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The genetics of resistance to the spotted alfalfa aphid
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Authors Powell, William Houston, 1926-
Publisher The University of Arizona.
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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
t a b l e o f c o n t e n t s :
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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-21 D- 2 0 7 7 30 13 23 20 30D-21 D- 5 6 6 9 25 19 29 6 32D-21 D-15 3 5 11 24 11 21 25 38D-21 D-16 0 7 7 7 17 10 52 30D-21 D-21 12 19 17 12 12 21 7 42D-21 D-27 4 17 21 26 7 21 4 28D-21 D-28 0 22 30 24 8 8 8 37D-21 D-40 0 12 15 34 24 15 0 33D-21 D-56 42 17 14 21 3 3 0 29D-21 D-57 13 16 18 21 16 13 3 32
D-27 D- 2 0 0 19 31 13 31 6 32D-27 D- 5 9 17 12 17 17 14 14 35D-27 D-15 4 0 10 17 14 39 16 50D-27 D-16 0 9 21 30 17 21 2 43D-27 D-21 0 6 6 15 15 40 18 34D-27 D-27 0 0 14 14 17 14 41 35D-27 D-28 10 26 16 23 6 6 13 31D-27 D-40 17 45 14 7 14 3 0 29D-27 D-56 34 26 7 7 7 15 4 27D-27 D-57 17 0 17 25 8 25 8 12
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
D-57 D- 2 6 31 9 21 6 18 9 33D-57 D- 5 54 30 10 3 3 0 0 30D-57 D-15 35 43 10 6 3 3 0 31D-57 D-16 21 21 21 11 11 15 0 28D-57 D-21 44 14 11 17 14 0 0 28D-57 D-27 25 17 0 17 17 17 7 12D-57 D-28 17 66 0 16 0 0 0 6D-57 D-40 19 14 19 24 14 5 5 21D-57 D-56 16 21 47 11 0 5 0 19D-57 D-57 32 35 9 12 9 3 0 34
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.
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5. Deal, Andrew S., Dickson, R. C., and Reynolds, H. T. Yellowclover aphid in state. Calif. Agri. 8(9):5. 1954.
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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.
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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.
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19. _____________, ______________ . Fungi on spotted alfalfa aphid.Calif. Agric. 11(2): 5, 14. 1957.
20. Harpaz, I. Bionomics of Therioaphis maculata (Buckton) in Israel.Jour. Econ. Ent. 48:668-671. 1955.
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.
36. Moore, J . B. Reactions of aphids to colored insecticides. Jour.Econ. Ent. 30:305-309. 1937.
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.
38. Nielson, M. W. and Barnes, O. L. Life history and abundance ofthe spotted alfalfa aphid in Arizona. Jour. Econ. Ent. 50: 805-807. 1957.
39. _____________ and C urrie, W. E. Biology of the convergent ladybeetle when fed a spotted alfalfa aphid diet. Jour. Econ. Ent.53:257-259. 1960.
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.
41. Padilla, A. and Young, W. R. El pulgon manchado de la alfalfa enMexico. Secretaria de A gricultura y Ganaderia, Oficina de estudios especiales, Mexico. Folleto Tecnico. 25:1-32.1958.
42. Painter, Reginald H. Insect resistance in crop plants. The Macmillan Company, New York, 520 pp. 1951.
43. Paschke, John D. and Sylvester, Edward S. Laboratory studies onthe toxic effects of Therioaphis maculata (Buckton). Jour. Econ. Ent. 50:742-748. 1957.
44. Pesho, G. R. and Lieberman, F. V. A biotype of the spotted alfalfaaphid on alfalfa. Jour. Econ. Ent. 53:146-150. 1960.
45. ___________ , , and Lehman, W. F. A biotype of thespotted alfalfa aphid, Therioaphis maculata (Buckton) on alfalfa. Special Report X -77 (Unpublished), Cereal and Forage Insects Section, ENT, ARS, USDA. 1959.
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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.
47. Peterson, George D., J r . and Deal, Andrew S. The spotted alfalfaaphid and its control in Im perial County. Univ. of Calif. Agric. Ext. Serv. May, 1960.
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.
50. Schaefer, C. W. Physiological conditions which produce wingdevelopment in the pea aphid. Jour. Agr. Res. 57:825-841. 1938.
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.
52. Simpson, Robert G. and Burkhardt, C. C. Biology and evaluationof certain predators of Therioaphis maculata (Buckton).Jour. Econ. Ent. 53:89-94. 1960.
53. Smith, Ray E. The spread of the spotted alfalfa aphid, Therioaphis maculata (Buckton), in California. Hilgardia 28(21: 647-685. 1959.
54. _____________and Hagen, Kenneth S. Enemies of spotted alfalfaaphid. Calif. Agric. 10(4):8-10. 1956.
55. Snelling, R. O. Resistance of plants to insect attack. Bot. Rev.7:543-586. 1941.
56. Tuttle, Donald M. The spotted alfalfa aphid. A riz. Agric. Ext.Rep. 131. 1956.
57. ______ , Barnes, O. L., Nielson, M. W., Roth, V. D., andSchonhorst, M. H. The spotted alfalfa aphid in Arizona. Ariz. Agric. Expt. Sta. Bui. 294. 1958.
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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.
59. van den Bosch, Robert. P arasites of spotted alfalfa aphid. Calif.Agric. 10(10): 6, 7, 15. 1956.
60. ____________________ . The spotted alfalfa aphid and its parasitesin the M editerranean region, Middle East and East Africa. Jour. Econ. Ent. 50: 352-356. 1957.
61. _____________________, Schlinger, E. I., and Dietrick, E. J.Imported parasites established. Calif. Agric. 11(7): 11,12. 1957.
62 . ___________________ , ,________________ _, Hagen,K. S., and Holloway, J. K. The colonization and establishment of imported parasites of the spotted alfalfa aphid in California. Jour. Econ. Ent. 52:136-141. 1959.
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.