THE AXKNIC CULTURE OF WHEAT AND FLAX RUST FUNGI
by
AMETAVA BOSE
3.Sc.(Agr.), Bihar Agricultural College M.Sc.(Agr.), Bhagalpur University M.Sc., University of British Columbia
A Thesis Submitted in Partial Fulfilment
of the Requirements for the Degree of
DOCTOR OF PHILOSOPHY
in the
Department of Plant Science
We accept this thesis as conforming to the
required standard
THE UNIVERSITY OF BRITISH COLUMBIA
October, 1973 In presenting this thesis in partial fulfilment of the re• quirements for an advanced degree at the University of
British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Depart• ment or by his representatives. It is understood that copy• ing or publication of this thesis for financial gain shall not be allowed without my written permission.
Department of Plant Science, -University of British Columbia, Vancouver 8, British Columbia.
1 Date im-M- ^ ABSTRACT
Rust fungi belonging to the order Uredinales have usually been considered the classical examples of obligate parasites amongst plant pathogenic fungi.
The development of our knowledge of the metabolism, nutrition and physiology of the rust fungi has been restricted because of our inability to grow these fungi in axenic culture.
In the past the physiological and biochemical investigations on rust fungi per se have been limited to studies of the ger• mination and development of germ tubes.
The cultivation in vitro of Puccinia graminis tritici
(Erikss. and .Henri. ) , Australian race ANZ 126-6,7, by Williams et al. (1966, 1967), together with the report of Turel (1969)
on the axenic culture of Melampsora lini (Ehrenb) Lev, race 3
promoted research on culturing rust fungi on artificial medium.
An artificial medium containing 3% glucose, Czapek's
mineral salts, 0.1% Evan's peptone, plus defatted BSA support•
ed growth and sporulation of Puccinia graminis tritici race
ANZ 126-6,7. Typical pigmented uredospores and teliospores
were formed after 6-3 weeks growth. The uredospores were
capable of infecting the mesophyll of wheat leaves exposed
by stripping back the lower epidermis. Scanning electron
microscopy revealed the presence of a coating of unknown chem•
ical composition around the uredospores developed in vitro and
not observed on uredospores grown on wheat leaves. iv
Two different strains of Melampsora lini (Ehrenb)
Lev were grown on solid media containing I+% sucrose, modified
Knop's tissue culture macronutrients, Berthelot's micronut-
rients, yeast extract and peptone. The mycelium was generally
binucleate. Spore-like structures were recorded in the stroma
which resembled uredospores and teliospores. Addition of 1%
defatted BSA to the medium described above greatly increased
the frequency of establishment of flax rust colonies. A de•
fined liquid medium, containing Czapek's mineralsy Ca++, glu•
cose, aspartic acid, glutathione and cysteine, and inoculated
with uncontaminated uredospores, supported good vegetative
growth and sporulation of wheat stem rust (Puccinia graminis
f. sp. tritici race ANZ 126-6,7). Of six North American races
of wheat stem rust fungus tested, only three grew vegetatively
on artificial medium.
Finally a chemically defined liquid medium contain•
ing sucrose, Knop's mineral salts, micronutrients, aspartic
(or glutamic) acid and cysteine supported the growth of vege•
tative colonies of Melampsora lini race 3 from uncontaminated
uredospores In a highly reproducible manner. The formation of
uredospores and teliospores of these colonies in the liquid
medium was controlled by the level of Ca++ (as CatNO-^)^ and
the number of colonies per flask. With 60-70 colonies per
flask, uredospore formation occurred on 60 to 70% of the
colonies at a Ca++ level 8.5 mM. A decrease in the Ca++
level to 4.5 mM or colony frequency to 10 per flask resulted V
in only infrequent sporulation. The uredospores produced in vitro infected intact cotyledons In a normal manner. This result with flax rust represents a substantial advance in our ability to control the growth of this important 'obligate* parasite In axenic culture. vi
TABLE OF CONTENTS Page
FRONTISPIECE Xiv ABSTRACT iii
• TABLE OF CONTENTS vi
LIST OF TABLES ix
LIST OF FIGURES x
ACKNOWLEDGEMENTS xiii
GENERAL INTRODUCTION 1
REFERENCES 16
CHAPTER I - Sporuiation and Pathogenicity of an Australian Isolate of-Wheat Rust grown in vitro.
ABSTRACT..; 20
INTRODUCTION 21
MATERIALS AND METHODS 22
RESULTS 24
1. Behaviour of Germinating Uredospores on Solid Medium 21+
2. Development of Colonies on Liquid Medium 24
3. Pathogenicity of Uredospores produced in Axenic Culture 30 4. Scanning Electron Microscopy,... 30 Page
.CHAPTER II. In vitro Culture of the Flax Rust, Melampsora lini.
ABSTRACT 36
INTRODUCTION 36
MATERIALS AND METHODS 37
1. Melampsora lini, Strain 1 37
2. Melampsora lini, Strain 2.... .- 40
3. Cytological Examination of the Cultures 41
RESULTS 41
1 • Melampsora lini , Strain 1. .. 41
2. Melampsora lini, Strain 2 43
3. Cytological Studies with Melampsora lini 1+6
DISCUSSION 51
REFERENCES.' 59
CHAPTER III. In vitro Growth of Wheat and Flax Rust Fungi on Chemically Defined Media.
ABSTRACT 60
INTRODUCTION •. . . 61
MATERIALS AND METHODS 68
1. Production of Uncontaminated Uredospores 68
2. Culture Media.. ' 69
3. Sources of Organic Nitrogen and Sulphur 70
4. Inoculation of Media 71
5. Assessment of Growth.. 72
RESULTS 72
Wheat Rust 72
1. Trials with Solid Media 72 VXll
Page
2. Trials with Liquid Media 73
A. Growth of races 15B4, 3#, 32-113 and ANZ 126-6,7 73 B. Effect of cAMP on growth of ANZ 126-6,7 73 C. Effect of aspartic acid, cysteine, gluta• thione and calcium on the growth of
ANZ 126-6,7 74
Flax Rust 79
1. Trials with Solid Media., 79
A. Effect of BSA 79 B. Reinfection of host using vegetative colonies grown on solid medium 80 2. Trials with Liquid Media 8l
A. Preliminary trials and effect of BSA 81 B. Effects of mineral nutrients 85 C. Effects of aspartic acid, cysteine, gluta• thione and calcium 88 D. -Reinfection of flax using uredospores grown in vitro 91 LIST OF TABLES
PAGE TABLE
I Growth of obligate parasites in host tissue 5 II In vitro development of rust fungi from germinating uredospores 8 III-I Effect of cyclic AMP and theophylline on growth of Puccinia graminis tritici (race ANZ 126-6,7) 77 III-II Effect of amino acid, glutathione and Ca(N0^)2 on the growth of Puccinia graminis tritici (race ANZ 126-6,7) 78 III-III Effect of different organic nitrogen sources on the growth of Melampsora lini (race 3 ) $k III-IV Growth and development of Melampsora lini (race 3) on completely defined liquid media... X
TiTS-T OF FIGURES
PAGE FIGURE
1-1 Uredospores with burst germ tube on basic medium 26 1-2 Two germinating uredospores and their germ tubes have crossed each other 26 1-3 Two nuclei in cytoplasm extruded from burst germ tube 26
1-4 Germ tubes originating from two uredospores showing anastomosis 26
1-5 Vegetative juvenile colonies and sporula- ting colonies developed on liquid medium 26 1-6 Developing uredospores in colony grown on 28 liquid medium
1-7 Developing teliospores and maturing uredo• spores from colony grown on liquid medium 28
1-8 Fully developed two-celled teliospores....,,... 28
1-9 Thick mat of hyphae at periphery of colony 28 grown on liquid medium,
1-10 Pustules developed on primary leaf of Little Club wheat as a result of inoculation with uredospores grown in vitro 28
1-11 Scanning electron micrographs of mature uredospores grown on intact wheat leaves 32
1-12 Scanning electron micrograph of mature uredospores grown in vitro. . '. 32
1-13 Scanning electron micrograph of spines on surface of uredospores grown on intact wheat leaf 32
1-14 Scanning electron micrograph of spines on surface of uredospores grown on basic medium... 32
1-15 Scanning electron micrograph of developing spines on surface of uredospores grown on intact wheat leaf 32
1-16 Scanning electron micrograph of a teliospore grown on basic medium 32 xi PAGE FIGURE
II-l A 12-day-old colony of strain 1(fourth
transfer)..,, 45
II-2 Same colony viewed from below... 45
II-3 An 8-week-old colony of strain 2 grown from aseptic uredospores 4$ II-4 An"8-week-old colony of strain.2 of small size consisting entirely of aerial mycelium... 48" II-5 Fusion of germ tubes ,.. 48"
II-6 Binucleate hyphae of an initial colony.. 4$
II-7 Binucleate mycelium of a transferred colony, 50
II-3 Uninucleate and binucleate hyphal cells from an old colony 50
II-9 Anastomosis of aerial hyphae of an old colony ' 50 11-10 Thin walled spherical cells from stromata . of an initial colony 50
11-11 Thin walled spherical cells interspersed by short hyphal cells 50
11-12 "Microsorus" structure from an initial
colony 53
11-13 Non-pigment ed uredospore like cells 53
11-14 Uredospore like cells joined by short hyphal cells 53 11-15 Dense spore cluster from stromata of a transferred culture. ;. . . 55
11-16 Spore like cells formed at terminal hyphae..... 55
II- 17 Spore like cells formed in chains. 55
III- l North American isolates of wheat stem rust showing vegetative growth on liquid medium 76 III-2 Growth and development of an Australian race of wheat stem rust on liquid medium 76
.'.'hsat s tern rust ANZ 126-6,7 developed on a chemicaliv defined medium 7o FIGURE III-4 Flax rust colony initials raised on solid medium
III-5 Colonies of flax rust originating as a re• sult of gelatin spore suspension seeding on solid medium
III-6 Ectoparasitic mycelium of flax rust de• veloped on excised cotyledon
III-7 Flax rust colonies developed on liquid medium
Ill-8 Flax rust colonies developed on liquid media surface. The medium contains either peptone plus BSA or amino acid mixture plus BSA as an organic nitrogen source..... -9 Ill Flax rust development under submerged condition..
Ill-10 Growth and development of. flax rust on chemically defined media
Ill-11 In. vitro developed uredospores of flax rust.a
111-12 In vitro developed tellospores of flax rust..
Ill-13 Vegetative and sporulating colonies of flax rust developed on a chemically defined medium 111-14 Axenically developed uredospores infecting the cotyledon when placed on exposed meso- phyll ,
111-1$ Axenically developed uredospores infecting intact cotyledon X1XX
ACKNOWLEDGEMENTS
I express my sincere appreciation to Dr. Michael Shaw for suggesting the problem and for advice and encouragement during the course of the research and in the preparation of the thesis.
Chapter II has been published in the-Canadian Journal of Botany with Dr. M. D. Coffey as the senior author. I thank
Dr. Coffey for his active co-operation and gratefully acknow• ledge his help. I also thank Dr. C.0. Person for help with
the photomicrography; Mrs. Gayle Smith for typing the manu•
script; and many other people who have helped me in various
ways.
This research was supported by grants to Dr. M. Shaw
from the National Research Council of Canada and the Canada
Department of Agriculture. till
FRONTISPIECE
"There is hardly a question in phytopathology so absorbing yet so elusive as the obligate parasitism of the rust fungi. Many investigators have sought in vain the philosopher's stone underlying the problem: a means of culturing rusts on nutrient media other than the living plants". (Chester 1946).
"Plant pathologists, plant physiologists, and others have tried unsuccessfully for about 100 years to culture viruses, downy mildews, powdery mildews and rusts on nonliving media". (Yarwood 1956).
Growth of flax rust Melampsora lini (Ehrenb.) Le"v on chemi• cally defined liquid medium containing inorganic salts, suc• rose, aspartic acid and cysteine. A. Colony initiation from germinating uredospores. X 5. B. Fully developed vegetative colonies. X 15. C. Fully developed colony with mature uredospores. X 15. D. Intact flax cotyledons inoculated with uredospores taken from an axenic colony like that in C and showing typical flax rust uredopustules. 1
GENERAL INTRODUCTION
DeBary (1387) defined obligate parasites as "species to which a parasitic life is essential for the attainment of
•their full development". He recognized that this category should be further divided into (1) strictly obligate para• sites, (2) species which as far as is known live only as para• sites, and (3) facultative saprophytes which can, though usually they do not, live as saprophytes for at least part of their life cycles. The rust fungi have long been regarded as strict obligate parasites of vascular plants, I.e. they
could not be cultivated axenically. Since precise studies
on the nutrition and metabolism of these obligate parasites
can hardly be carried out in the presence of host plant
tissue, repeated attempts were made by various groups to adapt
them to a saprophytic mode of existence. Most of these
attempts have not been recorded in publication and many re•
garded the task as Impossible. For example, Chester in 1946
wrote, "There is hardly a question in phytopathology so ab•
sorbing yet so elusive as the obligate parasitism of the rust
fungi. Many investigators have sought In vain the philoso•
pher's stone underlying the problem: a means of culturing
rusts on nutrient media other than the living plant". He
pointed out that it was believed that in the nutrition of
the rust fungus, lies the secret of many basic problems: the
obligate parasitism of the rusts, their alternation of hosts, 2
their narrow host ranges, their physiologic specialization, their dependence on activity and vigor of the host plant, and the mechanisms for resistance of host to rust.
Writing ten years after Chester, Yarwood (1956) was the first to consider the field of obligate parasitism in a broad comprehensive review. He stated that, "since all the
viruses, most of the chytrids, most parasitic higher plants,
and many miscellaneous fungi are also obligate parasites
according to the present knowledge, it seems likely that about
one-fourth of plant parasites are obligate parasites". These
obligate parasites include the causes of many of our most im•
portant plant diseases, e.g. rusts, mildews, etc. Most fungi
regarded as obligate parasites by DeBary are sti]1 so regard•
ed (Yarwood 1956) . Prior to the 1960's limited success v/as
achieved in culturing lichen fungi, and a number of chytrids
previously known to be obligates. This situation obviously
indicated the feasibility of culturing rust fungi on non•
living substrata. Yarwood expressed the opinion that it would
ultimately be possible to grow these organisms in axenic cul•
ture but questioned whether or not the successful axenic cul•
ture of obligate parasites of plants would aid in their con•
trol.
Ten years after Yarwood's review, P.W. Brian en•
titled The Leeuwenhoek Lecture for 1966 to the Royal Society
of London "Obligate parasitism in fungi" (Brian 1967). In
his lecture Brian also suggested that fungi considered to be 3 obligate pathogens would eventually be grown In axenic cul• ture once their metabolic peculiarities v/ere understood
(Brian 1967). In describing the physiology of obligate para•
sitism he mentioned that germinating uredospores of rust do not lack the capacity to synthesize protein. Adaptive, as well as constitutive enzymes, appear during germination and
exogenous amino acids can be incorporated into such proteins.
He emphasized the point that, as opposed to sporelings of
non-obligate parasites, no net protein synthesis takes place
during uredospore germination. New protein molecules formed
are mainly the result of turnover at the expense of existing
protein. RNA and DNA synthesis are similarly restricted.
Two approaches have been employed in attempts to
culture the rust fungi in vitro: (1) The use of dual-membered
callus cultures i.e. in vitro growth of infected host tissue
leading to the establishment of saprobic fungal colonies on
the tissue culture medium, (2) Axenic culture of the pathogens
directly from uredospores germinated on nutrient media.
Dual-membered callus cultures provide a simple sys•
tem for the study of the biochemical and ultrastructural
events that accompany growth of the pathogen which avoids
some of the physiological complexities of the infected whole
plant. Such studies may be able to define the events in
dual-membered systems which enable the parasite to develop
a saprobic mode of existence.
A summary of the successful attempts to establish
obligate parasites in axenic culture through the use of dual- 4 membered callus cultures Is presented in Table 1. These
earlier works greatly aided in understanding the basic prin•
ciples involved In culturing rust fungi.
It was recognized by Morel (194$) that the inocula•
tion of callus cultures by uredospores is difficult owing to
the high rate of contamination. He attributed his lack of
success in attempting to culture rust on undifferentiated
host tissue to an inadequate inoculation technique, and
suggested that successful cultures of rusts might best be
achieved starting from systemically infected plant tissue,
Which could be vigorously surface-sterilized. This was in
fact achieved by Hotson and Cutter (1951). Unfortunately
other workers have not been able to obtain axenic cultures of
the rusts cultured by Cutter and hence his work has not re•
ceived the recognition it is now considered to deserve.
However, it was realized that maintenance of the cul•
ture and development of the parasite depends on establishing
a proper balance between the growth of parasite and the host
tissue. For example, Turel and Ledingham (1957) reported,
that, if intact rust infected cotyledons are placed on solid
medium a great number of uncontaminated uredospores are pro•
duced but the host tissue fails to proliferate. On the other
hand if the cotyledons are cut in halves before culturing,
host callus growth occurs first, followed by the development
of masses of parasitic mycelium, which overgrows the host
callus. The situation was exploited by subsequent workers
to produce uncontaminated uredospores for starting axenic TABLE I. Growth of obligate parasites in host tissue cultures and axenic culture.
MEDIUM REMARK AUTHOR RUST FUNGUS Plasmopara viticola Gautheret's medium Successful establishment of Morel (1946: downy mildew of grape in cal• lus culture.
The first successful isolation Hotson & Gutter Gymnosporangium Gautheret's nutrient of rust fungus in axenic cul• (1951) .juniperi-virginianae solution, yeast ex• tract and ascorbic ture. Unconfirmed. acid
Gymnosporangium Gautheret's nutrient Both the rusts were isolated Cutter (1959, and subcultured. I960) .juniperi-virginianae solution, yeast ex• Uromyces ari-tri- tract and ascorbic phylli acid Fungus was not isolated Heim & Gries Erysiphe cichora- Sucrose, minerals, auxins, vitamins, ad• axenically. (1953) cearum enine sulphate and coconut milk
Turel & Melampsora lini Knop's tissue culture Fungus failed to develop ax• enically. Ledingham (1957) solution, Berthelot's micronutrients and coconut milk.
Nozzolillo & Puccinia helianthi Modified Heller's or Fungus failed to develop T Craigie (I960) White s.medium, yeast axenically. extract and peptone
Maheshwari, Hil- Puccinia antirrhini Modified Murashige Fungus failed to develop debrandt & Allen and Skoog's medium axenically. (1967) TABLE I. (Continued). REMARK AUTHOR RUST FUNGUS MEDIUM White's basal miner- Fungus developed axenically, Tewari & Arya Sclerospora gramini- als, casein hydroly- survived transfer Results
(1969) cola satg are very similar to Cutter's work.
Modified Barnes & Fungus developed axenically. Hollis et al. Cronartium fusiforme Naylor's minerals, Vegetative growth was predic- (1972) sucrose, yeast extract, table but sporulation was m- peptone and amino acid frequent. mixture.
Mineral salts, sue- Vegetative growth was highly Harvey & Grasham Cronartium ribicola rose, peptone, yeast reproducible only after the (1973) Unpub• extract or amino acid prolonged association of the lished mixture. fungus with its host main• tained in tissue culture. Culture sporuiated after a prolonged subculturing. Pathogenicity was recorded. 7
cultures of rust fungi.
A chronological list of published attempts to cul• ture rust fungi from uredospores is given in Table II. Follow• ing unconfirmed claims by Ray in 1901 (Phragmidium) and
Gretscushnikoff in 1936 (Puccinia spp.), the successful es• tablishment of axenic cultures of Puccinia graminis tritici from uredospores was reported by a group of Australian workers in 1966 and 1967 (Williams et al. 1966, 1967). They grew an Australian race of wheat stem rust on a relatively simple medium (Table II). Their report was quickly confirmed
(Bushnell 1968, Coffey et al. 1969) and marked the beginning of a new era in the study of obligate parasitism.
In 1969 Scott and Maclean published a comprehensive
review describing the work that led to the axenic culture of
Uromyces ari-triphylli, Gymnosporangium juniperi-virginiana
(Cutter 1959, I960) and Puccinia graminis tritici (Williams
et al. 1966, 1967). They discussed the implications of the
new results in relation to the concept of obligate parasitism,
and pointed out that a major problem in obtaining in vitro
development of rust colonies from uredospores was the erratic
behaviour of the spores from one experiment to the next under
apparently identical conditions. In a subsequent review
paper Scott (1972) critically assessed the current status of
the biochemical aspects of host-parasite relationships of
the rusts and mildews and the nutrition of rust fungi grown
in axenic culture. development of rust fungi from germinating uredospores, TABLE II. In vitro
MEDIUM REMARK AUTHOR RUST FUNGUS Gelatin medium plus Unconfirmed report. Ray (1901) Phragmidium subcor- ticium plant extract Casual claim: not confirmed. Gretschushnikoff Puccinia spp, Culture medium con• taining substances (1936) which adsorbed ammonia
Czapek's minerals, Simple technique, although Williams et al. Puccinia graminis glucose, yeast ex• considerable variability and (1966, 1957) tritici, ANZ 126-6,7 unpredictability was reported. tract Partly confirmed. Williams' Bushnell (1968; Puccinia graminis Czapek's minerals, tritici, ANZ 126-6,7 glucose, yeast ex• work. tract and peptone Unsubstantiated result. Singleton & Puccinia recondita Czapek's minerals, Young (1968) glucose, yeast ex• tract, peptone. Synchronus colony development Puccinia graminis Czapek's minerals, Coffey et al. was recorded, but increased (1969) tritici ANZ 126-6,7 yeast extract, pep• tone, glucose and lag phase preceded the develop BSA ment of the colonies. Sporu• lation was altogether absent.
Vegetative growth occurred Melampsora lini Modified Knop's tis• Turel (1969' with stroma formation. Fre• Race'3 sue culture medium, Berthelot's micro- quency of established culture nutrients, sucrose, was low. yeast extract. TABLE II. (Continued).
MEDIUM REMARK AUTHOR RUST FUNGUS Confirmed the above. Cyto- Melampsora lini Modified Knop's tis• Coffey et al. logical studies showed the Race 3 & Race 210 sue culture medium, (1969) Berthelot's micro- hyphae to be binucleate. nutrients, sucrose, yeast extract, pep• tone
7 Vegetative growth occurred Willets Puccinia graminis Czapek's minerals, Wong
Uninucleate cultures, evi- Maclean & Scott Puccinia graminis Czapek's minerals, (1970) tritici, ANZ 126^6,7 elucose yeast extract dence is presented that hyphal and peptone cells are diploid. Poorly documented claims remain unconfirmed.
Established colonies infected Melampsora lini Modified Knop's tis• Turel (197i: and overgrew the excised flax Race No. 3 sue culture medium, Berthelot's micro- cotyledons. Spore produced nutrients, sucrose, from these infected intact vitamin free casamino plant; Races are found to acid be the same as inoculum used to raise axenic cultures. TABLE II. (Continued)
MEDIUM REMARK AUTHOR RUST FUNGUS Uninucleate cultures did re• Puccinia graminis Czapek's minerals, Maclean et al. infect the host and subsequent tritici, ANZ 126 glucose, yeast ex• (1971) -6,7 tract, peptone sporulation occurred on the plant and those spores were found to be uninucleate. These authors believed the cultures remained haploid at uninucleate condition. Needs confirmation, although an un• natural biological phenomenon. Change of ploidy more common• ly seen in plant and animal cells grown in tissue culture.
Growth and sporulation was re• Bose and Shaw Puccinia graminis Czapek's minerals, ported. S.E.M. revealed an tritici, ANZ 126 glucose, yeast ex- (1971v 7 ) -6,7 tract, peptone and unknown coating around the in BSA vitro grown uredospores. Further detail studies are needed.
Growth was poor of these Bushnell and Puccinia graminis t Czapek's minerals, North American races. Stewart tritici, Race 38, glucose, yeast ex• (1971] tract and peptone
On defined medium growth was Kuhl et al. Puccinia graminis Czapek's minerals, tritici, ANZ 126 glucose, yeast ex poor for this isolate. (1971T -6,7 tract and peptone H o Hartley & Puccinia graminis Czapek's minerals, Variation was reported regard• tritici, ANZ 126 glucose, yeast ex- ing saprophytic ability be• Williams (1971 -6,7 (a) and (b)) tract and peptone tween different strains be• longing to the same race. TABLE II. (Continued).
MEDIUM REMARK AUTHOR RUST FUNGUS A given race had mixture of Hartley and two strains and one showed Williams (Con• different culture proper• tinued) ties when studied on in vitro.
Brief report on vegetative Puccinia graminis Czapek-'s minerals, Masin and growth of a Russian isolate t x^xt X C X glucose, yeast ex• Andreev (1971) tract and peptone of wheat stem rust. Needs Strain 21 (USSR) confirmation.
Czapek's minerals, In the light of present con• Vassil'ev and Melampsora pini- cept difficult to evaluate. Saplina torqua glucose, yeast ex• tract . Needs confirmation. Unconfirmed. Kuhl et al. Puccinia graminis Czapek's minerals (Unpublished) avenae Modified Knop's tis• Evidence was presented that Coffey and Shaw Melampsora lini established clones can adapt (1972) Race 3 sue culture medium, Berthelot's micro- to broad range of carbohy• nut rients, sucrose drates and amino acids. & amino acid mixture Vegetative growth was obtain• Jones (1972) Uromyces dianthi Czapek Dox Broth, pep tone, glucose, yeast ed. Sporulation absent. extract Pathogenicity trials were not reported.
A defined solid medium was Foudin and Wynn Puccinia graminis Czapek's minerals, (1972) tritici , ANZ 126-6,7 glucose and amino used. Sporulation was in• acid mixture frequent. Pathogenicity trials were not reported. TABLE II. (Continued).
MEDIUM REMARK AUTHOR RUST FUNGUS
Puccinia graminis Czapek's minerals, Established clones raised on Howes and Scott media containing- peptone etc (1972) tritici, ANZ 12b-6,7 glucose., citrate, cysteine, glutamme were used to study S nutri• tion. A range of S amino acids was suitable for rust growth, but inorganic S was not.
Growth of flax rust and sun• Melampsora lini Modified Knop's tis• Coffey and flower rust was poor in chem Race 3, 79 sue culture medium, Allen (1973) ically defined liquid medium Puccinia helianthi Berthelot's micro- Typical sporulation was ab• Race 1 nutrients, sucrose, yeast extract, BSA, sent. peptone
Vegetative growth occurred, Melampsora lini Modified Knop's tis• Turel (1973 sporulation absent in chemi• Race 3, 210 sue culture medium, Berthelot's micro- cally defined solid medium. nutrients, sucrose, aspartic acid, cys• teine, glutathione 13-
Following the initial report by Williams et al. several laboratories have published the results of successful attempts to grow rust fungi in axenic culture. In addition to the work of the Australian group, the most extensive pro• grams have been conducted by Turel at Saskatoon, Bushnell at
Minnesota, by Jones at Keele, by Coffey and -Allen at Wiscon•
sin and in the Department of Plant Science at U.B.C.
At U.B.C. work on the axenic culture of wheat
rusts was started by the author in Dr. Shaw's laboratory in
1967. The first new finding at U.B.C. was that when \% gela•
tin was used to suspend the uredospore inoculum vegetative
growth of an isolate of the Australian race ANZ 126-6,7 of
Puccinia graminis tritici occurred with greater frequency and
predictability in vitro than had been reported by the Austral•
ian group at that time (Coffey, Bose and Shaw 1969) . An in•
teresting feature of the results was that growth occurred
with a low density of uredospores (20-40 spores/mm ) when
the uredospores were suspended in gelatin before the plates
were seeded. Using dry uredospores Bushnell (1968) had found
earlier that colony development only -occurred in regions of
plates seeded at a high density (100-200 spores/mm^). Al•
though the gelatin technique described by Coffey et al.
(1969) led to the synchronous development of axenic colonies
of wheat rust on solid media, the colonies failed to sporu-
lat e. 14
In subsequent trials with Puccinia graminis tritici conducted using a liquid medium, typical uredopustules and teleutopustules developed on the axenic colonies. Reinfec• tion of the host was obtained using uredospores grown in vitro.
These results, which confirmed and extended those reported
earlier by Williams et al. (1967), are described in Chapter I
of this thesis, which has already been published (Bose and
Shaw 197D .
Chapter II describes the axenic culture and cytology
of two strains of Melampsora lini, the flax rust fungus.
These results confirmed Turel's original report of the axenic
culture of this autoecious rust oh a solid medium containing
sucrose, Knop.'s mineral nutrients and yeast extract. Chapter
II has also already been published (Coffey, Bose and Shaw
1970). Further studies on the carbohydrate and amino acid
nutrition of these flax rust cultures were published by Coffey
and Shaw (1972). Coffey and Shaw's results and further
studies by Turel (1973) laid the groundwork for the experi•
ments described in Chapter III.
Chapter III deals with the growth of both wheat stem
rust and flax rust on chemically defined liquid media con•
taining only a simple carbohydrate, mineral salts and 2 or 3
amino acids. Typical uredospores and teleutospores were pro•
duced by both rusts on the media employed and intact flax
cotyledons were successfully inoculated with flax uredospores
raised in vitro. 15
The three chapters in the thesis thus record our increasing ability to obtain reproducible vegetative and reproductive growth of the wheat and flax rust fungi. The illustrations of axenic cultures of the flax rust fungus in the frontispiece are striking testimony to the progress which has been made since the writer began his experiments in 1967. 16
REFERENCES
Bose, A., and M. Shaw. 1971. Sporulation and pathogenicity of an Australian isolate of wheat rust grown in vitro. Can. J. Botany 49: 1961-1964. Brian, P.W. 1967. Obligate parasitism in fungi. Proc. Roy. Soc., Ser. B, Biol. Sci. 1968: 101-118.
Bushnell, W.R. 1968. In vitro development of an Australian isolate of Puccinia graminis f. sp. tritici. Phyto• pathology 58: 526-527.
Bushnell, W.R., and D.M. Stewart. 1971. Development of Amer• ican isolates of Puccinia graminis f. sp. tritici on an artificial medium. Phytopathology 61: 376-379.
Chester, K.S. 1946. The Nature and Prevention of the Cereal Rusts as exemplified in the Leaf Rust of Wheat. Chronica Botanica Co., Waltham, Mass., U.S.A. 269 P.
Coffey, M.D., A. Bose, and M. Shaw'. 1969. In vitro growth of gelatin suspensions of uredospores of Puccinia graminis tritici. Can. J. Botany 47: 1291-1293.
Coffey, M.D., A. Bose, and M. Shaw. 1970. In vitro culture of the flax rust, Melampsora lini. Can. J. Botany 43: 773-776.
Coffey, M.D., and M. Shaw. 1972. Nutritional studies with axenic cultures of the flax rust, Melampsora lini. Physiol. Plant Pathology 2: 37-47.
Coffey, M.D., and P.J. Allen. 1973. Nutrition of Melampsora lini and Puccinia helianthi. Trans. Brit. Mycol. Soc. 60: 245-260.
Cutter, V.M. 1959(1961). Studies on the isolation and growth of plant rusts in host tissue cultures and upon syn• thetic media. I. Gymno sporangium. Mycologia 52: 248- 295. Cutter, V.M. 1960(1961). Studies on the isolation and growth of plant rusts in host tissue cultures and upon syn• thetic media. II. Uromyces ari-triphylli. Mycologia $2: 726-742.
DeBary, A. 1887. Comparative Morphology and Biology of the Fungi, Mycetozoa and Bacteria. (Eng. trans, by Garnsey, H.E.F.; rev. Balfour, I.B.). Clarendon Press, Oxford, 525p. 17
Foudin, A.S., and W.K. Wynn. 1972. Growth of Puccinia gram• inis f. sp. tritici on a defined medium. Phyto• pathology 62: 1032-1040.
Gretsehushnikoff, A.I. • 1936. Toxins of rust (Puccinia). Rev. Appl. Mycol. 15: 710 (Abstr.).
Hartley, M.J., and P.G. Williams. 1971. Morphological and cultural differences between races of Puccinia gram• inis 1. sp, tritici in axenic culture. Trans. Brit. Mycol. Soc. 57: 137-144. Heim, J.M., and G.A. Gries. 1953. The culture of Erysiphe cichoracearur. on sunflower tumor tissue. Phyto• pathology 43: 343-344.
Howes, N.K., and K.J. Scott. 1972. Sulphur nutrition of Puccinia graminis f. sp. tritici in axenic culture. Can. J. Botany 50: 1165-1170.
Hotson, H.H., and V.M. Cutter. 1951. The isolation and cul• ture of Gymnosporangium juniperi-virginianae Schw. upon artifical media. Proc. Nat. Acad. Sci. (U.S.A.) 37: 400-403.
Hollis, C.A.,. R.A. Schmidt, and J.W. Kimbrough. 1972. Axenic culture of Cronartium fusiforme. Phytopathology 62: 1417-1419.
Jones, D.R. 1972. In vitro culture of carnation rust, Uro- myces dianthi. Trans. Brit. Mycol. Soc. 58: 29-36.
Maclean, D.J., and K.J. Scott. 1970. Variant forms of sapro• phytic mycelium grown from uredospores of Puccinia graminis f. sp. tritici. J. Gen. Microbiol. 64: 19-27.
Maclean, D.J., K.J. Scott, and I.C. Tommerup. 1971. A uni• nucleate wheat-infecting strain of the stem rust fungus isolated from axenic culture. J. Gen. Microbiol. 64: 339-342.
Maheshwari, R., A.C. Hildebrandt, and P.J. Allen. 1967. Factors affecting the growth of rust fungi on host tissue cultures. Botan. Gaz. 128: 153-159.
Masin, V.V., and L.N. Andreev. 1971. Vegetative growth of the pathogen of wheat stem rust in vitro. Mikolo- giya i Fitopathologiya 5(2): 197-200.
Morel, G. 1948. Recherches sur la culture associee de para• sites obligatiries et de tissue vege'taux. Ann. Epiphyt. (Paris) 14: 1-112. 18
Nozzolillo, C., and J.H. Craigie. I960. Growth of the rust fungus Puccinia helianthi on tissue cultures of Its host. Can. J. Botany 33: 227-233.
Ray, J. 1901, Cultures et formes attenue'es des maladies cryptogamique des ve\getaux. C.R. Hebd. Seanc. Acad. Sci., Paris 133: 307-309. Scott, K.J. 1972. Obligate parasitism by phytopathogenic fungi. Biol. Rev. 47: 537-572.
Scott, K.J., and D.J. Maclean. 1969. Culturing of rust fungi. Ann. Rev. Phytopathology 6: 123-146.
Singleton, L.L., and Young, H.C. 1968. The in vitro culture of Puccinia recondita f. sp. tritici. Phytopathology 58: 1068 (Abstr.1T Tewari, M.M., and Arya, H.C. 1969. Sclerospora graminicola axenic culture. Science (Washington) 163: 291-293.
Turel, F.L.M. 1969. Saprophytic development of the flax rust, Melampsora lini race no. 3. Can. J. Botany 47: 821-
Turel, F.L.M. 1971. Pathogenicity and development differ• ences of three saprophytically growing isolates of flax rust fungus Melampsora lini, race 3. Can. J. Botany 49: 1993-1997. Turel, F.L.M. 1973. Growth of the flax rust Melampsora lini on chemically defined media. Can. J. Botany 51: 131- 134. Turel, F.L.M., and G.A. Ledingham. 1957. Production of aerial mycelium and uredospores by Melampsora lini (Pers.) Lev. on flax leaves In tissue culture. Can. J. Microbiol. 3: 813-819. ' Vassil'ev, O.A. and V.I. Saplina. 1971. Culture of the fun• gus Melampsora pinitorqua (A.BR.) Rostr. - the pathogen of the stem rust of pine. Mycologiya i Fitopathologiya 5(2): 182-183.
Williams, P.G., K.J. Scott, and J.L. Kuhl. 1966. Vegetative growth of Puccinia graminis f. sp. tritic in vitro. Phytopathology 56? 1418-1419. 19
Williams, P.G., K.J. Scott, J.L. Kuhl, and D.J. Maclean. 1967. Sporulation and pathogenicity of Puccinia graminis f. sp. tritici grown on an artificial medium. Phyto• pathology 57: 326-327.
Wong, A.L., and H.J. Willetts. 1970. Observations on growth of selected Australian races of wheat stem rust in axenic culture. Trans. Brit. Mycol. Soc. 55: 231- 23&\
Yarwood C.E. 1956. Obligate parasitism. Ann. Rev. Plant Physiol. 7: 115-142. 20
CHAPTER I
Sporulation and Pathogenicity of an Australian
Isolate of Wheat Rust grown in vitro
ABSTRACT
Colonies of Puccinia graminis Pers. f. sp. tritici
Erikss. & Henn., race ANZ 126-6,7 were grown from uredospores
on Czapek's mineral salts, 3% glucose, 0.1% Evans' peptone,
plus defatted bovine serum albumen. Dikaryotic vegetative
hyphae apparently developed from centers of germ tube anasto•
mosis, without the formation of typical infection structures.
Typical pigmented uredospores and teliospores were formed
after 6 to 8 weeks growth. Both spore forms were coated with
a layer of material which was visible under the scanning elec
tron microscope and was not observed on uredospores grown on
intact wheat leaves. The uredospores were capable of infec•
ting the mesophyll of wheat leaves exposed by stripping back
the lower epidermis. The possibility is considered that the
surface coating of uredospores grown in vitro is related to
their inability to infect intact leaves via the stomata. 21
INTRODUCTION
Williams et al. (1966, 1967) and Bushnell (1968) have described the production of uredospores by P. graminis
Pers. f. sp. tritici Erikss. & Henn., race ANZ 126-6,7 in vitro. Williams' group (1967) found that successful reinfec• tion of wheat leaves by uredospores grown in vitro only
occurred when the spores were placed on exposed mesophyll of
Little Club wheat. They considered that the inability of such
uredospores to Infect via stomata was due to a low potential
for Independent growth during the initial stages of infection,
rather than to a lack of pathogenicity per se. In further - ex•
periments with an isolate of the Australian rust obtained
from Dr. P.G. Williams, Coffey et al. (1969) obtained best
growth of vegetative colonies by seeding the uredospores onto
the medium in suspension of 15% gelatin. These colonies
eventually produced non-pigmented uredospore-like structures.
We have repeated our experiments with the Australian isolate
and have obtained the production of typical, pigmented uredo•
spores capable of reinfecting the host plant, as well as
typical pigmented teliospores. Since observations on the
axenic culture of rust fungi still cannot be described as
routine, our new results are reported here. 22
MATERIALS AND METHODS
Little Club wheat was grown in a controlled environ• ment cabinet set at 500 ft-c and 20°C for 16 h followed by 8 h in darkness at 15°C. The first seedling leaves of plants 1 week old were inoculated with uredospores of Puccinia graminis
Pers. f. sp. tritici Erikss. & Henn., race ANZ 126-6,7 suspend•
ed in water. The plants were sprayed with water both before
and after inoculation, covered with plastic bags to ensure
high humidity, and incubated for 20-24 h in darkness at 15°C.
The bags were then removed and the plants returned to the
growth cabinet. After visible infection flecks developed,
leaves were surface-sterilized, their cut ends planted in agar
in culture tubes, and incubated as described earlier (Coffey
et al. 1969). After 10 days, dry, contaminant-free uredo•
spores were harvested and used to inoculate solid or liquid
media.
The basic medium contained Czapek's mineral salts,
3% glucose, and 0.1% Evans' peptone (Evans Medical Ltd.)
(Bushnell 1968), plus 1% defatted bovine serum albumen (BSA;
fraction V, Armour Pharmaceutical Co..), added after autoclaving,
as described earlier (Coffey et al. 1969). Medium solidified
with 1.5% Difco Bacto Agar in plastic petri dishes was in•
oculated with a gelatin suspension of uredospores (Coffey et
al. 1969). The dishes were then covered and sealed with 23
masking tape. In experiments with liquid medium, 20 ml ali- quots were pipetted into 50 ml conical, pyrex flasks. The pH was adjusted to 6.4 before autoclaving and addition of BSA.
The pH of the medium did not change significantly after auto• claving. Uredospores were carefully placed on the surface of the liquid using a flattened needle and the flasks were closed with polypropylene stoppers.
Agar pieces (1 cm ) bearing vegetative colonies or sporulating colonies grown on liquid medium were stained on clear microscope slides with phloxine-KOH or on undiluted
Giemsa reagent for 3 to 4 min (Giatgong and Fredericksen 1969)
Photomicrographs were taken under bright-field or phase con•
trast, using a 35 mm camera.
For examination in the scanning electron microscope
(Cambridge II Stereoscan), uredospores grown in vitro were
first teased onto a dry microscope slide and kept at room temp
erature for 24 h. Uredospores harvested from intact plants
and those grown in vitro were cemented to specimen stubs with
silver paint and coated first with carbon and then with gold
at an angle of -40 to +50° on a continuous rotating stage,
using a shadow caster. Differences in treatment were elimin•
ated by processing both samples of uredospores at the same
time. 24
RESULTS
1. Behaviour of Germinating Uredospores on Solid Medium The germination and development of uredospores were
examined 8 days after the plates were seeded. It was found
that the germ tubes of single isolated germlings had frequently
burst, liberating the cytoplasm. This was observed repeatedly.
For example in one trial at least two burst germ tubes were ob•
served in each.of 10 consecutive microscope fields (10 to 20
spores per field; magnification X 100) after staining with
phloxine-KOH (Figs. I-l and 1-2). In a few Instances two
nuclei were seen in the extruded cytoplasm after staining with
Giemsa reagent (Fig. 1-3). No typical infection structures were
seen, but swellings or vesicles were observed in some instances.
In a few instances incipient branching was observed in single
isolated germ tubes, but these did not develop septa. Burst•
ing was not observed when two or more germ tubes had undergone
anastomosis. Anastomosed tubes frequently formed septa (Fig.
1-4). Infection structures did not develop after anastomosis
but there were a number of vesicular swellings associated with
each 'center' of anastomosis (Fig. 1-4). All the hyphae
examined were dikaryotic and there was no evidence for the
occurrence of the monokaryotic hyphae described by Williams
and Hartley (1971).
2. Development of Colonies on Liquid Medium Growth was strongly influenced by temperature. After
incubation for 4 weeks, growth was negligible at 11°C, slow at Figure 1-1. Uredospore v;ith burst germ tube (arrow) on basic medium plus BSA. Stained with phloxine-KOH. X 320.
Figure 1-2. Two germinating uredospores. The germ tubes have crossed each other, but have not anas• tomosed, and have burst (arrow). Medium and stain as for Figure 1-1. X 634.
Figure 1-3. Two nuclei (arrows) in cytoplasm extruded from burst germ tube. Basic medium. Stained with Giemsa-HCl. X 640.
Figure 1-4. Germ tubes originating from two uredospores showing anastomosis (upper arrow) and the development of a vegetative hypha with septum (bottom arrow). A third uredospore (ug), containing two nuclei, has not ger• minated. X 341.
Figure 1-5. Colony development on liquid medium. A, sporulating colonies with collapsing mycelium, grown-at 17°C. B, juvenile colonies in vegetative stage grown at 14°C. Photographed after 6 weeks growth.
1t 26 27
14°C, and excellent at 17°C. There was only a little growth at 21°C, which caused a conspicuous collapse of the mycelium.
At 11°C small colonies developed after 8 weeks and a few of these sporulated after 10 weeks. Sporulation and the forma• tion, of a brown pigment occurred after 8 weeks at 14°C.
Figure I-5A shows sporulating colonies grown on liquid medium for 6 weeks at 17°C and Figure I-5B shows vegetative colonies
of the same age grown at 14°C
At 17°C, 25 to 30 fluffy white colonies, which
tended to coalesce with each other, developed in each flask
(15 replicates). The results at this temperature were highly
reproducible. After 6 weeks the colonies had turned brown,
some brown pigment had diffused into the medium, and large
numbers of uredospores had developed in about 60% of the
flasks (Fig. I-5A). The developing uredospores were .formed
terminally on sporophores (Fig. 1-6), and, when mature, were
released by gentle teasing with a needle on a microscope
slide. The uredospores were similar in shape and size to those
formed normally in infected host tissue (Fig. 1-7). Thick-
walled, two-celled teliospores developed in clusters on sporo•
phores (Figs. 1-7 and 1-8) about 2 weeks after the formation
of uredospores, and the colonies turned dark brown in color.
Coffey et al. (1969) reported the occurrence of
groups of irregularly shaped non-pigmented bodies (5-10 u in
diam) in the medium at the periphery of colonies that developed
when a gelatin suspension of uredospores was seeded onto solid
medium. No such bodies were observed in liquid medium but 23
Figure 1-6. Developing uredospores in colony grown for 5 to 6 weeks on liquid medium. Phloxine/ KOH. X 793.
Figure 1-7. Developing teliospore (arrow) and maturing uredospores from colony grown on liquid medium for about 8 weeks. Phloxine/KOH. X 1587.
Figure 1-8. Fully developed two-celled teliospores. Phloxine/KOH. X 634.
Figure 1-9. Thick mat of hyphae at periphery of colony grown on liquid medium for 6 weeks. X 307.
Figure 1-10. Pustules developed on primary leaf of Little Club wheat about 10 days after in• oculation with uredospores grown in vitro. A, the upper surface; B, the lower sur• face, of uninfected leaf. Uredospores were applied to the lower surface of the leaf after the epidermis had been stripped back. The cellophane strip used to cover the inoculum is visible in B. 29 30 the peripheral hyphae v/ere thick and compact (Fig. 1-9).
Actively growing vegetative colonies (diam 2-4 mm) were transferred from liquid to solid medium (plus BSA) in test tubes. About 5% of these transfers v/ere successful, re• newed vegetative growth being followed by sporulation.
3, Pathogenicity of Uredospores Produced in Axenic Culture
Uredospores grov/n in vitro were apparently unable to
infect intact v/heat leaves. When such uredospores were placed
directly on mesophyll tissue,, which had been exposed by strip•
ping off the epidermis and covered with cellotape, normal
uredial pustules developed in about 10 days (Fig. 1-10).
Eighty percent of these trials were successful. The uredo•
spores produced in such pustules v/ere themselves able to infect-
normal, intact wheat leaves. Uredospores from pustules formed
under cellotape were also found to be free of bacterial con•
taminants. When seeded directly onto solid or liquid media at
17°C they produced fluffy white colonies which later sporulated.
4. Scanning Electron Microscopy
On examination by scanning electron microscopy uredo-
• spores grown in vitro were found to be covered with a layer
of material of unknown composition (Fig. 1-12), which was not
seen on uredospores taken from intact, infected plants (Fig. I-
11). The spines on uredospores from both sources were similar
in shape, size, and distribution (Figs/ 1-13, 1-14, and 1-15).
The. morphology of the uredospore surface v/as similar to that
reported recently for 'Puccinia coronata (Corlett 1970). 31
Figures 1-11 - 1-16. Scanning electron micrographs.
Figure 1-11. Mature uredospores grown on intact wheat leaves. X 21,805.
Figure 1-12. Mature uredospores grown in vitro. Note v/rinkled surface coating not present in ' Figure I-11. X 23,291.
Figure I-13. Spines on surface of uredospore grown on intact wheat leaf. X 10."'"
Figure 1-14. Spines on surface of uredospore grown on basic medium. X 25,875.
Figure 1-15. Developing spines on surface of uredo• spore grown on intact wheat leaf. X 10,797.
Figure I-l6, V/rinkled surface covering of teliospore grown on basic medium. X 2274. 32 33
Teliospores formed in vitro also appeared to have a surface coating like that of the uredospores grown in vitro, but telio• spores grown on intact leaves were not examined. The wrinkled appearance of this coating is clearly seen in Figure 1-16.
DISCUSSION
Our results suggest, that in the Isolate made avail• able to us by Dr. Williams, anastomosis of germ tubes is a necessary prerequisite for the formation of vegetative colonies but we have not been able to obtain conclusive evidence for this. Nevertheless all attempts to produce colonies from single
spore inoculations of the media used have given negative re•
sults. Our observations provide no evidence to support
Williams' (1971) recent demonstration that dikaryotic mycelia
develop only from uredospores which first develop clearly
differentiated infection structures. Further work is needed
to resolve this point. The uredospores and teliospores that
develop from the dikaryotic mycelium appear to be entirely nor•
mal, except for the existence of the coating material, which is
here reported for the first time. Under the microscope, pig•
mentation of both uredospores and teliospores was similar to
that of spores produced on intact plants. In our earlier ex•
periments (Coffey et al. 1969) uredospore-like structures
were found but were virtually colorless. It is possible that
a fall in the water content of the medium prevented sporulation
in our earlier attempts (Coffey et_ al. 1969); this would not 34 occur with the liquid medium used in the current experiments.
The pathogenicity of the uredospores produced on liquid me• dium also appears to be normal, except for the important fact that infection occurs only on exposed mesophyll tissue and not via the stomata on intact leaves. In this respect our obser• vations confirm those of Williams et al. (1967). It is temp• ting to suggest the possibility that the inability of uredo•
spores grown in vitro to infect Intact leaves is related to
the presence of the unknown material with which such spores
are coated. While this material does not inhibit germination
in vitro or on the intact leaf, it may well affect contact with
the intact leaf and the development of normal infection struc•
tures. We are of the opinion that the material Is secreted by
the spores during their development in vitro. The possibility
should be envisaged that it Is attacked by enzymes released
from the host cells when the epidermis of the leaf is stripped
off. These observations open a new area in the Investigation
of the'properties of uredospores produced in vitro. 35
REFERENCES
Bushnell, W.R. 1968. In vitro development of an Australian isolate of Puccinia graminis f. sp. tritici. Phyto• pathology 58: 526-527.
Coffey, M.D., A. Bose, and M. Shaw, 1969. In vitro growth of gelatin suspensions of uredospores of Puccinia graminis f. sp. tritici. Can. J. Botany 47: 1291- 1293 .
Corlett, M. 1970. Surface structure of urediniospores of Puccinia coronata f. sp. avenae. Can. J. Botany 48: 2159-2161.
Giatgong, P., and R.A. Fredericksen. 1969. Pathogenic varia• bility and cytology of monoconidial subcultures of Piricularia oryzae. Phytopathology 59: 1152-1157.
Williams, P.G. 1971. A new perspective of the axenic culture of Puccinia graminis f. sp. tritici from uredospores. Phytopathology 6TT"994-1002.
Williams, P.G., K.J. Scott, and J.L. Kuhl. 1966. Vegetative growth of Puccinia graminis f. sp. tritici in vitro. Phytopathology 56: 1418-1419.
Williams, P.G., K.J. Scott, J.L. Kuhl, and D.J. Maclean. 1967. Sporulation and pathogenicity of Puccinia graminis f. sp. tritici grown on an artifical medium. Phyto• pathology 57: 326-327.
Williams, P.G., and M.J. Hartley. 1971. Occurrence of diploid lines of Puccinia graminis tritici in axenic culture. Nature New Biol. 229: 181-182. 36
CHAPTER II
In vitro Culture of the Flax Rust, Melampsora lini
ABSTRACT
Two different strains of Melampsora lini (Pers.)
Lev. were grown on media similar to those described by Turel
(1969a). Alterations in organic and inorganic constituents did not, in general, prevent growth. Peptone and yeast ex• tract, singly or in combination, supported growth, but coco• nut milk did not. Maintenance of the ambient temperature below 17°C during transfer procedures Increased the number of colonies which grew. The mycelium was generally binuc• leate. Spore-like cells of varying shapes were found, some of which resembled uredospores and teliospores.
INTRODUCTION
The growth in axenic culture, that is, in isolation
from all other living organisms, of mycelium from germinated
uredospores of flax rust was reported by Turel ( 1969a). She
grew uredospores of race 3, Melampsora lini (Pers.) Lev. on a
medium containing Difco yeast extract, sucrose, and Inorganic
salts. The inability of the cultures to establish on a
similar medium in which Czapek's minerals were included as
a source of inorganic constituents led Turel to assume that
the balance of inorganic salts played an important part in 37
determining growth of this fungus. In addition, she found that raising the temperature above 17°C led to the eventual death of the cultures (Turel 1969b). Her cultures were
capable of transfer under certain defined conditions and in
ageing parts of the stroma produced a range of spore-like
cells, including some which resembled one-celled teliospores
(Turel 1969a). An attempt was therefore made to confirm
Turel's work using two different strains of M. lini'1'. The
influence of inorganic, and more particularly that of organic
constituents, of the culture medium on growth was examined.
Nuclear condition of the mycelium and of spore-like cells
formed in culture was determined where possible and the mor•
phology of the spore-like cells compared with typical spore
types.
MATERIALS AND METHODS
1. Melampsora lini, Strain 1
Seven-day-old Bison flax cotyledons were inoculated
with an aqueous suspension of uredospores of Melampsora lini.
The inoculated seedlings were kept in the dark under high
humidity for 21+ h and then grown under fluorescent lighting
''"The material used in these experiments was thought to be race 3 (strain 1) and race 210 (strain 2). Hov/ever, sub• sequent testing of this material on host differentials has revealed that the races are not pure. 33
(1000 ft-c for 16 h). Cotyledons were removed after 6 days, when flecking had occurred, and placed in a solution of
Gramicidin (Nutritional Biochemicals; 10 mg per liter) for
2-3 h. They were then removed, surface sterilized for 10
min with 10% Javex (5.25% available chlorine) and a drop of
Tween 80, and washed three times in sterile glass-distilled
water. The cotyledons were then placed in culture tubes on
agar slants with their adaxial surfaces facing upwards. Each
tube contained 6 ml of the tissue culture medium used by
Turel (1969a). Cultures were placed under continuous fluores
cent lighting (500 ft-c) at 17°C. After 13 days, contaminant
free cotyledon cultures "were used as a source of aseptic
uredospores. - Small numbers of flax rust spores were placed
directly on the surface of media in pyrex culture tubes (125
x 16 mm- with 'Bacti Capall' polypropylene closures). Four
different media were used, all based on Turel's medium II
(1969a), but with different organic constituents:
(i) with 0.1% Difco yeast extract,
(ii) with 0.1% Evans' peptone,
(iii) with 0.1% Difco yeast extract and 0.1%'Evans'
peptone,
(iv) with 10% coconut milk. 39
Micronutrients''^ v/ere supplied to all four media at a level of 0.8 ml per liter. Six ml of medium were placed in each tube; 100 tubes v/ere used for each medium. The pH after autoclaving v/as in the range 4.9 to $.1. All culture tubes
Containing media and all instruments used to place the uredo•
spores were chilled at 3°C before use. Culture tubes v/ere
incubated at 17°C in the dark.
At periods between 4 and 6 weeks after culturing had
been initiated, colonies v/ere transferred to the medium with
0.1% peptone. After 3 weeks colonies were again transferred
to a medium with peptone plus modified inorganics consisting
of KN03, 1.0 g; MgS04'7H20, 0.25 g; KH2P0^, 0.25 g; NH^N03,
0.04 g; per liter of glass-distilled water (pH adjusted to
6.5 with 0.1 N NaOH before autoclaving). Micronutrients were
added either at 0.8 or 0.5 ml in later transfers. A further
five transfers were made to media containing the modified in•
organics and either peptone, or both peptone and yeast extract,
at Intervals of 3 to 6 weeks.
''"Micronutrients were Sequestrene, 13% NaFe (Geigy), 10 .g;
MnS04-7H20, 447 mg; KI, 10 mg; NiCl2-6H20, 18* mg; GoCl2-6H20,
18 mg; Ti(S04)2'9H20, 42 mg; ZnSO^, 35 rag; CuSO^H^O, 15 mg;
BeSO^, 20 mg; H^PO^, 10 mg; H2S0^ concentrated, 1/5 ml;
glass-distilled water, 200 ml. Micronutrients added either
at 0.5 or 0.8 ml per liter of medium. 40
To determine if colonies could be initiated on the medium with modified inorganics, an experiment was carried out with Melampsora lini using a liquid culture system.
Small amounts of aseptic uredospores were placed in 10 ml of the culture medium (modified inorganics with micronutrients,
4% sucrose, 0.1% yeast extract, and 0.1% peptone) in 50 ml
culture flasks. The flasks were incubated at 17°C in the
dark on a reciprocating shaker (about 100 cycles per min).
Shaking was stopped after 2 weeks and the cultures then grown
under still conditions.
2. Melampsora lini, Strain 2
Aseptic uredospores were produced by methods already
described in the previous section. Inoculation procedures
were similar to those used for strain 1, media and Instru•
ments being kept below 17°C and spores placed in small quan•
tities directly on the agar surface. A modified version of
Turel's medium I (1969a) was used with 0.1% Evans' peptone
and 0.1% Difco casamino acids as additional ingredients.
Tubes were examined after 6 weeks and again at 8 weeks. At
8 weeks colonies were transferred to five different media:
(i) Turel's I inorganics with 0.1% peptone,
(ii) Turel's I inorganics with 0.1% yeast extract,
(iii) modified inorganics (see page 39) with 0.1%
peptone, 41
'" (iv) modified inorganics with 0.1% yeast extract,
(v) modified Inorganics with 0.1% casamino acids.
In all five media micronutrients were added at 0.8 - ml per liter.
3. Cytological Examination of the Cultures
Tease-mount preparations of initial and transferred
colonies of both strains were stained with phloxine/KOH or
haematoxylin for examination of cells in cultures of different
ages, and of their nuclear condition.
RESULTS
1. Melampsora lini, Strain 1
Treatment with Gramicidin before conventional sur•
face sterilization markedly reduced bacterial contamination
in the cotyledon cultures. In addition, no adverse effect on
the subsequent growth of the cultures was observed. During
the first 3 days in culture no evidence of bacterial contam•
ination of the cotyledons could be found. After 13 days con•
tamination was only found in 3S out of 126 tubes. In earlier
experiments in which the antibiotic was not used, contamina•
tion was sometimes as high as 75% even at 3 days.
Only very small quantities of uredospores were
placed at any particular point on the agar surface. Large
quantities of uredospores of the order required to produce
mycelial growth of Puccinia graminis (Bushnell 1968, Williams
et al. 1966, 1967) resulted in no growth with Melampsora lini 42
because germination was completely inhibited. Estimations of the number of established colonies of Melampsora lini after
4 weeks in culture revealed 80% success on media with yeast extract or peptone, or both together. No distinction could be made in growth on the three different media. Of the total, about 5% showed very good growth at this stage (colonies up to 1 cm in diam) with profuse white aerial mycelium. With coconut milk, however, only germ tubes were produced as a loose mat on the agar surface. At this stage, transfer of all the small colonies (1-2 mm in diam) was attempted. The faster growing colonies v/ere left a further 2 weeks, during which time they produced more aerial mycelium. With the smaller colonies, survival on transfer was about 0.5%; with the faster growing colonies It was about 30%. In subsequent transfers of the latter an average of 50% survival was ob• tained. Entire colonies could easily be lifted from the agar
surface and It was apparent that only the stromata and not
the mycelium were in contact with the medium.
In preparing Turel's medium II it was noted that
precipitation of calcium phosphate occurred, as a result of
the reaction of the dibasic potassium phosphate with cal•
cium nitrate. A medium with modified inorganics (see
page 39) was therefore used for the transferred colonies.
Good growth was obtained on this modified medium (Fig.
II-l) and this should prove useful In future nutritional
studies with different races of Melampsora lini. In the initial colony an orange stroma formed be• neath the mycelium after $ to 8 weeks. In transferred colonies this original stroma turned brown and a new orange stromal region developed around it (Fig. II-2). The initia• tion of fresh mycelial growth was always associated with this latter region. Successful transfer of the rust was only ac• hieved when large stromal segments (from 5 to 10 mm in diam) were included in the fungal material.
With the liquid culture system shaking was stopped after 2 weeks as it was found that the loose mat of germ tubes tended to become attached to the walls of the culture flasks and removed from the medium. After 5 weeks (3 weeks
in still culture) small amounts of mycelium appeared in 23
out of 24 flasks. In about 2$% of these colonies development
was good (3-6 mm diam). Colonies grew on the surface of the
medium and had a similar appearance to those formed on solid
media, having orange stromata and profuse aerial mycelium.
A few flasks had three or four individual colonies. It is
evident, therefore, that both initial and transferred col•
onies can be grown on the medium with modified inorganic
salts.
2. Melampsora lini, Strain 2
Growth of strain 2 was achieved on the medium des•
cribed (see Materials ar.d Methods) . After 6 weeks about 60%
oi the tubes had mycelial growth; 15% of the cultures were
larger (3-5 mm) and had orange stromata. By 8 weeks about 44
Figure II-l. A 12-day-old colony of strain 1 (fourth transfer) growing on a medium with in• organics, sucrose, yeast extract, and peptone. "White aerial mycelium covers a central stromatal region, the latter projecting through the colony at one point. X 9.
Figure II-2. Same colony viewed from below. Arrows denote a visible demarcation between the old -central brown stromatal region (ori• ginal inoculum) and an orange zone which formed subsequent to transfer. X 6.
46
30% of the colonies had orange stromata and extensive aerial mycelium (Fig. II-3). In the other 50% of established cul• tures, colonies were white and fluffy (1-2 mm) but without stromata (Fig. II-4); 20% of the tubes contained nothing be• yond a network of germ tubes. An interesting observation made on these germ tubes was that anastomosis had occurred
wherever they were in contact with each other (Fig. II-5).
After 10 weeks a large proportion of the small colonies had
grown and developed orange stromata. At this time half the
cultures which showed no growth at 8 weeks had now produced
aerial mycelium.
Colonies transferred at '8 weeks grew on all the
media tested. It was not possible to determine consistent
differences in growth between media because the variability
in colony size was too high,
3. Cytological Studies with Melampsora lini
Staining with haematoxylin revealed binucleate,
septate mycelium in both initial and transferred colonies
of both races (Figs. II-6 and II-7). In about 20% of the
cells only one nucleus was noted. This could have been due
to migration of one of the nuclei (Fig. II-8). Anastomosis
of hyphal cells was observed in a few cases (Fig. II-9).
Examination of the orange stromata of the initial
colonies revealed large numbers of thin-walled rounded
bodies (Fig. 11-10) with two nuclei not visible in the
figure. In a small number of cases these same types of 47
Figure IT-3 . An 8-week-old colony of strain 2 grown from aseptic uredospores. X 13.
Figure II-4. An 8-week-old colony of strain 2 of small size (1-2 mm) consisting entirely of aerial mycelium. X 14.
Figure II-$, Fusion of germ tubes of uredospores of strain 2. Arrow indicates a region of germ tube fusion. Phloxine/KOH. X 768.
Figure II-6. Binucleate hyphae of initial colonies of strain 2. Hyphal swellings are pro• minent. Haematoxylin. X 768.
49
Figure 11-7. Binucleate mycelium of a transferred colony of strain 1. A mitotic figure (anaphase) is visible, indicated by an arrow. Haematoxylin. X 2307.
Figure I1-8. Hyphal cells from an 8-week-old colony of strain 2. One cell has two nuclei, others have one. The arrow denotes a cell in which the two nuclei are separ• ated. Haematoxylin. X 1920.
Figure II-9. Hyphal anastomosis. An 8-week-old colony of strain 2. Haematoxylin. X 480.
Figure 11-10. Thin-walled spherical spore cells from stromata of an Initial colony of strain
inoculum. Phloxine/KOH. X 234.
Figure 11-11. Thin-walled cells interspersed by short hyphal cells. Initial colony of strain 1 (8 weeks old). Phloxine/KOH. X 560. 50 51
structures were seen interspersed by short hyphal cells (Fig.
11-11). These cells were sometimes formed in chains organ• ized into a 'microsorus'-like structure (Fig. 11-12). There were also small numbers of thick-walled, non-pigmented cells with verrucose walls (Fig. 11-13), and with dimensions similar to those of typical uredospores (18.6 to 31.0 by 24.0 to 37.2 microns), though no germ pores were observed. Some of these were connected by hyphal structures (Fig. 11-14). In trans• ferred colonies in which the original uredospores were no
longer present, thin-walled spherical cells and thick-walled
cells with elongated and variable shapes v/ere located in the
brown stromata (Fig. 11-15). Attempts to stain the latter with haematoxylin to differentiate their nuclear condition
usually failed, but in a few preparations two nuclei were ob•
served. Some of these spore-like cells formed terminally and
others in chains (Figs. II-16, 11-17, and II-18). Their dimensions were similar to those of typical teliospores
(24.8 to 31.0 by 43.4 to 60.0 microns).
No cytological differences were noted between the
two strains.
DISCUSSION
Growth of both strains on media based upon those
used by Turel (1969a) confirms her findings. Furthermore,
the use of low temperatures (3-I7°C) during the inoculation
and transfer procedures improved the consistency of growth 52
Figure 11-12. 'Microsorus* structure from an initial colony of strain 1 (7 weeks old). Phloxine/KOH. X 768.
Figure 11-13. Non-pigmented, thick-walled cells. No germ pores visible. Verrucose. From initial colony of strain 2. Haematoxy- lin. X 1200.
Figure 11-14. Same, showing two of these cells joined by short hyphal cells. Haematoxylin. X 480. 53 54
Figure 11-15. Dense spore cluster from stroma of a trans• ferred culture of strain 1. Arrows denote thick-walled cells with variable shapes. Phloxine/KOH. X 307.
Figures II-16 and 11-17. Spore-like cells formed ter• minally and in chains. Some resemble telio- spores, other are atypical. From a trans• ferred colony of strain 1. Phloxine/KOH. x 307.
Figure 11-18. A cell which probably most closely resembles a typical teliospore. Phloxine/KOH. X 768. 55 56
(cf. 80-90% growth with 20% growth obtained in earlier work). This finding also supports Turel's (1969b) work. However, there was still a great deal of variability in growth with
some cultures exhibiting a lag period as long as 8 weeks be•
fore the initiation of mycelial growth from the original
spore inoculum. This contrasts with the high consistency and
more rapid growth obtained with Puccinia graminis f. sp.
tritici, ANZ 126-6,7 using gelatin suspensions of uredospores
(Coffey et al. 1969) . The ability of both strains to grow on media with
different organic constituents and some changes in inorganics
suggests that their nutritional requirements may be relatively
non-specific.. The precipitation which occurred during prepara• tion of one of the media is a warning that care must be exer•
cised in Interpreting the nutrition of microorganisms in terms
of the contents of the culture medium. A modified medium prepared without the precipitation of inorganic salts supported
growth of Melampsora lini and will be used in future work. The
fact that casamino acids supported growth of transferred
colonies of strain 2 suggests that amino acids may be the active component of the organic complexes used. However,
transferred colonies are not suitable material for nutritional
studies since there could be a large carry-over of nutrients
from the previous medium. If there were any differences in
growth rates between the two strains, these were not apparent
because of the high inherent variability in the cultures
obtained. This variability is in part due to variation in 57 the length of the lag phase. Genetic or physiological factors or both may be involved here. It is likely that the strains used contain a heterogenous mixture of spores (Fincham and
Day 1963), possibly with a wide range of abilities for sapro- phytism. Cytological evidence showed that the mycelium is
generally binucleate and septate.
Similar spore-like cells were found in both Initial
and transferred material of the two strains. Many of these
cells were thin-walled and spherical but cells with shapes
and dimensions similar to uredospores and teliospores were
recorded. They were certainly not typical spores, and their
formation in a tough leathery zone rendered them unavailable
for germination studies. It is probable that the physical
and chemical conditions necessary for normal sporulation pro•
cesses were not achieved. Ideally spore formation should
occur superficially on the surface of the mycelial network as
it did with Puccinia graminis (Williams et al. 1967) but even
with this species it was not always found (Bushnell 1968,
Coffey et al. 1969).
Early work of Hotson and Cutter (1959, I960, 1951)
reported the growth of strains of Gymno.sporangium juniper!-
virginianae Schw. and Uromyces ari-triphylli Schw. on artificial
media. More recently, the growth of representatives from
two different rust families, Melampsoraceae and Pucciniaceae,
and of different physiological strains within a single
species, Melampsora lini (Pers.) Lev., suggests that in time
all rust fungi will be grown in axenic culture. The sensi- 53 tivity to temperature and other, as yet undetermined, environ mental factors has obviously been a contributory factor in the many previous abortive attempts at culturing the
Uredinales. 59
REFERENCES
Bushnell, W.R. 1968. In vitro development of an Australian isolate of Puccinia graminis f. sp. tritici. Phyto• pathology 5W: 526-527.
Coffey, M.D., A. Bose, and M. Shaw. 1969. In vitro growth of gelatin suspensions of uredospores of Puccinia graminis f. sp. tritici. Can. J. Botany 47: 1291- 1293.
Cutter, V.M., Jr. 1959(1961). Studies on the isolation and growth of plant rusts in host tissue cultures and upon synthetic media. I. Gymnosporangium. Mycoiogia 51: 248-295.
Cutter, V.M., Jr. 1960(1961). Studies on the isolation and growth of plant rusts in host tissue cultures and upon synthetic media. II. Uromyces ari-tryphylli. Mycoiogia 52: 726-742.
Fincham, J.R.S., and P.R. Day. 19-63. Fungal genetics. In Botanical Monographs. Vol. 4. Blackwell Scientific Publications, Oxford, p. 254.
Hotson, H.H. , 'and V.M. Cutter, Jr. 195L The isolation and culture of Gymnosporangium iuniperi-virginianae Schw. Proc. Nat. Acad. Sci. U.S. 37: 400-403.
Scott, K.J. 1968, The initiation of saprophytic growth of Puccinia graminis. Abstract of papers. First International Congress of Plant Pathology, London.
Turel, F.L.M. 1969a. Saprophytic development of the flax rust Melampsora lini, race No. 3. Can. J. Botany 47: 821-823.
Turel, F.L.M. 1969b. Low temperature requirement for sapro• phytic flax rust cultures. Can. J. Botany 47: 1637-1638.
Williams, F.G., K.J. Scott, and J.L. Kuhl. 1966. Vegetative growth of Puccinia graminis f. sp. tritici in vitro. Phytopathology 56: 1418^1419.
Williams, P.G., K.J. Scott, J.L. Kuhl, and D.J. Maclean. 1967. Sporulation and pathogenicity of Puccinia graminis f, sp. tritici grown on an artificial medium. Phyto• pathology 57: 326-327. 60
CHAPTER III
In vitro Grov/th of Wheat and Flax Rust Fungi
on Chemically Defined Media
ABSTRACT
A chemically defined, liquid medium containing
Czapek's minerals, Ca++, glucose, aspartic acid, glutathione
and cysteine and seeded with uncontaminated uredospores,
"supported good vegetative growth and sporulation of an Aus•
tralian isolate of wheat stem rust (Puccinia graminis Pers.
f. sp. tritici (Erikss. & Henn.), race ANZ 126-6,7). Only one
of six North American isolates tested (Race 38) formed colon•
ies which approached those of race ANZ 126-6,7 in final size
and general morphology. 5'AMP had no effect on growth, but
cyclic AMP inhibited growth after uredospore germination.
Highly reproducible growth of flax rust (Melampsora
lini (Ehrenb,) Lev., Race 3) was obtained routinely by seed•
ing uncontaminated uredospores on a solid medium containing
Difco bacto-agar, sucrose, Knop's mineral salts and micro•
nutrients plus yeast extract, peptone and bovine serum al•
bumen (BSA). BSA was shown to overcome the initial inhibi•
tion of growth and to promote the formation of uredospores.
It could not be replaced by putrescine. Vegetative cultures
could be maintained indefinitely by subdivision at the 61
vegetative stage and transfer to the same medium minus BSA.
Surface sterilized flax cotyledons became infected when placed with their cut ends in contact with these colonies.
A chemically defined liquid medium containing suc• rose, Knopts mineral salts, micronutrients, aspartic (or glu• tamic) acid and cysteine supported the growth of vegetative colonies of Melampsora lini from uncontaminated uredospores in a highly reproducible manner. The formation of uredospores and teliospores by these colonies was controlled by (a) the
level of Ca++ (as Ca^O-^^) and (b) the number of colonies per
flask. With 40 to 60 colonies per flask, uredospore formation
occurred in 60 to 70% of the colonies at a Ca++ level of 8.5 mM
(2.0 g/l Ga(N0Q)0). A decrease in the Ca++ level to 4.25 mM
or in the colony frequency to 10 per flask resulted In only
infrequent sporulation. The uredospores produced in vitro
infected intact, week-old flax cotyledons in a normal manner.
INTRODUCTION
The successful growth of an isolate of Puccinia
graminis Pers. f. sp. tritici (Erikss. & Henn.), Race ANZ 126-
6,7 by Williams et al. (1966, 1967) from uredospores seeded on
a simple medium indicated the feasibility of culturing some
of the rust fungi traditionally known as obligate parasites
and stimulated research in this field in several laboratories.
A number of heteroecious and autoecious rusts belonging to
the Pucciniaceae and Melampsoraceae have subsequently been 62 grown in culture free from their hosts (Bushnell 1968, Coffey et al. 1969, Turel 1969, 1971, Maclean and Scott 1970, Wong and Willets 1970, Kuhl et al. 1971, Masin and Andreev 1971,
Bose and Shaw 1971, Jones 1972). Recently Vassil'ev and
•Saplina (1971) v/ere able to establish axenic culture of Mel• ampsora pinitorqua (A. BR) Rostr., the causal organism of stem rust of pine, starting from basidiospores, but their account is too brief to allow a critical assessment of their results, which require confirmation.
The requirements for axenic growth of these fungi
Include a suitable carbohydrate source, mineral nutrients In
the 'correct' balance (Turel 1969, Coffey and Shaw 1972,
Coffey and Allen 1973) and an organic nitrogen source such as
peptone, bovine serum albumen and yeast extract. The calcium
level appears to be particularly important (Coffey and Allen
1973). Saprobic growth of the rusts is characterized by a
lag phase followed by vegetative growth and the formation of
a stroma. The formation of uredospores and teliospores is
irregular. Indeed, under certain cultural conditions, some
strains remain persistently vegetative (Maclean and Scott
1970, Howes and Scott 1972). Successful re-infection of the
host with mycelium has been achieved by several workers
(Williams et al. 1967, Bushnell 1968, Hartley and Williams
1971, Turel 1971). Re-Infection by uredospores produced in
axenic culture has only been reported by two groups (Williams
et al. 1967, Bose and Shaw 1971). 63
Currently, attempts are being made in several labora• tories to develop defined media for the axenic growth of rust fungi by substituting mixtures of amino acids for more complex sources of organic nitrogen. Thus Kuhl et al. (1971) reported that Puccinia graminis tritici Race ANZ 126-6,7 showed some growth on a medium in which yeast extract and peptone were re• placed by a sulphur amino acid such as cysteine. As measured by the total nitrogen content of the mycelium growth on this medium v/as about half that on yeast extract or peptone, and the authors concluded that no medium or method of inoculation
-was completely reliable. Foudln and Wynn (1972) achieved a
greater degree of success with the same strain of wheat rust
on a defined medium consisting of 1% agar, 3% glucose, Czapek's
minerals, Burkholder and Nickell's trace elements and a mix•
ture of 16 amino acids in the proportions found in purified
casein hydrolysate. Growth on this medium was equal to that
in the presence of casein hydrolysate and superior to that on
Evans' peptone - a brand of peptone which Williams et al. (1967)
had found to be particularly suitable for the growth of this
strain. Both uredospores and teliospores were formed on
. Foudin and Wynn's (1972) medium, but. there is only a passing
reference to sporulation in their paper and pathogenicity
trials with spores produced in axenic culture were not re•
ported .
Coffey and Shaw (1972) reported that a mixture of
eleven 1-amino acids (found by analysis to be common to both
Evans' peptone and Difco yeast extract and equivalent in 64
amounts to those supplied by 0.1% peptone and 0.1% yeast ex• tract), plus sucrose and mineral nutrients, supported the sustained growth of an established isolate of their strain 1 / of Melampsora lini (Ehrenb.) Le V. Unfortunately the percen• tage of successful transfers to the amino acid medium was only
25% compared with 80% in transfers to the basic medium plus peptone and yeast extract. In addition the "final size of the colonies was only about half that obtained with peptone and yeast extract. Further trials indicated that only 1-alanine and 1-methionine were essential for the growth of transferred cultures, but, once again, the results were irregular and growth was poor.
In subsequent work at Wisconsin, using Coffey and
Shaw's strain'1 and race 79 of Melampsora lini, Coffey and
Allen (1973) reported that the requirements of primary (i.e.
derived directly from uncontaminated uredospores) and estab•
lished (i.e. transferred) cultures were different. The de•
velopment of mycelial initials leading to the formation of
primary cultures did not occur on as wide a range of carbo•
hydrates as would support the growth of established cultures
and a combination of glutamic acid and cystine was the best
substitute for peptone or casamino acids. Methionine did not
support the growth of primary cultures at the level tested.
Established cultures grew in the presence of any one of ala•
nine, glycine, serine, aspartic acid, asparagine, glutamic
acid, glutamine or arginine, In combination with either 65 cysteine or cystine. Methionine could only be substituted for cysteine or cystine if established colonies were first conditioned on the 11-amino acid mixture used by Coffey and
Shaw. Coffey and Allen (1973) also reported the first success• ful axenic growth of Puccinia helianthi Schw. on sucrose, in• organic salts, bovine serum albumen (BSA) and peptone or cas- amino acids or tryptone. Cysteine in combination with either aspartic or glutamic acids or alanine could be substituted for peptone, but no substitute for BSA was found.
It must be emphasized that in all the experiments of
Coffey and Shaw (1972) and Coffey and Allen (1973), the growth of Melampsora lini on defined media solidified with Difco-Bacto agar was not predictable. The number and final size of the colonies which developed from mycelial initials v/ere much smaller than in the presence of a complex organic substance such as peptone. Moreover, the morphogenetic pattern of development was far from normal with respect to the forma• tion of a stroma and the differentiation of uredospores and teliospores. Indeed, sporulation was rare, and the only di•
rect reference to it in these two papers is Coffey and Allen's
(1973) statement that the stroma of some cultures of Puccinia
helianthi contained typical, thick-walled teliospores.
More recently Turel (1973) has achieved good sapro•
bic growth of both primary (i.e. derived directly from sterile
uredospores) and established (i.e. transferred) cultures of
race 3 of Melampsora lini on defined media containing Difco
Bacto agar, modified Knop's minerals, micronutrients, sucrose 66
and aspartic acid plus either cysteine or glutathione (pri• mary cultures) or cystine (established cultures). Alanine and methionine supported only slow and sporadic growth of primary cultures. Established cultures started in the pre• sence of yeast extract did not survive on transfer to the basic medium with alanine and methionine, confirming the ob• servations of Coffey and Allen (1973). Turel (1973) reported great variation in the development of primary cultures on her defined media, both within and between isolates from one spore harvest to another. She did not record the development of uredospores; conspicuous clusters of teliospores formed in the older parts of established cultures In one experiment, but not in another.
Several years prior to Williamss (1966, 1967) work,
Cutter (1959, I960) had demonstrated by the use of rust-
infected callus cultures that rusts could be isolated axenic•
ally, following a period of adaptation on host tissue cultures maintained in Gautheret's medium. He was, In fact, able to
isolate several strains of Gymnosporangium juniperi-virginianae
and Uromyces ari-triphylli. Although his technique suffered
from a lack of reproducibility in terms of successful isola•
tion, a few of the isolates were maintained and subcultured
for a number of generations on solid and liquid media. Accord•
ing to Cutter the appearance of the rust in vitro was far from
normal and the incidence of sporulation was very low. This
situation loo gutter ~ - • Vc -c- th?t typical sporulation is 67
a host-induced phenomenon (Hotson and Cutter 1951). However,
re-infection of host callus tissue as well as the intact host was achieved successfully, even after prolonged subculturing
on artificial medium.
The validity of Cutter's approach, using dual-mem•
bered callus cultures v/as confirmed recently by Hollis et al.
(1972). Starting with rust-infected tissue "cultures they re•
ported the sustained axenic culture of Cronartium fusiforme
Hedge, and Hunt ex Cumm, the fusiform rust of slash pine, on
a complex defined medium containing 22 amino acids, glutamine,
"glutathione, vitamins, growth substances, citric acid, glucose,
and agar. One culture produced an aecial sorus with typical,
mature aeciospores in a medium containing yeast extract and
peptone. Similarly, working in our laboratory, Harvey (un•
published) was able to isolate Cronartium ribicola, the white
pine blister rust fungus, from tissue cultures of its host.
Vegetative growth occurred after isolation of the fungus on
a broad range of substrates.
It will be evident from this brief review that al•
though much progress has been made in the axenic culture of
rust fungi, nevertheless all the results described suffer from
a satisfactory degree of reproducibility, particularly in rela•
tion to sporulation. Moreover the experiments with 'defined'
media are marred by the inclusion of bacto agar, which may well
serve as a source of unidentified materials. The primary ob•
jective of the work to be described in this paper was to de•
velop fully defined liquid media for the wheat and flax 68
rusts - media which could be sterilized by millipore filtra• tion only and which would support growth and the formation of uredospores capable of reinfecting host tissue,
MATERIALS AMD METHODS
1. Production of Uncontaminated Uredospores
Trials were conducted with an isolate of race ANZ
126-6,7 of Puccinia graminis Pers. f, sp. tritici(Erikss. &
Henn.) originally supplied by Dr. P.G. Williams and with 6
North American isolates, 15B4, 56, 17, 32-113, 15B-1L and 38
supplied by Dr. Gordon Green. Inoculum of all these isolates was raised on the first seedling leaf of Little Club wheat
grown in a controlled environment cabinet set at 500 ft-c.
and 20°C for 16 h followed by 8 h darkness at l6°C. All hand•
ling procedures were carried out in glove boxes to prevent
cross-contamination between isolates. Uncontaminated uredo•
spores were obtained as described by Bose and Shaw (1971).
Race 3 of Melampsora lini (Ehrenb.) Lev. was pro•
pagated on Bison flax. Uncontaminated uredospores were
raised on cotyledons on Gamborg*s B5" medium without auxin
(Gamborg et al. 1968), Better results were obtained when the
cotyledons were dried on a sterile filter paper after surface
sterilization before transfer to Gamborg's medium. Using
this technique at least two batches of uredospores can be ob•
tained from each cotyledon. 69
2. Culture Media
The basic components (per liter) of the four media used are given below. In addition, each contained a source of organic nitrogen. All chemicals used were of reagent grade.
MI. Czapek1s-glucose, for wheat rusts: Glucose, 30 g;
NaN03, 2.0 g; KC1, 0.5 g; MgS04-7H20, 0.5 g; KH2P04,
1.0 g; FeS0^-7H20, 10 mg.
Mil. Czapek's-glucose plus micronutrients, for wheat rusts:
Components of MI plus 0.5 or 0.8 ml micronutrient solu•
tion containing: sequestrene, 13% NaFe (Geigy), 10 g;
MnS04«7H20, 447 mg; KI, 10 mg; NiCl2'6H20, 18 rng;
CoCl2«6H20, 18 mg; Ti(S0^)2'9H20, 42 mg; ZnSO^, 35 mg;
CuSO^«'5H20, 15 mg; BeSO^, 20 mg; KB^PO^ concentrated.
0.2 ml; glass distilled water, 200 ml. (Modified
Berthelot's solution; Turel 1969).
Mill. Knop's-sucrose plus micronutrients, for flax rust:
sucrose, 40 g; Ca(N03)2, 2.0 to 6.0 g; KN03, 0.25 g;
MgS04'7H20, 0.25 g; KH2P04, 0.25 g; NH^JTC^ , 40 mg;
K2HP04, 0.75 g; 0.B ml micronutrient solution as in
Medium II. (With solid media the Ca(N03)2 was added
later, just before the plates were poured).
MIV. For flax rust: as for Medium III except sucrose, 50 g;
and NH,_N0^ , 20 mg. 70
When required, media were solidified with 15 g/l
Difco Bacto agar, sterilized by autoclaving at 15 psi for 20 minutes, and 10 ml aliquots poured into plastic petri dishes
(Labtek 60 x'20 mm). After inoculation, plates were sealed with parafilm to minimize evaporation.
3. Sources of Organic Nitrogen and Sulphur
Evans' peptone (0.1% w/v, Evans Medical Ltd., U.K.),
Difco yeast extract (YE 0.1% w/v), and bovine serum albumen
(BSA, 1% w/v; Fraction V; defatted according to Chen 1967) served as sources of organic nitrogen in undefined media.
Yeast extract and peptone were always added before final ad•
justment of the pH and before autoclaving. BSA was sterilized
by millipore 'filtration (pore size 0,22 jj,) and added after autoclaving and cooling the medium.
In defined media either the 11 1-amino acid mixture* of Coffey and Shaw (1972) or combinations of aspartic acid, cysteine and glutathione were substituted for YE, peptone and
BSA. Fully defined media contained no agar and were not auto-
claved. All amino acids and glutathione were obtained from
Sigma Chemical Company.
''Composition of amino acid mixture (Coffey and Shaw 1972).
1-glutamic acid, 770 mg/l; 1-leucine, 160 mg/l; 1-phenylal-
anine, 150mg/l; 1-alanine, 120 mg/l; 1-isoleucine, SO mg/l;
1-aspartic acid, 80 mg/l; 1-valine, 60 mg/l; 1-methionine,
60 mg/l; threonine, 50 mg/l; 1-serine, 50 mg/l; 1-glycine, 40 mg/l. 71
Liquid media were sterilized either by autoclaving
or, when complex organic nitrogen sources (YE and peptone)
were replaced with amino acids mixtures, by filtration through
Nalgene Millipore filters (pore size 0,22 u.) . When used,
1 MP, CAMP and theophylline were added to the cool medium after
millipore filtration. Unless otherwise stated, 10 ml aliquots
were dispensed into $0 ml pyrex culture flasks (4.5 cm internal
base diameter) which were closed with bacticapall polypropy•
lene caps. The pH of all media v/as adjusted to 6 to 6.4 for
wheat stem rust and 5.5 for flax rust, except in trials with
high levels of calcium, in which the pH was kept strictly at
5.0 to avoid precipitation.
4. Inoculabion of Media
All inoculations v/ere conducted on an airflow bench
under aseptic conditions at 19 + 2°C. Normally uredospores
were dusted on to the surface of the medium at a density
corresponding to 1000 to 2000 per mm . In those experiments
in which flax rust uredospores v/ere suspended in gelatin for
inoculation of solid medium the spore density v/as 400 to 900
per mm^. Ten replicate plates were .used with solid media and
5 replicate flasks with liquid media. Trials with liquid
media were repeated twice. All plates and flasks were in•
cubated at 17°C in darkness. 72
5. Assessment of Growth Growth of both Melampsora lini and Puccinia graminis was rated according to one of the following five types.
I no growth beyond germ tubes.
II sparse mycelial development, stroma not developed.
Ill small white tufted colonies; yellowish stroma on under surface.
IV small white 'button' colonies; orange stroma for flax rust; brownish stroma for wheat rust.
V • large white 'button' colonies with prominent stroma; under the proper conditions these colonies developed sporulating pustules on their upper surfaces.
Microscopic observations were made using phloxine/
KOH stain and photographs were takren with a Carl Zeiss photo-
microscope using bright field illumination and a 35 nun camera.
RESULTS
Wheat Rust
1. Trials with Solid Media Petri plates containing solid medium MI with 0.1% YE,
0,1% Evans' peptone and 1% w/v BSA were inoculated with dry
uredospores of each of races 15B4, 55, 17, 32-113, 15B1L, 33
and ANZ 126-6,7 of Puccinia graminis tritici. ANZ 126-6,7
was included to provide a standard by which the growth of the
North American races could be judged. Races 15B-1L, 17, and
56 turned pale brown and showed distinct signs of senescence
after 1 week. Races 15B4, 38, 32-113 and ANZ 126-6,7 73 developed colonies composed of septate aerial hyphae and a leathery stroma. At this stage the colonies of race 38 and
ANZ 126-6,7 were of type 4. Growth of race 32-113 was less advanced and-the stroma was much thinner. Race 15B4 remained vegetative for about 2 months and microscopic examination in• dicated that the hyphae were composed of small circular cells.
These trials showed that growth approaching that
characteristic of race ANZ 126-6,7 v/as obtained with race 38
and there v/ere differences in the degree of development of
the different races on the same medium.
2. Trials with Liquid Media
A. Growth of races 15B4, 38\ 32-113 and ANZ 126-6,7
On .the basis of the preliminary trials with solid
medium, liquid Mil medium with peptone and BSA plus 0.5 g/l
Ca(NO ) was inoculated with each of races 15B4, 38, 32-113
and ANZ 126-6,7. Race 1$B4 developed fluffy white colonies,
but growth was very slow and no stroma developed (Fig. III-l).
Race 32-113 developed profuse aerial hyphae but did not form
a stroma (Fig. III-l). After 4 weeks race 38 developed a
brown stroma submerged in the medium (Fig. III-l) and growth
appeared to be equivalent to that of race ANZ 126-6,7 with
respect to both the final size and the general morphology of
the colonies which were of type 4.
B. Effect of cAMP on growth of ANZ 126-6,7
Since cyclic AMP is implicated In hormone action and
the control of enzyme synthesis and morphogenesis in micro- 74
organisms (Robison _et al. 1971) it was considered worthwhile to test its effect on the growth of the wheat rust fungus.
The results are presented in Table III-I. Low concentrations
(0.1 mM and 1 mM) had no apparent effect, but 10 mM cAMP com•
pletely inhibited growth of ANZ 126-6,7. Complete inhibition
of growth was also observed'with 1 mM cAMP in the presence of
1 mM theophylline (Fig. III-2). The latter inhibits phospho•
diesterase and thus increases the effective level of cAMP.
5'AMP alone or in combination with theophylline had no effect
on growth.
C. Effect of aspartic acid, cysteine, glutathione and cal•
cium on the growth of ANZ 126-6,7
In 'our attempts to develop a completely defined
medium for the growth of Puccinia graminis using only a carbo•
hydrate source, amino acids and mineral nutrients we found that
it was essential to provide a source of calcium. This element
is not a constituent of the standard Czapek's mineral nutrient
solution used in earlier attempts to culture the wheat rust
fungus. Using liquid medium Mil containing various combina•
tions of aspartic acid, cysteine and glutathione plus calcium
nitrate, best results were obtained when both amino acids and
the peptide were combined with from 2 g/l (8.1 mM) to 6 g/l
(25.4 mM) Ca(N03)2, as shown by the data in Table III-II. At
concentrations of Ca(N03)2 less than 2 g/l (8.1 mM) the length
of the lag phase was increased and the reproducibility of the
results was markedly decreased. In our experience the growth 75
Figure III-l. North American isolates of wheat stem rust showing vegetative growth on liquid medium Mil. Photographed after 6 weeks. (A) Race 32-113, (B) 15B-4, (C) 38. X 1.0. Figure III-2. Growth and development of wheat stem rust race ANZ 126-6,7 on liquid medium MI. (A) 1 mM cAMP, (B) 1 mM theophylline, (C) 1 mM cAMP plus 1 mM theophylline. Note: Ihe growth in (C) is the result of uredospore germination only. X 1.2.
Figure III-3. Wheat stem rust ANZ 126-6,7 developed on a chemically defined medium Mil having aspartic acid, cysteine and glutathione. Darker region at the top of the colonies indicate the position of developing uredopustule. X 2.4. 76 77
TABLE III-I. Effects of cyclic MP and theophylline on growth of Puccinia graminis (race ANZ 126-6,7).
Type of Colony on Medium MI with 0.1% peptone + 0.1% peptone 1% BSA
Control IV V cAMP 0.1 mM1 III V cAMP 1.0 mM III V cAMP 10 mM II II Theophylline 1 mM III IV Theophylline 1 mM and cAMP 1 mM II II Theophylline 1 mM and 5 tAMP 1 mM III V 5T AMP IV IV
Identical results were obtained with dibutyryl cAMP.
s TABLE III-II. Effect of amino acids, glutathione and Ca(N03)2 on the growth of Puccinia graminis tritici (race ANZ 126-6,7).
Weeks to Development Type -of of Colonies Staling Growth Remarks Organic Nitrogen Source Level of Ca(N0o)2 S/
1. Aspartic + glutathione 6 4 III No sporu• lation
2. Aspartic + cysteine 6 2-3 4 IV No sporu• lation
3. Aspartic + cysteine + glutathione 6 6-7 V Uredospores formed after 8 weeks
4. Aspartic + cysteine + glutathione 6-7 V Uredospores formed after 8 weeks
All treatments contained the basic medium Mil (see text). Aspartic acid 4.5 mM (600 mg/
100 ml); cysteine 4.6 mM (56 mg/100 ml); glutathione 3.3 mM (101.4 rng/lOO ml). 03- ' • 79
of ANZ 126-6,7 was better and more easily reproducible on this
new defined medium (Treatments 3 & 4, Table III-II) than on media containing peptone, BSA and YE (Fig, III-3) . In par•
ticular the cultures In about 50% of the flasks produced uredo•
spores after 8 weeks. There was no release of brown pigment
into the medium as in our earlier experiments (Bose and Shaw
1971). Teliospore formation was infrequent. The morphology
of the uredospores v/as very similar to that of those described
earlier (Bose and Shaw 1971). These spores were capable of
reinfecting wheat seedlings when placed on exposed mesophyll
tissue of the first seedling leaf.
Flax Rust
1. Trials with Solid Media
A. Effect of BSA
Medium Mill with 2 g/l Ca(N03)2 plus 0.1% Evans' pep•
tone, 0.1% YE and 1% defatted BSA was used routinely for the
culture of flax rust. Uredospores were seeded in clumps (1000
to 2000 per mm ) either directly onto the surface of the
medium or onto pre-sterilized Millipore filters lying on the
agar surface (Quick and Cross 1971). Type 5 colonies with a
luxurious fluffy mycelial mat and an underlying orange stroma
developed, usually in about 5 weeks (Fig. III-4). In the pre•
sence of BSA, the aerial parts of these colonies often de•
veloped in to uredosori. Subculturing was therefore done at
the vegetative stage by division and transfer to the same
medium minus BSA. In our experience cultures of this type
can be subcultured independently. Continued incubation of 80
the plates resulted in the development of secondary colonies
from the residual germ tubes at the sites of the original pri• mary colonies which had been removed for transfer. At the
same time colonies also developed in regions of the plates which had originally been seeded at lower densities of 400- o 500 uredospores per mm .
These observations suggested that the Mill medium
plus BSA might be interacting in some manner with the inhibi•
tors released from uredospores which prevented germination in
our earlier experiments (Coffey et al. 1970). In order to test
this possibility uredospores were therefore suspended in 15% w/v gelatin and seeded onto solid medium Mill with and without
BSA. After eight weeks large colonies had developed over the
inoculated zones on the plates with BSA (Fig. III-?). These
colonies were about 50% larger in diameter than the colonies which developed on plates seeded directly with dry uredospores.
They remained vegetative and collapsed after 12 weeks. Negli•
gible growth occurred on the control plates lacking BSA. This
result confirms the conclusion that BSA overcomes the initial
inhibition of growth.
B. Reinfection of host using vegetative colonies grown on
solid medium
Surface sterilized cotyledons of Bison flax were
placed with their cut ends in contact with month old colonies
of flax rust on solid Mill medium plus peptone, BSA and YE.
After 4 weeks rust mycelium developed ectoparasitically on 8l the upper surface of the cotyledons, which became completely covered by the fungus. The ectoparasitlc mycelium then pro• duced- masses of orange coloured uredospores (Fig. III-6). Nor• mal erumpent_rust pustules developed on the lower surfaces of the cotyledons. Uredospores from these pustules infected
intact flax plants in a normal manner.
2. Trials with Liquid Media
A. Preliminary trials and effect of BSA
With liquid media it was found that, after the de•
velopment of a stroma, isolated flax rust colonies have a
marked tendency to sink as a result of slight disturbances
during handling of the culture flasks (Fig. III-7). This pro•
blem was never encountered with wheat rust colonies. It was
overcome by seeding flax rust uredospores as evenly as possi•
ble over an area at least 3 cm in diameter. Anastomosis and
interlocking of the germ tubes formed a mat which, presumably
due to surface tension effects, held the developing colonies
above the liquid surface.
The results in Table III-III show that Mill medium
containing either 0.1% Evans' peptone plus YE or Coffey and
Shaw's (1972) 11-amino acid mixture, but lacking BSA,
supported only poor growth with respect to both the percentage
number of flasks in which growth occurred and to the type of
colony formed (Type 3). Addition of 1% BSA markedly increased
the percentage of flasks showing growth and resulted in the
formation of type 5 colonies (Fig. 8). Normally when 82
Figure III-4. Flax rust colony initials raised on medium Mill having YS, peptone and BSA. Photographed after 6 weeks.
Figure III-5. Colonies of flax rust originating as a result of gelatin spore suspension seed• ing on medium Mill. (A) medium contain• ing peptone, note very little visible growth. (B) peptone plus BSA, photographed after 8 weeks,
Figure III-6. Ectoparasitic mycelium of flax rust de• veloped on excised cotyledons. Arrows indicate the region of uredospores. Photographed after 6 weeks. X 1.2.
x
TABLE III-III. Effects of different organic nitrogen sources on the growth of Melampsora lini (race 3).
Weeks to Develop• % of Flasks show- Type of Nitrogen Source ment of Colonies ing Growth Colony
0.1% Evan's peptone 7 20 III
0.1% Evan's peptone + 1% BSA 5- 6 50 V
1 8 Amino acids 35 II to III
1 60 Amino acids + 1% BSA 6- 7 V Amino acids1 + 40 1 mg/l putrescine 6-7 III Amino acids1 + 10 mg/l putrescine 50 III
All trials with liquid medium Mill containing 2 g/l Ca(N0^)2
1 Amino acid mixture of Coffey and Shaw (1972^ 85
colonies became submerged in the medium, development stopped, but when, as in a few isolated cases in the presence of BSA,
submerged colonies did continue to grow, development was ab• normal and was characterized by fragmentation and the forma-
'tion of several subcolonies which remained completely devoid
of pigmentation (Fig. III-9).
Since it is possible that one of the effects of BSA may be due to its polycationic properties, and since the poly-
amines 'and the closely related diamine, putrescine, are now
considered to have a relationship to nucleic acid metabolism
(Smith 1972) an attempt was made to replace BSA by putrescine.
The results of these trials are given in Table III-III and in•
dicate that while putrescine does not inhibit growth, it does
not substitute for BSA.
B. Effects of mineral nutrients
Flax rust showed only very poor growth on media con•
taining Czapek's mineral salts and micronutrients (i.e. medium
Mil, for wheat rust) together with aspartic acid and cysteine
i.e. when Czapek's were substituted for Knop's macronutrient
salts. One effect of substituting Czapek's for Knop's mineral
salts is the elimination of calcium. In other experiments the
growth of flax rust on Knop's minerals plus micronutrients (I.e.
medium MIV) together with aspartic acid and cysteine was con•
spicuously reduced when the calcium level was less than 1.5
gm/l Ca(NO^)9 (Table III-IV. These effects of varying the
levels of calcium are described in greater detail in Section C. 86
Figure III-7. Flax rust colony developed on liquid medium Mill containing peptone and BSA. (A) initial floating colonies, (B) isolated single colony is partially submerged. X 1.1.
Figure III-8. Flax rust colonies on liquid medium Mill (A) peptone plus BSA, (B) amino acid mixture plus BSA, photographed after 7 weeks growth, X 1.6.
Figure III-9. Flax rust development under submerged condition. Note the fragmented clustered hyphae. X 1.2.
Figure 111-10. Growth and development of flax rust on chemically defined medium MIV. (A) aspartic acid, cysteine plus gluta• thione, (B) aspartic acid plus cysteine, (C) aspartic acid plus glutathione. X 1.1. 87 88
Growth was also strongly influenced by the micronutrient solu• tion used. Thus, no growth occurred when all the components of the modified Berthelot's micronutrient solution was omitted from media Mill and MIV. On the other hand, the simultaneous
elimination of Ti(SO,)„, BeSO,, KI and HoP0, from the micro- 4 2 4 J 4 nutrient solution had no effect on growth and development.
These components were used routinely because it was observed that when they were included the stock solution remained sterile for long periods at room temperature. When Berthelot's micro•
nutrients were replaced with Turel's (1973) STE solution
(Fe(N03)2-9H20, 139 mg; EDTA-2Na, 134 mg; MnSO^, 895 mg; all
In 80 ml distilled H20) growth was highly unpredictable, with
only 10% of the culture flasks developing vegetative colonies.
C. Effects of aspartic acid, cysteine, glutathione and
calcium
The effects of aspartic acid, cysteine, glutathione
and calcium nitrate on the growth of flax rust in medium MIV
are summarized in Table III-IV. At concentrations similar to
those used by Turel* (1973) aspartic acid (22.5 mM), cysteine
(2.3 mM), glutathione (1.65 mM) and Ca(N0^)2 (2.0 g/l) re•
sulted in the development of vegetative type 3 colonies after
6 weeks in 20 to 50% of the flasks (Treatments 1 to 3, Table
III-IV). This is an unsatisfactory level of predictability.
*Turel used 28 mg/100 ml cysteine-HCl, i.e. I.65 mM cysteine. Growth and development of Melampsora lini (race 3) on completely defined TABLE III-IV. liquid media. of Colony Developed Flasks Aspartic Cys• Gluta• Calcium Within 4 After b showing Growth Remarks Trial Acid teine thione Nitrate Weeks Weeks (g/D No sporulation + + 2 III 50 1 + 30 No sporulation 2 + + 2 III + 2 III 20 No sporulation 3 + 50 No sporulation 4 + + + 4 III 4 III 40 No sporulation 5 + + No sporulation ++ 0 III 20 6 ++ 50 No sporulation ++ ++ 0.5 IV 7 80 Uredospores infre• 8 ++ ++ 1 IV quent. No teliospores. 100 Uredosporess& telio• 9 ++ ++ 1.5 V spores formed with high frequency. 2 100 Uredospores & telio• 10 ++(G) •++ V spores formed with high frequency. 2 100 Uredospores & telio• 11 ++ + • V spores formed with high frequency. 1 Uredospores infrequent 12 + ++ 2 IV 60 90 Uredospores & telio• 13 ++ ++ 6 V spores formed with high frequency. 100 Uredospores & telio• 14 ++(G) ++ ++ 6 V spores formed with high frequency. 90 Uredospores & telio- 15 ++ ++ 6 V spores infrequent.
All trials on liquid medium MIV with Ca(N0Q)2 levels as noted + denotes aspartic acid, 22.5 mM (300 mg/lOO ml); cysteine 2.3 mM (28 mg/100 ml); gluta• thione 1 65 mM (50.7 mg/100 ml); ++ denotes twice the foregoing concentrations, (a) denotes that in otherwise similar trials 48.5 mM (68.5 mM (684 mg/l00 ml) glutamic acid was substituted for aspartic acid with identical results. 90
Doubling the level of Ca ) did not improve growth or lead (N0 9 3 <~ to the development of spores (Treatments 4 & 5).
In trials 6 to 10 the concentrations of aspartic acid and cysteine were doubled to 45 and 4.6 mM respectively and the level of Ga(N0^) was varied from to g/l. In the 2 0 2 absence of Ca(N0^)2 (trial 6) growth was the same as in treat• ment 2. As the level of calcium nitrate was increased to 2 g/l vegetative growth was improved and accelerated and the percentage number of flasks shewing growth increased. Thus with 1 g/l of Ca(N0^)2> bype 4 colonies developed in four weeks in 80% of the flasks but uredospore formation occurred only infrequently (Trial 8). In trials 9 and 10, at CaUO^^ levels of 1.5 and 2.0 g/l, type 5 colonies developed in four weeks in 100% of the flasks and uredospores and teliospores were formed by 60 to 70% of the colonies in each flask.
Trials 11 and 12 show that a high level (45 mM) of aspartic acid is necessary for good growth and sporulation while the concentration of cysteine can be decreased to 2.3 mM without adverse effect. Trials 13 to 15 show further that the level of calcium nitrate can be increased to 6.0 g/l and that cys• teine can be completely replaced by-glutathione without ad• versely affecting vegetative growth. Sporulation was, how• ever, markedly decreased in the complete absence of cysteine.
No growth occurred when methionine (4.6 mM) was substituted for cysteine In otherwise similar trials.
In the media used in trials 9, 10, 11, 13, 14 and
15 growth was highly reproducible. Type 5 colonies developed 91 in virtually 100% of the flasks (Fig. 111-10). A most impor• tant feature of these results is that when 40 to 60 colonies grew in a synchronous manner in a single flask orange uredo• spores developed on the upper surfaces of the colonies and produced masses of dry uredospores after 5 weeks (Fig. III-ll).
One to two weeks later dark brown teliosori had appeared and developed a crusty mass of teliospores (Fig. 111-12). These results are in striking contrast with the situation that de• veloped when there were less than 10 colonies per flask. Under these conditions each colony developed a leathery stroma on the under surface and uredopustules never developed (Fig. III-
13). Such colonies remained vegetative even after the original medium was removed by pipette and replaced with fresh medium, although they did increase in size.
When the original medium was replenished in flasks with 40 to 60 colonies, renewed vegetative growth occurred and the mycelium overgrew the sporulation pustules. Successive replenishments of the medium did not result in re-sporulation.
D. Reinfection of flax using uredospores grown in vitro.
Uredopustules taken from colonies grown routinely on the medium used in trial 10 (Table III-IV) were placed on the exposed mesophyll of flax cotyledons and covered with cellotape. After 14 days a large pustule developed on both sides of each cotyledon and produced normal uredospores (Fig.
111-14) . Since it was possible that reinfection v/as accomp• lished by mycelium in the uredopustules rather than by germin- 92
ating uredospores, the following procedure was adopted. Axen•
ically grown colonies bearing uredopustules were dried at room
temperature (20 + 2°C) for 43 h and held in gelatin capsules
in the refrigerator for about one week. The dry uredospores were then dusted onto slides under the dissecting microscope
and placed in small clumps on exposed mesophyll tissue which
was covered with cellotape as before. Normal uredopustules
developed on the cotyledons inoculated in this way in 10 days.
In other trials mature, dry uredospores were care•
fully harvested directly from the tops of uredopustules on
colonies grown in vitro. When intact flax cotyledons were
Inoculated directly with water or 1%, gelatin suspensions of
uredospores harvested in this way, normal uredopustules de•
veloped after'two weeks (Fig. 111-15).
In some trials mature dry uredospores, taken direc•
tly from the tops of uredopustules on colonies grown in vitro,
showed less than 10%0 germination after 24 h on solid Mill
medium plus Evans' peptone, BSA and YE. In other trials such
uredospores showed up to 60% germination. After 2 months a
few of the seeded zones which had shown a high percentage of
germination developed into type 3 colonies. This result shows
that it is possible to establish axenic cultures from flax
rust uredospores which v/ere themselves produced in axenic cul•
ture. Unfortunately only a few trials of this kind have been
made to date and further experiments of this kind are urgently
required. 93
Figure III-11. Matured uredospores in cluster tease mount preparation. Phloxine/KOH. X 660.
Figure 111-12. Tease mount preparation of teleuto- spores. Phloxine/KOH. X 660.
Figure 111-13. Growth and development of flax rust on chemically defined medium MIV con• taining aspartic acid and cysteine. (A) Vegetative colonies after stroma formation medium was replenished after four weeks. (B) Sporulating colonies with pustule formation. U indicates the position of uredopustule, T denotes teleutosori. X 1.5.
Figure III-1A. Axenically developed uredopustule in• fecting the cotyledon, when placed on exposed mesophyll. Note the produc• tion of uredospores on both sides of cotyledon.
Figure 111-15. Axenically developed uredospores in• fecting intact cotyledon and the pro• duction of normal uredospores. Photo• graphed after two weeks. 94 95
DISCUSSION
Bushnell and Stewart (1-971) reported that 25 Ameri• can isolates of Puccinia graminis tritici showed wide varia• tions in growth in axenic culture and that their techniques v/ere not adequate for the long term culture of most of the isolates tested. Two isolates (races 56 and 23) did, however, survive subculturing for nine months. Of the six races tested by us three (15B4, 38, and 32-113) developed colonies but only one (race 38) formed type 4 colonies with a stroma approaching the final size of the colonies obtained routinely with the iso• late of the Australian race ANZ 126-6,7 available to us. 'The formation of irregular thick-walled cells by cultures of race
38 on both liquid and solid media reflects an unfavourable physico-chemical environment in spite of the presence of pep• tone and BSA. Our observations support the view that the ability of different isolates to develop in axenic culture depends on inherent characteristics of the isolates (Bushnell and Stewart 1971).
The growth and development of primary cultures of rust fungi from uredospores in vitro is often variable and unpredictable (Scott and Maclean 1969). The addition of BSA to the medium has been shown to improve the frequency with which colonies of wheat rust are established and their subsequent growth (Coffey et al. 1970, Kuhl et al. 1971). The results
of our trials with both solid and liquid media show that BSA also increases the frequency of establishment of flax rust 96
colonies as well as their differentiation to form a stroma.
Possibly BSA inhibits or compensates for the loss of metabo• lites by the germinating spores. Whether its effect Is rela• ted to its properties as a. polycation is not known but the results in Table III-III show that it cannot be replaced by the diamine, putrescine, which stimulates growth in a number of microorganisms (Smith 1972). Addition of cAMP to the medium inhibited the growth of race ANZ 126-6,7 after germination of uredospores (Table III-I. It might therefore be rewarding to investigate the levels of cAMP in resistant and susceptible host plants in the light of the current concept that the in•
creased hormonal level causes a concomitant increase in cAMP
(Pollard 1970).
The results in Tables III-II and III-IV show that
primary cultures of both wheat and flax rust grew and sporu-
lated on simple, completely defined media. Czapek's minerals
have been used In the culture medium for wheat rust whereas
Knop's minerals plus modified Berthelofs micronutrients have
been used for flax rust. The relative concentrations and comp•
osition of the macronutrient salts required are therefore
apparently quite different for the two rusts. One important
difference between Czapek's and Knop's mineral nutrient solu•
tions is that the latter includes Ca(N0^)2. In our experi•
ments best growth of wheat rust was obtained on Czapek's
minerals supplemented by the addition of the same levels of
n< Ca(N0-3)2 a- 3 micronutrients and containing the same amino
acids that gave the best growth of flax rust (Tables III-II 97 and III-IV). These observations suggest that the nutritional requirements for axenic growth of primary cultures of Puccinia
and Melampsora are not dissimilar, in several important res• pects .
Calcium has been implicated as a micronutrient in the nutrition of fungi by Steinberg (1948), but it is not really known whether the calcium requirement of fungi is
specific or not (Cochrane 1958). Calcium exerts a protective
effect against a number of agents (Lindeberg 1944, Olsen 1972) and has a stabilizing effect on cell membranes (Benedetti and
Emmelot 1968). The relatively high level of calcium found to be necessary for the sporulation of flax rust (Table III-IV)
suggests that the effects of calcium on the growth of rust fungi in vitro may be related to its protective and stabiliz•
ing action.
Although glucose and sucrose are the preferred carbo• hydrate sources for wheat and flax rusts, respectively, both
rusts can utilize either of these two sugars and a wide range
of other carbohydrates (Williams et al. 1966, 1967, Coffey and
Shaw 1972). The two rusts also have similar amino acid re•
quirements. Aspartic (or glutamic) acid and cysteine in
combination supported both vegetative growth and sporulation
of flax rust. On the other hand, while wheat rust grew on
aspartic acid plus cysteine, sporulation was only obtained with the further addition of glutathione.
The new results obtained with flax rust (Table III-
IV, Trials 9 to 15) show that the use of higher concentrations 98
of amino acids than those employed by earlier workers (e.g.
Turel 1973) improves vegetative growth and permits sporulation.
The results with flax rust represent a distinct advance in our
ability to obtain apparently morphogenetically 'normal1 vege• tative development and sporulation in vitro with a high degree
of reproducibility. On the basis of animal cell culture work .
Harris (1964) suggested that there may be a greater loss of
certain metabolites in a chemically defined liquid medium.
This leaching effect can be counterbalanced by increasing the
external supply of certain essential ingredients of the medium.
It seems probable that sporulation is correlated with changes
in the chemical composition of the medium brought about by the
fungus itself, since renewed vegetative growth was induced by
replacement of the original with fresh medium. In addition,
the possibility should not be overlooked that interactions
between colonies may also be mediated by volatile metabolic
products and that these may also play a role in controlling
development and sporulation.
The results with wheat rust (Table III-II) do not
reflect the same degree of control and were less predictable
than those for flax rust. We cannot,, explain the difference in
performance between the two rusts in axenic culture at pre•
sent, but it is possible that further modifications in the
media used may improve the growth and development of the wheat
rust. One factor which may have some bearing on the results
is the procedure used to raise aseptic uredospores to provide
the inoculum for the experimental work. The procedure for 99 flax rust yields a friable mass of easily manipulated uredo• spores but that for wheat rust (Bose and Shaw 1971) does not.
The wheat rust uredospores have a strong tendency to clump together and .are more difficult to manipulate. "Whether these different characteristics of the uredospores of the two rusts have any effect on their subsequent growth in axenic culture is entirely a matter of conjecture at present.
The successful establishment of the rust fungi in axenic culture clearly involves a period of adaptation on the medium which supports growth. We are of the opinion that such adaptation occurs as a result of the close association and anastomosis of germ tubes following uredospore germination. In particular anastomosis may result in a multinucleate situation, analogous to that which occurs in the infection structures nor• mally formed on the host plant. In dual-membered (i.e. in• fected) callus cultures the necessary adaptation for subsequent saprobic growth presumably occurs during prolonged association
of the two organisms in vitro.
Despite our success in obtaining reproducible vege• tative and reproductive growth, particularly of flax rust,
under precisely defined conditions, the mechanism or nature
of the adaptive changes that permit saprobic growth are com•
pletely unknown. At the genetic level adaptation may involve
the derepression of a particular segment of the fungal genome
(Scott and Maclean 1969) or the exchange of chromosome segments
between adjacent fungal nuclei (Hartley and Williams 1971).
The diffusion of cytoplasmic factors or even volatile compon- 100 ents between germlings, in the ease of primary cultures de• rived from uredospores, or between host and parasite in dual- membered cultures, may also be involved. Induced adaptative changes involving cytoplasmic rather than nuclear elements are known in fungi and their transmissibility has been demon•
strated (Jinks 1957, 1958). Finally, adaptation may Involve, the selective removal or inactivation of inhibitors.. Inhibi• tors of uredospore germination are well known (Allen and
Dunkle 1971) but to our knowledge naturally occurring inhibi• tors of hyphal growth have not been reported. Whatever the mechanism of adaptation to saprobic growth, established axenic
colonies have been shown to utilize a broader range of sub•
strates than that which permits the Initiation of primary
colonies from uredospores or from infected callus (Cutter
1959, I960, Coffey and Shaw 1972). 101
REFERENCES
Allen, P.J., and L.D. Dunkle. 1971. Natural activators and inhibitors of spore germination. In Morphological and Biochemical Events in Plant Parasite Interaction. S. Akai and S. Ouchi (Eds.). Mochizukl Pub. Co., Japan. 415 p.
Benedetti, E.L., and P. Emmelot. 1968. Structure and function of plasma membranes isolated from liver. In The Membranes. A.J. Dalton and F. Haguenau (Eds.). Academic Press, Nev/ York, 223 P. Bose, A., and M. Shaw. 1971. Sporulation and pathogenicity of an Australian isolate of wheat rust grown in vitro. Can. J. Botany 49: 1961-1964. . Bushnell, W.R. 1968. In vitro development of an Australian isolate of Puccinia graminis f. sp. tritici, Phyto• pathology 58: 326-327. Bushnell, W.R., and D.M. Stewart. 1971. Development of Ameri• can isolates of Puccinia graminis f. sp. tritici on an artificial medium, phytopathology 61: 376-379. Cochrane, V.M. 195#. Physiology of Fungi. John Wiley & Sons, Inc., New York. 524 p. Coffey, M.D., and P.J. Allen. 1973. Nutrition of Melampsora lini and Puccinia helianthi. Trans. Brit. Mycol. Soc. 60: 2413-26707 Coffey, M.D., A. Bose and M. Shaw. 1969. In vitro growth of gelatin suspensions of uredospores of Puccinia gramin• is f. sp. tritici. Can. J, Botany 47: 1291-1293. Coffey, M.D., A. Bose and M. Shaw. 1970. In vitro culture of the flax rust, Melampsora lini. Can. J. Botany 48: 773-776. Coffey, M.D., and M. Shaw. 1972. Nutritional studies with axenic cultures of the flax rust, Melampsora lini. Physiol. Plant Pathology 2: 37-47. Cutter, V.M., Jr. 1959(1961). Studies on the isolation and growth of plant rusts in host tissue cultures and upon synthetic media. I. Gymnosporangium. Mycologia 52: 248-295. Cutter V.M., Jr. 1960(1961). Studies on the isolation and growth of plant rusts in host tissue cultures and upon synthetic media. II. Uromyces ari-triphylli. Mycologia 52: 726-742. 102
Foudin, A.S., and ¥.K. ¥ynn. 1972. Growth of Puccinia gramin- is f. sp. tritici on a defined mediura. Phytopathology h~2: 1032-lUlAT.
Gamborg, O.L., R.A. Miller, and K. Ojima. 1968. Nutrient re• quirements of suspension cultures of soybean root cells. Expt. Cell Res. 50: I5I-I58.
Harris, M. 1964. Cell Culture and Somatic Variation. Holt, Rinehard & Winston, Inc., New York, 547 p.
Hartley, M.J., and P.G. Williams. 1971a. Morphological and cultural differences between races of Puccinia gramin• is f. sp. tritici in axenic culture. Trans. Brit. Mycol. Soc. 57: 137-144.
Hartley, M.J., and P.J. Williams, 1971b. Genotypic variation within a phenotype as a possible basis for somatic hybridization in rust fungi. Can. J. Botany 49: 1085-1088.
Hollis, C.A., R.A. Schmidt, and J.W. Kimbrought. 1972. Axenic culture of Cronartium fusiforme. Phytopathology 62: 1417-1419. Hotson, H.H., and V.M. Cutter, Jr. 1951. The isolation and culture of Gymnosporangium juniperi-virginianae Schw. upon artificial media. Proc. Nat. Acad. Sci.(U.S.A.) 37: 400-403. Howes, N.K., and K.J. Scott. 1972. Sulphur nutrition of Puccinia graminis f. sp. tritici In axenic culture. Can. J. Botany 50: 1165-1170.
Jinks J L. 1957. Selection for cytoplasmic differences. ' Proc. Roy. Soc. B. 146: 527-540.
Jinks J.L. 1958. Cytoplasmic differentiation In fungi. Roy. Soc. B. ' Proc. 11+8: 314-321.
Jones D.R. 1972. In vitro culture of carnation rust, Uro- ' 'myces dianthi. Trans. Brit. Mycol. Soc. 58: 29'=J6".
Kuhl, J.L., D.J. Maclean, K.J. Scott, and P.G. Williams. 1971. ' The axenic culture of Puccinia species from uredo• spores- experiments on nutrition and variation. Can. J. Botany 59: 201-209.
Lindeberg G. 1944. Uber die physiologie ligninablauender bodenhymenomyzeten. Symb. Bot.Ups. 8(2): 1-183. 103
Maclean, D.J.., and K.J. Scott. 1970. Variant forms.of sapro• phytic mycelium grown from uredospores of Puccinia graminis f. sp. tritici. J. Gen. Microbiol. 64: 19-27. .
Masin, V.V., and L.N. Andreev. 1971. Vegetative growth of th pathogen of wheat stem rust in vitro. MIkologiya i Fitopathologiya 5(2): 197-200.
' Olsen, R.A. 1972. Triterpeneglycosides as inhibitors of fun• gal growth and metabolism. Physiol. Plantarurn 27: 202-208.
Pollard, C.J. 1970. Influence of gibberellic acid on the in• corporation of 8-lA-c adenine into adenosine 3S,5T- cyclic phosphate in barley aleurone layers. Biochim. Biophys. Acta 201: 511-512.
Quick, W.A., and S.L.C. Cross. 1971. The use of millipore filter discs in axenic culture of flax rust. Can. J. Botany 49: 187-188.
Roblson, G.A., R.W. Butcher, and E.W. Sutherland. 1971. Cycl AMP in lower organisms. In Cyclic AMP. Roblson, G.A R.W. Butcher and E.W. Sutherland (Eds.). Academic Press', New York, p. 422-453 .
Scott, K.J., and D.J. Maclean. 1969. Culturing of rust fungi Ann. Rev. Phytopathology 6: 123-146.
Smith, T.A. 1972. The physiology of polyamines and related compounds. Endeavour 31(112): 22-28.
Steinberg, R.A. 1948. Essentiality of calcium in the nutri• tion of fungi. Science (Washington) 107: 423.
Turel, F.L.M, 1969. Saprophytic development of the flax rust Melampsora lini race no. 3. Can. J, Botany 47: 821- 823 .
Turel, F.L.M. 1971. Pathogenicity and developmental differ• ences of three saprophytically growing isolates of flax rust fungus, Melampsora lini, race 3. Can. J. Botany 49: 1993-1997.
Turel, F.L.M. 1973. Growth of the flax rust Melampsora lini on chemically defined media. Can. J. Botany 51: 131- 134.
Vassil'ev, O.A., and V.I. Saplina. 1971. Culture of the fun• gus Melampsora pinitorqua (A. BR.) Rostr. - the patho• gen of the stem rust of pine. MIkologiya i Fito• pathologiya 5(2): 182-183. 104
Williams, P.G., K.J. Scott, and J.L. Kuhl. 1966. Vegetative growth of Puccinia graminis f. sp. tritici in vitro. Phytopathology $67 1413-1419.
Williams, P.O., K.J. Scott, J.L. Kuhl, and D.J. Maclean. 1967, Sporulation and pathogenicity of Puccinia graminis f. sp. tritici grown on an artificial medium. Phyto• pathology 57: 326-327.
Wong, A.L., and H.J. Willetts. 1970. Observations on growth of selected Australian races of wheat stem rust in axenic culture. Trans. Brit. Mycol. Soc. 55: 231- 238.
1 105
SUMMARY
1. Liquid mediurri containing Czapek's minerals, 3% glucose,
0.1% Evans' peptone and 1% defatted bovine serum albumin
supported the vegetative growth and sporulation of
Puccinia graminis tritici race ANZ 126-6,7.
2. Typical pigmented uredospores and teliospores were formed
after 6-8 weeks of growth.
3. The uredospores were capable of infecting the mesophyll
of wheat leaves exposed by stripping back the lower epi•
dermis.
4. Solid medium containing Difco .bacto agar, sucrose, Knop's
mineral salts and micronutrients plus yeast extract and
peptone supported the vegetative growth of two different
strains of flax rust Melampsora lini (Ehrenb.) Lev.
5. Incidence of establishment of the colonies sharply in•
creased when defatted BSA was added to the above media.
6. Surface sterilized flax cotyledons become infected when
placed with their cut ends in contact with these colonies.
7. Good vegetative growth was obtained when three North
American races (32-113, 15B4 and 38) of Puccinia graminis i tritici were tested on a liquid medium containing Czapek's minerals, 3% glucose, 0.1% Evans' peptone and i • . - •
BSA plus 0.5 g/l Ca(N03)2. ' ' ' '
8. A chemically defined liquid medium containing sucrose,
Knop's mineral salts, Berthelot's micronutrients, aspartic
(or glutamic) acid and cysteine supported the growth of 106
vegetative colonies of Melampsora lini race 3 from un• contaminated uredospores..
The formation of uredospores and teliospores occurred at a relatively high frequency and sporulation seems to be dependent upon the level of Ca++ and number of colonies originating per flask.
The uredospores of flax rust developed in axenic culture infected healthy week old flax cotyledons in a normal manner.