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The genetics of resistance to the spotted alfalfa aphid

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Authors Powell, William Houston, 1926-

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THE GENETICS OF RESISTANCE TO THE SPOTTED ALFALFA APHID

. , - W ■ - - , ■

W illiam Houston Powell

A Thesis Submitted to the Faculty of the

DEPARTMENT OF AGRONOMY

In P artia l Fulfillment of the Requirements

For the Degree of

MASTER OF SCIENCE. . '

In the Graduate College

STATEMENT BY AUTHOR

This thesis has been submitted in partial fulfillment of requ irements for an advanced degree at The University of Arizona and is deposited in The University Library to be made available to borrowers under ru les of the Library.

Brief quotations from this thesis are allowable without special perm ission, provided that accurate acknowledgment of source is made. Requests for perm ission for extended quotation from or reproduction of this m anuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in their judgment the proposed use of the m aterial is in the in terests of scho larship. In all other instances, however, perm ission must be obtained from the author.

SIGNED:

APPROVAL BY THESIS DIRECTOR

This thesis has been approved on the date shown below:

MELVIN H. SCHONHORSTAssociate P rofessor of Agronomy

Date

, ACKNOWLEDGMENTS ;' ' '

.This spotted alfalfa aphid inheritance study of alfalfa was con

ducted under the guidance of Dr„ Melvin H„ SchoiihorsL I take this oppor

tunity to express My sincere gratitude to Dr. Schonhorst for his invalu

able assistance.

. My-; sincere' thanks' are Also extended to M r. Frank V. Lieberman

who provided me with spotted alfalfa. aphids, made available a green

house, and gave advice and suggestiohs that helped in barrying on the

research . ■ . - ' ; k ; '̂ ' - : \

I wish to extend sincere thanks to Dr s. M. W. Nielson? R. T.

Ramage, and L». S. Stith for their advice and suggestions in connection

with the research and preparation of this m anuscript.

To all others who contributed in any way and are not mentioned

here, I am sincerely gratefuL

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IV.

Table

LIST OF TABLES

L The level of resistance of ten pareht plants selected id r the genetic study..... * . . . . . . . . . . . . . . . . . . .

» o d o o o o o o o o q o o o e o o o e o o o o © o e o © o © o o o o o o o clevel of re s istane e . . . . .

th e to ta t number of pb F 1 and S. diploid alfalfa plants falling into each level

a o o o o o o o o o o e o o o o o o o o o o o o o o o o o o o o o o o o o o d o o o o o o o o o o

2.

3.:

,4 .;percent of progeny in each class when using the plant as either the male or female paren t. . . . . . . . . . . . . . . . . . . . . . . . .

5. Possible loci contained by the various p l a n t s .. . . . . . . . .

Page

20'

25

. 38

.32

: 37

v

' : ; M T -O F H G U ? ^ ;- ' ' ■ V

Figure / v ; : ; ̂ , Page

method

'In t r o d u c t io n

The spotted alfalfa aphid (TfaerioapMg inaculata FBucktonl) is one

of the most serious insect pests attacking alfalfa in the United S tates.

Estim ated losses from this pest, f irs t reported in the United States in

1954, have mounted into millions of dollars annually. Alfalfa breeders

and entomologists at many state experiment stations in cooperation with

personnel of the United States Department of A griculture have expanded

their alfalfa research program s to include resistance to the spotted

■ alfalfa aphid, ' ■ ; , ' . - :■

"When attempting to transfer a genetic factor such as spotted

alfalfa aphid resistance from one line to another, it is important to know

its mode of inheritance. Such knowledge will help to determ ine the m ost

efficient and tim e-saving breeding procedure.

This study was initiated to determ ine the mode of inheritance of

resistance to the spotted alfalfa aphid in diploid alfalfa. Such inform a

tion would be useful to breeders in developing new varieties or improv

ing existing varieties of alfalfa.

REVIEW OF LITERATURE

An attempt has been made to review the litera tu re pertaining to

the spread of the spotted alfalfa aphid in the United States, methods of

controlling this pest, and work being done on developing resistan t vari-

. eties. L iterature pertaining to each is presented under a separate head-.

' ■' . ing. . ■ ■ ' ' ; ' ■; ' ■ ,

Introduction into the United States

The spotted alfalfa aphid was f irs t found in eastcentral New Mex

ico in February of 1954. (7, 10, 58). This pest is an Old World species ,

having previously been reported in India, Israe l, Italy, and other Medi

terranean countries (57) o After becoming established in alfalfa in the

irrigated desert regions of New Mexico, the aphid spread so rapidly that

by the spring of 1954 it was present in areas from southern California

east to w estern Texas (7, 58). At present this pest occurs in most

alfalfa-producing regions of the United States, except the New England

states. Of all the economically important but harmful insect pests in tro

duced into North America, not one has spread so rapidly or caused such

destruction in so short a tim e (53). The phenomenal spread was due to

2

'̂ ' ' : A : ^ : : -':

its ability to reproduce under a wide range of environmental conditions,

to produce large numbers of alate (v/inged) forms when conditions become

unfavorable (4 3) , and to its parthogenetic reproduction (53) „

D e s c r i p t i o n t ' ' ' - v

The spotted alfalfa aphid is L40 to 1.95 mm long (41, 47), gray-

' ish or pale yellow,;: with four to ̂ M rpw s; of sm all black ■ spots and,spine -

like setae on the dorsal surface of the thorax and abdomen (5, 29, 57).

Mature female spotted alfalfa aphids may be either alate (winged) or

apterous (w ix ^ le ^ l '.WiEgSjtwheh-preseiit,. are netted or spotted and

usually lie roof-like over the body whep at re s t. Apterous females are

usually' slightly la rger than - alate fem ales, but do not differ'appreciably

in color. The young, or nymphs, are ivory-colored when newrborn.

Normally, the nymph goes through four instars or molts during its

development. One or two additional molts may occur due to pathological

changes in the host plant (20).

Therioaphis maculata has a life cycle sim ilar to other w arm -

climate aphids; apterous and alate parthenogenetic fem ales have been

observed throughout the year. Dickson pt al. (7) reported finding no

evidence that apterous oviparous fem ales laid eggs; other workers (32)

have found eggs. . \ . / - A - • ̂ : ■ ■■; ■ / ■ v.:

Tem perature, humidity, and condition of the host plant influence

the ra te of parthenogenetic reproduction. In June; under warm Arizona

' ■ \ ; : r . .. V : 4

conditions, nymphs reached m aturity in about five days with an average

reproductive life of eleven days for the apterous form and fifteen days

for the alate form s (38) „ Length of active reproduction was determined

mainly fey. female fertility and rate of reproduction (20). ■ Usually, half

of the offspring was born in the f irs t th ird of the reproductive period.

Generally, a female did not die immediately after the end of her rep ro

ductive period. The period of senescence tended to occupy proportion

ately more of the fem ale’s life in sum m er than in winter (20).

Nielson and Barnes (38) reported that at least thirty-five genera

ations of the spotted alfalfa aphid occurred in southern Arizona in 1055.

This study showed that; 28% alate and 15% apterous fem ales lived beyond

thirty days, and apterous females produced more living young than did

the alate fem ales. Harpaz (20) reported forty-five generations per year

under field conditions in Israe l as compared with forty-one generations

per year under laboratory conditions. . ■'

Tuttle e t al. (57) found that th irty to forty generations per year

may be produced in parts of Arizona. Optimum tem perature for m axi

mum reproduction ranged between- 90°;and 100° F. When tem peratures

fell within this range aphids produced an average of four to five young

per day» Some adults produced up to fourteen young in one day. Total

production varied from twenty-five to one-hundred nymphs per female (1)

;■ ;: Pesho and Lieberman (44) and Pesho et ah (45) reported the

existence of two biotypes of the spotted alfalfa aphid in southern

California. The new biotype, called Ent. A, was capable of survival and

reproduction On four of the nine parent clones of Moapa. The new bio~.

type was unable to survive on the remaining five parent clones of Moapa,

or on any of the five parent clones of Lahontan. All parent clones in

both Moapa and Lahontan were resis tan t to the original population of the

spotted alfalfa aphid. ,

Damage, . . ■; . .

The spotted alfalfa aphid prefers alfalfa, but will attack bur

clover, sour clover, and bersedm (1, 8, 47, 48, 56, 57). It also feeds

on crim son clover, button clover, straw berry clover, yellow-blossom

sweetclover, and alsike clover, but will not live on legumes such as red ,

clover, ladino clover^ white Dutch clover, rose clover, Hubam.clover,

subterranean clover, lespedeza, common vetch, purple vetch, birdsfoot

trefoil, sesbania, and sweetclovers other than yellow-blossom (1, 8).

Spotted alfalfa aphid infestations s ta r t with m igrating alate

females on the upper portion of the alfalfa plant where they produce the ir

young (57). L ater, m ost of the nymphs m igrate down the stem s and begin

feeding on the under side of the lower leaves (1, 29, 46, 47, 57, 58).

When plants become heavily infested the aphids move up the stem and

feed on the upper leaves and buds (1, 29, 57) . As the plant becomes

heavily infested the young aphids go through a biological change and

eventually develop into winged fem ales (43).

V :r ;F irs t signs of spotted attalfa aphid damage usually are found in

the leaves near the base of infested plants. Several days after infesta

tion, depending upon the tem perature and humidity, light colored veins

can be observed on the lower leaves. Continued feeding causes these

leaves to curl, turn yellow, die, and drop from the plant. Continued

heavy feeding eventually kills the plant (1, 29, 3,7, 43, 46, 48, 57),

The spotted alfalfa aphid inserts its stylet inter cellular ly and

feeds'-'within the mesophyll parenchyma and phloem. ■ Considerable, .

quantities of saliva containing toxic substances are injected into the

tissues during feeding (9)j In addition, the aphid sucks large amounts

of cell sap from the host plant and secre tes a sticky honeydew. The

honeydew becomes infested with a black sooty mold that discolors the

plant, lowers protein and carotene content, and thereby reduces the

feed value and yield of the hay. This sticky fluid also in terferes with

harvesting procedures (1, 29, 47, 57). :

Maxwell and Painter (34) found that the ra te of honeydew deposi

tion from the spotted alfalfa aphid was affected significantly by (a) changes

in tem perature; (b) the part of plant being fed on; (c) varie tal differ

ences; and (d) amount of light reaching the host plant.

Biological Control

Several kinds of native p redato rs, introduced parasites , and

fungus diseases attack the spotted alfalfa aphid. Normally, these do

Snot destroy enough aphids to control a serious infestation, but they

may hold down light infestations and delay or prevent reinfestations (1,

29 , 47, 57) . ; . ' . '; :

Native predators of the spotted alfalfa aphid are the ladybird

beetles and their larvae, Collops adults, minute pirate bugs, big-eyed

bugs, and larvae of syrphid flies (1, 14, 29,. 39, 52, 54, 57). Nielson

and C urrie (39) reported that convergent lady beetles reared under

laboratory conditions lived longest when fed a diet of sixty aphids per

day for m ales, and ninety p e r .day for fem ales. F ie ld -reared adults

lived longest on thirty aphids per d ay .. Smith and Hagen (54) reported

that a ladybird beetle may eat over 1, 000 aphids during its life.

Three parasitic wasps of the spotted alfalfa aphid were imported

from the M editerranean and Middle E astern areas during 1955 and 1956.

These species were Aphelinus semiflavus (Howard), Praon palitans

(Muesebeck)> and Trioxys utilis (Muesebeck) (1, 12, 16, 51, 60, 61, 62).

Barnes (2) and Tuttle et al„ (57) reported that T. utilis helped to control

the spotted alfalfa aphid in Arizona. The larvae of these wasps destroy

aphids by consuming their internal organs and body fluids (59). Studies

in northern California from 1955 to 1957 have indicated that native

predators played a significant part in governing the density of the spotted

alfalfa aphid (54) .

Fungus d iseases, presumably native, have been recovered from ■

diseased spotted alfalfa aphids. The f irs t fungus observed on aphids was

found in Riverside County, California, in December 1954. During wet

spells, or following irrigation, a large percentage of the aphids may be

Killed by several species of this fungus. These a re Entomorphthora

obscura, E. ignobilis, E. ex itia lis, E. coronato, and E. virulenta (1,

17, 18, 19). ■

Chemical Control

The spotted alfhlfa aphid has been economically controlled with

properly applied chemical treatm ents. Field applications can be applied

as a spray or dust by the use of either ground equipment or airplane,

while treatm ent of seedlings can be made by field applications or p re

planting seed treatm ents. Areas such as m argins, fence areas, and

ditch banks containing host plants left untreated will harbor aphids that

will reinfest treated fields (1, 29, 47, 57).

Resistance - ,

Painter (42) defined plant resistance to insect attack as the r e la

tive amount of heritable qualities possessed by the host plant which

'influenced' the degree' of’damage done by the specific • insect. Resistance;,

is a relative m easurem ent Of damage using a susceptible variety as a

standard. Normally, a resis tan t variety suffers injury, but always less

than a susceptible one under the same conditions.

Howe et al„ (24) divided resistance of alfalfa to the spotted alfalfa

aphid into seven classes as follows:

(1) +-H-+ 100% m ortality of introduced nymphs before

m aturity and reproduction.

(2) 4--H- At least one introduced nymph matured to

v ' reproduce. • ' . — ' ,

(3) ++ Light reproduction with partia l m ortality of

newly born nymphs. No substained build-up

' in population.

(4) + Light reproduction with light m ortality of

newly born nymphs. Small build-up in

population. ■ • . :

(5) I (Intermediate) Moderate build-up in popula

tion. Newly born nymphs are. found in congre

gated colonies.

(6) S (Susceptible) Average or more than average

' build-up in popula,tion. • ;■

(7) HS (Highly susceptible) Plants with more than

average build-up in insect population.

Insect resistance has been grouped into three general areas: (a)

nonpreference of insects, (b) inability of the insect to maintain life on

the host plant, and (c) ability of the plant to grow and reproduce itself

while supporting an insect population.

■ v v; : ■ ■. ■ ; . ■ 10

Insect preference may resu lt from physical, chemical- or physi- •

ological plant factors o Painter (42) stated that pubescence and tough

ness of plant tissue in some alfalfa varieties were responsible for

resistance to leafhoppers. Toughness of plant tissue could interfere

with entrance of the spotted alfalfa aphid’s stylet into feeding sites.

Moore (36) reported that increased light intensity due to light

reflection in cabbage attracted pea aphids. Cody (3) found that dark

green colored peas had m ore pea aphids than did light green plants.

. Schonhorst found both dark green and light green colored alfalfa plants

resis tan t to the spotted alfalfa aphid.

Painter (42) stated that high soil fertility increased the level of

resistance in plants to insect attack. I t has been postulated that com

position of the available food in the host plant possibly plays the most

important role in determining its resistance to aphid attack. Induced

resistance can be obtained sometimes by applying fe rtilize rs (42).

, M altais and Auclair (31) found that to ta l soluble nitrogen content

was 11.5 to 37.1 percent lower in pea aphid resistant: pea varieties than

susceptible plants for corresponding stages of growth and types of plant

samples. They also found wide and consistent differences in sugar con

tent between susceptible and resis tan t varie ties. F ree sugars occurred

Dr. Schonhorst reported this in a personal interview with the author. ' ^

in la rger quantities in resistan t varieties than in susceptible ones.

Varieties susceptible to the pea aphid contained m ore nitrogen and less

sugar than the resis tan t varieties, and the sugar-nitrogen ratio (8 /N)

.was 23,4 to 63.7 percent higher in the resis tan t plants. This supports

Schaefer’s (50) hypothesis that aphids m ust take in large quantities of

plant sap in order to obtain the supply of nitrogenous food necessary for

reproduction. The excess m bisture and carbohydrates are then voided

as honeydew. M altais (30) found, with one exception, that the level of

amino acids was lower in pea plants resis tan t to the pea aphid than in

susceptible peas. \

M arble et ah (33) reported that alfalfa plants susceptible to

spotted alfalfa aphids were high in 0 -alanine and ethanolam ine.. Seven

teen amino acids were detected in honeydew from spotted alfalfa aphids,

, These workers stated that no conclusive difference in amino acid com

pounds of alfalfa were apparent between resis tan t and susceptible plants.

Physiological resistance may resu lt from (a) deleterious effects

of specific chemicals including toxins, (b) lack of a specific m aterial

in parts of plant fed upon, (e) differences in quantities of food present,

(d) presence of m aterial repellent to the insect, and (e) frequent r e s t

lessness of insects and other peculiar behavior patterns (42).

The inability (antibiosis) of the spotted alfalfa aphid to maintain

life on resis tan t alfalfa plants may resu lt in (a) death of insects, often

in the f irs t instar, (b) abnormal length of life /(c ) reduction in food

resulting in sm aller size in sects, and (d) decreased fecundity of the

insect. : ' ' ' • ■ ■■ ' - . ' ' : • ,

The ability of half-grown nymphs to feed on resis tan t plants is

not evidence that new born nymphs can do so. Therefore, the length of

te s t should be sufficient to insure aphids an opportunity to complete

their life cycle. / / ’ / . ■■ ■ ' '

P lant tolerance to insects is difficult to analyze. Vigor of a

plant greatly affects itstolerance to insect attack. Snelling (55) reported

that first-generation hybrids between two susceptible varieties of

sorghum remained green, and flourished long after the parents had

been killed by chinch bugs. .

Insect tolerance is of little value in resistance to the spotted

alfalfa aphid. This is because the spotted alfalfa aphid" excretes honey-

dew that in terferes with harvesting of alfalfa for either hay or seed.

The study of insect resistance is a long-term project. Develop

ment of a new variety, which involves hybridization followed by selec

tion, usually requires a.minimum of: six to ten years, although this may

be shortened when m ore than one generation is grown per year.

According to Painter (42), ’’Some of the best evidence concern

ing the cause of resistance comes from a study of; segregating popula

tions in crosses between resis tan t and susceptible p lan ts.’’ Whether

- . . v - - ' r \ ■■ ■ ' ; ; : ; ; ;

the apparently resis tan t plant is actually resis tan t can be discovered

by progeny testing. Progeny testing of plants by the antibiosis cage

te s t described by Howe and Smith (25) gave the best information on

resistance to the spotted alfalfa aphid.

Plants can be progeny'-tested in the field for resistance by one

of three methods: (a) individual plants caged with a definite number of

aphids, (b) several plants caged at one tim e, and (c) plants infested

under field conditions. The: last;method is the easiest; however,' due to - '

environmental conditions and variations in natural insect population

build-up, it is not always possible to use this method effectively (25,

40). . ; " ' ; ' . ’ ; j ; ' . : '■ ■ ■

Alfalfa plants can be tested for resistance from the seedling

stage to m aturity. / However, seedlings a re more easily stunted and

killed than mature plants. When working with young plants, either use

sm all insect populations or else leave the pests for a shorter period

of time; unless killing of susceptible seedlings is desired (23) .

When spotted alfalfa aphids are placed on resis tan t plants, they

usually become res tle ss in one to four hours, and the aphids die in

forty-eight to seventy-two hours. This period may be shortened at

higher tem peratures (25, 35). Dahms and Painter (4) also found this

to be true of the pea aphid. They stated that:

The pea aphid reproduced more rapidly and the m ortality was less on alfalfa plants that appeared to

be susceptible under field conditions than on thosethat appeared to be resistanto- ' ; * ' ; :

Harvey and Hackerott (21) reported that spotted alfalfa aphid

survival and reproduction appeared unaltered when placed on either

scions or stocks of reciprocally grafted resistan t and susceptible alfalfa

clones. This indicated that the factors responsible for resistance were

not transferred . Grafting resis tan t plants upon susceptible stocks was

suggested as a possible technique for subjecting antibiotic or nonpre

ferred plants to aphid-injected toxin.

Diallel Crosses

The diallel cross offers a means of rationalizing certain resu lts

while keeping the amount of work at a manageable level. The diallel

analysis provides (a) estim ates of the over-all degree of dominance, if

present, (b) degree of heterozygosity of loci showing dominance, and

(c) the allel frequency at such loci (6). Jinks (28). reported that if within-

family variances of the segregating generations were included in the

analysis, it was possible to detect linkage and estim ate its effect. Jinks

(27) and Hayman (22) suggested the diallel cross may prove a powerful

method for obtaining a rapid, over-all picture of the genetical structure

of a large number of parental lines.

Gilbert (13) stated that many of the twenty-eight interactions :

from an eight by eight cross were expected to be ’’significant” but none

so large as to affect the prediction for main effects of a particu lar cross.

This does not mean that the two parents with the highest level of r e s is t

ance would necessarily combine to give the best resistance; they may ,

show a negative interaction, or another cross may show a la rg er posi

tive interaction.

In using a diallel analysis, plants are crossed in all combina

tions . When evaluating the different parental lines that a re normally

cross -fertilized, it is frequently of value to use reciprocal crosses and

the self-pollinated populations.

It should be expected, within lim its of experimental e rro r, that

the main effects w ill correspond to the parental resistance; some p a r

ents may be m ore potent when crossed than would be expected by their

own level of resistance. In practice, heterogeneity of potence does

occur; this can be of the magnitude found in interactions. Heterogeneity

is unpredictable in the regression of main effects on parental lines,

The diallel cross does contribute information that cannot be obtained

from the parents as such (13). >

The analysis of quantitative data from a diallel cross is based

on the partitioning of second degree sta tistics such as variance and

covariance. The purpose is to estim ate certa in param eters of the pop

ulation from which the parents were derived (15).. Specific combining

ability is always associated with the preserice of nonallelic interactions,

. while generial combining ability is the resu lt of uncomplicated domin

ance (2 )/ : ,

■ /METHODS

Diploic! alfalfa seed was obtained from Dr. M. H. Schonhorst.

Associate Agronomist at the University of Arizona. This seed was p ro

duced from crosses between diploid form s of Medicago falcata L. and

Medic-ago sativa L. The parental plants came from sev era l sources-

The seed was scarified on July 13, 1961,, placed on moist filter

paper; in petri dishes, and stored in a dark desk draw er. After 24 hours

all swollen seeds were removed, treated with ceresa,n, and planted in .

sterilized 18. x 24 inch flats containing a mixture of three parts sand to

one part soil. Care was taken not to coyer seeds with m ore than a

quarter inch of soil. Shortly after emergence, seedling damping off was

noted. A light layer of ceresan was dusted on the so il surface to control

this disease:.:, ’ .V.- . ̂ ’

A female spotted alfalfa aphid was obtained from plants growing

at the University of A rizona's Campbell Avenue Farm and placed upon a

susceptible Hairy Peruvian alfalfa plant in a 12 x 24 x 18 inch cage. An

aphid colony was thus obtained from a single female. Additional Hairy ;

Peruvian plants, were placed in the cage as needed in order to maintain

a sufficient food source for the aphid colony. ' .

18

Seven 18 x 14 x 6 inch screen cages were constructed. Cages

were made from 1 x 1 inch lumber and covered with a 32 x 32 megh

plastic screen. Metal straps were fastened inside the bottom of cages

to prevent aphids from escaping through gaps between cages and flats.

Four days after unifoliolate leaves appeared, fifty spotted alfalfa

aphids were caged in each flat. The cages were to be left on the flats for

seven days until the aphids destroyed all Susceptible plants. However,

due to the extremely high humidity inside the cages, all of the seedlings

damped-off and as a resu lt the aphids were unable to survive.

A 9 x 12 foot screenhouse was built and covered with a 32 x 32

mesh plastic screen. The building was covered with a 16 mil. plastic to

hold the heat in.

Flats of diploid alfalfa plants were planted again according to the

method discussed previously. After the Seedlings em erged the flats were

moved from the greenhouse to the screenhouse. Four days after the

unifoliolate leaves appeared, fifty ■ spotted .alfalfa aphids were placed on

plants in the flat. Because of cool weather and poor distribution of aphids,

,the killing of seedlings was not uniform and this method of testing.dis

continued. All plants that survived this te s t were established in number

10 tin cans and tested individually to determ ine the level of resistance

by the antibiosis cage method described by Howe and. Smith (215).- Ten

th ird-ins ta r nymphs were caged on the leaflets of a single stem . At the

end of seven days the cage was removed and the aphids;were counted.

This te s t was repeated three tim es for a total of four te s ts per plant.

When a te s t appeared to have been influenced by: adverse conditions, it

was discontinued and the plants re tested . In addition to the plants des - ,

.cribed above, three diploid alfalfd plants resistan t to the pea aphid.• ■ . . r . , 2' ■ ' ; , ,: - , . v •: ; - s'y •' ' ' ' "

(Macrosiphum p is i, Kltb.) were included in the antibiosis test. All

three 6f these plants were found to be of the three-plus level of re s is t

ance to the spotted alfalfa aphid. Two of these plants were selected and

included with eight plants selected from the screeiihouse te s t for the

genetic study. The plants and their reaction to the spotted alfalfa aphid

are shown in Table 1. Prelim inary testing lasted from M arch 3 to June

12, 1961. : ;".V ■ :A..; - ; : . ■ ;

To obtain an estim ate of environmental effects, three susceptible

tetraploid plants were used as checks.

For the next phase of the study , the ten parent plants were

crossed in all possible combinations, including Reciprocals,and self-

pollinated. This gave a total of ten self-pollinated and ninety c ro ss-

pollinated combinations. . "

'nr ' .' ' ' .. . ' -.. M r. V. D. Roth tested these plants while with the Entomology

Research Division, ARS, USDA, and stationed at the University ofArizona Experimental Farm at Yuma.

20

Table 1; • The level of resistance of ten parent plants selected for the genetic study.

. Plant lf' . . . . Level of resistanceL/OtiLv/ HO e y 1961 te s t 1 1962 testD-56 " -+++ .++4- '

' ' D--57 . .. .+■++; ' ■■ ■ - ' - •{—{"i-D-5 ■■+ . " : 4-4-D-15. ■ ■ S 4

, D-40 : H S' D-2 : ; , ■+ .; ' ■ ' • - I ■' ' . ■ : ■

• D-21 ++ • I ': D-27 . ■S

■ D-28 s ■■■, , s .... D-16 - - a s ' ; < ; : ' : V HS '

On June 14, 1961, seed from twenty-five crosses were scarified ,

placed on m oist filte r paper in petri dishes for forty-eight hours. One. ■

hundred swollen seeds from each cross were planted in 18 x 24 inch flats

containing three parts sand to one part soil that had been autoclaved for

eight hours to kill soil organism s. The seeds were treated with ceresan

and planted at a depth of one-quarter inch .... Of'the 2,500.seeds planted,

twenty-six germinated between June 21 and 26. Later all of these seed

lings .damped-dff. . . V ‘

\ On June 26, additional seeds were placed in two petri dishes on

m oist filte r paper. One dish was placed in a desk draw er while the other

was left on top of the desk. Water was added to the dishes each day in

order to keep the seeds moist. The Seeds took in w ater, but remained

in the dishes for fourteen days without germinating. It was then decided

that some form of m ature-seed dormancy was present in this m aterial.

Lack of germination initiated a review of lite ra tu re for seed

dormancy in alfalfa. After finding no information on seed dormancy in

alfalfa, it was concluded that m ature-seed dormancy was a problem

only to some wild alfalfa types. Various tests were then conducted tp

find some method of breaking this dormancy. The resu lts are listed in

Appendix Table 1. .■ , :

As a resu lt of the information obtained from these tests , seeds

were Scarified, placed in te s t tubes of water in the refrig era to r at 57°. F.

for twenty-four hours, germinated in petri dishes for forty-eight hours,

treated with ceresan, and then planted in 18 x 24 inch flats filled with

sand and soil. In using this procedure germination of seed and percent

emergence of seedlings was greatly increased. However, damping-off

after seedling emergence was high, survival percentage was less than

10 percent. With plant survival low, and seed difficult to obtain it was

decided that some other method of planting must be tried . Two fla ts ,

one filled with perlite and the other with washed sand, were used to

grow the seedlings. A nutrient solution containing necessary plant e le

ments was applied to the plants.

The seedlings planted in perlite and watered with the nutrient ,

solution had one hundred percent survival. Plants in the sand had only

thirty-five percent survival.

v Y - ; ; ' ; / : v - : w / - : . " , :;;.22:;;

From this point on, the seeds were scarified, placed in test;

tubes of water in the refrigera to r at 57° F. for twenty-four hours, placed

on moist filte r paper in petri dishes for. forty weight hours, and then the

seedlings were planted in perlite and watered with a nutrient solution

until they became established. About a week after the unifoliolate leaves

appeared, the seedlings were transplanted into 16-ounce tin cans contain

ing sand. When the plants became established, they were transferred to

a plastic greenhouse at the Campbell Avenue Farm .

' The plants were placed bn an open bench and each was infested ;YY \ ■. ̂ A;with ten aphids (Figure 1)., Susceptible H airy Peruvian plants were,used

, as checks. Extreme variation in aphid reproduction on these check

plants indicated a position effect was present in the greenhouse. T here

fore, the information obtained in the f irs t te s t was not used in the analy-

/ s is .' Benches . were moved to minimize-this, effect. ’ A

Testing to determine levels of resistance of the plants in the 100

populations was started in M arch, 1962. Fourteen groups of plants were

tested successfully. Unfortunately, three additional groups of plants

being tested were exposed to formaldehyde and died. '

A 9.x 12 foot glass greenhouse with cooler was obtained from

M r. Frank V.- Lieberman of the Entomology Research Division, ARSy :

- ' ■ 3 ' . ' - - ' ■ ' ' : . ' ' ' - '

This greenhouse was made available through the courtesy ofDr. W. P. Bemis of the Horticulture Department, University of Arizona.

23

Figure 1. View showing plants being tested by the open bench method.

24

USDA. The house was constructed at the Campbell Avenue Farm , and

its use restric ted to spotted alfalfa aphid testing. All further tests for

determining level of resistance to the spotted alfalfa aphid were carried

on in this greenhouse. The aphids used in these tests were provided by

Mr. Lieberman.

Upon completion of the testing in August, 1962, all possible

combinations, including reciprocals and self-pollinations (one cross and

reciprocal were m issing),were analyzed to determine main effect and its

interactions of resistance.

After the antibiosis tests were completed, the plants were moved

to the campus greenhouse and treated with 5 percent Malathion, 15 p e r

cent DDT, and 40 percent sulfur insecticide dust to kill all surviving

aphids. These plants were later planted at the Campbell Avenue Farm

by Dr. M. H. Schonhorst for further genetic studies.

RESULTS

A diploid alfalfa population consisting of sixty-four plants was

screened in 1961 by using the antibiosis test, each plant was tested four

tim es. The purpose of this test was to determine the magnitude of v a r

iability for resistance to the spotted alfalfa aphid that existed among

diploid alfalfa plants. After the antibiosis te st had been completed,

plants were grouped into the various levels of resistance. The number

of plants in each class is shown in Table 2.

Table 2. The number of diploid alfalfa plants falling into each level of resistance.

T

Item T Classr ++++ * +++ T ++ ’ + ’ I ' S ' HS

No.aphids 0 1-15 16-30 31-50 51-70 71-100 100-250

No.plants 0 3 9 15 16 13 8

No plants with complete resistance (++++) to the spotted aphid were

found. Three pea aphid resis tan t plants obtained from M r. Roth were

25

26

higher in resistance to the spotted alfalfa aphid than were the other

sixty-one plants. Since at least one introduced nymph matured to

reproduce on each plant, they were classified as three-p lus. Two of

these three-plus plants and eight additional plants were selected for

the genetic study. Of the eight additional plants selected, two came

from each of the following groups: two-plus, one-plus, susceptible,

and highly susceptible. Plants used and the level of resistance of each

is listed in Table 1.

These ten plants were tested again in 1962. The second test of

the parent plants was conducted concurrently with that of their progeny.

The level of resistance in six of the parent plants varied from the 1961

test. The largest difference was found in plant D-40, which gave a

highly susceptible reaction in 1961 and one-plus in 1962. Two other

p lan ts--D -5 and D -15--also gave a higher reaction. Both D-21 and

D-27 dropped in their reaction from two-plus to susceptible and in te r

mediate, respectively; D-2 dropped from one-plus to interm ediate.

The remaining plants gave the same reaction in both te s ts . P art or all

of the difference may have resulted from testing at different seasons

of the year, at different age of p lants, and unfavorable environmental

conditions resulting from sharing a greenhouse with personnel of the

Department of Horticulture. The resu lts obtained in 1962 appeared to

fit the segregation pattern of cross-pollinated and self-pollinated

progeny better than did the 1961 resu lts.

27

The ten parent plants, representing the various levels of reaction,

were self-pollinated and crossed in all possible combinations, including

reciprocals. About one hundred seeds from each combination were

planted in anticipation of obtaining twenty-five or more seedlings.

Because of varying germination ra te s , damping-off of seedlings, and

dying of plants during testing, the population size of plants tested ranged

from six to sixty-four, with an average of 29.7. One cross-pollinated

group, D-40 x D-56, and its reciprocal were m issing completely.

After establishment, the plants were moved to greenhouses at

the University of Arizona Campbell Avenue Farm for testing. Each plant

was infested with ten th ird -insta r aphids before being placed on an open

bench. At the end of about a week all aphids present on each plant were

counted and the number recorded. Susceptible Hairy Peruvian plants

were used as checks.

Upon completion of the test for resistance, the resu lts were

converted into percent in order to have a common bases for comparing

populations containing unequal plant numbers. Results of the test are

listed in Table 3.

All self-pollinated (S^) populations segregated and gave a d is tr i

bution ranging from more resis tan t to more susceptible individuals than

their respective parents. This would indicate that all parent plants were

heterozygous for at least one locus. When the three-plus parent plants

28

Table 3. The total number of plants tested and the percent of and diploid alfalfa plants falling into each level of resistance.

tParents '

tr

T

T

++++ , t

+++

rT

t ++ t

T 1

t tr + tt t

»

t

i ,f

S

t

! HSt

' Total ’ popu- T lation ' tested

D- 2 D- 2 0 0 2 12 18 38 30 40D- 2 D- 5 6 22 11 24 17 14 6 36D- 2 D-15 3 11 14 31 20 14 7 35D- 2 D-16 3 16 19 30 16 13 3 31D- 2 D-21 10 23 30 27 10 0 0 30D- 2 D-27 3 6 12 28 9 18 24 34D- 2 D-28 4 4 18 28 28 14 4 28D- 2 D-40 17 8 8 29 11 20 7 35D- 2 D-56 20 36 16 12 4 12 0 25D- 2 D-57 6 27 6 13 10 19 19 31

D- 5 D- 2 7 13 10 27 3 27 13 30D- 5 D- 5 19 22 14 31 14 0 0 36D- 5 D-15 0 20 24 16 8 12 20 25D- 5 D-16 0 4 4 31 22 17 22 23D- 5 D-21 3 3 10 32 14 24 14 29D- 5 D-27 3 21 18 25 6 12 15 34D- 5 D-28 7 26 14 29 5 14 5 44D- 5 D-40 4 18 21 14 11 14 18 28D- 5 D-56 11 16 25 16 16 11 5 19D- 5 D-57 50 25 0 6 13 0 6 17

D-15 D- 2 0 9 18 18 9 37 9 11D-15 D- 5 4 0 11 26 19 33 7 27D-15 D-15 0 40 0 40 0 20 0 10D-15 D-16 0 0 0 22 0 67 11 9D-15 D-21 3 3 10 32 23 10 19 31D-15 D-27 3 3 6 44 16 19 9 32D-15 D-28 4 7 19 19 11 25 15 54D-15 D-40 6 32 28 17 11 6 0 19D-15 D-56 24 28 20 20 8 0 0 25D-15 D-57 20 40 4 12 16 8 0 25

D-16 D- 2 2 13 7 28 22 17 11 46D-16 D- 5 4 10 10 23 15 21 17 48D-16 D-15 5 10 12 15 28 18 12 40

29

Table 3. (Continued).

Parents T T

, ++++ f +++ + + t HS

T Total T popu- r lation T tested

D-16 D-16 0 0 6 21 24 18 31 34D-16 D-21 2 20 20 20 14 10 14 41D-16 D-27 9 5 9 9 9 36 23 23D-16 D-28 3 3 10 36 3 20 25 29D-16 D-40 0 17 28 24 7 14 10 29D-16 D-56 23 7 7 20 20 20 3 30D-16 D-57 28 34 12 12 5 7 2 41



D-28 D- 2 0 11 4 11 21 25 28 28D-28 D- 5 0 8 8 16 16 20 32 25D-28 D-15 0 11 8 13 26 18 24 38D-28 D-16 3 11 8 20 20 30 8 64D-28 D-21 7 9 17 27 14 17 9 43D-28 D-27 5 19 10 28 14 24 0 21D-28 D-28 0 20 25 25 15 5 10 20D-28 D-40 6 6 0 22 27 22 17 18

30

Table 3. (Continued).

Parentsr t tt r rr + + + + , + + + ,

t T T

+ + HST Total r popu- T lation 1 tested

D-28 D-56 58 23 5 0 5 0 9 22D-28 D-57 8 36 4 16 0 16 20 25

D-40 D- 2 7 29 17 25 7 11 4 28D-40 D- 5 20 20 16 24 16 4 0 25D-40 D-15 9 28 9 24 9 15 6 33D-40 D-16 12 30 8 8 27 15 0 26D-40 D-21 20 16 9 21 4 16 4 25D-40 D-27 0 4 4 20 20 40 12 25D-40 D-28 0 3 0 23 20 31 23 26D-40 D-40 2 16 14 34 16 10 8 37D-40 D-56 No progeny available for testingD-40 D-57 4 14 14 43 11 7 4 27

D-56 D- 2 29 14 8 20 14 8 7 35D-56 D- 5 60 24 12 4 0 0 0 25D-56 D-15 56 10 14 17 0 3 0 29D-56 D-16 29 19 14 19 19 0 0 27D-56 D-21 25 4 8 34 12 17 0 24D-56 D-27 54 20 10 10 3 3 0 30D-56 D-28 26 8 8 12 12 22 12 24D-56 D-40 No progeny available for testingD-56 D-56 43 8 8 33 4 4 0 24D-56 D-57 70 19 11 0 0 0 0 27


31

were in tercrossed , the segregation of their progeny differed from that

of either parent’s self-pollinated progeny. This would mean that these

plants were not alike at all loci. When any of the parent plants were

in tercrossed, the range of reaction of the resulting progeny exceeded

that of either parent. This indicated that transgressive segregates might

be present.

When parent plants giving the highest reaction (+++) for re s is t

ance were used as one parent, the percent of the progeny found in the

three-plus and four-plus levels of resistance was high. When those that

were more susceptible (S and HS) were used as one parent, the reaction

of the progeny found in the susceptible classes also was high.

The average level of resistance of each parent and the percentage

of progeny in each class is found in Table 4. From this table it was

apparent that there are differences in the reciprocals--D -5 is an example--

but these differences may have been due to sm all populations or environ

mental differences arising between te sts .

32

Table 4. The average level of resistance of each parent and the percent of progeny in each class when using the plant as either the male or female parent.

’ Level ' c la ssParent ’ of ' v la ssfT

R esis t- ' ance '

i++++ ,

i+++ ,

T+ + ,

t+ !

tI ,

ts , HS

D-56 * +++ 44 14 11 16 7 6 231 20 15 16 8 8 2

D-57 +++ 28 27 14 13 9 7 2

<? 24 26 10 16 9 9 6

D-5 $ ++ 9 18 40 25 10 13 1117 16 11 20 14 14 8

D-15 + 7 14 13 24 14 19 9

11 14 12 18 14 18 13

D-40 + 7 18 11 26 15 16 7

(? 7 19 16 24 15 12 7

D-21 % I 8 13 15 21 13 17 13

& 11 12 15 23 11 16 9

D-2 ? I 7 15 11 24 15 17 119 12 9 23 13 23 11

D-27 $ s 7 12 13 19 13 23 13

10 11 11 23 11 19 15

D-28 ? s 7 14 9 18 17 20 15

6 15 16 24 11 16 12

D-16 ? HS 8 14 12 21 15 16 14

cP 6 13 12 20 18 18 13

DISCUSSION

At the time this study was initiated, no diploid plants were avail

able with known reaction to the spotted alfalfa aphid. Therefore, the

f irs t objective was to screen a diploid alfalfa population to determine

the amount of variation present. Four individually caged (antibiosis)

tests were conducted on each plant in order to determine more precisely

the levels of resistance. Some differences were found in aphid numbers

between tests repeated on the same plant. Because of the large amount

of work involved in rearing, applying, and counting of aphids and other

problems related to greenhouse culture of diploid alfalfa plants, the

initial screening process lasted approximately four months. It was felt

that tem perature differences between early and late te s ts may have been

partially responsible for variation in the number of aphids which

developed.

Because of large quantities of plant m aterial in the second phase

of this study, the tests were conducted by putting ten th ird -in sta r aphids

on each plant before placing on an open bench. It was realized that when

using spaced tin cans on an open bench that aphids may jump or fly from

plant to plant. Although there were some alate females present, very

little movement was observed.33

34

Since the tests were conducted on an open bench, plant popula

tions were sm all, and cool night tem peratures could not be controlled

it was not possible to assign the definite number of genes that gave

resistance to the spotted alfalfa aphid.

With unequal number of plants in each population, it was neces

sary to convert the data to percent in order to have a common basis for

comparison. All self-pollinated populations segregated to give progeny

that ranged from more resis tan t to more susceptible individuals than the

parents. Highly resistan t (++++) progeny had no aphids present at the

end of the test, while the most susceptible progeny had up to 290 at the

end of the test period. This information indicated that none of the ten

parent plants was homozygous at all loci. When homozygous plants are

self-pollinated, their progeny will have a level of resistance sim ilar to

that of the parents.

When the three-plus plants were in tercrossed, the reaction for

resistance of some of the F^’s was increased over that of either parent.

Segregation of cross-pollinated (F^) populations obtained from crosses

between two one-plus, two interm ediate, or two susceptible plants was

found to vary from that of the self-pollinated progeny of the parent

plants. This lead to the conclusion that although the parents were sim i

lar phenotypic ally, they differed genotypically.

35

Progeny of all plants being cross-pollinated with three-plus

plants were checked. The progeny increased in resistance over the

more susceptible parent. This would indicate that there was dominance

or partia l dominance in one or more factors conditioning plant resistance

to this insect. The amount of increase in resistance depended upon the

level of resistance of the geneotype of the two parent plants involved.

The variation in the amount of increase between different crosses

further substantiated the hypothesis that the inheritance of resistance

was controlled by several ra ther than a single gene.

Distribution of the progeny of different crosses was studied and

the conclusion drawn that three or more loci were involved in resistance.

Three le tte rs (B, C, D) representing genes then were assigned to plants

D-56 and D-57. Because of the increase in plant resistance obtained

when both D-56 and D-57 were crossed with the other plants, it was

believed that they had one gene in common which was homozygous

dominant for resistance. This gene was designated as B. These plants

were not heterozygous at the same loci; therefore, the second and third

le tte rs were varied when assigned.

Distribution of the progeny from D-2 x D-56 and D-2 x D-57

crosses were examined and le tters assigned to represen t loci that might

be found in D-2. This procedure was repeated for other parent plants

until all had been assigned le tters to represen t genes; these are shown

36

in Table 5. Distributions of all cross-pollinated populations were

checked to determine the closeness of fit after assigning the le tters to

the parent plants. All those not fitting expected ratios were re-evaluated.

It was found that, in general, those which did not fit expectations were

those that had been assigned one or two dominate D genes. Next, date of

testing was checked with the available weather information. It was found

that most of the populations not fitting expected ratios were tested during

cool weather. Although no heating system was provided, maximum day

tem peratures were within, or above, the optimum range for best aphid

reproduction; therefore, it appears that low night tem peratures influenced

the reaction of locus D. This tem perature influence helped explain some

of the variation observed in the test. Also, it appeared that locus (D)

was influenced by tem perature when dominate. When allowances were

made for the variation in night tem peratures, the resu lts closely fit

expected ratios.

In an effort to determine if tem perature did influence resistance,

the parent plants were checked again in August of 1962. The resu lts

were not conclusive, as the power supply to the greenhouse cooler was

disrupted during this te st and the resulting high day-tim e tem peratures

damaged some of the plants and aphids. Because of damage to the

parent plants the tests were not repeated. P art of the plants undamaged,

but containing assigned D locus, changed in their reactions to the

spotted alfalfa aphid.

37

Table 5. Possible loci contained by the various parent plants.

Parent», Loci

D-2 bb Cc DdD-5 bb Cc DDD-15 bb Cc ddD-16 bb cc DdD-21 bb Cc DdD-27 bb Cc DdD-28 bb Cc ddD-40 bb Cc ddD-56 BB CC DdD-57 BB Cc DD

Dominate B locus indicated that it added more resistance than

did C or D. Locus C may have added more resistance than D, since C

was affected less than D by cool night tem peratures.

A number of conditions can influence resu lts obtained from tests

of insect resistance. Season of testing may prove im portant in d e te r

mining the level of resistance in plants. This factor may account for

the differences reported in aphid resistance due to tem perature changes.

Acidity of plant tissue was reported (11) associated with diurnal and

seasonal fluctuations in light intensity and tem perature. The pH of

plant tissue also may effect the level of aphid resistance.

38

Soil fertility and available plant nutrients have helped to d e te r

mine insect resistance in some plants. Available nitrogen and the

nitrogen-sugar ratio were reported to influence the ra te of pea aphid

reproduction in peas (34, 35). Cody (5) reported that plant colors affect

aphid preferences. It is felt, by this author, that the amount of phos

phorus and the nitrogen-phosphorus ratio should be examined; it may

help provide answers involving spotted alfalfa aphid resistance.

Changes in resistance of six of the ten parent plants between

1961 and 1962 may have been influenced by the soil fertility or other

environmental factors. Small amounts of a 16 percent nitrogen and 20

percent phosphorus fe rtilizer were added to the pots at approximately

two-month intervals, but it was difficult to keep soil fertility at a uni

form level.

It is felt by the author that all plants used in this study should

be subjected to antibiosis tests using the individual cage method. The

te st should be repeated a minimum of two tim es on each plant.

SUMMARY

An experiment to determine the genetics of resistance to the

spotted alfalfa aphid was conducted between July, 1960, and August,

1962. A diploid population of alfalfa plants was used because previous

information showed that aphid survival and reproduction could be

influenced by certain environmental factors. Thus, sm aller diploid

populations would be required and diploid segregation ratios would be

easier to in terpret than ratios obtained from tetraploid plants.

Seeds were obtained from hybridization between plants of

Medicago sativa and Medicago falcata. The plants obtained from

these crosses were started in the greenhouse. After seedling emergence

the flats were moved to a screenhouse. Sixty-one plants became estab

lished and were transferred into number 10 tin cans and tested four

tim es by the antibiosis method to determine their level of resistance to

the spotted alfalfa aphid.

In addition to these sixty-one plants, three pea aphid resistan t

diploid alfalfa plants, obtained from Mr. V. D. Roth, were tested four

tim es for reaction to the spotted alfalfa aphid. Two of the plants

obtained from Mr. Roth and eight from the original diploid population

39

40

were selected as representatives of the various levels of resistance for

a genetic study. These ten plants were self-pollinated and cro ss-po l

linated in all possible combinations.

The seeds obtained from these crosses and selfs were scarified

and planted. About a week after the unifoliolate leaves appeared, the

seedlings were transplanted into cans filled with sand. Later, the plants

were transferred to the University of Arizona’s Campbell Avenue Farm

for testing.

In testing the and plants, ten th ird -insta r aphids were put

on the young plants before placing them on an open bench. At the end of

about a week the number of aphids on each plant was counted.

Because of the method of testing, size of the population, and the

inability of controlling night tem peratures it was difficult to analyze the

data. The number of plants found in each class was converted to percent

to have a common bases for comparison. Host plant reactions to the

spotted alfalfa aphid indicated that the plants were heterozygous for

factors for resistance; also, resistance in these plants was conditioned

by three or more genes, and an additive effect was present. It was also

evident that tem perature influenced the reaction of alfalfa plants to the

spotted alfalfa aphid.

LITERATURE CITED

1. Anonymous. United States Department of Agriculture. The spottedalfalfa aphid. U. S. Dept. Agric., Agric. Serv., Leaflet 422.1957.

2. Barnes, O. L. Establishment of imported parasites of the spottedalfalfa aphid in Arizona. Jour. Econ. Ent. 53:1094-1096.1960.

3. Cody, C. E. Color preferences of the pea aphid in W estern Oregon.Jour. Econ. Ent. 34:584. 1941.

4. Dahms, R. C. and Painter, R. H. Rate of reproduction of pea aphidon different alfalfa plants. Jour. Econ. Ent. 33:482-485. 1940.

5. Deal, Andrew S., Dickson, R. C., and Reynolds, H. T. Yellowclover aphid in state. Calif. Agri. 8(9):5. 1954.

6. Dickinson, A. G. and Jinks, I. J . A generalized analysis of diallelcro sses. Genetics 41:65-78. 1956.

7. Dickson, R. C., Laird, Edward F., J r . , and Pesho, George R. Thespotted alfalfa aphid (yellow clover aphid) on alfalfa. Hilgardia 24:93-118. 1955.

8. _____________ and Reynolds, H. T. The yellow clover aphid onalfalfa. Calif. Agri. 9(7):4, 15. 1955.

9. Diehl, S. G. and Chatters, R. M. Studies on the mechanics of feeding of the spotted alfalfa aphid on alfalfa. Jour. Econ. Ent. 49:589-591. 1956.

10. Dobson, R. C. and Watts, J . G. Spotted alfalfa aphid occurrence on seedling alfalfa as influenced by systemic insecticides and varieties. Jour. Econ. Ent. 50:132-135. 1957.

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11. Emery, W. T. Temporary immunity in alfalfa ordinarily susceptible to attack by the pea aphid. Jour. Agr. Res. 73:33-43. 1946.

12. Finney, Glenn L., Puttier, Benjamin, and Dawson, Louis. Rearingof three spotted alfalfa aphid Hymenopterous parasites for m ass release . Jour. Econ. Ent. 53:655-659. 1960.

13. Gilbert, N. E. G. Diallel cross in plant breeding. Heredity 12:477-492. 1958.

14. Goodarzy, Karim , and Davis, Donald W. Natural enemies of thespotted alfalfa aphid in Utah. Jour. Econ. Ent. 51:612-616.1958.

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16. Hagen, Kenneth S., Holloway, Jam es K., Skinner, F. E., andFinney, G. L. Aphid parasites established. Calif. Agric. 12(2): 3, 15. 1958.

17. Hall, Irvan M and Dunn, Paul H. A rtificial dissemination ofentomophthorous fungi pathogenic to the spotted alfalfa aphid in California. Jour. Econ. Ent. 51:341-344. 1958.

18. _____________, ______________ . Entomophthorous fungi parasiticon the spotted alfalfa aphid. Hilgardia 27(4): 159-181. 1957.

19. _____________, ______________ . Fungi on spotted alfalfa aphid.Calif. Agric. 11(2): 5, 14. 1957.

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21. Harvey, T. L. and Hackerott, H. L. Spotted alfalfa aphid reactionand injury to resis tan t and susceptible alfalfa clones rec ip rocally grafted. Jour. Econ. Ent. 51:760-762. 1958.

22. Hay man, B. I. The theory and analysis of diallel c rosses. Genetics39:789-809. 1954.

23. Howe, W. L. and Pesho, G. R. Influence of plant age on the s u r vival of alfalfa varieties differing in resistance to the spotted alfalfa aphid. Jour. Econ. Ent. 53:142-144. 1960.

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24. ____________ , _____________ , and Scrivener, J. W. The use ofvaried testing procedures for selection of alfalfa resistan t to the spotted alfalfa aphid. Special Report X-34 (unpublished), Cereal and Forage Insects Section, ENT, ARS, USDA. 1956.

25. ___________ , and Smith, O. F. Resistance to the spotted alfalfaaphid in Lahontan alfalfa. Jour. Econ. Ent. 50:320-324.1957.

26. Jinks, J . L. A survey of genetical basis of heterosis in a varietyof diallel crosses. Heredity 9:223-238. 1955.

27. __________. The analysis of continuous variation in a diallel crossof Nicotina rustica varie ties . Genetics 39:767-788. 1954.

28. __________. The Fp and backer os s generations from a set of diallelcrosses. Heredity 10:1-30. 1956.

29. Knowlton, George F. Alfalfa aphids. Utah Agr. Expt. St a. Leaflet57. 1959.

30. M altais, J. B. The nitrogen content of different varieties of peasas a factor affecting infestation by Macrosiphum pisi (Kltb.) (Hemoptera: Aphididae) A prelim inary report. Canad. Ent. 83:29-32. 1951.

31. ____________ and Auclair, J. L. Factors in resistance of peas tothe pea aphid, Acyrthosiphon pisum (Harr.) (Homoptera: Aphididae). I. The sugar-nitrogen ratio. Canad. Ent. 84:

32. Mangletz, G. R., Bergman, P. W., Howe, W. L., and Calkins, C.O. Overwintering in the egg stage by the spotted alfalfa aphid in Nebraska. Jour. Econ. Ent. 55:292-294. 1962.

33. M arble, V. L., Meldeen, John C., M urray, Hazel C., and Zscheile,F. P. Studies of free amino acids in the spotted alfalfa aphid, its honey dew, and several alfalfa selections, in relation to aphid resistance. Agron. Jour. 51:740-743. 1959.

34. Maxwell, Fowden G. and Painter, Reginald H. Factors affectingrate of honeydew deposition by Therioaphis maculata (Buckton) and Toxoptera graminum (Rond). Jour. Econ. Ent. 52:368-373. 1959.

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35. McMurtry, J. A. and Stanford, E. H. Observations of feeding habitsof the spotted alfalfa aphid on resistance and susceptible alfalfa plants. Jour. Econ. Ent. 53:714-717. 1960.

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37. Nickel, John L. and Sylvester, Edward S. Influence of feeding tim e,stylet penetration and developmental instar on toxic effect of the spotted alfalfa aphid. Jour. Econ. Ent. 52:249-254.1959.

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40. Ortman, Eldon E., Sorensen, E. L., Pain ter, Reginald H., Harvey,T. L., and Hackerott, H. L. Selection and evaluation of peaaph id-resistan t alfalfa plants. Jour. Econ. Ent. 53:881-887.1960.

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46. P e te rs , Don C. and Painter, Reginald H. Studies on the biologiesof three legume aphids in relationship to their host plants. Kansas Agric. Expt. Sta. Tech. Bui. 93. 1958.

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48. Reynolds, H. T., and Anderson, L. D. Control of the spotted alfalfaaphid on alfalfa in Southern California. Jour. Econ. Ent. 48: 671-675. 1955.

49. Roth, Vincent D. Alfalfa seed treatm ents for spotted alfalfa aphidcontrol in Southern Arizona. Jour. Econ. Ent. 52:654-658.1959.

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51. Schlinger, Evert I. and Hall, Jack C. A synopsis of the biologiesof three imported parasites of the spotted alfalfa aphid.Jour. Econ. Ent. 52:154-157. 1959.

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58 . and Butler, G. D. J r . The yellow clover aphid--A new alfalfa pest in the southwest. Jour. Econ. Ent. 47: 1157. 1954.

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60. ____________________ . The spotted alfalfa aphid and its parasitesin the M editerranean region, Middle East and East Africa. Jour. Econ. Ent. 50: 352-356. 1957.

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APPENDIX

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Appendix Table 1. Treatm ents used for determining a method for breaking m ature-seed dormancy in diploid alfalfa.

t T Number 1 NumberTreatm ent ’ Time 1 of ’ of seeds

T exposed T seeds ' that ger-T ' exposed ' minate*

Boiling water 5 min. 10 0Boiling water 10 min. 10 076° F. water 18 h rs. 10 376° F. water 40 h rs. 10 157° F. water 18 h rs. 10 657° F. water 40 h rs. 10 5Check 10 1Red light 30 min. 10 0Red light 60 min. 10 0No seed coat 5 0Sulfuric acid V 5 min. 10 2Sulfuric acid V 10 min. 10 2Sulfuric acid V 20 min. 10 0Sulfuric acid V 30 min. 10 3Sulfuric acid 2/ 5 min. 10 0Sulfuric acid 2/ 10 min. 10 0Sulfuric acid 2/ 20 min. 10 0Sulfuric acid 2/ 30 min. 10 1Heat (200° F.) 3/ 5 min. 10 0Heat (200° F.) 3/ 10 min. 10 0Coldness (0° F.) 5 min. 10 0Coldness (0° F.) 10 min. 10 0

After treatm ent the seeds were placed on moist filte r paper in petri dishes for seven days.

1 The seeds were soaked in acid, then placed in hypochlorite for five minutes and then washed in distilled water.

The seeds were f irs t scarified, soaked in acid, then placed in hypochlorite for five minutes and washed in distilled water.

oThe seeds were placed on a m etal pie plate over a hot plate and the pie plate was kept in constant motion to evenly expose the seeds to the heat.

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