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Studies on ustilago hordei. Holmwood, Michael Arthur 1970

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STUDIES ON U3TILAG0 HORDSI by MICHAEL ARTHUR HOLMWOOD B.Sc, University of B r i t i s h Columbia, I 9 6 6 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Botany We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , '1970 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree tha permiss ion fo r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed without my w r i t t e n p e r m i s s i o n . Mlchaei A. Holmwood Department of Botany The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8 , Canada Date A p r i l 27. 1970 i ABSTRACT N u t r i t i o n a l mutants of Ustllago hordel* were used to demonstrate that parasexual recombination occurs within the host plant (Hordeum vulgare) p r i o r to the production of t e l i o s p o r e s . The n u t r i t i o n a l mutants were also used to show that resistance of the newly-germinated seedling °f H. vulgare to U. hordel and of subsequently formed t i l l e r s to i n f e c t i o n was not correlated, and was probably not co n t r o l l e d by the same gene or genes. v The a p p l i c a t i o n o f . g i b b e r e l l i c a c i d to H. vulgare was found to cause an increase i n the o v e r a l l t i l l e r height of healthy plants by increasing the elongation of Internodal regions 0-1, 1-2, 2-3, and 3-4. There was no Increased elongation of internodal regions 4-5 and 5-6. The healthy t i l l e r s of diseased plants showed no Internodal elongation when g i b b e r e l l i c acid was applied. Diseased t i l l e r s , which are usually shorter than healthy t i l l e r s , , were also unaffected by the presence of g i b b e r e l l i c a c i d . The i n j e c t i o n of both mating types of U. hordel into the young developing spike of a normally r e s i s t a n t s t r a i n of H. vulgare resulted In the production of diseased spikes. This would Indicate that blockage to normal Infection occurs at the time of seedling penetration, at the l e v e l of t i l l e r primordia development, or at the time of spike prlmordla development. i i TABLE OF CONTENTS Page INTRODUCTION 1 MATERIALS AND METHODS .5 A. General Techniques 5 1 Barley v a r i e t i e s 5 2 Ustllago horde! cultures 5 3 Media 6 i complete medium 6 i i minimal medium 6 i i i s a l t s o l u t i o n 6 i v trace element s o l u t i o n 7 v vitamin s o l u t i o n 7 v i n u t r i t i o n a l supplements 7 4 Maintenance of cultures 8 5 N u t r i t i o n a l t e s t i n g 9 6 Seed preparation 9 7 Mating type te s t 1 0 8 Planting 1 0 9 Bleach treatment of spores before germination 1 1 1 0 I s o l a t i o n and i d e n t i f i c a t i o n of teliospores 1 1 B. S p e c i f i c Techniques 1 3 1 Multiple i n f e c t i o n 13 TABLE OF CONTENTS CONTINUED 2 E f f e c t s of g i b b e r e l l i c a c i d on Ustllago horde! host-parasite system 3 Injection of r e s i s t a n t s t r a i n s RESULTS AND DISCUSSION A. Multiple Infection B. The Ef f e c t s of G i b b e r e l l i c Acid on Inoculated and Uninoculated T i l l e r s 1 Plants not treated with g i b b e r e l l i c acid 2 E f f e c t s of g i b b e r e l l i c a c i d on Inoculated and uninocilated t i l l e r s C. The Percentage of Diseased T i l l e r s Following Two Different Inoculation Procedures with Susceptible and Resistant Barley V a r i e t i e s SUMMARY BIBLIOGRAPHY i v LIST OP TABLES Page-Table I Table II Table III Table IV Table V Table 1 VI Table VII Table VIII Table IX Table X Table XI Table XII Table XIII Table XIV Table XV Table XVI Percentage of Plants with Following Percentage of Infected T i l l e r s 18 Percentage of Plants with 0 Percent and 100 Percent of T i l l e r s Infected 1 9 Ratio of Smutted Plants to Smutted T i l l e r s 20 Corre l a t i o n C o e f f i c i e n t f o r Diseased T i l l e r s Versus Diseased Plants 22 Growth of Mutant Cultures on Various Media ' 2 5 Resultant S p o r i d i a l Types Obtained f o r the Cross E^a X A 24 Tests on Three Multiple Mutants 3 0 T i l l e r Length i n Centimeters 3 2 t-Test to Determine Equality of the Population Means of Smutted and Non-Smutted T i l l e r s 34 G i b b e r e l l i c Acid Treatment Categories 37 T i l l e r Lengths i n Centimeters 39 T i l l e r Categories 40 Relationship Among T i l l e r Heights 42 Relationship of G i b b e r e l l i c Acid Treated No-smut Plants to Non-gibberellic Acid Treated No-smut Plants 43 Relationship of Healthy T i l l e r s of Diseased Plants with and without G i b b e r e l l i c Acid Treatment 44 Relationship of Smutted T i l l e r s of Diseased Plants with and without G i b b e r e l l i c Acid Treatment 45 V LIST OP TABLES CONTINUED Page Table XVII Table XVIII Table XIX Table XX Relationship of Non-inoculated (Healthy) plants with Inoculated (Healthy) plants Comparison of Healthy T i l l e r s of Diseased and Healthy Plants Internodal Length Presence of Smut i n Various V a r i e t i e s of Barley 46 47 4 9 64 v i LIST OF FIGURES Figure 1 Ustilago hordei L i f e Cycle Figure 2 Diagrammatic Representation of Hordeum vulgare -Figure 3 Comparison of Internodal V a r i a t i o n of G i b b e r e l l i c Acid Treated, Control Plants with Nongibberellic Acid Treated, Control Plants Figure 4 Comparison of Internodal V a r i a t i o n of Healthy T i l l e r s of Nongibberellic Acid Treated, Diseased Plants with Nongibberellic Acid Treated, Control Plants Figure 5 Comparison of Internodal V a r i a t i o n of Nongibberellic Acid Treated, Smutted Plants with Nongibberellic Acid Treated, Control Plants Figure 6 Comparison of Internodal V a r i a t i o n of G i b b e r e l l i c Acid Treated, Control Plants with G i b b e r e l l i c Acid Treated Smutted Plants Figure 7 Comparison of Internodal V a r i a t i o n -of G i b b e r e l l i c Acid Treated, Smutted Plants with Nongibberellic Acid • Treated, Smutted Plants Page 3 4 55 56 58 59 60 ACKNOWLEDGMENT I wish to express my appreciation to my supervisor Dr C»0o Person f o r his assistance offered throughout the work. The suggestions and help of Dr K.M. Cole, Dr R.J. Bandoni, and Dr J.J. Stock are also. g r a t e f u l l y acknowledged. I also wish to thank Margaret Shand f o r her technical assistance throughout the course of study,, and f o r her preparation of the Figures,, A s p e c i a l thanks to my mother and s i s t e r f o r the typing and proof-reading of t h i s t h e s i s . 1 ;INTRODUCTION Covered smut i s a disease of c u l t i v a t e d barley (Hordeum vulgare L.) caused by Ustilago hordel (Pers) Lagerh. With covered smut of barley, as with most other plant diseases, the i n f e c t i o n process and subsequent events which take place during the development and expression of the disease are poorly understood (Fisher and Holton 1 9 5 7 ) . The culmination of the covered smut disease of barley i s the production of d i p l o i d t e l i o s p o r e s . In the normal course of events these are disseminated to healthy seeds and the disease process i s reinitiated,, Immediately following germination each teliospore produces an ordered tetrad from which four haploid s p o r i d i a l cultures can be developed. The haploid cultures can be grown In v i t r o and compatible pairs brought together to produce a dlkaryon, which I n i t i a t e s the Infective phase (Figure 1). The dlkaryon cannot normally be grown, as such, i n v i t r o . Infective hyphae or "suchfaden" are formed by the dlkaryon to f a c i l i t a t e i t s entry into the seedling during germination of the seed. A number of UV-induced biochemical mutants were produced from U.hordel by Hood ( I 9 6 6 ) and are being maintained i n the Botany Department at the University of B r i t i s h Columbia. The use of biochemical mutants affords an opportunity f o r in v e s t i g a t i n g the n u t r i t i o n a l needs of U.hordel during i t s i n f e c t i v e (dlkaryotic) stage. The use of ."marker" genes„ (one or more mutant genes ca r r i e d by . -either or both n u c l e i of the i n f e c t i v e dlkaryon), also affords an opportunity f o r determining whether parasexual recombination occurs within the host plant p r i o r to the production of te l i o s p o r e s . By using marker genes i t should be possible to determine whether multiple infections can occur i n t h i s s p e c i f i c disease system. Parts of the present study were directed to the problems of n u t r i t i o n a l requirement and multiple i n f e c t i o n . Farrar (1958) has reported that the use of g i b b e r e l l i c a c i d (500 PPm) on Sorghum vulgare inoculated with Sphaoelotheca  sorghl resulted i n a decrease i n the percentage of smutted plants. The effects of g i b b e r e l l i c acid on the U.hordel / H.vulgare system were investigated i n another part of t h i s study. 3 Figure 1 Ustilago horde! L i f e Cycle I n t e r c e l l u l a r mycelium Figure 2 D i ^ r a m m t i c R e p r e s e n t a t i o n Of Hordenm v u l g a r e 5 MATERIALS AND METHODS  A« General Techniques 1. Barley V a r i e t i e s Pour v a r i e t i e s of barley were used i n t h i s study. These, l i s t e d i n order of Increasing resistance to the parental s t r a i n of U.hordel used i n t h i s study, are as follows: Vantage, Himalaya, Conquest, and Exc e l s i o r (Shand, personal communication). 2. Ustilago hordel Cultures The two wild-type (prototrophic) strains are designated E-^ a and I^A (Hood 1966)» As the mutant strains were a l l derived from one or the other of these two parental s t r a i n s , E^a and I^A are maintained as "standard" s t r a i n s f o r te s t i n g f o r auxotrephy and f o r mating type 0- Mating occurs between "A" and "a" stra i n s to produce the dlkaryon which, i f It i s i n f e c t i v e , can i n i t i a t e the disease. The autotrophs used in, the experiments a l l had a single n u t r i t i o n a l requirement not present i n the parental s t r a i n s . They a l l grew on complete medium and on minimal medium with the addition of the required supplement. The mutants normally showed no growth on minimal medium or on minimal medium that had been supplemented by a n u t r i t i o n a l f a c t o r other than the one required.. Only those n u t r i t i o n a l 6 mutants f o r which both mating types were a v a i l a b l e were chosen f o r t h i s study. •3. Media The media used were of two types; complete and minimal, 1_ Complete; medium 20 ml s a l t s o l u t i o n 1000.ml d i s t i l l e d H 20 50 ml tryptophane" 5 gm casein hydrolysate (vitamin and s a l t free) 5 gm yeast extract 10 gm dextrose 20 gm agar 10 ml vitamin solu t i o n - added a f t e r autoclaving media to prevent vitamin breakdown mixture steam autoclaved at 121°C f o r 15 minutes 11 Minimal medium 20 >ml s a l t s o l u t i o n 1000 ml d i s t i l l e d H20 20 gm agar 10 gm dextrose mixture steam autoclaved at 121°C f o r 15 minutes n u t r i t i o n a l supplements were added a f t e r autoclaving i l l S a l t s o l u t i o n '• 123 gm Na^ c i t r a t e 0 2H20 250 gm KH2P0ij,- monobasic 100 gm NH/jNO^- anhydride 10 gm MgSOj/'pHpO 5 gm CaCl 2»2H|o 5 nil trace element s o l u t i o n 750 ml d i s t i l l e d H2Q 2 ml chloroform 7 i v Trace element solut i o n 5 gm c i t r i c a c i d IH2O 5 gm ZnSOij.»7H20 1 gm Pe(NH4)2 ,(S01| .)2 , 6H20 0 .25 gm CuS04«5H20 0 .05 gm MnSCVlH 2 0 0 .05 gm H 3 B O 3 - anhydride 0 .05 gm NaMo04»2H 2 0 1 ml chloroform 95 ml d i s t i l l e d H 2 0 Vitamin s o l u t i o n 100 mg thiamin 50 mg r i b o f l a v i n 50 mg pyridoxlne 200 mg Ca pantothenate 50 mg v p-amlno-benzoic ac i d 200 mg n i c o t i n i c a c i d 200 mg choline chloride 400 mg i n o s i t o l 50 mg f o l i c a c i d 1000 ml d i s t i l l e d H20 v i N u t r i t i o n a l supplements Stock solutions; amino acids 500 mg added to 75 ml H2O and.25 ml ethanol bases 50 mg added to 75 ml H20 and 25 ml ethanol vitamins 5 nig added to 100 ml H20 The above solutions were passed through a s t e r i l e m i l l i p o r e f i l t e r (0.22u) and stored i n a s t e r i l e f l a s k at 4°C. For use as n u t r i t i o n a l additives, 2 ml of stock so l u t i o n was added per 100 ml of hot, f r e s h l y autoclaved minimal medium. 8 4. Malntenace of Cultures Cultures were stored on s i l i c a gel following the method of Perkins ( I 9 6 2 ) as modified by Shand (personal communication). Fresh cultirres of the s t r a i n desired were obtained by placing several c r y s t a l s of s i l i c a gel on a slant of complete medium and incubating at 22°C. After approximately 14 days, the culture had grown s u f f i c i e n t l y to permit tr a n s f e r to supplemented minimal medium (here-a f t e r c a l l e d i l n ( + )). A f t e r one week of growth these could be stored on sla n t s , as stock cultures, at 4°C f o r up to s i x weekso To obtain the culture f o r inoculation, a portion of the stock culture was transferred to 50 ml of min(+) medium i n a 250 ml Erlenmeyer f l a s k and allowed to grow at 22°C on a shaking platform. When the c e l l count reached approximately 10^ c e l l s per ml, the c e l l s were d i l u t e d , r a p i d l y shaken, and the l i q u i d spread on min(+) agar plates to obtain Isolated colonies a r i s i n g from a single c e l l . A colony was then selected, tested f o r i t s deficiency, placed on a sla n t , allowed to grow f o r one week and transferred to l i q u i d medium from which the inoculum was obtained. 9 5• N u t r i t i o n a l Testing To determine whether or not the mutant had remained constant 0 or to determine the requirements of an unknown mutant p n u t r i t i o n a l t e s t i n g was c a r r i e d out. The mutant to be tested was grown on complete medium f o r 5 days and then transferred by the r e p l i c a p l a t i n g method to minimal medium and to the various, test plates„ A single-mutant culture should show no growth on minimal, and growth on only one of the min(+) PLATES, Multiple mutants must be tested on a series of plates i n which the suspected combinations of the n u t r i t i o n a l requirements are represented. 6» Seed Preparation -Seed to be inoculated was f i r s t given an anaerobic/ formaldehyde treatment to eliminate contamination by unwanted microorganisms. An Erlenmyer f l a s k was h a l f f i l l e d with barley seed and the f l a s k f i l l e d with water. A f t e r s i x hours the water was drained o f f . A rubber balloon was placed over the neck of the f l a s k and the fl a s k incubated at 22°C f o r 48 hours. The conditions In the f l a s k are such that aerobic organisms such as loose smut (Ustilago nuda) are able to germinate. The conditions however soon became anaerobic with the subsequent death of aerobic organisms. Following t h i s treatment the seeds were dried, Immersed i n a 1/400 formaldehyde/water 10 so l u t i o n f o r one hour, washed i n running tap water f o r § an hour, dried on paper towels, and stored i n glass jars f o r l a t e r use. 7. Mating Type Test To test the mating type a l i q u i d culture of each of 7 8 / the two parental types was prepared, (10'-10 c e l l s / m l ) . Each l i q u i d culture was then spread evenly over the surface of a separate minimal or a minimal (+) plate, and the c e l l s were allowed to grow f o r 2 days at 22°C, creating a lawn of growth over the surface of the plate. Several separate 7 8 drops of the culture to be tested (liquid-cone 10 r-10 cells/ml) were.placed on the plates and incubated at 20°G f o r 72 hours. The presence of i n f e c t i o n hyphae ( a e r i a l hyphae) indicated a compatible reaction (I.e. that the two cultures were of opposite mating type). 8. Planting F i e l d ; The seeds i n the f i e l d s were planted i n 10 foot rows, 120 seeds per row, then thinned to 100 plants per row. Seeding was done either by hand or with a V-belt planter. Greenhouse: Pots with a diameter of 6^ inches were planted with, twelve seeds, then thinned to six plants per pot. The pots were placed under l i g h t s and the day length adjusted to correspond to that of the normal growing season. 11' 9. Bleach Treatment of Spores Before Germination Bleach treatment i s a method f o r surface s t e r i l i z a t i o n of spores to prevent b a c t e r i a l contamination of the media. The spores to be treated were suspended f o r 1 hour i n 2 ml of s t e r i l e water. A f t e r t h i s period, 0.5 ml of a 5 percent solu t i o n of chlorine bleach was added, the spores were shaken f o r 3.0 seconds, and then the s o l u t i o n was poured onto a f i l t e r paper i n a Buchner funnel where they were washed f o r 2-3 minutes with s t e r i l e d i s t i l l e d water. The f i l t e r paper with the spores was then added to a f l a s k of complete medium and allowed to shake f o r 1 hour, a f t e r which time the f i l t e r paper was removed and the f l a s k allowed to continue shaking. 10. I s o l a t i o n and i d e n t i f i c a t i o n of tellospores Teliospores vrere removed from each panicle a f t e r the head had been allowed to dry f o r several days placed into complete l i q u i d medium, and grown f o r 48 hours i n a New Brunswick Psycrotherm R-27 shaker Incubator at 23°G. The medium contained t e t r a c y c l i n e at a concentration of .01 mg per ml to I n h i b i t b a c t e r i a l growth. At the end of 48 hours the r e s u l t i n g culture (I.e. a mixture of t e l i o -spores and haploid s p o r i d i a derived from them), was d i l u t e d to 500 sporidia per ml and spread at a concen-t r a t l o n of 50 spo r i d i a per plate on complete medium. Aft e r 72 hours the colonies a r i s i n g from i n d i v i d u a l 12 spor i d i a were transferred to complete media and were allowed to grow f o r 48 hours. They were then r e p l i c a t e d onto the various media f o r t e s t i n g (colonies which produced suchfa'den were presumed to have ar i s e n either from a tell o s p o r e or from a compatible pair of s p o r i d i a ) . 13 B. S p e c i f i c Techniques 1. Multiple Infection To demonstrate the e f f e c t of inoculating barley seed with several separate mutants, ten mutant cultures showing the highest pathogenicity on Vantage In the 1967 University of B r i t i s h Columbia f i e l d tests (Shand personal communication) were crossed, i n compatible combinations, with both wild and mutant partners. The r e s u l t i n g dikaryons were heterozygous (with wild partner) or homozygous (with mutant partner) f o r the mutant def i c i e n c y . In a second series of inoculations, a l l the mutant cultures of a single mating type were mixed together and t h i s mixture was used i n compatible combination with the wild-type culture to produce an inoculum capable of producing a number of d i f f e r e n t dikaryons. The tellospores produced from these crosses were c o l l e c t e d and an attempt was made to i d e n t i f y the n u t r i t i o n a l d e f i c i e n c i e s of the r e s u l t i n g s p o r i d i a . The r e l a t i o n s h i p between the percentage of plants smutted and the percentage of t i l l e r s smutted was also investigated. 2. Effects of G i b b e r e l l i c Acid on Ustilago hordel Host-parasite System To test the e f f e c t of the fungus upon the o v e r a l l 14 length of the t i l l e r s of the barley plant, one hundred seeds of Vantage barley were infected with I^A and E-^a. The seeds were planted i n rows, 15 seeds per row, and thinned to 10 plants per row (approximately one foot between each p l a n t ) . A f t e r the plants had matured they were pulled up and the length of the t i l l e r s measured from the crown to the base of the spike. The e f f e c t of g i b b e r e l l i c a c i d (500 ppm) was determined by using four experimental categories of plants. Each category contained 100 plants. . 1. c o n t r o l : uninoculated seeds; no g i b b e r e l l i c a c i d 2. g i b b e r e l l i c a c i d c o n t r o l : uninoculated seeds; plants sprayed with g i b b e r e l l i c acid throughout growth. 3. I4A and E3a: seeds inoculated with the fungus: plants not treated with g i b b e r e l l i c a c i d . 4. Inoculated g i b b e r e l l i c : plants from seed inoculated with the fungus; sprayed with g i b b e r e l l i c acid through-out growth. -The percentage of smutted t i l l e r s and smutted plants was determined. Using t and P tests the e f f e c t of g i b b e r e l l i c acid on t i l l e r length and Internodal elongation was measured. 3* Injection of Resistant Strains Seeds of Vantage, Himalaya, Excelsior, and Conquest 15 barley were divided each into three groups and planted i n the greenhouse., The f i r s t group of seeds was planted a f t e r inoculation with I^A and E^a, and acted as a control group to show whether or not i n f e c t i o n occurred through the seed of each v a r i e t y . The second group of seeds was planted and a f t e r germination the plants were injected every second day with a mixture of I^A and E^a; t h i s group served to show whether Inoculation by i n j e c t i o n could r e s u l t In development of the disease. The t h i r d group contained uninoculated seeds that were allowed to grow-and the r e s u l t i n g plants were not Injected with the fungus; t h i s group served as the uninoculated control group. RESULTS AND DISCUSSION A» Multiple Infection In order to determine whether an infected t i l l e r can carry more than one genotype of the pathogen, plants were inoculated with a mixture of haploid (gametic) cultures and either of the wild mating types. A l l haploid cultures were of a single mating type but each c a r r i e d a d i f f e r e n t genetic marker. As the inoculum was capable of forming a number of heterozygous dikaryons, each with a d i f f e r e n t marker, the recovery from a singl e infected t i l l e r of two or more genetic markers would indicate that two or more dik a r y o t i c i n f e c t i o n s had taken place. The r e l a t i o n s h i p between i n f e c t i v i t y taken on the basis of t o t a l plants and i n f e c t i v i t y taken on the basis of t o t a l t i l l e r s was also determined. Virulence of a smut culture i s usually estimated on the basis of percentage of smutted t i l l e r s per row or p l o t . Thus, an estimate of f i f t y percent smutted t i l l e r s could mean either that f i f t y percent of the plants were healthy and f i f t y percent completely diseased, or that a l l plants were diseased with an average of f i f t y percent smutted t i l l e r s $>er diseased plant. The procedures i n t h i s series of experiments,were designed with t h i s problem i n view. A high percentage of the t o t a l number of inoculated seeds usually gave r i s e to plants that had no smutted 17 t i l l e r s . The f i r s t three cross types of Table I show the kind of r e s u l t that i s usually obtained: approximately h a l f the plants showed no evidence of the disease; about 1/5 of the plants showed disease i n a l l t i l l e r s ; and the remaining plants showed both healthy and diseased t i l l e r s . Plants that have been infected with a heterozygous dlkaryon (e.g. I^A X pantothenic acid V359&) showed a varied percentage of t i l l e r s infected, depending on the cross (Table I ) . The percentage of plants with no infected t i l l e r s (Table II) may be as high as 70 percent, as with pantothenic V359a X I^A, Adenine V175a X I^A, argenine X52a X I^A, n i a c i n X95a X I^A, and n i a c i n X95A X E^a, or as low as 20 percent with h i s t i d i n e V466a X I^A, and leucine U50a X I/^ A. The v a r i a t i o n s may have been due to differences i n i n f e c t i v i t y or to differences i n l a t e r stages of the p a r a s i t i c phase, but further studies would be necessary to e s t a b l i s h which was the case. Crosses made to E^a i n a l l cases but one ( h i s t i d i n e V466) gave a much higher percentage of t o t a l l y infected plants than did crosses to I/^ A. The crosses made to IjjA varied from zero to 20 percent of the plants t o t a l l y infected (except h i s t i d i n e V466) whereas the same crosses made to the E-^ a varied from 25 percent to 50 percent of the plants being t o t a l l y infected (except py.ridoxine V26, serine V5, and n i a c i n X95). This may indicate that the nucleus of the A mating type c a r r i e s a greater genetic 18 Table I Gross Type Percentage Of Plants With The Following Percentage Of Infected T i l l e r s 0 1-19 20-39 40-59 60-79 80-99 100 e a X A* 1 43 6 13 - 6 9 8 16 I A X a * 2 44 5 12 11 8 2 18 A* X a* 57 6 13 7 3 2 12 E a X Pan V359A 25 13 12 - -E a X Pdx V26A 57 28 - 15 - -E_ X Ad V175A 57 - - - 43 E X Pro V-234A SI 56 - 11 - - - 33 E 0 X Ser V5A EL 60 7 - 13 13 7 Ea X Arg X52A 28 - - 15 15 42 E a X His V466A 40 20 10 10 - 20 E a X Leu U50A 42 30 - - 28 E a X Nia X95A 67 - 17 8 - • - 8 E Q X Met V241A 42 - 15 15 - 28 I A X Pan V359a 70 - 10 10 - - 10 I A X Pdx V26a 50 16 - • 16 18 I A X Ad V175A 70 10 - 10 10 I A X Pro V324a 57 15 28 - • -I A X Arg X52a 67 11 11 - 11 - -I A X Ser V5a 57 15 14 14 • - -I A X His V466a 20 10 10 20 10 30 1 A * = mixture of a l l A type mutants 2 a* = mixture of a l l a type mutants 19 Table II Percentage Of Plants With 0 Percent And 1 0 0 Percent Of T i l l e r s Infected Mutant Wild type Percentage Percentage of plants of plants v with no • with 100$ diseased of t i l l e r s t i l l e r s diseased pantothenic ac i d 7359 A E 3a 31 50 a I £ A 70 10 pyridoxine V26 A E 3a 6o 0 a I^A 50 20 adenine V175 A E ?a 65 40 a IijA 70 15 proline V324 A E„a 60 35 a IZJA 60 0 argenine X52 A Eoa 30 40 a 70 0 serine V5 A E^a 60 10 a I Z £ A 60 0 h i s t i d i n e V466 A E^a . 40 20 a IijA 20 30 leucine U 5 0 , A E~a 40 30 a 20 20 n i a c i n X95 A Eoa 70 10 a IJJA 7 0 0 methionine V24l A E^a 40 30 a I^A 50 20 Table III Ratio of Smutted Plants to Smutted T i l l e r s Gross Percentage Percentage Percentage Ratio-of Percent Plants Plants Smutted Smutted Plants with Smut with a l l T i l l e r s of to the Percent T i l l e r s Smutted Smutted T i l l e r s Smutted Plants E a X pantothenicV359A 7 5 50 60 •1.25 E a X pyridoxineV26A 43 0 8 5 . 3 8 Ea X adenineV175A 43 43 4 6 0 . 9 4 E a X prolineV324A 4 4 3 3 16 2.92 E a X arginineX52A 71 43 52 1.36 Ea X serineV5A 40 6 18 2.21 E a X h i s t i d i n e V 4 6 6 A 60 20 19 3 . 1 0 E a X leuclneU50A 5 7 28 27 . 2.09 E a X n i a c i n X 9 5 A 3 3 8 12 2 . 6 8 E a X methlonineV24lA 5 7 28 3 7 1 . 5 3 IA X pantothenicV359a 30 10 28 1.07 IA X pyridoxineV26a 50 12 17 2 . 9 8 I A X adenineV175a 30 10 21 1 . 4 5 I A X prolineV324a 43 0 9 4 . 9 2 IA X a r g i n i n e X 5 2 a 3 3 0 6 5.72 IA X serinev"5a 43 0 . 7 5 o 8 8 IA X h i s t i d i n e V 4 6 6 a 80 30 3 9 2.07 IA X leucineU50a 86 14 50 1.72 IA X n i a c i n X 9 5 a 2 9 0 14 2 . 0 8 I A X m e t h i o n i n e V 2 4 l a 50 1 4 22 2.32 2 1 p o t e n t i a l f o r pathogenicity. The v a r i a b i l i t y i n the percentage of diseased plants, using d i f f e r e n t heterozygous dikaryons, i s also shown by the data of Table I I I . The percentage of t o t a l t i l l e r s smutted was nearly always less than the percentage of t o t a l plants smutted (Table I I I ) . Using the data of Table I I I , the c o r r e l a t i o n between i n f e c t i v i t y on the basis of plants ( i . e . percentage of t o t a l plants with one or more infected t i l l e r s ) and on the basis of t i l l e r s (I.e. percentage of infected t i l l e r s i n plants with at. le a s t one t i l l e r known to have been infected) was cal c u l a t e d . The re s u l t s are given i n Table IV. The resultant c o r r e l a t i o n c o e f f i c i e n t (0,35) i s low and i t can be concluded that the percentage of diseased t i l l e r s and percentage of diseased plants are not interdependent. The low c o r r e l a t i o n would Indicate that i f resistance of the host to U. hordel was g e n e t i c a l l y determined, the resistance to i n f e c t i o n of the seed and subsequent i n f e c t i o n of the t i l l e r were not related, and probably were not con t r o l l e d by the same gene or genes. The a b i l i t y of the fungus to i n f e c t the secondary primordla seemed not to be correlated with the a b i l i t y to penetrate the o r i g i n a l primordla. This could, be a r e s u l t of the presence of d i f f e r e n t genes governing the two events. The fungus may possess a gene (or genes) f o r i n f e c t -i v i t y that counteracts the gene(s) f o r resistance. V a r i a b i l i t y i n both these genetic systems may account 22 Table IV Correl a t i o n C o e f f i c i e n t For Diseased T i l l e r s Versus Diseased Plants Xj_ = percent plants with smut X 2 = percent smutted t i l l e r s of smutted plants EXi • • = 980 EX 2 - 567 ExiX2/0>l) = covariance bl,2 = regression c o e f f i c i e n t b2,l = regression c o e f f i c i e n t r l , 2 = c o r r e l a t i o n c o e f f i c i e n t E x i x 2 = E X 1 X 2 - E X 1 X 2 = 30,895 .00 -555.660/20 = 3,112.00 n bl,2 = E x ^ / C n - l ) = 163.78 = 0.23 Ex§/(n-l) 711.08 b2,l = Ex 1X2/(n-l) = 163.78 = O.53 Ex|/(n-l) 308.42 r l , 2 = (bl ,2 ) (b2,l) = (0.23)(0.53) = 0.12 =0.35 Confidence Limits; P=0.95 (-0.1 to +O.65) 23 f o r some of the c o r r e l a t i o n . Because the c o r r e l a t i o n was po s i t i v e one itfould expect that a culture that i s going to i n f e c t more seedlings i s also going to give a larger number of infected t i l l e r s per diseased plant following t h i s i n f e c t i v e event. To investigate the p o s s i b i l i t y of mitotic recombination i n U. hordel, ten biochemical mutants were chosen (Table V). To insure that these mutants were stable the tests shown i n Table V were c a r r i e d out. In addition to the crosses l i s t e d i n Table I, crosses were also made to produce the homozygous dikaryon of each mutant. Smutted t i l l e r s were produced only a f t e r Inoculation with heterozygous dikaryons (Table I ) ; these t i l l e r s were c o l l e c t e d and a random s e l e c t i o n of teliospores was germinated and the r e s u l t i n g s p o r i d i a were tested fo r biochemical d e f i c i e n c i e s . The expected s p o r i d i a l types were recovered i n a l l cases. In the cross Ej X A several recombinant sporidia were also recovered (Table VI). •* * -it-Testing of the crosses I^A X a and A X a yielded the two wild mating types only. The i s o l a t i o n of both mating types from both crosses demonstrates that recombination had occurred,.as only a singl e wild mating type was used during I n f e c t i o n of the seeds. I s o l a t i o n of the various biochemical mutants from these two crosses was not successful, ;probably because of the i s o l a t i o n techniques used. The I s o l a t i o n involved randomly selected 24 Table VI Resultant S p o r i d i a l Types Obtained f o r the Gross E^a. X A ; Mutants showing absolute requirement f o r supplements. 1 adenine 2 adenine, serine, pyridoxine 3 adenine, proline, h i s t i d i n e 4 adenine, pyridoxine Mutants that show slow growth unless c e r t a i n requirements added -requirements e s s e n t i a l f o r any growth shown before the brackets, requirements that must be added to achieve growth equivalent to that demonstrated by the parental types shown i n brackets. 1 adenine, pantothenic ac i d ( n i a c i n , proline, serine, and pyridoxine) 2 adenine, serine ( n i a c i n , proline, pantothenic acid, pyridoxine and arglnine) 3 adenine, proline (serine, and h i s t i d i n e ) 4 adenine, proline (serine, pantothenic acid, arginine, and h i s t i d i n e ) Table V Growth of Mutant Cultures on Various Media Mutant Growth on Complete Medium Growth on Minimal Medium Growth on Minimal Plus N u t r i t i o n a l Requirement Mating with-E3a Mating with pantothenicV359a -f V 3 5 9 A pyridoxineV26a V26A adenineV175a V175A p r o l i n e V 3 2 4 a V324A a r g i n i n e X 5 2 a X 5 2 A s e r i n e V 5 a V5A hlstidineV466a V466A leucineU50a U50A niaclnX95a X95A methionineV24la V241A E3a + indicates growth - indicates no growth + - + - + + - • + -+ - + - . + + - + + -+ - + . -+ - + — + - + • — . + + - + — + - + - + + - + — + - + + + - - + + -1 . - - + + - + : + — + + + + - + + - + + + . - • + ' • — + V - + + + - + + -+ + - + + . + + 26 i n d i v i d u a l teliospores which had been allowed to germinate and produce s p o r i d i a . The s p o r i d i a were then i s o l a t e d and allowed to grow on plates. Resultant colonies were chosen and i d e n t i f i e d by n u t r i t i o n a l t e s t i n g . The wild type colonies probably outgrew the mutants e and thus prevented the i s o l a t i o n of mutants by t h i s method. This problem could be overcome by the early separation,, by micromanipulator, of the products of a single t e t r a d . Grossing each of the mutants In d i v i d u a l l y with the opposite wild mating type yielded non-mutants of both mating types as well as the o r i g i n a l mutant; however, no recombinant mutant of the opposite mating type was recovered. The micromanipulator was used to separate the products of the tetrads produced by the E^a X A cross. Teliospores p were Isolated and placed onto small (2 cm ) blocks of complete agar medium, allowed to germinate and then, using a micromanipulator, i n d i v i d u a l s p o r i d i a were i s o l a t e d and placed on i n d i v i d u a l agar blocks. These s p o r i d i a were allowed to grow and the resultant colonies were i d e n t i f i e d as to t h e i r n u t r i t i o n a l requirements. •a-Using the micromanipulator on the E^a X A cross, eight mutant spo r i d i a were i s o l a t e d from germinating teliospores; the eight r e s u l t i n g cultures were from eight d i f f e r e n t t e l i o s p o r e s , and represented a very small 2 7 sample of the p o t e n t i a l v a r i a b i l i t y of t h i s cross, The v a r i a t i o n i n the amount of growth of these eight cultures when re p l i c a t e d to d i f f e r e n t media should be noted. As an example of t h i s v a r i a t i o n , mutant VIb 9 (Table VII) grew well on media that lack methionine, leucine, or h i s t i d i n e ; the mutant had the enzyme systems av a i l a b l e to produce Its own supply of these requirements. There was no growth on media d e f i c i e n t i n adenine or serine; these nutrients must be supplied. The media that were d e f i c i e n t i n pyridoxine, pantothenic acid, proline, niacins, or arginine, support very l i m i t e d growth. There were several possible explanations f o r t h i s r e s u l t . Gene i n t e r a c t i o n may be involved whereby the presence of one gene was suppressing enzyme production of another gene; a l t e r n a t i v e l y , a dosage e f f e c t might allow only a small amount of a c e r t a i n compound to be produced. A recombination could have resulted In the organism receiving only a c e r t a i n f r a c t i o n of the entire gene dosage needed f o r adequate production of the f i n a l compound. The l i f e cycle of U. hordel involves the formation of a "double haploid" hypha c a l l e d a dikaryon (Figure 1). One nucleus of the dikaryon may exert greater influence on the growth of the fungus than the other nucleus. If the c o n t r o l l i n g nucleus was d e f i c i e n t f o r a gene, the secondary nucleus may be able to provide a small amount of the d e f i c i e n t product, r e s u l t i n g i n minimal growth of 28 the fungus In the absence of an external supply of the product. The i s o l a t i o n of multiple mutants indicated that a series of recombinations had taken place between the two nucl e i of the dikaryon. It may also indicate that fusion of independent dlkaryons had occurred either i n the inoculum p r i o r to i n f e c t i o n or within the infected plant. The linkage relationships of the ten markers used i n t h i s experiment are not known. If i t i s assumed that the recombinational event involved linked markers, such a process would require formation of a d i p l o i d nucleus followed by mitotic crossing over. To obtain a t h i r d marker i n the same nucleus, a mitotic crossing over would need to occur between the haploid nucleus with tvjo markers and another haploid nucleus. To obtain a t h i r d haploid nucleus, some type of mycelial fusion and nuclear transfer would have to occur. There i s however, no evidence that mycelial fusion occurs i n basidiomycetes. The p o s s i b i l i t y that transformation has occurred i s also present. During the inoculation of the seeds, or i n the plants, some c e l l s may be destroyed, l i b e r a t i n g free deoxyribonucleic acid that could be taken up by the young growing hyphae. This experiment has also provided the beginnings of a system for studying competition. Table VI shows the mutants that have resulted from the cross E-^ a X A*, 29 each mutant c o n t a i n s an a d e n i n e d e f i c i e n c y . The h i g h f r e q u e n c y o f a d e n i n e - d e f i c l e n t mutants would i n d i c a t e a low s u r v i v a l r a t e f o r t h e gene o r genes i n v o l v e d i n a d e n i n e s y n t h e s i s . T h i s f r e q u e n c y , a t w h i c h t h e v a r i o u s mutants appear i n t h e p o p u l a t i o n , c o u l d be used as a means o f d e t e r m i n i n g genome f r e q u e n c y f o r t h e mutant genes» 30 Table VII  Tests on Three Multiple Mutants Growth on complete plates minus the following nutrients Nutrient Mutant VIb 12 VIb 9 Via methionine 3 3 3 leucine 3 3 3 n i a c i n 1 1 2 h i s t i d i n e 2 2 2 adenine 0 0 0 proline 1 1 3 serine 1 0 0 pantothenic a c i d 0 1 2 arginine 2 1 2 pyridoxine 1 1 0 Growth on minimal plates with the following single nutrient added. Nutrient Mutant VIb 12 VIb 9 Via 1 methionine 0 0 0 leucine 0 0 0 n i a c i n 0 0 0 h i s t i d i n e 0 0 0 adenine 0 0 0 proline 0 0 0 serine 0 0 0 pantothenic a c i d 0 0 0 arginine 0 0 0 pyridoxine 0 0 0 complete 4 4 4 minimal 0 0 0 4 = wild type growth 0 = no growth 1-3 = v a r i a t i o n i n amount of growth between none and wild ..type. 31 B,, The E f f e c t s of G i b b e r e l l i c Acid on Inoculated and  Uninoculated Barley Plants The f i r s t part of the experiment was a study of t i l l e r elongation following treatment with g i b b e r e l l i c a c i d . The e f f e c t of U. hordel on the t o t a l t i l l e r length of plants that were not treated with g i b b e r e l l i c a c i d was f i r s t studied. By measuring the internodal distances on healthy and diseased, treated and untreated plants the e f f e c t of g i b b e r e l l i c a c i d on t i l l e r elongation i n barley was also determined. The second objective of the experiment was to determine i f g i b b e r e l l i c acid treatment reduced the percentage o f diseased t i l l e r s on barley plants inoculated with U. hordei. 1. Plants not treated with g i b b e r e l l i c a c i d . Using a table of random number, 108 non-smutted t i l l e r s were chosen from one hundred plants with both smutted and non-smutted t i l l e r s . When a nonsmutted t i l l e r had been chosen, a smutted t i l l e r from the same plant was selected, again by random numbers. As a student t t e s t was to be used It was e s s e n t i a l that the smutted and non-smutted t i l l e r s be matched as c l o s e l y as possible and that no genetic v a r i a b i l i t y affected the ca l c u l a t i o n s of the matched t i l l e r s . Lengths of the 108 pairs of t i l l e r s are recorded i n Table VIII, which al s o shows the v_ value (the difference between non-smutted Table VIII T i l l e r Length i n Centimet ers itted Nonsmutted Y Smutted Nonsmutted Y Smutted Nonsmutted Y 70 84 14 40 75 35 55 75 20 53 64 6 50 75 25 50 55 5 52 76 24 40 60 20 40 60 20 52 78 26 55 75 20 50 80 30 30 48 18 40 60 20 50 70 20 52 66 14 45 53 8 65 70 5 55 42 13 30 ^5 15 40 46 6 40 69 29 48 55 7 45 60 15 50 80 30 40 60 20 50 80 30 48 56 8 56 70 14 50 55 5 6o 80 20 60 85 25 60 65 5 65 80 15 30 53 23 • 34 55 21 45 65 20 30 53 23 60 68 8 50 82 32 60 78 18 30 45 15 40 45 5 50 66 16 35 70 35 38 55 17 35 55 20 55 65 10 60 60 0 45 65 20 70 80 10 50 45 5 55 90 . 35 20 53 38 60 75 15 40 50 10 30 52 22 40 42 2 60 75 15 40 65 25 45 70 25 50 60 10 44 55 11 45 66 21 40 46 ' 6 50 60 10 43 48 5 60 33 23 60 86 26 52 70 18 55 75 20 34 45 11 33 60 27 30 50 20 30 45 15 57 70 13 45 60 15 30 46 16 Table VIII continued Smutted Nonsmutted YX 42 53 11 55 70 15 60 73 13 60 84 24 40 65 25 55 80 25 40 40 0 65 73 8 40 40 0 50 60 10 Smutted Nonsmutted Y 45 53 8 55 80 25 42 65 23 50 60 10 60 70 10 40 35 5 40 55 15 48 70 22 60 76 16 30 50 20 Smutted Nonsmutted Y 40 65 25 55 70 15 50 70 20 30 45 15 55 70 15 30 22 8 35 46 11 26 46 11 36 65 29 40 65 25 34 Table IX t - t e s t to Determine Equality of the Population Means of Smutted and Nonsmutted T i l l e r s n = 108 E smutted = 5030,00 E nonsmutted = 6775 E y = 1745 The n u l l hypothesis i s that the presence of U. hordel does not e f f e c t the t i l l e r length of thr barley plant and therefore the two population means (smutted and nonsmutted) w i l l be equivalent,, u = 0 ( E y ) 2 = 3,045,025.00 SS = 9,633.00 n = 108 (Ey) 2/n = 28,194.67 S 2 = 90.02 Ey = 1745 Ey 2 = 37,827.00 S 2/n '= 0.83 f = 16.15 S V n = 0.83 .= 0.911 t = y - 0/ S 2/n - 16.15/0.911 := 17.74 with 107 degrees of freedom At 0.5 percentage point, of the t - d i s t r i b u t l o n with 107 degrees of freedom, t 2.625 ; since t - c a l c . = 17.74, the hypothesis that the population mean i s equal to zero or that the presence of .U. hordel does not ef f e c t t i l l e r length i s rejected. 35 and smutted t i l l e r s ) . To test the e f f e c t of U. hordel. the n u l l hypothesis was taken to represent that there was no difference i n the lengths of smutted and non-smutted t i l l e r s . As Is shown i n Table IX, the calculated value of t (1?„74) was considerably greater than the t table value (2.625). From t h i s i t was concluded that the null, hypothesis did not apply, and that smutted t i l l e r s are shorter than those which show no smut. (No completely smutted plant was used i n t h i s c a l c u l a t i o n . However i t may be mentioned that plants that were completely smutted were always v i s i b l y dwarfed). 36 2. The e f f e c t of G i b b e r e l l i c . a c i d on smutted and non-smutted t i l l e r s * The f i r s t objective of the experiment was to determine whether the presence of g i b b e r e l l i c acid reduces the percentage of smutted t i l l e r s . The data of Table X would indicate that the g i b b e r e l l i c acid treatment had reduced the percentage of smutted plants*. However»; the percentage of smutted plants (85$) In the non-treated rows i s much higher than normal. The conclusion here i s that i n t h i s p a r t i c u l a r case, the presence of g i b b e r e l l i c a c i d reduced the percentage of smutted plants from 85 to 50 percent. In a s i m i l a r way, the g i b b e r e l l i c a c i d treatment reduced the percentage of" smutted t i l l e r s from 35 to 28 percent. Thus, whether measured In terms of plants or t i l l e r s , the g i b b e r e l l i c acid treated plants showed lower l e v e l s of the disease. If g i b b e r e l l i c a c i d treatment resulted i n a decrease i n the percentage of smutted t i l l e r s , i t could possibly r e l a t e to the ; difference i n r e l a t i v e growth rates of the fungus and the merlstem of the host i n which i t resided. The second objective of the experiment was to determine the ef f e c t of g i b b e r e l l i c acid on t o t a l t i l l e r height. For measurement purposes, the ten categories i n Table X were used. As d i f f e r e n t categories contained d i f f e r e n t t o t a l t i l l e r counts, a table of random numbers was used to produce categories with less v a r i a b i l i t y i n 37 Table X G i b b e r e l l i c Acid Treatment Categories 1 control plants: seeds not infected 2 g i b b e r e l l i c acid c o n t r o l : seeds not infected 3 seeds infected: plants treated with g i b b e r e l l i c acid -no smut plants 4 seeds Infected: plants treated with g i b b e r e l l i c acid -plants "with some s m u t - t i l l e r count of unsmutted t i l l e r s seeds infected plants treated with g i b b e r e l l i c acid .-plants t o t a l l y smutted-no plants i n th i s category 6 seeds infected plants treated with g i b b e r e l l i c acid -plants *with some s m u t - t i l l e r count of smutted t i l l e r s 7 seeds infected no g i b b e r e l l i c acid-no smut plants 8 seeds infected no g i b b e r e l l i c acid-plants with some s m u t - t i l l e r count of. unsmutted t i l l e r 9 seeds infected no g i b b e r e l l i c acid-plants a l l smut 10 seeds in f e c t e d no g i b b e r e l l i c acid-plants with some s m u t - t i l l e r count of smutted t i l l e r s Total percentage of smutted t i l l e r s on infected g i b b e r e l l i c acid treated plants 97/34-3 28$ Total percentage of smutted plants i n Infected g i b b e r e l l i c acid rows 17/34 50$ Total percentage of smutted t i l l e r s on infected no g i b b e r e l l i c acid plants 74/210 35$ Total percentage of smutted plants i n infected no g i b b e r e l l i c a c i d rows 17/20 85$ 38 t i l l e r number., Classes 7 ,8,9 and 10 had very few t i l l e r s and the t o t a l t i l l e r number for these four classes was used. In a l l cases only the longest t i l l e r of an i n d i v i d u a l plant, smutted or unsmutted, was used to provide data f o r Table XI. Using an.F test the relationships between these categories were examined. The f i r s t tests (described i n Table XIII),, indicate that there was no r e l a t i o n s h i p among the groups. An o v e r a l l increase i n the maximum t i l l e r length over the control as a r e s u l t of g i b b e r e l l i c a c i d i s shown In Table XIV. If a plant has been inoculated, and some of i t s t i l l e r s develop smut, then g i b b e r e l l i c a c i d has no e f f e c t on the length of the smutted or non-smutted t i l l e r s ( i . e . the difference i n t i l l e r length due to smut i s retained Table XV)). A s t a t i s t i c a l test was not needed to show that the lengths of smutted and non-smutted t i l l e r s were d i f f e r e n t since y of the non-smutted t i l l e r s was 88.11 (Table XII ) and y of the smutted t i l l e r s was 62.00 (Table X I I ) . The control plants, and plants that remained healthy a f t e r inoculation'were not s t a t i s t i c a l l y d i f f e r e n t (Table XVII). The uninfected t i l l e r s of plants showing some i n f e c t i o n however were not i n the same s t a t i s t i c a l population as the healthy t i l l e r s of the healthy plants (Table XVIII). The experiment showed that g i b b e r e l l i c acid treatment resulted i n an increase i n the maximum length of uninfected plants. If a 39 Table XI T i l l e r Lqngths In Centimetres 1 2 3 4 6 7 8 9 10 100 115 95 99 75 105 92 44 75 106 8 a 110 95 68 90 95 60 76 90 120 125 99 44 90 97 75 42 105 110 112 86 66 90 95 70 72 85 112 110 85 70 88 43 62 110 105 115 66 68 90 73 100 110 112 118 70 90 60 105 90 90 85 80 90 44 100 115 110 100 65 92 36 85 100 105 60 70 75 •' 60 90 100 120 86 65 90 85 110 100 105 70 60 104 100 105 70 45 90 114 110 85 60 90 106 100 80 60 100 115 100 102 80 93 120 105 80 66 105 115 80 86 55 100 110 106 80 65 100 120 10? 85 75 Table XII T i l l e r Categories Sample number T i l l e r Length Sample t o t a l T 1 2 3 4 6 7 8 9 10 100 115 95 99 75 105 92 44 75 106 80 110 95 68 Q0 95 60 76 90 120 125 99 44 90 . 97 75 42 105 110 112 86 66 90 95 70 72 85 112 110 85 70 88 43 62 110 105 115 66 68 90 73 100 110 112 118 70 90 60 105 90 90 85 80 90 44 100 115 110 100 65 92 36 85 100 105 60 70 75 60 90 100 120 86 65 90 85 110 100 105 70 60 104 100 105 70 45 90 114 110 85 60 90 106 100 80 60 100 115 100 102 80 93 120 105 SO 66 105 115 80 86 55 100 110 106 80 65 100 120 107 85 75 .968 2157 2122 1717 1307 375 994 292 685 Table XII Continued Sample 1 2 3 4 6 7 8 9 10 number Size of sample n 20 20 20 20 20 4 11 5 11 Sample . mean y 98.40 107.85 106.10 85.85 65.35 93.75 90.36 58.40 62.27 T 2/n • 193, 232, 225, 147, 85, 35, 89, 17, 42, 651.20 632.45 144.20 404.45 412.45 156.25 821 .45. 052.80 656.81 Grand t o t a l = 9,333 En = 9 5 General mean y = 97.05 E(T 2/n) = 924,487.31 Table XIII Relationship Among T i l l e r Heights 4 2 G = 9 , 3 3 3 En = 9 5 y = 97»05 E(T 2/n) = 9 2 4 , 4 8 7 . 3 1 G 2 = 8 7 , 1 0 4 , 8 8 9 o 0 0 (I) = G 2/En = 916.893 0 5 6 ( I D = E(T 2/n) = 924 ,487*31 (III) 2 = Ey^ = 933 ,055.00 among samples (II-I) 7593 .75 within ; samples (III-II) 8567069 t o t a l (III-I) 16161.44 K ~ l = 5 En~K = 89 2-= 1,518.75 2 s p = 96.26 P 2 . 2 = ns y/s p = 1,518.75/96.26 P at 5% " ^ 8 9 d f = 2.3683 15*77 ) > 2.3683 = 15.77 Therefore the population means were not the same, 43 Table XIV Relationship Of G i b b e r e l l i c Acid Treated No-Smut Plants To Non-Gibberellic Acid Treated No-Smut Plants G = 6247.00 En = 60 y = 104.11 E(T 2/n) = 651,427.85 G 2 = 390,025,099.00 (I) = G 2/En = 650,416.81 (II) E(T 2/n) = 651P427.85 (III) = E y 2 = 656,575.00 among samples (II-I) = 1011.04 within samples (III-II) = 5i.47.i5 K-l = 2 En-K = 57 2-ns y = 505.52 2 s p = 90.30 P 2 2 = ns y/s p = 505.52/90.30 P at 5% - 2/^^df = 3.20 5.60 > 3.20 = 5.60 Therefore the population means were not the same. <8 Table XV Relationship Of Healthy T i l l e r s Of Diseased Plants  With And Without G i b b e r e l l i c Acid treatment G = 2711.00 En =31 y. . = 880II E(T 2/n) = 237,255.90 G 2 =7,349,521.00 (I) = G 2/En = 237,081.32 2 (II) = E(T /n> = 237,255»90 (III) = Ey 2 = 241,155.00 among samples (I'I-I) = 174.58 within samples (III-II) = 3,899.10 t o t a l (III-I) = 4,073.68-K=l =1 En=K =30 ns 2y = 174.58 s 2 p = 129.97 P =• n s 2 y / s 2 p = 174.58/129.97 = 1.34 P at 5% - 1/ df = 4.1709 1.34 \ 4.1709 Therefore the population had the same means. Table XVI Relationship Of Smutted T i l l e r s Of Diseased Plants With And Without G i b b e r e l l i c Acid Treatment G = 2 2 8 4 En = 36 f = 62.00 E(T 2/n) = 1 4 5 ,122.06 G 2 = 5,216,656. 00 (I) = G 2/En = 1 4 4,907.11 (II) = E(T 2/n) •= 1 4 5,122.06 (III) = Ey 2 = 150,260.00 among s amples (TI-I) 2 1 4 . 9 5 within samples (III-II) 5,137.74 t o t a l (III-I) 5,352 . 8 9 K-l = 2 En-K =33 ns 27 = 107.4? 2 s p. = 155.68 P - 2- 2 = ns y/s p = 107.47/155.68 P at 5% - 2 / 3 3 d f = 3o3000 0 e69 < ' 3.3000 = 0.69 Therefore the populations had the same means, Table XVII Relationship Of Non-Inoculated (Healthy) Plants  With Inoculated (Healthy) Plants G = 23^3.00 En = 24 f = 96,07 E(T 2/n) = 228,807.45 G 2 =5,489,649.00 (I) = G 2/En = 228,735.37 (II) =• E(T 2/n) = 228,807.45 (III) T7 2 = Ey = 230,151.00 among samples (II-I) = 72.08 within samples (III-II) = 1,343.55 K-l = 1 En-K = 22 ns y = 72.08 = 61.07 p 2 2 = ns y/s p = 72.08/61.07 = P at 5% - ^22" = 4.3009 1.18 < / = 4.3009 1.18 Therefore the population had the same means, Table XVIII  Comparison Of Healthy T i l l e r s Of Diseased And Healthy Plants G = 2962 En = '31 y = 95.6 E(T 2/n) = 284,472s65 G 2 = 7,772,444 (I) = G 2/En = 250,724 (II) = E(T 2/n) = 284,472.65 (III) = Ey 2 = 284,982.00 among samples ( I I - D within samples (III-II) K-l = 1 En-K = 30 2-ns y ='. 33,748.65 B 2P = 16.95 33,748.65 F •''= ns 2/y/s 2p = 33,748.65/16.95 = 4.170 F at 5% l / 3 o d f = 4.170 Therefore the population means were not the same. 48 plant was diseased the maximum t i l l e r length of the g i b b e r e l l i c a c i d treated and untreated t i l l e r s was not s t a t i s t i c a l l y d i f f e r e n t . Healthy t i l l e r s of diseased plants were not as t a l l as the healthy t i l l e r s of healthy plants. G i b b e r e l l i c a c i d seemed to produce an e f f e c t that was countered by the development of the disease. Smutted t i l l e r s were usually shorter than non-smutted t i l l e r s , whether or not they have been treated with g i b b e r e l l i c a c i d . To determine the e f f e c t of g i b b e r e l l i c a c i d on internodal elongation i n the barley plant, the six categories shown.in Table XIX were used. Prom the data, bar graphs were prepared that showed the spread of the internodal distances i n the six categories (Figure 3 to Figure 7). The f i r s t bar graph. Figure 3» compares g i b b e r e l l i c a c i d treated with untreated (control) plants. The g i b b e r e l l i c a c i d treatment resulted i n increased elongation i n internodal regions 0-1, 1 -2 , 2-3. and 3-4, (crown and f i r s t node, f i r s t and second node, second and t h i r d node, and t h i r d and fourth node), whereas regions 4-5 and 5-6 showed no increased elongation. The average internodal distances f o r the f i n a l two nodes of both the g i b b e r e l l i c a c i d treated and untreated controls were very s i m i l a r (Figure 3)° The next two graphs I l l u s t r a t e the i n t e r a c t i o n 49 Table XIX  Internodal Length Row Type Internodal Distance Node Number 1 2 3 4 5 6 2 5 7 12 23 33.5 5.5 8 10 13 25 38 2.5 6 10 13 20.5 31.5 5.5 11.5 13.5 17.5 28.5 38 1.5 5.5 10 12.5 22.5 36.5 •3.5 :6.5 9 12.5 18 34 4.5 10 11 12 17.5 22 6 9 10.5 12 19.5 29.5 5.2 10.5 12.5 15.5 24 .5 38.5 3 8 13 17.5 30 44 4 10.5 14.5 18.5 31.5 35 2 7.5 16.5 17.5 29 41 .5 6.5 9.5 11.5 12 22 23.3 3.5 7 9.5 12.5 20 32.5 5 10 14.5 16.5 25 38.5 /4.0 /8.3 /U.5 /14.3 /23.8 /34.* Control average 50 Table XIX 1 2 3 5 6 Smutted Infected 3 10 13 17.5 20 14 No G i b b e r e l l i c Acid 4 9 15.5 16 13.5 16.5 5.5 12 14.5 20 12 17 4 11.5 x 5 17.5 16 13 3 10 • 14 15 12 4 3 6.5 7 7.5 7.5 6 2 9 14.5 17.5 13.5 5 2.5 9 11 14 18 10.5 2 7 9.5 17.5 16 10 3 10.5 14 17 17 10 average /3.2 /9.5 /12.8 /16 /14.6 /10.6 51 Table XIX 1 2 3 4 5 6 No Smut Infected 3 10.4 11.5 12.5 10 , 30 No 26 34 G i b b e r e l l i c Acid 3 6 11.5 16 4 11 13.5 !5 23 29 5.5 13 16.5 18 20 21 4 10 14 i e > 21 28 4 11 13 .. 15 21 23 3.5 9.5 14 16.5 24 26 4.5 11.5 12.5 14 15 18 3 8 12 14 23 34.5 3.5 10 13 15 21 27 average /3.8 /10 /13.2 /15.1 /20.4 /27 52 Table XIX 1 2 3 4 5 6 3 9 13 15.5 17 ; 10 3 8 15.5 18 22 8 2.5 5.5 15 19.5 26.5 • 15 1 4 .5 14 18 21 23 3 6.5 14.5 18.5 21 12 2 4 9 ^ 17.5 13 .,4.5 10.5 15 18 11 8 3 6.5 13 13 8.5 3 3 4 .5 x 3 17 22.5 21 3 2 11 15.5 16 8 1 7 13 14 15.5 7 4 .5 5.5 8 17 21 8 3-5 5 15 17.5 19.5 9 4 .5 11.5 15.5 17.5 20 12.5 2.5 4 10.5 17 17.5 12 /2.9 /6.3 A 3 /16.7 /18.4 / l l . 3 Smutted Infected G i b b e r e l l i c Acid average 53 Table XIX No Smut Infected G i b b e r e l l i c Acid average 1 2 3 4 5 6 7 13 18 20.5 20.5 17.5 5 11 15 18.5 27.5 41.5 6,5 10 14,5 16 24 ,30 7 12 16.5 17 24 38.5 2.5 9 • 16 18.5 23 30 4.5 11.5 14.5 17 22.5 19 4.5 8 13 17 16.5 30 7.5 14.5 16 18.5 22 25.5 4.5 10.5 15 17 21 40 8 12 !5 17.5 21.5 38 9 12 16 17.5 17.5 20.5 9.5 13.5 19 21.5 27 20.5 7 11.5 16 18 23 32.5 8 10 14 18 26 39 6.5 12 15.5 18 25 31.5 /6.5 / l l . 4 A 5 . 6 /18 /22.7 /30.3 V 54 Table XIX G i b b e r e l l i c Acid Control average 1 2 3 4 5 6 3*5 7.5 11 16 14 .5- 29.5 5*5 10.5 14 16 23 33 5.5 10.5 14 18 22.5 ,30 8 13.5 18.5 18.5 28 . 37.5 3 11 15 . 17.5 26 45.5 2.5 10 15.5 20 26 36 9.5 14 19 20 26 35 4 11 16.5 20.5 28 39 7 13 19.5 20.5 25 26 9 15 17 20 22 28 ,5 6.5 12.5 16.5 18 26.5 37.5 2 10 15.5 19.5 26 32 4.5 9.5 14.5 16.5 19 36.5 7 12.5 17 17.5 19.5 20.5 6 10 14 17.5 18 29 /5.6 / l l . 4 /15.8 /18.4 /23.3 /33. Explanation of Facing Figure G i b b e r e l l i c Acid Treated, Control Plants Nongibberellic Acid Treated, Control Plants Average Elongation of Each Internodal Region Figure 3 Comparison of Internodal V a r i a t i o n of G i b b e r e l l i c Acid  Treated, Control plants .with Nongibberellic Acid Treated, Control Plants 4 5i 4 0' S5-W U CD -p CD 0 •H •P CD O C •H CD O ctf -P w •H P 30-25-20-15-10-I I I 7 7 2\ 0-1 1-2 2-3 3-4 4-5 5-6 Internodal Regions Explanation of Facing Figure Nongibberellic Acid Treated, Diseased Plants Nongibberellic Acid Treated, Control Plants Average Elongation of Each Internodal Region 56 Figure 4 Comparison of Internodal V a r i a t i o n of Healthy T i l l e r s of  Nongibberellic:.Acid~ Treated c .Diseased Plants with.. „ Nongibberellic Acid Treated, Control" Plants j- • 45" .40-K5" 30-W u <y •P 0) B C 0) o c <H 20 0) o <3 •P w Q 15 •10 7 / 7" X / 2 .Zl o-i 1-2 2-3 3-4 4-5 5-6 Internodal Regions 57 between the disease and g i b b e r e l l i c acid treatment. Figure 4 compares the non-treated control with the healthy t i l l e r s of diseased plants. The Internodal distances were s i m i l a r except f o r the upper two, where the control plants exhibited greater elongation. The f a i l u r e of elongation of internodes 4-5 and 5-6 of the infected plants was possibly due to the presence of U. horde! i n the healthy t i l l e r s . The fungus could be using some of the nutrients of the plant thereby r e s u l t i n g i n f a i l u r e of elongation. The i s o l a t i o n of U. horde! from healthy t i l l e r s would support t h i s conclusion. A l t e r n a t i v e l y , the presence of the fungus i n the plant, but not necessar-i l y i n the healthy t i l l e r s , may be causing a reduction i n the a v a i l a b i l i t y of metabolites needed by the healthy t i l l e r s , thus r e s u l t i n g i n f a i l u r e to elongate. The c r i t i c a l point, so f a r as f a i l u r e of elongation i s concerned, appeared to be the time when the plant was undergoing elongation of internodes 4-5 and 5-6. When the t i l l e r s that have smutted spikes were compared with those of non-inoculated and non-glbberelllc a c i d treated plants, internodes 4-5 and 5-6 were again those that f a i l e d to elongate. Figure. 5 shows a small overlap between internodes 4-5 of smutted and non-treated, healthy plants. The average elongation of Internodes 5-6 of the smutted t i l l e r s compared more c l o s e l y with the elongation of Internodes 4-5 of non-inoculated non-g i b b e r e l l i c a c i d treated plants. Internodes 5-6 of Explanation of Facing. Figure Nongibberellic Acid Treated, Smutted Plants Nongibberellic Acid Treated, Control Plants Average Elongation of Each Internodal Region Figure 5 Comparison of Internodal V a r i a t i o n of • Nonglbberellio Acid Treated, Smutted Plants with Nonglbberelllo Acid Treated, Control Plants. 45" 4 0-•35-30-co U CO •p CO . & •H .. ,. .p 25 -S CO o •H 0-1 1-2 2- 3 3-4 4-5 5-6 Internodal Regions Explanation of Facing Figure G i b b e r e l l i c Acid Treated Control Plants G i b b e r e l l i c Acid Treated Smutted Plants Average Elongation of Each Internodal Region = — Figure 6 Comparison of Internodal V a r i a t i o n of G i b b e r e l l i c Acid  Treated ? Control Plants with G i b b e r e l l i c Acid Treated, Smutted Plants. • J . 1 1 1 1 _ #- 1 ; 1 . H 2-R ?-4 4-5 Internodal Regions E x p l a n a t i o n o f F a c i n g F i g u r e G i b b e r e l l i c A c i d T r e a t e d , Smutted P l a n t s N o n g i b b e r e l l i c A c i d T r e a t e d , Smutted P l a n t s Average E l o n g a t i o n o f Each I n t e r n o d a l R e g i o n 60 Figure 7 • Comparison of Internodal V a r i a t i o n of G i b b e r e l l i c Acid- Treated, Smutted Plants with Nongibberellic Acid Treated, Smutted Plants. 45 40 -35 CO u <D P 25 " •H •P <D O In 20 -O d a •p CO .H 15 Q 10. 5 -1 1 I i 0-1 1-2 2-3 3-4 4-5 5-6 Internodal Regions 61 smutted t i l l e r s showed a marked reduction In elongation. The average elongation of internode 5-6 of the smutted t i l l e r s i s s i m i l a r to the elongation of Internodes 1-2, and 2-3 of either the untreated control or the smutted t i l l e r s o One e f f e c t of the disease was to i n h i b i t elongation of internodes 4-5 and 5-6 of barley plants. The a p p l i c a t i o n of g i b b e r e l l i c a c i d to diseased plants f a i l e d to cause elongation of t i l l e r s that are normally shortened by the presence of U, hordel i n f e c t i o n . A comparison of diseased t i l l e r s with those of g i b b e r e l l i c a c i d treated non-inoculated control plants (Figure 6) showed that there was a reduction i n t i l l e r length of a l l internodres-, indicating'- that- there was no increased; internodal elongation of smutted g i b b e r e l l i c a c i d treated t i l l e r s as compared with e i t h e r the control plants or the diseased t i l l e r s of the i n f e c t e d non-gibberellic a c i d treated plants (Figure 7). When a t i l l e r was infected, the presence of U. hordel seemed to prevent increased internodal elongation that would normally take place a f t e r treatment with g i b b e r e l l i c a c i d . . The presence of g i b b e r e l l i c a c i d produced an elongation of internodes 0-1, . 1-2, 2-3 and 3-4, but had no e f f e c t on the internodes. 4-5 and 5-6. Diseased t i l l e r s showed normal elongation (as compared with the control) of internodes 0-1, 1-2, 2-3 and 3-4. Internodes 4-5 and 5-6 showed a f a i l u r e of elongation as compared with the / 62 c o n t r o l . Diseased t i l l e r s that were treated with g i b b e r e l l i c a c i d showed no e f f e c t . A possible explanation f o r t h i s r e s u l t itfas that the presence of the disease and g i b b e r e l l i c a c i d both i n t e r f e r e d with the normal process of internodal elongation, and that the presence of the disease (whose ef f e c t was to i n h i b i t elongation) i s not counteracted by g i b b e r e l l i c acid (whose e f f e c t was to promote elongation). 63 C. The Percentage of Diseased T i l l e r s Following Two  Different Inoculation Procedures with Susceptible  and Resistant Barley V a r i e t i e s . The a p p l i c a t i o n of a mixture of the two haploid str a i n s (I^A and E^a) to the barley seeds at or just before t h e i r germination was the usual method of ino c u l a t i o n . The "standard" dlkaryon generated by t h i s mixture produced the disease i n the barley v a r i e t i e s Himalaya and Vantage, but not i n the v a r i e t i e s Excelsior and Conquest. Whether the f a i l u r e of disease induction on the l a t t e r two v a r i e t i e s was the r e s u l t of f a i l u r e of i n f e c t i o n or of f a i l u r e at some l a t e r stage i n the p a r a s i t i c phase of the l i f e cycle Is unknown. Table XX shows the re s u l t s of experiments which were designed to elucidate t h i s point. Following inoculation, by i n j e c t i o n of the.growing plants, the va r i e t y Conquest showed the disease, suggesting that the block to disease development on the variety Conquest occurred early. Perhaps t h i s blockage occurred at the time of I n i t i a l penetration of the dlkaryon, or p r i o r to the establishment of the "crown" node, or l a t e r , at the time of development of t i l l e r s from the crown node. The establishment and maintenance of the dlkaryon i n the meristeraatlc tissues of the host was necessary at a l l the preceding develop-mental stages as f a i l u r e to do so at any one stage would r e s u l t i n the dlkaryon being l e f t behind, and the spike 64 Table XIX Seed Variety. Presence Of Smut In Various V a r i e t i e s Of Barley Infected seed Injected plant Control Number of Total Number of Total t i l l e r s number of t i l l e r s number of infected t i l l e r s infected t i l l e r s Himalaya 25 42 15 58 E x c e l s i o r 0 68 0 6l Vantage 41 55 35 67 Conquest 0 70 6 62 65 p r i m o r d i a . would t h e n be a b l e t o d e v e l o p f r e e o f t h e disease,, The t i m e a t which t h e f a i l u r e o c c u r r e d , and t h e r e a s o n s f o r i t , r e m a i n unknown. The t i l l e r s o f E x c e l s i o r d e v e l o p e d a t a much e a r l i e r s t a g e i n p l a n t growth t h a n d i d .those o f t h e o t h e r v a r i e -t i e s s t u d i e d . The e a r l y development and r a p i d growth o f t h e t i l l e r and s p i k e p r i m o r d i a may p r e v e n t e n t r a n c e of t h e f u n g u s . 66 SUMMARY A, Multiple Infection - the resistance of the newly-germinated seedling of H. vulgare to U. hordei and the resistance to i n f e c t i o n of subsequently formed t i l l e r s are not correlated and are probably not cont r o l l e d by the same gene or genes. - haploid sp o r i d i a with several genetic markers were obtained from infected plants, i n d i c a t i n g recombination as a r e s u l t of hyphal fusion followed by mitotic recombination or as a r e s u l t of some other event, (for example transforma-t i o n ) , which has not been i d e n t i f i e d . B. The Effects of G i b b e r e l l i c Acid on Inoculated and  Uninoculated Barley Plants - the a p p l i c a t i o n of g i b b e r e l l i c . a c i d increases the o v e r a l l t i l l e r height of healthy plants by e f f e c t i n g the elongation of the internodal regions 0-1, 1-2, 2-3, and 3-4. - g i b b e r e l l i c acid does not ef f e c t elongation of the internodal regions 4-5 and 5-6. - healthy t i l l e r s of diseased plants are uneffected by the a p p l i c a t i o n of g i b b e r e l l i c a c i d . - diseased t i l l e r s are usually shorter than hea]»thy t i l l e r s - the a p p l i c a t i o n of g i b b e r e l l i c acid does not eff e c t the t i l l e r length of diseased t i l l e r s . 67 C. The Percentage of Diseased T i l l e r s Following: Two Different Inoculation Procedures with Susceptible  and Resistant Barley V a r i e t i e s - the i n j e c t i o n of U. hordel (compatible mating types) into the developing spike of a normally r e s i s t a n t s t r a i n o f > H o vulgare resulted i n the production of smut,, i n d i c a t i n g that blockage to normal i n f e c t i o n occurs at the time of penetration of the newly;germinated seedling, at the l e v e l of t i l l e r primordla development, or at the time of spike primordla developmento 68 BIBLIOGRAPHY Aamodt, O.S., and W;H;."Johnston. 1935. Reaction of barley v a r i e t i e s with covered smut. (Ustllago hordel Pers. K and S). Canadian Journal of Research 12 : 590-613. Alexopoulos, C.J. 1962. "Introductory Mycology" 2nd. Ed. John Wiley and Sons, Inc. New York. Bever, W.M. 1945. Hybridization and Genetics i n Ustllago hordel and Ustllago nigra. Journal of A g r i c u l t u r a l Research 71 : 41-59. Cochran, W.A. and G.M. Cox. 1957. "Experimental Designs" Chapter 3, pp 4 5 - 9 2 . John Wiley and Sons, Inc. N.Y. Cook, A.M. Ed i t o r . I 9 6 2 . "Barley and Malt" Academic Press, New York. Paris, J.A. 1924 a. Physiologic S p e c i a l i z a t i o n of Ustllago  hordel. Phytopathology 14 : 537-557* 1924 b. Factors Influencing Infection of Hordeum sativum by Ustllago hordel. American Journal of Botany 11 : 189-214. 69 Farrar, L.L. 1958. Control of Covered Kernal Smut (Sphaoelotheca sorghl) of Grain and Black Loose Smut (Ustllago avenae) of Oats with G i b b e r e l l i c Acid i n Greenhouse Studies. Plant Disease Reporter November-15, 1958. Fisher, G.tf. and C.S. Holton. 1957, "Biology and Control of the Smut Fungi" Ronald. Press,, N.Y.. Goulden, C.H. 1952. "Methods of S t a t i s t i c a l Analysis" Chapter 7, PP 122-133« John Wiley and Sons, Inc. London. Hare, R.C. 1966. Physiology of Resistance to Fungal Diseases i n Plants. Botanical Review ^2. : 95-137. Hood, C.H. 1966. U.V.-irradiation S e n s i t i v i t y and Mutation Production i n Haploid Sporidia of Ustllago horde!. Ph.D. Thesis, University of Alberta. Kline, D.M., D.M. Boone, and G.W. K e i t t . 1957. Venturla inaequalls (Cke) Went XIV. N u t r i t i o n a l Control of Pathogenicity of Certain Induced Biochemical Mutants. American Journal of Botany 44 : 797-803. 70 P e r k i n s , D.D. 1962. P r e s e r v a t i o n o f Neurospora S t o c k C u l t u r e s w i t h Anhydrous S i l i c a Gel„ Cana d i a n J o u r n a l o f M i c r o b i o l o g y 8. : P o n t e c o r v o , G. 1958. "Trends i n G e n e t i c A n a l y s i s " Columbia U n i v e r s i t y P r e s s , New Yor k . Selby, S.M. I 9 6 5 . " S t a n d a r d M a t h e m a t i c a l T a b l e s " . pp 268, 269, .and 273. The C h e m i c a l Rubber Co., C l e v e l a n d , O h io. S t e e l , R.G.D. and J.H. T o r r i e . i960; " P r i n c i p l e s and Pr o c e d u r e s o f S t a t i s t i c s " C h a p t e r 13, pp 252-276. M c G r a w - H i l l Book Company I n c . T o r o n t o . Thomas, P. 1965^ V i r u l e n c e i n U s t l l a g o horde1 ( P e r s . ) Lagerho M.Sc. T h e s i s . U n i v e r s i t y o f A l b e r t a . Van A n d e l , O.M. I 9 6 6 0 Amino A c i d s and P l a n t D i s e a s e s . Annual Review o f P h y t o p a t h o l o g y 4 : 349-368. 

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