Reaction of Genes in Alfalfa Conditioning Resistance and Susceptibility to Common Leaf Spot (Pseudopeziza medicaginis (Lib). Sacc)

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PU R D U E UNIVERSITY

THIS IS TO CBRTIFT THAT THE TH ESIS PREPARED U N D ER MT SU PER VISIO N

BY

Ralph L an ier Davis

ENTITLED R eactlon of Genes in A lfa lfa C onditioning R e sista n c e and S u s c e p b ib ility to Gonimon Leaf Spot (Pseudopesiza M edica^inia (L ib ). S acc). COMPLIES WITH THE UNIVERSITY REGULATIONS O N GRADUATION T H E SE S

AND IS APPROVED BY ME A S FULFILLING THIS PART O F THE REQUIREM ENTS

FOR THE DEG REE OF

D octor of Philosophy

H. H. Kramer

XT, B, P ete rso n

P

H

r o f e s s o r in

ead of

S

Charge

chool or

D

of

Th

e s is

epa rtm en t

February 1________ 50

TO THE LIBRARIAN:---THIS TH ESIS IS NOT TO BE REGARDED A S CONFIDENTIAL.

pb o f e s s o h

G R A D . S C H O O Ii F O R M 9 —3 . 4 9 —I M

m

o ra ro b

REACTION OF GENES IN ALFALFA CONDITIONING RESISTÂI'ÎGE AND SUSCEPTIBILITY TO CŒMON LEAF SPOT (PSEUDOPEZIZA MEDXCAGINIS (LIB ). SACC). A T hesis Subm itted to th e F a c u lty of Purdue U n iv e rsity

by Ralph L an ier Davis In P a r t i a l F u lfillm e n t of th e R equirem aits f o r th e Degree of D octor of Philosophy February, 19 $0

ProQuest N um ber: 27712228

All rights reserved INFORMATION TO ALL USERS The q u a lity of this re p ro d u c tio n is d e p e n d e n t u p o n the q u a lity of the co p y su b m itte d . In the unlikely e v e n t that the a u th o r did not send a c o m p le te m a n u scrip t and there are missing p a g e s, these will be n o te d . Also, if m a te ria l had to be re m o v e d , a n o te will in d ic a te the d e le tio n .

uest P roQ uest 27712228 Published by ProQuest LLO (2019). C o p y rig h t of the Dissertation is held by the A uthor. All rights reserved. This work is p ro te cte d a g a in s t u n a u th o rize d co p yin g under Title 17, United States C o d e M icroform Edition © ProQuest LLO. ProQuest LLO. 789 East Eisenhower Parkway P.Q. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346

ÂCKNO^ÆEDGaæNTS

The au th o r i s g r e a tly in d eb ted t o Dr. H. H. Kramer f o r h is generous h e lp , p e rso n al i n t e r e s t , and encouragement; and he is l i k e ­ w ise in d eb ted to Dr. H. R. A lb rech t who suggested the problem and aided in th e i n i t i a l phases*

Ill

TABLE OF CONTENTS

V ITA ..............................................................................

Vi

ABSTRACT.......................................................................................................................... v i i INTRODUCTION ...............................................................................................................

1

LITERATURE REVIEW

............................................................................................

2

MATERIALS AND METHODS............................................................................................

6

RESULTS AND CONCLUSIONS.......................................................................... V a r ie ta l D i f f e r e n c e s Hybrid S tu d ie s

...............................

...........................

13 15

Fj^ D a t a ..................... R e su lts o f th e F2 Progeny

13

15 .............................

24

SUMMARY.............................................................

30

LITERATURE CITED......................................................................................................

32

APPENDIX......................................................................................................................* 35

IV

LIST OF TABLES

Table

Page

1.

S eg reg atio n of F^ and s e lf e d p r o g e n y .......................................... ..

17

2*

Average ra tin g s o f F^ and S-j^ progeny rows ......................................

19

3*

A nalysis o f v a ria n c e o f r a tin g s o f F, and s e lf e d p r o g e n y ........................................................................................... ...................

22

R egression of th e F^ progeziy on th e mean of th e s e lf e d progeny ................................................................................................

22a

The mean and v aria n ce o f Fg p o p u latio n s from s e le c te d F^ p la n ts ..........................................................................................................

26

The comparison o f th e means o f S _ 's , S p 's and re s p e c tiv e checks ................................................................................................................

27

7.

Means and stan d ard e r r o r of P ^ 's , P ^ 's , F ^ 's , and F g ^ s

29

8.

P ercentage of Fg progeny in grouped c la s s e s in v o lv in g r e s i s ­ t a n t X s u s c e p tib le cro sses ......

30

4. 5* 6.

For in d iv id u a l p la n t d a ta see Appendix.

LIST OF FIGURES

F ig u re

Page

1* T y p ical leav es showing th e f iv e c la s s e s ofin f e c tio n

..............

9

2. Class

1 . R e sista n t r e a c t i o n ...............

3« Class

2 . R e sista n t r e a c t i o n ..............................................................

10

4* C lass

3« Both s p o ru la tin g and n o n -s p o ru la tirg l e s i o n s

10

5. Class

4 . S u sc e p tib le r e a c t i o n .................................................

11

6.

5o S u sc e p tib le r e a c t i o n

11

Class

9

.....................................

7. Leaves showing th e range of in f e c tio n in th e progeny of r e s i s t a n t X s u s c e p tib le cro ss ......................................

a

8. Leaves showing th e range of in f e c tio n when a r e s i s t a n t p la n t i s s e lfe d ..............

21 21

Vi

VITA Ralph L. Davis was born September 10, 1921 on a farm in F a y e tte County, Alabama.

He re c e iv e d h is elem entary and high sch o o l tr a in in g a t

Kennedy High School, g rad u atin g in 1939.

In 1939 he en te re d Alabama

P o ly tech n ic I n s t i t u t e , Auburn, Alabama and re c e iv e d a B. S. degree in A g ricu ltu re in 1943*

In A p ril, 1943 Mr. Davis en te re d th e U nited S ta te s

Naval R eserve Midshipmen’s School and was commissioned an E nsign in th e United S ta te s Naval R eserve fo u r months l a t e r .

He served aboard th e USS

P leiad es from O ctober, 1943 u n t i l December, 1945, f i r s t as a s s is ta n t deck o f f ic e r , th e n F i r s t L ieu te n a n t, and l a t e r E xecutive O fficer*

In 1946 he

was granted an Indiana Farm Bureau R esearch G rant fe llo w sh ip to pursue grad­ u ate s tu d ie s in p la n t breed in g and g e n e tic s a t Purdue U n iv e rs ity , receiv ed h is M. S. degree in 194S.

Mr. Davis

He was appointed J u n io r A s s is ta n t in

Agronomy, Purdue U n iv e rs ity , June 15, 1948. Mr. Davis is a member of Sigma X i, Alpha Z e ta , Gamma Sigma D e lta , and th e American S o c ie ty of Agronomy* He is co -au th o r of th e follow ing p u b lic a tio n s : H olt, E. C. and R. L. D avis,

D if f e r e n t ia l Responses of A rlin g to n and Norbeck

Bent G rasses to Kinds and R ates of F e r t i l i z e r s . 40: 282-284.

J o u r. Am. Soc. Agron.

1948*

Kramer, H. H. and R. L. D avis. Computed Y ields of A lf a lf a .

The E ffe c t of Stand and M oisture C ontent on Agron. J o u r. 41: 470-473*

T is d a le , S. L ., R. L. D avis, A. L. K ingsley, and E. T. M ertz.

1949. M ethionine

and C ystine Content of Two S tra in s of A lf a lf a as In flu en ced by D iffe re n t C oncentrations o f th e S u lfa te Io n .

Agron* Jo u r, ( in P r e s s ) .

vil

ABSTRACT A su rv ey had shown t h a t by working w ith la rg e p o p u la tio rs , a few a l f a l f a p la n ts couM be found i n n e a rly any s t r a i n or v a r ie ty th a t were r e s i s t a n t to common le a f s p o t, Pseudopeziza m edicaginis*

A program

was i n i t i a t e d t o determ ine (a) th e e x ten t th e r e s is ta n c e e x h ib ite d in v ar­ ious s e le c tio n s wag g e n e tic a lly c o n tro lle d and, in a d d itio n , (b) th e n a tu re of th e r e a c tio n of th e se fa c to rs* Two le a f - s p o t r e s i s t a n t clones and four s u s c e p tib le ones were crossed in a l l p o s sib le com binations, in c lu d in g r e c ip r o c a ls .

The F^’ s along

w ith S^’s were grown in th e greenhouse and in o c u la te d w ith c u ltu r e s of th e organism.

The in o c u late d progeny were c l a s s i f i e d and ra te d as fo llo w s:

Class 1,

N o n -sp o ru latin g le s io n s - few in number

Class 2*

N o n-sporulating le s io n s - many

Glass 3*

Both n o n -sp o ru la tin g and s p o ru la tin g le s io n s

C lass 4*

S p o ru la tin g type o f le s io n s - few in number

Class 5.

S p o ru latin g type o f le s io n s - many

C lasses 1 and 2 a re r e s i s t a n t r e a c tio n s ; c la s s e s 3 , 4, and 5 a re su sc e p tib le re a c tio is * An a n a ly sis of v a ria n c e d is c lo s e s t h a t th e re i s no s ig n if ic a n t d iffe re n c e between r e c ip r o c a ls w ith in c ro s s e s aid between d u p lic a te s e lfs * Of the two r e s i s t a n t clo n es, clone 1699 i s more r e s i s t a n t th a n 170$.

S elfed progeny of 1699 have a mean r a t i n g and a v a ria n c e th a t in d ic a te

i t is approaching h aaozygosity f o r f a c to r s c o n d itio n in g r e s is ta n c e .

The

mean r a tin g s of r e s i s t a n t X s u s c e p tib le h y b rid s a re s ig n i f i c a n t l y low er th a n any of th e su sc e p tib le X s u s c e p tib le c ro s s e s , in d ic a tin g t h a t th e r e s is ta n c e of th e hybrids is co n d itio n ed by th e r e s is ta n c e o f th e p a re n t.

The mean

V lll

r a tin g s of th e r e s i s t a n t X s u s c e p tib le h y b rid s a re always low er when 1699 i s used a s th e r e s i s t a n t p a re n t.

Hybrids between th e s u s c e p tib le clones

have mean r a tin g s and v a ria n c e s of th e same magnitude as th e s e lf e d progeny o f th e s u s c e p tib le p a re n ts . The mean r a tin g s and v a ria n ces o f th e

and

progeny su g g est t h a t

th e g en etic f a c to r s c o n d itio n in g s u s c e p t i b i l i t y a re r e c e s s iv e , A number of F^ and

p la n ts were s e lf - p o ll in a t e d and th e progeny

grown in th e greenhouse and in o c u la te d .

R ath er than s e l f an u n se le c te d pop­

u la tio n of F .* s , s e le c te d r e s i s t a n t and s u s c e p tib le p la n ts from th e r e s i s t a n t X s u s c e p tib le hyb rid s were s e lf - p o ll in a t e d to o b ta in th e F^ p o p u la tio n . p la n ts s e le c te d f o r s e l f ipg i n th e

progeny to o b ta in th e

The

p o p u la tio n

ranged frcm th e most r e s i s t a n t in d iv id u a ls to th e most s u sc e p tib le in d iv id u a ls . R esu lts in the F^ were of th e n a tu re expected i f a r e a c tio n is con­ d itio n e d by a number of g e n e tic f a c t o r s .

The p o p u la tio n s in th e F gis and S g 's

could be s h if te d by s e le c tio n tow ard e i th e r r e s is ta n c e or s u s c e p t i b i l i t y . The prepotency of th e more homozygous r e s i s t a n t clone 1699 is mani­ fe s te d in the F^ ju s t as i t was in th e F^.

Thus, i t can be concluded t h a t ,

ir r e s p e c tiv e of th e g e n e ra tio n , clone 1699 tra n s m its th e se g e n e tic f a c to r s to th e progeny.

REACTION OF GENES IN ALFALFA CONDITIONING RESISTANCE AND SUSCEPTIBILITY TO COMMON LEAF SPOT (PSEUDOPEZIZA MEDIGAGINIS (LIB). SACC).

INTRODUCTION Coramon le a f sp o t has lo n g been known as a d e s tr u c tiv e d ise a se of a lfa lf a .

As e a r ly as 1891, Combs (4 ), in Iowa, s ta te d " a t t h i s tim e

th e le a f sp o t caused by P. m edicaginis i s th e most d e s tr u c tiv e d is e a s e in a lfa lfa " .

In 1908, S tew art, e t . a l . (23) r e f e r r e d t o th e d is e a s e as th e

most im portant fungus a tta c k in g a l f a l f a in New York.

The e a r ly method of

c o n tro l was to cu t th e a l f a l f a p rem aturely b e fo re d e f o lia tio n . w ill not p rev en t th e lo s s o f le av es a f t e r cu ttin g *

However, t h i s

Such tre a tm e n t over a

period o f tim e can only r e s u l t in reduced v ig o r and lo s s of stan d due to de­ creased food re se rv e s ( 6 ) .

Since th e d is e a s e r a r e l y causes a d ir e c t re d u c t­

ion in sta n d , i t has been co n sid ered one of th e unavoidable e v ils of a l f a l f a . At tim es, especialijy in th e s p rin g and f a l l , th e r e is as much as 50 per cen t d e f o lia tio n , o f te n more.

This r e s u l t s n o t on ly in a d ecrease in

y ie ld , b u t a ls o , and more s ig n if i c a n tl y , in a low er q u a l ity .

Approximately

tw o -th ird s of th e p ro te in and 80 t o 90 p er cen t of th e caro ten e are found in th e le a v e s ,

K ingsley (17) found th a t in fiv e s tr a i n s of a l f a l f a examined,

the a l f a l f a leav es co n tain ed approxim ately f iv e tim es more m ethionine and th re e tim es more c y stin e th an th e stem .

The lo s s w ith as much as $0 p er cent

d e f o lia tio n i s obvious » With th e in c re a s in g s e rio u sn e ss o f b a c t e r i a l w ilt in a m a jo rity of th e a l f a l f a growing re g io n s , i t i s im p erativ e th a t an a l f a l f a be r e s i s t a n t to t h i s d is e a s e .

U n fo rtu n ate ly , th e group of a l f a l f a s th a t c a rry th e h ig h e st

degree o f w ilt r e s is ta n c e , T u rk ista n , (12, 23, 24, 29) i s th e most s u s c e p tib le

2 to le a f sp o t (2 2 ).

At p re s e n t th e re a re no ex p erim en tal d a ta to in d ic a te

t h a t a p la n t cannot be r e s i s t a n t to b a c t e r i a l w ilt and a ls o c a rry a high degree of r e s is ta n c e to P. m e d icag in is. Most o f th e p re s e n t s e le c tio n s of a l f a l f a , though r e s i s t a n t to w i l t , a r e q u ite s u s c e p tib le t o common l e a f s p o t.

However, i t seems d e s ir ­

ab le to develop an a l f a l f a th a t is adapted t o areas where lo n g e v ity i s the prim ary r e q u i s i t e , and i s a ls o r e s i s t a n t to l e a f spot*

Under p r e s e n t con­

d itio n s growers must depend on th e W estern s ta t e s f o r seed supply; th e r e f o r e , i t is im p ra c tic a l to have an a l f a l f a v a r ie ty adapted only to lo c a l con­ d it io n s . In o rd er to breed le a f - s p o t r e s is ta n c e in to th e b e t t e r v a r i e t i e s most e f f i c i e n t l y , i t i s n e c e ssa ry t o know th e r e a c tio n o f h e re d ita ry f a c to r s th a t co n d itio n r e s is ta n c e and s u s c e p t i b i l i t y .

With t h i s in mind, a program

was i n i t i a t e d t o determ ine (a) the e x te n t to which th e r e s is ta n c e e x h ib ite d in v a rio u s s e le c tio n s i s g e n e tic a lly c o n tro lle d , and (b) th e n a tu re o f th e re a c tio n of th e se f a c t o r s . LITERATURE REVIEW P. m edicaginis i s an As cornyce t e w ith no known ase x u a l s ta g e .

The

as cospores, g erm in atin g on m oist le a v e s , send out a germ tu b e which p e n e tra te s th e epiderm al c e l l .

From h ere i t sp read s by branching of th e myoolium u n t i l

th e re i s a mass of strom a (1 1 ). Jones (11) su g g ests t h a t r e s is ta n c e i s not due to any m echanical b a r r i e r , b u t t o something w ith in th e epiderm al c e l l which in h i b its grow th. When v is ib le in f e c tio n i s n o t ev ident fo llow ing in o c u la tio n , o b serv atio n s in d ic a te th a t th e growth of th e germ tu b e ceases prom ptly upon e n te rin g th e epiderm al c e l l .

3 According to Jones (1 1 ), two c h a r a c te r i s t ic s ten d to d is tin g u is h P. m ed icag in is from "sp o ts" caused by o th e r fu n g i. a re c i r c u la r in shape and of lim ite d s i z e . m illim e te rs in d ia m ete r.

F i r s t , th e s e le sio n s

They r a r e l y exceed two t o th re e

Second, a sm all r a is e d d is k appears in th e c e n te r

of th e sp o t when i t reach es f u l l development (F ig . 6 ) .

The r a is e d d is k may

appear a s a j e l l y - l i k e drop o f exudate under m o ist c o n d itio n s . The fungus rem ains in th e f a l l e n le a v e s where th e a p o th e c ia over­ w in te r.

Spores are abundant the fo llo w in g sp rin g and th e r e is u s u a lly an

adequate su p p ly o f inoculum d u rin g th e e n t i r e growipg seaso n . V a r ie ta l d iffe re n c e s have been observed in a l f a l f a i n th e r e a c tio n to a number of d is e a s e organisms (3 , 12, 13, 23, 24, 27, 2 9 ).

Of th e v a r ie ­

t i e s te s te d , T u rk ista n showed the most r e s is ta n c e to b a c te r ia l w ilt (12, 23, 24, 2 9 ).

F ield o b serv atio n s (10, 27, 28) and greenhouse in o c u la tio n s (26,

27) showed s tr ik in g d iffe re n c e s in th e v a r i e t a l re a c tio n s to th e le a f spot and stem b lig h t, Ascochyta im p e rfe c ta . Jones and Weimer (15) found a marked d iffe re n c e among v a r i e t i e s in o c u late d w ith Stagnospora m e l i l o t i , an o th er le a f - s p o ttin g organism .

There

were d iffe re n c e s in r e s is ta n c e to a l f a l f a r u s t , Uromyces s t r i a t u s m ed icag in is. among tw elve Medicago sp e c ie s and v a r i e t i e s and s e le c tio n s o f M. s a t i v a .

M.

ru th e n ic a c a rrie d th e h ig h e st r e s is ta n c e ; W iereas, Ladak was th e most r e ­ s is ta n t o f th e s a tiv a form s (1 9 ), Clonal li n e s in th e breeding n u rsery (14) in fe c te d w ith P. medica­ g in is showed d if f e r e n c e s ranging from alm ost com plete freedom of the d ise a se to more th a n 50 p er cen t d e f o lia tio n .

F i f t y per c e n t o f th e p l a i t s w ith in

th e v a r ie ty Ladak c a r r ie d a high r e s is ta n c e ; however, none showed complete freedom .

In a v a r ie ty and s tr a i n s n u rse ry in f e c te d w ith Stemphylium b o try o -

sum, d iffe re n c e s ran g in g from a l i g h t in f e c tio n t o com plete d e f o lia tio n were

4 re c o rd e d . P e l t i e r and Tysdal (24) dem onstrated t h a t , w ith o u t ex c e p tio n , th e h ig h er th e b a c t e r ia l w i l t r e s is ta n c e of th e p a r e n t, th e h i ^ e r th e r e s is ta n c e of th e progeny.

B rink, e t . a l . (3) found from progeny perform ance th a t

s e le c te d r e s i s t a n t in d iv id u a ls d i f f e r g r e a tly in t h e i r a b i l i t y to tra n s m it re sista n c e # In studying th e r e a c tio n o f p ro g en ies o f s e le c te d p la n ts in o c u la te d w ith A. im p e rfe c ta , R e itz , e t . a l . (2?) observed th a t " in a l l of the v a r ie ­ t i e s and sp e c ie s re p re s e n te d in t h e f i r s t g en era tio n of in b ree d in g , th e re was a s ig n if ic a n t tendency f o r th e in b red p o p u la tio n from each p a re n t p la n t to re a c t t o b la ck stem, in th e same manner a s th e p a r a i t had r e a c te d ."

In b reed ­

ing follow ed by s e le c tio n and h y b rid iz a tio n proved valu ab le in r a is in g th e le v e l of r e s is ta n c e .

However, th e re was evidence t h a t th e h ig h e st le v e l of

r e s is ta n c e achieved by s e le c tio n among in b red s could be matched by s e le c tio n in o p en -p o llin ated s to c k s . Atwood and Grun ( l ) p o in t out t h a t in 34 g e n e tic s tu d ie s , in c lu d in g 22 c h a ra c te rs , only disom ic r a t i o s have been proposed.

This seems scmewhat

unusual sin c e a l f a l f a i s co n sid ered a te tr a p lo i d (2N»32).

Evidence (16)

in d ic a te s t h a t th e two M. s a tiv a genomes a re v ery s im ila r and in t e r p a ir f r e e ­ ly ,

There is a ls o in t e r p a ir i n g between th e genomes of M. s a tiv a and M. f a l -

c a ta , in d ic a tin g homology of t h e chromosomes. r iv a le n ts would be expected a t m etaphase.

As a r e s u l t a number of quad-

From c y to lo g ic a l in v e s tig a tio n s ,

Ju len (16) re p o rte d th a t th e most common c o n fig u ra tio n a t metaphase I was 16 b iv a le n ts , b u t both u n iv a le n ts and q u a d riv a le n ts o c cu rred .

Ledingham

(20) rep o rte d t h a t th e most fre q u e n t type of p a irin g in crosses between M. f a lc a ta and M. s a tiv a was 16 b iv a le n ts * I t was p o in ted out by T ysdal, e t . a l . (32) th a t soiæ of th e com-

5 p le x g e n e tic r a t i o s might be ex p lain ed on th e b a s is of te tra s o m ie in h e r i­ ta n c e .

They used d a ta on le a f shape and f i t t e d i t to a te tra so m ie se g re ­

g a tio n assuming chrom atid s e g re g a tio n .

A P v alu e of .7 to .8 was o b ta in e d .

However i t is th e w r i t e r ’s opinion th a t w ith homologous genomes and f r e e in te r p a ir in g plus a high freq u en cy of 16 b iv a le n ts , chrcmosome r a th e r th a n chrom atid s e g re g a tio n would be expected provided in te r p a ir in g was at random. A number of c h a ra c te rs have seg reg ated in such a way as to su g g est a m u ltip le f a c to r in h e rita n c e ( 4 ) .

These a re a s fo llo w s: (a) stem le n g th

of 6-week old p la n ts , (b) p la n t h e ig h t, (c ) number o f stems p er p la n t, (d) le a f shape, (e) l e a f s i z e , ( f ) p er cen t of leav es 3rd h a rv e s t, (gj) d a te of blooming, (h) ro o t ty p e , and ( i ) seed y i e l d . A few in v e s tig a to r s have attem pted t o determ ine th e number cf genes c o n d itio n in g r e s is ta n c e and s u s c e p t i b i l i t y to b a c t e r ia l w i l t .

P e ltie r

and Tysdal (24) su g g ested t h a t as many as th re e major g en e tic f a c to r s were involved.

B rink, e t . a l . (3) s ta te d t h a t " r e s is ta n c e t o b a c t e r ia l w ilt in

a l f a l f a behaves in in h e rita n c e as an in te rg ra d in g c h a ra c te r and probably r e s t s upon a complex g e n e tic b a s is , time im p o ssib le".

A f a c t o r i a l in t e r p r e ta tio n i s a t t h i s

On th e o th e r hand, ¥ils