Proof of pathogenicity of Fusarium slani (Mart.) Appel & Wr. to sainfoin (Onobrychis viciifolia Scop.)
and development of procedures to select for resistance
by Dick Lindsey Auld
A thesis submitted in partial fulfillment of the requirements for the degree of DOCTOR OF
PHILOSOPHY in Crop and Soil Science
Montana State University
© Copyright by Dick Lindsey Auld (1976)
Abstract:
Root and crown rot limits the production of sainfoin. This disease reduces longevity and forage yield.
The objectives of this study were to: 1) determine the causal organism(s) of root and crown rot; 2)
further characterize the host-parasite relationships; and 3) develop and initiate screening procedures to
detect potential sources of disease resistance.
The results of this study indicate that the root and crown rot of sainfoin usually observed in Montana is
caused by Fusarium solani. This pathogen is not seed transmitted but probably occurs in most
agricultural soils as a pathogen of other legumes.
Sainfoin can be successfully screened for disease resistance by soaking injured roots in concentrated
suspensions of microconidia of F. solani. Initial screening to locate potential sources of disease
resistance has shown differential disease resistance in both the world collection of Onobrychis and
current breeding material. Selection within these populations should increase disease resistance. PROOF OF PATHOGENICITY OF Fusarium solani (Mart.)' APPEL & WR-.. TO
SAINFOIN (Onobrychis viciifolia Scop.) AND DEVELOPMENT. OF
PROCEDURES TO SELECT FOR RESISTANCE
by
DlCK LINDSEY- AULD
A thesis submitted in partial fulfillment .
of the requirements for the degree
of
DOCTOR OF PHILOSOPHY
in
Crop and Soil Science .
Approved.:
Gha^rm
&
'j-j?^
Head, Major Department
Graduate -Dean
MONTANA STATE UNIVERSITY
Bozeman, Montana
February, 1.976
iii
ACKNOWLEDGMENTS
I wish to express, my sincere appreciation to the following:
Dr. R. L . Ditterline for his professional guidance and friend­
ship while serving as my major professor during the. course.of this
study.
Dr. D, E . Mathre for his patience arid, personal support in the
pathological investigations..
Dr. C. S . Cooper for. his professionalism and contributions to
this study.
■ Professor R. F. Eslick, Dr. L. E. Wiesner,.and H. E. Holen for
serving on my graduate committee.
My wife. Sherry, for. her sacrifice.and encouragement through
iiiy graduate work.
And to my two daughters. Shannon and Shelly>.. for
their love and trust.
The Montana Agricultural. Experiment Station and the Plant and
Soil Science Department for providing me. with an assistan.tship.
With­
out this assistance, attending graduate school, would have been impos­
sible.
iv
TABLE OF CONTENTS
Page
VITA
. . . . . . . . . .
ACKNOWLEDGMENTS
.........
. . . . . .
. . . . . . ............
Ta b l e o f c o n t e n t s
. . .. . . .
LIST OF TABLES
. . . . ................
.
. .
LIST OF FIGURES
.. ■
ABSTRACT
. . . .. . . . . . . .
. . .
INTRODUCTION
;. .
LITERATURE' REVIEW
...
. . . . '. . .
.-. .
. . . ..
.. .
.
..
..
• .
. . . .
.
> . . .
• . iv
.
■ - Vi
.:■
..
.
iii
. : .Vix■
. . . .
. :. . . .
.. . . ...
ii
>
. ' ; ■■ i.
... . .
.. ■
2.
Description of Onobrychis yiciifolia Scop.
. . ... . ... ..
2
History and Distributions of Onobrychis
viciifolja S c o p .
■. . . .
. ..■.
. .. . . ... . . . . ..
2
Agronomic Characteristics of Onobrychis
vxcrxfolia Scop.
. ..... .
. ..... . .
. . .- . . . :.
3
Diseases o f Onobrychis viciifolia S c o p ; . . . . . . . .
. '., .: '5
Folxar dxseases . . ■ . . . . -* . .. . ... • '. ... .■.■. . .
.5
Soil-borne diseases . . .
. .. „
- - ..... ^ . .:
6
Seed and seedling diseases
. . .. . . .
.. . . . .
... . . .
7
Development of Root and Crown Rot in Onobrychis .
viciifolia S c o p .
.. ...
. .. . . .. . . . . . . . . ..
. 8
.. Occurrence of the disease
. .. . . . . .
...■, . . .. .
8
The suspected pathogen . . . .
....
. . . . . .... .9
Crown Rot in Other Forage Legumes .. .. . .
. . . . ... .. . .
11
Development o f .Selection Procedures . . . . .
. . . . . . ..,
12
Chapter I . . . . '. .
.. . .
. ... .
. .. . . .
. .. •
MATERIALS AND METHODS . . .
. .. . . ... . . ■. . .
General . . . . . .
. .
....
Experiment I
. . . . ... ... .. . . . .. . . . .
Experiment II . . ...... ..... .. . . . . . . .
.
■ Experiment III
. . . . . . . . . . . .
. .. . . ..
RESULTS AND DISCUSSION
. .
. . . .. .. . .
. . .
Experiment I
. . . •. .
. . ... . . . - . . . .
- • .. .
14
. .., . .'. . 14
.... .
14
... .
14
. . .
16
. . .. .; .
17
. . .. -,
. I9
. . .
19
■ Page
Experiment I I ............. .. . .
Experiment I I I ......... ...................
CONCLUSIONS . . .
. . .... .
. .. . .
Chapter LI
.
.. .
..
. .. . . . .
. V
. . . .
.....
.
. i. . . .. . . .
.. .
. 24
26
. :■' 28
..
29
29
30
31
31
31
33
35
MATERIALS AND METHODS
General . . . . .
Experiment I
. .
Experiment II . .
RESULTS AND DISCUSSION
Experiment •
.I
.' .
Experiment II . .
CONCLUSIONS . . . . '.
Chapter III . .............
.. . . . .
. : . . .. . . .
. ;* .. .
LITERATURE CITED
APPENDIX
36
36
36
36
.40 .
40
41
42
44
44
48
52
54
57
61,
MATERIALS AND METHODS
General . . . .
Experiment I
. .
■Experiment Il ... .
. Experiment III
Experiment IV . .
Experiment V .. .
RESULTS AND DISCUSSION
Experiment I - . ,.
Experiment II .
Experiment III
Experiment IV . ^
Experiment. V
; .
conclusions . . .
SUMMARY . . . . .
29
. . ...
, . •
. . . . . . . . . . . . . . . . . . . . . . .
62
64
72
vi
LIST OF TABLES
Table
'
1— 1.
1-2.
1-3.
1-4.
1-5.
1-6.
The visual rating system used to assign nodulation
scores to sainfoin seedlings
. . . .
. . . .
17
The date of sampling and the indices measured bn
the soil fumigation and seed treatment experiment.
at the Field Research Laboratory, Bozeman,. Montana
20.
. .
The effect of pod removal and surface-sterilization .
of seed On the percentage of. seed contaminated.
with either Alternaria spp. or F. oxysporunv from
eight commercial seed, lots Of sainfoin , . . . . . ,. .
21
Sainfoin seedling emergence as affected by seedborne Alternaria spp. . . . . . . . . . . . . . .
,
23
. . .
The effect of pod removal and seed surfacesterilization on the percentage of seedlings .
infected with Alternaria spp. and percentage of
seedling emergence
. . . . . . . ............... . . . .
The effect of inoculating sainfoin seedlings with '
either microconidia or macroconidia of F. solani
' .on the mean disease severity score.and- the
percentage of plants infected . . . . . ..' . . . . . . .
1-7.
1- 8.
,Page
The e f f e c t .of inoculating sainfoin seedlings, with
four isolates, of F y solani on the. mean disease
severity score, and the percentage of plants'
infected.
Also shown is the percentage of micro­
conidia produced.in Snyder and Nash's spotulatioh
medium (56) ....................... .................. ..
The effect of four fungicide treatments oh sainfoin
plants grown at the Field Research Laboratory,
Bozeman, Montana
. .. . . . . . . . . . . . . . .
23
■ 24
25
. :
27
2- 1. . The pathogenicity of IT.
oxyspprum
and
F. solani on
sainfoin seedlings as measured by the mean disease
.
severity score and.the percentage of plants
infected
. . . . . .. . . . . . ..
.. ... ..
. ... .
32
vii
Table
2-
3-
3— 2.
3-3.
3-4.
3-5.
Page
2 * The pathogenicity of three isolates of F\ solani on
peas, beans, alfalfa, and sainfoin as measured by
the mean disease severity score and the percentage
...........
. .
of p l a n t s ■infected
1.
34
The effect of four inoculation techniques oh the
disease severity score, the percentage of plants
infected.with F\ solani, and mortality of sainfoin
seedlings inoculated in the greenhouse
. . . .. . . . .
45
The effect of fouf inoculation techniques on the
disease severity score, the percentage of plants
'infected with F. sol a ni, and mortality of sainfoin
seedlings inoculated 83 days after emergence at
the Field Research Laboratory, Bozeman, Montana . . . . ■
51
The response of the vegetative cuttings of 10
sainfoin clones inoculated with F. solani using
the root-cut-soak technique . . . . . . ... ...........
53.
The response of 11 species of Onobfychis to
inoculation with F. solani as measured by the
mean disease severity score ............
58
1
The response of three cultivars of sainfoin to
inoculation with F. solani in two trials as
measured by the mean, variance, and range of.
the disease severity scores .. . . . . . . . . . . . .. .
60
Appendix
1.
2.
The composition of the media used for fungal isolations
and increase, and the composition of nutrient
solutions used in this study
. . * ............. ..
.
73
The response of the progeny of 39 entries of sainfoin
to inoculation wifh £. solani as measured by the
percentage of plants infected; and the m e a n ,
variance, and range of the disease severity score . . .
77
Viii .
'
Table
3.
4.
Page
The response of 181 accession of. Onobrychis to
inoculation with IT. solani as measured by the
mean, v a r iance, and range of the disease
severity s c o r e ............. ^
The response of 113 accessions of Onobrychis to
inoculation with E\ solani as measured by the
mean, variance, and range of the disease
severity scores.
Data were based on unreplicated
........................... .. * ...........
observations
.
79
85
ix
LIST OF FIGURES
■
Figure
3-1.
3-2.
Page
The visual scoring system used to assign disease ■
severity scores to the aerial spray, crown
injection, and toothpick techniques is shown
above and the .root-cut-soak technique is shown
b e l o w ............ ..
J. . . . . . . i
The effect of four inoculation techniques on the
percentage of plants infected with F. solani
when inoculated two weeks and six weeks after
emergence. . Isolations were made oh PCNBA (57)
.•■■■
3-3
3-4.
3-5.
3-6.
3-7.
■
.•
;
.....
39.
46
.
The effect o f four inoculation techniques on the
mean disease severity score assigned to sainfoin
seedlings inoculated with F. solani at two weeks
and six weeks of age
. . . . . . . . . . . . . . . . .
47
The effect of four inoculation techniques on the
percentage of plants infected with F-. solani
when inoculated 83 days after emergence at the
Field Research Laboratory, Bozemany Montana.
Isolations were made on PCNBA (57)
. . . . . . . . . .
49
The effect of four, inoculation techniques on the
mean disease severity score assigned to sainfoin
seedlings inoculated with F_. solani at the Field
Research C e n t e r ,- Bozeman, M o n t a n a .. . ... . . . . . . .
The response of the open-pollinated seed of 39
entries to inoculation with F_. solani as measured
by the m e a n ■disease severity score of. the entries . . .
The response of 181 accession of the world collec­
tion of Onobrychis ■to inoculation with F_. solani
as measured by the mean disease severity score
of each accession . . . . .
.. .. . . . . . . . . . . . .
50
55
59
X
ABSTRACT
Root and crown rot limits the production of sainfoin.
This
disease reduces longevity and forage yi e l d . The objectives of this
study were to:
Ij determine the causal organism(s) of root and crown
rot; 2) further characterize the host-parasite relationships; and 3)
develop and initiate'screening procedures to detect potential sources
of disease resistance.
The results of this study indicate that the root and crown rot
of sainfoin usually observed in Montana is caused by Fusarium solani.
This pathogen is not seed transmitted but probably occurs in most agri­
cultural soils as a pathogen of other legumes.
Sainfoin can be. successfully screened for disease resistance
by soaking injured roots in concentrated suspensions of m'icroconidia
of F. solani. Initial screening to locate potential sources of dis­
ease resistance has shown differential disease resistance in both the
world collection of Onobrychis and current, breeding material.
Selec­
tion within these populations should increase disease resistance v
INTRODUCTION
Sainfoin
..
(Onobrychis yiciifolia S c o p .) is a non-bloating per-
ennial legume which has been used for hay production and pasture in
■ »
Europe for several centuries. Cultivation of sainfoin began in Montana
in 1964 w i t h the release of lE s k i l.
Sainfoin rapidly gained popular­
ity because of its excellent forage quality, winter hardiness, drought
tolerance, palatability, and resistance to the alfalfa weevil
postica Gyllenhal).
(Hypera
It offered an excellent alternative to alfalfa
(Medicagp sativa L . )•
In recent years sainfoin has been attacked by a root and crown
rot which limits persistance and reduces forage production.
This dis­
ease is found statewide and is thought to be caused by Fusarium spp.
Sainfoin's.popularity has declined because of root and crown rot.. Dis­
ease resistant varieties must be developed if sainfoin is to remain an
economic forage c r o p .
The objectives of this study were to:
I) determine the causal
o r g a n i s m (s) .Of root and crown rot; 2) characterize the host-parasite,
relationships; and 3) develop and initiate sqreening procedures to de­
tect potential sources of disease resistance.
LITERATURE REVIEW
Description of Onobrychis viciifolia Scop.
Sainfoin is a deep rooted, perennial, forage legume.
It has a
branched tap root system which may extend to a depth of several meters
and be 5 cm in diameter
(60).
roots and the young tap roots
Modulation occurs on the fine lateral
(3,54,69).
Multiple erect stems, 90-150 Cm tall, arise from a branched, ■
prostrate crown.
These, stems are usually hollow, and bear pinnately,
compound leaves with 13-15 leaflets per leaf (18,60).
Sainfoin's influorescence is an erect raceme with from 5-80
florets.
Each floret gives rise to single seeded pod
(6,42).
The
pods are b r o w n , indehiscent, lenticular, and reticular on the surface
(60).
The seed are brown to black in color and are approximately
2.5 m m long, 2.0-3.5 m m wide, and I.5-2.0 m m thick (6).
The seed pod
comprises approximately 30% of the weight of the pod-intact seed
(6,16).
History and Distributions of Onobrychis viciifolia.Scop..
. Sainfoin has b e e n utilized as a forage crop in Russia for
over 1,000 years and in Europe for over 400 years
(60,68).
Early im­
migrants brought plants.of this species to the North American Conti­
nent with introduction intp Montana dating back to before 1900
(18).
■ V. ■.
•
’ ..
.
Sainfoin was evaluated as a potential.forage crop in the early
part of this century
(60), but failed to develop into a major, crop
3
because of disease problems; failure, to recognize its forage quality,
palatability, and non-bloating characteristics; and due to testing in ^
areas not well adapted to this crop.
In recent y e a r s , the increasing
losses of alfalfa to the alfalfa weevil,rand the need for a higher
yielding, non-bloating forage legume, has increased the ^interest in
sainfoin.
In 1964 the variety Eski was released by the. Montana Agricul­
tural Experiment- Station.
ductions from Turkey. (19).
This variety was selected from plant intro­
Other varieties such as lO n a r l and 'Mel­
rose' are selections from plant introductions from Russia
(28).
Cur­
rently, plant breeders working in sainfoin are continuing to '.evaluate
both new plant .introductions and the earlier introductions which have
become adapted to this country.
.
•
.
"
Agronomic Characteristics of Onobrychis. viciifolia.S c o p .
Sainfoin is drought tolerant, winter hardy, and adapted to
coarse calcarpus soils, which are not well suited for other legumes
(31,37,60).
It. has been utilized for hay production, seed production,
and pasture under both dryland and irrigated conditions.
However,
many researchers report there -is a lack of effective nodulation and.
that stand persistence is poor under, certain conditions
69) .
(3,48,54,66,
4
Sainfoin has high quality forage and has never been reported
to cause bloat
(6,16,18,28,60).
Feeding trials conducted in Canada,
M o n t a n a , Idaho, and Nevada have shown sainfoin hay to equal alfalfa
hay in nutritive value and to have even greater palatability
28,36,55).
(4,7,27,
Sainfoin seed also has the potential of becoming a protein
supplement in animal diets
(16).
Hay yields of sainfoin have been variable.
When grown under
proper, conditions, yields of sainfoin are competitive. With the best
adapted alfalfa varieties
(4,7,27,28,36,55).
Best yields are obtained
on well-drained, basic soils with a good moisture-holding capacity
(4,18,28,60).
Sainfoin is sensitive to frequent cuttings.
Most ex­
isting varieties produce maximum forage in the first cutting (4,18,55).
Many stands begin to fail in the third year when irrigated > and in the
fourth or fifth year under dryland conditions
(4,28,31,62).
This
stand depletion is thought to be the result of a disease which se- /
verely damages the crowns and roots o f mature plants
(48,66).,
Seed yields often approach 1,500 kilograms of clean seed per
hectare
(27,55).
In colder climates, seed is usually not harvested
until the second year of establishment.
Maximum seed yields are. ob­
tained if the first cutting is allowed to ■set seed
(6,28,55).
Sainfoin's role as a pasture legume is enhanced by its lack of
bloat, good feed value, and broad range of adaptation (18,28,43).
can either be pastured alone or in mixtures with grasses such as
It
5
Russian wild rye-grass or crested wheatgrass.
However, it does not
seem to compete well with aggressive, rhizomatous grasses such as
brome grass or pubescent wheatgrass
(28,43).
The use of sainfoin in
the hay-stockpile management regime distributes forage production and
allows an increase in total carrying capacity (12).
Diseases of Onobrychis viciifolia Scop;
Foliar, diseases.
in E u r o p e .
Mathre
...
Most work on foliar diseases has been done
(48) in a review, found reports of several foliar :
diseases of sainfoin.
A leaf spot caused by the fungus Ramularla
onobrychidis Allescher, forms dark brown spots on the upper surface of
leaves.
This disease is most severe under moist conditions and occurs
in large areas of Europe
(33,48).
Another leaf spot, which occurs primarily in England, is
caused by the fungus Septoria orobina Saac.
spots on both the leaves and young pods.
nated by contaminated pods
It forms fawn-colored
This disease may be dissemi­
(34,48).
Ring spot, which occurs in both England and Montana, is caused
by the fungus Pleospora herbarum (Pers.) Rabenh.
ilar to those of Septoria leaf spot.
tions to promote rapid spread.
economic damage in Montana
The symptoms are sim­
Gdhidia form under moist cdndi-
The disease does not appear to cause
(32,48).
6
The fuiigus Aschochyta onobfychidis forms spots on both the
leaves and stems.
It is found in Europe and Montana, where it occurs
primarily as blackened stem lesions.
borne and persists on crop residue.
ing economic damage
This .fungus appears to be seed
If is not considered to be caus­
(32,48).
Other diseases attacking the foliage of sainfoin are s
a rust
caused by Uromyces Qnobrychidis; Chocolate spot caused by Botrytis:
conerae; and powdery mildew caused by Erysjphe polygon! D.C.
(32,48).
None of these foliar diseases are thought, to be of economic importance ■
in Montana
(48).
Soil-borne diseases.
Diseases, that attack the crowns and
roots of sainfoin, are the primary cause of stand reduction.
Iium wilt of sainfoin
Verticil-
(Verticillium albo-atrum Reinke and Berth) has.
been observed in Europe and causes wilting and vascular discoloration
(35).
It is most severe during the warmer months when the plant js
often under moisture stress.
This pathogen has not been isolated.from
diseased sainfoin plants in Montana
(48,66).
The fungus, Sclerotinia trifoliorum Erikss, causes root ,crown,
and stem rot in Montana and Europe
(32,48),
Infected plants wither and
turn brown during the middle of the growing, season and sclerotia can
be found in the pith tissue.
Under high moisture conditions, white or
tan mycelial mats appear near the soil line.
The damage in infested
fields is usually light in M o n tana, with only 1% of the plants showing
symptoms
(48).
Rhizotonia solani Ku e h n. has been isolated from secondary roots
and darkened lesions on tap foots.
R. solani has never been isolated
from the internal portion of the crown but could possibly act as a
source of entry for other pathogens
(66).
Sainfoin is extremely susceptible to the northern root-knot
nematode, Meloidogyne hapla Chitwood.
temperatures.
are susceptible
Damage was more severe at higher
Unlike alfalfa, both mature and young roots of sainfoin
(24)..
The most serious disease of sainfoin in Montana is root and
crown rot though to be caused by Fusarium S p p . (48,66,67).
Seed and seedling diseases.
The seed and seed pod of sainfoin
has been shown to carry Alternaria, Fusarlum, Muc o f , and numerous bac­
teria
(74).
(66,74).
Many of the bacteria were antagonistic to modulation
Contaminated seed have been shown to spread Fusarjum pathogens
in beans
(Phaseolus vulgaris L . ) and m i m o s a . (Albizia julibrissin)
(22,58).
Sainfoin seedlings are susceptible to Alternaria s p p . ,
Rhizbctohia solani and Bythium spp.
classical 1damping o f f 1*.
(66).
Symptoms often appear as
• I:
8
Development of Root and Crown Rot in Onobr^cchis. viciifolia Scop.
Occurrence of the disease.
Sears
(.66,67) in an extensive study
of Montana found the root and crown rot of sainfoin in nine of ten sam­
pled locations.
While both F . sdlani
(Mart.) Appel & W r . and F. oxy-
sporum Schlecht. were isolated, F. solani was present in over 90% of
the diseased plants.
This disease also causes severe losses in New
Mexico and may occur in other areas where sainfoin is grown
(52).
When Eski was grown under normal field conditions, E\ solani
was isolated from 60% of the seedlings by the end of the first growing
season.
The rapid entry of this fungus was thought to be enhanced by
the seed pod wounding the young tap root as it began to thicken and
elongate
(66).
The seed pod reduces emergence in the field and germi­
nation in the laboratory (5).
may be beneficial
Removal of the pod prior to planting
(5,66).
Symptoms begin to appear during the second year.
The crown
appears to be unable to support the increasing number of stems and
splits.
This tearing may be a source of entry to pathogens.
has been consistently isolated from this area.
F . solani
The disease appears to
develop first in the stem, just above the crown where it forms a dar­
kened streak which extends into the vascular tissue of the crown.
As
the disease progresses, the crown tissue is.destroyed resulting in the
eventual death of the plant
(66).
Fumigation of field plots increased
vigor and regrowth, and reduced the frequency of root and crown rot in
9
sainfoin
(66).
The. crown splitting does not result in destruction of
crown tissue in the absence of pathogens.
Occurrence and severity o f ■root and crown rot of sainfoin
could be aggravated by root damage caused, by nematodes or insects. In
alfalfa, the infection of F. oxysporum was increased from 15 to 90% by
the addition of northern root-knot nematodes
wood) (51) .
In red clover
(Meioidbgyne hapla Chit-
(Trifolium pratense L . ) insect root injury
increased the incident of crown rot
(23) .
Noh-parasitic nematodes
such as Diplogaster lheritieri Maupas may spread plant pathogens.
These nematodes injest spores of IT. oxysporum hnd Verticillium s p p .
without reducing their v i a b i l i ty•(37).
The suspected p a t hogen.
most agronomic crops
(2,17,47).
crop residue as chlamydospofes.
F\ solani is a common pathogen of
This fungi exists in the soil and in
Chlamydospores are resting structures
produced by hyphae and conidia through the condensation of their con­
tents and the formation of a thick wall
(56,72).
Root exudates of
susceptible plants stimulate the chlamydospores to germinate and pro­
duce Aycelium which seek the surface of the root
(11).
The fungus
forms a small surface thallus and penetrates the plant through the
middle lamella at a junction of epidermal cells
(9,11).
vades roots through mechanical and natural wounds.
It also in­
Once established,
hyphae invade the. living cortex intercellularly until they are stopped
by the endodermis
(11).
. .
10
When the hyphae are exposed to daylight they produce macroconidia and microconidia.
Macroconidia are large sickle-shaped condia
with three to seven cells and are usually formed on spprodichia.
Microconidia are produced on the aerial mycelium and are usually com­
posed of only one or two cells
(72).
The principle function of the
spores is dissemination,.but they may also be directly infective (57).
When macrbconidia are added to the soil they form a short hyphae which
then forms chlamydospores
(11,56).
Isolates of F.. solani may produce a hormonal growth response
in plants t h a t .a r e .grown in infested soil
(11,25,56,66)
The asexual
forms are capable of genetic recombination, through the processes of
heterokaryosis and parasexuality
(50,72).
The genus F u s a r i u m .is divided into nine taxonomic species in S
system developed by Snyder and.Hansen
(72).
This system uses the
shape and morphology of macroconidia as its principle index.
Other
characteristics such as colony morphology and pigmentation are; unde­
pendable and subject to rapid mutation
(72).;
F. solani has been.di­
vided into nine formae species bn the basis of selective pathogenici­
ties to their hosts
(50). ..This selectivity is subject to rapid muta­
tion and may be simply inherited (73),
Some investigators feel the
division into formae species is highly artificial and question its
validity
(61).
11
F. solan! has been a severe pathogen on beans
(Pisium sativum L . )(47,49), and many other legumes
fungus produces a. sexual stage
(Hypomyces)
(2,49,56), peas
(20,49,56).
This
that is composed of indi­
viduals which are Self-sterile, interfertile, and usually hermaphro­
ditic.
Ascospores are found in perithecia which develop in nature
only on mulberry leaves under very humid conditions.
Recently induced the sexual stage in vitro
Investigators
(49,50).
Crown Rot in Other Forage Legumes
Red clover has historically been limited by a lack of stand
persistence due largely to root and crown diseases
(15,45).
Isolates
of Pyth i u m , Fusarium, Rhizoctonia, P h o m a , and Gliocladium have been
associated with this disease, but Fusarium s p p . a r e the prevalent
pathogens.
Strain's of these pathogens exhibited a wide range, of viru­
lence when tested on 'Midland' red clover
thought to occur in the seedling stage
(40,41).
(41)..
Stands often fail to re­
cover after the first cutting of the second year.
rot was more severe in years
with
Infection is
The root and crown
high temperatures,
limited soil
moisture, and when the plants were grown under nutritional stress
(8,17) .
.Pathogens associated with root and crown rot of alfalfa are
Phytophthora
(63), Aphanomyces
(65), and Fusarium
(46,65), Pythium (65), Rhizoctonia
(1,13,65,70).
moisture and nutritional stress
Root and crown rot is aggravated by
(10,55).
12
Nitrogen fixing bacteria, Rhizobium, are less effective in the
presence of Fusariuiu S p p i (38,45,53).
The relationship between the
bacteria and fungus is dependent upon soil pH, soil microflora, and :
time of inoculation
(53).
Plants with diseased roots have fewer nod­
ules which may lead to.a nitrogen stress and increased disease sus-■ V
ceptibility.
'
■ ■■
■■
■
■
:
.' ■ .
Development of. Selection Procedures •
Techniques do not exist for selecting sainfoin plants with re­
sistance to root and crown rot caused, by Eusarium spp.
After.six
years, only 3 of 100,000 plants were free of disease.symptoms i n d i - .
eating that resistance is rare in existing populations
(66).
There­
fore, selection procedures m u s t .be found that will screen large number
■of plants in short periods of. time.
Early work on alfalfa root diseases was done on.bacterial wilt,
Corynebacterium insidiosum (McCull.) Jensen
(14,39).
Techniques de­
veloped to screen for resistance to this organism have been adapted to
screen for other diseases caused by fungi such as Fusarium (13,14,21,
30,44,64).
Techniques which appear favorable are inoculation of.sin­
gle roots, bare-root-soak, root^ball-sdak; crown-soak, and inoculation
of cut stems
(13,14,45,63).
The percentage of infected plants can be
increased by wounding the roots prior, to, or in. conjunction w i t h , in­
oculation
(13,14,59,64).
Because of.the slow action of most root rot­
ting pathogens, researchers visually score the spread of infection
13
_
three to six months after inoculation. , This scoring system is usually
based bn five to seven categories and is used as the.principal index
of disease resistance
(13,14,45).
It is' possible to control the severity of a test by manipu­
lating environmental factors.
at time of inoculation
These factors include:
(2,14,47), water status
(14,21), concentration
of inoculum (14,45), the length of the trial period
temperature
(14,21,45).
age of plants
(14,21,47), and the
These factors should be manipulated to create
severe .disease symptoms in inoculated sainfoin plants in a minimum
■amount of t i m e . .The development of the proper combination of these,
factors will require extensive investigation..
...
• Chapter I
MATERIALS AND METHODS
General ■■■ ■•'
The purpose of. these experiments was to determine if:
I ) the
pathogens causing root and crown rot were seed^borne,.2) microconidia
of F.' solani could be used as inoculum, and 3) fungicides or seed
treatments could, reduce the. severity of. root and crown rot in sainfoin.
Plant's were grown either in the greenhouse or at the Field Research
Laboratory, Bozeman, Montana.
Plants.in the greenhouse were grown,
with a 16-hour photoperiod provided by supplemental lighting from in­
candescent lights and Were watered with a nutrient solution
Table I).
(Appendix
Seed and root pieces used for isolation were surface-
sterilized by soaking in a 0.5% NaOCl solution for three minutes.
Data were analyzed by analysis of v a r i a n c e :and means were
separated by Duncan's New Multiple Range Test.
Correlations were de­
termined among indices within experiments.
Experiment I :
.
Determination of potentially pathogenic fungi associated w i t h sainfoin seed
Samples of eight commercial seed lots of sainfoin were ob­
tained from the Montana State Seed Testing Laboratory to determine if
seed-rborne pathogenic fungi were affecting sainfoin.
The seed of each
sample were divided into three groups.and treated as follows:
Group I:
Pod-intact - no treatment.
15 ■
G r o u p .H :
Group III:
Pod-intact - surface-sterilized
Pod-removed - surface-sterilized
.
Eighty seed of each group were placed in petri dishes contain­
ing acidified potato dextrose sugar
(HPDA)(66)(Appendix Table I).and
80 seed were placed in petri dishes containing pentachlofonitrobenzene
agar
(PCNBA) (57) (Appendix Table I) . HPDA was used for general fungal
isolations and PCNBA was used to isolate■Eusarium S p p .
The HPDA and
PCNBA plates were incubated at 27 C for four arid eight days, respec­
tively.
nation
Fungal cultures were then ideritified by microscopic exami­
(72).
T h e .percentage of seeds contaminated with Alternaria
S p p . and/or Bh oxysporum were analyzed by contingency 'chi square.
On the basis of laboratory isolations, two seed lots were ,
chosen for further evaluation in the greenhouse.
The seed pods were,
removed from one-half of the seed of both the seed lot heavily con­
taminated (#1538) and the seed lot lightly contaminated (#405) with
.Alternaria spp.
One-half of both the
"pod-intact" and the "pod-
removed " seed were then surface-sterilized.
One hundred seed from
each of.the four treatments of both seed lots were incubated for nine
days at 25 C. in germination trays to determine if treatments had af­
fected germination.
Treated seed were planted in Bozeman silt loam ..
that had been, either autoclaved at 121 C for six hours, or pasturized
for 16 hours with unpressurized steam.
The experiment was conducted
in a Split-split-plot randomized complete block design with four
16
replications.
The two seed lots were assigned to main p l o t s , the two
soil treatments t d 'subplots, and the four seed treatments to sub­
Each sub-subplot contained 50 seed.
subplots .
After 24 days, seedling emergence was determined and the roots
of four seedlings from each sub-subplot were harvested.
•
■ '
-
-
■■
;
■■
■
■
■
■
■
■
.■
..
A root piece
;
.
was removed I cm below the crown of each seedling, surface-sterilized
for 45 seconds, and placed on HPDA (66) to determine, the percentage,
of seedlings infected with Alternaria spp.
Experiment I I ;
An evaluation of the relative pathogenicity of.
microconidia and macroconidia of four isolates
of F . splahi
Three surface-sterilized; pod-removed 1Remont1*
4 seed were
planted in 10.2 cm square plastic pots which contained Bozeman silt
;loam soil, that had been autoclaved at 121 G for six h o u r s .
were thinned to one p e r pot at.emergence.
Seedlings
Eighty-three days after,
emergence the seedlings were inoculated with
soI a n i ■by piercing .
the crown with a sterile dissecting needle and applying approximately
4 x IO6 conidia to the wound.
: The experiment was conducted in a split-plot randomized com­
plete block design with three replications.
Inoculation with either
micro- or macroconidia was. assigned to main plo t s .
produced in Modified Eckert's Broth
Microconidia were
(66.) and a mixture of micro-, and
macroconidia were produced in Snyder and Nash's sporulation medium (56)
17
Appendix Table I).
Inoculations with each of the four isolates of
F . ■solani and with sterile water were assigned to subplots.
There
were 10 seedlings in each subplot. . .
Ninety days after inoculation, roots of seedlings were har­
vested, split longitudinally, and visually scored for disease severity.
A single section of each taproot was removed 2.cm below the crown,
surface-sterilized, and placed on HPDA
(66) to determine the.percent­
age of plants infected with F. solani.
Experiment I I I :
Evaluation of soil fumigation and seed treatments for
the control of root and crown rot of sainfoin.
A split-plot randomized complete block design with three repli­
cations waS used to evaluate the effect of planting either pod-intact
or pod-removed Seed into pots which had been treated with fungicides.
Surface-sterilized Remont seed with either the pod-intact or the podremoved were randomly assigned to main plots.
ments were assigned to subplots.
Cont r o l :
The fungicide treat­
These were:
N o .treatment.
Chloropicrin Soil Fumigation:
.
Chloropicrin
(trichloronitro-
methane) was injected 10 cm into the soil in a 15.4 cm
grid pattern with a Fumigun at the rate of 647 kg/ha.
The
treatment was applied on June 10, 1974, and the plots were
sealed with plastic tarps for seven days.
18
Berilate Soil D r e n c h :
A 50% active-ingredient, wettable powder
of the fungicide 'Benlate - (Methyl-(butylcarbanoyl)-2 benzi­
midazole carbamate) was incorporated 5 cm into the soil
with a rake at the rate, of 340 k g / h a T h i s
treatment was
applied on July I, 1974.
Benlate Seed Treatment;
Benlate was dusted on the seed at t h e .
rate of 2.5 g of wettable powder of. the fungicide per 1000 g
of seed immediately prior to planting.
The study was seeded at the Field Research Laboratory on July 9,
1974, at the fate of 24 and 34 kg/ha for the pod-removed and the podintact seed, respectively.
Plots were 3.1 m long, 1.8 m wide, and
contained 12 rows spaced 15.4 cm apart..
The border surrounding each
plot was 92 cm wide and seeded to crested wheatgrass.
The plots were
irrigated 20 minutes daily for 12 days, to permit seedling.establish­
ment.
Thereafter, irrigation water was applied as needed. .
Five times during the course of the. study a minimum of eight .
seedlings were removed from the margins of each plot and scored for
nddulation
(Table 1-1) and disease severity.. A section of root from
each plant was removed 3 cm below the crown, surface-sterilized, and
placed on PCNBA
(57) to determine the percentage of seedlings infected
with Fusarium spp.
Fusarium isolates were identified to species by
cultural characteristics on slants of fresh potato dextrose agar (FPDA)
(Appendix Table I) and microscopic examination of macroconidia
(72).
19
Table 1-1.
The visual rating system used to assign npdulation scores
to sainfoin seedlings.
Score
Description
1
No nodules formed
2
Nodules on 1% of the secondary roots
3
Nodules on 5% of the secondary roots
4
■Nodules on 25% of the secondary roots
5
Nodules on 50% of the secondary roots
6
Nodules on 75% of the secondary roots
Forage yield, plant height, stand density, and seedling vigor
were measured on a I m wide strip in the center of each p l o t .
Table
1-2 lists the indices and the dates on which they were measured in
this study.
.
■E x periment.I :
RESULTS AND DISCUSSION
Determination of potential pathogenic fungi associated
with sainfoin seed.
Helminthosporium, Nigrospora, Fusarium, Alternaria, and numer­
ous saphrophytic fungi were found on sainfoin seed.
Fifty-seven per­
cent of the untreated seed w e r e .contaminated with Alternaria spp.
(Table 1-3).
Other fungal species occurred on less than five percent
of the seed. ■ Seed treatment reduced contamination with Alternaria spp.
(X2 = 594i2; P
< .005). . With the exception of one seed lot
(#1538) ,
20
Table 1-2.;
The date of sampling' and the indices measured oh the soil
■ fumigation and seed treatment experiment at the Field .
.Research Laboratory, B o z e m a n M o n t a n a .
Date
1974
Index
Aug: 26
Number of p l a n t s .showing visual wilt symptoms
Number of plants with visual root symptoms.
Percentage of plants infected with F. spp.
Aug.
30
Stand density
Seedling vigor scored.
Sept. 13
Number of plants with visual root rot symptoms
Percentage of plants infected with F . oxysporum
Percentage of plants infected with F. sdlani
Oct. 8 .
Nodulation scored.
Percentage of plants infected with F_. Oxysporum
Percentage of plants infected with F. s o l a n i .
1975
June 5 -
Nodulation scored
Disease severity scored
Percentage of.plants infected with F . oxysporum
Percentage of plants infected with
July 3
Forage yield
Aug. 13
Plant height
S e p t . 29
Forage yield
Oct. 8
Disease severity scored
solani
'
.•
Percentage of plants infected with F. oxysporum
Percentage of plants infected-with Fr solani
Table 1-3.
The effect of pod removal and surface-sterilization of seed on.the percentage ■
of seed contaminated with either Alternaria spp. or.F. oxysporum from eight
commercial seed lots of sainfoin.
Contamination with Alternaria spp. I/
; PT 4/
Untreated
Seed
lot
PR
Surface
sterilized
%
0.0
0.0
42.5
2.5
0.0
1.3
0.0
0.0
405
23.8
0.0
. 0.0
1.3
0.0
0.0
469.
42.5
0.0
0.0
li3
0.0
0.0
.509
61.3
1.3
0.0
3.8
0.0
0.0
699
11.3
0.0
9.8
0.0
0.0
1005
75.0
0.0
O
. ■ 0.0
0.0
1538
100.0
51.3
403
.
H
0.0
■
„
O
4.8
Based bn isolations made oh HPDA (66).
2
';■■■■;.
■
Based bn isolations made On PCNBA agar
o.o
O
0.0
98.8
007
O
1.3
O
%
■ pi
Surface
.sterilized
%
O
'
PI
Untreated.
PR 5/
Surface
sterilized
%
CO
CO
%
' PI
3/
Surface. —
sterilized
%
Contamination with F. oxysporum 2/
(57).
^Seed surface sterilized by soaking in a 0.5% NaOCl solution for 3 minutes.
4
Seed p o d -intact.5
5
Seed pod removal.
0.0
removing the pod and surface-sterilizhtion of the seed eliminated this
•fungus
(Table 1-3).
Two percent of the untreated seed were contaminated with E,.
oxysporum
(Table 1-3).
Surfacersterilization of the seed pod com­
pletely eliminated this fungus.
treatments
Seed lots responded the same to seed
(X2 = 3.4; P > .995).
the pod or seed of sainfoin.
F. solani was never isolated from
This pathogen does not appear to be seed
transmitted.
When planted in the greenhouse, seedling emergence of the seed .
lot heavily infested with Alternaria spp.
(#1538) was.reduced
(Table
.1-4). • The degree of seedling infection was. proportional to the amount
of Aiternaria spp. contamination on the seed.
Since both seed lots
had very similar germination in the laboratory, the. reduction in etner, gence was probably due to seed rot or pre-emergence damping^off caused
by Alternaria spp.
Seedling response was the same in both the pasteurized and auto-r
claved so i l s .
Sears
(66) reported sainfoin losses to Alternaria oc-
curred only in heat treated soils.
More information would have been
. gained, if. an ..untreated soil had been used as. one of the soil treat- ...
meats.
Surface-sterilizing the pod, removing the pod, and surface-
sterilizing the seed reduced the percentage of seedlings infected with '
Alternaria spp. but did not result in increased seedling emergence
, (Table 1-5). '
'
23
Table 1-4.
Sainfoin seedling emergence as affected by seed-borne
Alternaria spp.
Seedling
emergence
24 days
%
Seed lot
68.8 a 2/
1538
■ 405
Seedlings
infected with
Alternaria spp.—
■
%
Seed
germination
laboratory^/
% •
11.7 a
85.8
4.7 b
86.0
80.3 b
.
"''Based on isolations made on H P D A . (66) .
2
Means based on 400 seed.
3
I
Means within a column not followed b y .the same letter differ
at the .05 level of probability.
Table, 1-5.
The effect of pod removal and seed surface-sterilization
on the percentage of seedlings infected with Alterharia
spp. and percentage of seedling emergence.
Seed treatments
Pod-intact
.
Pod-intact
Surface-sterilized 2/
Seedling
emergence
24 days
%
18.8 a 2/
79.6
■ 70.4
Seedlings
infected with
Alternaria spp. I/
%
■
7.8 b
Pod-removed ■
Surface-sterilized
76.2
1.6 b
Pod-removed
72.2
4.7 b
1
Based on isolations made on HPDA
(66).
2
Surface-sterilized by soaking in a 0.5% NaOCl solution for
three m i n u t e s .
^Means within the column not followed by the same letter differ
at the .05 level of probability.
24
Experiment I I :
An evaluation of the relative pathogenicity of
microconidia and macroconidia of four isolates
of F. solan!.
The microconidia! inoculum infected a higher percentage of
plants than did the macfocpnidial inoculum, but both inocula produced
similar disease symptoms
(Table 1-6).
Both types of bonidia are i n - .
fectious, but the smaller microconidia may have more readily entered
the wounds caused by the inoculation technique.
The four isolates of
F. solani produced different amounts of macroconidia in the Snyder and
■Nash's sporulationmedium (56) (Table 1-7).
The macroqoriidial inoculum
was a composite of both microconidia and macroconidia, and probably
did hot accurately estimate the performance of an inoculum composed
entirely of macroconidia.
Table 1-6.
The effect of inoculating sainfoin seedlings with either
. microconidia dr macroconidia of F_. solani on the mean
•disease severity score arid the percentage of plants
• infected.
••
Mean disease
severity score i/
Inoculum
2.05 a V
Microconidia
Macroconidia plus
microconidia
X
1.87 a
Plants infected
with F . solani 2/
30.0 a
. 21.6 b
-
'
Disease severity scored as follows: I = no spread of discol- .
oration; 2 = spread of discoloration less than I cm; 3 =
■spread of discoloration more than I cm; .4 = extensive vascular
2 necrosis'; and 5 =. dead plant
■
^Based on isolations made on HPDA (66).
Means within a column not followed by the Same letter differ
at the .05 level of probability.
25
■The isolates caused similar disease symptoms and.infected a
similar number of seedlings
(Table 1-7)'.
Inoculation with any of the
isolates resulted in more severe disease symptoms.than inoculation
with sterile water, but only one isolate infected a higher percentage
of the seedlings than, that observed in the control.
Greater differ­
ences in the percentage of plants infected could probably h a v e 'been
detected had a more efficient inoculation procedure been u s e d .
Table 1-7.
isolate
Mean disease
severity scorel/
. 2.25 a y
.11
. 07
T h e ■effect of inoculating sainfoin seedlings with four .
isolates of F. solani on the mean disease severity score
and the percentage of plants infected.
Also shown is
the Percentage of microconidia produced in Snyder and
Nash's sporulationmedium (56).
' 2.18 a
.
.
Plants infected
with F. solani!/
%
%
.
35
16.7 ab
50
18.3 a
01
1.98 a
11.7 ab '
Control
1.20 b
. " ;■
‘
16.7 ab
2.17 a.
06
Micrdconidia
1.7 b
5
• • 94
0
'
Disease severity scored.as follows:
I = no spread of discol­
oration; .2 = spread of discoloration less than .1 cm; 3 =
Spread of discoloration more than I cm; 4 = extensive vascular
necrosis; and 5 = dead plant.
^Based on isolations made on HPDA
(66).
.
'
Means within a column not followed by the same letters differ
at the .05 level of probability.
26
In future studies more resolution could be obtained by:
I)
increasing, the inoculum.level from 4 x , l b 6* to 4 x I O 8 conidia/plant;
2) using the root-cut-soak inoculation technique;
3) including the
composited isolates as a treatment; 4) isolating the treatments to re­
duce contamination; and 5) developing procedures to obtain inoculum
composed entirely of macroconidia.
Experiment .III:
Evaluation of soil fumigation and seed treatments for
the control of the root and crown rot of sainfoin
Field fungicide, treatments affected only the number of seed­
lings with wilt symptoms, seedling vigor, stand density, and plant
height.
The Benlate soil drench caused several emerging seedlings to
wilt and reduced both stand density and seedling vigor
(Table. 1-8)<
This indicates that sainfoin is sensitive, to high rates of Benlate.
During the second year plant, height differences were Observed.
Plants
grown in the soil treated with Benlate and Chloropicrih were signifi­
cantly taller than the control.
of long-term effects.
This could be the initial expression
.
This study should be continued.to determine
other possible effects of the Benlate and Chloropicrin treatments.
No differences were detected in the isolation of Fusarium spp.,
disease severity, nodulation, or forage yield.
This could have been
the result of limited sample size; rapid invasion of the small plots
by soil microbes, or lack of sufficient time for effects to be ex­
pressed.
In future studies, the use of larger plots would allow the
Table 1-8.
The effect of four fungicide treatments on sainfoin plants grown at the
Field Research Laboratory, Bozeman, Montana.
. Treatments
Number of wilted
plahts/plot
,Stand density
plants/meter rowi/
18.5 a
Mean seedling
vigor score2/
Plant height
.. cm V
Chloropicrin
soil fumigation
.50 a I/
Benlate seed
treatment
.33 a
. 16.3 ab
Control
.17 a
15.9 ab
3.2 ab '
62.8 b
13.8 b
1.7 b
69.3 a
Benlate soil
drench
2.17 b
4.8 a
' 3.2 ab
70.1 a
67.6 ab
^Means based on 10 samples of I m row/plot.
2
Seedling vigor scored as follows: I = very poor growth; 2 = poor growth; 3 =
fair growth; 4 = good growth; and 5 = excellent growth.
3
Means based on 5 random samples per plot.
^Means within a column not followed by the same letters differ at the .05 level
of probability.
28
sampling of more plants and reduce the possibility of invasion of m i ­
crobes into treated p l a n t s .
CONCLUSIONS
Alternaria spp. are common contaminates of sainfoin seed.
F.
oxysporum contaminated the pod of two percent of the seed evaluated.
F-. solani was not found on the seed of sainfoin,
The presence of p o ­
tentially pathogenic fungi on: sainfoin seed may .require seed treat­
ments to insure good seedling emergence in heat treated soils.:
Both microconidia and macroconidia infected and caused disease
symptoms in sainfoin.
Microconidia were more effective in entering
the small wounds caused by the inoculation technique.
The four iso- '
lates were equally pathogenic.
The addition of fungicides to the seed and soil did not reduce
the expression of the root and crown rot of sainfoin during the first
18 months of this stu d y .
Benlate fungicide was phytotoxic to a few
seedlings indicating, that sainfoin may be sensitive to this fungicide.
This study should be monitored to detect possible long-term effects of
the fungicide treatments.
Chapter II
MATERIALS AND METHODS
General
The purpose of these experiments was to determine:
I) the
causal organism.(s ) responsible for root and crown rot of sainfoin,. and
2) the host range of the o r g a nism(s).
following procedures were standard.
In all studies reported the
Seed were Surface-sterilized by.
soaking in a 0.5% NaOCl solution for three minutes.
Three seeds were
planted in each pot and seedlings were thinned to one per pot after
emergence.
Pots were .15.4 cm deep and 10.2 cm wide and contained a
soil mixture of two parts Bozeman silt loam and one part river washed
sand.
The mixture had been autoclaved at 147 C for 85 minutes.
Plants were grown with a 16-hour photoperiod provided by supplemental
lighting from incandescent lights and were watered with, a nutrient
solution
(Appendix I).
. Single, spore cultures of Fusarium spp. were increased in Modi­
fied Eckert's Medium
(66) in shake culture.
Isolates of F. solani f.
sp. pisi and F.. solani f. sp. phaseoli were obtained from Washington
State University
(26).
After inoculation, those plants inoculated .
with different pathogens Were separated to prevent contamination.
At
the termination of each trial, roots were visually scored for disease
severity.
Two pieces of each root were removed I and 3 cm below
the c r o w n , surface-sterilized, and placed on HPDA (66) to determine
the percentage of seedlings infected with Fusarium spp.
Isolates of
Fusaritim were identified to. species on the basis, of cultural character­
istics on slant tubes of fresh potato dextrose agar
dix I ) .
(FPDA) (72) (Appen-^
Disease severity scores and the percentage of plants infected
with Fusarium were analyzed by analysis of variance.
Duncan's New
.
Multiple Range Test was used for mean separation.
' Experiment I i ■ A test of pathogenicity of F. solani and F. pxysporum '
on sainfoin.
The purpose of this test ,was to determine if F_. solani and Fv
oxysporum. were the pathogens responsible for the root and Crown rot Cf
sainfoin.
Remont sainfoin seedlings were grown in a growth chamber :
with diurnal temperatures of 15.5
(night) and 25 C
(day) ,for 83 days.
' Seedlings were then inoculated by severing their roots 5 cm below the
cr o w n ; soaking the. injured plants in inoculum for 15: m i n u t e s , and then
transplanting the plants back into their pots.
Plants were inoculated .
with either F. solani f. sp. p i s i , F. solani f . sp. phaseoli, F.
■solani isolated from sainfoin, F. oxysporum isolated from sainfoin, of
sterile water.
used.
A completely random design with five replications was
Inocula of E^. solani f. sp. p i s i , F. solani 1sainfoin1 , and F.
oxysporum 'sainfoin-1 contained 2.65. x IO6 , 2.70 * IO6 , and 22.50
microcohidia/ml, respectively.
XlO6
The inoculum of Fv solani f . s p . phas­
ed Ii contained mycelial mats instead of conidia.
Ninety-five days
after inoculation, roots were visually scored for disease severity and
isolations made to determine the percentage of plants infected.
Experiment I I :
Evaluation of the host range of three isolates of .
F. sblani isolated from bea n s , p e a s , and sainfoin..
■The purpose of this experiment was to determine if the three
isolates of F . splani had common host ranges.
lLadaK 65' alfalfa and.
Kemont sainfoin ,'seedlings, were 65 days, old and the 'Alaskan' pea and.
red kidney bean seedlings were 2.8 days old at the time of inoculation.
Seedlingb were inoculated by piercing the hypocotyl with a sterile
dissecting needle and applying 2.0 ■x I O 8 micrbcohidia to the w o u n d .
.;
Twelve plants of e a c h ’species were inoculated, with either F. s o lani'f .
■ sp. p i s j ,. F- solani f - ■s p 'phaseoli, F. solani 'sainfoin'-, or sterile
water:
The plants were arranged in a split-plot' randomized complete
block design with three replications;
The.four pathogen treatments
.
'■
were assigned to main plots and the four host species' were assigned to
subplots.
■Each subplot contained four seedlings.
Thirtyrtwb days
after inoculation, roots were visually scored for disease severity and
isolations were made to determine the.percentage of plants infected.
A pooled error term was used to compute the mean separation
(71).
' RESULTS AND DISCUSSION
•. :
■'. • ■
■.
.■ .
■
•
••• •
Experiment I :;. A test of the pathogenicity of F. solani and F .
oxysporum on sainfoin. •
'
•
The isolate of F. oxysporum infected sainfoin but produced only
sporatic vascular discoloration of limited severity
(Table 2-1).
This
32
limited symptom development indicates that F. oxysporum is hot respon­
sible for the root and crown rot of. sainfoin.
Table 2-1.
The pathogenicity of Eh oxysporum and F., solani on
sainfoin seedlings as measured ‘b y 'the mean disease
severity score and the percentage of plants infected.
■Pa t h o g e n s '
Mean disease
severity score I/
Control
1.4 a 4/
F. oxysporum 1sainfoin1 2/
2.0 a
F. solani f . sp; p h a s e d !
4.2 b
F . solani
4.2 b
'sainfoin'
F. solani f. s p . .pisi,
4.4 b
.Percentage of
plants' infected 2/
0
.
' 100
loo
.
100
'
. 100
1
Disease severity scored as follows: I = no spread of discol­
oration; 2 = spread of discoloration less than I cm; 3 =
: spread of discoloration more: than I cm; 4 = extensive vascular
■necrosis; and 5 = dead plant.'
2
Based on isolations made on HPDA
(66),
. Isolated from diseased sainfoin plants.
^Means within a column not followed by the same letter differ
at the .05 level of probability.
The three.'isolates of F. solani infected, sainfoin and produced
disease symptoms of equal severity
(Table 2-1).
Inoculated plants had
extensive vascular necrosis very similar to disease symptoms observed,
in naturally infected sainfoin plants,.
These results provide evidence
that E\ solani i s 'responsible for the root.and crown rot of sainfoin.
33
The tests with F_. solani f. sp. pisi and E. solani f . sp.
phaseoli indicate that sainfoin may be. susceptible to many isolates of
this organism.
.Because peas, beans, and other legumes have been grown
for many years in Montana, it is possible that isolates of F_. solani
.
have spread and proliferated in agricultural lands. . This could ex- .
plain the rapid development of root and crown, rot of sainfoin in areas
where this crop has never been grown.
.Experiment I I ;
Evaluation of the host range of three isolates of
F. solani isolated from b e a n s , peas; and sainfoin. -
The three isolates o f 'F. solani were equally pathogenic bn
sainfoin*and beans
(Table 2-2). ■F. solani 1sainfoin' was not highly
pathogenic to.peas and was more pathogenic of alfalfa than F. solani f .
sp.. phase o l i .
tested.
F. solani f. sp. pisi was pathogenic on all hosts
Crop rotations which include beans ,..peas, or alfalfa would
not control sainfoin root and crown rot.
Stands of sainfoin estab­
lished in fields with a recent history of these crops would be more
rapidly attacked by root and crown rot than.stands established in areas
without a prior history of these crops. ■
.
■
•
.
- '
'
:
•
,
.
\
The bean plants inoculated with sterile water were discolored
and infected with F. solani
(Table 2-2).
This was probably due to. the
slow, formation of callus tissue by bean seedlings following inoculation
with the sterile dissecting needle.
lower percentage of infection.
Controls of other species had a
34
Table 2-2.
The pathogenicity of three isolates of F. solan! on peas,
beans, alfalfa, and sainfoin as measured by the mean
disease severity score and the percentage of plants
infected.
Host
Pathogen
Mean disease
severity score—
.Plants infected
with F . solani?/
Peas:
. F . s o l a n i .'f. s p . pisi
F . solani f. sp. phasebli ^ ,
F . solani 'sainfoin'
.Control.
5.00
4.33
2.50
1.42
100.0
83.3
' 8.3
0.0
a y
ab
fg
h
a
abc
e
e
'
Beans:
F. solani f. sp. pisi
■F. solani f . sp.. phaseoli
'F. solani 'sainfoin*
Control
.
3.42
3.25
2.92
2.42
cde
cdef
def .
f .
3;58
3.08
2.67
1.33
bed
cdef
ef
h
3.83
. 3.42
3.25
I .67
be
cde
cdef
gh
91.8
. 100.0
100.0
■ . . ' 41.8
ab
a
a
d
Alfalfa:
F. solani 'sainfoin'
F . solani f . sp. pisi
.F. solani f. sp. phasebli
Control .
83.3
100.0
. 83.3
. 8.3
abc
a
abc
e
:67.7
58.3
75.0
8.3
be
cd
abc
e
Sainfoin:
F. solani 'sainfoin'
F. solani •f. sp. phaseoli
F. solani f. sp. pisi
Control
I
'
■
■
Disease severity scored on spread, of discoloration from point
of inoculation as follows:
I = 0.1 - 0.9 cm; 2 = l.'O - 1.9 cm;
3 = 2.0 — 2.9 cm; 4 = 3.0 - 5.0 cm or extensive vascular
necrosis; 5 = dead plant.
Based on isolations made on EPDA (66).
^Isolated from diseased sainfoin plants.
4Means within a column not followed by the same letters differ
at the .05 level of probability.
35
The lack of specificity of the isolates evaluated in this
study question the concept of 1formae speciales' in fungi such as FV
solani.
It is possible that this lack of specificity is an artifact
of injecting pathogens into host species and. that many of these isolates would not be pathogenic under natural conditions. Further,
studies are needed to test this concept and to define the host range
of F. solani 1sainfoin*.
These s tudies.should include a wide range of
native and cultivated legumes.
CONCLUSIONS
.
.
F. solani f . sp. pisi, F. solani f . S p i phaseoli, and F .
solani
1sainfoin' caused root and crown rot in sainfoin.
did not appear.to be responsible for root and crown rot. .
E. oxyspoium
Chapter III
MATERIALS AND METHODS
General
The purpose, of these studies was to develop and initiate
screening procedures to detect potential sources of resistance to root
and crown rot of sainfoin caused by .F. solani.
All plants were grown
either in the greenhouse or at the Field Research Laboratory < Bozeman,
M o n tana.
Plants in the greenhouse were grown with a 16-hour photo-
period provided by supplemental lighting from incandescent; lights and
were watered with a nutrient solution
(Appendix Table I).
Root pieces
used for isolation and seed were surface-sterilized by soaking in a
0.5% NaOCl solution for three minutes.
All data were analyzed by analysis of variance.
Harvey's (29)
l e a s t .squares analysis was used when, there were unequal numbers in
each replication.
Test.
Means were separated by Duncan's New Multiple Range
Mean separations were computed with a p o o l e d .error term in
those experiments with multiple error terms
(71).
Correlations w e r e .
determined for the indices within experiments.
Experiment I ; . Greenhouse evaluation of four techniques of inoculating
sainfoin seedlings with F. solani.
...
Two- and six-week-old sainfoin seedlings were inoculated with
either F_. solani or sterile Water using four techniques.
The experi­
ment was conducted in a split-split-plot randomized complete block de­
sign with four replications.
Seedling ages were randomly assigned to
main p l o t s ; inoculation with F. solani or sterile water to subplots;
and. the four techniques, of inoculation to sub-subplots.
Within each .
age group, one half of the.seedlings were inoculated with sterile water
instead of -F. solani
to
allow an assessment of the physical injury
resulting from the inoculation techniques.
Each inoculation technique
was applied to 10 seedlings in each sub-subplot.
Root-Cut-Soak:
Techniques .were:
Seedlings were removed from the pots and the
roots severed 5 cm below the crown..
ThCy were then soaked
in inoculum for 15 minutes and transplanted,back into the
.
pots.
Grown Injection:
The crowns were pierced With a sterile
dissecting needle and 5 ml of inoculum applied to the
' wound.
Aerial Spray:
'p.'s.i.:
Seedlings were sprayed with inoculum at 14
The foliage was removed 5 cm above the crown of
the six-week-old seedlings prior to inoculation, to in­
crease penetration of the inoculum into the crown area.
Toothpick:
Wooden toothpicks' were placed in 20 Cm test tubes, .
soaked in Modified E c k e r t 1s Broth
(66), autoclaved at 154 C
for 15 m i n u t e s , and then incubated with F. solani for 14 ■■
days.
These toothpicks were pierced through the seedling's
Crowns where they remained until the termination of the
study.
..'
Sterile toothpicks were used as the c o n t r o l . T h i s
technique' was not used on two-week-old'seedlings because
. their crowns were too small.
'
. Three Remdnt seed were planted in each pot and seedlings were
thinned to one per pot. after emergence.
The plastic pots were 10.2 cm
square and contained a Bozeman silt loam ,soil that had been autoclaved .
for six hours at 97 C.
■The inoculum was a composite of four isolates of F. solani
isolated from sainfoin at different Montana locations.
was increased individually on Modified Eckert's Broth
Each isolate
(66) in shake
culture and equal proportions of conidia were combined prior to inocu­
lation.
The composited inoculum contained 9.1 x T O
rriicroconidia/ml.
'Ninety days after inoculation, counts, were made to determine
seedling mortality.
The roots were then harvested, split longitudin­
ally, and; visually scored for disease, severity
(Figure 3-1).
A sec­
tion of each root was removed from the crown, surface-sterilized, and
placed on PCNBA
(57) to determine the percentage of plants infected
with F . solani.
The mean disease severity score and the percentage of plants
infected were used as indices of inoculation effectiveness.
Data ob­
tained from inoculating six-week-old seedlings were analyzed separately
in a split-plot randomized, complete block design to evaluate the tooth­
pick inoculation technique..
The toothpick data were omitted When the •'
indices were analyzed over both dates of inoculation.
'
39
Figure 3 - 1 ; The visual scoring system used to assign disease severity
scores to the aerial spray, crown injection, and toothpick techniques
is shown above and the root-cut-soak technique is shown below.
Arrows
denote point of inoculation.
Disease severity scored as follows: I =
no spread of discoloration; 2 = spread of discoloration less than I cm;
3 = spread of discoloration more than I cm; 4 = extensive vascular
necrosis; and 5 = dead p l a n t .
40
Experiment I I :
Field evaluation of four techniques of inoculating
sainfoin seedlings with F_. solani.
Remont seed were planted in the field in rows 60 cm apart and
12 m long.
Sixty days after emergence, seedlings were thinned to one
plant every 15 cm.
Plants were irrigated as needed.
The experiment was conducted in.a split-plot randomized.com­
plete block design with four replications;
Inoculation treatments,
either with F_. solani or sterile water, were assigned to maihplots;
and the four previously described inoculation techniques were assigned
to subplots.
A minimum of 25 seedlings in each subplot were inocu­
lated 83 days after emergence.
The domposited inoculum contained
11.3 x I O 7 microconidia/ml.
One year after inoculation, plants were counted to determine
mortality.
Roots were scored for disease severity and the percentage
of plants infected with F. solani determined by isolations o n 'PCNBA
■(57)-
. ;V
Experiment I I I : ■An evaluation of the repeatibility and resolution of
the root-cut-soak technique of inoculating replicated
genotypes of sainfoin.
Vegetative cuttings were made of 10 sainfoin clones that had
been selected on the.basis of being either solid crowned
of visual disease symptoms
(DR)..
in the mist bench for 45 days.
(SC) of free
Crown buds of each clone were placed
Those buds which initiated root growth
were transplanted into plastic pots 15.4 cm deep and 10.2 cm wide.
41
Pots contained a soil mixture of perlite, washed sand, and Bozeman
silt loam.
flowered.
These cuttings were grown in the greenhouse until they had
The foliage was then removed and they were inoculated with
F_. solani using the root-cut-soak inoculation technique.
The com­
posited inoculum contained 3.0 x I O 7 microconidia/ml.
One hundred and four days after inoculation, the plant roots
were harvested and scored for disease severity (Figure 3-1).
Two sec­
tions of each root were removed I arid 3 cm below the crown, surfacesterilized, and placed on HPDA (66) to determine the percentage of
cuttings infected with E\ solani.
Experiment I V :
An evaluation of the disease resistance of the open- .
pollinated progeny of 31 clones of sainfoin.
Open-pollinated seed of 31 clones o f sainfoin which had been
selected for longevity under field conditions was tested for resis­
tance to root and crown rot caused by F_. solani.
Remont were check cultivars.
Melrose > fiski, and
Also included in the study were:
an ex­
perimental cultivar developed in New Mexico; open-pollinated seed from
a 12-year-old stand of Eski near Creston
(Population I); and open-
pollinated seed ,from ah eight-year-old stand near Bozeman
(Population
II) .
Ninety seeds of each entry were planted into greenhouse
benches containing plaster-grade Vermiculite.
Seedlings were inocu-
.
Iated 42 days after emergence with F_. solani using the root-cut-soak .
42
inoculation technique.
They,were then transplanted into benches con­
taining an untreated Bozeman silt loam, soil in rows 9 cm apart with
plants 7 cm apart within rows.
The foliage was removed 5 cm above the.
crown after transplanting to increase uptake of the inoculum.
composited inoculum contained 8.8 X l O . micrbconidia/ml.
The
Each entry
.
was represented by 10 seedlings in each of the three replications ar­
ranged in a randomized complete block design.
Twenty-four days after inoculation, counts were made to deter­
mine seedling mortality,
bead plants were considered to have died
from transplant shock and were not considered in the disease evalu­
ation.
severity
Ninety days after inoculation, foots were scored for disease
(Figure 3-1) and the range, variance, and mean for each entry
was calculated.
Isolations from each plant were made on PCNBA (57) to
determine the percentage of plants infected with
solani.
The mean
disease severity score and the percentage of plants infected were used
as indices of disease susceptibility.
Experiment V :
Screening 296 accessions of the world collection of
Onobrychis for sources of resistance.to crown and root
rot caused by F. solani.
Seed of 296 accessions of Onobrychis obtained from the USDA
Plant Materials Center, Pullman, Washington, were planted into green­
house benches filled with plaster-grade vermiculite.
The seedlings
were inoculated with F. solani 105 days after emergence using the .
root-cut-soak inoculation technique. . The composited inoculum
43
contained 7.3 x 10
microconidia/ml.
The seedlings were then trans- .
planted into greenhouse benches as previously described.
The foliage
was removed from the seedling's 8 cm above the crown.
Those accessions with less than 15 healthy seedlings were
evaluated in ah unreplicated trial in which each accession was repre­
sented by eight seedlings.
Accessions with 15 or more healthy seed­
lings were evaluated in a randomized complete block design with three
replications.
lation.
Seedling mortality was determined 16 days after inocu­
Dead seedlings were assumed to have died from transplant
shock and were hot considered in the disease evaluation.
The unreplicated trial was terminated 89 days after inocula­
tion.
Replications I, II, and III were harvested 110, 112, and 123
days after inoculation, respectively.
At time of harvest, seedlings
were scored for disease severity and a random sample of 50 root pieces
from each replication were surface-sterilized and placed on HRDA
to determine the percentage of plants infected with F. splani.
(66)
In
replications II and III ah expanded scale was used to assign disease
severity scores.
Scores of 3-, 3, and 3+ were assigned numeric values
of 2.5, 3.0, and 3.5, respectively.
This expanded scale was necessary
to differentiate among the broad range of symptoms withih .the 3 cate^
:gory.
The range,, variance, and mean disease severity scores were cal­
culated for each accession.
'
'
44
RESULTS AND DISCUSSION
Experiment__I:
Greenhouse evaluation of four techniques of inoculating
sainfoin seedlings with F . s o l a n i .
The ;inoculation
techniques differentially affected the per­
centage of sainfoin plants infected with F_. soIani,. the severity of
disease symptoms, and seedling mortality
(Table 3-1).
An effective
inoculation technique should cause a high percentage of infection,
produce severe disease symptoms, and result in a low seedling mortal­
ity from physical injury.
The aerial spray on 'two-week-old and six-
week-old seedlings, and the crown injection, on two-week-old seedlings
failed to meet these criteria
(Figures 3-2 and 3-3).
The crown injec­
tion and toothpick techniques on six-week-old seedlings produced mod­
erate disease symptoms, but infected only 58 and 48 percent of the
seedlings, respectively.
Inoculation of two-week-old and six-week-old sainfoin seed­
lings with the root-cut-soak technique resulted, in 77 percent, of the
.
•
plants becoming infected with F_. solani
(Table 3-1) .
H o wever, the six-
week-old seedlings had the most severe disease symptoms and the lowest ..
seedling mortality.
For these reasons, the root-cut-sqak technique
applied to six-week-old seedlings were selected as the best mass
screening procedure.
The mean disease severity score arid the percentage of plants
infected with F . solani Were highly correlated
(r .= .86 **).
This
45.
Table 3-1.
The effect of four inoculation techniques on the disease
severity sc o r e , the percentage of plants infected with
IT. solani/ and mortality of sainfoin seedlings inoculated
in the greenhouse.
Age of seedlings
&
inoculation
technique
Disease severity
score I/
Plants infected
with F. solani .
%
Seedling
mortality
%
Inoculation with F. solani
2 weeks:
Root-cut-soak
Crown injection
Aerial spray
,
-
'
. 2.36 b
. 1.92 cd
1.43 de
/.
78.3 a
19.5 c
10.0 e
63
8
0
77.5 a
57.5 b
47.5
20.0 c
. io ..
0
. . 0.
0
6 weeks:
Root-cut-soak .
Crown injection.
Toothpick
Aerial spray
. 3.13 a
2.58 b
2.73
1.28 ef
Inoculation with sterile H,0
2 weeks:
Root-cutT soak
. Crown injection
Aerial spray
\
1.67 cd
.1.42 de
1.08 f
. 6.0 f
12.5 d
5.0 f
50
5
3
.
6 weeks:
Root-cut-soak
Crown injection
Toothpick ■
Aerial spray
1.73 Cd
1.38 de
1.56
1.18 ef
10.0
' .2.5
5.0
0.0
e
g
.
h
0
0
0
0
^Disease severity scored as follows:
I = no spread of discol­
oration; 2 - spread, of discoloration less than I cm; 3 = spread
of discoloration more than I cm; 4 = extensive vascular
gnecrosis; and .5. = dead p l a n t .
gBased on isolations made on .PCNBA (57).
Means with a column not followed by the same letter differ at
the .05 level of probability.
IOO RCS-Roct-Cut-Sook
C l* Crown Irtfoctfon
T P * Toothpick
A S * Amria ! S p ra y
0 0
—
With F Sofani
With s te rile H^O
2 WEEKS
Figure 3-2.
6 WEEKS
The effect of four inoculation techniques on the percentage of plants in­
fected with F. solani when inoculated two weeks and six weeks after emer­
gence.
Isolations were made on PCNBA (57).
DISEASE SEVERITY SCORE
RCS
3.0
RC SsR oot- C u t- Soak
Ct=C row n Injection
TP = Toothpick
A S mA e r ia I S p ra y
RCS
□
With F S olani
Cl
2.0
m
With sterile
AS
1.0
Figure 3-3.
2 WEEKS
6 WEEKS
The effect of four inoculation techniques on the mean disease severity score
assigned to sainfoin seedlings inoculated with F. solani at two weeks and
six weeks of age.
Disease severity scored as follows:
I = no spread of dis­
coloration; 2 = spread of discoloration less than I cm; 3 = spread of discolor­
ation more than I cm; 4 = extensive vascular necrosis; and 5 = dead plant.
48
strong association indicates that plants could be screened on the sin­
gle index of the mean disease severity score.
The coefficient of variability of the percentage of plants in­
fected with F. solani was. high
(CV - 45.3%).
This index was based on
isolations from a single root piece from each plant.
In future.
studies, this index should be based on multiple root pieces to mini­
mize this source of variability.
Experiment I I :
Field evaluation of four techniques of inoculating
sainfoin seedlings with F_. Solani.
The. inoculation techniques differentially affected both the
mean disease severity score and the percentage of plants infected with
F. solani.
The physical injury resulting from the inoculation tech­
niques was responsible for these differences since inoculation with
both F_. solani and sterile water produced similar disease expression
(Figures 3-4 and 3-5).
This indicates that either the natural inbcu-
Ium was at a high level or that microconidia of -F. solani are ineffec­
tive as inoculum under field conditions.
The root-cut-sdak technique produced the most severe disease
symptoms and caused the highest percentage of infection
Jand 3-5).
(Figures 3-4
This technique, however, was so severe that 75 percent of
the seedlings inoculated died during the year-long trial period
3-2).
Seedling mortality may have resulted, from physical injury
(Table
:
50
o
RCS
F 40
RCS - Root'- Cut'- Soak
C l- Crown Injection
TPs Toothpick
A S -A e ria / Spray
Lu
O
Ul
i
I
With F. sotani
20
VD
U
I
QC
Ul
CL
AS
With sterile H2 O
INOCULATION TECHNIQUE
Figure 3-4.
The effect of four inoculation techniques on the percentage of plants in­
fected with F. solani when inoculated 83 days after emergence at the Field
Research Laboratory, Bozeman, Montana.
Isolations were made on PCNBA (57).
LU
o:
o
o
cn
3 .0
>
K
OC
LU
>
LU
CO
LU
CO
2.0
<
LU
CO
Q
1.0
INOCULATION TECHNIQUE
Figure 3-5.
The effect of four inoculation techniques on the mean disease severity score
assigned to sainfoin seedlings inoculated with E\ solani at the Field Re­
search Center, Bozeman, Montana.
Disease severity scored as follows: I = no
spread of discoloration; 2 = spread of discoloration less than I cm; 3 = spread
of discoloration more than I cm; 4 = extensive vascular necrosis; and 5 =
dead plant.
51
Table 3-2.
The effect of four inoculation techniques on the disease
severity score, the percentage of plants infected with
F. solani, and mortality of sainfoin seedlings inoculated
83 days after emergence at the Field Research Laboratory,
Bozeman, Montana.
Inoculation
technique
Disease severity
score L '
Plants infected
with. F. solani 2/
;
,
.
.Seedling
Mortality
%
Inoculation with F . solani
Root-cut-soak
3.40 a
. 39.5 a
Grown injection . 2.37 b
. 9.1 b
Toothpick
2.33 b
14.6 b
Aerial spray
2.21 b
10.6 b
2
75
..
79
: .26
"
17
Inoculation with sterile H ?0
Root-cut-soak
3.31 a
35.0 a
. Crown injection
2.21 b
8.8 b
Toothpick
2.20 b
' .• 11.3 b
Aerial spray
2.36 b:
15 .
• 20
8.9 b .
11
Disease severity scored as follows:
I = no spread of discol­
oration; 2 = spread; of discoloration less than I cm; 3 =
spread of discoloration more than I cm; .4- = extensive vascular
necrosis; and 5 = dead plant.
2
' ■ ' ■ ■ ■
Based on isolations made on PGNBA
■■■
•
-
(57).
3
Means within a column not followed by the same, letter differ
■ at the .05 level of probability.
52
rather than from disease.
This technique has potential for mass
screening but must be refined to reduce seedling mortality.
The mean disease severity score and the percentage of plants
infected with JF. solani were highly correlated
(r = .98 .**).
Thus,
mass screening in the. field could be based on the single index of dis­
ease severity without direct isolation from every plant.
The percentage of plants infected with EV solani was much lower
in the field than in the greenhouse.
plants at time o f inoculation.
This could, be due to the age of
Plants in the field were 41 days older
than those in the greenhouse at time of inoculation.
Further inves­
tigations are needed to refine field inoculation techniques before
screening is initiated.
.
!
.
.
Experiment I I I :. An evaluation of the repeatibility and resolution of
the root-cut-soak technique of inoculating replicated
genotypes of sainfoin with IV solani.
An effective inoculation technique applied to cuttings of the
same: clone should produce disease symptoms of a constant severity.
■
■
.
■
■ '
' ■
This would produce disease severity scores with a narrow range and a
low variance which permits detection of genotypic differences among
clones.' Except for three clones, the range was within plus or minus
one scoring unit of the mean
ranged from .20 to 1.00.
(Table 3-3).
The homogeneous variances
T h u s , the disease severity scores assigned
to cuttings of the same clone were repeatable.
Both the range and
53
Table 3-3.
The response of the vegetative cuttings of 10 sainfoin
clones inoculated with P.. solani using the root-cut-soak
■ technique.
1
, Cuttings
evaluated
Clone
Mean .
SC 8
5
2.60 a —
DR 5 .
3
2.67 ab
SC 10
3
3.00 abc
DR 2
;
.
'• 3
■' /
Variance
.. ■
Range.
-30
2 - 3
.33
2 - 3
1.00 .
. 2 - 4
3.33 abed
.36
' 3 - 4
3 - 4
SC 11 .
5
3.60 abed.
.30
SC 3
4
3.75 bed
.92
DR 5
5
3.80 Cd
.70
SC 7 '
5
3.80 Cd
.20
SC 4
7.
4.00 cd
. ' .67
SC -9 •
8
4.25 cd
.50
;
3 - 5
■3.-5
3. - 4:
' 3 - 5
Disease severity scored as follows:
I = no spread of discol­
oration; 2 — spread of discoloration less than I cm; 3 =
spread of discoloration more than I cm; 4 = extensive vascu­
lar necrosis; and 5 = dead plant
'
I'
Means not followed by the same letter differ at the .05 level
of probability.
54
variance might be reduced if the visual scoring system (Figure 3-1)
were expanded from five to seven categories.
Such an expansion would
require a longer trial period to increase the severity of disease .
symptoms and a more rigid scoring index.
'
•
;
The clones had different mean disease severity scores.
.
Only
those differences of one scoring unit or greater were significant
(Table 3-3).
This limited resolution was due to small sample sizes.
Resolution might be improved by increasing the number of scoring cate­
gories, increasing the length of the trial p e r i o d , and increasing the
number of plants evaluated.
The. root-cut-soak technique was effective.
F_. solani was iso­
lated from 95 percent of the cuttings and obtained an evaluation of
each clone's disease susceptibility.
Current breeding material and
other sources of germplasm should be evaluated for potential sources
of disease resistance using the root-cut-soak inoculation technique.
Experiment I V ;
An evaluation of the disease resistance of the openpollinated progeny of 31 clones of sainfoin. ■
Disease severity scores differed significantly among entries.
Scores ranged from 2.82 to 4.22
tion .approximated a normal curve
(Appendix-Table 2) and their distribu­
(Figure 3-r6) .
This indicates that
genetic differences do exist among t h e .entires in their resistance to
2.7
2.9 3.1
3.3 3.5 3.7 3.9 4.1 4.3 4.5 4.7
MEAN DISEASE SEVERITY SCORE
Figure 3-6.
The response of the open-pollinated progeny of 39 entries to inoculation with
F . solani as measured by the mean disease severity score of the entries. Dis­
ease severity scored as follows: I = no spread of discoloration; 2 = spread of
discoloration less than I cm; 3 = spread of discoloration more than I cm; 4 =
extensive vascular necrosis; and 5 = dead plant.
56
root and crown rot caused by F. solani.
Selection within this popula­
tion should increase disease resistance.
Population I, which had undergone 12 years of natural selec­
tion, was not significantly better than its parental v ariety, Eski
(Appendix Table 2).
Population II and many of the clones which had
been selected for longevity in the field were very disease susceptible.
Natural selection appears to be ineffective, in developing resistance
to the inoculated pathogen.
It is possible, that naturally selected
populations would be attacked by
solani less under field conditions
due to the development of morphological barriers which prevent infec­
tion.
The development of true physiological resistance probably could
be accomplished by screening with the root-cut-soak inoculation, tech­
nique.
Melrose was the most disease resistant cultivar evaluated and
may have some resistance to F. solani
(Figure 3rd).
It is possible
that, selection could b e practiced within this and other existing culti­
var s as demonstrated by the wide range and large variance in their
disease severity score
(Appendix Table 2).
Twenty-four days after inoculation, 45 percent of the seed- ,. .
lings had died because of severe transplant shock.
In future studies,
mortality could be reduced by inoculating older seedlings, removing
the foliage at 8 cm instead of 5 cm above the crown, and by pasturizing the soil benches, prior to transplanting.
■Experiment V : ' Screening .296 accessions of ■the world .collection of:
Onobrychis for, sources of resistance to crown and
root rot caused by
solani. ' . .
The 11 species of Ohobrychis and the accessions within the
species of tanaitica, transcaucasica, arenaria, sibirica,, and y IcIifolia differed in disease susceptibility
(Table 3-4).
Mean disease
severity scores of the accessions ranged from 2.79 to '4.62 and their
distribution d loseIy approximated a normal curve
(Figure .3-7). . Most,
accessions■had d i s e a s e .severity scores.with a wide range and a large
variance
(Appendix Table 3)
.Thus, one could select for disease re­
sistance among species, among accessions within a species, or among
plants within an accession... Most rapid progress could be obtained by
selecting the best plants Within the best accessions of the most dis­
ease. resistant species..
The.mean disease severity scores of the check cultivars, Mel­
rose and.Remont were similar to the scores observed in Experiment IV.'
E s k i 's mean disease severity score was ..48 scoring units lower in
this trial than in the previous trial
(Table 3-5).
This disparity may
have been due to the small, sample used in the earlier experiment or
because different seed lots of Eski were used in the trials.
In fu­
ture studies, seed of check cultivars should be .taken from, the same
seed lot.
.Vv' '" 'v,
. -v-
'
.
-
'
:: V.'''VV-;v -
The mean disease severity, scores of the 113 accessions of
Ohobrychis evaluated in the unreplicated trial ranged from 2.88 to
58
5.00
(Appendix Table 4).
Values presented were based on an evaluation
of eight or fewer plants,. and should be used only.as an initial evalu­
ation.
•
Table 3-4;
The response of 11 species, of Onobrychis to inoculation
with F . solani as measured by the mean disease severity .
score.
Species
Mean disease
severity score i/
0. tanaitica
3.31 a
0. vulgaris
Accessions
Evaluated
Plants
evaluated
14
233
3.32 ab
8
124
■
O. iberica
3.38 abc
2
■ 30.
.
0. inermis
3.47 abc
14
273
3/
Check Cultivars —
3.49 abc
3
60
70
1187
0. transcaucasica
0. biebersteinii
0. antasiatica
•
,
' 3.51 be
3.61 bed .
3
2
; 3.61 bed
0. arenaria
3.64 cd
38
0, altissima ..
3.64 cd
-4
0. sibirica
3.65 cd
0. viciifolia
3.80 d
I
.
50
'
,
'
40
;
704
66
2
■ 24
36 .
383
■
■
Disease severity scored on spread of discoloration from point
of inoculation as follows: I = no spread; 2 = 0 . 1 - 0 . 9 cm; .
2.5 = 1 . 0 - 1 . 9 cm; 3 = 2.0 - 2.9 cm; 3.5 = 3.0 - 3.9 cm; 4 =
extensive vascular necrosis; and 5 = dead plant.
^Means not followed by the same letters differ at the .05
level of probability.
3
Check cultivars■consisted of E s k i , Remont, and Melrose.
MEAN DISEASE SEVERITY SCORE
Figure 3-7.
The response of 181 accessions of the world collection of Onobrychis to inocu­
lation with F. solani as measured by the mean disease severity score of each
accession.
Disease severity scored on spread of discoloration from point of
inoculation as follows : I = no spread; 2 = 0 . 1 - 0 . 9 cm; 2.5 = 1.0 - 1.9 cm; 3 =
2 . 0 - 2.9 cm; 3 . 5 = 3 . 0 - 3 . 9 cm; 4 = extensive vascular necrosis; and 5 = dead
plant.
Table 3-5.
The response of three cultivars of sainfoin to inoculation with E\ solani in
two trials as measured by the mean, variance and range of the disease severity
scores.
Experiment IV
Number
Cuitivar'
Melrose
Eski
Remont
I
■ ■of
plants
Experiment V
I/
Disease Severity Score —
Mean
Variance
Range
. Number
of
plants
2/
Disease severity score —
Mean
24
3.29
.65
2 - 5
18
3.31
■ 11
3.82
•.36
3 - 5
22
3.34
20
■ 3.75
1.04
.2-5
20
3.83
Variance
.80
'
Range,
.
2.5-5
.34
2.5-5
.74
2.5-5
. ■
‘"
Disease severity scored as follows:
I = no spread of discoloration; 2 = spread
of discoloration of less than I cm; 3 = spread of discoloration of more than I cm;
4 = extensive vascular necrosis; and 5 = dead plant.
Disease severity scored on spread of. discoloration from point of inoculation as
follows:
I = no spread; 2 = 0.1 - 0.9 cm; .2.5 = 1.0’- 1 . 9 cm; 3 = 2.0 - 2.9 cm;
3.5 = 3.0 - 3.9 cm; and 4 = extensive vascular necrosis; and 5 = dead plant.
' • ■ '. 61
■
CONCLUSIONS
The root-cut-soak inoculation technique is effective, for
screening sainfoin plants for resistance to the root and crown rot
caused by F. splahi.'. Current breeding material, released, cultivars,
and introduced accessions differ in their disease resistance but.none
of these sources are immune to the disease.
Most rapid.’progress
could be obtained by selecting the best plants from the most disease
resistant sources.
These selections should be recombined arid.their
. progeny screened in repeated cycles of selection.
Improved popula-
. ■■■'
■
/ ■ .■ '
.■■■■'■
;'V.'
tions should be evaluated under field conditions to insure that selec
tioh in the greenhouse is improving longevity in the field. . Care
should, be taken to insure agronomic qualities are maintained during
the selection procedure.
Studies should be. initiated to characterize the mechaniSm(s)
of d i s e a s e ’resistance, and its inheritance.
SUMMARY AND CONCLUSIONS
'
Root and crown rOt. limits the production of sainfoin..
This
disease reduces the longevity, of the stand arid severely reduces forage
yield.
The Objectives of this study were to:
I) determine the causal;
o r g a n i s m (s) of root and crown rot; 2) further, characterize the hostparasite relationships; and 3) develop and initiate screening proced­
ures to detect potential sources of disease resistances
.F . oxysporum infected sainfoin but caused only limited symptom
,development.
Several 'formae speciales' of F. solani infected sairifOin
and caused disease symptoms very similar to those observed urider natu­
ral conditions.
This explains the presence of this disease in areas
where sainfoin had never been cultivated.
Due. to the longevity of
chlamydospores in the soil, it is unlikely that crop rotations with
.
legumes would effectively control this disease.
MicrocOnidia and macroconidia of F . solani are capable of infectirig and causing disease symptoms in sainfoin.
The four isolates
of E\ solani evaluated in this study are .equally pathogenic.
The results, indicate that F. solani is probably not seedborne.
seed.
F_. oxysporum and Alternaria spp. were found ori the untreated
Removing the seed-pod arid surface-sterilization eliminated ;
these fungi from most of the seed lots.
reduce emergence in autoclaved soil.
Alternaria spp. were shown to
-
63
• Soil fumigation with chloropicrin, soil treatments with the
fungicide Benl a t e , and seed treatment with Benlate failed to reduce
disease development.
•Development of cultivars of sainfoin which are resistant or
tolerant to root and crown rot caused by F. solani offer the greatest
potential for control of this disease.
T h e .root-cut-soak inoculation
technique w a s ■used to successfully screen plants in the greenhouse.
This technique had good repeatibility and is capable of detecting
differences in disease resistance.
H owever, this technique needs to
be. refined before it is used for screening in. the field.
Differences were found in current breeding material, the
world collection.of Onobrychis, and released cultivars.
No source of
resistance was immune to the disease, but several sources appeared to
have some resistance. . Selection and recombination of these plants .
should result in increased disease resistance.
Future studies should concentrate on I) defining the mechanism
and inheritance, of disease resistance; 2) refining selection proced­
ures; and 3) developing highly disease resistant lines of sainfoin.
■LITERATURE' CITED
LITERATURE'CITED
1.
Armstrong, G. M . , and J. K. Armstrong.
1965.
Further studies of
three forms of Fusarium oxysporum causing wilt of alfalfa. .
Pit. D i s . Reptr., 49:412-416..
,
2.
B u r k e , D. W.. 1965.
Plant spacing and Fusarium root rot of b e a n s .
Phytopathology,. 55:757-759.
3.
Burton, J. C., and R, L. Curley... 1968.
Modulation and nitrogen
fixation in sainfoin (Onobrychis sativa Lam.) as influenced by .
strains of rhizobia.
p. 3-5.
In C. S. Cooper and A. E .
Carleton (ed.) Sainfoin symposium.
Mont. Agr.. E x p . S t a . Bull. .
627.
4.
Carleton, A. E., C . S . Cooper, C. W. Roath, and J. L. Krall.
1968.
Evaluation of sainfoin for irrigated hay in Montana,
p. 44-48.
Ih C. S- Cooper and A. Ev Carleton (ed.) Sainfoin
symposium.
Mont. A g r . Exp. S t a . Bull. 627.
5.
_________ , _________ , and L. E. Wiesrier. 1968.
Effect of seed pod
and temperature on speed of germination and seedling elonga- .
tion of sainfoiri (Onobrychis yiciaefolia Scop;). Ag r o n . J.
60:81-84.
6.
________ R. H . Delaney, A. L. Dubbs, and R. F.. Esli c k .
1968.
Growth and forage quality comparisons Of sainfoin
(Onobrychis yiciaefolia Scop.) and alfalfa (Medicago sativa
L.) . . A g r o n . J. 60.:630-632,
7.
_________ , and L. E. W i e sner. 1968.
Production of sainfoiri seed.
p. 71-73.
Iri-C. S . Cooper and A; E. Carleton (ed.) Sainfoinsymposium.
M o n t . A g r . Exp. S t a . Bull. 627.
8.
Chi,. C. C ., and E . W. Hansori. 1961.' Nutrition in relation to
the development of wilts and root rot incited by Fusarium in
red clover.
Phytopathology, 51:704-711.
9.
_________, W. R. Childers, and E. W. Hanson.
1964.
Penetration
and subsequent development of t h r e e •Fusarium species in alfal­
fa and red clover.
Phytopathology, 54:434-437.
10.
'
. 1968.
Nutrition stress in relation to Fusarium wilt
and root rot of alfalfa.
Pit. D i s . Reptr., 52:939-943.
66
11.
Christon, T., and W. C . Snyder.
1962.
Penetration and the hostparasite relationships of Fusarium solani f . phaseoli in the
bean plant.
Phytopathology, 52:219-226.
12.
Cooper, C. S.
1973,
Sainfoin-birdsfoot trefoil mixtures for
pasture, hay-pasture, and hay-stockpile management regimes.
Agron. J., 65:752-754.
13.
C o r mack, M. W.
1937.
Fusarium s p p , as root parasites of alfalfa
and sweet clover in Alberta. Can. J. Res., 15:493-510.
14.
_____ ___ , R. W, Peake, and R. K. Downey.
Studies on methods a n d .
materials for testing alfalfa for resistance to bacterial wilt.
C a n . J . A g r . R e s . , .37:1-11.
15.
Cressman, R. M.
1967.
Internal breakdown and persistence of red
clover.
Crop Sci., 7:357-361.
16.
Ditterline, R. L.
1973.
Yield and yield components of sainfoin
(.Onobrychis viciaefolia Scop.) seed and evaluation of its use
as a protein supplement.
Ph.D. Thesis.
Montana State Univer.sity.
17;
Elliot, E. S., R. E. Baldwin, and R-.' B. Carroll. : 1969.
Root
rots of alfalfa and red clover.
W. V a . Agr. Exp. S t a . Bull.
385-T.
18.
Eslick, 'R. F. . 1968.
Sainfoin— Its possible role as a forage
legume in the West.
p. 1-2.
In C. S. Cooper and A. E .
Carleton (ed.) Sainfoin symposium.
Mont. Agr. Exp. Sta. Bulli
627.
19.
_________ ,. A. E . Carleton, and G. P. Hartman.
for Eski sainfoin.
Crop Sci., 7:402-403.
,20.
Ford, R. E. ■ 1959.
Fusarium blight of birdsfbot trefoil..
Phytopathology, 49:481-485.
21.
Frosheiser, F. I,, and D. K. Barnes.
1973.
Field and greenhouse .
selection for Phytophthora root rot.
Crop Sci., 13:735-738.
22.
Gill, D. L.
1968.
Mimosa wilt Fusarium carried in Seed.
Dis. Reptr., 52:949-951.
1967.
Registration
Pit.
67
23.
Graham, J. H., and R. Ci Newton.
1959.
Relationship between
root feeding insects and incidence of crown and root rot in
red clover.
Pit. D i s . Reptr., 43:1114-1116.
24.
Griffin, G. D.
1971.
Meloidogyne h a p l a .
25.
Griffiths, D. A., and W. C. L i m . 1965.
"Overgrowth" in Malayan
. . crop plants following infection by Fusarium solani and
F. decemcellulare. Pit. D i s . Reptr., 49:979-980.
26.
Hadwiger, L.
1975.
Personal communications.
University. . Pullman, Washington.
27.
Hanna, M. R., and S. Smoliak. 1968.
Sainfoin yield evaluations •
in Canada,
p. 38-43. In C. S . Cooper and A. E. Carleton (ed.)
Sainfoin symposium.
Mont. A g r . Exp. S t a . Bull. 627.
28.
_________, _________, and B. P. G o plin. 1972.
Canada.
Canada Dept, of A g r . P u b l . 1470.
29.
Harvey, W. R.
1960.
subclass numbers.
D.C.
30.
Hawn, E. J., and M. W. Cormack,.
Phytopathology, 42:510-511.
31.
Holden, J. L.
1968.
A producer's evaluation of sainfoin.
p. 53-54.
Iri C. S. Cooper and A. E. Carleton (ed.) Sainfoin
symposium.
Mont. A g r . Exp. S t a . Bull. 627,
32;
.33.
34.
Susceptibility of common sainfoin to
Pit. D i s . Reptr., 55:1069-1072.
Washington State
Saitifoin for western
Least squares analysis of data with unequal,
A.R.S. Bull. 20-8 U.S.D.A., Washington,
1952.
Crown bud rot of alfalfa.
Hughes, S. J.. 1945.
Studies on diseases of sainfoin (Onobrychis
saliva) . I.
Ring-spot caused by Pleospora herbarum (Pers.).
Rab e n h .. Trans. British M y C o l . Soc., 28:86-90.
■
______ . 1949.
Studies of some diseases of sainfoin.
II.
. life history of Ramularia onobrychidis Allescher. Trans.
British Mycol^ Soc., 32:34-59.
The
'
________ . 1949.
Studies on some diseases of sainfoin (Onobrychis
sutiva),.
IV.
Leaf-spot caused by Septoria orobinia Saac♦
Trans. British M y c o l . S o c ., 32:60-62.
68
35.
Isaac, I.
1946.
Verticillivim wilt of sainfoin.
B i o l . , 33:28-34.
Ann. A p p l .
36.
Jensen, E. H., C. R. Tore ll, A. L. Cosperance, and C. E. Speth.
1968.
Evaluation of sainfoin and alfalfa with beef cattle,
p. 97-99.
In C . S . Cooper and A. E. Carleton (ed.) Sainfoin
symposium.
Mont. A g r . Exp. S t a . Bull. 627.
37.
Jensen, H. J.
1967.
Do saprozoic nematodes have a significant
role in epidemiology of plant diseases? Pit. Dis Reptr;,
' 51:98-101.
38. . Johnson, H. W.
1967., Potential of the Rhizobium-Fusafium inter­
actions on the incidence of alfalfa root rot.
Ph.D. Thesis.
Uni v e r . R. I.
39.
K e h r , W. R., F i I . Frosheiser, R. D. Wilcoxson, and D v K. Barnes.
1972.
Breeding of disease resistance.
In Al f a l f a , Science
and Technology. C. H. Hanson (ed.),. pp. 335-354.
40.
Kilpatrick, R. A., E . W. Hanson, and J. G. Dickson.
1954.
Rela­
tive pathogenicity of fungi associated with rots of.red clover
in Wisconsin.
Phytopathology, 44:292-297.
41.
_____
., _________ and
,■ . 1954.
Root and crown rots of
red clover in Wisconsin and the relative prevalence of associ­
ated fungi.
Phytopathology, 44:252-259.
■42. . K h i p e , W. J . . 1972.
Reproduction and genetics of sainfoin
(Onobrychis viciaefolia Scop.) as they relate to its breeding.
Ph.D. Thesis.
Montana State University.
43.
K r a l l , J. L., C. S. Cooper, C. W. Crowell, and A. J . Jarvi.
1971.
Evaluation of sainfoin for irrigated pasture.
Mont.
A g r . Exp. Sta. Bull. 658.
44.
Kreitlow, K . .W.
1963.
Infecting seven-day^old alfalfa seedlings,
with wilt bacteria through wounded cotyledons.
Phytopathology,
53:800-803.
45.
Death, K. T., F. L. Lukenzie, H. W. Crittendon, E . S. Elliot,
P. M. H a l isky, F. L. Howard, and S. A. Ostazeski. 1971.
The
Fusarium root - rot complex of selected forage legumes in the
northeast.
Penn. A g r . Exp. Sta. Bull. 777, 64 pp.
69
46.
Leb e a u , J. B., and J. G. Dickson.
1955.
Physiology and nature
of disease, development in winter crown rot of alfalfa.
Phytopathology, 45:667-672.
47.
Lockwood, J. L.
1962.
A seedling test for evaluating resistance
of pea to Fusarium root rot.
Phytopathology, 52:557-559.
48. , M a t h r e , D. E.
1968.
Diseases of sainfoin,
p^ '66-67.
Copper and A. E. Carleton (ed.) Sainfoin symposium.
A g r . Exp. S t a . B u l l . 627.
In C. S .
Mont.
49. .M a t u o , T . ,. and W. C. Snyder.
1972.
Host virulence and the
■ .Hypomycex stage of Fusarium soIani f .. sp. p i s i . Phytopathology.
62:731-735. .
50.
_____ __ , and _____
. 1973.
Use of morphology and mating
populations in the identification of formae speciales in
Fusarium solani. Phytopathology, 63:562-565.
.
51.
McGuire, J. M., H. H. Walters, and D. A. Slack.
1958.
The rela­
tionship of root - rot nematodes to the development of
Fusarium wilt in alfalfa.
(Abst). Phytopathology, 48:344.
52.
Melton, B . A., 1973.
Evaluation of sainfoin and cicer m i Ikvetch .
in New Mexico.
New. Mexico A g r . Exp. Sta-. Rep. 255. .
53.
Mew, T. W.
1968.
Root rot of soybeans (Glycine m a x ) in relation
to antagonism of Rhizobium japonicum and Fusarium oxysporum.
M.S. Thesis.
Uni v e r . R. I.
Cited in "The Fusarium.root - rot
complex of selected forage legumes in the northeast." 1971.
Penn. A g r . Exp. S t a . Bull. 777.
54.
M u r r a y , G. A.
1968.
Plant growth and nodulation of alfalfa and
sainfoin plants -in relation to manganese concentrations,
p. 6-7.
In C. -S. Cooper and A., E. Carleton (ed.) Sainfoin
symposium.
Mont. A g r . E x p . S ta. Bull. 627.
55.
_________ , and E . A. Slinkard.
1968.
Forage and seed, production
potential o f sainfoin in northern Idaho,
p. 74-76.
Iri C. S .
Cooper and A. E. Carleton (ed.) Sainfoin symposium.
Mont.
A g r . Exp. S t a . Bull. 627.
56.
Nash, S. M., T. Christen, and W. C. Snyder.
1961.
Existence of
Fusarium solani f . phaseoli as chlamydospores fn soil.
Phyto­
pathology, 51:308-312.
70
57.
_____
,. and W. C. Snyder.
1962.
Quantitative estimates by.
plate counts of propagui.es of the bean root rot Fusarium in
. field soil.
Phytopathology, 52:567-572.
58.
______
, and _________ . 1964.
Dissemination of the root rot
■Fusarium with bean seed.
Phytopathology, 54:880.
59.
O ’Rourke, C. J., and R. L. Miller.
1966.
Root rot and root
microflora of alfalfa as affected by potassium nutrition.,
frequency o f cutting and leaf infection.
Phytopathology,
56:1040-1046.
60.
Piper, C. V.
1914.
Forage Plants and Their Culture.
MacMillian Co. ,. New York.
pp. 559-562.
61.
Reinking, 0. A.
1950.
Fusarium strains causing; pea and bean
. root rot.
Phytopathology, 40:664-687.
62. . Roa t h , C. W.
p. 26-28.
symposium.
63.
The
1968.
Sainfoin for dryland hay in western Montana.
In C. S. Cooper and A. E. Carleton (ed.) Sainfoin
Mont. A g r . Exp. S t a . Bull. 627.
Schmitthenner, A. F., and L. E. Williams. .1957.
injectioninfestation technique for studying root rot pathogens.
Phytopathology, 47:30.
64.
'
_____ _, and J. W. H i l t y . 1962.
A method for studying postemergence, seedling root rot.
Phytopathology, 52:177-179.
65.
'.
■
1964.
Prevalence and virulence of Phytpphora,'
Apanomycetes, P y t hium, Rhizoctonia, and Fusarium isolated from
diseased alfalfa seedlings.
Phytopathology, 54:1012-1018.
66.
Sears, R. G.
1974.
The crown - root rot complex in sainfoin.
(Onpbrychis viciaefolia Scop.).
M.S. Thesis.
Montana State
Univ e r s i t y . .
67.
_ _ _ _ _ _ _ _ R-' L: Ditterline, and D. E. Mathre. 1975.
Crown and
root rotting organisms affecting sainfoin (Onobfychis
■ viciaefolia) in Montana.
Plant Dis:. Reptr. , 59:423-426.
68..
Sh a i n , S, S.
1959.
(Moscow State Publishing House of Agricultu­
ral Literature), Description of.Sainfoin in Agitechniques of
Perennial Forages,.
51-54.
T r a n s . R. P. Knowles.
Cited in
"A compilation of. abstracts of sainfoin literature." 1968.
A. E. Carleton and C. S. Cooper (Processed).
71
69.
Sims, J. R. , N. K. M i u m 7 and A. E. ..Carleton. 1968.
Evidence of
ineffective. Rhizobia and its relation to the nitrogen nutrition ■
of sainfoin (Onobrychis viciaefolia) . p. 8-11.
In C. S.
. Cooper and A. E. Carleton (ed.). Sainfoin symposium.
Mont.
A g r . .Exp. Sta. Bull. 627.
70.
Stat e n , G.,. and P. J. Leyendecker. 1949.
A root disease of
alfalfa caused by Fusarium solani. Pit. D i s . Re p t r ., 33:254255.. .
71.
Steel, G. D., and
H. Torrie. 1960,
Principles and procedures
of statistics.. McGraw-Hill Book C o . , New York,
p^ 99-249.
72:
Toussoyn, T. A., and P. E. Nelson. , 1968.
A pictoral guide to
the identification of Fusarium species according to the tax­
onomic system of Snyder and Hansen,
The P e n n . State U n i v .
Press.
University Park and London.
73.
Yank, S. M., and D. J. Hagerdorm.
1965.
Pathogenicity studies
■ with Fusarium solani from beans and peas.
Phytopathology,
55:1085.
74.
Zare m b a , V. P.
1961.
Osobennosti vzaimootnos-Shenii Kulbenkovykh
bakterii s mikrofloroi semyan espartseta (Characteristics of
the interrelationships of the root nodule bacteria with micro­
flora of the sainfoin family.
Trudy Inst. MikfobiOl.
Akad.
N a u k . SSSR., 11:188-197),
Cited in "A compilation of ab­
stracts of sainfoin literature:".. 1968.
A. E . Carleton and,
. C. S. Cooper (Processed).
APPENDIX
•J-"•
X
\.
x
Appendix Table I.
I.
The composition of the media used for f u n g a l ■isola­
tions and increase, and the composition of nutrient
solutions used in this study.
Fungal Media:
A. Acidified. Potato Dextros Agar
(HPDA):
Difco potato dextros agar
39.0 g
Distilled water
Lactic acid
1000.P ml
2.0 ml
(25%) *
A general purpose growth media for many fungi.
B. Pentachloronitrobenzene Agar
(PCNBA)(57):
20,0 g
Agar
5.0 g
Difco p e p t o n e ■
. 1.0 g
K H 2P O 4
0.5 g
M g S O 4-VH2O
1000.0 ml
Distilled water
Pentachloronitrobenzene
(PCNB)., 75% W.P. *
■
300.0 mg
Streptomycin *
Preparation:
1.0 g '
streptomycin and PCNB are added after
autoclaving and medium cooled to 42 - 45 C .
C.
Modified Eckert's Medium:
Glucose
18.0 g .
Yeast extract
3.P. g
Difco peptone
5.0 g
K H 2P O 4
1.7 g
74
C. Modified Eckert's Medium:
(continued.) .
• .K2HPO4 ■
; 1.4 g
: ' o:,;,'
. M g S p 4-VH2O'
Distilled water .
1000.0 ml
A general.purpose growth medium for fungi •
D.
Fresh Potato Dextrose Agar
(FPDA)(72):
Fresh white potatoes
■
.
■
'
Distilled water Agar.
.•
. .. Dextrose
.
' .'
20.0 g
20.0 g
■
Prep a r ation:
. 250.0 g
•• ....
1000.0 ml
Peeled potatoes'steamed for I hour in ah'
; •.
autoclave with the exhaust o p e n , filtered through 2 layers,
of cheesecloth, and agar and dextrose added to filtrate. ..
' Solution is then tubed,, autoclaved, and slanted for. use.
A medium for fungal identification.
E.
.SnydSr and Nash's Speculation Media: (56)
. Peptone,
. Dextrose '
K H 2P O 4
.
2.5 g
'5 ip g
7.5 g ,
.MgSO *VH2Q
Distilled water
Adjust to pH 5.5 - 6 . 5 after/autoclaving
.. ■
■
A medium which results in abundant sporulation.
1000.0 ml
75
'.N u t r i e n t .Solutions: ■
■A. Normal Nutrient Solution:
,
2.3 g
N
2.3 g
KgO
2.3 g ;
Tap water
18.9 I
Used for general plant.growth.
B. Nitrogen-Free Nutrient Solution:
I.- Salt stock m i x t u r e :
10.0 g
MgSO'TH.O
2.5. g
C a S O 4..*2H2b
2.5 g
C a j(PO4)2
2 . 5
FePO,,
2.5 g
y
.
2. Micronutrient stock solution:
H 3B0 3
2.9 g
M n C l 2M H 2O
1.8 g
ZnSO4*7H20
.2 9
C u S O 4*SH2O
H 2M o O 4-H2O .
C o C l *GH2O
.Distilled water
■. y .1 mg
1000.0 ml
76
■Preparation;
Add 1.5 g of the salt stock mixture to 2.0.
ml of the micronutrient stock, solution.
and I liter, of water..
_
_
_
—
'
Added after autoclaving.
Add 2 g of C a C O 3 :
Mix well and apply to plants.
...
Appendix Table 2.
The response of the progeny of. 39 entries' of sainfoin to inoculation with
F. solani as measured by the percentage of plants i n f e c t e d a n d the mean/
variance, and range of the disease severity score.
Plants
evaluated
Entry
27
20.
17
20
15
19
17
21
24
16
. 1 6
18
20
.
'
11
20
15
16
19
12
10
21
24
24
.. 13
■ . .14
.
95.3
100.0
100.0
:. ioo. p
76.7
91.7
90.3
84.0
93.3
100.0
83.3
100.0
100.0
100.0
' , 76.0
100.0
•
91.7
95.0
100.0
100.0
.96.0
.100.0
100.0
100.0
100.0
:
..
Disease Severity Score 2/
.■'Mean
Variance
Range
2.82
. 1,08
.3.00
'■
.84
'. 3.18
..15
3.20
.38
3.20
.46
3.21
.84
3.24
.69
3.29
.61
3.29 ■
.65
3:31 .
.36
3.31
1.03
3.33
1.06
3.40
1.31 .
3.46 .
I* 67
I; 42
3.50
3.53
.55
.
■ 3.56
1.06
.3.58
.
1.15
. 3.58
.63
.
3,60
.71
3.62
.55
.
3.63
,59 :
3.67
■ .58 3.69
.90
! 3.71
0 .53
■*
1
I
3
2
2
1
2
2
2
3
2
- 5
- 5
- 4
- 5
- 5
- 5
- 5
- 5
- 5
- .5
- 5
in
I
<x
3000-161
3000-112
3000-59
3000-16
3000-23
3000-155
3000-58
3000-18
Melrose
3000-148
3000-116
3000-90
.3000-41
3000-124
SS - 6
3000-102
3000-7
3000-141
3000-20
3000-55
Population. I
3000-30
New Mexico
3000-86
Population II
P l a n t s .infected .
with F . solani —
%
2 -r S
2
2
3
2
2
3
3
3
3
3
3
2
- 5
- 5
- 5 .
- 5
5
- 5
-• 5
- 5
- 5
- 5
- 5 ■
- 5
Appendix Table 2 (continued)
Plants
evaluated
. Entry
Remont
3000-8
3000-66
3000-22
Eski
3000-37.
3000-129
30.00-68
3000-5
3000-133
3000-14
3000-126
3000-89 .
3000-60
.
Plants infected
with F. solani —
%
20
20
17
21
11
19
14
10
. 8
14
12
10
12
9
1
Based on isolation made on HPDA
100.0
■ 100.0
100.0
94.3
100.0
l o oj o
100.0
83.0
100.Q
' 100.0
1.00.0
100.0
66.7
100.0
■
Disease Severity Score 2/
Mean
Variance
Range
3.75 .
3.75 '
3.77
3.81
3.82
3.84
3.86
. 3.90
4.00
4.07
.4.08
4.10
. 4.17 ,
4.22 . .
1.04
.62
.94
.66
.36
.81
.75
>99 .
.86
.53
.81
.77
.88 .
. .94
2 - 5
3 - 5
3 --5
3 :- 5
3 - 5
2-5.
3.-5
3 - 5
3 - 5
3 - 5
3 - 5
3 - 5
2. -! 5
3 - 5
(66).
2
Disease severity scored as follows:
I = no .'spread of discoloration? 2 - spread of
discoloration less than I cm.; 3 = spread of discoloration more than I cm;
4 = extensive vascular necrosis; and 5 = dead plant.
.
Appendix Table 3.
The response of 181 accession of Onobrychis to in-.
oculation with F. solani as measured by the mean,
variance, and range of the disease severity scores.
0. tanaitica
P.I.
'Origin
312963
U.S.S.R.
312958 .
U.S.S.R. .
312962
U.S.S.R:
U.S.S.R.312970
U.S.S.R.
312969
U.S.S.R.
312960
U.S.S.R.
312959
206905 .
Turkey'
312971
U.S.S.R.
312966
■ U.S.S.R.
312949
U.S.S.R.
. U.S.S.R.
312950
312961
U.S.S.R. '■
312955
U.S.S.R.
Numberof plants
evaluated
14 ■
. 16
18
16
15
23
20
' 12
■ 17
17
'
.20
13
18
' 14 ■
Disease Severity Score I/
Mean
Variance
Range
2.79
3.06
3.11 •
3.16
3.23
- 3.24
. 3.25 :
3.29
3.29
' 3.47
3.43
3.54
3.86
3.93
.45
,30
.13
.12
- .57
.36
.14
.34
■ .31
.67
.85
'.64
.67
.80
I — 4‘ '2-4
■2.5-4 .
2.5-4
2.5-5
2.5-5
■
3 - 4
3 - 5
3..- 5:
2.5-5
2 - 5.
'■ 2 ,5-5
2.5-5
'3 - 5
0. vulgaris
178988
170585
171725
170583
167236
170582
171726
110404
■
.
3.12
3.13
3,17
3.29
' 3.40
3.41
3.46
3.50
.26
2.5-4 ,
.
2.5-4
•25 :
.33 2 - 4
.54 ,
.2-5
.38
2.5-5
.34
3 - 5
.44
2.5-5
.36
.3-5
Turkey
Turkey.
Turkey
Turkey
Turkey
Turkey
Turkey .
Armenia. •
13
16 •
12
19
19
16
14
15
U.S.S.R.
U.S.S.R.
19
11
3.29
3.50
.59
.25
2.5-5
3 - 5
■ 22
23
22
3.11
' 3.17
3.23
.31
.60
. .54
2.5-5
2 - 5
2 - 5
.
0. iberica
312934
315085
. 0. inermis
372807
372808
372806
■
Czechoslovakia
Czechoslovakia
Czechoslovakia
80
Origin
•p.I..
0. inermis
312944
312942
312936
372805
312940
312939
312937
325438
312941
312938
312943
'
(continued)
U.S.S.R.
.
U.S.S.R.
U.S.S.R.
Czechoslovakia
U.S.S.R.
U.S.S.R.
U.S.S.R.
U.S.S.R.
U.S.S.R.
U.S.S.R..
.U.S.S.R.
1C
C
D
Disease Severity
■ Mean
Variance
%
Number
of plants
evaluated
CO
O
Appendix Table 3 (continued)
Range
..
17
15
16
23
14
16
19
24 ..
19
. 23
20
.44
.23
■ ,96
.59
.38
.52
.45 ■
1.01
.26 .
.70
.35
. 3.32
3.27
3.44
3.46
3.50
3.53
3.58
3.58
3.74
3.74
3.78
■
•2.5-5
2.5-4
2 - 5
2 - 5
3 - 5 .
3 - 5
. 2.5-5
• 2 - 5
3 - 5 .
3 - 5.
■
3
~
5
Check Varieties
Melrose
Remont
Eski
18
20
22
3.31
3.83
3.34
.80
.14
. .34
.
2.5-5
2.5-5
2.5-5
Qr transcaucasica
Russia
273760
372818 .
Czechoslovakia
U.S.S.R.
312983
U.S.S.R.
■:
313037
U.S.S.R.
312979
U.S.S.R.
312991
U.S.S.R.
313013
U.S.S.R.
312982
313030'
U.S.S.R. .
U.S.S.R.
313019
312978
U.S.S.R.
.. Russia
273762
Russia
273754
312980
Turkey
Czechoslovakia
372819
U.S.S.R.
313020.
U.S.S.R..
313021
. 15 .
22
17
...
22
.
23
13
22
20
18 •
24
.14
.14
• 17
20
. 12
19
14
.
2.87
2.93
. 3.00
3.02
' 3.04
3.12
3.16
3.18
. 3.19
3.19
. 3.21
3.21
3.27
3.28
3.29
.
3.29
3.29
'
.52
.2-5
.36 .
2 - 5
2 - 4
.31
■ 2 - 4
' •34
2-5.
.57
.46
2.5-5
2-5.
.65
.30.
2 - 4 .
.80
1 - 5
,63
2 - 5
.41 • • '2.5-5
2 - 5
.80
2 - 5
.53.
2. - 5
.80 .
2.5-5
• .48, .:•
. 2.5-5
: .68
- .95 '•
.2 - 5
Appendix Table 3 (continued)
P.I.
Number
of. plants
evaluated
Origin
0. transcaucasica
Disease Severity Score I/
Mean.
,Variance
Range
(continued)
372827
'Czechoslovakia
313026 .
U.S.S.R.
273755
. Russia.
Russia
.273763
U.S.S.R.
■313022
312998
U.S.S.R..
Czechoslovakia
372816
•
,U.S.S.R.
312973 •
273768
Russia
313036
U.S.S.R.
Russia
273756
U.S.S.R.
313011
Czechoslovakia
372825
Russia
273783
313034
U.S.S.R. ■
Russia
273764
U
.S .S •R.
313031
.. U.S.S.R. .
313007
3130.39
U •S-.S .R*
U.S.S.R.
313032
Czechoslovakia
372820 .
Russia
25169.8
U.S.S.R.
312974
372826.
. Czechoslovakia
U.S.S.R.
313010
273757. . . Russia
273767 • Russia
.313008
U.S.S.R.
U.S.S.R.
3-13009
313035
U.S.S.R.:
’
U.S.S.R.
•'
■313001
■ U.S.S.R.
313012
Czechoslovakia
372817
U.S.S.R.:
.312976
Czechoslovakia
372814 ■
314097
U.S.S.R.
.
18 .
14
... 20
23 .,
16.
21 .
15
.15 . •
10
24 ... .
22
18
..19
23
23 ■
■ 15
23
12
20
18 '
11
17
.
8
. 14..
22 ■ .
15.
24 .
■
11
23
21 ■■
. ■ 14
. ■ 14
22
10
18
19
'
'
.
3.31
2 - 5
• .74 .
3.32
.72
2 - 5
.77
2.5-5 .
3.33 ..
.76.
I - 5 "
3.33
2.5-5
3.34
.59 .
.55
. 2.5-5. ■
3.36
2 - 5
.70
3.37
3.37
.62
2 - 5
3.40 .
.38
3 - .5 ■.
3.40 ■
.48
' 2 - 5 :
.52
3 - 5
3.41
3.42
. .54
2.5-5
3.42
.67
2.-5
,71
3.44
.2 - 5
2 - 5
3.46
.57
.62
■.. 2.5-5 ,
’ 3.47 '
.72
2.5-5
3.48
1,05
2 - 5
3.50
... 2 - 5
1.07
.. 3.53
.93
.2-5
3.53
1.07
\. 2 - 5
3.55
3 - 5 •
.75,
3.56
3.56
. .82
3 - 5 ■.
3.57
2.5-5 ■
... ' 1.03
;
2.5-5
■
3.57 ,
.65 ..
. 3.57
1.07
■ 2 - 5
3.58
.99
2 - 5
3.59
.94
. . 2.5-5
2.5-5 .
.75
3.61
.86 .
• 3.67
2 ' 5.
..2.5-5 ■
' 3.68
. .91
■2 - 5
3.68
1.02
■ 2.5-5
■ 3.68
. .75
.68 '
. 3 - 5 '
3.70
.65
2.5-5
3.70
...
.73
•
:
3 - .5,
■ 3.71
•
■
_ '
"
82
Appendix Table 3 (continued)
P.I.
Origin
•
273758
■ Russia
372815
Czechoslovakia
313018
U.S.S.R.
314098 '
U.S.S..R.
312975
U.S.S.R.
273780
■Russia
273753
. Russia
312989
U.S.S.R. ■
312992
U.S.S.R..
273766
Russia
U.S.S.R.
312990 ■
■ U.S.S.R.
313029
372821
Czechoslovakia
312999
U.S.S.R.
273765
Russia
312993
U.S.S.R.
■ U.S.S.R.
313017
Number
of plants
evaluated '
20
18
11
17
.10
17
21
15
15
18
12
10
10
12
21
14
8
Disease Severity Score I/
Mean
Variance
Range
.
3.73
3.75
3.81
3.82
3.85
3.85
3.93
4.00.
4.03
4.03
4.08
4,10
4.10
4.13
. 4.29
4.36
: 4.43
14
15, .
21
3.50
3.60
3.69
1.038
.76
•61
21
.19
.3,57
3.66
.31
.22
20
21
23
24
17
■ 14
22
19
3.18
3.21
3.26
3.33
3.35
3.36
3.36
3.42
.51
.39
.27
. .56
.34
.86
.50
.40
.
.93
1.04 ■
.81- '
1.06
1.50
.74
.66
1.10
.95
■ .90
.99
.99
.99
1.32
.80 .
.86
.67
3 - 5
2 - 5
2.5-5
2.5-5
2.5-5
3 - 5
3 - 5 .
2 - 5
3 - 5
'3-5
3 - 5
3 - 5
3 - 5
2.5-5
3 - 5
3 - 5
3 - 5
2 - 5
2.5-5
2.5-5
0. biebersteinii
22737.7
312932
312931
Iran
U . S .S.R..
U.S.S.R.
O. antasiatica
314541
316294
U.S.S.R.
China
.
3 - 5
•3'- 4.
O. a f e n a r i a .
LO
I
■
. 2.5-5
2.5-5
CM
Russia
U.S.S.R.
Turkey
U.S.S.R.
Czechoslovakia
Czechoslovakia
Czechoslovakia
Czechoslovakia
m
228155
312919
368033
312922
372802
372803
3728.04
372798
2.5-5
3 - 5
2.5-5
2.5-5
2.5-5
83
Appendix Table 3. (tiontinued)
Origin
P.I.
0. arenaria
312929
312911
3:12926
273751
273745
312923
312917
312914
312930
273744
368032
273749
.273750
273746
372800
312921
273748
250977
372801
312925
312912
312918
312924
372799.
3129.13
27375.2
312927
273.743
273747
312928.
.
■
.
.
•
Number
of plants
evaluated
Disease Severity .Score I/
Mean
Variance
Range
(continued)
U.S.S.R.
.U .S'.S .R. ■
U.S.S.R;
Russia
. Russia
■ U.S.S.R.
U.S.S.R.
U.S.S.R.
U.S.S.R.
Russia
Turkey
Russia
Russia
Russia
Czechoslovakia
U.S.S.R. ■
Russia
Yugoslavia
Czechoslovakia
U.S.S.R.
U.S.S.R.
U.S.S.R.
U.S.S.R-. /
.Czechoslovakia
U.S.'S.R.'
Russia
U.S.S.R.
Russia
Russia
u .s .s .r . ;
14
11
20
21
19
24
23
19
16
13
21
20
19
17
20
. 11
24
17
15
20
’ - 14
22
■ 15
22
18
20
'' 17
18
19
15
• :
.
'
■
\
.
.
3.43
3.46
3.48
3.52
3.53
3.58
- 3.59 ■
3,63
3.66
3.65
3.67 ■
3.68
3.68
3.68
3.68
3.68
3.69
3.77 '
3.77
3.78
'3*79,
3.82
: 3.83
3.91
3.92
3.93
4.00
.4.11
4.16
4.17
.53 .
3 - 5
2.5-5
. .47 .
1.01
2.5-5
.79
2.5-5
.82
.2.-5
.77
' 2 -.5
.54
. 2.5-5
.77 •
.2 - .5
.79
2.5-5
UOl
2.5-5
. 1.08
2.5-5
■ .67
.. 2.5-5
.76
■2.- 5
.59
. 3 - 5
.59
2.5-5
.71
2.5-5.
.67
2.5-5
.53
3 - 5
.57
3 - 5
.70
3 - 5
.64
3 -.5
.52 .
3 - 5
,85
3 - 5
3 - 5
,49
.98
2.5-5
1.14 .
2.5-5
■2 - -5
1.00
• .55
’3 - 5
.92
. 3 - 5
.85
• 2.5-5
■0. altissima
312906
312908
273742
312907
U.S.S.R.
U.S.S.R.
Russia .
U.S.S.R.
14
15
15
22
3-. 25
3.63
3.77
3.82
.
.34
.34
.57
.51 ;
3
3
3
3
- 5:
- 5
- 5
- 5
84
Appendix Table 3 (continued)
•
Origin
P.I.
Number
of p l a n t s ■
evaluated
Disease Severity Score i/
Mean
Variance
Range
0. sibirica
369283
371534
U.S.S.R.
U.S.S.R.
14
22
3.29
3.89
.41
.86
2.5-5
2.5-5
3.27
3.28
3.28
3.33
3.43
3.50
3.50
3.53
3.59
3.71
3.74
3.78
3.85
3.85
3.92
3.94
' 4.04
4.10
' 4.15
4.19
4.42
.4.50
4.61
4.67.
■ .53
1.13
.83
.95
.46
.65
1.41
.95 ■
'
.82
1.34
.66
.80
.56
1.08
. 1.54
.91 .
1.66
.58
1.06
, 1.00
.95
'.64
.36
.44
2.5-5
2 - 5
2 - 5
2 - 5
2.5-5
2.5-5
2 - 5
2.5-5
2 - 5
2.5-5
3 - 5
2.5-5
.3-5
2.5-5
2.5-5
2.5-5
2 - 5
3 - 5
2.5-5
, 2.5-5
2.5-5.
3 - 5
2.5-5
3.5-5
0. viciifolia
318604
204594
368036
229612
372828
273787
313061
273784
368034
258768
273786
263159
338651
243227
258773
258777
258770
205202
258769
186520 :
182247
258776
192993 .
200872
Switzerland
Turkey
Turkey
Iran
■
Czechoslovakia
Russia
U* S •S •R.
Russia
Turkey
Russia
Russia
Russia
Morocco
Iran
Russia
Russia
Russia
Turkey
Russia
Spain
Turkey
Russia
Spain •
Turkey
17
9
16
23
21
14
17
17
17
12
17
18
. 23
20 ..
13
. 18
12
15
20
16
.18
12
9
.
9
2
Disease severity scored as follows: I = no spread of dis­
coloration; 2 = spread of discoloration I cm or less;
2.5 = spread of discoloration 1.1 - 2.0 cm; 3 = spread of
discoloration 2.1 - 3.0 cm; 3.5 = spread of discoloration
3.1 - 5.0 cm; 4 = extensive vascular necrosis; 5 = dead
plant.
9
85
Appendix Table 4.
P.I. .
0. viciifolia
The response of 113 accessions of Onbbrychis to
inoculation with F. solani as measured by the mean,
variance, and range of the disease severity scores.
Data were based on unreplicated observations.
Origin
Number
of plants
evaluated
Disease Severity Score
Mean
Variance
Range
(51)
204595 ' ■ Turkey
Turkey ■
206459
319062 .
Spain
313048
UySiS .R.,
.'
... U.S.S.R...
313053
313054 ■
U .S .S .R.
Czechoslovakia.
372829
372830
Czechoslovakia
225728
Turkey
U.S.S.R.
313060 .
318605 .
Switzerland
313058 ;
U.S.S.R., .
212241
Washington
Czechoslovakia
372832 .
258774
. Russia
U.S.S.R.
-313056
234644
Spain
250024.
.Iran
Poland
.
313049
U.S.S.R.
",
313050
313065
U.S.S.R.
23.4822
Switzerland
Turkey
.205200
227038
Iran
273791 .
Russia .
313046
Spain
. Czechoslovakia
372833
205201
• Turkey
Iran
228289
I r an .
228402
Spain
302939.
Italy.
306693
8
7
7
7
8
8
6
.7 . ■
6
6
8
3.00
■■■• .00
3 .
3.00 . .
2 - 4 .•
.33
3.00
. .oo
3 ,
3.00
. .00
• 3. .
• 3.00
.29
2 - 4
3.00
.00
3 \
3.00
3
.00
. 3.00
3.
.00 . .
3.17
- 1 - 5
2.57 ■
3.17
.97
• 2-5.
3.25
.21
3 - 4
3.29
■ . .57
.3 - 5.
I.
7
3.43
.62 ...
3 -.5
7
3.43
.62
3 - 5
7
. 3.57
3 - 5
.
.95
7
. 3.5.7
3 - 5 .
.62 .
3-67
1.03
3 - 5
6
•3.71
.90
3. -. 5 ■
7
;
. 3.71
3 - 5 '
.
.90
. 7 ...
3 - .5
.■ 3.71
. • .90
I
3.71
3 - 5
7
.90
. 3.83
• . : .97
6
.3 - 5
3.86
.81 - . 3 - 5 •.
7
. 3 - 5
3.86 .
■ 7
i81
3.86
3 - 5
7
1.14 ..
3.86
.81 3 - 5
7
.81 ■
3.86
3 - 5 .
. ■ '7. . ..
4.00
7
1.00
3 - 5
3 - 5
4.00
7
1.00
3 - 5
7
4.00
1,00
3 - 5
.67
7 • • . 4.00
3 - 5
4.00
■ 7. . •
. .67
.
86
Appendix Table 4 (continued)
Origin
P.I. ■
0. viciifolia
Disease Severity Score I/
Mean
Variance
Range.
(continued)
313064: ■ Italy
273787 ■
Russia
Czechoslovakia
372831 .
372835
Czechoslovakia
Iran
227373 . ,
■U.S.S.R.,
313062
Russia
273788
Spain
192995
206458
Turkey
258772
Russia
- Russia
263159
Russia
273786
319713
Rumania
313063
. U.S.S.R.
192944
Spain
258771
Russia
258767
Russia
263158
Russia
Russia
258775
0. transcaucasica
313005
273778 273775
372813
273773
273779
273777
372822
313041
273782
312986
313000
313003
312985
313040
Number
of plants
evaluated
•
7
•
7
7
7
7 ■
.7 '
8
7
■7
7
7
7
7
. 5
6 ■
6
7
7
7
4.00
4.14
4.29
4.29
■ 4.43
4.43 ■
4.50 '
.. 4.57:
■ 4.57
■4.57
■ 4.57 .
4.57
4.57
4.60
4.67
. 4.67 .
4.86
4.86
■ 5.00 .
1.00
3 - 5
. .81
3 - 5 .
.90
3 - 5
.57
3 - 5
.29
' 4 - 5
.
■ .62 .
3 - 5 "
.57
3 - 5
.29
:4 -• 5
.62
3 - 5
,62
3 - 5
.62.
3 - 5
.62
3 - 5
.62
3 - 5
.
.80
3 - 5
•67
.■ 3 - 5
.67
3 - 5
.
4 - 5
• .14 '
4 - 5 . '
• •■14
:5 ' .
.00
6 ..
8.
8
7 ,.
6
6
.6
6
7
6
. 7
7
7
7
•7
2 i83
• 2.88 :
.3.13
3.14
3.17 .
3.17
■ 3.33
3.33
' ' 3.43 .
3.50
3.57
3.57
3.57
3.71
3.71
. 2 :- 3
.17 ■
2 T- 3 •
.13 .
3 - 4
• .13
.14 .
'3 — 4
.97
2 - 5
.97
2 - .5
.67
■ 3.-5 :
.67
. 3 - 5
■
.29
3 — 4
.2 - 5
1.50
.62
'3-5
1.29 ■
2 - 5
.62
3 - 5
.90
' 3 - 5
.90
3 - 5
(38)
U.S.S.R.
•Russia
Russia
Czechoslovakia
Russia •
Russia
Russia
Czechoslovakia
■ U.S.S.R.
Russia
U.S.S.R.
U.S.S.R.
U.S.S.R.
U.S.S.R. ■
U.S.S.R.
87
Appendix Table 4 (continued).
•
p. I.
•
. , '■ Origin
O., transcaucasica
313043
251697
273776
273781
312981
312995
313023
313025
278154
273769
312977
31.3016
313033
.372812
.273770
312.984
31.3004
273759
312988
318601
273772
273771
313044
312956
312964
312967.
312968
312972
312954
312957 '
312953
Disease Severity.Score i/
•Mean
Variance
Range ■
(38)
'
U.S.S.R.' U.S.S.R.
;
" 3.71 ..
.90
7
'
- 3 - 5 ..
. 6
.
. 3.83
1.77
■ 2 - 5
' 3.83
6 ■■
3 - 5
. .97 ;
1.14
7
; 3.86 ■
3 - 5
7
3.86 .
.81 .■■■ . 3 - 5 '
3.86
7
. 1-48
.■ .2 - 5. •
3.86
1.48.
.7 :
2 - 5
. 1.14
7
.
3.86
3 - 5
■ ■7
'
4.00
1.00
3 - 5
4.14
.
1.14
3 - 5
7
4.14
.81
3 - 5
.
7
'. 4.14'
. 1.14
. 7
3 - 5
4.14 ■
.48
7
3 - 5
4.14
.81,
. 3 ■•- 5 '
7 :
7
. .4.29 , ■
: : .90 .
.3 - 5
4.29
.90 ■ . 3 - 5
7
4.29
.90
3 - 5
. 7 ;
.90 ; 4.29 .. ■.
'
7
•
3 - 5/
4.43 - . 1.07 ;■ . '. 3 - 5
' 6
'
3 - 5 •
6. ■'
4.43
■ ' 1.07 ■■■
' .95 . - 3 - 5
7
. ■ 4.43V
.00 . ■' 5 .
' ;:5.00
7 '
5.00
.00
6
■
.. 5
. 3.00
. .00
,7
' 3 .. ■ ■
3 - 4 .
3.29
.24
' :"■■■ 7 .
1.07.
3 - 5 .
3.67 .
'6
3 - 5 ■'
; 3 . 83
' .97
6
.81
' 3.86
■
.3-5 .
7
3.86 '
3 - 5
.81
■ 7
.si
■ 4.14
7
' ,3-5
.57
3 - 5
.
4
.
2
9
.
' 7 ' ■;
U.S.S.R. ■
Russia .
Russia
Russia
Turkey
U.S.S.R.
U.S.S.R.
Hungary .
Russia
.Russia
Iran
■U.S.S.R.
■ U.S.S.R.
Czechoslovakia •
Russia
'U.S.S.R.U.S.S.R.
’
Russia
U.S.S.R.
Switzerland
Russia
Russia"
U.S.S.R. '
;
U.S.S.RU.S.S.R. :
• U.S.S.R. .
U.S.S.R.
U.S.S.R. .
.U.S.S.R.
U.S.S.R.
U.S.S.R.
O . 'tanaitica .(8)
312956..
312964 '
■Number
of plants
evaluated
_
■7
7 "
3.00
■ 3.29
.00
3
.24.
3 - 4
88
A p p e n d i x .Table 4 ,(continued)
Origin
P.I. . .
0. tanaitica
312967
312968
312972
312954
312957
31.2953
(8)
Number
Of plants
evaluated
-' ,
(continued)
U.S.S.R.
U.S.S.R.
U.S.S.R.
U.S.S.R.
U.S.S.R.
U.S.S.R.
0. arenaria
312909
312915
315084
312910
312920
(5)
■
'i
6
' 7 :
7
7
7
.
..
Ot inermis ■(2) .
4.14
.
6
7
6
7
6
.
0. iberica
(2)
314931
219602
U.S.S.R.
Pakistan
'
',
'
;
3 - 5
■, 3 - 5
3 - 5
. ■. 3 - 5
3 - 5
. .3 - 5
3.14
' 4.33
7
7
3.14
5.00
•
■■
7 '•
U . S .S.R.. .
7
.'
.67
' -95
.
.57
.62
.67
: 3 - 5 :
. 3 - 5
3 - 4
■3 - 5 ,
. 3 - 5
.48"
2 - 4 ■
• i.07 ; ". ;a. - 5
.14
;00
, 3 - 4
:::5
5.00 '
4.00
.
• .00
'
■■
■
1 .00
■
.
Spain .....
5 .;
■V
3 - 5
/■/
0. argentea ■ ■
280259
•■ 4
:
. ■ ■■■■ .
■
/
•
■
0. kamulariae
312464
1.07
. .97
■ ,81
.81
.81
.57
'
7
6
(I)
Iraq.
;
4.29
3.33
. 3.57
4.17
■ 4.43
4.67
..
- '*;■
.. U.S.S.R.
U.S.S.R.
215344■ ■
.
.
U.S.S.R. ..
U.S.S.R.
U.S.S.R.
U . S .S.R,.
0. Ianata
.. 3.67
3.83
3.86
3,86
6
■U .S .StR.
325439 '
312935
.Disease Severity Score i /
Mean
Variance
Range
:• 4 . 0 0 .
1.33 .
■
' 3 - 5
..
89
Appendix Table 4
(continued)
Origin
■ P.I.
Number
of plants
evaluated
Disease Severity Score I/
Mean .. . Variance
Range
0. argyrea .
Turkey
288255
7
. .5.00
.00
5
6
5.00
.00
5
7
3.71
.00
3 - 5
6.
7
6.
3.50
3.86
4.00
1.90
.90
1.20
0. vavinalis
U.S.S.R.
325444
0. bierbersteinii
U.S.S.R.
325446
Check Cultivars
Melrose
Eski
Remont
Turkey
.
.2-5
3-5.
3 - 5'
0. altissima
325448,
.
U.S.S.R.
. • .
7
-
. 3.00 .
.00
3 - 5
.
I
Disease severity' scored as follows; I = no' spread of discoloration; 2 = spread of discoloration of I cm or less; 3 = spread
of discoloration of I cm or m o r e ; 4 = extensive vascular ne­
crosis; 5 = dead plant.
MOWr»U» ____
3 1762 10005120 8
D370
Au52
c o p .2
A u l d , Dick L
Proof of pathogenicity
o f F u s a r i u m sola n ! ...
DATE
ISSUED TO
Ag-KAcxni
>
^
5--Z