Hypnotheca graminis gen. et sp.nov., perfect state of Monochaetiella themedae

Trans. Br, mycol. Soc. 55 (3),463-475 (1970) Printedin Great Britain

HYPNOTHECA GRAMINIS GEN. ET SP.NOV., PERFECT STATE OF MONOCHAETIELLA THEMEDAE By INEZ C. TOMMERUP*

Department

ofBotany,

University

of Queensland

(With Plate 28 and 4 Text-figures) The perfect state of Monochaetiella themedae is described as Hypnotheca graminis gen. et. sp.nov, which belongs in the Dothioraceae and is characterized by small orange, basin-shaped ascocarps, bitunicate, cylindrical, sessile, octosporous asci and uniseriate, subglobose, hyaline, one-celled ascospores. H. graminis is the cause of a common leaf spot of Heteropogon contortus and Themeda australis in Queensland. Initiation and development of ascocarps are described. Conidia germinate in water with simple or branched germ-tubes which penetrate hosts through stromata or between epidermal cells of leaves or inflorescence bracts. Hyphae are intercellular and hyaline. Acervuli form symphogenously in the subepidermal spaces and produce blastospores. Conidia and mycelium are monokaryotic.

The genus Monochaetiella was erected by Castellani (1943). The type species M. hyparrheniae was collected in Erytrea, on lesions of Hyparrhenia tufa. Kandaswamy & Sundaram (1956) described M. themedae on Themeda tremula from India. A third species M. cymbopogonis occurs on leaves of GJmbopogon wintereanus in India (Punithalingam, 1969). Tommerup & Langdon (I969a) have reported the occurrence of M. themedae on leaves of H. contortus (L.) Beauv. and T. australis (R. Br.) Stapf. in Queensland, and have showed it has a distribution extending to the United States of America. Host plants are dormant during winter and long dry periods in summer (Tothill, 1966). The fungus is a biotroph the conidia of which are shortlived (Tommerup & Langdon, I969a). Moreover it forms localized lesions in which the mycelium dies when host cells become chlorotic. A search for a resistant state has been made and sclerotia or chlamydospores have not been found. However, in some lesions bearing M. themedae, small ascocarps were found, which proved to be the perfect state of M. themedae. MATERIALS AND METHODS

Development

ofMonochaetiella themedae in host tissues

Laboratory study. Plants of H. contortus were placed in a Sherer growth cabinet with fluorescent lighting of 27-32 klx (2500-300 f.c.), There was an alternation of 16 h light and 8 h darkness with a corresponding alternation of temperature of 30 and 20°C; relative humidity was 60 %. Plants

*

Present address: Botany School, Cambridge CB2 3EA.

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were enclosed in plastic bags for periods of up to 24 h to maintain the relative humidity at 100 %. Plants were inoculated by placing several masses of conidia on leaves at different heights in the plants and spraying water over the surfaces for every alternate 2 min for 20 min. After 16, 18, 24 h and then at intervals of 24 h for 2 I days, material was examined. Some leaves were examined directly. Segments were fixed in absolute alcohol-glacial acetic acid ( I : I) and cleared by a modification of the lactophenol chloral hydrate method (Riker & Riker, 1936). Leaves were examined by whole mounts of the segments in lactophenol with aniline blue. Other leaf segments were fixed in lactic-acetic-alcohol (I: I : 6) for 12 h and stored in 70 % alcohol a14 0 • Segments were hydrated prior to sectioning with a freezing microtome. Sections were stained with aniline blue in lactophenol, by an acid-Giemsa method (Tommerup & Langdon, 1969b), a propionic-irou-haematoxylin technique or a Feulgen method. In the propionic-irori-haematoxylin method the sections were washed in N HCI for 5 min, hydrolysed at 60° in N HCI for 8 min, washed three times in water and three times in 50 % propionic acid, washed in two changes of solution B, stained in solution A: solution B, I: I (Henderson & Lu, 1968), destained in 50 % propionic acid and then mounted in 50 % propionic acid. In th e Feulgen method, sections were washed in N HCI, hydrolysed at 60° in N HCI for 8 min, stained with Feulgen reagent for 3 h, washed and mounted in water. Field Study. Material was from two sources, namely particular plants at Esk and in the St Lucia plots of the University of Queensland. Leaves with a large number of recently formed conidial masses were collected on wet days. Pieces of leaves were harvested from immediately below or adjacent to acervuli which had formed conidial masses on the surface. Collection times and the methods of examination were the same as above. The morphology of M. themedae and the macroscopic and microscopic symptomsof the disease have been studied in many specimens from Queensland. Immediately after collection leaf pieces with lesions of M. themedae were fixed, stored and cleared or stained as described previously.

Ontogeny of the perfect state Details of ascocarp ontogeny and the structure of dormant ascocarps have been elucidated from studies of longitudinal and transverse sections of lesions in H. contortus. Lesions were collected 3-24 days after periods of rainfall, lasting for a minimum period of 16 h, during which natural inoculations occurred. The sources of plants were Esk, St Lucia, Samford and Gatton. Leaf pieces with ascocarps were fixed, sectioned and stained by the methods described above. Development of ascocarps in which dormancy had been broken was followed. At daily intervals after ascocarps had been placed in a suitable environment (20-26°, 100 % r.h.), some ascocarps were squashed and others sectioned. In both cases some were stained by the Feulgen technique, the remainder being mounted in water or in lactophenol with aniline blue.

Hypnotheca graminis. Inez C. Tommerup DEVELOPMENT OF MONOCHAETIELLA THEMEDAE IN HOST TISSUES

Compared with laboratory studies, observations of field material showed that the mode of germination of conidia, infection processes and development of M. themedae in host leaves was the same. Germination of conidia resulted in one, or occasionally up to three, lateral, usually uninucleate, unbranched germ-tubes (Text-fig. I D). In branched germ-tubes there were sometimes more than one nucleus.

Text-fig. I. Monochaetiella themedae. A, Young blastogenous conidia; B, conidiophores which have produced several blastospores ; C, conidia; D, germinated conidia.

Leaf penetration was both by infection hyphae pushed down between epidermal cells or through stomata. On inflorescence bracts where stomata were absent penetration was directly through the epidermis. On young leaves intercellular penetration was more frequent than on old leaves where usually it was stomatal. Penetration occurred directly from the germ-tube which was sometimes swollen at the point of contact. The

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diameter of the germ-tube at the actual point of penetration was reduced slightly in the case of stomatal penetrations and by approximately onethird in the case of intercellular penetration. After penetration a hypha regained the diameter it had outside the host. From the point of penetration, simply branched, thin-walled, hyaline, monokaryotic hyphae spread in all directions through the intercellular spaces, Hyphae grew through the leaf tissues from one surface to the other. Acervuli developed symphogenously in the intercellular spaces between the epidermis and mesophyll. Observations indicated that acervuli were initiated adjacent to the sites of penetration. During early stages of development of acervuli the interwoven hyphae of the stroma, like hyphae that invaded other tissues of the host, were formed as cells 40-60 Jtm long and 3-5 Jtm wide. At later stages when mature conidia were present the stroma cells were short and isodiametric. In cross-section the stroma, two to six cells deep, was pseudoparenchymatous and filled the outer lacunae of the mesophyll. Conidiophores arose from the outer layers of stromatal cells. The conidiophores formed a compact palisade. They were short, 8-10 x 2-4 Jtm, cylindrical to elliptical, simple when first-formed, later becoming one to three times dichotomously branched. Conidia were sympodial blastospores (Text-fig, I A, B). As the result of the formation of a number of conidial scars and new growing points, some conidiophores had an irregular form. Conidia commenced growth as apical or lateral, thin-walled protrusions of the conidiophores. The basal width of the swellings varied from 3-4 to 1'5-2'0 ust»: On any conidiophore the first conidium to form arose as an extension of the whole apex of the parent conidiophore, Conidia rapidly increased in length relative to their diameter and became tapered at the distal end. Following mitotic division of the conidiophore nucleus, a daughter nucleus migrated into the elongated conidium which was separated by an inwardly growing septum, this occurring about the time spores attained their mature size. Septa appeared to have uniform thickness across their width. The primary wall of the conidium was continuous with that of the conidiophore. As far as could be determined, rupture of the connecting walls occurred by anticlinal splitting. In mature conidia an apical appendage develops after the basal septum has formed (Text-fig. I B, C). Usually development of the appendage commenced while the conidium was attached to the conidiophore by contraction of the cytoplasm enclosed by the plasma membrane, contraction commencing prior to disjunction of conidia from the conidiophore. A comparison of the lengths of the appendages of attached conidia, of conidia which had just detached, of those which had collected above the acervulus beneath the epidermis, and of conidia which had exuded from the same acervulus showed that contraction of the plasma membrane continued after the conidia were released from the conidiophore, but probably terminated prior to conidia exuding on to the host surface. The results of observations made in a similar way showed that the acytoplasmic basal segment present in some conidia was formed as a result of contraction of the cytoplasmic membrane from that region of the conidia.

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The acytoplasmic basal zone of variable length (I -4 p,m) developed after the conidium was detached from the conidiophore. Mature and developing conidia frequently were longer than the distance between the mesophyll and epidermis and they became curved. Developing conidia which formed beneath stomata or splits in the epidermis and projected through the opening were straight. The conidial sizes were 25-26 x 2-4 p,m, the apical appendage contributing 8-20 p,m of the total length. The macroscopic symptoms were not as obvious on T. australis as on H. contortus. They were mainly in the form of orange conidial masses on the surface, but there were usually no striking changes in infected green leaves. Lesions had a watery appearance when first visible, later becoming chlorotic and eventually necrotic. In senescent leaves infection sites appeared as' green islands' in the centre of which a yellow area formed and subsequently became necrotic. Adjacent lesions sometimes coalesced forming chlorotic regions of different sizes. In extreme cases most of a leaf became yellow. Lesions which formed on leaf blades, leaf sheaths and bracts of inflorescences had a size range of 1-5 x 2-20 mm on H. contortus and 1-2 x 2-10 mm on T. australis. They were elongate to oval in outline with the long axis parallel to the longitudinal axis of the host, of a local nature and amphigenous. The amorphous spore mass varied from 1 to 5 mm. Fungal invasion of the host tissue did not extend beyond the region which was yellow. Spore masses did not form on the surfaces of some infection sites when conditions which brought about a high water deficit in plants were operating, for example low soil water or high transpiration rate. The lesions became chlorotic before the spores exuded and the epidermis above the spore mass was raised. ONTOGENY OF THE PERFECT STATE

Hypnotheca gen.nov. Pseudothecia crateriformia, solitaria vel gregaria, pariete externo crassitie cellulae unae. Asci super stromate basali, cylindrici, sessiles, bitunicati. Ascosporae continuae. Paraphyses mullae. Status conidicus, Monochaetiella. Species typic a : Hypnotheca graminis.

Hypnotheca graminis sp.nov. Pseudothecia crateriformia, solitaria vel gregaria, subepidermalia vel erumpentia, 60-150 X 70-200 pm. Asci bitunicati, sessiles, cylindrici, octospori, 20-25 x 6-8 pm. Ascosporae uniseriatae, subglobosae, hyalinae, continuae, 2-4 x 3-6 pm. Status conidicus Monochaetiella themedae Kandaswamy et Sundaram. In foliis Heteropogonis contorti. Esk, Queensland, 25.ii.1g67, leg. I.e. Tommerup (BRIU 2412, Typus).

Pseudothecia basin-shaped, often gregarious, subepidermal or erumpent and 70-200 p,m in width, wall yellowish, one cell thick. Basal stroma pseudoparenchymatous, the upper plane surface subtending a fascicle of aparaphysate asci. Asci project into a broad, flat locule filled mostly with lipid during the dormant phase lasting 5-8 months, when each ascus has a diploid nucleus. Asci bitunicate, thickened at the apex, sessile, cylindrical, 6-8 ust: in width and 20-25 p,m in length. Asci become 60-150 p,m high

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eight-spored and exposed simultaneously by rupture of the apical wall at its junction with lateral walls. Ascospores uniseriate, subglobose, hyaline, thin-walled, one-celled, 2-4 x 3-6 psn.

Initiation ofthe perfect state Initials of ascocarps developed where mycelium from two lesions of M. themedae invaded the same area of a leaf, but they did not always develop when two colonies became confluent. The number of lesions with

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B

C

Text-fig. 2. Hypnotheca graminis. A, Enlarged hyphae (e) and conidiophore (e) arising from the same hypha (h); B, fusion oftrichogyne with antheridium; C, septation of enlarged hyphae of ascostroma; D, branches arising from groups of dikaryotic cells, (m) mesophyll cells; E, a group of multinucleate branches.

ascocarps compared with lesions having acervuli was small. Ofmore than leaves having acervuli only 145 bore one or more ascocarps. The first evidence of ascocarp initiation is the development in subepidermal 10000

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intercellular spaces of hyphae which differed morphologically from vegetative mycelium of M. themedae in being twice the diameter of vegetative cells (Text-fig. 2A-C). From a single hypha, some branches were seen differentiated into conidiophores and others produced ascocarp initials. This change often occurred prior to mycelium from two acervuli intermingling or occupying th e same intercellular spaces.

Development of an ascostroma Ascocarps, and ascostromata in which the former were borne, developed simultaneously. At the time of dikaryotization and centrum initiation in the first-formed ascocarps, the interwoven hyphae at the base of these ascocarps became septate. The hyaline, thin-walled, isodiametric, uninucleate cells so formed constituted the ascostroma. Cells in the ascostroma proliferated, resulting in the development of pseudoparenchymatous tissue which filled the intercellular spaces between the epidermal cells and the mesophyll cells, with pockets of mycelium growing into the outer lacunae of the mesophyll, Ascostromata were composed of three to six layers of cells. Cells of ascostromata above and adjacent to ascocarps were formed by septation of enlarged hyphae (Text-fig. 2C). Early stages in development oj ascocarps Enlarged hyphae were uninucleate, unicellular and continuous with the parent cell when first formed (Text-fig. 2A ). In some enlarged hyphae septa formed, separating uninucleate cells of th e ascostroma. From other enlarged hyphae narrow protrusions developed in th e apical region fusing at th eir points of contact with the api ces of adjacent, morphologically similar, enlarged hyphae (Text-fig. 2B). Just pri or to or following hyphal fusion, nuclei in th e two enlarged cells underwent one or more mitotic divisions and a tot al of four to six nuclei were found in the two cells. Dikaryotization and centrum initiation followed migration of nuclei through the fusion tube (T ext-fig. 2D; Pi. 28, fig. I ). Pairs of nucl ei migrated to the base of each enlarged hypha and into the parent cell. From the cells having pairs of nuclei a group of dikaryotic branches arose. The centrum of an ascocarp resulted from the fusion of either one pair or a group of a few pairs of enlarged hyphae. In the latter type, the groups of dikaryotic cells arising from each pair of enlarged hyphae coalesced and formed a narrow paradermal zone in the ascostroma. A palisade of branches arose from th e groups of dikaryotic cells (Text-fig. 2 E; PI. 28, fig. 2). In each branch th e nuclei divided until th ere were six to nine nuclei (T ext-fig. 3A, B; PI. 28, fig. 3). Septa then formed in a branch so that there were two to four uninucleate cells at the apex , a dikaryotic rectangular cell in the centre and three to five uninucleate cells at the base (Text-fig. 3 C; PI. 28, fig. 4). All uninucleate cells were isodiametric. The dikaryotic cell was th e ascus initial. At the same time as the branches were elongating and becoming septate, the y also increased in diameter forming a compact tissue. In longitudinal sections the centrum seen at this stage of development had a columnar form.

Transactions British Mycological Society

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Hyphal branches at the periphery of the centrum differentiated to form the longitudinal wall of an ascocarp. Septa formed in the branches separating uninucleate cells. The outer layer of uninucleate cells which had been separated from the apices of branches of the centrum formed the paradermal wall of an ascocarp. As the walls of the cells forming the ascocarp wall thickened, they also became pigmented.

Hypnotheca graminis. Inez C. Tommerup

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Asci and locule During elongation of an ascus the wall thickened and its bitunicate structure was discernible. The pair of nuclei remained in the apical third of each ascus. When an ascus had almost reached its mature length the nuclei fused, the chromosomes synapsed and a resting diploid nucleus formed. Subsequent to completion of the processes of nuclear fusion the ascal wall, in particular the apical portion, continued to thicken. At the same time, cells which had been separated from the branch apices and which formed a layer between asci and the paradermal ascocarp wall gradually broke down (Text-fig. 3D, E). Simultaneously, large drops of lipid accumulated in the cavity and in the interascal spaces. When a cavity had formed in an ascocarp it had reached its dormant stage of development (Text-fig. 4A). At this stage both diploid and haploid nuclei were in resting phase. There were many large vacuoles in the ascal cytoplasm. Asci were cylindrical to clavate and formed a fasciated palisade which filled the central part of an ascocarp. In tangential or horizontal sections very narrow interascal spaces were observed. The bases of the asci were continuous with cells of the outer, almost horizontal layer of the basal stroma. The lateral walls of asci were thin and those of peripheral asci were closely pressed to the ascocarp wall. The much thickened apical walls of asci formed a continuous layer above which was a locule filled mostly with lipid. Dormant ascocarp In any lesion the number of gregarious orange, basin-shaped dormant ascocarps varied from one to twenty-eight, and usually they formed in rows (Text-fig, 4D). Ascocarps developed in the intercellular spaces between the epidermis and mesophyll, these spaces being in parallel rows. Spread of the fungus was restricted by vascular bundles so that the lesions became elongate to oval in outline. Ascocarps grew towards the surface of leaves and by the time they had reached a dormant phase, splits usually had formed between the rows of cells of the overlying epidermis. Some ascocarps were erumpent. When leaves senesced and died, the ascocarps usually remained attached to ascostromata in the leaf tissue. Dormant ascocarps could be easily dislodged from an ascostroma. Abscission of ascocarps occurred at the junction of cells of the ascostroma with cells of the basal stroma. Ascospores

Ascocarps in which dormancy had been broken under natural conditions were placed in an atmosphere of 100 % r.h.. Meiosis of the diploid nuclei in asci occurred after 7-10 days. In the asci in any ascocarp, nuclear divisions were synchronous. Eight uninucleate, uniseriate ascospores differentiated in the following I or 2 days (Text-fig. 4B). During the next day or two, the thick outer apical wall of the ascus ruptured and the endoasci elongated into the locule. The paradermal wall of the ascocarp, a onecelled layer, tore at its junction with the side wall and the endoasci rapidly (in 1-2 h) elongated above the ascocarp wall until their total

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length was approximately twice that of unexpanded endoasci (Text-fig. 4E). Simultaneously, the contents of all asci were violently ejected into the air and the endoasci collapsed (Text-fig. 4F). Usually the torn paradermal wall was also shot away from the ascocarp. Ascospores were hyaline, thinwalled, sub-globose, aseptate and measured 2-4 x 3-6 /lm (Text-fig. 4C).

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Text-fig. 4. Hypnotheca graminis. A, Distribution of tissues in a dormant ascocarp, (a) asci, (b) basal stroma, (l) locule, (w) wall of ascocarp, (s) ascostroma, (m) mesophyll cell; B, bitunicate asci with eight ascospores; 0, ascospores; D, ascocarps in a lesion; E, elongation of endoasci prior to ascospore discharge; F, ascocarp immediately after ascospore discharge. DISCUSSION

The morphology of M. themedae and the symptoms of the disease which it causes in H. contortus and T. australis are similar to the descriptions recorded by Kandaswamy & Sundaram (1956) of this fungus on T. tremula. A more detailed study of the mode of development of the organism on the

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two Queensland grasses has resulted in different interpretations of some conidial characteristics. In their description of the type specimen of M. themedae Kandaswamy & Sundaram (1956) have used the term cilia for the acytoplasmic filiform apices of conidia and noted that they formed only when the spores were mature. Using the Queensland material, it was shown that the appendages form as a result of contraction of cytoplasm from the apical region of conidia. Since the appendages were not a separate structure formed by growth and differentiation of the mature conidia but were present in young conidia still attached to their conidiophore, the appendages were interpreted as being an integral part of the conidia. Accordingly in this study, and contrary to the method of Kandaswamy & Sundaram (1956) who have given two figures for the length of spores, one for the length of appendages and the other for the remainder of the spore, appendages have been included in the overall conidial measurements. The range of spore lengths obtained by addition of the two measurements for the fungus from India are in close agreement with the lengths of conidia of M. themedae from Queensland. The bases of some conidia from Queensland collections were acytoplasmic and developed in the same way as the acytoplasmic apices. Similar acytoplasmic areas in the bases of most of the conidia of a sample from the type specimen were found, no thickened bases were observed and the outer spore wall was uniform in width. Kandaswamy & Sundaram (1956) have described the base of the conidium as being much thickened. It is suggested that they interpreted the acytoplasmic base as a much thickened base. The description of M. themedae should be amended to read 'some conidia acytoplasmic at the base' in place of' thickened at the base' and to read 'appendage formed by contraction of the cytoplasmic membrane at the apex of the mature spores' in place of' cilia formed at the apex of mature spores'. Observations of developing conidia showed that curvature of their apices probably resulted from physical factors rather than being an inherited characteristic. The interpretation of curvature would support the suggestion that the degree of curvature in conidial populations is not of taxonomic significance. Mycelium of M. themedae in lesions of H. contortus and T. australis was entirely intercellular and no haustoria were observed. In T. tremula both inter- and intracellular hyphae were found by Kandaswamy & Sundaram (1956) but they made no reference to haustoria, or to the relative frequency of inter- and intracellular invasion, or to which of the plant tissues were invaded. In Hyparrhenia rufa although all cells, particularly those of the epidermis, were invaded by hyphae of M. hyparrheniae, the mycelium was intercellular and rarely intracellular (Castellani, 1943). Conidia and hyphae of M. themedae examined in this study were monokaryotic. The number of nuclei in cells of the various fungal tissues of this organism collected in India are not known. However, conidia of M. hyparrheniae (Castellani, 1943) and M. cymbopogonis (E. Punithalingham, personal communication) are uninucleate. The ascal state found in association with the conidial state belonging in 31

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the form-genus Monochaetiella is the sexual phase of lv/. themedae. Developmental studies have shown that from the same hypha some branches differentiated as sex organs or as part of the ascostroma, while other branches formed conidiophores bearing conidia of M. themedae. The continuity of hyphae from a conidial stage with hyphae forming an ascocarp shows that the two structures are genetically linked and are two stages of the life cycle of one fungus. Ontogenetic studies of the globular, uniloculate ascocarps show that they are pseudothecia sensu Luttrell (1965). Luttrell (1965) has critically re-examined the current concept of the Loculoascomycetes. Taxonomically the fungus belongs in the Loculoascomycetes and would be referred to the Dothideales on the basis of its clavate, aparaphysate asci which are produced in a fascicle forming a continuous layer in a broad, flat locule exposed by rupture of the overlying wall. It is placed in the Dothioraceae which is characterized by clavate, aparaphysate asci which form a continuous layer in the broad, flat locule exposed by rupture of the overlying stroma. Few developmental studies enable an analysis to be made of the origin of hyphae and tissues of fructifications of the Dothioraceae. The family is typified by Dothiora which, although it may be considered apothecioid, has an internal structure related to the Dothidea developmental type (Luttrell, 195 I) in which asci push up in compact groups into the disintegrating stromatal tissue (Luttrell, 1960). In Dothiora schizospora the locule forms by disintegration of a parenchymatous centrum in which aparaphysate asci arise in a single large cluster (Luttrell, 1960). The amerosporous members of the Dothioraceae have been revised by Arx & Muller (1954). Hypnotheca can be distinguished from all genera hitherto described by its sessile, cylindrical asci which are formed fasciculately, all at one time, on a plane stroma, and the consistently unilocular condition. The conclusion that the ascomycete Hypnotheca graminis is parasitic on H. contortus, with M. themedae as its conidial phase has been reached from studies involving visual evidence of a morphological link between the conidial phase and the initials of the ascal state. There is, however, evidence of several other kinds to support this conclusion (Tommerup, 1969). Cytological studies showed that the structure and mode of division of haploid nuclei in the perfect state was the same as in the conidial form. In cross-inoculation experiments, the perfect state was parasitized by the same isolate of the mycoparasite Hendersonula monochaetiellae as is parasitic on M. themedae. Ascospores of H. graminis were responsible for the primary infections of young leaves of H. contortus with the conidial state and the perfect state. Hypnotheca graminis may possibly be heterothallic. The mycelium and conidia of M. themedae were monokaryotic and observations indicated the two hyphae which fused to initiate a dikaryophase arose from different colonies of M. themedae. By comparison with the number of colonies which were found to be confluent, only a few pairs gave rise to ascocarps, suggesting that two mating-strains were required for initiation of ascocarps. The two sex organs which fused were morphologically different in that one, the trichogyne, produced a narrow tube which fused with the other, the antheridium. If there are two mating-strains it is not known whether

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Vol. 55.

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(Facing p. 4-75)

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one type always produces antheridia and the other only ascogonia or wh ether all thalli can form both mal e and female gametangia. The first differentiation of hyphal branches of an ascocarp seemed to occur in advance of hyphae from two compatible colonies invading the same intercellular spaces, indicating th at a diffusible substance may possibly have stimulated morphogenesis in the hyphae. I am grateful to Dr R. F. N. Langdon for much helpful discussion and for preparation of the Latin diagnosis. REFERENCES

Aax.}, A. VON & M ULLER, E. ( 1954). Die Ga ttungen der amerosporen Pyrenomycet en. Beitr. KryptogFlora Scluoeiz II ( I) , 1-434. CASTELLANI, E . (1943). Monochaetiella hyparrheniae nobis n.sp, Nuooo G. bot. ital. 49, (1942),487. HENDERSON, S. A. & Lu, B. C. (1968). The use ofhaematoxylin for squash preparations of chromosomes. Stain Tcchnol, 43, 2'.n-216. KANDASWAMY, M . & S U NU ARAM, N. V . ( IY5G). All Interesting di seas e of 'Ih cmcda ti'¢mula. Indian Ph)!topath. 9, 202- 203. LU'ITRELL, E. S. ( 1951). Taxonomy of the Pyr en om ycetes. Univ. M o. Stud. 24 (3), 1- 120. L UTTRELL, E. S. (1960). The m orpholo gy of an undescr ibed species of Dothiora. My cologia 55 , 64-79· L UTTRELL, E. S. (1965). Cl assification of th e L oculoasomycetes. Phytopathology 55, 828-833. P UNITHALINGAM, E. (1969). N ew species of Monochaetiella and Septaria. Trans. Br, mycol. Soc. 53, 311- 3 15. RIKER, A. J. & RIKER, R. S. (1936) . Introducti on to research of plant dise ases. N ew York: John S. Swift Co., Inc. T OMMERUP, 1. C . (1969). Studies of some biotrophic fungi associ ated with Heteropogon contortus. Ph.D. Thesis, Universit y of Queen sland. T OMMERUP, 1. C . & L ANGDON, R . F. N. (I 969a). The biology a nd geog rap h ic distribution of M onochaetiella themedae. Aust. ]. Sci. 31, 371. T OMMERUP, 1. C. & LANGDON, R. F. N. ( 1969b). Studies ofCintractia axicola. 1. D evelopm ent of the sorus. Aust. ]. Bot. 17, 25-29. TOTHILL, J . C. (1966). Phenological variation in Heteropogon contortus and its relation to climate. Aust. ]. Bot. 14, 35- 47. EXPLANATION OF PLATE

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Stages in the development of ascocarps of Hypnotheca graminis stained with propi onic iron haematoxylin Fig. I. Proliferation of mycelium having pairs of hybrid nuclei prior to initiation of group s of branches. Fig. 2 . Columnar branches each with 3-5 haploid nuclei. Fig. 3. Columnar branches each with 6-g haploid nuclei. Fig. 4. Differentiation of ascocarp. (m) Mesophyll cell, (e) epidermis, (n) hapl oid nucleus, (I) point of branching of columnar cell, (b) region of columnar cells giving rise to basal stroma, (h) pair of haploid nuclei in a differenti ating ascus, (I) region giving rise to locule, (p) paradermal wall of ascocarp, (w) side wall of ascocarp, (s) ascostroma.

(Accepted for publication 14: July 1970)

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