Surface Characteristics of Conidia from Monosporous Cultures of Penicillium Digitatum and Aspergillus Nidulans Var

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Theses and Dissertations

1969-08-01

Surface characteristics of conidia from monosporous cultures of Penicillium Digitatum and Aspergillus Nidulans Var. Echinulatus

Raymond Kunito Fuji Brigham Young University - Provo

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BYU ScholarsArchive Citation Fuji, Raymond Kunito, "Surface characteristics of conidia from monosporous cultures of Penicillium Digitatum and Aspergillus Nidulans Var. Echinulatus" (1969). Theses and Dissertations. 8055. https://scholarsarchive.byu.edu/etd/8055

This Thesis is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. SURFACE CHARACTERISTICS OF CONIDIA FROM MONOSPOROUS

CULTURES OF PENICILLIUM DIGITATUM AND I

ASPERGILLUS NIDULANS VAR. ECHINULATUS ✓ /) v

A Thesis

Presented to the

Department of Botany

Brigham Young University

In Partial Fulfillment

of the Requirements for the Degree

of Master of Science

Raymond Kunito Fujii

August 1969 ACKNOWLEDGEMENTS

Sincere appreciation is expressed to Dr. Wilford

M. Hess of the Botany Department, Brigham Young University 9 for his encouragement, support, and assistance in the

completion of this investigation and manuscript.,.

Additional thanks are expressed to Dr. Dayna L.

Stocks arid to Dr. Joseph R. Murdock of the Botany

Department, Brigham Young University, and to Dr. David Mo

Donaldson of the Microbiology Department, Brigham Young

University, for their helpful suggestions in writing the thesis; and to Mrs. Connie Swensen for her technical

assistance.

Special appreciation is expressed to my parents,

Mr. and Mrs. Sadayoshi Fujii, for their encouragement and

support.

This research was supported in part by the

National Science Foundation Grant GB 7002.

iii TABLE OF CONTENTS

ACKNOWLEDGEMENTS. • • • • • • • • • • • • • • • • •• iii LIST OF FIGURES •• • • • • • • • • • • • • • • • • • .v-vi INTRODUCTION• • • • • • • • • • • • • • • • • • • • • 1 REVIEW OF LITERATURE• • • • • • • • • • • • • • • • • 4 MATERIALS AND METHODS. • • • • • • • • • • • • • • • • 8 Single Spore Isolation Procedures. • • • • • • 8 Penicillium digitatum. • • • • • • • • • 8 Aspergillus nidulans var. echinulatus •• 10 Culturing Techniques for Freeze-etching •••• 11 f• digitatum ••••••••••••• o 11 A. nidulans var. echinulatus •••••• 11 Freeze-etching Procedures• • • • • • • • • • o 12 Electron Microscopy and Photography •••••• 16

RESULTS• • • • • • • • • • • • • • • • • • • • • • 0 17

P. digitatum Single Spore Isolate 1 ••••• o 17 P. diqitatum Single Spore Isolate 2 •••••• 20 f• digitatum Single Spore Isolate 3 •••••• 25 A. nidulans var. echinulatus Single Spore Isolate 1 •••••••••••••••• 28 A. nidulans var. echinulatus Single Spore Isolate 2 •••••••••••••••• 33 A. nidulans var. echinulatus Single Spore Isolate 3 ••••••••••••••• • 36

DISCUSSION AND CONCLUSIONS• • • • • • • • • • • • • • 43 SUMMARY • • • • • • • • • • • • • • • • • • • • • • • 48

BIBLIOGRAPHY• • • • • • • • • • • • • • • • • • 0 • 0 49

iv LJ:ST OF FIGURES Figure Page lA. E.• digitatum single spore isolation 1, spore 1 • • • • • • • • • • • • • • • • • • 19 1B. E.• digitatum single spore isolation 1, spore 2 • • • • • • • • • • • • • • • • • • 19 1c. E.• digitatum single spore isolation 1, spore 3 • • • • • • • • • • • • • • • • • • 21 1D. E.• digitatum single spore isolation 1, spore 4 • • • • • • • • • • • • • • • • • • 21 2A. P. digitatum single spore isolate 2, -spore 1. • • • • • • • • • • • • • • • • • • • 22

2B 0 E.• digitatum single spore isolate 2, spore 2. • • • • • • • • • • • • • • • • • • • 22" 2c. E.• digitatum single spore isolate 2, spore 3. • • • • • • • • • • • • • • • • • • 0 24 2D. E,. digitatum single spore isolate 2, spore 4. • • • • • • • • • • • • • • • • • • 0 24 3A·. E.• digitatum single spore isolation 3, spore 1 • • • • • • • • • • • • • • • • • • 26 3B., !• digitatum single spore isolation 3, spore 2 • • • • • • • • • • • • • • • • • • 26 3C. E.• digitatum single spore isolation 3, spore 3 • • • • • • • • • • • • • • • • • • 27 3D. E.• digitatum single spore isolation 3, spore 4 • • • • • • • • • • • • • • • • • • 27 4A. -·A· nidulans var. echinulatus as cos pore • • • • 29 4B. !• nidulans var. echinulatus single spore isolation 1, spore 1 • • • • • • • • • • 30 4.C. A• n;i.g~J.an~ var. echinulatus single spore isolation 1, spore 2 • • • • • • • • • • 30

V Figure I?age

4D. nidulans var. echinulatus single ~-spore isolation 1, spore 3 • • • • • • • • • • 32 4E. nidulans var. echinulatus single ~-spore isolation 1, spore 4 • • • • • • • • • • 32 4F. nidulans var. echinulatus single ~-spore isolation 1, spore 5 • • • • • • • • • • 34 SA. nidulans var. echinulatus single ~-spore isolation 2, spore 1 • • • • • • • • • .. 34 SB. !£• nidulans var. echinulatus single spore isolation 2, spore 2 • • • • • • • • • • 35 sc. nidulans var. echinulatus single ~-spore isolation 2, spore 3 • • • • • • • • • • 35 so. nidulans var. echinulatus single ~-spore isolation 2, spore 4 • • • • • • • • • • 37 SE. nidulans var. echinulatus single ~-spore isolation 2, spore 5 • • • • • • • • • • 37 GA. nidulans var. echinulatus single ~-spore isolate 3, spore 1 • • • • • • • • • • • 38 GB. !:.• nidulans var. echinulatus single spore isolate 3, spore 2 • • • • • • • • • • • 38 6C. !• nidulans var. echinulatus single spore isolate 3, spore 3 • • • • • • • • • • • 40 6D. ti.• nidulans var. echinulatus single spore isolate 3, spore 4 • • • • • • • • • • • 40 6E:;. b_. nidulans var. echinulatus single spore isolate 3, spore 5 • • • • • • • • • • • 41 6F. nidulans var. echinulatus single ~-spore isolate 3, spore 6 • • • • • • • • • • • 41

vi INTRODUCTION

Fungal ultrastructure is the topic of many research articles; however, as noted by Hess, Sassen, and Remsen (1968) very few investigators have studied the fine structure of fungal spore surfaces. It was noted by Bracker (1967) that dormant fungal spores are difficult to fix chemically. Consequently, the usual methods employed in electron microscopy investigations cannot be adequately used to investigate fungal spore-wall ultrastructure.

In order to overcome the difficulties encountered by chemical fixatives, Hess, Sassen, and Remsen (1966) employed a relatively new technique developed by Moor, .!£ .sl.• (1961). This technique, called freeze-etching, was reported to give good resolution and contrast; and surface characteristics of Penicillium conidia have been described (Sassen, Remsen, and Hess, 1967; Hess, Sassen and Remsen, 1968). Later work was done with conidia of

several species of Aspergillus. (Hess and Stocks, 1967 9 1969).

Hess, Sassen, and Remsen (1968) investigated the surface characteristics of conidiospores of ten species of Penicillium. In their report it was written that

"the surfaces of the Penicillium conidia studied are

1 2 covered with ordered arrays of 'rodlets' which appear to vary in pattern and distribution from species to species". Variation was also noted within the same culture of a species. Therefore, the authors concluded that within a single culture there possibly exists different strains of a particular species. If different strains exist within a single culture, then heterokaryosis or the parasexual cycle or both may occur. With either of these two processes genetic variation is possible,. thereby giving probable phenotypic variation in the "rodlet" patterns~ Thus, it was concluded by Hess, Sassen, and Remsen (1968) that heterokaryosis and the parasexual cycle or both may have important significance and may cause variation of "rodlet" patterns within the same culture of a partic¥lar species .. of Penicillium.

Classification of species of Penicillium can be done with the light microscope; however, a good method for the classification of Penicillium on the subspecies and strain level does not exist. A correlation between the specific! ty of different "rodlet'' patterns and different strains and subspecies should be possible. The possibility of heterokaryosis and the parasexual cycle must be minimized before any conclusive deducti.on can be made concerning the possible taxonomic significance of the

"rodlet" patterns. The following study was designed to examine the 3 possible taxonomic significance of the spore wall patterns on conidiospores. To lower the probability of heterokaryosis, single spore cultures of Penicillium digitatum were used. To minimize the probability of heterokaryosis and the effect of the parasetual cycle, single ascospore cultures of Aspergillus nidulans var. echinulatus were made. The monosporous cultures of these fungi were frozen-etched and replicas of. conidia were studied in this investigation. LITERATURE REV:IEW,

Because of the rapid development of many basidio- carps and because there are so many fungal depredations 9 the fungi must have been of interest since prehistoric times. Ainsworth (1965) pointed out that men have been interested in fungi throughout written history. These early observations were obviously only macroscopic in nature until the development of the light microscope.

But even with the light microscope, much of the information about microfungi was not elucidated. Moore (1965) reported that, 0 much of fungal morphology lies beyond the limits of resolution of the light microscope". This problem was somewhat eliminated with the development of techniques of electron microscopy as reviewed by Hawker (1965). Fungal ultrastructure has since become of great interest as is exemplified by review atticles on the subject (Hawker, 1965; Bracker, 1967).

One aspect of fungal ul tras,.tructure which only recently became of interest is the fine structure of fungal spores. Because of the relative thickness and impermeability of the intact spore wall to either the fixative or the embedding material, very little could be done on spore ultrastructure using the usual electron microscopy procedures. Ultrastructural studies of fungi

4 5 were done initially by use of KMn0 or fixation. 4 Oso4 During the last five or six years ~ost of the ultrastructure studies of fungi have involved the use of glutaraldehyde for fixation. Even with glutaraldehyde fixation and other fixatives now available such as para- formaldehyde, fungus spores are still generally very difficult or impossible to fix for thin-sectioning studies. Methods other than chemical fixatives and stains haye been used by investigators to examine fungal spore surface structures. Bigelow and Rowley (1968) reported the use of single and two-stage carbon replicas of basidio- spores to investigate several species of basidiomyceteso

Bracker (1967) mentioned the use of alternative replica methods and further indicated the possible usefulness of the scanning-reflection microscope in investigating fine structure of fungal spore surfaces. During recent months several publications have appeared which have demonstrated the use of scanning-reflection microscopy to investigate finer details of spore surfaces (Hawker, 1968; Hawker and

Gooday, 1968; Iizuka, 1968). Works by other investigators

(Hess and Stocks, 1967; Hess, Sassen, Remsen, 1968) have demonstrated that freeze-etching gives better resolution and contrast than other replica techniques.

The method of freeze-etching was first introduced by Steere (1957) and later simplified by Steere (1969). Moor .!!.t .!!• (1961) later improved Steere's original idea 6 by the construction of a complex freezing ultramicrotome in a vacuum evaporator. The value of this modification was delineated by Moor and Mnhlethaler (1963), by their work with yeast cells. Many other investigators have worked to modify the freeze-etching procedure as reviewed by Steere {1969). Later reports by Hess, Sassen, and Remsen (1966,

1968) further illustrated the usefulness of freeze- etching with an ultramicrotome as developed by Moor~ .sl• {1961). The unique fracturing process of this method revealed "rodlet" patterns on the spores of several species of Penicillium.

As was noted earlier the report of these "rotllet".- patterns indicated possible taxonomic significance. ·-,

However, the works of Hess, Sassen, and Remsen (1968) did not include the examination of monosporous cultures of Penicillium conidiospores. Also later investigations of Aspergillus conidiospores did not include the use of monosporous cultures {Hess and Stocks, 1967; 1969).

For reasons indicated above, monosporous cultures of fungal spores must be examined before any taxonomic importance of the "rodlet" patterns can be concluded. To eliminate the effects of heterokaryosis and to minimize the implication of the parasexual cycle, single spore isolations are necessary. In other types of investigations monosporous cultures are commonly used.

Consequently, there are many methods described for iso- 7 lating single cells or spores on culture media. Keitt (1915), LaRue (1920), Hanna (1928), and Dickinson (1933) are a few of the investigators who have introduced various methods of obtaining cultures originating from single spore isolations. MATERIALS:,AND METHODS

The organisms used in this study were Penicillium digitatum Saccardo and Aspergillus nidulans Eidam var. echinulatus Fennell & Raper. Single conidiospores of

~- digitatum and single ascospores off!• nidulans var. echinulatus were used to start monosporous cultures of each fungus. Conidiospores from three monosporous cultures of each fungus were frozen-etched and examined in an electron microscope.

Single Spore Isolation Procedures

A. Penicillium digitatum

l. Cultures.--stock cultures of this fungus were maintained on malt extract agar 1 , and conidiospores were obtained by growing the fungus on malt extract agar at room temperature for one to two weeks.

2. Harvest of Spores.--Conidiospores were harvested by a method similar to that described by Hess, Sassen, and Remsen (1968). A detergent solution of one drop of Teepol 2 to 3 ml. of sterile distilled water was used to

1Difco malt extract agar containing: maltose, Tech- nical 12.75 gm.; Dextrin, Difeo 2.75 gm.; Glycerol 2.35 gm.; Bacto-Peptone 0.78 gm.; and Bacto-Agar .15 gm. in one liter of distilled water. '·

2Teepol is a commercial detergent manufactured by Shell Chemical Corp.

8 9 wet the spores. Approximately 20 ml of this detergent solution was used to cover the agar surface containing conidia. A flamed inoculating needle was used to scrape

the agar surface to detach the conidia from the fungal mycelium. The resulting spore suspension was filtered

through glass wool to separate the hyphal cells from the spores. The filtered solution, now containing mainly

the spores, was centrifuged in the International Clinical Centrifuge at approximately 3000 RPM for 5 to 10 minutes.

3. Washing.--The supernatant was discarded and the spores were resuspended in 10 ml of sterile distilled water. The suspension was again centrifuged at approx.

3000 RPM for 5 to 10 minutes and the supernatant was discarded. This procedure was repeated 2 times. 4. Dilutions.--After the last washing the spores were resuspended in 10 ml,of sterile distilled water, and then were aseptically diluted (by 10-fold dilutions) to 1:100,000. Each dilution mixture was shaken vigorous- ly to separate fused conidiospores. s. Plating.--A 1 ml sample of the 10- 5 dilution was found to be a suitable dilution for isolation of spores and was dispensed onto a thin layer of malt extract agar in a Petri plate. It was evenly dispersed with a sterile inoculating loop.

6. Incubation.--These plates containing the 10 -5 dilutions were incubated approximately 10 hours at room temperature. The incubation period allowed germination to 10 occur; however, this length of time did pot allow the

spores to lose their identity by proliferating hyphae

from the germinated spores.

7. Single Spore ~solation.--Well isolated spores were selected under the low power of the light microscope

and they were marked with a glass needle mounted on an Aloe Scientific Pneumatic Micro-manipulator (De Fonbrune).

The spots that were marked were then cut with a flamed

"microbiscuit" cutter fashioned from a teasing needle and the pieces of cut agar were lifted up and transferred

to other Petri dishes containing malt extract agar. The

Petri dishes which contained the single spore isolates were immediately sealed with Parafilm and they were incubated at room temperature. This method of isolating

a single fungus spore was described by Keitt (1915).

a. Aspergillus nidulans var. echinulatus 1. Cultures.--stock cultures of this fungus were grown and maintained on malt extract agar. Conidiospores and ascospores were obtained by growing the fungus on

malt extract agar at 37°C in an oven for 2 to 3 weeks. 2. Harvesting Spores.--The spores were harvested with a 1eepol detergent solution (see step 2 of procedure

A above). However, before filtering through a glass wool

filter, the cleistothecia were squashed against the Petri dish with a transfer needle to expel the ascospores. The

detergent solution had a reddish tinge from the abundance of ascospores which were expelled from the cleistothecia. 11.

3. Washing.--The spores were washed with distilled water as described in step 3 of procedure A above.

4. Dilution.--The spores were diluted according

to step 4 of procedure A above.

5. Plating.--The plating of the 10- 5 spore dilu-

tions was identical to step 5 of procedure A above. 6. Single Spore Isolation.--The larger ascospores

were not difficult to distinguish from the smaller conid- - iospores and they were readily visible under the low power

objective. Immediately after the spore dilution was

plated and after the water was absorbed into the agar, single ascospores were isolated by the same procedure

listed in step 7 of procedure A above.

Culturing Techniques For Freeze-Etching

A. ~• digitatum. Temperature and Length of Incubation.--Two of the

cultures examined were incubated at room temperature (26°C) for, 2 to 3 months. The third culture was incubated

at room temperature (26°C) for 11 months before the conidiospores were harvested and frozen-etched. The reason

for the differences in incubations times was to determine

whether the age of the colonies affected the ttrodlet"

patterns.

B. !:.• nidulans var. echinulatus Temperature and Length of Incubation.--All three

cultures of~- nidulans studied were incubated at 37°C 12

for approximately 7 months.

Freeze-etching Procedures

Both fungi were treated identically with the Freeze- etching process as follows:

1. Spore harvest.--The conidiospores from each

fungus were harvested in Teepol detergent solution as described in step 2 of procedure A,above.

2. Washing.--The conidiospores were washed in

distilled water (see step 3 of procedure A above).

3. Copper Disc Preparation.--A small sheet of copper was cleaned thoroughly with chloroform to remove any oil. Three mm copper discs were then cut from this sheet with a hole punching tool. These copper discs were scratched with a razor blade to. insure greater adherence of the frozen specimens on the discs. Scratched discs were stored in a clean covered Petri dish.

4. Specimen Freezing.--The washed spores were centrifuged into a pellet and the supernatant was poured off, then the walls of the centrifuge tube dried with

Kimwipes. Using a pipette drawn from pyrex glass tubing,

a drop of water containing a concentration of spores was placed on the prepared 3 mm copper discs. While holding

the copper discs with watchmaker's tweezers, the spore

concentrations on the copper discs were immersed immediately into liquid Freon 22 for 5 to 10 seconds; then

they were transferred to a dewar containing liquid nitrogen. Better results were obtained when specimens were 13 frozen-etched the same day they were frozen. If the

spores were frozen for several days prior to freeze- etchirtg they shattered more easily. s. Freeze-etching Technique. The freeze-etching techniques described below are

similar to those described by Moor and Mnhlethaler (1963). a. Electrodes.--The two carbon rods were sharpened in a special sharpener so that one was pointed while the other was blunt. The blunt rod was attached

to the non-insulated part of the electrode. The pointed rod was attached to the movable part of the electrode so that the spring tension was tight and the point was centered on the blunt surface of the other rod.

The platinum electrode was fashioned by coiling a

0.1 mm dia x 7 cm platinum wire around a small metal rod. This coiled wire of platinum was just large enough to slip snugly on a cylinder constructed on a blunt carbon rod. The resulting carbon rod with the platinum coil was fastened to the non-insulated part of the platinum electrode and centered. A slightly flattened, pointed carbon electrode was centered on the carbon rod containing platinum and tightened so that it would depress the platinum coil and have part of the coil snug around it.

The platinum evaporator was attached to the freeze- etch apparatus so when fired it would shadow the object from a 45° angle. The carbon evaporator was attached 14 directly over the specimen so that its shadow would be perpendicular to the object.

b. Cuttinq.--'fhe frozen specimen on the copper disc was transferred quickly from the liquid nitrogen to the pre-cooled (-100°C), Freon dampened specimen table of the Balzers Freeze-Etching Device. The liquid nitrogen cooled, specimen holder was then screwed onto the stage or table and touched with liquid Freon 22 to remove frost. The bell jar was closed and a rough vacuum was pulled on it. Schick single edge stainless steel razor blades which were previously washed with chloroform were used as chipping kn~ves. This knife was lowered until it touched the object. When the pressure within the jar was .1 mm Hg, the high vacuum was switched on and th~ knife was cooled to approximately -195° with liquid nitrogen. The knife was lowered a quarter of a turn each cut until a smooth, shiny surface was visible on the object. When the vacuum reached approximately 1-2 x 10- 6 mm Hg, the specimen was ready for etching. c. Etching.---The colder knife {-195°C) was placed over the object (-100°C) for 60 seconds to sublime the surface of the object. d. Shadowing.--With the vacuum at 1-2 x 10= 6 mm Hg and the knife moved away to the back, the etched object was shadowed. The platinum electrode was fired first for 4 to 5 seconds and immediately after the carbon was evaporated on the object for 8 to 10 seconds. 15

e. Loosening the Replica.--Air was allowed to enter the jar. With the bell jar open and the specimen holder removed, the object was picked up with watchmaker's tweezers and held until thawed. Then the object was immersed into distilled water at an angle which allowed the replica to float on the surface of the water. 6. Treatment of Replicas.--The replicas were transferred on films of water with a platinum loop to 70% sulfuric acid for 1 hour. The replicas were rinsed I, three times with distilled water, then they were transferred to 5.25% sodium hypochlorite (Clorox) for one hour. After the clorox treatment the replicas were transferred to a dish of distilled water for 5 minutes.

After an additional distilled water rinse (approximately one hour) the replicas were picked up on plastic coated microscope grids. Throughout this process the replicas were kept floating. 7. Formvar Grids.--Formvar coated grids were used to pick replicas up. The Formvar solution was made by dissolving approximately 0.3 gm Formvar in 100 ml of chloroform. Clean microscope slides were used to make the films which were approximately 80 mp thick (silvery) color). Grids were cleaned prior to plastic coating by sonicating in chloroform for 3-5 minutes, and then they were air-dried. These grids were then placed on the films. The films along with the coated grids were picked up on clean filter paper and air-dried before used. 16

Electron Microscopy and Photography The replicas were examined in Hitachi electron micro-

scopes. The models used were the HS-7 and the HU-llE.

Pictures were taken at magnifications of 15 9 500 to 25 9 800 9 on Kodak contrast grade projector slide plates. These

plates were developed in Kodak D-11 developer diluted lg:l with water. Prints were made on Kodak single weight Kodabromide

paper and developed in Kodak Dektol developer in a li;l

dilution with water. The micrographs found in this thesis are of conidio-

spores unless otherwise specified. RESUL:l'S,

In both Penicillium digitatum and Aspergillus nidulans var. echinulatus the "rodlet" pattern sizes were, in general, in agreement with those reported by previous investigators (Hess, Sassen, and Remsen, 1968;

Hess and Stocks, 1969). Hess, Sassen, and Remsen (1968) reported the ttrodlet" groupings of!:• digitatum to vary

0 from a single ttrodlet" to.2,000 A.in width. The average lengths of the rodlets were reported as averaging 2,500 A• in length. Hess and Stocks (1969) reported the ttrodlet" groupings of!• nidulans var. echinulatus to vary from

100 ( a single "rodlet") to 2,300 A:,• in width with indi- vidual ttrodlets" averaging 2,100 A• in length.

In the following descriptions "rodlets" will be discussed only when length or width varies considerably from the average given above, and the spore replicas within each isolate have been selected as the most variable in surface morphology.

g. digitatum Single Spore Isolate l Figure 1-A--The characteristic interlacing pattern of the "rodlets" is slight with most of the groupings of "rodlets" near parallel to each other. The underlying surface of the spore is devoid of "rodlets'' in places.

17 18 The irregularly raised areas are smaller and scattered sparsely when compared with figure 1-B and 1-D. The raised areas on the surface are primarily scattered and have the appearance of isolated dome-shaped projections with an occasional ridge-like projection. These raised

0 areas average 2,100 A, in diameter, at the base. The

0 0 "ridges" average 3,700 A by 1,000 A. In comparison with conidia on figures 1-B to 1-D, the conidium in figure 1-A is relatively smooth with very few raised areas. Figure 1-B,..-The interlacing patterns of the "rodlets" are bolder than the "rodlet" patterns of figure 1-A. The raised areas are more numerous than the conidium of fig. 1-A• however, they are not as prominent as figures 1-C and 1-D. Whole groups of "rodlets" are slightly raised giving a uniform rough surface of "ridges"~ Under- lying spore surface areas which are devoid of "rodlets 0 • 0 are present. The "ridgesn average 4,400 A by 1,400 A in size. Figure 1-C-.,.."Rodlet" patterns are characteristically interlaced. The large groupings of "rodlet" patterns are interestingly raised into interconnecting 0 ridges" with

0 average widths of 1,100 A over the entire spore exterior. Beneath the 0 ridges" are areas devoid of "rodlets 0 or when the "rodlets" are present they are in small groupings of one to five. The lengths of these "rodlets" are shorter

0 (average 1,200 A) and less prominent than those on the "ridges". 19

Figures lA-1B. �- digitaturn single spore isolate 1. Fig. lA (upper) spore 1. x 35,000. Fig. 1B (lower) spore 2. x 35,000. 20 Figure 1-D--Large groups of "rodlets" (average width

0 of 2,200 A) are prominently interlaced over the spore surface. Underlying areas devoid of "rodlets 0 appear minute or absent. The surface is covered with prominent dome- like projections averaging 3,850 A• in diameter at the baseso The surface is considerably more irregular and lumpy when compared with the previous micrographs of spores from single spore isolate l (figures 1-A to 1-C). Summary.--The surface of the conidium from isolate 1 of E.• digitatum showed differences in "rodlet" lengths as well as overall surface configuration. Figure 1-A illustrates a comparatively smooth surface with "rodlets" which are considerably longer than usual. The interlacing of "rodlet" groupings as well as the raised areas are very prominent in fig. 1-D. The sequence of micrographs from fig. 1-A to fig. l-D9illustrates the variation ...,,of the roughness of the spore surfaces.

!• digitatum Single Spore Isolate 2 Figure 2-~ .....The interlacing patterns of the "rodlets" are slightly raised. The spore surface is relatively smooth. Some of the."rodlets" are longer (up to S,000

0 A in length) than those reported for the species. Small underlying surf aces devoid of ttrodlets'' are present but sparse. Part of the spore wall has been chipped away exposing the inner layer of the spore wall and the invaginated membrane. Figure 2-B--The surface of this conidium is 21

Figures lC-lD. f• digitatum single spore isolate 1. Fig. lC «upper). spore 3. x 35,000. Fig •. lD (lower) spore 4. x 35,000. 22

Figures 2A-2B. �- digitatum single spore isolate 2. Fig. 2A (upper) spore 1. x 35,000. Fig. 2B(lower) spore 2. x 35,000. 23

covered with an ordered array of 0 rodlet" patterns and

ttrodlet" bundles are interlaced over the entire spore

surface. Again areas without "rodlets" are present. The

projections gradually rises to form dome-shaped protuber- ance and "ridges" giving a slightly rough appearance to

the spore. These raised or dome-like areas average 0 • 2,200 A in diameter and the ridges average 3,750 A by

0 1,300 A. Figure 2-C--Groupings of "rodlets" are raised to

form "ridges" which interconnect over the entire spore

0 0 surface. These "ridges" averaging 3,840 A by 1,440 A~ cause the spore to be rougher in appearance than spores

of either fig. 2-A or fig. 2-B. A remnant of a membrane-

like structure that originally covered the entire spore and masked the ttrodletn pattern is present and visible

(lower left). The fracturing process used with freeze-

etching usually removes this membrane.

Figure 2-D-The "rodlet" patterns are very similar

to the "rodlet" patterns of the conidium in fig. 2-C. They are raised to form interconnecting "ridges". How- o ever, the ridges (averaging 1,625 A in width) are con-

siderably more prominent than those shown in fig. 2-C. Areas without "rodlets" are present •.

Summaryr-4'he sequence of micrographs as seen in

figures 2-A to 2-D illustrates the great variation of spore appearance of spores from single spore isolate 2. 24

Figures 2C-2D. �. digitatum single spore isolate 2. Fig. 2C (upper} spore 3. x 35,000. Fig. 2D (lower} spore 4. x 35,000. 25

!• digitatum Single Spore Isolate 3 Figure 3-A--The "rodlet" patterns are character- ,istically interlaced over the entire surface. 'I'he sizes of the "rodlets" comply with the sizes reported for the

0 0 1 species ( varying between 1,000 A and 4,000 A) • The raised areas are relatively large ( average width is 2, 600:, 0 A) and several "rodlet" groupings are situated on each , raised area. The depressions between the ridges are

0 sharp but very narrow. They average 330 A in width. Again the remnant of the surface membrane str~cture is visible as shown in fig. 2-C (lower right). Figure 3-B..,.-Large groupings of "rodlets" (approxi-

0 mately 2,600 A) are present over a large portion of the

0 conidium. Each of these (2600 A) rodlet groups are slightly raised giving a rolling hill appearance to the spore surface. Figure 3-C--Interlacing ttrodlet" patterns vary 0 considerably in size over the entire spore surface (200 A

0 to 3,000 A. in width). Each of these "rodlet" groupings are raised to give the spore a rough appearance. The variations in the sizes of the raised areas are great 0 0 ( averaging 800 A to 3,000 J.ILin width). The appearance of this conidium is rougher than the conidium in fig. 3-B. Figure 3-D--The "rodlet" patterns are interlaced with larger groupings of "rodlets" considerably raised to form prominent, interconnecting "ridges". The ridges are much more prominent than on other conidia of spore 26

Figures 3A-3B. �. digitaturn single spore isolate 3. Fig. 3A (upper) spore 1. x 35,000. Fig. 3B (lower) spore 2. x 35,000. 27

Figures 3C-3D. �- digitatum single spore isolate 3. Fig. 3C (upper) spore 3. x 35,000 o Fig. 3D (lower) spore 4. x 35,000. 28 0 0 isolate 3. These ridges average 8,200 A by 2,200 A.

Summary.--Each of the conidia in figures 3-A to 3-D have characteristic interlacing "rodlet'' patterns; however, there is a considerable amount of variation in

the roughness of the spore wall surfaces from spore to

spore.

Aspergillus nidulans var. echinulatus

Single Spore Isolate 1 Figure 4-A--Figure 4-A is a micrograph of an ascorm

spore of~. nidulans var. echinulatus showing the characteristic spine-like projections. The presence of these echinulations or projections on the ascospore resulted

in the classification of this variety of Aspergillus as

echinulatus. There are no "rodlet" patterns on the ascospore, which can be easily distinguished from the

conidiospores.

Figure 4-B--The groupings of "rodlets" vary from single isolated "rodletsn to groups of "rodlets'' averaging

0 400 A in width. The lengths of the ttrodlets" vary from

0 0 200 A to 1,600 A. Consequently, there are numerous short, small groupings of "rodlets" interlacing over the entire

spore surface. The spore surface is smooth with no prominent raised areas.

Figure 4-C--The "rodlet" patterns are character-

istic and similar to the other conidia of this single spore isolate. No prominent "ridges" are present; however, numerous dome-like projections are scattered 29

Figure 4A. �- nidulans var. echinulatus ascospore. X 35,000. 30

Figures 4B-4C. 1• nidulans var. echinulatus single spore isolate 1. Fig. 4B (upper) spore 1. x 35,000. Fig. 4C. (lower) spore 2. x 35,000. 31 on the surface of the spore. These projections average

0 1,100 A in diameter at the base.

F-igure 4-D--Interlacing "rodlet" patterns are characteristic for this single spore isolate and the underlying spore surface is visible in some places. Raised areas are visibly scattered over the spore surface as

0 isolated cones averaging 1,200 A.at the base to small

0 0 ridges averaging 3,900 A by 725 A.

Figure 4-E'--The interlacing "rodlet" patterns are normal in appearance when compared with other!• nidulans var. echinulatus conidiospores. The overall surface is much rougher in appearance than the conidia shown in figures 4-B to 4-D. "Ridges" vary from approximately

0 0 0 770 A in width (narrow "ridges") to 10,000 .If, by 1900 A..

(larger raised areas). Isolated dome-like projections of

0 approximately l,400 A in diameter are also scattered on the spore surface.

Figure 4-Fw-"Rodlets" are characteristical~y grouped and interlaced on the entire spore surface. Raised area• are very prominent with dom~-like projec- o tions (averaging 2,600 A in diameter at the base), which are considerably larger than those found in figures 4-C to 4-E:. The overall spore surface is highly irregular and very rough in appearance.

Summary.-- 11Rodlet" patterns of spores from this isolate are relatively consistent from spore to spore, however, the overall surface of each spore varies con- 32

Figures 4D-4E� �. nidulans var. echinulatus single spore isolate 1. Fig. 40 (upper) spore 3. x 35 9 000� Fig. 4E (lower) spore 4. x 35,000. 33 siderably from smooth to extremely rough.

!• nitjulans var. echinulatus Single Spore Isolate 2

Figure 5-A--The "rodlet" patterns are characteristic of -A. nidulans var. echinulatus. The surface is relatively smooth with an occasional dome-like projection

0 of approximately 1 1 500 ~ Note that part of the spore wall has been fractured away exposing the characteristic invaginated plasma membrane (lower right) with particles

0 which average 100 A\in diameter.

Figure 5-B--Small groups of 11rodlets" are interlaced over the entire spore surface. Raised areas appear as dome-like projections with some fusing to form "rid- o 11 ges • The raised dome-shape areas average 2 1 000 A in 0 diameter at the base, while the "ridges" average 4,000 A,

0 by 900 A,.

Figure 5--C..--No major differences occur in the

"rodlet" patterns from previous figures (5-A and 5-B) • .. Ridges" are distributed over the spore surface and they

appear as though they have been contracted from desicca-

0 tion. Numerous 1,100 A', cone projections are visible and

give the spore wall a relatively rough texture. Figure 5-D--Bands of ''rodlet" groupings appear to vary in width from a single rodlet to 1,400 A~• The groups of rodlets appear to be slightly larger than has been described for the species. The surface is better described as having shallow depressions distributed over 34

Figure 4F (upper). �- nidulans var. echinulatus single spore isolate 1, spore s. x 35 7 000. Fig e SK (lower) A. nidulans var. echinulatus single spore isolate 2, spore 1. x 35,000. 35

Figures 5B-5C. �- nidulans var. echinulatus single spore isolate 2. Fig. SB (upper) spore 2. x 35,000. Fig. SC (lower) spore 3. x 35,000. 36

the spore surface rather than ridges. These depressions

0 average 300 A in width.

Figure-5-E--The interlacing groups of "rodlets"

are raised and depressed over the entire spore surface.

0 The surface is very irregular with 800 A projections, and 0 0 s,ooo A by 1,800 A ridges. Crevice-like depression

0 0 measure approximately 3500 A by 400 A. Summary.--The "rodlet" patterns in and of themselves are relatively stable in appearance from one conidium to

the next. The surface, on the other hand, varies con-

siderably from smooth to rough.

!• nidulans var. echinulatus Single Spore Isolate 3 Figure 6-A:;...-The "rodlettt groupings are interlaced with some groups of "rodlets" appearing is<;>lated. The

surface of the spore is re,latively smooth with no notice= able raised areas.

Figure 6-B...... S:ome of the "rodlet" groupings are slightly longer than has been reported for the species.

0 They are approximately 5500 A in length. The spore surfaces appear smooth with large dome-like projections

0 (average basal diameter of 1,600 A) protruding from the

spore surface.

Figure 6-C~-Extensive interlacing of "rodlet" patterns with visible underlying areas which are ' devoid of "rodlets". The relief appears as tiny projections

0 (average diameter 1000 A0 scattered over the entire spore 37

Figures SD-SE. �- nidulans var. echinulatus single spore isolate 2. Fig. 5D (upper) spore 4. x 35 9 0000 Fig. SE (lower) spore 5. x 35,000. 38

Figures 6A-6B. �. nidulans var. echinulatus single spore isolate 3. Fig. 6A (upper) spore 1. x 35�0000 Fig. 6B (lower) spore 2. x 35 9 000. 39

surface.

Figure 6-D--Interlacing "rodlet" patterns are

present over the entire spore surface. Underlying areas

without "rodlets" are scattered on the spore surface.

Cone shape projections are distributed at random and they O 0 vary from 300 A to 2,500 A. Figure 6-E--The conidium in fig. 6-E clearly

exhibits the outer membrane-like covering characteristic of the conidiospores investigated in this study. This membrane-like structure is usually removed during the

fracturing process of freeze-etching.

0 On this spore where rodlet" patterns are exposed 9 they comply with the descriptions reported for~• nidulans var, echinulatus. Spore surfaces contain ridges (averag- o 0 ing 800 A by 3 9 100 A) and isolated cone protrusions. Figure 6-F--The spore surface is relatively smooth with a portion of the wall fractured away during the

0 chipping process of freeze-etching. Note the 100 A

particles characteristic of plasma membranes. Also of

interest is that no invaginations occur on the membrane, which is a characteristic of dormant spore membranes as

is shown,. in fig. 5-A. The spore wall also appears

smoother in fig. 6-F than in fig. 5-A. The membrane in

fig. 6-F has no invaginations which implies that the spore

is swollen and in comparison to the conidium in fig. S-A·1>

the spore wall also appears expanded and consequently it is somewhat smoother. 40

Figures 6C-6D. �- nidulans vare echinulatus single spore isolate 3. Fig. 6C {upper) spore 3 o x 35 p 000 e Fig. 6D (lower) spore 4. x 35 9 000. 41

Figures 6E-6F. �- nidulans var. echinulatus single spore isolate 3 o Fig o 6E (upper) spore 5. x 35 p OOO. Fig. 6F (lower) spore 6 e x 35 p 000 o 42

Summarv.--"Rodlet" patterns do not vary considerably. Spore surfaces do vary from smooth to rough as

indicated in other single spore isola.tes of h,.. nidulans var. echinulatus. Of interest is the micrograph showing the swollen conidium which seems to indicate that

swelling of conidia may have some influence on the roughness of the spore.

• DJ:SCUSSION AND CONCLUSIDNS:

The results of this investigation reveal that the

"rodlet" patterns and surface configurations of conidiospores from monosporous cultures of~• digitatum and~• nidulans var. echinulatus vary in appearance. In general, the spore wall patterns of both of these fungi were highly variable. The "rodlet" patterns were slightly more variable on conidia of single spore isolates of E.• digitatum than they were on spores of single spore isolates of~- nidulans var. echinulatus. However, the differences in the variation of the "rodletn patterns between these two fungi are not significantly different from those reported in previous investigations (Hess, Sassen, and Remsen, 1968; Hess and Stocks, 1969). Consequently, it is not possible to reduce the amount of variation of the nrodlet" patterns significantly by use of single conidium isolations or single ascospore isolations.

Since the environment appears to influence the spore wall configuration and because of the possible influences of mutation and cytoplasmic inheritan~e, the spore wall patterns are not significantly stable to be of taxonomic importance on the strain and subspecies level. The possibility of a relationship between mutation or

43 44

extranuclear influences or both and spore wall patterns warrants further investigation.

A conidium may contain more than one nucleus.

Ascospores on the other hand, are sexual spores resulting directly from meiosis and have single haploid nuclei.

Therefore, genetically, cultures from single ascospore isolations should be less variable. However, when

considering the spore wall patterns of!• nidulans var.

echinulatus, there is a considerable amount of variation. The "rodlet" patterns appear to be slightly more stable on conidia of -A. nidulans var. echinulatus than on conidia of.!:• digitatum; which suggests that "rodlet" patterns of conidia which come from single ascospore cultures are not more stable than conidia from mass spore cultures.

The amount of variation found on spore wall patterns of single spore cultures of both~. digitatum

and!• nidulans var. echinulatus can possibly be explained. The shadowing angle may vary the size of the relief structures on the spore surfaces. Hess, Sassenp and Remsen

(1968) stated that, "variations between spore surface patterns may not be as great as they appear because the platinum shadow angle, used in freeze-etch procedures 9 may exaggerate or de-emphasize surface configurations."

Examination of P. digitatum does not rule out the possibility of genetic variation expressing itself as a phenotypic variation in the spore wall patterns. Hetero- karyotic conidiospores, having nuclei of different geno- 45

types could have been used as the single spore isolates

with!:• digitatum. According to Pontecorvo (1956) heterokaryons may undergo fusion of the nuclei; they

may then undergo mitotic crossing over and finally they may undergo haploidization to complete the parasexual

cycle. The completion of the parasexual cycle allows for genotype variation and thereby possible phenotype

variation.

With the use of the single ascospore isolation of

~- nidulans var. echinulatus, heterokaryosis is essen= tially eliminated. Since heterokaryosis is a necessary part of the parasexual cycle (Pontecorvo, 1956), and

since~. nidulans is a homothallic sexually reproducing fungus, the parasexual cycle is of less importance in

this species (Fincham and Day, 1956). Despite the use

of single ascospore cultures, there is still considerable variation in the spore wall pattern of !• ·,nidulans var. echinulatus. Because of the great variation that exists

in spores from single ascospore cultures of this fungus~

the implication is that environmental factors may contri- bute greatly to the variation of the spore wall patterns.

The micrographs of figure 6-F and 5-A illustrate clearly a possible influence of environmental factors

on the spore wall pattern. In figure 6-F the plasma

membrane is stretched and devoid of any invaginations

characteristic of dormant fungal spores. The conidium

is probably swollen with water and possibly shows a pre- 46 germinated state. The wall of this spore is relatively smooth and the conclusion is that just prior to germination the spore swells with the accumulation of water causing both the plasma membrane and the spore wall to expand and appear somewhat smoother than the spore shown in figure 5-A. Dormant conidia of Penicillia {Sassen 9 Remsen, and Hess 1967) and conidia of other species of

Penicillia and Aspergilli {unpublished results 9 w. Me Hess and o. L. Stocks, Brigham Young University) characteristically have invaginations in the plasma membrane.

Figure 5-A.shows a dormant spore with the invaginated membrane and a relatively rough spore wall configuratione

This phenomenon indicates that conidia which are swollen may have a different surface appearance than conidia which are not swollen.

Although the results of this investigation indicate that the environment may greatly influence the vari= ation in "rodlet" patterns and spore wall configurations 9 the role of direct genetic influence cannot be completely ruled out. Alexopoulos (1964) stated that besides germination of heterokaryotic spores or anastomoses of different homokaryons, heterokaryons can originate from muta= tions. Since we had no way to detect mutations during this investigation, mutations cannot be ruled out as a possible mechan1sm influencing the spore wall pattern.

Furthermore, the possibility of a relationship between spore wall configuration and extra-nuclear inheritance 47 was not contended with during this study.

The membrane-like structure covering the "rodlet" patterns of the spore shown in figure 6-E characteristic~ ally covers conidia of both Aspergillus and Penicillium species. The presence of this membrane-like covering obviously contributes to the poor resolution of "rodlet" patterns when other replica techniques are used. In freeze-etching the fracturing process apparently removes this membrane-like structure and clearly exposes the ,"rodlets" to shadowing with platinum and carbon. The presence of this membrane often greatly hinders conidia surface studie&. This investigation may have contributed more information if both conidia and ascospores of~. nidulans var. echinulatus would have been used for single spore isolation studies of conidia surface configurations. This approach would provide a means to determine whether conidia from ascospore isolates differed significantly from conidia from conidial isolates. The value of this approach did not become evident until this investigation was almost complete. In addition, although every precaution possible was taken during the single spore isolation process 7 the possibility still exists that some of the single spore isolates became contaminated. Three cultures from single spore isolations were used for each of the fungi studied and it is very unlikely that all single spore isolations were contaminated. SUMMARY

The results of this investigation indicate that

the spore wall patterns of conidiospores from monosporous

cultures of~- digitatum and~- nidulans var. echinulatus vary considerably. Because of the great variation of the

spore wall configurations, no taxonomic significance on the strain and subspecies level can be related to the spore wall patterns.

Environmental factors and possible genetic influ-

ences (cytoplasmic inheritance and mutations) may con- tribute greatly to the variation of the spore wall

patterns.

Freeze-etching is probably the best procedure to 'use in investigating spore surfaces. The reason is because freeze-etching removes a membrane-like covering

found on conidia from both Penicillia and Aspergilli.

48 BIBLIOGRAPHY

Ainsworth, G. c. 1965. Historical introduction to mycology. In: The Fungi. Vol. I (G. c. Ainsworth and A. s. Sussman, editors), pp. 3-20. Academic Press, New York. Alexopoulos, c. J. 1964. Introductory mycology .. John Wiley & Sons, Inc., New York. pp. 3-613 ..

Bigelow, H. E• and J. R. Rowley. 1968 .. Sbrface replicas of the spores of fleshy fungi. Mycologia 60; 869-887. Bracker, c. E. 1967. Ultrastructure of fungi. Ann. Rev. of Phytopathology 5: 343-374. Dickinson, s. 1933. The technique of isolation in microbiology. Phytopathology 23: 357-367. Fincham, J. R. s .• and P. R. Day. 1965. Fungal genetics .. Vol. 4 of Botanical Monographs (w. o. James F • Re s:.• editor), PP• 1-326. Blackwell Scienti- fic Publications, Oxford.

Hawker, Lilian E. 1965. Fine structure of fungi as revealed by electron microscopy. Biol. Rev. 40:. 52-92.

Hawker, Lilian E. 1968. Wall ornamentation of ascospores of species of Elaphomyces as shown by scanning electron microscopy. Trans. Brit. Mycol. Soc. 51: 493-498. Hawker, Lilian E. and Margaret A. Gooday. 1968. Development of the zygospore wall Rhizopus sexualis (Smith) Callen. J. Gen. Microbiol. 54: 13-20. Hanna, w. F. 1928. A simple apparatus for isolating single spores. Phytopathology 18:. 1017-1021. Hess, w. M., M. M.A. Sassen, and c. c. Remsen. 1966. Surface str~cture of frozen-etched Penicillium conidiospores. Die Naturwissenschaften 52i708.

49 50 Hess, w. M., M. M.A. Sassen, and c. c. Remsen. 1968. Surface characteristics of Penicillium conidia. Mycologia 60: 290-303. Hess, w. M. and D. L. Stocks. 1967. Surface structure and organelle characterization of frozen-etched Aspergillus conidiospores. Amer. J. Bot. 54~: 637-638 (Abstr.). Hess, w. M. and D. L. Stocks. 1969. Surface characteristics of Aspergillus conidia. Mycologia 61 ::: Ctn Press). Iizuka, H. 1968. Use of a scanning electron microscope for the examination of Aspergilli. J. Electron Microscopy 17: 264 ( abstract BII 23). Keitt, G. w. 1915. Simple technique for isolating single spore strains of certain, types of fungi. · Phytopathology 5: 266-269. LaRue, c. D. 1920. Isolating single spores. Bot. Gaz. 70: 319-320. Moor, H. and K. Mllhlethaler. 1963. Fine structure of frozen-etched yeast cells~ J. Cell Biol. 17i 609-62B. Moor, H., K. Mnhlethaler, H. Waldner, and A. Frey- Wyssling. 1961. A new freezing ultramicrotome. J. Biophys. and Biochem. Cytol. 10: 1-130 Moore, R. T. 1965. The ultrastructure of fungal cells. In: The Fungi. Vol. I (G. C. Ainsworth and A. S. Sussman, editors), pp. 95-118. Academic Press, New York. Pontecorvo, G. 1956. The parasexual cycle in fungi. Ann. Rev. Microbiol. 10: 393-400. I Sassen, M. M.A., c. c. Remsen, and w. M. Hess. 1967. Fine structure of Penicillium megasporum conidiospores. Protoplasma 64: 75-88. St.eere, R. L. 1957. Electron microscopy of structural detail in frozen biological specimens. J. of Biophy. and Biochem. Cytol. 3: 45-60. Steere, R. L. 1969. Freeze-etching simplified. Cryobiology 5: 306-323. ABSTRACT

The purpose of this investigation was to eval- uate the possible taxonomic significance of the "rodlet" patterns and spore wall configurations of conidia from monosporous cultures of Penicillium digitatum Saccardo and Aspergillus nidulans Eidam var. echinulatus Fennell

& Raper. Freeze-etching the conidiospores from single spore isolates of these fungi revealed that spore wall patterns and configurations vary much too greatly to be of taxonomic importance on the strain and subspecies level. Environmental factors and genetic influences are discussed as possible mechanisms causing the variations of the spore wall configurations. A membrane-like structure that covers the "rodlet" patterns and which hinders spore surface investigations of conidia of these fungi is discussedo