A NU!RITIONAL AND CYTOLOGIOAL STUDY OF ROSBLLINIA LIMONIISPORA.
David s. Hayman
A theais submitted to the Faoulty of Graduate Studies and Researoh in partial fulfilment of the requirements for the degree of Master of Science.
Department of Botany, KoGill University, Montreal.
April 1963. ACKNOWLEDGEMENTS
I am very grateful to Dr. Charles M. Wilson for
providing me with a culture of Rosellinia and for the benefit of his direction during this investigation, to Dr. Howard Whisler
for his interest and suggestions on various occasions, to Dr. Roy
~. Cain, Department of Botany, University of Toronto, for the
identification of the species studied, to Dr. Robert ~. Craig for instruction in photomicrography, and to all fellow graduate
students and members of the Botany Department at McGill who have
been a constant source of encouragement during my research programme. Special thanks are due to McGill University for a University
Scholarship during the first academie session and a Graduate ~aoulty
~ellowship during the second, and to the National Research Council
of Canada for assistance through Dr. Wilson during the summer months.
------·····-· CONTENTS -PAGE
INTRODUCTION
The Problem••••••••••••••••••••••••••••••••••••• 1
TaxonoiD.J. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 1
PART I - NUTRITION INTRODUCTION AND LITERATURE REVIEW...... 3
MATERIALS AND METHODS••••••••••••••••••••••••••• 9 EXPERIMENTS AND RESULTS••••••••••••••••••••••••• 16 DISCUSSION AND CONCLUSIONS •••••••••••••••••••••• 21 PART II - CYTOLOGY
INTRODUCTION AND LITERATURE REVIEW•••••••••••••• 26
MATERIALS AND METHODS••••••••••••••••••••••••••• 34 CYTOLOGICAL OBSERVATIONS •••••••••••••••••••••••• 38
DISCUSSION AND CONCLUSIONS•••••••••••••••••••••• 46
SUMMARY. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 55
BIBLIOGRAPHY •••••••••••••••••••••••••••• • • • • • • ••• • • ••• • • 57 PHOTOGRAPHS ••••••••••••••••••••••••••••••••••••••••••••• 67
------...... --~· INTRODUCTION
The Problem The number of cultural and cytological etudies in the fungi has witnessed a marked upsurge in the past two decades. The use of modern cytological techniques has made possible detailed etudies of fungus nuclei with important consequences in the fields of cytotaxonomy and cytogenetics in particular. Xuch work bas also been undertaken to elucidate some of the factors affecting fruiting by investigating the nutrition and metaboliaœ of fungi which fruit in pure culture, either for its intrinsic value or with a view to providing the fungus cytologist and morphologist with a means of obtaining a ready supply of material with which to work. Such material contains all stages of growth, which are often lacking in field collections, and certain morphological characteristics may be seen to be due to variations in nutrition and therefore are not always strict criteria for distinguishing species and varieties. The following investigation is concernsd with soms of the factors intlusncing the production of perithecia in purs culture by Rosellinia limoniispora, together with details of crozier formation and the nuclear changes in the developing ascus. Taxonomy The genus Rosellinia Css. & De-lot. is a pyrenomycste and was formerly placed in the family Sphaeriaceae (Saccardo 1892, Lindau 1897, Glumann and Dodge 1928, Bessey 1950). Most authorities now place - 2 -
it in the family I7lariaceae (Arnaud 1925, Miller 1928 and 1949, Luttrell 1951, Glumann 1952, Cain- persona! communication). tiller in 1928 considered Rosellinia as having perithecia consieting of a definite wall plus a thin peeudoparenchymatous layer equivalent to
the stroma of Hypoxylon, and the dark ascospores suggested a close relationship with the Xylariaceae. In 1949 he emphasised the ascua
crown character of Rosellinia as showing further affinity with the Xylariaceae. The aaci of Rosellinia limoniispora Ellis & ETerhart appear to have lost the apical apparatus characteristic of the Xylariaceae, and this species probably representa one of the many linea of evolution leading to groups of Ascomycetes with evanescent
asci (Cain - personal communication). The imperfect stage, lacking
well defined conidiophores, defies identification.
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PAR! I - IUTRITION
INTRODUCTION AND LITERATURE REVIEW
Oulturing fungi creates many problems, and ~though perhaps the saprophytic ascomycetes are more readily cultured than most, there are several factors which require investigation in as many genera, species, and even strains as possible in order that certain generalisations may be subetantiated and new ones evolved. Many pyrenomycetes require certain conditions before they will produce abundant peritheoia for study. One such pyrenomyoete is Rosellinia limoniispora which fails to fruit unless several factors are taken into acoount. Since a multitude of factors are involved in sexual re production, and sinoe nutrition is an important factor, the nutri tional experimenta that can be performed on any organism are prao tically unlimited. One therefore has to analyse those factors which appear to play major roles in the organism selected for study. One may experiment, for example, with temperature, light, and the cul tural medium itself where auch factors as pH, growth substances, carbohydrate source, carbon: nitrogen ratio and nitrogen source are perhaps the most important. In addition to testing one or several variables at a time, one may also take a more radical approach and study the medium during growth to see which compound& are utilised by the fungus and in which order and quantity. - 4-
In a survey of the Tast literature on fungus nutrition only the most pertinent examples are oited. A. Environmental laotors (1) Light is usually needed before perithecia will be pro duced. Tylutki (1958) for example, working with Gelasinospora calospora var. autosteira, f'ound that few or no peritheoia or protoperithecia were produced in the dark. Sometimes not a great deal of light is necessary, as in the case of Ascobolus magnificus where Yu (1954) f'ound that apothecie. were produced in the presence of light at an optimum of' one hour per day. Sometimes different species of' the same genus vary in their response to light. Suoh a genus is Nectria where Hanlin (1961) f'ound that N. gliocladioides and N. peziza would not form perithecia if kspt in continuous dark neas, whereas I. ipomoeae normally formed perithecia in cultures kept in constant darkness although not as abundantly as those kept in light.
{~~) ~eaperature Aa.s a marked effect on fruiting. There is usually a broad range of temperature within which fruiting can take place, but fruiting bodies are normally produced in large quantities only at temperatures very close to the optimum. This optimum temperature is usually between 20 and 25°0. Gelasinoapora calospora var. autosteira produces ascospores beat at 20°0 (Tylutki 1958), and apecies of Nectria fruit beat at 24°0 (Hanlin 1961); - 5-
Neurospora crassa fruits well at 18°0 (Westergaard and
Mitchell 1947). Unusually high optimum temperatures tor growth may occur as in the case of Sordaria destruens and Sordaria timicola (Hawker 1951) although this optimum may vary with the carbohydrate source (Hawker 1947a).
(lll) pH is important, and its ettects may be modified by altering the concentrations of various growth factors present in the medium. Lilly and Barnett (1947), for example, found that a pH of 3.6 - 3.8 inhibited perithecial production in Sordaria fimicola, but the addition of thiamin overcame this. Although thiamin itself was not an essential growth factor, culture media containing it reverted to an optimum pH !aster than media without it.
B. Nutritional Factors
(1) Growth substances. It is well known that many fungi are unable to synthesize certain necessary vitamine or are capable only of a limited synthesis. The two vitamine most commonly deficient, either singly or together, are biotin and thiamin. No perithecia are produced in Sordaria fimicola in the absence of biotin and mycelial growth is eparse (Barnett and Lilly 1947a). Hackbarth and Collins (1961) tried various combinations of ten vitamine on three species ot Gelasinospora and found that biotin was the factor li miting growth. Ceratostomella timbriata is heterotrophic with respect to thiamin (Barnett and Lilly 1947b). Sordaria (Melanospora) destruens - 6 -
needs both biotin and thiamin for perithecial production (Hawker
1939a). The proportions of vitamine are also important. :Barnett and Lilly (1947b) showed that sexual reproduction occurs in
Ceratostomella fimbriata only when the ratio of thiamin to the amount of nutrients in the medium is rela~ively high, i.e. the formation of perithecia is inhibi~ed as the amount of thiamin is reduced whilst the nutrient level is kept constant. Their resulta with thiamin correspond to their resulte with biotin on Sordaria fiaicola where the number of perithecia produced is proportional to the concentration of biotin (Barnett and Lilly 1947a). Similarly
Hawker (1942) found that the concentration of glucose optimal for fruiting rose with an increasing concentration of thiamin.
The major effect of a vitamin may sometimes be on sugar uptake. For example, Bretzloff (1954) showed that where no biotin was added to a medium in which Sordaria fimicola was growing, very little sugar was used. (11) Sugar source. In general mycelial growth increases and perithecial production decreases as the concentration of sugar in. the medium is increased. For example in Sordaria destruens the production of perithecia falls off rapidly when the concentration of hexose is greater than 0.5~ (Hawker 1947b). In Sordaria fimicola 2% glucose produces high vege~ative growth and poor fruiting, whereas - 7 -
1.5% sucrose and 0.5% invert sugar produce meagre vegetative growth and a high and relatively rapid fruiting (Bretzloff 1954). This contrasta with Iectria gliocladioides where Hanlin (1961) found that with increasing concentrations up to 2% of glucose, maltose and fructose, the number of perithecia rose and the time for fruiting decreased; with sucrose this was true up to a concentration of 1%. In most cases the optimum concentrations for fruiting are probably low in glucose and fructose, higher in disaccharides auch as maltose, and highest in the disaccharide sucrose. Thus the response of fungi to various sugars varies botb qualitatively and ~uantitatively. Galactose is usually a poor sugar source (Horr 1936, Brook 1951), although Aspergillus nidularis appears to be an exception (Agnihotri 1962). Glucose, fructose, and maltose are generally better sources tor growtb and truiting. Oombinations of more than one sugar are sometimes preterred by a tungus. ~or example, some species ot Penicillium grow better with galactose and glucose together than with glucose alone (Mehrotra and Kumar 1962). In mixtures of two sugars preference is often shown tor one of the two as in Ohaetomium globosua where fructose uptake is completely inhibited in a medium oontaining fructose and glucose in a 5al ratio (Walsh and Harlay 1962). In certain boletes also, substitution of two or three sugars for single sugars gives increased
Y~•lds possibly because of the parallel enzymatic reactions thus - 8 -
necessitated (Anderson 1941). The above etudies support Hawker's view (1950) that it may be easier for some fungi to secrete several enzymes in small quantitiea than one single enzyme in large quantity. Hence fungi sometimes grow better on a mixture of several sugars than on a similar total concentration of one sugar alone. There is some evidence that polysaccharides are broken down to hexoses before the fungus can utilise them efficiently. In the case of sucrose, the responae of a fungus is correlated with the rate at which it inverts sucrose (Hawker and Chaudhuri 1946). lhen a high concentration of aucrose is present, this is not usually inhibiting to fruiting since inversion occurs at a speed allowing a favourable (low) concentration of glucose to be maintained for much of the growing period (Hawker 1939b). The addition of a dilute solution of invertale to a young culture with a high concentration of sucrose may stimulate perithecial production (Hawker l947b).
Sucrose per ~ seems substantially unavailable to a fungus, but some hydrolysis may occur when autoclaving (Bretz1off 1954). The hydrolytic products of disaccharides and polysaccharides may be detected in the medium in which a fungus is grcwing, together with new ones synthesised by the fungus (Tandon and Chandra 1962). The formation of phosphoric esters of sugars may govern the rate of growth and fruiting of a fungus; for example, Hawker (1948.) found with Sordaria destruens that the addition of D-l-phosphate to - 9 -
5% sucrose or 0.25% glucose plus 0.25% fructose le4 to increased fruiting. (111) The literature is also extensive on other nutritional
aspects such as the nitrogen source, the carbon& nitrogen ratio,
and mineral and trace elements, but whese aspects are not embraced by the present study.
The following nutritional study on Rosellinia limoniispora attempts to reveal some of the effects of different sugars at different concentrations, both singly and in combination. It also illustrates the importance of biotin and thiamin for this fungus.
MATERIALS AND METHODS The isolate of Rosellinia limoniiapora Ellie & Everhart used in the present study is from Dr. C.M. Wilson'a culture
collection where it bas been growing in pure culture on PDA slants for about ten years and still frui1sreadily. It was originally isolated from the soil around corn roots. Media used A 2% agar base was used in each case. A. Batural media i.e. those media containing certain ingredients of which the exact cheœical composition is not known.
(l) PDA - Potato dextrose agar
2% dextrose plus juice extracted from 400 gm. of potatoes boiled until soft in 500 ml tap water, plus distilled water to - 10 -
make l litre. (11) M- Malt extract 2% and 0.2% malt extract {Difco).
(111) CM - Corn meal 2% corn meal agar (B.B.L) (lV) eMY - Corn meal + yeast extract (Carr and Olive 1958) 2% Corn meal agar (B.B.L) + 0.1% yeaet extract (Difco) (V) YpSs - Yeast - starch agar (Emerson 1941)
4 gm powdered yeast extract 0.5 gm MgS0 4 1 gm K HP0 2 4 15 gm soluble starch 1 litre H2o (2/3 distilled 1 1/3 tap). (Vl) T - Tryptone medium (Whisler 1962)
5 gm Bacto-Tryptone (Difco) 3 gm glucose 200 pgm tl:liamin P0 stock - O.OOlM KH P0 + O.OOlM X HP0 + 0.0001K(BH ) HP0 4 2 4 2 4 4 2 4 Oalg stock - 0.0005M CaC12• 2H20 + 0.005M MsCl Trace elements: no-3 ouso4• 5H2o 0.39 gm Il KnC12• 4H2o 1.80 gm (1H4)6M0 7°24• 4H20 0.31 gm " ZDS0 4• 7H20 0.44 gm " CoC1 • 6H o 0.81 gm 2 2 " H :so 2.86 gm 3 4 " :Pec1 • 6H 0 4.84 gm 3 2 " - 11 -
(Vll) C - Cantino 1 s medium (Cantino 1954) ) 1.25 gm Peptone (or Tryptone) in 1 litre of l0-2M NaHC0 1.25 gm yeast extract 3 3.0 gm glucose l B. Defined media i.e. those media of known chemical composition. (1) CzS and CzD - Czapek's
1 gm K HP0 2 4 3 gm :tlaN03 0.5 gm Mgso 4 0.5 gm KCl 0.01 gm Feso4 2% Sucrose or Dextrose H 0 to 1 litre 2 (11) X ! B ! T + Sugar The basal medium (X) consista of the quantities of minerale as formulated by Ryan (1950) minus the ammonium tartrate, plus trace elements in the proportions used by Whisler (1962) Basal medium:
~ sm NH4!lo3 1 gm KH Po 2 4 0.5 gm MgS04• 7H20 0.1 gm liaCl 0.1 gm CaC12 trace elements {aee Tryptone med~ua). Biotin (B), when added, was present at a concentration of 8 pgm/litre.
Thiamin (T), when added, was present at a concentration of 200 pgm/litre of thiamin hydrochloride. - 12 -
Sugars used were glucose (dextrose) (D), fructose (d(-) levulose) (F), galactose (D(+) galactose)(G), sucrose (s) and maltose (K).
The different media tested, based on combinations of the basal medium with or without biotin and/or thiamin plus a sugar source, are summarised in the following table.
MEDIUM HISTOGRAM
x + 0.2% D x + 1~ D x + 2% D x + 4% D x + 1~ D + B x + 1% D + T x + 1~ D + B + T Dl x + 2% D + B x + 2% D + T x + 2% D + B + T D2 x + 4% D + B + T D4 x + 0.2% M x + 1% M x + 2%• I + 4%14 x + 1%14 + B J.. x + 1% 14 + T Mor x + 1~ M + B + T Ml x + 2% M + B x + 2% M + T M~ x + 2% 14 + B + T M2 x + 4% M + B + T M4 x + 0.5% D + 0.5% M + B + T Di~ x + 1~ D + 1% M + B + T Dli x + 1% D + 1~ 14 + ! x + 2% D + 2% M. + B + T D2M.2 - 13 -
MEDIU1l HISTOGIWl
x + 1% F 'X + 2~ ., x + 1~ J + B x + 1% F + T x + 1~ B + B + T F1 x + 2% F + B x + ~ F + T x + 2% J + B + T -.2 x + 0.2% G x + 1% G x + 2% G x + 1% G + B x + 1% G + T x + 1% G + B + T G1 x + 2~ G + B x + 2% G + T x + 2% G + B + T G2 x + 0.2% s x + 1% s x + 2% s x + 1% S + B x + 1% S + T x + 1% S + B + T s1 x + ~ S + B x + 2% S + T x + 2% S + B + T 82 x + 2% D.M.FSG) x + 2% DMJISG~ + B x + 2% DMJiiSG + f x + 2% DDSG) + B + T DIIFSG x + 2% DUS) + B + T DUS x + 2% DDG~ + B + T DMFG x + 2% DMSG + B + T DIISG x + 2% DFSG) + B + T DFSG x + 2% (.M.FSG) + B + T USG
Blanka in the co1umn marked "histogram" mean that no peri thecia were produoed. - 14 -
Conditions of environment The main experimenta were done in a non-controlled environment where fluctuating temperatures and light appeared to be beneficial. Temperature fluctuations were from 20 to ;o0 o, averaging around 2; - 24 0 o. Light fluctuations were alternating darkness with daylight plus or minus fluorescent lighting. pB measurementa were not made - changes in pB could account for some of the differences obtained on the various media. Inoculations were made using a transfer needle with the flattened end bent into a hook. The inoculum consisted of a small piece of mycelium which was approximately the same aize in each case. This piece of mycelium was always taken from a 4 - 5 day old culture on a petri-dish where it was still expanding rapidly in a radial direction. There may have been a carryover of eome nutrients either in the hyphae or in traces of agar sticking to the hyphae, but the effects of this were considered negligible since : (a) The amount of nutrients carried over was exceedingly minute. (b) The amountsof biotin anà thiamin teeted were constant - there were no tests using different concentrations of these vitamine. (c) The resulte are discussed in relative, not absolute, - 15 -
terme. Apparatua Stock cultures were maintained on PDA elants in test·- tubes with cotton plugs.
•ruiting experimente were made on petri-dishes 50 mm in diameter and 15 mm deep. •or each replicate of each mediua 11 al of mediua were used in each dish. !bis was measured by marking a rack of test-tubes at the 11 ml mark, pouring the media into these, and autoclaving a second time. AutoclaTing was at 15 lb for 15 minutee. The sterile media in the test-tubes were then poured into the reapectiTe sterile petri-dishes. Each solid medium was tested in triplicata. fhe petri-diahes were first washed thoroughly with a detergent, rinsed in water, and then rinsed three timea with double-distilled water. fhey were then 4~-sterilized in aluminum foil tor three hours at 170°0.
Por the d~1 weight measurements, 250 ml Erlenmeyer flaaks were ueed, each containing lOO ml of the liquid medium. Jour days after ino~ulation, the flasks were placed on a horizontal-type rotary shaker where the media were kept continually swirled at a 0 rate of 140 ehakes per minute for 22 daye at 25-28 c. Each was then filtered, washed, dried on aluminum pans at 95-1000 C for 48 hours, and then weighed. - 16 -
lleasurements
(a) Length of time required for fruiting was based on the observation of masses of ripe ascoaporea being extruded from the ostioles of about half the perithecia. This was visible to the naked eye and was checked under a lens. (b} Bumbers of perithecia in the petri-dishes are the numbers actually counted. The large aize of the perithecia made it possible to count them with the naked eye under an oblique light source. Tabulation of resulte In this study, it was decided that the use of histograma would greatly facilitate comparisons of the resulte from the different media. !he gross effects, which are the important ones here, are thereby quickly and clearly discernible.
EXPERIMEN!S ABD RESULTS Rosellinia limoniispora was found to fruit abundantly on standard natura.J. media auch as PDA, YpSe and 1laJ. t extract. However, ; weeks were needed for the production of mature perithecia on ~malt extract and 4 - 4i weeks on the other two. As it is obviously more convenient to obtain perithecia containing asci ready for cytological study in a shorter time, nutritional factors were studied in more detail. - 17 -
Environmental factors were first studied in order to ascertain favourable conditions under which to grow the fungus when the different nutritional factors were being studied. (1) Light Inoculations were made on PDA and YpSs media in petri- dishes and test-tubes, and replicates were p1aced in continuous darkness, continuous 1ight, and alternating 1ight and darknese, approximate1y 14 : 10 hours respectively per day. In continuous darkness profuse vegetative growth was observed after 2 weeks, but there was no sign of perithecia nor perithecial initiale. In continuous 1ight perithecial initiale were visible atter a month, but mature perithecia were not diseernible until atter nearly two months. A yellowish exudate was seen on the mycelium in the PDA cultures. Under conditions of alternating light and darkness mature perithecia were produced atter one month on both PDA and YpSs. It was eoncluded that light is necessary tor fruiting, but continuous light is de1eterious. (11) temperature Cultures were grown on PDA and YpSs at 16°0, 20°0, 24°0, 30°C, and at room temperature (fluctuating by about 5°0 on either aide of the mean of 24°C). The optimum controlled temperature was 24 0 c, but a fluctuating room temperature gave better resulta. - 18 -
(111) Filter paper was placed in some test-tubes and the perithecia were seen to mature faster in groups at its base.
This was probably due to a drying-out effect since perithecia were also observed to form !aster on petri-dishes where the agar was not poured too deeply. In this latter case there was probably a eombined effect of drying out of the media plus the quicker using up of food resourees.
(lV) A trial with PDA, using different concentrations of dextrose (0.2%, 0.5%, 1%, 2%) showed a differential response with a 1% concentration appearing to give the fastest fruiting time.
In the nutritional experimente that followed, 11 ml of medium were poured into each petri-dish. This is in agreement with
Tylutki (1958) who also found !aster perithecial production with smaller amounts of medium. The dishes were set up in the same room under conditions of fluctuating light and temperature, rather than under strictly controlled conditions, since room conditions approached the optimum for fruiting more closely.
Experiment I Several natural and defined media with different con centrations of various sugars were tested, with biotin and thiamin together or separately in the media containing dextrose and maltose. The necessity of biotin and thiamin for fruiting was proved. - 19 -
lxperiment II
All 5 sugars were tested singly in varying con centrations, all togethert and in all possible combinations of 4, with the addition of biotin and/or thiamin. Some media from expertment I were repeated here since room conditions were slightly lesa optimal in the later experiment II. !hese repeats were used to calculate a conversion factor so that the resulta from both experimenta could be reduced to the same ecale for representation on histograms to enable relative comparisons to be made. Vegetative growth
Measurements were made of the diameters of each colony. Also several media were tried as liquid cultures in order to provide material for the recording of dry weights. The broad implications of these tests were considered with regard to the response of the fungus to the different media.
Resulte A. Description of colonies The vegetative mycelium first spread over the whole dish and then perithecia tormed and matured at more or lesa the same time, all over the dish,not in centritugal succession. The aize of the perithecia varied from very large in high sugar concentrations, to medium where the highest production of peritheci• occurred, to small on media where few perithecia - 20 -
developed.
There was a wide range of black stroma formation, varying from about lOO% in the highest sugar concentrations (4%) to nearer 5o% in the lower sugar concentrations, and to none at all with sucrose, corn meal, and where biotin and/or thiamin were lacking. In those media where most perithecia were produced, i.e. where 4 or 5 sugars were present, the black stroma extended over 85 - 90% of the colony. The diameters of colonies with biotin alone were usually about twice that of those with thiamin alone. Perithecia were not produced on the following : PDA containing 0.2% dextrose, the tr~ptone medium (!),Cantino's medium (c), and all synthetic media with all the sugaJBwhen biotin and/or thiamin were lacking; maltose was the single exception to this last case. B. Vegetative growth (1) Liquid media: a 4% concentration ot both dextrose and maltose gave nearly twice as much growth as 2% concentration which in turn gave nearly twice that of the l% concentration. Sucrose gave the lowest yield, The mixture of sugaiB(2% total concentration) gave about the same growth as 2% maltose. The order of sugars giving best growth, starting with the highest, is: 4% D, 2% D + 2% K, 4% M, 1% D + 1% M, 2% D, 2% M, 1% F, 2% G, NUMBER OF DAYS FOR FRUITING
40 1 NATURAL MEDIA DEFINED MEDIA ..... ~ - r-- r-- - r-- r-- 30 r-- r-- r-- r-- r-- 1-- r-- 1 1-- ~ ~ 1-- 1-- )-.) 0 p ~ 1
20 . 1--
1
10
0 0 D 0 D M - o-t D' 02. M pt pt M2. F2. 51 G, M M M F F y p" c CY ,..( M•D' D2. D'*' M' M•~ M~M1 F' s' G' F M' ~ s F F s s s G s G G G G
0 - 21 -
0.5% D + 0.5% K & 1% M & the sugar mixtures, 2% s, 1% G, 1% s. (ll) Colony diameter measurements showed K and D to be clearly better than F, G and s. o. Histograms showing the numbers of perithecia produced and the number of daye for fruiting summarize the final resulte. With regard to the number of daye for fruiting, an increase in sugar concentration tended to make a longer period of time necessary. As for the number of perithecia produced, dextrose, maltose and fructoae appear to be good sugar sources, and sucrose and galactose poor ones. Using a mixture of 4 or 5 different sugars produced the largest number of perithecia during a period of time not signiticantly greater or lees than that with the single sugars. lone of the synthetic media,however, could match 2% malt extract.
DISCUSSION ABD CONCLUSIONS When analysing the resulte from the nutritional experimenta, as recorded in the histograms, it is important to realize that minor variations are not significant. It is the broad relative differences that are meaningful. In general, fruiting is slightly faster where the sugar concentration is around the 1% level, though the differences between l% and 2% sugar concentrations are not areat. Kore perithecia tend to be produced in a longer period of time as - 22 -
the sugar concentration is increased to around the 2~ level in maltose and the 4% level in dextrose. Larger peritheoia are also produced with higher sugar concentrations. Dextrose is clearly the beat sugar for vegetative growth, and is the beat sugar for perithecial production when used as the sole sugar source. Maltose and fructose are also good sugar sources, but galactose and sucrose are not. Galactose is not usually a good sugar eource for fungi, probably because most fungi cannot produce the right enzymes in large enough amounte to deal with it effectively. Sucrose is a poor sugar source since it is probably inverted to glucose and fructose by the fungus sufficiently slowly for this to be a limiting factor in growth and fruiting. The greatest number of perithecia was produced on defined media containing mixtures of sugars. This is in agreement with Hawker's view (1950) that it may be easier for some fungi to grow and fruit better on auch media due to the production of several enzymes in small quantities rather than one enzyme in bulk. !his is also more similar to natural conditions where the substrate will usually contain more than one carbohydrate source. fhe effects of the mixture of 5 sugars and of all possible combinations of 4 did not differ significantly.
fhe amounts of biotin and thiamin used in the defined - 23 -
media were high, being 8 pgm and 200 pgm per litre respectively. This is probably near the optimum concentration. Biotin and thiamin, together with other growth substances, are also present in agar in small amounts (Robbins 1939), but these amounts did not appear to affect the experimental resulte which are based on high concentrations of the vitamin. The amount of thiamin in agar is about 0.1 mp mole per gm (Day 1942). This is 0.675 pgm in 20 gm of agar, i.e. in 1 litre of medium, which is negligible compared to the 200 pgm of thiamin ueed in each litre of medium. The production of perithecia on a medium eontaining maltose and thiamin but lacking biotin is possibly due to minute traces of biotin in the maltose itself, but is more probably due to the presence of an enzyme system that can utilise maltose sufficiently well without requiring biotin. This latter suggestion is feasible since the effect of biotin may well be on sugar uptake
(Bretzloff 1954) and therefore a difference in uptake of different susars is to be expected. Also sterile perithecial initiale were obeerved on media containing fructose and thiamin but lacking biotin, and on the media containing galactose and thiamin but lacking biotin indications of perithecial initiale were present.
All these three colonies were irregular in shape and stroma formation.
The absence of any traces of perithecial initiale in the - 24 -
media containing only biotin as a vitamin, and the presence of such initiale where thiamin replaeed biotin, does not belie Hawker's suggestion (1942) that thiamin seems to affect fruiting. She found the concentration of dextrose optimum for fruiting increased as the concentration of thiamin was increased. Also the very restricted vegetative growth on most thiamin-containing media as compared to the larger amount of growth on biotin containing media implies that thiamin does not stimulate sugar uptake and therefore it would influence another metabolic process in the fungus. Ho tremendous differences were found in the effects of different media on fruiting time such as Hanlin (1961) found in Hectria which bad a range of 10 to lOO days. Various fungi grow better on different natural media. Gelasinospora calospora var. autoste1ra, for example, grows beat on corn meal (Tylutki 1958). Many fungi, bowever, includ1ng
Rosellinia 11moniispora, grow well on standard PDA and lees wel~ on auch media as corn meal. The work of Hawker (1939b,l941), Bretzloff (1954), and others tends to support the theory originally proposed by Klebs (1899) tbat sexual reproduction in fungi is greatest under conditions of reduced nutrition, Hanlin (1961) suggests that his resulta with spec1es of Hectria indicate that a re1atively higb carbohydrate .~ 25-
requirement is more common than is generally believed, and the resulte with Rosellinia limoniispora tend to support this suggestion. The production of a very extensive stroma on media with a high concentration of nutrients also supports Hanlin's work.
Sinee 2% malt extraet produced a sufficient crop of perithecia in less time than other media and is much more convenient to prepare than the complex defined media, it was used to produce the peritheoia for cytological etudies in later stages of the present work. Malt extract is rich in pentoses, and may also contain vitamine in addition to biotin and thiamin which the fungus can synthesize for itself only at sub-optimum levels. A continuation of nutritional studies with this fungus along the lines of testing different pentoses and more vitamine might yield further enlightening information. - 26 -
PART II - CYTOLOGY
INTRODUCTION AND LITBRATURE REVIBW
Modern cytological techniques now applied to the study of nuclear behaviour in fungi have made possible in recent years several detailed etudies whioh help to shed light on some of the problems confronting the fungus taxonomist and genetioist. The modern squash technique enables the oytologist to observe nuolear details much more olearly than did the older more elaborate sectioning techniques where the staining methode usually caused severe shrinkage of the chromosomes and stained them so deeply that details of oentromeres, heteroohromatic regions, etc., were obsoured. Also the present-day use of photographe, sometimes accompanied by interpretative drawings, illustrate the facts the investigator cla~s to have observed. The Ascomycetes are particularly suited to cytological study due to the large aize of the fusion nucleus in the asous and its series of divisions leading to the formation of ascospores which can be readily followed. Such etudies, in addition to their intrinsic value, help to provide elues to auch phenomena as heterothallism and homothalL1sm, and since the fungi follow the laws of inheritance that apply to higher organisme, the ascospores are proving to be important tools in genetio research.
The first cytological study of an Ascomyoete made along - 27 -
the lines of higher plant cytology was the brilliant cyto!enetic account of Neurospora crassa by McClintock (1945). McClintock began a morphological description of the seven chromosomes and worked out the approximate positions of their respective centro meres. She found that synapsis occurred early between the contracted chromosomes which then elongated during a prolonged pachytene, during which time the ascus increased markedly in aize. She also obeerved the initiation of the eight ascoepores by fibres emerging from the centrioles. fhis work complemented the genetic etudies of Beadle and Tatum (1945) on this fungus. Singleton (195;) continued the cytological study of leurospora crassa and gave a very impressive aocount of nuclear division and chromosome morphology. His photographe and drawings of the extended pachytene chromosomes document very well his maps of chromosome morphology showing the positions of the chromomeres and the probable positions of the centromeres. His illustrations of the enormous increase in aize of the centrioles by Telophase
III and their radiating fibrils are convincing evidence of the harperian mechanism (Harper, 1900) of spore delimitation in this species. He also determined spindle orientation and its effect on the relative positions of the ascospores within the as eus. Another species in the Sordariaceae, of which Beurospora is a member, is Sordaria fimicola. Carr and Olive - 28 -
made an excellent study of this species in 1958. Here again precooious synapsis was found to ocour and a detailed investigation, supported by photographe and drawings, of the morphology of the sevan elongated paohytene chromosomes and of the centriole-fibril mechanism in delimiting the ascosporea, was undertaken. Olive's (1956) genetic etudies with this species also help place the interdependance of cytological and genetic etudies on virtually the same plane as in oytogenetic research on higher plants. The French workers Heslot (1958) and Doguet (1960) also examined this species. They did not obtain chromosome detail, but concentrated more on the centrosomes. He:Slot observed centrosomes in Interphase II and tbeir cleavage in Prophase III. Doguet olaims to have seen two centrosomes in the Prophase I nucleus. Both suggest the centrosomes play an active role in ascospore delimitation. Doguet disagrees with Carr and Olive in that he doea not support the occurrence of nucleolar fusion in the fusion nucleus, but offers no clear photographie evidence one way or the other. Some controversy waa encountered with the species Hypomzces solani f. oucurbitae, a member of the Hypocreales. This fungus has male, female, hermaphrodite and neuter thalli. Hansen and Snyder (1946) obtained aome intereating genetic resulte with it when crossing male and female thalli. From the ratios they obtained of the four different types of thalli they believed - 29-
that the genes determining sex were not alleles but were located at different loci in homologous chromosomes; crossing over may then have taken place between them to give the unexpected types.
Hirsch (1949) made a cytological study of this ~pecies, using all possible crosses, and concluded that the four types of thalli were due to differences in chromosome number, the hermaphrodites and neuters arising as a result of occasional non-disjunction of the two medium-sized chromosomes to produce nuclei with four and two chromosomes respectively. However, El-Ani (1954) made a genetic analysis of the asci from a male x female cross, and obtained ratios which could not be explained by the cytological picture offered by Hirsch; he suggested that the explanation of Hansen and Snyder (1946) should be reconsidered and the cytology reinvestigated. He took up this suggestion and in 1956 published a detailed study of the cytology of this fungus using male x female and hermaphrodite x hermaphrodite Olf.oase.s,, and found that the chromosome number in the ascus nuclei was always four. He emphaeized this by counting four chromosomes in the somatic nuclei of male and neuter thalli. He therefore rejeeted Hirsch1 s expla nation for the origin of the four different strains and regarded sex determination as a function of specifie loci, not of entire chromosomes. El-Ani oontinued his oytogenetio etudies in the Hypocreales - 30 -
With Gibbere1la cyanogena (195&~»and Bectria peziza (1959) with a view to obtaining cytological information bearing on the taxonomy and phylogeny of this order. With Gibberella cyanogena he crossed two compatible hermaphrodites, and also a hermaphrodite with a compatible male, and found the chromosome number to be four in all caaes. He regarded the mutation of hermaphrodite to male as not due to the losa of a chromosome but merely a single gene mutation. In Bectria peziza he found the meiotic divisions and ascus development to be simi1ar to that in BypO![Ces. He also found the chromosome number to be five, and measured their maximum lengths in pachytene. Ho reference is made to centrioles or fibres in the delimitation of the eight ascospores which he says are eut out by cleavage of the cytoplasm. Trichometasphaeria turcica, the perfect stage of Helminthosporium turcicum, is another fungus in which a detailed study of the nuclear cycle in the developing ascus has been made. Knox-Davies and Dickson (1960) documented their findings in this species with excellent photographe of the elongated paehytene chromosomes, the heteropycnotic regions frequent1y seen in the nuclei, and the rod-like centrioles. The centrioles did not appear to play major roles in ascospore delimitation which they observed to occur usually late in the fourth division when cleavage planes were deteoted in the ascus cytoplasm. Also they found spindle - 31 -
orientation haphazard in Division III, with the daughter nuclei distributed irregularly in the ascus, a fact that may well affect the serial order of the ascospores and henoe genetic interpretations based on this order. Another perfeot stage of Helminthosporium, Cochliobolua sativus, has been studied by Shoemaker (1955) and Hruahovetz (1956). Both found preoocioussy,napsis to occur. Hrushovetz worked specifi cally on aseus development in this fungus, and although he observed centrioles and astral fibres, the ascospores were not usually delimited until atter the eight nuclei had entered Division IV. Hrushovetz also observed prominent chromomeres in the extended pachytene chromosomes. Venturia inaegualis, a relative of Cochliobolus, has also been studied cytologically, Backus and Keitt (1940) found suggestions of beaked nuclei and astral radiations in a few asci, and also thought that changes in the serial order of the spores may occur before they are tully matured. Day, Boone and Keitt (1956) found precocious synapsis of the sevan chromosomes in the young asci, and observed no significant change in the shape and aize of the ascus between full pachytene and the uninucleate spore stage - a different condition to Neurospora crassa where the ascus doubles in length during this period. This early pairing of the seven chromosomes was likewise observed by Julien (1958) who also - 32 -
suggested that the central non-eister nuclei in Interphase II could pass each other in the ascus, an. occurrence that would be of genetic significance if it really took place and hence changed the linear order of the aecoepores. Another relative of Venturia, Sporormia obliguieeptata, was studied cytologically by Wells (1956). She produced good photographie evidence of chiasmata in this species, together with linear centriolea, asters, and spindle fibres. She believed that ascospore delimitation was initiated at the eight-nucleate stage by the centriole-astral ray mechanism. The arrangement of the spores in the ascus, i.e. uniseriate or biseriate, depended on the position of the spindles in Division III. Other lese recent etudies of the Ascomycetes could be cited here, but most lack critical detail of nuclear changes within the aecus and were conoerned mainly with refuting the brachymeiosis theory of Gwynne-Vaughan (1937). Examples are Wood'e
(1953) etudy of Ascobolus magnificue, Olive 1 s (1950) study of Patella melaloma, and Mcintoeh 1 s (1954) study of Pyromeaa eonfluens. Wheeler et al (1948) suggested early synapsis in Glomerella. Morgan-Jones (1953) observed the frequent development of asci in Gnomonia directly from bi- or multinucleate cella, and also believed centrosomes and astral raye to be lacking. Some cytological details are aleo available for one epecies of Roeellinia. Greis and Greis-Dengler (1940) in their stud7 of Rosellinia reticulospora nov. sp. observed little bodies inside and outside the fusion nucleus which the7 likened to chondriosomes (mitochondria), and noted astral rays in this species. Greis (1936) also noted a similar occurrence in Sordaria fimicola. The Belgian school has provided details of the nuclear condition of the cella of the ascogenous hyphae but no critical nuclear details in the developing asci. Less than 50% of the cella of the ascogenoua hyphae were dikar7otic in lectria flava (Gilles, 1948), leurospora tetrasperma (Gilles, 1950), and Ohaetomium !lobosum {van der Weyen, 1954), though all the cella appeared to be dikaryotic in Gelasinospora calospora {Meyer, 1957).
The gene~ical work of Ames (1934) explained the homothalliia of the binucleate ascospores of Sordaria (Pleurage) anserina. He cult~d the uninucleate ascospores which are occasionallT formed instead of the binucleate ones, and found that the homothallism of this fungus is due to the two nuclei in the binucleate spores representing the two self-sterile but cross-fertile hermaphrodite strains. Moreau and Moreau (1951) described ascus development and spore formation in Pleurage anserina and Pleura~e setosa. The spores were first cylindrical, then became more or lesa tadpole-shaped. The spores were initially binucleate in P.anserina, - 34 -
and uni - and multinucleate in P.setosa. Pollowing a conjugate nuclear division in the spore, with the spindle of the lower nucleus parallel to the spore ~, one daughter nucleus is found in the "tail" or future primary appendage of the spore, and the other nuclei are located in the bulging centre. A septum forma isolating the nucleus in the appendage and this nucleus soon degenerates. Rosellinia limoniispora is well suited for a study of details of the nuclear changes within the developing ascus. The following investigation attempts to follow the course of events from crozier initiation to the production of ascospores and compares nuclear behaviour in this species with that in other Pyrenomycetes which have been subjected to similar analysis.
MATERIALS AJD METHODS
Cultural Methode Stocks of Rosellinia limoniispora from Dr. O.K. Wilson1 s culture collection were maintained on PDA slants, and transferred to small petri-dishes to furnish material for cytological study. The media for the production of the perfect stage in the petri dishes were PDA and !pSs. The perithecia were harvested after 4- 4! weeks. Later in the investigation 2% malt extract was used exclusively and the perithecia were harvested after 3 weeks. - 35 -
Perithecia of this age contained all stages of ascus development from crozier initiation to mature ascospores. The different media did not affect the aize of the asci or the nuclei.
Spore Germination
Two lota of spore suspensions were made in glass vials, one lot containing water and the other 1% BaOH. Both were heated for five minutes at 60°0. A drop of the suspension was then spread over a drop of agar solidified on a slide and a cover-slip added.
Only the elides kept constantly moist by pipetting water under the cover-slips at intervals showed germinating spores. Germ - tubes were seen after about 40 hours. These conditions were not sterile, but in the short time necessary for germination no difficulty was encountered with contaminants. After various stages of germination were visible in about half the spores, stain was pipetted under the cover-slip which was then sealed with a paraffin wax - gum-mastic preparation. Observations, photographe, and drawings could then be made.
~ixation
Two types of fixative were tried.
(a) Weak Chrom-Acetic: (Johansen, 1940)
This solution, although "weak", was found to decompose the asci, even after fixing for only li hours at room temperature.
(b) Carnoy's Jluid: - 36-
3:1 absolute ethyl aloohol: glacial acetio aoid. 'rhis was found to work Tery well wi th a fixing tim.e of both·:. li and 24 hours at room temperature. The material was then stored in test-tubes in 70~ ethanol in a refrigerator where it remained suitable for study for up to three months, atter wbiob time the contents of the perithecia hardened. Studies were also made of fresh material in aceto - carmine and aceto-orcein smears. Hydrolysis Perithecia, pierced with a needle to ensure penetration by the solution, were heated for 5 minutes at 60°0 in small glass Tials in B HCl. They were then washed, and stained in the usual way.
Stains Various stains were tested, of which the beat were found to be 2% orcein (Edward Gurr, Ltd., London, U.K.) in 75% acetie aeid, and 2~ carmine (Allied Chemical & Dye Corporation, B.Y.) in 45~ acetic acid with added iron. Both stains were prepared by refluxing 2 gm of the powdered etain in 100 ml of aoetic acid tor 6 hours, after which they were filtered. A few drops of saturated terrie acetate solution were added to the aceto-oarmine solution until it became plum-coloured. - 31 -
Preparation of Slides
Pour or five perithecia were placed on a elide, in a drop of 70% ethanol to prevent them from drying out, and their contents were dissected out under a dissecting microscope with an arrowhead needle and an ordinary needle. Perithecial walls and other debris were removed from around the ascue elumps.
A drop or two of etain was added once the alcohol had almost evaporated from the elide. A cover-elip was next added, and the elide was heated to just below boiling over an alcohol lamp. The elide was then pressed heavily between sheets of absorbant paper to remove excess etain and to flatten the asci, after which the cover-elip was ringed With a paraffin wax-gum mastic preparation. Slides prepared in this way showed contracted chromosomes to be stained after a few hours, though the extended pachytene chromosomes of Prophase I showed up best after 6 - 7 daye. Later the cytoplasm became too densely stained for clear observations and etain had seeped into the karyolymph. All stages were etained reaeonably well with either etain, but the aceto-orcein was found to stain the contracted chromosomes more deeply, whereas the aceto-carmine gave better detail in the longer prophase chromosomes and also stained the nucleolus clearly. No improvement was obtained using both stains together, either by mixing them before use, or by drawing them in turn under the cover-slip with filter paper. - 38 -
Microscopy
Observations and photographe were made with a Spencer microscope fitted with B.A •• 25, B.A •• 66 and I.A. 1.25 objectives and lOX oculars. A No. 74 green Wratten filter was used to improve the contrast. Photographe were taken with a Kodel L Bausch & Lamb
Photomicrographie ''~ipment under èil ~ersion (N.A. 1.25 objective) on Kodak Contrast Process Panchromatic lilm (4" x 5" plates), with the exception of tigs. 1 and 46 taken on Kodak Royal Pan Film (medium contraat). !hey were developed in Kodak Dll developer, and printed on F4 or l5 Kodabromide paper.
CY!OLOGICAL OBSERVATIONS
All stages of aacus and ascospore development can be found in a single perithecium in squash preparations (tig. 1). !he sequence of nuclear divisions was followed in detail in Rosellinia limoniispora from crozier initiation to the formation ot mature ascospores. As in the accounts of Neuroapora (Singleton, 1953) and other species, the nuclear divisions in the ascus, starting with the first division of meiosis, are referred to respectively by number I to IV.
Crozier initiation ie quite clearcut in this epecies. lirst of all the tips of the ascogenous ~yphae are seen to be - 39 -
binucleate (fig. 2, a). The tip benda over with one nucleus appearing on either side of the bend. These two nuclei then undergo a mitotic division more or lesa simultaneously (fig. 3). Six chromosomes were counted in the haploid nucleus in this division. The hook tben contains four nuclei (fig. 4, a).
Two septa next arise, cutting off a uninucleate terminal cell and a binucleate penultimate cell (fig. 4, b). The four nuclei in the hook were once observed to have a hollow ·appearance, also observed by Wells (1956) in Sporormia, probably due either to the sentral nucleolus failing to take up the etain or to the chromosomes being arranged in an arc. The two nuclei come together in the penultimate cell and fuse either at once or when the cell attains a clavate shape as it begins to elongate, forming the young ascus (fig. 5, c). The two nuclei come together in the small dark-staining interphase condition (fig. 6, a) and the two nuclear membranes in contact break dawn. The chromatic masses then expand, come together, and fuse (figs. 6, b, and 2, b). The tip cell usually benda around and touches the basal cell below the young ascus, the separating walls break down, and the nucleus from the tip cell passes into the basal cell to pro duce a new dikaryon (fig. 5, b). Then the crozier-ascus cycle occurs again, and this may happen several times along an ascogenoua hypha so that a long row of young asoi may be seen with the asci - 40 -
pointing outwards in all directions• This proliferation of the croziera, in which the ascogenous hyphae appear to grow sympodially, is readily seen in the two ascogenous hyphae in fig. 5 where the asci are being produced in basifugal succession, the youngest being neareat the apex.
The young ascus, containing the fusion nucleus with a large nucleolua and a maas of chromatic material, is very small at first as can be seen in fig. 1. Probably there are two nucleoli which fuse since a young ascus has been observed with two smallish oval nucleoli not yet fused (fig. 8). In fig. 9 a large nucleolus is visible, equivalent in size to the two in fig. 8 combined, and the chromosomes appear as thick double threads.
kfter nuclear fusion the young ascus expands extremely rapidly in both length and breadth. The ascus in fig. 10 shows a maas of chromosomes, which appear thick and double, suspended in a clear karyolymph (the nucleolus is not stained here). The chromosomes atill appear thick and double in fig. 11 where the aacus has inoreaeed in aize. The asous in fig. 12, the tip of which has been distorted in aquashing, shows a very large nucleolus with a number of chromosome threads forming a sort of sphere around it; some of these threads are clearly in pairs with a small space between each of the paired chromosomes. It would seem that the paired chromosomes pull apart slightly along their entire length as they elongate and beoome thinner than in the earlier - 41 -
paired stages of figs. 9 and 10. One of the longest chromosomes is attached to the nucleolus which is very large at this stage (fig. 13). Fig. 14 shows some of the paired chromosomes sligbtly separated except where there are lumps or chromomeres along their length. In fig. 15 the long thin chromosome pairs with a narrow space between each two members of a pair are clearly visible. Sometimes the chromosomes appear fuzzy in outline which may be due to coiling of the chromosomes (fig. 16, right-hand ascus). The ascus meanwhile is still increasing in aize. The varying widths of the asci are mostly due to variation in squashing pressure. Figs. 17, 18 and 19 show paired chromosomes and the large nucleolus. The chromosome pairs at times appear twisted once or twice around each other (figs. 15, 16 and 20) and soma times they appear untwisted but pulled apart except at chiasmata (tige. 20 and 21). The nucleolus begins to decrease in aize once diplotene is reached (fig. 21). As the nucleolus shrinks still further, the chromosomes etain unevenly and contain heterochromatic regions before they start to contract (figs. 22 and 2;). The chromosomes contract quickly to short dark-staining rode. Fig. 24 shows the six contracted bivalents, four large and two small ones, and is a late Prophase I diakinesis figure or an early Metaphase I. Fig. 25 is similar except that the lower two rode appear to be two sets of two roda each overlapping, thus - 42 -
making six bivalents when added to the top two bivalents in
the figure. No clear metaphase plate was observed here comparable to those observed in the crozier {fig. 3, a),
Metaphase II (fig. 30, b) and Metaphase IV {fig. 41). This
suggests that the stage is very short-lived. In fig. 26 the bivalents appear to be disjoining in an early anaphase seen from an oblique angle.
Anaphase I must take place very rapidly since this stage was observed only once (fig. 27) where the chromosomes were seen separating in two masses, and a linear centriole could be distinguished at one pole. The division is longitudinal with respect to the ascus length. Two nuclei result which appear close together in the centre of the ascus, either one above the other (fige. 28, 29, right-hand ascus, and 31) or almost aide by aide (fige. 29, left-hand ascus, and 30).
Telophase I and Interphase I are passed through quickly, and the two nuclei go into Prophase II simultaneously where they are still extremely large and the chromosomes first appear as very thin attenuated threads (fig. 29, left-hand ascus). This and the following stages are probably short-lived, judging by their infrequency as compared to the abundant Prophase I's.
In Metaphase II six chromosomes can again be counted
(fig. 30, b). The chromosome masses then separate more or lesa - 43 -
conjugately (fige. 31 and 32) to form four nuclei (fig. 33). The plane of division of the top nucleus is clearly parallel
to the ascus length (longitudinal) in fig. 31, a. The lower
two nuclei in fig. 32 appear to be in late Anaphase II, but no spindle fibres show up. These four nuclei do not at first appear
to be in a straight line (figs. 32 and 33) and the middle two non-sister nuclei might conceivably reveree their positions, eapecially in fig. 32. However, this possibility of switching
places may be because observations were made on asci which bad been squashed and the nuclei may have been pushed out of their original order. When Prophase III occurs the nuclei are again in one row (fig. 34), although an ascus was seen with four nuclei probably in Metaphase III arranged in two more or lese horizontal pairs (fig. 35). The nuclei in Prophase III appear very large, similar in size to those of Prophase II. Eight nuclei result from Division III, but show no arrangement in any defi~te pattern in the ascus (fig. ~6). Spindle orientation in this division must therefore be haphazard. The nuclei appear rather odd in fig. 36, their hollow appearance probably being due to the unstained nucleolus in the centre of the nucleus which most likely reforma in interphase, or to the chromosomes being arranged in an arc, or both reasons together; this configuration was also noted in the four nuclei in the crozier. - 44 -
Interphase III also seems to be short-lived and the nuclei take on an early Prophase IV configuration just before the outlinee of the young ascospores are visible (fig. 37 - the top two nuclei are overlapping). These early Prophase IV nuclei tend to be situated at one or other end of the faintly delimited ascospores (fige. 38, 39 and 40). In fig. 39 the third and fourth nuclei from the top are being enolosed by one spore outline. No oentriole-astral ray mechanism was observed, but a lining up of small vacuoles along the demarcation lines of the young ascospores was observed (fig. 38). The eight nuclei may be in one line (fig.
39) or more (fig. 38). The Prophase IV nuclei are smaller than the nuclei in
Prophases II and III. The ascospore outlines are cle~rly visible as Prophase IV proceeds (fig. 40). No beaked nuclei (Harper 1900) were seen. The asci in early Prophase IV are not noticeably larger than they were in late pachytene-early diplotene. The nuclei divide inside the delimited ascospores, and at this t~e the young spore outlines are very sharp and clearly mark off the epiplasm (fig. 42). The nuclear divisions within the eight spores in an ascus are not quite conjugate and not in serial order. In fig. 41 six chromosomes can be counted in the metaphase plate inside one of the young ascospores. Telophase IV ends with one nucleus remaining more or lees in the centre of the - 45 -
spore and the other nucleus situated in the little bulge at
one end of the JOung spore (fig. 42) where it is eut off by
a ~eptum (tig. 43i b) and completely tilla this bulge. !he
other nucleus increases in aize in the main body of the spore where it becomes more diffuse when it is presumably in Interphase
IV (fig. 43, b). This nucleus may subsequently undergo one more
division, but this could not be verified since the spores were
increasing in aize and beooming filled with oil vacuoles whieh
were ooalesoing (fig. 43, b) and the wall was becoming pigmented
(fig. 45). The spores may be partially biseriate (fig. 44) but
. beeome uniseriate as their walls thicken and they increase in
width and become more rounded (fig. 45).
There is no apical pore in the ascus (fig. 47}. The
mature spores, in which the vacuoles have coalesced to form one large one, have a bulge at each end and a ridge along opposite
aides stretehing from pole to pole (figa.46 and 48). The spores do not put forth a germ-tube from either end, i.e. the nucleus in either or both polar bulges is not used in germination; they put forth one or several germ-tubes or vesicle·a through the wall
where it bas eraeked open along the ridge (fig. 48). - 46 -
DISCUSSION AND CONCLUSIONS
The ob~ect of this cytological study has been to provide details of the nuclear cycle in Rosellinia limoniispora, to reveal the significant tacts diseovered, and to compare the similarities and differences in this species with those described inoother higher Aseomyeetes. Possible genetie and taxonomie impli cations are also considered. !he asci of Rosellinia limon11spora have proved to be good material tor such a study.
The typical crozier formation, as a prelude to aseus formation, can be tollowed clearly in this speeies. lor this reason the material could be used quite conveniently in the classroom to demonstrate this phenomenon.
The nucleolus takes up the aceto-earmine etain quite readily. Two nucleoli were seen in a young ascus after the two chromosome masses bad tused, and at a slightly later stage a very large single nucleolus was frequently observed. Disintegrating nucleoli were never seen. In this species, therefore, it is highly probable that a fusion nucleolus is formed shortly after fusion of the chromosomes in the very young ascus. This nucleolus is very large until late pachytene-early diplotene when it quickly shrinks to half its former volume and disappears at the end ot
Prophase I. The nueleolar chromosome is one ot the longest, or perhaps the longest, of the six chromosomes. - 47 -
There seems to be no general pattern for the behaTiour of the nucleolus in an ascus, and different workers haTe reported
Tarious occurences according to the species they haTe inTestigated.
In the young ascus the two nucleoli may disappear and a new nuoleolus form in some species (Wells, 1956), though in most cases the two nuoleoli fuse shortly after the two ohromatio masses haTe fused
(Jones, 1926, Wheeler et ~' 1948, HrushoTetz, 1956, Carr and OliTe,
1958, and Knox-DaTies and Dickéo~;: ~, 1960). The nucleolar chromosome is usually the longest one (Wheeler et ~' 1948, and El-Ani, 1956 a) or the second longeat (McClintock, 1945, and Carr and Olive, 1958).
In several species the nucleolus disappears in late pachytene or early diplotene and does not reappear in later divisions (Hirsoh,
1949, Wheeler et ~' 1948, El-Ani, 1956 a, and Knox-Davies and Dickson, 1960), although in many ether cases it may persist in the cytoplasm through Metaphase I or eTen longer (McClintock, 1945, and Singleton, 1953, Mcintosh, 1954, OliTe, 1950, Carr and OliTe,
1958, Julien, 1958, Bagchee, 1925, and Day, Boone and Keitt,
1956 ). In diplotene the chromosomes were occasionally seen to have a fuzzy appearance. This may be due to the presence of coiling of the two homologous chromosomes around one another. It has been observed by Singleton (1953) in Neurospora crassa, Carr and Olive
(1958) in Sordaria fimicola, and Knox-Davies and Dickson (1960) - 48 -
in Trichometasphaeria turcica. Its absence was reported in
Nectria peziza by El-Ani (1959).
Precocious synapsis of the homologous chromosomes in the fusion nucleus in the young ascus was observed in
Rosellinia limoniispora. The still contracted chromosomes in the young ascus had a thick appearance and seemed to consist of paired threads. The chromosomes were clearly in pairs before maximum extension of the chromosomes and asci, although the exact stage when pairing started was not seen. This early pairing of the chromosomes whilst they are still in the contracted state after fusion of the nuclei in the very young ascus has been reported several times recently in the higher Ascomycetes, notably by McClintock (1945) and Singleton (1953) in Neurospora crassa, Wheeler ~ al (1948) in Glomerella, Shoemaker (1955) and
Hrushovetz (1956) in Cochliobolus sativus, El-Ani (1956~a) in
Hypomyces solani f. cucurbitae and (1959) in Nectria peziza, and
Day, Boone and Keitt (1956) and Julien (1958) in Venturia inaequalis. One reported exception is Sporormia obliquiseptata
(Wells, 1956), but this may be due to misinterpretation of her photographe. It would seem that the concept that this is probably a general phenomenon in the Ascomycetes (Day et al, 1956) is becoming well verified by the evidence that is now accumulating.
It is not known to occur in the higher plants however.
In Rosellinia limoniispora the chromosome number is six, - 49 -
as counted in the metaphase plate in the mitotic division in the crozier, the diakinesis or early Metaphase I stages, the Metaphase II plate in the developing ascus, and the Metaphase IV plate in one ot the ~oung spores. It is theretore clear that brachymeiosis does not occur in this species. Chromosome counts together with genetic etudies have rejected the theory in all species studied in the past two decades. Chromosome counts are beat made in diakinesis or metaphase before disjunction of the bivalents haa commenced. Counta in anaphase are hazardoua and bence frequently incorrect due to the ditticulty ot distinguishing univalents from bivalents {Olive, 1953). Chromosome numbers may be important in providing cytotaxonomic evidence of relationships between certain speciea and groups of Ascomycetes, as in the case of Allomyces in the Phycomycetes (Emerson and Wilson, 1954) and the higher plants. In most higher Ascomycetes the nuclear divisions within the aecus give rise to a linear row of eight nuclei and consequently a linear row of eight ascospores. In Rosellinia limoniispora, however, Division II in the ascus gives rise to tour nuclei which were otten aeen to be lacking a linear arrangement. The two central non-aiater nuclei were sometimes observed to lie alaost aide by aide and could conceivably slip paat one another, although of course - ?0 -
this position may be an unnatural one as a result of squashing. If these two central nuclei could switch places, this would affect genetic etudies based on the serial order of the spores - false inferences might be drawn from the isolation of ascospores in order from individual asci. Singleton (1953) never observed auch nuclear passage in Neurospora crassa, though Mcintosh (1954) believed it to be possible in Pyronema confluens, as did Julien (1958) in Venturia inaequalis. The eight nuclei arising from Division III in the asous were observed to be distributed apparently at random in the central region of the ascus, and here again the nuolei oould conceivably slip past each other before becoming lined up in the cytoplasm prior to spore delimitation. A similar irregular distribution of the eight nuclei in the ascus of Trichometasphaeria turcica was -observed by Knox-Davies and Dickson (1960)i Graff (1932) also noted that it is difficult to trace the relationships of the eight nuclei when they are massed in the centre of the ascus in Meliola circinans. As in the case of the four-nucleate stage, genetio conclusions based on the linear order of the eight ascospores might have to be modified. The plane of division of the nuclei in the ascus was often observed, though actual spindles were not seen even after keeping in fixatives for long or short periode of time, or after - 51 -
hydrolysing inN HCl (Singleton, 1953). It would eeem there-
fore that different species of fungi vary in the ability of their
spindle fibres, and also centrioles, to take up stains. In
Rosellinia limoniispora, Division I is longitudinal, but Divisions
II and III were observed in longitudinal, oblique and almost
t~ansverse directions. Divisions have been reported in all three
directions in diverse fungi. Colson (1934) and Dodge (1936)
considered the genetic significance in Neurospora tetraeperma where oblique spindles govern whether two like or unlike nuclei are enclosed in each of the four ascoepore outlines. In most
Ascomycetes the first nuclear division in the ascus is longitudinal, whereas the second and third divisions may be in all the three
directions as described for Rosellinia limoniispora. This bas
been reported by such workers as Carr and Olive (1958), Knox
Davies and Dickeon (1960), Singleton (1953), Mcintosh (1954), El-Ani (1956 a and 1959), Zickler (1953), and Lloyd and Wilson
(1962). Wells (1956) ehowed that in Sporormia obliquiseptata a unieeriate or biseriate arrangement of the ascosporee depended on spindle orientation in Division III. There is much discussion in the literature on the
eubject of aecospore delimitation. No sign of a centriole-astral ray mechaniem delimiting the young aecosporee wae observed in
Boeellinia limoniiepora. This may have been due to the stains - 52 -
employed but probably not since simple cleavage of the cytoplasm has been reported in many species. There is much controversy
over which mechaniam actually occurs, and many workers elaim
to have demonstrated conclusively that one or the other is the
correct interpretation. The present writer believes that either mechanism can operate, depending on the species in question. Sometimes fibrils radiating out from the centriole of the beaked nuelei have been convinoingly demonstrated, especially by
Singleton (1953)1 also by Wells (1956) and Carr and Olive (1958). The drawings of earlier cytologiste, auch as Harper (1900) and
Bagchee (1926), are open to doubt in the light of later work. Simple cleavage of the cytoplasm into eight masses around the eight ascospores (Heim, 1952, and El-Ani, 1959), sometimes accompanied by the lining up of amall vacuoles along the delimiting lines (Jones, 1926, and Jenkins, 1934), bas been shown to occur in several cases. ~inally, centrioles and radiating fibres (astral rays) have been observed that do not appear to play a significant role in ascospore delimitation. In these cases the nuclei are already in Division IV before the outlines of the spores beoome apparent (Knox-Davies and Diokson, 1960). In Rosellinia limoniispora the eight nuolei enter Prophase IV before the ascospore outlines are visible. When they appear, the outlines are often emphasized by the presence of lines of little oil vacuoles. - 53 -
Roaellinia reticulospora, according to Greis and Greis-Dengler
(1940)'1 haa centrioles and astral rays in the divisions in the ascus, but they offered no photographe in support of their observations. It is obvious from all these conflicting reports that more study is required, and that either interpretation may be right for the species studied. It is interesting to note, however, that Heim (1952) examined various fungi including
Pyronema confluens in which Harper (1900) claimed to have observed the centrioles and radiating fibrils so clearly, yet concluded that simple cleavage of the cytoplasm occurred here.
The division of the nucleus within the young spore in
Rosellinia limoniispora and the cutting off of one daughter nucleus by a septum, followed by its degeneration or at least ceasing to function as determined by germinating the spores,is rather peculiar and does not appear to have been reported elsewhere. The most similar case is in Pleurage anserina and Pleurage setosa where
Moreau and Moreau (1951) found degeneration of one daughter nucleus, following nuclear division within the young ascospore, after its being eut off in the young primary appendage of the spore by a septum. This similarity may be important taxonomically since the
Sordariaceae, which includes the genus Pleurage, and the Xylariaceae, which includes the genus Rosellinia, are believed to be fairly closely related. - 54-
The nuclei in the maturing ascospores of Rosellinia limoniispora divide at elightly different times and without this being related to their serial order. This is somewhat similar to the asynchronous maturation at random of the ascospores in Hypomyces solani f. cucurbitae (El-Ani, 1956 a), though different to Neurospora crassa (Singleton, 1953).
The aize of the ascus does not change noticeably between early diplotene of Prophase I and the beginning of spore delimitation. This is similar to Venturia inaegualis (Day et el,
1956) though different to Neurospora crassa (Singleton, 1953) where the ascus approximately doubles in length during this period. The change is slight in Sordaria fimioola (Carr and Olive, 1958).
This investigation of the nuelear cycle in the ascus of
Rosellinia limoniispora has shown a general similarity to ether pyrenomycetes recently investigated, particularly with regard to crozier formation, precoei.oiW!sym.apsis of the oontracted chromosomes in Prophase I, and the formation of the eight nuclei. Ascospore delimitation resulta from simple cleavage of the cytoplasm. The degeneration of one daughter nucleus atter Division IV in the maturing spores is striking and, as far as is known to the present writer, has not been reported elsewhere. A similar investigation of other species in the Xylariaceae would certainly provide useful information pertaining to this point. - 55-
SU'lllü.RY
1. Rosellinia limoniispora, a pyrenomycete, fruits readily in pure culture under room conditions.
2. The Vi tamins bio tin and thiamin are required for frui ting to talee place.
3. Growth and fruiting vary widely according to the amount and type of sugar; at a 2% concentration, which is favourable.
for fruiting, more perithecia are produced by using a mixture of sugars than by using one sugar alone.
4. !he best medium for growth and frui ting was found to be 2% malt extract.
5. Nuclear details in the asous are readily seen in aceto-oroein and aceto-carmine smears.
6. Spindles are not revealed by these stains, even after pre
treatment by fixing or hydrolysing. 1. The haploid chromosome number is six, one of the longest chromosomes being attaohed to the nuoleolus. s. The nuclear cycle in the developing asous follows the general pattern for the higber Ascomyeetes: crozier formation, early synapsis of the chromosomes in the fusion nucleus followed by
nucleolar fusion, rapid elongation of chromosomes and ascus
up to late pachytene of Prophase I followed by contraction of
the chromosomes and nucleolus, and the formation of eight nuclei - 56 -
as a result of the two meiotic and one mitotic divisions.
9. The orientation of nuclei in Divisions II and III was
haphazard, a phenomenon that would affect genetic anal7ses
based on the linear order of the eight ascospores.
10. Ascospore delimitation is by simple cleavage of the cytoplasm;
no centriole-astral ray mechanism was observed.
11. A nuclear division occurs in each young ascospore, one
daughter nucleus degenerating.
12. Spores germinate by means of a crack along the ridge in the
spore coat. BIBLIOGRAPHY
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PHOTOGRAPHS
All the photographe are at a magnification of
X 3000 diameters unless otherwise stated in the legend accompanying eaoh photograph. The etain used in each case is indicated in brackets after the figure number s (o) is aceto-orcein (c) is aceto- carmine (o.e.) is a combination of both stains. ~ig. 1 (o) Portion of contents of perithecium showing asci in various stages of development. x 300. •
Fig. 2 (o) Group of yo~ asci showing various early stages of development. a. Two nuclei present in tip of ascogenous hypha. b. Two nuclei fusing in the young ascus. c. Young ascus with fusion nucleus rapidly increasing in aize. a b
Fig. 3 (o) Young crozier at two different focuses. a. Shows a metaphase plate in which 6 chromosomes can be counted. b. Shows the second nucleus at early anaphase where the two chromosome masses have just begun to separate.
Fig. 4 (c) Croziers at different stages of development. a. 4 - nucleate stage in the hook. b. Two septa visible cutting off the binucleate penultimate cell. c. & d. Two more croziers at different angles. Fig.5 (c) Two lines of proliferating croziers. a. Crozier with two nuclei just before fusion in the pen ultimate cell. b. Nucleus of terminal cell of crozier has migrated into the basal cell. c. Young ascus. Asci grow out in all planes. Growth appears to be sympodial, and the youngest asci are towards the tip.
b ~ - •
Fig. 6 (o) Fusion of the two nuclei in the young ascus (nucleolus not stained). a. Two nuclei in contact; chromatic masses emall and the nuclear membranes have not yet broken down. b. The nuclear membranes have broken down between the two nuclei and the enlarged chromatic masses are beginning to fuse. Fig. 7 (c) Young ascus with fusion nucleus containing a large nueleolus and a maas of chromatin.
Fig. 8 (c) Young ascus with two nucleoli. (fusion apparently delayed here) and a maas of chromosome threads which appear to be paired. Fig. 9 (c) Expanding young ascus. Nucleus contains large nucleolus and thick • double chromosome atrands •
' '
Fig. 10 (o) Young ascus with nucleus containing double chromosome threada in a clear karyolymph (nucleolus not stained). •
Fig. 11 (c) Ascus with large fusion nucleolus and double chromosome threads.
Fig. 12 (c) Aecus with large fusion nucleolus enclosed in the spherical maas of chromosome; some of the threads are in pairs with a narrow space between the homologous chromosomes of each pair at early Prophase I. Fig. 13 (c) Long ascus with very large nucleolus and the long nucleolar chromosome attached to it.
Fig. 14 (c) Fusion nucleus with large nucleolus and the paired chromosomes . of Prophase I. The two chromosomes of each pair are separated by a narrow space except at certain points which may be chromo centres. Fig. 15 (c) Ascus with large nucleolus and long thin chromosomes clearly paired with a marked space between the two chromosomes o:f each pair.
Fig. 16 (c) The le:ft-hand ascus shows a mass o:f dis organised chromatic material plus a nucleolus. The right-hand ascus is unusually rounded and contains a nucleolus plus paired chromosomes whioh have a rather :fuzzy appearance; the two chromosomes in some o:f the pairs seem to be twisted round each other a :few times. Fig. 17 (c) Ascus containing large nucleolus and double chromosome threads.
Fig. 18 (c) Another ascus containing large nucleolus and thick double chromosome threads.
• Fig. 19 (c) Ascus containing large • nucleolus and thick double chromosome threads.
Fig. 20 (c) Ascus with nucleus probably in early diplotène of Prophase I. The nucleolus is still large, and the chromosome threads are in pairs with each member of a pair twisted round ita partner; chiasmata appear to be present. The greater width of this ascus is due to heavier squashing. Fig. 21 (c) Ascus with nucleus probably in mid-diplotene. The nucleolus is diminishing in aize and the double chromosome threads are beginning to contract.
Fig. 22 (c) Ascus with nucleus in late diplotene where the nucleolus is shrinking and the chromosomes are becoming heterochromatic. • •
Fig. 23 (c) Nucleus in late diplotene with the nucleolus fairly small and the chromosomes heterochromatic.
___ ...
Fig. 24 (o) Diakinesis or early Metaphase I. 6 bivalents are visible - 4 large ones and 2 small ones. This ascus is abnormally narrow due to shrinkage of the cytoplasm. Fig. 25 (o) Diakinesis or early Metaphase I •. 6 bivalents are not clear since the 4 bottom ones are overlapping one another in 2 double pairs.
Fig. 26 (o) Late Metaphase I or early Anaphase I from an oblique angle. Disjunction of the bivalents is just beginning. The cytoplasm · has cont~acted, making the ascus appear narrower than it really is. Fig. 27 (o.c.) Anaphase I. Note linear centriole at 'a'.
Fig. 28 (c) Interphase I. •
Fig. 29 (o) Prophase II. The large aize of the early Prophase II nuclei is evident in the left-hand ascua.
a b Fig. 30 (o) Metaphase II, seen in two different focuses. 6 chromosomes can be counted in the metaphase plate in1 b 1 • a b Fig. 31 (o) Late Metaphase II or early Anaphase II. The two chromosome masses are beginning to separate in the top nucleus.
Fig. 32 (o) Late Anaphase II or early Telophase II. Fig. 33 (o.c.) Interphase II. Note the alignment of the 4 nuclei as compared to fige. 32, 34 and 35. Fig. 34 (o) Prophase III. The nuclei at this stage are very large. The fourth nucleus is out of focus.
Fig. 35 (o) The four nuclei appear to be at Metaphase III and are seen obliquely in the photograph. The bottom-right nucleus may be in early Anaphase III. ·'
a b
Fig. 36 (o) Interphase III. The eight nuclei are apparently arranged at random in the centre of . .. ·· .. .. . the ascus. The nuclei • ....·...... have an odd hollow •• :· 0 • • appearance, due probably 0 •• • •• to the chromosomes . . • :... .• .:0 ·. ·.· . ·. · .. . being arranged in a ring ·.. ·.. . around a (non-stained) :. ::-. ·.. . ·. : . : : : ..·: . nucleolus • . . . . :·. ·.: .· ::-: ·. ·-: ::. ·: .•. · : _: ... ~ -:: :·::..:: .....
: ... • •• :. - : : • •• • 0 • •• . .· .. . ·: .·
.... 0 ••• :: : : :· ·.·: .· Fig. 37 (o) Late Interphase III or early Prophase IV. No spore outlines are visible although the eight nuclei aeem to be entering Prophase IV. The top nuclear maas is probably two nuclei overlapping. Fig. 38 (o) Prophase IV. The eight nuclei are not beaked. The spore outlines are faintly visible in the cytoplasm which is full of small vacuoles, some of which are lined up along the outlines of the young ascospores. •
Fig. 39 (o) Prophase IV. Ascospore outlines are clearly visibl e in the vacuolated cytoplasm. ~e third and fourth nuclei from the top appear to be enclosed in the same spore. Fig. 40 (c) Prophase IV in clearly delimited young asoospores.
Fig. 41 (o) Nuclei dividing in the young spores. Note the Metaphase IV plate, in the third nucleus from the top, where 6 chromosomes can be counted. \
Fig. 42 (o) Nuclear division is taking place non conjugately in the young ascospores. Anaphase IV and Telophase IV can be seen. One daughter nucleus becomes located in the bulge at one end of each spore. Fig. 43 (o) Three asci show the young developing ascospores. a. Eight young spores in Prophase IV. b. Eight spores with nuclei in Interphase IV. Note the septum cutting off the nucleus in the bulge in the top spore, and the vacuoles in the spores which are coalescing to form fewer larger ones. c. Spores with vacuoles becoming fewer and larger. Fig. 44 (o) Eight young spores in ascus with nuclei in various stages of division. The arrangement of the spores is semi biseriate here. x 2260.
Fig. 45 (o) Spores becoming more rounded as they mature. Their arrangement is becoming uni eeriate as in the mature ascue. One terminal bulge is present in each spore. X 1440. Fig. 46 Mature and almost mature ascosporee. The oil vacuoles coaleece to form one large one before the black pigment is laid down on the spore walls. Note the ridges on spores a and b which connect the two bulges which are now present at opposite ends of the spores. X 1350.
Fig. 47 (o) Overexposed photograph to show the end of the mature ascus. No pore or other dehiscence structure is visible. x 2260. Fig. 48 Germinating spores. No germ-tubes were ever seen emerging from either terminal bulge. The spore coat cracks along the ridge; a vesicle emerges, and grows out into a filament which soon branches. X 1350.