , mostly a character of obligate parasites, is an indication of tendencies towards high adoption . The present study was undertaken to study in detail the haustorial development in T. maculans and its parasitic relation with the host.

Material and methods

Young uninfected leaves of turmeric plants grown in isolated chambers were smeared with the culture suspension of T. maculans (Pavgi and Upadhyay 1964) and incubated in moisture

1 Award of a Jr . Research Fellowship by the Council of Scientific and Industrial Research, New Delhi is gratefully acknowledged. 1967 Development of Haustorium in Taphrina maculans 263

chambers. Peelings from the inoculated leaf surface taken at different intervals after 24hrs incubation were observed for spore germination, initial infection processes and progressive development of the haustorium. Leaf pieces collected from these inoculated plants as well as from the field at progressive stages of development were fixed in FAA (rect. spirit 50ml, formalin 10ml, glacial acetic acid 5ml and dist. water 35ml) and chrom-acetic (weak) sol. (10% aqueous chromic acid sol. 2.5ml, 10% aqueous acetic acid 5ml, and dist. water 35ml), washed thoroughly and preserved in 70% alcohol. Free hand sections lightly stained with aniline blue, acidic carbol fuchsin and Heidenhain's hematoxylin (Johansen 1940) and microtome sections

(8-10ƒÊ) stained with Heidenhain's hematoxylin and counterstained with light green or Orange G were prepared as necessary for comparative or complementary observations.

Observations Developmentof haustorium. Entry of the pathogen into a host cell is effected by an aseptate hyphal peg from a germinating blastospore or subcuticular or inter cellular hyphae. The tip of the peg or the hypha bulges a little with accumulation of dense protoplasm after entering the host cell (Fig. 1) and soon bifurcates into 2 short branches (Fig. 2), which again bifurcate in turn further (Figs. 3, 4). New branches also arise from the point of bifurcation of the stem branching dichotomously further (Figs. 5, 6, 7). The haustorial base slightly thickens with branching and growth (Figs. 5, 7). Numerous short branches resulting from the profuse dichotomous branching increase the size of the haustorium imparting it a 'cystolith'-like appearance (Figs. 8, 9, 10). Continued branching at short intervals makes it a compact mass of a single unit (Figs. 11, 12), finally obliterating identity of individual branches (Fig. 13). The length of its stem is variable. In some haustoria it is short and stumpy (Figs. 4, 7, 10) and in others it is rather slender and long (Figs. 6, 8). The haustorium is hyaline in the beginning but changes to pale amber yellow with maturity. Members of Peronosporales (Fraymouth 1956), Erysiphales (Smith 1900) and Uredinales (Rice 1927) develop branched haustoria, but none is reported to possess a multiple dichotomously branched haustorium as in T. maculans. Morphologically it has attained a new form not duplicated in any other fungus species. Haustoria in epidermis. Formation of haustoria in the epidermal cells is conveni ently observed in peelings taken from inoculated/infected leaf surfaces. A thin slimy coat around the wall of the blastospores appears to help them adhere firmly to the waxy leaf surface. It has been repeatedly observed that culture spores smeared onto glass slides adhere them firmly on drying and resist washing away even by a water jet. The infection peg from the germinating blastospore penetrates directly the cuticle and subsequently the epidermal cell. On reaching inside it branches terminally forming the haustorial initial (Fig. 17). The germinating blastospore on branching into a functional haustorium in the epidermal cells grows further into a branched subcuticular mycelium (Figs. 18, 19, 20). The hyphal branches coming in contact with fresh epidermal cells penetrate through the walls similarly develop ing haustoria in them. Epidermal cells in surface view show them penetrated by a subcuticular net of hyphal branches (Figs. 21 to 25) as also in vertically 264 M. S. Pavgi and R. Upadhyay Cytologia 32

Figs. 1-16. Development of haustorium in Taphrina maculans. 1, 2, haustorial peg penetrat ing an epider mal cell and bifurcating at the tip. 3-5, further bifur cation and dichotomous branching. 6-8, young hausto ria borne on long and short stems. 9-12, progressive stages in haus torial develop ment with paired nuclei in the stem. 13-16, mature haustorium giving 'cys tolith'-like appearance. 14, young haustorium ensheathed by a membrane originating from the stem base. Note membraneous sheath in all cases. 15, epi dermal cell in surface view with hausto rium from subcuticular hypha. Note 2 nuclei in the mother cell.

sectioned cells (Fig. 29). The hyphae travelling down in the leaf through the radial epidermal walls also send haustoria sideways in the neighboring epidermal cells (Figs. 30, 31). They are sent in from any one or all sides of the cell by the enveloping hyphae, often enclosing more than 1 haustorium in a cell (Fig. 26). The hyphae generally grow in the intercellular spaces sending haustoria sideways in the neighboring cells. Frequently they are observed to permeate walls of the epidermis and also penetrate the cells forming haustoria in them. 1967 Development of Haustorium in Taphrina maculans 265

Figs. 17-34. Haustorial de velopment in the host leaf in surface and transverse views. 17-20, development and dichotomous bran ching of a haus torium from the germinating blas tospore and sub cuticular hyphae in close proximity with the host nucleus. 21, two haustoria close to the nucleus borne on long and short hyphal branches. 22, haustorium enveloping the host nucleus. 23-26, stretching and swinging the host nucleus on proto plasmic strands by 2-3 haustoria from opposite sides. 27, 28, host nuclei misshapen by haustorial contact. 29, haustorium from a subcuticular hypha in transverse view. 30, 31, pene tration by peg and haustorial forma tion in epidermis through side walls. 32-34, T. S. leaf with and without hypodermic show ing haustoria in epidermis, hypo dermis and palisa de. Note intralame llar haustorial development.

Haustoria in mesophyll. Further mycelial growth brings it in contact with the hypodermis, where it ramifies sending in flanking haustoria both in the epidermis and the hypodermis (Figs. 32, 34). Their growth between the radial walls of the hypodermis is restricted only to the upper side of the palisade cells (Fig. 34). Not infrequently the haustoria are formed in cells of the palisade, but never in the depth further. In the absence of a well differentiated hypodermis, they are often observed in the palisade cells of the mesophyll (Fig. 33). When the lower leaf surface is infected, only 1-2 layers of spongy parenchyma are permeated by the hyphae and haustoria. 266 M. S. Pavgi and R. Upadhyay Cytologia 32

Two types of wall penetration by the hyphae are observed. In one of them the haustorial peg produced by the intercellular hyphal branch penetrates the cell directly, while in the other type the hypha first establishes in the wall of the invaded cell before entering it to form a haustorium. Thus the haustorial mother cell lies in the intercellular spaces in the former and in the cell wall in the latter case. Haustorium: host cell nucleus association. The haustorial initial formed by the germinated blastospore is invariably seen in the vicinity of the host cell nucleus while differentiating the infection processes (Figs. 17, 18). Growth of the haustorium usually establishes a contact with host cell nucleus (Figs. 19, 20). Allen (1928) studying the haustorium: host nucleus association in Puccinia striiformis West. suggested possibility of movement of the host nucleus towards the haustorium, but in the present case the host nucleus does not appear to move towards the haustorium. Hyphal branches from the subcuticular mycelium are seen to elongate and extend to reach the host nucleus (Figs. 21, 22). The hypha nearer to the host nucleus produces a short branch penetrating the cell or it extends the hyphal branch to reach the host nucleus (Fig. 21). This partially confirms earlier observations by Cunningham (1931) that the haustoria invariably grow in the direction of the host nucleus. The haustorium developing in the vicinity of the host nucleus touches it at a point and gradually embraces it around. When 2 haustoria develop from opposite sides, the nucleus becomes stretched to oval-elongate losing its usual round shape (Figs. 23, 24). In other cases, when they lie a little away from the nucleus, they remain in contact with it by cytoplasmic strands. These appear to have formed by stretching the cytoplasm surrounding the nucleus (Fig. 25). Cytoplasmic connections are likewise discernible between distantly situated haustoria swinging the nucleus on them (Fig. 26). The nuclei embraced by the haustoria become deformed and appear smaller than nomal (Figs. 27, 28). Rice (1927) stated that the host nucleus did not show any abnormality due to the haustorial contact in Puccinia sorghi Schw.; abnormalities observed by her in few of the nuclei were not due to the fungal attack as they occurred in healthy condition also. Colley (1918) although figured the host nuclei dented by the haustoria of Cronartium ribicola J. C. Fisch. did not mention any alteration in nuclear morphology due to fungal invasion. The morphological change in the host nucleus by T. maculans appears due to mechanical pressure and possible depletion of food material from the former. Membraneous sheath around the haustorium. Soon after the peg penetrates the cell wall, a thin membraneous sheath is laid around the peg, later preventing the haustorium from direct contact with the host protoplasm. Fraymouth (1956) studying the haustoria of Peronosporales considered formation of the sheath as a reaction of host protoplasm against penetration by the haustorium of the invading fungus. A semiliquid sheath surrounded the organelle during its growth; slowly the sheath became solid bursting 1967 Development of Haustorium in Taphrina maculans 267 with increase in growth rate of the haustorium forming a collar around its base . In the present case the sheath surrounds and also expands with increase in size of the haustorium exhibiting elastic nature of the sheath (Fig . 14). A membraneous sheath similar in nature has been observed around the haustorium in Erysiphe gyaminis in advanced growth stages (Ehrlich and Ehrlich 1963 a). Chemical nature of the membrane could not be studied in the present fungus. Nuclei in the haustorium. The profusely branched haustorium is a unicellular organelle enclosing 2 conjugate nuclei in it. In the earlier stages of development, the 2 nuclei remain in the mother cell (Fig. 15) and later move in the stem of the haustorium with its advanced growth (Figs. 8, 11, 12, 13, 16). The sinuous branches of the haustorium, however, remain anucleate. Green islands. Green islands appear in the leaves yellowing with maturity late in the season or due to late supply of irrigation water. The infected cells become discolored after some time, but the chlorophyll in the cells around the spot is retained longer forming a green island. Similar green islands have been reported in several rust infections (Rice 1927) and are considered an illustration of longer life imparted to infected host cells under adverse conditions. Their development in turmeric leaves indicates tendencies of T. maculans towards mutualism rather than complete parasitism.

Discussion

The haustoria are specialized organs mainly of the obligate parasites to absorb nourishment from the host tissues with the least harm. Size and shape of the haustorium depend upon the necessity and availability of food material. The epidermal and hypodermal cells in the maturing turmeric leaves contain a relatively small amount of protoplasm forming a thin layer along the cell wall leaving a portion around the nucleus. The dichotomously branched haustorium is an ada ptation by this pathogen to obtain maximum benefit from the host cell by increas ing the surface of absorption. Haustoria are not formed in most of the Taphrina species except in T. laurencia Giesenh., T. osmundae Nishida and T. higginsii Mix (Mix 1947) and T. maculans is the only species forming specialized haustoria in Curcuma longa (Butler 1911). Gaumann and Wynd (1952) described the absence of haustoria as characteristic of the members of Taphrinales indicating the general trend, but T. maculans presents evidence of its tendency towards refined parasitism. Facultative pathogens (fungi) killing the host tissues postinfection and obtaining nutriments through intracellular mycelium do not form any hasutoria. However, some of the species of Phytophthora and Cercospora produce haustoria besides their facultative nature (Butler 1918). Latham (1934)working on Mycosphaerella cruenta (Sacc.) Latham (Cercospoyacruenta Sacc.) inciting the leaf spot of cowpea, observed that the pathogen absorbed at least a part of the nutrient from the surrounding cells by means of haustoria. T. maculans also produces 268 M. S. Pavgi and R. Upadhyay Cytologia 32 haustoria in the host cells without killing them, although it passes a part of the cycle saprophytically. Absence of intracellular mycelium shows an advan cement in its transition from facultative to obligate parasitism. The haustoria are formed in the epidermal and hypodermal layers of the leaf and rarely in the palisade cells. They are surrounded by a membraneous sheath, whose origin and nature have been controversial yet. Fraymouth (1956) observed in Peronosporales that the plasma membrane was not penetrated by the haustorium but remained intact around it. The sheath was produced by the host protoplasm in response to penetration by the haustorium. The sheath boundary was believed to be the thin protoplasmic membrane of the host cell which had thickened and hardened due to presence of calcium in the infected cells (Hirata and Kojima 1962). Ehrlich and Ehrlich (1963 a) showed that the sheath was formed between the invaginated plasma membrane and the haustorium. They suggested it as an invasive structure rather than a protective buffer between host and parasite. The sheath appears as function of both host and parasite originating from the stem base. In this fungus it resembles that of E. graminis in its membraneous nature in surrounding the haustorium and differs from that in members of Peronosporales in which it forms a collar at the base of the haustorium. Association of haustorium with the host cell nucleus is an interesting feature. Such associations have been reported in the rust fungi which are obligatory in their parasitism. T. maculans appears to have an affinity towards the host nucleus, as both stem and branches of the haustoria elongate in the direction of a distant nucleus. The haustorium exerts a pull on the host nucleus evidenced by the stretch and elongate shape of the latter when 2 haustoria enter the cell from opposite sides. The nuclear deformity suggests furtherance of food absorption as the purpose of contact with the nucleus. This observation is at variance with those by Rice (1927) reporting healthy condition of the host nuclei attacked by Puccinia sorghi.

Summary The subcuticular hyphae developed from the blastospores of Taphrina maculans Butler germinated over the leaf surface of turmeric (Curcuma longa L.) send in aseptate hyphal pegs in the epidermal cells which branch dichotomously many times forming cystolith-like haustoria. They are frequently formed in the hypodermis and sometimes in the mesophyll also. Contact of the haustorial peg and its branches with the host cell protoplasm is always associated with the layering of a colloidal membraneous sheath of unknown chemical composition separating the entire organel from the protoplasm. The haustorium is unicellular though profusely branched enclosing 2 conjugate nuclei in its stem. Its develop ment has been frequently observed to establish contacts with the host nucleus often deforming it to some extent. The nature of this relationship is not known precisely. 1967 Development of Haustorium in Taphrina maculans 269

Literature cited

Allen, Ruth F. 1928. A cytological study of Puccinia glumarum on Bromus marginatus and Triticum vulgare. J. Agr. Research 36: 487-513. Bulter, E. J. 1911. The leaf spot of turmeric (Taphrina maculans sp. nov.). Ann. Mycol. 9: 36 39. - 1918. Fungi and disease in plants. Thacker Spink & Co. Clacutta. 547 pp. Colley, R. H. 1918. Parasitism, morphology and cytology of Cronartium ribicola. J. Agr. Research 15: 619-660. Cunningham, G. H. 1931. The rust fungi of New Zeland. John McIndoe. Dunedin. N. Z. 261 PP. De Bary, A. 1863. Researches sur le developpment de quelques champignons parasites. Ann. Sci. Nat. Bet. IV 20: 5-148. Ehrlich H. G. and Ehrlich, Mary A. 1963. Electron microscopy of the host-parasite relationships in stem rust of wheat. Am. J. Botany 50: 123-130. - and - 1963 a. Electron microscopy of the sheath surrounding the haustorium of Erysiphe graminis. Phytopathology 53: 1378-1380. Fraymouth, J. 1956. Haustoria of Peronosporales. Trans. Brit. Mycol. Soc. 39: 79-107. Gaumann, E. A. and Wynd. F.L. 1952. The Fungi. Hafner Publishing Co. New York. 420 pp. Hirata, K. and Kojima, M. 1962. On the structure and sack of the haustorium of some powdery mildews, with some considerations on the significance of the sack. Trans. Mycol. Soc. Japan 3: 43-46. Johansen, D. A. 1940. Plant micro-technique. McGraw Hill Book Co. Inc. New York. 523 pp Latham, D. H. 1934. Life history of a Cercospora leaf spot fungus on cowpea. Mycologia 26: 516-527. Mix, A. J. 1947. Taphrina osmundae Nishida and Taphrina higginsii sp. nov. Mycologia 39: 71-76. Pavgi, M. S. and Upadhyay, R. 1964. Artificial culture and pathogenicity of Taphrina maculans Butler. Sci. & Cult. 30: 558-559. Rice, Mabel A. 1927. The haustoria of rusts and their relation between host and pathogen. Bull. Torrey Bot. Club 54: 63-153. - 1935. The cytology of host parasite relations. Bot. Rev. 1: 327-354. Sappin-Trouffy, 1892. Les sucoirs chez les Uredinees. La Botaniste S. 3: 215-219.- 1896. Researches histologiques sur la famille des Uredinees. La Botaniste S. 5: 59-244. Smith, G. 1900. The haustoria of the Erysiphaceae. Bot. Gaz. 29: 153-183.