Studies and Ascus in Ascocarp

J. Gen. Appl. Microbiol., 16, 251--258 (1970)

ANIXIELLA INDICA -STUDIES IN ASCOCARP DEVELOPMENT AND ASCUS CYTOLOGY

J.N. RAT AND K. WADHWANI

Botany Department, University of Lucknow, Lucknow, India

(Received January 26, 1970)

The paper deals with the development of ascocarp and ascus cytology in Anixiella indica. The ascocarp arises by the coiling of a hyphal branch. The septate ascogonial coil soon became invested with vegetative hyphae forming a spherical ball of pseudoparenchymatous tissue, which, as the development proceeds, becomes distinguishable in two layers of peridium and the central sporogenous tissue. The asci developed from typical croziers, which were produced by binucleate ascogenous cells, that developed from the basal compressed sporogenous tissue. The haploid number of chromosomes is 4. Chromosome behaviour during ascus development has been described.

In an earlier paper from this laboratory (1) a new species of Anixiella, A. indica, has been reported. Genus Anixiella was erected by SAITO and MINOURA (2) and placed in family Perisporiaceae, while CAIN (3) showed its close resemblance with Gelasinospora and placed it in family Neurosporaceae. Some members of this family, viz., Neurospora and Gelasinospora have been subjected to detail and exhaustive study with regard to sexual reproduction, cytology and genetics of segregation. However, so far, no detailed account of the ascocarp development and ascus cytology of Anixiella have been reported, hence a detailed investigation of the morphological development of the ascocarp and ascus cytology was undertaken and results are presented inn the following pages.

MATERIALS AND METHODS

The early stages of cleistothecial development were obtained by growing the fungus, Anixiella indica RAT, WADHWANI cat TEWARI (1), on a slide, previously coated with a thin layer of oat meal agar under aseptic conditions. The cultures were placed in a moist chamber and examined microscopically, fixed for desired stages of development and stained with aceto-iron-hemato xylin (prepared as aceto-iron-carmine according to SASS (4) substituting carmine with hematoxylin). For microtomed sections, the fungus was grown on oat meal agar, 251 252 RAI ANDWADHWANI VOL. 16

material was fixed in form-acetic-alcohol and sections of 5-10 t thickness were cut and stained in 0.1% solution of Haidehain's iron hematoxylin. Nuclear details of ascogenous hyphae, crozier formation and ascus development were studied by aceto-iron-hematoxylin squash preparations, after bleaching black cell walls of cleistothecia and ascospores by the method described by STALL (5).

OBSERVATIONS

Vegetative hyphae and hyphal fusion. The aerial hyphae formed the bulk of the mycelium. These hyphae contained 2-5 nuclei in each cell with densely stained cytoplasm and showed frequent hyphal anastomosis and fusion (Figs. 25, 26). The hyphae growing in contact with the substratum could be clearly distinguished from the aerial hyphae. These were relatively thick walled, at times with swollen cells and each cell containing 5-9 nuclei. Development of ascocarp. The ascocarp initial first appeared as a stout branch, arising laterally from aerial mycelium. It was wider in diameter and stained dark. These initials were cut off from parent hyphae by a septum and turned into a hook like structure having 3-5 nuclei. Elongation and septation of the hook took place leading to the formation of a coil of 4-5 spirals (Figs. 1-7, 26, 27). At this stage all cells of coil appeared similar with vacuolated, finely granular cytoplasm and many small nuclei. From basal cells of the coil slender hyphae were given out forming a spherical ball of pseudoparenchymatous cells around the coil (Figs. 8-15). These septate slender hyphae continued growing until a several layered pseudoparenchymatous wall was formed (Fig. 16). At this stage ascocarp was clearly distinguishable in two regions, the wall and the central core of coil (Fig. 28). As development proceeded wall was differentiated in two distinct regions-outer wall layer consisting of globose or polygonal thick walled cells 2-3 cell thick, inner wall 6-9 cell thick with hyaline tangentially elongated with markedly flattened multinucleate cells (Figs. 29-34). Centrum. After the differentiation of peridium layers, cells of central core became meristematic and multinucleate. These divided in all directions producing central sporogenous tissue (Fig. 29). As development proceeded central core broke to some degree and formed a loose mass of large multinucleate cells (Fig. 30). At the same time from inner cells of inner peridium slender, branched and septate hyphae protruded downward and were arranged almost in vertical rows pushing and compressing large multinucleate sporogenous cells towards the base (Fig. 31) thus establishing a sort of polarity in the ascocarp development (Figs. 31-34). Few of the cells from compressed basal tissue became meristematic and produced binucleate ascogenous cells (Fig. 35). Whether the binucleate condition was initiated by nuclear division or nuclear migration could not be ascertained so far. Crozier formation. Young binucleate ascogenous cells appear develop- 1970 Anixiella indica-Ascocarp Development and Ascus Cytology 253

Figs. 1-24. Anixiella indica, stages in ascocarp development, abnormal asci and ascospores. ing from the basal region of the centrum (Figs. 31-34). The upper part of binucleate cell produced a small branch, which immediately curved around and developed into a crozier. The method of crozier formation and ascus proliferation was very similar to that found in other Ascomycetes (Figs. 34, 35). Conjugate division occurred, typically forming a uninucleate tip cell (Fig. 35) where nuclear fusion generally occurred very quickly. At the same time uninucleate stalk and terminal cells of crozier fused immediately and this newly formed binucleate cell may give rise to another crozier (Fig. 35). Ascus development. Asci were produced on croziers showing lateral proliferation, as a result of which all stages from young croziers to mature ascus were found in a fully developed cleistothecium (Figs. 33-35). After fusion of nuclei in penultimate cell, it enlarged to form an ascus. The chromosomes of fused nucleus increased greatly in size (Figs. 39-41). In early prophase I, the nuclear membrane disappeared and chromosomes became un- coiled and elongated (Figs. 40, 41), following the phase of elongation in late prophase I (Figs. 40-44) and chromosomes gradually became shorter (Fig. 44). In well stained preparations four bivalents could be counted (Fig. 47). Maxi- 254 RAT AND WADHWANI VOL. 16 1970 Anixiella indica-Ascocarp Development and Ascus Cytology 255 256 RAI ANDWADHWANI VOL. 16

mum contracted chromosomes were seen at metaphase I, when the nucleolus completely disappeared and after which usual stages of anaphase I could be seen (Fig. 45). After anaphase I, it appears likely that chromosomes attained metaphase II position without undergoing prophase II stage (Fig. 46). The morphology of individual chromosome became more evident and each chromosome showed a sharp bent orienting themselves at metaphase III (Fig. 47), after which asci became 8-nucleate, each one with a nucleolus of its own. At the end of division III, new nuclear membrane did not appear and

Figs. 1-16. Showing early stages in ascocarp development x 750.

17-22. Abnormal asci with abnormal number of ascospores x 750.

23-24. Normal asci with 8 ascospores x 600.

Figs. 25-37. Anixiella indica, stages in ascocarp development. 25, 26. Showing hyphal anastomosis, 25 x 550, 26 x 790.

27. A coil of five spirals x 790.

28. Young ascocarp differentiated into central sporogenous core and pseudoparenchymatous peridium x 320. 29. Division in sporogenous core x 355.

30. Dissolution of centrum cells x 132.

31. Developing young pseudoparaphyses and basal ascogenous system x 130.

32. Development of young asci in basal fascicles x 130.

33. Young ascocarp with scattered pseudoparaphyses in between developing asci x70.

34. Basal fascicle of croziers and young asci developing from binucleate ascogenous cells x 560.

35. A group of young asci showing ascus proliferation x 848. 36. Mature ascocarp discharging ascospores in a globose mass x 36.

37. Abnormal ascus with three ascospores x 585.

Figs. 38-50. Anixiella indica, stages in ascus and ascospore development.

38. A crozier at conjugate division x 2,400. 39. Two nuclei just before fusion in penultimate cell x 1080.

40-44. Stages in prophase I, 40 x 1200, 41 x 1500, 42 & 43 x 480, 44 x 1050.

45. Anaphase I x 1090.

46. Young ascus showing chromosomes oriented at metaphase II x 840.

47. 4-nucleate ascus chromosomes oriented at metaphase III x 390.

48. 8-nucleate ascus showing delimitations of ascospores x 425.

49. An ascus showing irregular division II x 425.

50. Young ascospores showing final nuclear division within the ascospores x695. 1970 Anixiella indica-Ascocarp Development and Ascus Cytology 257

chromosomes at different stages of mitotic nuclear division were observed, and a cleavage in the cytoplasm of ascus appeared, delimiting the eight spores (Fig. 48). Subsequently one more final nuclear division in ascospore resulted in producing binucleate, one celled ascospores (Fig. 50). Normally mature ascospores were uniseriately arranged. In early stages they often exhibited biseriate or irregularly biseriate arrangement (Fig. 23). In such cases during delimination of ascospore walls, spores were formed around nuclei which were irregularly arranged due to irregular second or third nuclear division (Fig. 49), but became uniseriate at maturity (Fig. 24). Rarely an ascus was found with a giant single ascospore (Fig. 18). At times two- to seven-spored asci have also been observed (figs. 19-22, 37). How actually these abnormalities occurred and the nuclear condition of these asci and ascospores was not determined. In older cultures of A. indica ascospores were extruded out in a thin, deliquescent mucilage sheath by breaking away irregularly the cleistothecial wall (Fig. 36).

DISCUSSION

CAIN (3) placed genus Anixiella along with Gelasinospora which showed its close affinity with Neurospora, Melanospora and Sordaria, (6) on the basis of the dark coloured perithecia and sculptured walls of the ascospores, inspite of the absence of ostiole. The development of ascocarp initials in A, indica was similar to those as reported in Gelasinospora adjuncta (6), Neurospora tetrasperma (7), Sordaria fimicola (8) and Gelasinospora calospora (9). In these forms the young ascogonial coil generally arises as a result of coiling of a hyphal branch, containing irregular number of nuclei. The septate coil soon becomes invested with vegetative hyphae forming several-layered pseudoparenchymatous peridium. Plasmogamy has been reported in many species of Neurospora (7), Sordaria fimicola (8) and Gelasinospora (9, 10), either by the anastomosis or by fusion of two vegetative hyphae. Mycelial fusion has frequently been observed in A. indica and hyphae with perforated septum have also been observed as reported earlier (1). But, how exactly the nuclear migration takes place has not been determined so far. The paraphyses in A, indica arise, before the development of asci, as vertical rows of septate, slender multinucleate branched hyphae from the in- nermost layer of the peridium. Such structures have been termed as " Pseu- doparaphyses " by LUTTR ELL (11). In nature and development these are very much different from those described as interstitial connecting strand or compressed centrum after the development of asci. Though the investing hyphae made it difficult to follow the development of centrum and ascogenous system, it was observed from sectioned material that deeper stained binucleate cells developed from compact fertile tissue ly- 258 RAT ANDWADHWANI VOL. 16 ing at the base of centrum very much similar to Sordaria fumicola (8) and G. calospora (8, 9). The rest of ascus development, ascus cytology and spore formation was similar to those described for the species of Neurospora, Mela- nospora, and many other members belonging to this family. Haploid number of chromosomes is 4 in Anixiella indica. In Gelasinospora tetrasperma and Neurospora tetrasperma each spore has got two nuclei representing two sexual strains. Ascospores in A. indica are also binucleate. Although the sexual nature of the two nuclei has not been investigated it is likely that these may be representing the two sexual strains.

REFERENCES 1) J.N. RAT,K. WADHWANIand J.P. TEWARI,Can. Jour. Bot., 45, 479 (1967). 2) *K. SAITO and K. MINOURA,Jour. Ferment. Tech., 26 (1948). 3) R.F. CAIN, Can. Jour. Bot., 39, 1667 (1961). 4) E.J. SASS,Botanical Microtechnique, pp. 228 (1940). 5) RE. STALL,Amer. Jour. Bot., 45, 657 (1958). 6) R.F. CAIN, Can. Jour. Res., C 28, 566 (1950). 7) B. CoLsoN, Ann. Botany, 48, 211 (1934). 8) A.J.H. CARR and L.S. OLIVE, Amer. Jour. Bot., 45, 142 (1958). 9) J.J. ELLIS, Mycologia, 52, 279 (1960). 10) ES. DOWDLINGand A.H.R. BULLER,Mycologia, 32, 471 (1940). 11) ES. LUTTRELL,Brit. Mycol. Soc. Trans., 48, 135 (1965).

* not seen in original .