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ABSTRACT

MECHANISM OF INCREASED ROOT ROT
IN VIRUS-INFECTED PEAS

by Marvin K. Beute

The mechanism of increased fungal root rot induced
by virus infection in peas was studied, Two mechanisms
are possible: 1) The inherent susceptibility of root
tissue is increased by virus infection; 2) Increased leak—
age of nutrients from roots of virus-infected plants
increases the inoculum potential of the fungi in the rhizo-
sphere.

Increased Fusarium root rot in peas (Pisum sativum
L. cv. Miragreen) infected with bean yellow mosaic virus
(BYMV) could not be explained by changes in inherent
susceptibility, When roots were subjected to hourly leach—
ing, Fusarium root rot was equally severe in both virus-
infected and non—virus—infected plants. Addition of exudates
from roots of virus-infected plants to plants inoculated
with E, solani f. pi§i_gave more severe root rot than did
root exudates from healthy plants,

Roots of BYMV—infected pea plants growing in water

Marvin K, Beute

or sand cultures released more electrolytes, nucleotides,
amino acids and carbohydrates than did healthy roots,
Increased exudation began about 4 days after virus infec-
tion and ended after 12—16 days. Total amino compounds
in the exudate increased more than did total carbohydrates
or nucleotides. Similar results were obtained with pea
infected with common pea mosaic virus (PVM) and pea
enation mosaic virus (PEMV). Increased root rot induced
by virus infection in peas occurred only during the period
of increased exudation from roots of virus-infected peas.
Root exudates from virus-infected plants contained
more substances supporting growth of E, solani than
occurred in exudates from healthy plants. Also, germination

of Helminthosporium victoriae conidia on the surface of

 

natural soil was greater adjacent to roots of pea plants
infected with BYMV 8 to 16 days previously than adjacent
to healthy plants, This period of increased germination
coincided with the period of increased root exudation from
roots of virus—infected plants.
Each of 10 identified amino acids was released in

greater quantities from roots of BYMV-infected plants than
from healthy roots. Alanine exudation was increased the

most following virus infection. Threonine was the most

Marvin K. Beute

prominent amino acid in root exudates from both virus—
infected and healthy Miragreen plants, Phenylalanine was
present in root exudates from virus—infected plants, but
not from healthy plants. The increase in exudation of
amino acids from roots of virus-infected plants could not
be explained by changes in total quantities or in specific
amino acids within root tissue,

Roots of BYMV-infected Miragreen peas immersed in
a buffer solution containing 32PO4 accumulated more 32P
than did healthy roots. No selective decrease in accumula—
tion of radioactivity was noted with a 2 hour pretreatment
of roots with 2,4—dinitrophenol, an uncoupler of oxidation
from phOSphorylation. Increased uptake by roots of virus-
infected plants suggests an increase in plasma membrane
permeability, which may also account for the increased
leakage of substances from roots of virus-infected plants,

Fusarium root rot was increased by adding the
prominent amino acids found in virus—infected root exudates
to fungus-inoculated plants, but neither glucose nor
selected organic acids increased disease.

The following explanation is proposed for the
mechanism of increased root rot in virus—infected pea:

Virus infection causes changes in membrane permeability

Marvin K. Beute

of root cells, resulting in increased exudation of many
kinds of compounds, including nutrients utilizable by the
fungus, This increased supply of nutrients increases the
inoculum potential of the pathogen in the rhizosphere,
which in turn, results in increased root rot,

Two experimental pea varieties, each possessing
moderate resistance, one to Aphanomyces root rot, and the

other to both Aphanomyces and Fusarium root rots, released

 

more carbohydrates but less amino acids from their roots
than did the susceptible variety Miragreen. Root leachings
from varieties resistant to either fungus failed to increase
root rot when added to Miragreen plants inoculated with the
same fungus, However, when root leachings from the Aphano-
myces resistant variety were added to Fusarium—inoculated
Miragreen plants, severity of root rot was increased to the
level of disease in plants receiving root leachings from
Miragreen plants, These results suggest that the level of
resistance observed in the experimental pea varieties may
be, in part, a reflection of the influence of root exudates
on inoculum potential in the rhizosphere,

Infection with E, solani f. pig} caused an early
increase in infectivity of BYMV or PMV in pea leaf sap, A

2—3 fold increase in local lesions was noted using BYMV +

Marvin K. Beute

E, solani—infected leaf sap beginning 5 days after inocula-
tion. Beyond 12 days from initial inoculation, however, no
differences in virus titer were found. Infection with E,
solani resulted in a 2—3 fold increase in PMV infectivity
between the third and fourth day after inoculation. There-
after, leaf sap from virus + fungus-infected plants were

equally infective.

MECHANISM OF INCREASED ROOT ROT
IN VIRUS—INFECTED PEAS
by

I

v
. I
.

Marvin K.TBeute

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Botany and Plant Pathology

1967

ACKNOWLEDGMENTS

Deep appreciation is expressed to Dr. J. L.
Lockwood for his guidance and assistance during the course
of this investigation and in preparation of the manuscript.

Thanks are also due to Dr. E. S. Beneke, Dr. A. H.
Ellingboe, Dr. R. P. Scheffer and Dr. A. R. Wolcott for
their critical evaluation of the manuscript.

Appreciation is expressed to the Department of
Botany and Plant Pathology for awarding to me the Ernst A.
Bessey Award in recognition of high scholarship. The

award was used to purchase books utilized in this work.

ii

TABLE OF

ACKNOWLEDGMENTS . . . . . .

LIST OF TABLES . . . . . .

LIST OF FIGURES . . . . . .

INTRODUCTION . . . . . . .

LITERATURE REVIEW . . . . .

CONTENTS

METHODS AND MATERIALS . . .

Source and maintenance of fungus and virus cultures
Growing plants . . . . . . . . . . . . . . . . . .
Inoculation procedures . . . . . . . . . . . . . .
Evaluation of disease . . . . . . . . . . . . . . .
Collection of root exudates . . . . . . . . . . . .
Chemical analyses of exudates . . . . . . . . . . .
Bioassays of exudates . . . . . . . . . . . . . . .
Phosphate uptake . . . . . . . . . . . . . . . . .
Assay of virus infectivity in pea leaf sap . . . .

RESULTS . . . . . . . . . . . . . . . . . . . . . . .

External vs. internal effect of virus infection on
root rot disease . . . . . . . . . . . . . . . .
Influence of root exudates on disease development .
Changes in exudation patterns . . . . . . . . . . .
Relationship between exudation period and disease
Biological activity of root exudates . . . . . . .
Chromatographic analysis . . . . . . . . . . . . .
The effect of virus infection on phosphate ion
uptake . . . . . . . . . . . . . . . . . . . . .
Effect of glucose, organic acids and amino acids on
root rot . . . . . . . . . . . . . . . . . . .

iii

Page

ii

vii

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l3
14
15
l6
17
18
23
25
25

27

27
28
36
46
52
56

63

64

TABLE OF CONTENTS (Continued) Page

Effect of the fungus on virus infectivity . . . . . 70
Role of root exudation in varietal resistance to
root rot . . . . . . . . . . . . . . . . . . . . 71

DISCUSSION.....................76

LITERATURE CITED . . . . . . . . . . . . . . . . . . 83

iv

LIST OF TABLES
Table Page
1. The effect of removal of Fusarium solani

inoculum from pea roots on virus-induced
rOOt rot O O O O O O O O O O O O O O O O O O O 29

 

2. The effect of injecting inoculum into cortical
tissue on development of Fusarium root rot
in Miragreen pea . . . . . . . . . . . . . . . 30

3. Relationship between Fusarium root rot severity
and the increase in root exudation beginning
4-6 days after virus inoculation . . . . . . . 51

4. The effect of root exudates from healthy and
virus-infected plants on growth of E, solani
f. pisi germ tubes . . . . . . . . . . . . . . 53

5. Amino acid composition of exudates from roots of
healthy and virus—infected Miragreen peas
grown in silica sand . . . . . . . . . . . . . 57

6. Total carbohydrates and free amino acid pools in
healthy and BYMV—infected Miragreen pea root
tissue . . . . . . . . . . . . . . . . . . . . 6O

7. Free amino acid pool in 20-day-old root tissue
of healthy and virus-infected Miragreen pea
after virus inoculation . . . . . . . . . . . 61

8. Organic acids in exudates from roots of healthy
and virus-infected Miragreen peas grown in
silica sand . . . . . . . . . . . . . . . . . 62

9. The effect of virus infection on uptake of 32P04
by root tissue of Miragreen pea . . . . . . . 65

LIST OF TABLES (Continued)

Table Page
10. The effect of 2,4-dinitrophenol on uptake of

32P04 by root tissue of virus-infected and
healthy Miragreen pea . . . . . . . . . . . 66

vi

Figure

1.

LIST OF FIGURES

Apparatus used for collecting root exudates of
pea growing in silica sand . . . . . . . . . .

Effect
BYMV
rot

System
from
ment

Effect
root

Effect

of leaching roots of plants infected with
or PMV on susceptibility to Fusarium root

used to test effects of root exudates
pea roots on Fusarium root rot develOp-
in pea . . . . . . . . . . . . . . . . .

of leachates from pea roots on Fusarium
rot of Miragreen peas . . . . . . . . . .

of BYMV-infection on amount of electro;

lytes exuded by roots of pea plants growing in
water cultures . . . . . . . . . . . . . . . .

Effect

of BYMV—infection on amount of nucleotides

exuded by roots of pea plants growing in water
cultures . . . . . . . . . . . . . . . . . . .

Effect

of BYMV-infection on amount of carbohy—

drates exuded by roots of pea plants growing
in water cultures . . . . . . . . . . . . . .

Effect

of BYMV-infection on amount of amino acids

exuded by roots of pea plants growing in water
cultures . . . . . . . . . . . . . . . . . . .

Relative increased exudation of electrolytes,
nucleotides, carbohydrates and amino compounds
by virus—infected Miragreen pea plants growing
in water culture . . . . . . . . . . . . . . .

vii

Page

20

32

34

35

37

38

39

4O

42

LIST OF FIGURES (Continued)

Figure Page

10. Effect of BYMV-infection on amounts of amino
acids exuded by roots of pea plants growing
in sand culture . . . . . . . . . . . . . . 43

11. Effect of BYMV-infection on amount of carbo—
hydrates exuded by roots of pea plants
growing in sand culture . . . . . . . . . . 44

12. Relation between root rot severity and time
interval between inoculation of Miragreen
peas with BYMV and Fusarium solani . . . . . 48

 

13. Relation between root rot severity and time
interval between inoculation of Miragreen
peas with BYMV and Fusarium solani before
increased exudation occurs in virus-infected
plants . . . . . . . . . . . . . . . . . . . 50

 

l4. Germination of Helminthosporium victoriae
conidia on soil adjacent to roots of healthy
and virus—infected plants . . . . . . . . . 51

 

15. Fusarium root rot development on Miragreen
peas receiving glucose, organic acid or
amino acid amendments . . . . . . . . . . . 68

16. Effect of glucose, amino acids and organic
acids on Fusarium root rot of peas . . . . . 69

17. Relative increased exudation of electrolytes,
carbohydrates and amino acids by 3 pea
varieties with different resistances to
Aphanomyces or Fusarium root rot . . . . . . 72

 

18. Effect of root leachates from 3 pea varieties
with different resistance to Aphanomyces or
Fusarium root rot on root rot severity in
Miragreen peas . . . . . . . . . . . . . . . 74

 

viii

LIST OF FIGURES (Continued)

Figure Page

19. Severity of Fusarium root rot as a function of
inoculum density . . . . . . . . . . . . . . 79

ix

INTRODUCTION

Previous studies at Michigan State University
indicated that viruses may play a role in development of

the fungus root rots of pea (Pisum sativum L.) (12, 13).

 

Over a wide range of greenhouse environmental conditions,

3 pea varieties were more susceptible to Aphanomyces or

 

Fusarium root rots when plants were inoculated previously
with either common pea mosaic virus, bean yellow mosaic
virus, alfalfa mosaic virus or pea enation mosaic virus.
Similar results were obtained with different strains of 2
of the viruses, plants of different ages, and when different
time intervals between virus and fungus inoculation were
used.

My research problem was to study the mechanism of
increased root rot associated with virus infection. Two
mechanisms are possible: 1) The inherent susceptibility of
root tissue is increased by virus infection; 2) Increased
leakage of nutrients from roots of virus-infected plants
increases the inoculum potential of the fungi. Farley (12)
found that when residual fungal inoculum was removed from

1

surface of the roots at the time of virus inoculation 3-5
days after fungal inoculation, no increase in root rot
occurred above that in fungus-only-infected plants treated
in the same manner. This suggested that susceptibility of
virus-infected root tissue to the root rot fungi was not
changed.

Therefore, most of the research reported herein was
directed toward evaluating the role of root exudates as the
primary factor in the increased root rot. Root exudates
from healthy and virus-infected plants were analyzed to
determined any changes in quality or quantity of nucleo-
tides, carbohydrates, amino acids or organic acids brought
about by virus infection. The effect of the root exudates

on growth of Fusarium solani f. pisi (F. R. Jones) Snyder

 

and Hansen and on disease development was also studied.

LITERATURE REVIEW

INTRODUCTION: Exudation of soluble organic sub—

 

stances is a phenomenon common to root systems of all
higher plants. The importance of root exudates in influ-
encing soil microorganisms, both pathogens and saprophytes,
has been demonstrated for many plants. A major source of
exudates from uninjured seedlings appears to be intact
cells located in the region of highest enzymatic activity
in the root, i.e., in the vicinity of root tips (47, 49).
Other major sites of exudation appear where breaks occur
in the epidermis (58). The picture emerging from knowledge
of the types of substances found in root exudates is that
most substances involved in the cellular metabolism of
higher plants are released by exudation.

Results with pea and oat seedlings indicate that
exudates are a more important source of organic materials
in the rhizosphere than cell debris. However, as the plant
develops, exudation declines and decomposition of moribund
root hairs, and epidermal and cortical cells contribute a
substantial portion of the nutrients to the rhizosphere

3

(50). Amino acids form the most studied group of compounds
in root exudates. The zone of rapid cell elongation imme—
diately behind the growing tip is the most active amino
acid-exuding area (47, 58). The following amino acids are
known to be liberated by pea roots: glutamic and aspartic
acids, proline, leucine, alanine, cysteine, glycine, lysine,
phenylalanine, asparagine, serine, glutamine, homoserine,
tryptophane, methionine/valine, Y-amino butyric acid, and
threonine (25, 50).

The sugars glucose and fructose were found in pea
root exudate during the first 10 days of growth (50). How—
ever, when sand and root washings of plants were examined
after 21 days of growth, the sugars were no longer detected.
Although organic acids are found inside the root at signi-
ficant levels, there are few reports of these materials
occurring in root exudates (53). The presence of vitamins
in root exudates has long been suspected (34), but the
number and level of vitamins reported have been very low.
Meshkov (41) reported biotin and thiamin in the exudate
from pea and corn roots. The purines adenine and guanine
(37), the nucleotides uridine and cytidine (15) as well as
3-4—dioxyflavonone have been found in the root exudates

from pea (37). Krasil'nikov (28) found that exudates from

peas contained the enzymes invertase, amylase and protease.
Root exudation is affected by many factors.
Katznelson et al. (25, 26) showed that successive drying
and remoistening of peas, soybean, wheat, barley and toma-
toes growing in sand and soil—sand mixtures greatly
increased the release of amino acids from roots. Exudation
mixtures greatly increased the release of amino acids from
roots. Exudation was also greatest under high temperatures
and light intensity conditions (51). Addition of fertilizer
increases the rhizosphere populations of most plants, indi—
cating a greater leakage of nutrients from roots of well
fertilized plants (52). Agnihotri (1) reported increases
in the exudation of glutamine, Y-amino butyric acid and
other amino acids from roots following the foliar application
of urea sprays. Urea sprays also caused increased glucose
and fructose exudation, but organic acid exudation decreased.
Rovira postulated that microorganisms can affect
exudate patterns by altering the permeability of root cells,
by modifying the metabolism of roots, and by modifying some
of the materials released from roots (53). The amino acid
patterns in exudates from tomato and clover roots were
shown to be altered by microorganisms in the rhizosphere

(51). Exudation is also influenced by disease, and

exudation of nutrients from diseased tissues may in some
cases make infection autocatalytic or progressive (57).

EFFECTS 93 VIRUS INFECTION 95 ROOT EXUDATION:

 

 

Lesions resulting from infection by either Fusarium solani

 

f. phaseoli or tobacco necrosis virus on hypocotyls of bean

 

or COWpea exuded high levels of amino acids and reducing
sugars (57). Such lesions are necrotic and would be expected
to release cellular constituents. However, systemic virus
infection withmfizlocal lesions may also result in changes

in root exudation. In Dolichos lablab infected with

 

dolichos enation mosaic virus, changes in physiology of
virus infected tissue were accompanied by a change in
rhizosphere microfloras (31, 54). Virus infection altered
not only the rate of increase of organisms in the rhizo-
sphere, but also the time of manifestation of maximum
rhizosphere effect. Between 1 and 5 days after inoculation,
fungal numbers tended to increase. Bacteria and actino—
mycetes increased between 5 and 15 days after virus
inoculation. Twenty days after virus infection, the total
microbial numbers were considerably reduced as compared
with those from the healthy plant. In an investigation

of the physiological changes which precede tobacco etch

virus—indiced wilt of Tabasco pepper, Gabrial (16) showed

that a marked release of electrolytes from roots occurred
24-36 hr before the onset of wilt and 12 hr before respira-
tory changes could be detected. No such release of
electrolytes occurred with noninoculated plants nor with
wilt resistant, but virus susceptible plants infected with
tobacco etch virus. Stained sections of roots of infected
Tabasco seedlings showed no phloem or cambium necrosis at
the time of electrolyte release.

VIRUS-FUNGUS INTERACTIONS: Associative effects of

 

virus infection on fungus disease develOpment can be out—
lined as follows: 1) Fungal infection is increased,
decreased or not effected in virus—infected plants. 2)
Virus infection is increased, decreased or not effected in
fungus—infected plants.

Fungus infection is increased: A number of reports

 

 

indicate that fungal infections of foliage and roots of
plants are increased when the plants are also infected with

a virus. Early blight (Alternaria solani) symptoms on

 

leaves of several potato varieties in the field were usually
more severe on mosaic-infected plants than on mosaic—free
plants (23). Field observations indicated that the late

blight fungus (Phytophthora infestans) was present in about

 

twice as many tubers of the potato variety Ulster Supreme

infected with the leaf—roll virus than in tubers of virus—
free plants (48). Rice plants infected with hoja blanca

virus were more susceptible to Cochliobolus miyabeanus than

 

non—virus—infected rice plants (30).
Post-emergence damping-off of cucumber caused by

Rhizoctonia sp. increased as a result of cucumber mosaic

 

virus infection (3). Similarly, sudden wilt of cucumber
in Israel was reported to be a result of synergism between

Pythium ultimum and cucumber mosaic virus (46). In 1964,

 

Farley and Lockwood found that 3 pea varieties were more
susceptible to 2 common fungus root rot diseases when plants
were inoculated previously with any one of 4 different
viruses (13). Shortly thereafter, Watson et al. (64)
described the importance of the virus—fungus interrelation-
ship in reproducing the clover decline—root rot syndrome as
it occurs in Idaho. They also reported a severe root rot
epidemic on pea associated with widespread virus infection

in the field. Fusarium and Rhizoctonia root rots of wheat

 

in New Zealand and Canada occurred predominantly in plants
infected with barley yellow dwarf virus (59). Williams and
Alexander noted that a higher incidence of root and stalk
rot occurred in corn infected with maize dwarf mosaic virus

(65). Virus-infected, greenhouse—grown Ohio W49 yellow

dent corn, Golden Bantam sweet corn and Lucas wheat were
more susceptible to root rots, stalk rots, and seedling
blights (45) than were plants free of virus.

Fungus infection }§_decreased: In contrast to the

 

 

positive interactions discussed above, Muller and Monro

(44) observed that the growth Phytophthora infestans was

 

retarded on leaves of potato plants previously inoculated
with virus X or virus Y. Similarly, reduced susceptibility

of cucumber to powdery mildew (Erysiphe cichoracearum) or

 

scab (Cladogporium cucumerinum) infection (24) occurred

 

when cucumber mosaic virus was present in the plant. Tobacco
mosaic virus infection reduced susceptibility of Pinto bean

(Phaseolus vulgaris) to bean rust (Uromyces phaseoli typica)

 

 

(67), and leaf-roll virus reduced susceptibility of grape to

powdery mildew (Uncinula necator) (19). Infection of pigeon

 

pea (Cajanus cajan) with sterility virus afforded protection

 

against Fusarium-wilting (9), and infection of red clover

(Trifolium pratense) with bean yellow mosaic virus retarded

 

subsequent infection by powdery mildew (Erysiphe polygoni)(27).

 

Fungus infection E§_not affected: Systemic infection

 

 

by tobacco mosaic virus did not alter the susceptibility of

Nicotiana tabacum var. Turkish leaf tissue to Thielaviopsis

 

 

basicola (21). However, a localized, nonspecific resistance

10

to E, basicola was induced in Samsum N N tobacco half—
leaves opposite half—leaves inoculated with TMV.

Losses from Fusarium oxysporum, F. roseum (E,

 

avenaceum), Rhizoctonia solani and Sclerotium bataticola

 

 

 

were neither increased or decreased on red clover infected
with bean yellow mosaic virus (39). Fusarium sp. and

Rhizoctonia sp. were isolated with equal frequency from

 

white clover plants infected with bean yellow mosaic virus,
red clover virus, pea mosaic virus, and from virus-free
plants (20). However, the actual susceptibility of the
plants to these pathogens was not determined by these in—
vestigators.

Virus infection is increased: The concentration of

 

 

11 viruses in sap from bean leaves infected with Uromyces
phaseoli appeared to be increased compared with that in non-
rusted tissue (18, 68). Based on local lesion assays,
addition of rusted bean tissue (0.003% - 0.01%) to TMV sap
increased local lesions by 77%. When 3-day old rust mycelia
in Pinto bean leaves were killed by heat and the treated
tissue inoculated with TMV up to 4 days after therapy, a
similar increase in virus concentration occurred. Sunflower

(Helianthus annuus) leaves infected with rust (Puccinia

 

helianthi) had higher concentrations of 6 viruses than did

 

ll

non-rusted leaves. Since Erysiphe polygoni or Colletotrichum

 

 

lindemuthianum, which enter cells of bean by direct penetra—

 

tion, did not favor virus infection, increased susceptibility
of rusted tissue to virus infection did not appear to be due
to the punctures made by rust haustoria.

Virus infection l§_decreased: Resistance against TMV

 

 

was acquired by tobacco plants (Nicotiana tabacum) previously

 

infected with Thielaviopsis basicola (5). Virus lesions were

 

smaller in size and fewer in number when the lower leaves of
Samsun tobacco were first inoculated with E, basicola, then
followed 7 days later by a challenge inoculation of the upper
leaves with TMV.

Tobacco plants (E, tabacum) with Thielaviopsis

 

basicola lesions on stem or leaf tissue developed systemic
resistance to tobacco necrosis virus (21). However, root
infection by the fungus did not alter susceptibility of
aerial plant parts to virus infection.

Based on the literature reviewed above, one can con—
clude that diseases caused by obligate parasites are often
decreased by virus infection of the host. Examples of this
generalization include rusts and powdery mildews, which are
considered to be the most specialized or advanced pathogens

(40). One the other hand, fungal diseases caused by

12

primitive parasites are commonly increased by virus infection
of the host. These include damping-off and root rot diseases.
In some cases, failure to detect any change in a fungal dis-
ease when the same plant is also infected with a virus was

due to use of inadequate methods for assessing such changes.

MATERIALS AND METHODS

Source and maintenance of fungus and virus cultures:

 

 

Fusarium solani f. pisi (F. R. Jones) Snyder and Hansen,

 

isolate 19 was obtained by isolation from a Michigan pea

field. Aphanomyces euteiches Drechs. isolate 4 was obtained

 

from W. T. Schroeder, Geneva, N. Y., and E, oxysporum f.

 

pi§l_(Linford) Snyder and Hansen, race 2, isolate 90 was
obtained from D. J. Hagedorn, Madison, Wisconsin. Bean
yellow mosaic virus (BYMV) was supplied by W. T. Schroeder,
common pea mosaic virus (PMV) was supplied by D. J. Hagedorn
and pea enation mosaic virus (PEMV) was supplied by J. Bath,
Michigan State University.

E, solani f. pisi, E, oxysporum f. pisi race 2, and

 

E, euteiches were maintained on potato—dextrose agar (PDA)

 

slants at 240C. All fungi were pathogenic on pea. Common
pea mosaic virus, bean yellow mosaic virus and pea enation
mosaic virus were maintained in a greenhouse (70—900F) by
periodic sap inoculation on pea variety Miragreen. Viruses
in infected, finely-chopped pea leaves were also stored
over CaCl in a freezer.

2
l3

14

Growing plants: A commercial pea (Pisum sativum L.,

 

 

cv. Miragreen) was used as a host plant unless specified
otherwise. Pea seeds were surface sterilized by soaking in
0.5% sodium hypochlorite (10%.Clorox) for 15 minutes,
followed by rinsing with distilled water. Seeds were planted
in silica sand or in an autoclaved mixture of equal parts
sand and ground peat. Usually, 10 seedlings were grown in
moist silica sand in 4 in. plastic pots (1,400 g sand/pot).
However, seedlings were also grown in 8 in. diameter glazed
crocks, each with a hole on the side near the bottom, to
allow drainage of excess fluid from the sand.

For collection of root exudates, plants were first
grown in silica sand in 4 in. plastic pots. When seedlings
were l—2zhL,high, plants were removed from the sand by flood—
ing the pots with water and gently agitating the sand until
plants were floated free. Seedlings were placed in 4 in.
plastic pots and a slurry consisting of acid washed silica
sand and water was used to fill the pots.

In experiments designed to remove external fungal
inoculum from plants, seedlings were first grown in moist
silica sand in 4 in. plastic pots or in rows in metal pans.
Healthy plants and plants inoculated with a fungus 3 days

previously were carefully removed from the sand, and roots

15

were rinsed several times in running tap water before trans—
planting to soil infested with E, solani or to non—infested
soil.

Inoculation procedures: Fungus inoculum was prepared

 

in the following way: E, solani was grown on PDA in petri
dishes at 240C for 14-21 days. Conidia were removed from
the agar surface by adding distilled water, followed by

gentle agitation. The number of conidia was adjusted to a

standard concentration. Inoculum of E, euteiches was pre-

 

pared from mycelia grown at 240C in 250 m1 Erlenmeyer flasks
containing 50 ml of maltose—peptone broth (33). Production
of zoospores was induced by allowing 4-6 day old mycelial
mats to stand in tap water for 2 hr, followed by aeration
in distilled water for 12 hr (33). Zoospores were adjusted

to a standard concentration. E, oxysporum was grown on PDA

 

in petri dishes at 24°C. After 14-21 days both mycelia and
conidia were scraped from the agar surface and ground with
a small volume of distilled water in a Waring Blendor for 1
minute.

Pea plants were inoculated with E, solani conidia

or E, euteiches zoospores by pipetting 10 ml of the inoculum

 

suspension uniformly on the surface of the sand or soil in

the pot or row in which the peas were growing. The surface

16

of the sand or soil was immediately watered to saturation

with distilled water. Inoculation with E, oxysporum was

 

accomplished in one of two ways: 1) Pea seedlings were re-
moved from the soil, their root systems dipped into the
inoculum suspension and the seedlings replanted into auto—
claved soil. 2) Twenty ml of inoculum suspension was added
to each pot by applying 4 ml portions in each of 5 positions
with a hypodermic syringe inserted to a depth of l in. below
the soil surface.

Virus inoculum was prepared by growing virus-infected
Miragreen pea plants in the greenhouse. Leaves of plants
inoculated 10—20 days previously were ground in a mortar
together with a small amount of 0.1 M phosphate buffer and
400 mesh carborundum powder. Two expanded leaves on each
plant were dusted with carborundum and rubbed with a poly-
urethane swab containing the infected sap. Control leaves
were dusted with carborundum and rubbed with a polyurethane
swab containing 0.1 M phosphate buffer.

Evaluation 9E disease: Disease was evaluated 2—3

 

weeks after fungus inoculation by estimating the severity
of disease in foliage, epicotyls and roots (35). Tops of
plants with only the lower one or two leaves wilted were

rated 1, completely wilted tops were rated 3 and intermediate

l7

stages were rated 2. Watersoaked E, euteiches—infected

 

collars (area immediately above and below seed attachment)
were rated 1 and completely collapsed collars 2. Slight,
moderate and severe infection of collars by Fusarium was
rated 1, 2 and 3, respectively. Slight, moderate and
severe root decay was rated 1, 2 and 3, respectively, for
all diseases. The disease index was the sum of ratings

for foliage, collars and epicotyls. Thus, disease severity
was expressed on a scale of increasing severity from 0-9

for Fusarium root rot and 0-8 for Aphanomyces root rot.

 

Usually, the ratings were made for the group of plants in
each pot and not for individual plants.

Collection 9£_root exudates: When seedlings grown

 

 

in silica sand were 3-4 in. tall, half of the plants were
inoculated with a virus, followed by immediate removal of

all plants from the silica sand. The plants were treated

in either of the following ways: 1) Four-six healthy and
virus-infected plants with cotyledons excised were maintained
in 100 ml distilled water from the time of virus inoculation
through a 12-16 day period. The cultures were grown for 16
hr at 720C under 520 foot candles of light, followed by an 8
hr dark period at 560C. The water was changed daily and

conductivity of the discarded culture solution was measured.

18

2) Seedlings were transplanted into acid washed silica sand
in 4 in. plastic pots. At 1 hr intervals, 10 ml of 10-1
diluted Hoagland's solution (33) was applied to the surface
of the sand in which 6—15 seedlings were growing. The
leachate drained from holes in the bottom of the container,
and was collected in a sufficient volume of ethanol to in—
sure a final concentration of not less than 50% ethanol
after a 24 hr collection period (Figure l). Ethanol was
used to prevent utilization of exudates by contaminating
organisms.

All solutions containing root exudates were passed
through 0.45u Millipore filters and evaporated to dryness
at 600C in a flash evaporator. The ethanol soluble fraction
of the residue was removed and evaporated to dryness. The
final residue was resuspended in 10 ml of glass distilled

water. Solutions were frozen for storage.

Chemical analyses 9£_exudates: The conductivity of

 

water culture solutions was determined with a conductivity
bridge. Results were expressed in mhos, the reciprocal of
the electrical resistance of the fluid in ohms. Nucleic
acid content of root exudates was determined by ultraviolet
absorption (64). Portions of the concentrated solutions

containing root exudates were placed in a 3 ml silica

l9

 

Figure 1. Apparatus used for collectinglroot exudates
of pea growing in silica sand. Ten ml of 10— diluted
Hoagland's solution or distilled water was pumped on the
surface of the sand hourly. Leachate drained from holes in
the bottom of the container, and was collected in a suffi—
cient volume of 95% ethanol to insure a final concentration

of not less than 50% ethanol after a 24 hr collection period.

 

l—\

.1

tr)

H]

O\

(I)

(n

(D

((7

(I)

20

absorption cell and absorption at 260 mg was recorded using
a Beckman DB spectrophotometer.

Total carbohydrates were determined using a modifi-
cation of Dreywood's anthrone reagent (43). The anthrone
reagent was made by dissolving 0.4 g anthrone in 200 ml of
9.3 M sulfuric acid (190 ml concentrated sulfuric acid + 10
ml water). One ml of the solution to be assayed was mixed
with 2 ml reagent in a boiling water bath for 3 minutes.
The solutions were cooled and the optical density read at
620 mu in a colorimeter. A standard curve was made by mea—
suring 1 ml samples containing 20, 30, 60, 80 and 100 ug of
glucose. Glucose content was determined with the Glucostat
reagent (Worthington Biochemical Corporation) prepared
according to the manufacturer's directions. Optical density
was read at 400 mu in a colorimeter and compared with a
standard curve prepared from samples containing 20, 40 and
80 ug glucose.

Total amino acids were determined by the method of
Moore and Stein (42). A standard curve was made by measuring
glycine at concentrations of 4, 8 and 16 ug/ml water. Pro-
tein was determined with the Folin phenol reagent (36).
This reagent is prepared in several steps. Reagent C con—

sists of a 50: l (v:v) mixture of stock solution 1) 2% Na2C03

21

in 0.1 N NaOH and stock solution 2) 0.5%‘CuSO4-5H20 in
1% sodium tartrate. This mixture must be made fresh daily
from stock solutions 1 and 2. One ml of sample solution
was mixed with 1 m1 reagent C and allowed to stand for 10
minutes. Then, 0.2 ml Folin—Cicocalteu phenol reagent
diluted to l N with water was added rapidly to the sample
solution and allowed to stand for an additional 30 minutes
before determining optical density at 500 mu in a colori—
meter. A standard curve was made by measuring 1 ml samples
containing 25, 50 and 100 ug of bovine serum albumin.
Concentrated solutions containing root exudates or
root tissue extracts were desalted for paper chromatographic
separation of amino acids, organic acids and sugars (60).
Three cm3 of cation exchange resin (Bio Rad AG 50W—X8, H+
form) was prepared by passing 20 ml of 33% HCl through resin
in a column of 1 cm inside diam. Three cm3 of anion ex-
change resin (Bio Rad AG l—X8, formate form) was prepared
by passing 20 ml of l N NaOH through resin in a column of 1
cm inside diam. A sufficient volume of distilled water was
then passed through both columns to remove all acid or base
from the resin beds. The sample solutions were first passed

through the cation exchange resin. The resin was then

rinsed with 10 ml glass distilled water. The effluent,

22

including the rinse, was immediately passed through the anion
exchange column. This resin was also rinsed with 10 ml glass
distilled water. The effluent from the latter column was
designated the neutral fraction. The cation exchange column
was eluted with 10 m1 of l N NH4OH. The anion exchange
column was eluted with 10 ml 6 N formic acid. The neutral,
cationic, and anionic fractions were evaporated to dryness
in a flash evaporator at 600C and resuspended in 2.5—6.0 ml
10% n—propanol for spotting on chromatographic paper. All
chromatography work was done at 24-27OC.

The amino acids present in the cationic fraction
were separated on Whatman No. l chromatography paper (18 x
22 in.). The mobile phases for the 2-dimensional descending
Chromatograms were n-butanol: acetic acid: water (3:1:1
v/v), followed by phenol: water (4:1 w/v). The chromatograms
were air dried for 8-12 hr between solvent systems. Amino
acids were detected by spraying 0.5% ethanolic ninhydrin on
the develOped chromatograms, then heating at 750C for 10
minutes. Standards made from known combinations of pure
amino acids were included with each run. The Spots were
cut from developed chromatograms, submerged in 3 ml 50%

ethanol for 30 min., filtered through Whatman No. 1 paper

into a colorimeter tube, and read at 570 mu in a colorimeter.

23

A standard curve for each amino acid (15 and 50 us) was pre-
pared on paper in a similar manner.

Portions of the concentrated exudate solutions
containing the sugars (neutral fraction) were applied uniformly
to Whatman No. 1 paper (9 x 22 in.).‘ Equal volumes of root
exudate from healthy and virus-infected plants were analyzed.
N-butanol: acetic acid: water (4:1:5 v/v) was used as the
solvent in descending one-dimensional chromatography. Chro-
matograms were dried 8-12 hr and aniline- phthalate spray
reagent (Brinkmann Instrument Inc.) was used to detect
reducing sugars.

Portions of the concentrated exudate solutions con—
taining the organic acids (anion fraction) were applied
uniformly to Whatman No. 1 paper (9 x 22 in.). Equal volumes
of root exudates from healthy and virus—infected plants were
analysed. N-pentanol: 5 N formic acid (1:1 v/v) was used
as the solvent in descending one—dimensional chromatography.
Chromatograms were air dried for 8-12 hr and sprayed with
0.04% ethanolic brom-phenol blue.

Bioassays of exudates: Several bioassays were

 

utilized: 1) The rate of bacterial multiplication in water
cultures of plant roots was studied. Samples from the water

culture solution were diluted in distilled water or 0.05 M

24

phosphate buffer (pH 7.0) to 10—5 - 10-6, and 1 m1 of each

serial dilution was added to 100 mm diam petri dishes.

Fifteen ml of soil extract agar (43°C) was added to the petri
dishes, the dishes were swirled and incubated at 240C. After
3-6 days, bacterial colonies were counted. 2) The effect of

diluted root exudates on Fusarium solani f. pisi germ tube

 

growth was measured. Washed conidia were added to 1.5 ml
diluted exudate (10.6 concentrated solution) in 60 mm diam
petri dishes and incubated for 12 hr at 240C. Germ tubes

in root exudates from healthy and virus-infected plants were

measured microscopically. 3) Germination of Helminthosporium

 

victoriae conidia on natural soil adjacent to roots of

 

healthy and virus—infected plants was determined. Root
systems contained within a cellophane membrane or nylon mesh
were placed in 150 mm diam petri dishes. Moist, nonsterile
loam soil was packed around the roots, the surface of the

soil was smoothed and conidia washed 3 times by centrifugation
in glass distilled water were applied to the soil surface.
After 10-13 hr the spores were stained with phenolic rose
bengal and recovered with a solution of polystyrene (32).

The dried plastic film was transferred to a drop of mineral
oil on a glass slide, covered with a cover glass, and then

observed microscopically.

25

Phosphate uptake: Roots of healthy and virus—infected
Miragreen peas grown in silica sand were immersed in 300 ml

- 2 .
phosphate buffer (10 3 M) containing H 3 P0 or in phosphate

3 4'
buffer containing 10-4 M 2,4—dinitrophenol for 2 hr before
eXposure to H332PO4. After l-2 hr exposure to 32P, the plants
were removed from the buffer and placed in non—radioactive
phosphate buffer (10—3M) for 5 minutes. This rinse was re-
peated 2 times to remove all exchangeable 32PO4 from the root
surface. Whole roots or root tips (2 cm) were removed from
both healthy and virus—infected plants. The tissue was weighed,
evaporated to dryness in scintillation bottles, and 50 ml
scintillation fluid containing 5 g PPO (2,5—diphenyloxazole)
and 0.3 g POPOP (l, 4-bis-2-(4—methylo—5—phenyloxazolyl)-
benzene)/liter toluene was added. Radioactivity was determined

with a Packard Tri Carb liquid scintillation spectrometer.

Assay 2E virus infectivity i2 pea leaf sap: Miragreen

 

 

seedlings were grown in an autoclaved mixture of equal parts
sand and ground peat. When seedlings were 8—9 days old,
plants were simultaneously inoculated with E, solani or F.

oxysporum and PMV or BYMV. Beginning 3 days after inocula—

 

tions, leaf sap was periodically assayed for virus infectivity.
For each leaf sap assay, a 10 mm diam disc of leaf tissue

from young, newly expanded leaves was punched from each of

26

10 plants growing in a pot. The discs were ground with pestle
and mortar in 10 ml 0.05 M phosphate buffer (pH 7.0). One ml
of the crude sap was then diluted in 9 ml buffer and used to
inoculate the leaves of a local lesion host, Chenopodium

amaranticolor, Coste and Reyn. Chenopodium plants were grown

 

under shade for 7 days previous to inoculation to produce
optimum lesion development. Sap from Fusarium-infected plants
and non—fungus—infected plants was assayed on single opposite

half-leaves on 10 plants.

RESULTS

External Kim internal effect 9E virus infection 93

 

 

root rot ggsease: Experiments were designed to determine
whether the increased severity of root rot induced by virus
infection was brought about: 1) by a change in the inherent
susceptibility of root tissue; or 2) by increased inoculum
potential resulting from altered root exudates. The trans-
planting experiment used by Farley (12) was repeated.
Immediately after virus inoculation of plants inoculated 3
days earlier with E, solani, roots of inoculated and uninoc—
ulated plants were washed, and plants were transplanted to
soil infested with E, solani or to non-infested soil. Disease
ratings were made 17 days after transplanting.

Virus + fungus-infected plants had no more root rot
than fungus-only-infected plants (5% level) when both were
transplanted into non—infested soil (Table 1). However,
virus + fungus-infected peas transplanted to infested soil

had more severe root rot than transplants infected with a

27

28

fungus only. These data confirm previous results using the

fungi E, solani and Aphanomyces euteiches and the virus PMV

 

(12). The results of these tests suggest that virus infec-
tion enhances the infectivity of the fungal inoculum at the
root surface, and that the increased root rot was not due
to increased susceptibility of virus-infected tissues.

In an attempt to circumvent the influence of an
altered external environment, fungal inoculum was placed
directly into cortical tissues of the stem. When Miragreen
peas grown in silica sand were 1-2 in. high, 3 of 6 plants
in each pot were inoculated with BYMV. Eight days after
virus inoculation, all plants were inoculated by injecting
a 1 ul suspension of E, solani (106 spores/ml) directly into
the epicotyl. Disease severity was determined 21 days after
fungal inoculation. Length and severity of lesions were the
same (5% level) for virus-infected and non—virus—infected
plants (Table 2). This is further support for the conclusion
that the increase in root rot was not due to a change in
susceptibility of root tissue.

Influence 2E root exudates 92_disease development: If
root exudates are involved in virus-induced increased root

rot, removal of the exuded substances should correspondingly

29

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30

Table 2. The effect of injecting inoculum into cortical
tissue on development of Fusarium root rot in
Miragreen peaa

 

 

Lesion .
Len th Disease
9 Indexb
mm
Virus—infected plants 1.33 1.93
Non-virus—infected plants 1.26 1.61

 

a . . .

A 1 ul suspenSIon of E, solani conida (106/ml)
was injected into epicotyls immediately after virus
inoculation.

bDisease indices were based on a scale of increas-
severity from 0—3. Each figure is the mean of 39 plants.
Values for each treatment did not differ (5% level).

31

reduce the severity of root disease. When Miragreen seed—
lings growing in silica sand were 1-2 in. tall, plants in
half of the pots were inoculated with BYMV on the 2 lower
leaves. Three days after virus inoculation, a 10 ml suSpen-

sion of E, euteiches zoospores (104/m1) or a 10 m1 suspension

 

of E, solani conidia (106/ml) was pipetted on the sand
surface. At hourly intervals, 20—40 ml of deionized water

or 10-1 diluted Hoagland's solution was added to each pot.
The solution saturated the sand and the excess fluid flowed
out of the pots through perforations in the bottom. Plants
grown in silica sand without leaching served as controls.
Fourteen days later, plants were removed and disease severity
determined.

Root rot severity in virus-infected and non-virus-
infected plants subjected to hourly leaching, and in non—leached
plants with Fusarium only, was the same (Figure 2) in 5 tests.
However, root rot severity in virus—infected plants not
subjected to leaching was increased. These results suggest
that root exudates play an important role in the mechanism of
increased root rot. If this assumption is valid, addition of
root exudates from virus-infected plants to plants inoculated

with a root rot fungus should result in increased disease.

32

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5 "' ‘—
>< 4 ‘ "
“J
0
Z
— 3 -
m x .
U)
<
Lu 2 P
m
C)
I P
O
BY+MV FUS. BYMV FUS. P§V FUS. P'fv FUS.
+
FUS. FUS. F US. FUS.

LEACHED NON-LEACHED LEACHED NON-LEACHED

Figure 2. Effect of leaching roots of plants infected
With BYMV or PMV on susceptibility to Fusarium root rot.
Fourteen days after fungus inoculation, plants were removed
and disease severity was determined on a scale of 0-9. The
Only increased disease occurred in virus + fungus—infected
plants not subjected to root leaching. Similar results
Were obtained with BYMV in 4 additional tests.

33

Four in. plastic pots containing 10 E, solani—
infected Miragreen seedlings growing in silica sand were
positioned below the drainage hole in crocks containing
healthy or virus-infected Miragreen plants (Figure 3). In
an experiment with BYMV, fungus—infected plants received
leachings from 10 BYMV-infected or healthy plants. Fifty
m1 of deionized water was added daily over a 10 day period.
In an experiment with PMV, fungus—infected plants received
10 ml of leachate hourly from 10 PVM-infected or healthy
plants over a 17 day period. In another experiment with
PEMV, fungus-infected plants received 10 ml leachate from
20 PEMV—infected or healthy plants over a 10 day period.
Fungus-infected plants receiving leachates from crocks con—
taining only silica sand, and fungus—infected plants
receiving no leachates served as controls. Disease severity
on fungus-infected plants was determined 17 days after
inoculation.

In all experiments, irrespective of the virus used,
those plants which received leachates from virus—infected
plants had more severe disease (5% level) than plants re—
ceiving leachates from healthy plants (Figure 4). Similar

results were obtained in 2 additional tests. This evidence

34

    

\

  

Figure 3. System used to test effects of root exudates
from pea roots on Fusarium root rot development in eas. At
hourly intervals, 20—40 ml ofdeionized water or 10‘ diluted
Hoagland's solution was added to the surface of each pot
containing healthy or virus—infected plants. Excess fluid
flowed out of the pots through perforations in the bottom.
Pots containing 10 E. solani—inoculated Miragreen seedlings
growing in silica sand were positioned below drainage hole
in crocks.

35

 

 

 

 

 

9 b _
umm
x a - Ezmsnmn CONTROL -4
cans. + HEALTHY mnxumm
g 7 __ mm. + vmus murmur d
36 _
5 r
I.“
m4 "
f, 3
m
- 2
C3
1
0
1A B (I D A» B C I) ,A B (I D
BYMV PEMV PMV

Figure 4. Effect of leachates from pea roots on
Fusarium root rot of Miragreen pea. In the experiment
with BYMV, plants received leachings from 10 BYMV-infected
or healthy plants which were watered with 50 ml of deionized
water daily over a 10 day period. In the experiments using
PEMV, plants received 10 ml of leachate hourly from 20 PEMV—
infected or healthy plants over a 10 day period. In the
experiment with PMV, plants received 10 ml leachate hourly
from 10 PMV—infected or healthy plants over a 17 day period.
Disease severity was determined on a scale of 0-9. In all
experiments, plants which received leachates from virus-
infected plants had more severe disease (5% level) than
plants receiving leachates from healthy plants. Similar
results were obtained in 2 additional tests.

36

is further indication that the mechanism of increased root
rot in virus—infected peas is related to root exudates.

Changes Eg_exudation patterns: Water cultures:

 

 

Direct evidence of increased release of substances from roots
was sought. Root exudates collected from virus—infected and
healthy plants grown in water were analyzed at 2-day inter—
vals for electrolytes, carbohydrates, amino acids and
nucleotides.

An increased release of electrolytes from roots of
BYMV—infected plants, as compared with healthy roots, began
4—6 days after virus infection (Figure 5). Comcomitant with
increased electrolyte release from virus-infected roots was
an increased nucleotide (Figure 6), carbohydrate (Figure 7)
and amino acid (Figure 8) content of the solutions in which
virus-infected plants had grown. Similar results were
obtained in 2 additional tests. The increased rate of
release of these substances continued until 14-16 days after
virus inoculation when levels returned to those for healthy
plants. The substances detected are assumed to be primarily
nutrients not yet assimulated by the microflora in the solu—
tions, and are considered to reflect a differential release
of substances by roots of virus—infected and non—virus-

infected plants. Of the 4 categories of substances assayed,

37

 

S

"I
O
I

BYMV‘ 4
./////,\\\\\._____.____..

.. HEALTHY,

&
O
I

U
0
I

1

 

CONDUCTIVITY (IO‘MHOS)

2 A 6 8 13 1.2 114 1‘6
DAYS AFTER VIRUS INOCULATION

 

 

Figure 5. Effect of BYMV-infection on amount of
electrolytes exuded by roots of pea plants growing in
water cultures. Conductivity of water culture solutions

was determined daily. Similar results were obtained in
2 additional tests.

38

 

 

 

 

:.
r- . -I
2 2 \
CD BYIAVWSK .
0 . o><2
N )- \.\ u
2 .
o o
g II- 0/ \0/ d
E
0- “HEALTHY
a: ,_ A
U)
:0
< O 1 l l 4 4 l l 1
2 4 6 8 10 12 14 16
DAYS AFTER VIRUS INOCULATION
Figure 6. Effect of BYMV—infection on amount of
nucleotides exuded by roots of pea plants growing in
water cultures. Nucleic acid content was determined

by ultraviolet absorption at 260 mu. Results are from
I of 3 experiments, all with similar results.

39

 

4° ' IIYMVSt \

3w /><. a, 03> I

 

 

GLUCOSE EQUIVALENT (HG)

 

° HEALTHY
20 - .
‘0 1- " d
o A 4 A L A A L L

1-2 3-4 5'6 7-8 9-10 11-1213'1415-16
DAYS AFTER VIRUS INOCULATION

Figure 7. Effect of BYMV—infection on amount of
carbohydrates exuded by roots of pea plants growing in
water cultures. Carbohydrate content of concentrated
exudate solutions was determined using amodified Dreywood's
anthrone reagent. Similar results were obtained in 2 addi—
tional tests.

40

 

 

 

 

 

0

:340 " 7

h— 0

2

11.130 ~ BYMvsk .o\ . ‘

u-l

< ./

Z

3 . .

02° .

LU

LL]

2 10 I .

- O

g.) oHEALTHYz\o__ __o o

_.. -—____——_=8

0 o J 4 I l
1-2 3-4 5- 6 7-8 9-10 11-12 13-14 15-16

DAYS AFTER VIRUS INOCULATION

Figure 8. Effect of BYMV—infection on amount of amino
acids exuded by roots of pea plants growing in water cultures.
Amino acid content of concentrated exudate solutions was
determined with ninhydrin reagent. Similar results were
obtained in 2 additional tests.

41

amino acids had the greatest relative increase in exudation
(Figure 9). Carbohydrate and amino acid content of water
cultures used to grow healthy Miragreen peas showed an
approximate release of 25 ug of carbohydrates and 8 ug of
amino compounds per plant each day. During the period
between 4 and 16 days after BYMV infection, a 100—150%
increase in amino acids exuded from roots of virus-infected
plants can be compared with a 20-40% increase in release of
carbohydrates and 30-40% for nucleotides (Figure 9).

Similar increases in exudation of electrolytes,
carbohydrates and amino acids were observed with peas in-
fected with PMV and PEMV. The increased release for PEMV
began 6-8 days after inoculation and for PMV, 6—10 days
after inoculation. The increased exudation had not declined
16 days after virus inoculation with either PEMV or PMV.

Sand cultures: The nucleotide, amino acid, carbohy-

 

drate and protein content of root exudates collected from
plants grown in acid washed silica sand was determined.

An increased release of amino compounds from roots
of BYMV-infected plants began 4—6 days after virus inocula-
tion (Figure 10). The rate of exudation decreased to the
level of healthy controls 13—14 days after virus inoculation.

Similarly, increased exudation of carbohydrates (Figure 11)

42

WATER CULTURE

 

    

 

 

._l
A: CONDUCTIVITY ..
O 250 B=ABSCRPTION AT 260 I41
0: C=AMINO ACIDS
'— D=CARNHYDRATES
Z
O
K) 200
>.
J:
l.—
_J
<( ISO
u;
I:
u.
0
59 100
A B C D A B C D A B C D
P MV 8 Y M V P E M V
Figure 9. Increased exudation of electrolytes, nucleo—

tides, carbohydrates and amino compounds by virus—infected
Miragreen pea plants in water culture relative to the
exudation by non—infected plants taken as 100%. Values are
the average daily release of each for the period from virus
inoculation through 16 days later. Similar results were
obtained for BYMV in 4 additional tests.

43

SAND CULTURE

 

 

 

 

A 400-0 4
0
3 . .
.—
55300. .
—l
< I- .
>
3200- .
8 {-BYMV

" '1
LU
Z100- .1
S
5 - /H£ALTHY?'\O— \:>-e

0 1

0-2 3-4 5-6 7-8 9-10 11-12 13-14
DAYS AFTER VIRUS INOCULATION

Figure 10. Effect of BYMV—infection on amounts of
amino acids exuded by roots of pea plants growing in sand
culture. Amino acid content was determined at 2 day inter-
vals with ninhydrin reagent. Similar results were obtained
in another test.

44

SAND CULTURE

1 t l T I I W

 

5?

_a

U!

0
I

§

 

 

VI
0
T

HEALTHY)

 

 

l L l l A L I

0 0'2 3'4 5'6 7-8 9-10 "-12 I344
DAYS AFTER VIRUS INOCULATION

 

GLUCOSE EQUIVALENT (116)

Figure 11. Effect of BYMV-infection on amount of
carbohydrates exuded by roots of pea plants growing in
sand cultures. Carbohydrate content was determined at
2 day intervals using a modified Dreywood's anthrone
reagent. Similar results were obtained in another test.

45

and nucleotides occurred during this period. However, the
relative increase in amount of these compounds released was
never as great as for the amino compounds. Similar results
were observed in another test.

Increased root exudation induced by PMV infection
began 6-8 days after virus inoculation. Roots of BYMV-infected
or PMV-infected plants released 4—5 ug of protein each day,
the same as that released by healthy plants (5% level). A
rather large quantity of all substances was released by both
healthy and virus—infected plants 0-2 days after transplant—
ing. This can be attributed to the shock of transplanting
into acid washed sand since it did not occur with plants
grown in water cultures.

Roots of healthy Miragreen peas growing in silica
sand exuded approximately 25 ug of both carbohydrates and
amino compounds per plant each day. During the period
between 4 and 14 days after BYMV infection, the average in—
crease in amount of nucleotides, carbohydrates and amino
compounds was 22%, 55% and 137 %, respectively. Similar
results were obtained in another tests using BYMV infection.

Although the actual quantity of substances exuded
by roots differed in sand and water cultures, the change in

root exudation induced by virus infection was similar in

46

both systems. The quantity of carbohydrates released per
plant was the same in both water and sand cultures. Also,
the periods of time during which increased exudation from
roots of virus-infected plants occurred were the same. How—
ever, the quantity of amino compounds released per healthy
plant in water culture was approximately 33% that of sand
culture. Possibly, this difference resulted from lower light
intensity or to increased utilization of nitrogenous compounds
by microorganisms in water cultures. Total amino compounds
in the exudate increased more than did total carbohydrates
or nucleotides in both water and sand cultures.

Relationship between exudation period and disease:
If the increased root rot in virus-infected peas is a result
of increased nutrients in the rhizosphere of virus—infected
plants, the increased root rot should occur only during the
period of increased exudation.

Ten Miragreen seeds were planted in a mixture of
equal parts ground peat and sand in 4 in. plastic pots at 3
day intervals. When seedlings were 8 days old, the plants
in half the pots were inoculated with BYMV. At the time of
virus inoculation in the last-planted series of pots, plants
in all pots were inoculated with a 20 m1 suspension of E,

solani conidia (106/ml). Thus, the intervals established

47

between virus and fungus inoculations were 0, 3, 6, 9, l2,
and 15 days. Disease severity was determined 17 days later.
In another experiment, plants sown at the same time were
inoculated with BYMV at different intervals of time. Plants
were equally old at time of fungus inoculation. The previous
intervals between virus and fungus inoculation were repeated.

The increased root rot induced by virus infection
occurred only when fungus infection followed virus infection
by 9 days or less (Figure 12). After this time, root rot
severity was the same in virus + fungus—infected peas and in
fungus-only-infected controls. Similar results were obtained
in 2 additional tests using BYMV.

Root washing experiments were designed to determine
whether the correlation between increased root exudation and
increased root rot could be extended to the early stages of
virus infection before increased root exudation was evident.
Eight—day old Miragreen seedlings growing in silica sand were
inoculated by pipetting a 10 ml suspension of E, solani
conidia (106/m1) on the sand surface. Plants in half the
pots were simultaneously inoculated with BYMV. Plants from
the virus + fungus treatment and fungus—only controls were
removed from the pots immediately after inoculation, and at

3 and 6 day later, then washed thoroughly under running tap

48

 

 

 

 

>< 6 A -
I“
0
E 5 -
LU
‘2 4 -
[U
u:
o 3 .
.Z
— 2 b
“J
u:
'< I -
“J
a:
‘2’
- 0
0 3 6 9 12
DAYS BETWEEN VIRUS AND FUNGUS INOCULATION
Figure 12. Relation between root rot severity and

time interval between inoculation of Miragreen peas with
BYMV and Fusarium solani. Values are expressed as the
increase in disease index of fungus + virus—infected plants
over fungus-infected plants of the same age. The increased
root rot induced by virus infection occurred only when
fungus infection followed virus infection by 9 days or less.
Disease severity was determined on a scale of increasing
severity from 0-9.

49

Water to remove adhering spores and mycelia. The plants were
replanted in non—infested silica sand. Disease severity was
determined 17 days later. In another experiment, plants sown
at the same time were inoculated with BYMV at different inter-
vals of time. Immediately after the last series of plants was
inoculated with BYMV, a 10 ml conidial suspension (106/m1) of
E. solani was pipetted on the sand surface. Three days later,
all plants were removed from the pots, roots were washed
thoroughly under running tap water, and plants were replanted
in non—infested silica sand. Disease severity was determined
21 days later.

The increased root rot did not occur if the fungus
was removed before the fourth day after virus inoculation,
when increased exudation was known to first occur (Figure 13).
However, if the fungus was present for an additional 1-2 days
after increased exudation began, i.e., through 6 days after
virus inoculation, increased root rot did occur (Table 3).
Thus, a good correlation exists between the time increased
exudation from virus—infected plants first occurs and the
time when virus-infected plants show increased root rot.

These data indicate that increased root rot induced
by virus infection in peas occurred only during the period of

increased exudation from roots of virus—infected peas.

50

 

INDEX

DISEASE

 

 

 

—
FUS. 0 3 6 9 12
DAYS BETWEEN VIRUS AND FUNGUS INOCULATION

 

Figure 13. Relation between root rot severity and time
interval between inoculation of Miragreen peas with BYMV and
Fusarium solani. Plants sown at the same time were inoculated
with BYMV at different intervals of time. When the last
series of plants was inoculated with BYMV, all plants were
inoculated with E. solani. Three days later plants were
removed from pots, roots were washed, and plants were replanted
in non—infested sand. Fusarium—only—inoculated plants (Fus.)
served as controls. Disease severity was determined on a
scale of increasing severity from 0—9. The increased root
rot did not occur if the fungus was removed before the 6th
day after virus inoculation, when increased exudation was
known to first occur. Bars joined by a common line did not
differ (5% level).

51

Table 3. Relationship between Fusarium root rot severity
and the increase in root exudation beginning 4-6
days after virus inoculationa

 

Disease indices for plants
infected with indicated

 

 

pathogensb
Time of fungal removal Fungus Fungus
from root surface only +BYMV
0 days 1.3 1.5
3 days 2.0 2.0
6 days 4.3 8.0

 

a . . . .

Plants were Simultaneously inoculated With Virus
and fungus. At the indicated intervals plants were uprooted,
roots were washed, then transplanted into non—infested sand.

Disease indices were based on a scale of increasing
severity from 0-9. Each figure is the mean of 3 pots, each
with 10 plants. The only increase in disease index (5%
level) with virus infection occurred at 6 days.

52

Biological activity 9§_root exudates: One indication

 

 

of an increased release of nutrient substances from roots of
virus—infected as compared with healthy plants would be the
amount of bacterial growth in water cultures of plants. Dilu—
tion plate counts made daily from such culture solutions
generally showed a 2—3 fold increase in number of bacteria
associated with BYMV or PMV infection. This increase in
bacteria became evident 6-8 days after inoculation and con-
tinued until the experiment was terminated 16 days after
virus inoculation. Similar results were obtained in 2
additional tests.

The effect of diluted root exudates on the growth
rate of germ tubes of E, solani f. pig} was measured. Con—
centrated leachate from roots of BYMV-infected and healthy
plants grown in sand was serially diluted to 10-6 the original
concentration. Germ tubes of E, solani grew 32% longer in
12 hr in exudates from roots of virus-infected plants than
in exudates from roots of healthy plants (Table 4). Thus,
the exudate from virus-infected plants contains more growth
supporting substances than exudates from healthy plants.
Similar results were obtained in another test.

The germination of Helminthosporium victoriae conidia

 

Was observed on natural soil adjacent to roots of both healthy

53

The effect of root exudates from healthy and

 

 

Table 4.
virus-infected plants on growth of E, solani
f. pisi germ tubesa
b
Germ tube lengths, u
' ' BYMV-infected
Test Healthy plants plants

 

1 280 379
2 242 312
3 252 318

 

aExudgtes from plants grown in silica sand were
diluted to 10- original concentration. Duplicate plates
were used in each test, and germ tube lengths of 20 spores

were measured per plate.

bGerm tubes grew longer in root exudates from virus-
infected plants (5% level).

54

and virus-infected plants. Roots of 24-day-old plants in—
fected with BYMV for 4, 8, 12 and 16 days were compared with
healthy roots in their influence upon conidial germination
over a 4-7 cm distance from the roots (Figure 14). Roots of
plants infected with BYMV for only 4 days stimulated germina—
tion of conidia to the same extent as did healthy plants.
When plants were used 8 days after virus inoculation,
germination of conidia increased over that observed on soil
adjacent to healthy controls. Moreover, the stimulation
extended over a 3 cm distance from the roots. The highest
germination of conidia and the greatest distance of stimulation
occurred with plants 12 days after virus inoculation. At 16
days after virus inoculation, germination and distance of
stimulation were markedly reduced. The periods of greatest
stimulation of conidial germination coincided with the period
of increased root exudation from virus—infected plants.
Similar results were obtained in 3 additional tests using BYMV.

These data indicate that virus infection may result
in an increased stimulation of fungal germination and growth
over a longer distance from the root surface as compared with
healthy plants. The actual distance over which stimulation
was observed in these tests may be an artifact, however,

resulting from the continual moisture film produced by

 

60 1-

TIME AFTER VIRUS
so - ‘12 DAYS INOCULATION .
4o - .

30 - I
A'SDAYS \
I

20 - o a
K16 DAYS \
10 -.’ AEMJMHS ::::::2

AO< A \ /0\
HEALTHYq" ‘3 L§§5§3
l J

o 1

DISTANCE FROM ROOTS (cm)

GERM I NATION (9;)

 

 

 

 

 

 

”b
uni)

Figure 14. Germination of Helminthosporium victoriae
conidia on soil adjacent to roots of healthy and virus—
infected plants. Roots of plants infected with BYMV for 4,
8, 12 and 16 days were contained within a cellophane membrane
in petri dishes. Soil was packed around the roots, and
conidia were applied to the smoothed soil surface. Highest
germination of conidia and greatest distance of stimulation
coincided with the period of increased root exudation from
virus—infected plants. Similar results were obtained in 3
additional tests.

 

56

smoothing the soil surface.

Chromatographic analy§is: Amino acids: Portions of

 

 

the concentrated cationic fraction collected through a 6-12
day period after virus infection were chromatographed.

Based on the total quantity of ninhydrin positive compounds
in the root exudate, equivalent quantities of the amino acid
fractions of healthy and BYMV-infected rooI exudate solutions
were applied to the paper.

Seventeen ninhydrin-positive spots appeared on
chromatograms of virus-infected root exudates while 14 spots
were detected on chromatograms of healthy root exudates.

Ten of these were identified on each and the amounts deter-
mined (Table 5). The unidentified spots on the chromatograms
were estimated to represent less than 5% of the total
ninhydrin positive substances present. All amino acids were
released in greater quantities from roots of virus—infected
plants than from healthy roots. The total increase was 3-4
fold that of healthy plant exudates. The concentration of
alanine increased the most in relation to other amino acids.
Alanine was 4% of the total amino acids in healthy plant
exudates but increased to 21% of the total in virus-infected
plant exudate. Thus, the absolute amount of alanine increased

20-fold in root exudates from virus-infected plants. The

57

Table 5. Amino acid composition of exudates from roots of
healthy and virus—infected Miragreen peas grown in
silica sanda

 

BYMV-infected

Healthy plants plants

 

 

ug/plant/ % of ug/plant/ % of

 

Amino acids dayb total dayb total
asparagine 1.3 12 2.9 7
aspartic acid 0.2 l 0.4 1
glutamic acid 0.2 l 0.4 1
glutamine 0.6 4 1.5 4
serine 0.2 l 0.7 2
tryptophane 0.8 7 3.6 9
Y—amino butyric acid 3.8 35 5.8 - 15
alanine 0.6 4 8.4 21
threonine 3.8 35 13.1 33
phenylalanine 0.0 0 2.9 7

 

aAmino acids were detected with 0.5% ethanolic ninhy—
drin, spots were cut from developed chromatograms, eluted in
50% ethanol and compared with a standard curve for each amino
acid prepared on paper in a similar manner.

bEach value represents the average daily amount
exuded by 15 plants during the period 6-12 days after virus
infection. Similar results were obtained using exudates
from another experiment.

58

respective relative concentrations of Y—amino butyric acid

and asparagine were 35% and 12% of the total in healthy plant
exudates and 15% and 7% in virus-infected plant exudate.
However, the total amounts of Y-amino butyric acid and aspara—
gine in root exudates from virus-infected plants were
increased by 53% and 123%, respectively. Threonine was the
most prominent amino acid in root exudates from BYMV-infected
plants. Although its concentration in relation to other

amino acids was the same in both exudates, the increase in

the exudate from virus—infected plants was 263% as compared
with healthy root exudates. A striking difference was the
presence of phenylalanine in root exudates from virus-infected
plants and its absence in root exudates from healthy plants.
The results show that, in addition to a general increase in
exudation of all amino acids from roots, selective increases
in specific amino acids also occur. Similar results were
obtained with exudates from another experiment.

Amino acid content of root tissue was examined to
determine if changes in exudation of these compounds reflected
changes in the free amino acid pool in the tissue resulting
from virus infection. Root tissues from virus-infected and
healthy pea were extracted with distilled water on hot 80%

ethanol. Tissue extract was prepared and chromatographed by

59

the same method used for root exudates. Analyses of total
carbohydrates and amino acids indicated no differences (5%
level) in total soluble pools of either class of compounds
over a 20 day infection period (Table 6). Similar results
were obtained in 3 tests. Virus-infected and healthy Mira—
green plants both contained approximately 2.4 mg free amino
acid and 7.1 mg carbohydrate per root. In this test, amounts
of individual amino acids were similar in virus-infected and
healthy tissue. Phenylalanine was present in root tissues

of both virus—infected and healthy pea (Table 7). The 3—4
fold increase in amount of amino acids exuded from roots of
virus-infected plants cannot be explained by changes in total
quantities or in specific amino acids within root tissue.

Ogganic acids: Four major groups of organic acids

 

were separated (Table 8). The only detectable increase in
organic acids released from virus-infected plants occurred
among those compounds having an Rf value of 0.02. This group
includes gluconic, galacturonic and glucosaccharic acids (7).
No further attempt was made to identify these compounds.
Samples of the concentrated solutions were titrated with
0.0144 N NaOH using phenolphthalein as an indicator (60).

The acid equivalent in root exudate from virus—infected plants

was 140% that of healthy exudate. Similar results were

60

Table 6. Total carbohydrates and free amino acid pools in
healthy and BYMV-infected Miragreen pea root
tissue

 

mg/g/pea root tissuea

 

Infected with

 

virus for Amino acids Carbohydrates
4 days 2.4 6.0
8 days 2.1 7.7
12 days 2.9 10.2
16 days 2.5 6.5
20 days 2.3 6.0
Healthy 2.3 6.3

 

aValues are the average for 10 plants. Amino acid
content was determined by the ninhydrin reagent, carbohyl
drate content by the anthrone reagent. The quantities of
amino acids or carbohydrates in root tissues of healthy
plants did not differ from those in plants infected with
BYMV (5% level). Similar results were obtained in 2
additional tests.

61

Table 7. Free amino acid pool in 20-day-old root tissue of
healthy and virus—infected Miragreen pea after
virus inoculationa

 

BYMV—infected

Healthy plants plants

 

 

ug/plgnt/ % of ug/plant/' % of

 

Amino acids day total dayb total
asparagine 2.3 30 2.3 26
aspartic acid 0.2 2 0.3 4
glutamic acid 0.1 1 0.2 2
serine 0.1 l 0.1 l
tryptophane 0.3 5 0.4 5
Y-amino butyric acid 0.1 l 0.1 l
alanine 0.1 l 0.2 2
threonine 2.8 41 4.5 52
phenylalanine 0.3 4 0.6 6

 

aAmino acids were extracted by grinding 2 g root
tissue in a mortar with 10 ml of 80% hot ethanol. Amino
acids wereébtected with 0.5% ethanolic ninhydrin, spots
were cut from developed chromatograms, eluted in 50% ethanol
and compared with a standard curve for each amino acid pre-
pared in a similar manner.

bValues are the average for 10 plants. The average
plant root weight was 3 grams.

62

 

 

 

Table 8. Organic acids in exudates from roots of healthy
and virus-infected Miragreen peas grown in
silica sand

Diameter of spot on
chromatogram, mma
Rf _ possible Healthy BYMV-infected
value Compounds plants plants
.02 gluconic, glucosaccharie 12 18
and galacturonic acids
.45 glycolic, diglycolic, 15 15
and ketoglutaric acids
.69 pyruvic, aconitic, 17 17
and citraconic acids
.93 furoic, mesaconic, 8 8

and sorbic acids

 

inoculat
tests.

aEach value represents the average content from 2.3
plants each day during the period 6-12 days after virus

ion.

Similar results were obtained in 2 additional

63

in 2 additional tests and with root exudates from PMV-

infected pea.

Reducing suggrs: When root exudates from 16-20 day

 

old healthy or virus-infected Miragreen seedlings were chro—
matographed, reducing sugars were either not present or were
present in such low concentrations that they could seldom be
detected. Results with Glucostat reagent indicated an average
release of 0.88 ug of glucose by healthy plants each day
during this period, whereas roots of virus—infected plants
exuded an average of 1.76 ug per plant each day during this
period.

The effect 9£_virus infection 9g phosphate ion uptake:

 

 

 

The similarity in the size and composition of the free amino
acid pools of healthy and virus—infected plants indicated
that the increased release of amino acids from roots of virus-
infected plants was not due to alterations in the concentrations
of these substances within the root tissues. The possibility
that the increased exudation was related to increased perme-
ability of cell membranes to small molecular compounds was
investigated.

Roots of Miragreen peas infected with BYMV for 12
days accumulated two times more radioactivity than did healthy

roots, when both were immersed in a buffer solution containing

64

2
H33 PO4 for 2 hr (Table 9). In two other experiments, plants

32
exposed to P04 for 1 hr accumulated 24—26% more radioactiv-
ity than did healthy roots. Two explanations for increased
uptake were considered: 1) Permeability of plasma membranes

was increased, or 2) active uptake of P0 was increased due

4
to virus infection. Pretreatment of roots with 2,4-dinitro-
phenol (10-4M), an uncoupler of oxidation from phosphorylation,
reduced accumulation of 32P04 in both virus—infected and
healthy roots by 11-14% as compared with buffer control.

Since the reduction in each was proportional, no selective
decrease occurred in the case of virus-infected plants

(Table 10). These data indicate that PO4 accumulation by

pea roots is not energy dependent and suggests that the
increased uptake of phosphate by roots of virus—infected
plants was due to increased permeability of plasma membranes.
Probably, the increased leakage of substances from roots of
virus—infected plants is also due to increased membrane per—

meability.

Effect 92 glucose, amino acids and ogganic acids 93

 

root rot: Increased quantities of carbohydrates, organic
acids and amino acids were released by roots of virus-infected
plants as compared with those of healthy plants. When root

exudates containing these substances were added to plants

65

. . . 2
Table 9. The effect of Virus infection on uptake of 3 P by
root tissue of Miragreen peaa

 

CPM/g dry weight of

 

Treatment roots (x 1,000)b
Virus-infected plants 2,182
Non-virus—infected plants 1,064

 

aRoots were exposed to 32F in 10.3 M PO buffer (pH 7.0)

for 2 hr. Plants were inoculated 12 days previously with
BYMV.

bValues are the mean of 3 replicates. Roots of virus-
infected plants accumulated more radioactive 32F (5% level)
than did healthy roots. Similar results were obtained in 2
additional tests.

66

Table 10. The effect of 2,4-dinitrophenol on uptake of
P by root tissues of virus-infected and
healthy Miragreen peaa

 

CPM/g wet weight of
roots (x 1,000)b

 

 

Pretreatment Healthy BYMV'lnfeCted
plants plants
PO4 buffer 36 44
DNP in PO4 buffer 31 39

 

aPlants inoculated 8 days previously with BYMV were
placed in 10'3 M P04 buffer (pH 7.0) containing 2,4—dini—
trophenol (10"4 M) for 2 hr or in buffer alone for 2 hr
before a 1 hr exposure to 32F in 10"3 M P04 buffer.

bValues are the mean of 2 replicates of 3 plants each.
Roots of virus-infected plants accumulated more radio—
activity (5% level) than did healthy roots.

67

inoculated with E, solani or E, euteiches, increased root

 

rot occurred. Therefore, the prominent compounds occurring
in the root exudate from virus-infected plants were used to
prepare a simulated root exudate which was applied to
inoculated plants. Miragreen seedlings growing in moist
silica sand were inoculated with a 10 ml suspension of E,
solani conidia (106/m1). Daily for 8 days after inoculation,
each pot received a 10 ml water solution containing: 6 mg
glucose; 3 mg galacturonic acid and 3 mg glucuronic acid;

3 mg of each of the following amino acids: asparagine,
alanine, Y—amino butyric acid, tryptophane, threonine, and
phenylalanine; and a combination of all these compounds.
Thus, each pot containing 10 plants received approximately
the amount of each substance released by 40 virus-infected
plants (based on analysis of exudates from plants grown in
sand).

Disease severity was the same (5% level) in plants
receiving glucose or organic acids as in plants receiving
only water (Figure 15). Disease severity of plants receiv-
ing amino acids or a combination of all compounds was 2—
fold greater than plants receiving only water (Figure 16).
Similar results were obtained in 2 additional tests.

Substitution of glutamine for phenylalanine did not alter

68

 

Figure 15. Fusarium root rot development on Miragreen
peas receiving glucose, organic acid or amino acid amend—
ments. Daily for 8 days after fungus inoculation, 6 mg of
glucose (left), 3 mg of each of 2 selected organic acids
(center), and 3 mg of each of 6 amino acids (right) were
pipetted in 10 ml of water, individually or in combination
on 10 plants growing in silica sand. Symptoms on plants
receiving only water did not differ from those on plants
receiving glucose.

69

 

INDEX

DISEASE

 

 

   

G L U. CON T. O.A. A.A. COMB.

Figure 16. Effect of glucose, amino acids and organic
acids on development of Fusarium root rot of peas. Daily
for 8 days after fungus inoculation, 6 mg of glucose (G1u.),
3 mg of each of 2 selected organic acids (O.A.), and 3 mg
of each of 6 amino acids (A.A.) were pipetted in 10 ml of
water, individually or in combination (Comb.) on 10 plants
growing in silica sand. Similar results were obtained in
2 additional tests. Bars joined by a common line did not
differ (5% level). Disease severity was determined on a
scale of increasing severity from 0—9.

70

the stimulatory effect of this group of amino acids on root
rot severity; therefore, phenylalanine was not a critical
factor in increased inoculum potential. Thus, the increase
in inoculum potential of E, solani can be explained on the
basis of a general increase in amino acids exuded from roots
of virus—infected plants.

Effect 9£_fungus infection 9E plants 93 the virus:

 

 

In view of the striking effect of virus infection on develop-
ment of fungal root disease of pea, it was of interest to
determine whether fungus infection would in any way alter
virus infection or virus content in the plant.

Infection with E, solani f. pE§E_caused an early
increase in infectivity of BYMV or PMV in pea leaf sap. A
2-3 fold increase in local lesions was noted using BYMV + E.
solani-infected leaf sap beginning 5 days after inoculation.
Beyond 12 days from initial inoculation, however, no differences
in virus titer could be determined. Infection with E, solani
resulted in a 2-3 fold increase in PMV infectivity between
the third and fourth day after inculation. Thereafter, leaf
sap from virus-infected or fungus + virus—infected plants
was equally infective.

Infection of pea with E, oxysporum f. pisi failed to

 

influence the infectivity of virus in pea leaf sap. Moreover,

71

observations from a single test indicate that BYMV infection

had no effect on Fusarium wilt disease.

Role of root exudation in varietal resistance to root

 

 

 

£93: Data presented in this research indicate that severity
of root rot in pea is increased when the pathogen is exposed
to increased amounts of root exudates before or during infec-
tion. This concept of inoculum potential as a factor in root
rot severity was extended to 2 experimental pea varieties
being used in a program of breeding for disease resistance
(Lockwood and Beute, unpublished results). In greenhouse

tests, variety 333 has moderate resistance to Aphanomyces

 

root rot, and variety GW—4 possesses moderate resistance to

both Aphanomyces and Fusarium root rots. Root exudates from

 

Miragreen pea, which is susceptible to both diseases, were
compared with those of the 2 experimental varieties. Plants
were grown in glass-distilled water.

Both experimental varieties released fewer electrolytes
than did Miragreen (Figure 17). Total amino acids in culture
solutions of the variety GW-4 were only 53%, and those of
variety 333, 75% of those from Miragreen. However, both
resistant varieties released 24—31% more carbohydrates than
did Miragreen. Thus, if nutrients in the rhizosphere are a

factor in the relative susceptibility of these experimental

72

 

140 ~ -
120

A 100

I:

V so
so
40

 

 

 

A B C A B C A B C

CONDUCTIVITY CARBOHYDRATES AMINO ACIDS

Figure 17. Relative exudation of electrolytes, carbo—
hydrates and amino acids by healthy A) Miragreen pea, which
is susceptible to both Aphanomyces and Fusarium root rot,
B) Variety 333, resistant to Aphanomyces root rot, and C)
Variety GW~4, resistant to both pathogens. Plants were
grown in water culture. Values are the average daily
exudation over a 16 day period expressed as a percentage
of Miragreen.

73

pea varieties to root rot disease, the decreased release of
amino compounds, but not carbohydrates, would be implicated
in decreasing the inoculum potential in the root area. The
specific compounds present in these exudates were not deter-
mined.

The effectiveness of root leachings from each of the

3 pea varieties on Aphanomyces or Fusarium root rot was deter-

 

mined. Each pot containing 5, euteiches-infected Miragreen

 

seedlings received root leachings from 20 plants of one of
the 3 pea varieties, while each pot of E, solani—infected
Miragreen seedlings received root leachings from 30 plants
of one of the 3 pea varieties.

The disease index for E, euteiches in Miragreen

 

seedlings receiving root leachings from healthy Miragreen
seedlings was more than double that of seedlings receiving
no root leachings (Figure 18). The disease index for E.

euteiches in Miragreen seedlings receiving root leachings

 

from either variety 333 or GW-4 did not increase (5% level)
above plants receiving no leachings.

Similarly, the disease index for E, solani in Miragreen
seedlings receiving root leachings from healthy Miragreen
plants was almost double that of seedlings receiving no leach-

ings (Figure 18). The disease index for E, solani in Miragreen

74

 

INDEX

DISEASE

 

 

 

A B C D A B C D
A.EUTHCHE$ F SOLANI

Figure 18. Effect of root leachates from 3 pea
varieties with different resistances to Aphanomyces or
Fusarium root rot on root rot severity in Miragreen
peas. A) Plants received leachates from sand. B)
Plants received root leachates from Miragreen plants.
C) Plants received root leachates from Aphanomyces
resistant variety 333. D) Plants received root leach—
ates from variety GW—4, resistant to both pathogens.
Fusarium root rot was increased (5% level) by leachates
from both Miragreen plants (B) and Aphanomyces resistant
plants (C). Aphanomyces root rot was increased only by
leachates from Miragreen pants (B). Disease severity
was determined on a scale of increasing severity from
0—9.

75

seedlings receiving leachings from healthy GW—4 plants was
not increased (5% level) above fungus-infected plants
receiving no leachings. However, F. solani-infected Miragreen

seedlings, receiving root leachings from variety 333 which is

resistant to E, euteiches only, were as severely diseased as

 

plants receiving root leachings from Miragreen seedlings.

The levels of resistance observed in the experimental
pea varieties may be a reflection of the influence of root
exudates on inoculum potential in the rhizosphere. The effect
of root exudates from the 3 pea varieties on root rot severity
are compatible with this interpretation.

The fact that root exudates from Aphanomyces resistant

 

plants failed to increase root rot (5% level) on Aphanomyces-

 

infected Miragreen plants but did increase root rot on
Fusarium-infected plants indicates that qualitative as well

as quantitative differences in root exudates may be responsi-
ble for changes in inoculum potential. Exudates from
resistant plants never decreased root rot when used as amend—
ments to fungus-infected plants, indicating that root exudates

are not acting in a defensive role.

DISCUSSION

Little is known about the metabolic interactions
occurring when severity of a fungal disease is altered by
virus infection of the host. Several suggestions have been
made. Plant constituents essential for pathogenic develop-
ment may have increased or decreased (2, 8, 9, 44, 68).
Increased resistance to powdery mildew in leaf roll-infected
grapes has been suggested as due to increased osmotic
pressure of the host cells (19). Decreased resistance to
late blight in potato infected with leaf roll virus has been
attributed to a more favorable microclimate for infection in
leaves malformed due to virus infection. However, no evidence
has been presented to support these suggestions.

The virus-induced increased root rot observed in pea
(12, 13, 64) apparently is not due to changes in inherent
susceptibility of pea roots. This is indicated by several
lines of evidence: 1) the typical virus—induced increase
in pea root rot was not observed when all inoculum exterior
to the root surface was removed before fungus-infected

plants were inoculated with the virus. 2) Little or no

76

77

alternation in susceptibility was evident on the basis of
lesion length and severity when E, solani conidia were in—
jected directly into the hypocotyl. 3) Removal of exudates
from healthy and virus-infected plants by periodic leachings
of their roots resulted in plants being equally susceptible
to root rot. These results indicated that the critical
factor in the mechanism of increased root rot was operating
exterior to the root surface, i.e., in the rhizosphere.

One of the early effects observed in some host—parasite
interactions involving fungi is the enhanced release of
intracellular materials from host cells. This effect may
either be the result of physical diSruption (29, 57), or
the pathogen may alter cells at a considerable distance in
advance of its actual invasion (ll, 55). Wood and Braun
reported that crown—gall tissue took up more exogenous
solutes than did healthy tissues (67). They concluded that
permeability of one or more of the membrane systems of the

cell was increased in some way by the bacterium, Agrobacterium

 

tumefaciens.

 

Virus infection may also enhance the release of
intracellular materials from cells. Infection of Tabasco

pepper by tobacco etch virus resulted in a marked release

of electrolytes from roots of these plants (16). Also,

78

changes in rhizosphere microflora of Dolichos lablab infected

 

with dolichos enation mosaic virus suggest changes in root
exudation (31, 54).

In the present study, analysis of the nature and
quantity of substances in root exudates, as well as the time
of increased exudation from roots of BYMV-infected plants,
revealed that carbohydrates, amino acids, organic acids and
nucleotides were exuded in larger amounts from roots of BYMV-
infected plants than from healthy plants. Increased release
began 4-6 days after virus inoculation and continued until
14—16 days after inoculation. Although some amino acids
increased more than did others, the increased release of amino
acids from roots was the most pronounced effect observed in
virus—infected plants.

The amount of amino acids released by roots of virus—
infected peas was 2-3 fold that released by healthy roots. By
contrast, the quantity of exudates released by virus-infected
roots may have a direct effect on inoculum potential.

Indeed, addition of root exudates from virus—infected plants

to plants inoculated with E, euteiches or E, solani resulted

 

in more severe root rot than did addition of root exudates
from healthy plants. Moreover, increased root rot induced

by virus infection in peas occurred only during the period

79

 

6'
~1

d
O
0

d
O
U

 

_n
C)
A

 

 

FUSARIUM CONIDIA PER ML

1 l A A l l

3 4 5 6 7 8
DISEASE INDEX

 

Figure 19. Severity of Fusarium root rot as a
function of inoculum density. Four pots, each contain—
ing 10 Miragreen plants growing in silica sand, were
used for each inoculum concentration. A 10 ml conidial
suspension was pipetted on the sand surface. Disease
severity was determined on a scale of increasing severity
from 0-9.

K 11a! l'"

 

80

of increased exudation from roots of virus-infected peas.

Chromatographic analyses of amino acids released
from virus-infected roots showed a general increase of most
compounds identified. The 3-4 fold increase in exudation
of amino compounds could not be explained by increased quan-
tities of these compounds within virus—infected root tissue,
since concentration of individual amino acids within roots
were changed very little by virus infection. Specific
changes in amino acid release, such as the presence of
phenylalanine in virus-infected root exudate and its absence
in healthy root exudates, suggest alternation of membrane
permeability of these root cells. More 32PO4 was taken up
by roots of virus-infected plants than by healthy roots.
This increased uptake was not affected by exposure to
2,4-dinitrophenol, indicating that permeability of the
membrane, rather than active uptake by the membrane, was
increased.

Although many substances were increased in root
exudates from virus-infected pea plants, amino compounds
showed the greatest increase. In tests with a synthetic
root exudate added to E, solani—infected peas, amino acids
as a group, but neither glucose nor organic acids, were

able to increase disease. However, differences in

81

stimulatory capacity of root exudates observed in these
studies cannot be interpretated in terms of defense by roots
against pathogenic invasion, since exudates never decreased
root rot below levels occurrring with no additional exudate.
Further work is needed before the role of exuded amino acids
in resistance or susceptibility of these pea varieties can

be fully clarified. Possibley, differential root exudation
may play a role in quantitative resistance to nonspecialized
pathogens, such as root and stalk rot fungi. If so, suscep-
tible roots may inadvertantly contribute to their own disease
severity by their relative success or failure in retaining
within their root tissue small molecular compounds which are
stimulatory to potential pathogens in the rhizosphere. The
fact that seed exudation has been shown to be a factor govern—
ing susceptibility of peas and beans to pre—emergence . ‘_
damping-off (14) supports this conjecture.

The mechanism of increased virus infectivity in plants
infected with E, solani and BYMV or PMV was not studied.
Possibly, the rates of translocation of BYMV and PMV are
increased due to the physiological stress imposed by E, solani
invasion. In systemic viral infections, invasion of newly
formed healthy tissue must occur either by rapid translocation

through the vascular system or by slow passage from cell to

82

cell through plasmodesmata. Virus movement and translocation
into newly formed pea leaf tissue are known to be affected by

host nutrition and temperature (38).

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