ANAEROB§OSIS AS 5? AFFECTS THE fiUDATlGN
AND lNFECTIOi‘é OF PEA ROOTS GROWN
IN AN ASEPTEC MEST CHAMBER

Thesis for the Degree of Ph. D.
MECMGAN STATE UNfVERSHY
ALWN J. M. SMUCKER

‘ 1971

fit‘i“' Y

This is to certify that the

thesis entitled

Anaerobiosis as it Affects the Exudation
and Infection of Pea Roots Grown
in an Aseptic Mist Chamber

presented by

Alvin J. M. Smucker

/

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Soil Physics

LIBRARY

Michigan State
University

 

 

Major professor

Date June 14, 1971

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ABSTRACT

ANAEROBIOSIS AS IT AFFECTS THE EXUDATION
AND INFECTION OF PEA ROOTS GROWN
IN AN ASEPTIC MIST CHAMBER

BY

Alvin J. M. Smucker

The effects of anaerobiosis on exudation and infection
of pea roots were determined by growing seedlings in an
aseptic root chamber filled with mist of a mineral nutrient
solution. Gaseous compositions simulating those found in
flooded soils were flushed through the mist chamber for
periods up to 17 days. Root exudation was related to gas
composition and light intensity by measuring the accumulation
of plant metabolites in the circulating nutrient solution.
Pathogenesis was related to anaerobiosis by ranking the
severity of disease on the treated plants.

Ethanol, an indicator of cellular oxygen deficiency,
was the primary constituent of exudates from roots subjected
to anaerobic conditions. Essentially no ethanol was detected
in the exudates of aerobic roots. Conversely, up to 295 mg
ethanol was produced per gram of dry weight by roots sub-
jected to 70% N2 with 30% CO2 for 6 days. There were 125 mg

ethanol produced per gram dry weight by roots treated with

Alvin J. M. Smucker

N2 for 6 days. Ethanol was detected in root exudates a few
hours after the onset of anaerobiosis.

The silylated derivatives of amino acids and carbo-
hydrates in the root exudates were quantitatively measured
by gas chromatography. Anaerobiosis increased the quantity
of both amino acids and carbohydrates. An atmOSphere of
70% N2 and 30% C02 caused roots to lose the most amino acids
and carbohydrates. Air with 30% C02 caused the next highest
loss. An atmosphere of 100% N2 appeared to have the least
effect upon exudation except for the air control.

Anaerobic treatment of pea roots inoculated with
Fusarium promoted spore germination, fungal growth, and
disease. There was a 5-fold increase in the fungal growth
with a concomitant 4-fold increase in the disease of roots
treated with anaerobic as compared to aerobic conditions.

Bioassay experiments indicated ethanol alone had no
effect upon Spore germination. In contrast, alanine promoted
Spore germination. Both ethanol and alanine promoted the

growth of Fusarium.

 

 

ANAEROBIOSIS AS IT AFFECTS THE EXUDATION
AND INFECTION OF PEA ROOTS GROWN

IN AN ASEPTIC MIST CHAMBER

BY

. I,
L be

Alvin J. M? Smucker

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of CrOp and Soil Sciences

1971

TO BETTY

This thesis is dedicated to my wife.
For without her interest, patience
and unfailing support this study

could not have been completed.

ACKNOWLEDGEMENTS

Sincere appreciation is expressed to Dr. A. E. Erickson
for his assistance and guidance during this investigation.
His enthusiasm in all phases of research and teaching has
been a true inspiration.

Special thanks are due to Dr. B. D. Knezek, Dr. J. M.
Tiedje, Dr. A. R. Wolcott, Dr. R. S. Bandurski, and Dr. J. L.
Lockwood who served on my guidance committee.

Thanks are also due to Dr. C. M. Harrison, Dr. B. G.
Ellis, and Dr. M. L. Lacy for their assistance in the
preparation of this thesis.

Appreciation is extended to Dr. R. K. Hammond and other
members of the mass spectrometer laboratory for their
assistance in identifying some of the root exudates.

Financial assistance granted by the Agricultural

Experiment Station is gratefully acknowledged.

TABLE OF CONTENTS

INTRODUCTION . . . . . . . . . . . . . . .
LITERATURE REVIEW . . . . . . . . . . . . .

Root Exudates

Root Physiology
Fusarium Physiology
Exudate Measurements

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

Description of Plant Growth Chamber
Description of Root Chamber System
Preparation of Seedlings

Treatment of Seedlings

Collection of Root Exudates

Collection and Measurement of Root Gases
Plant Measurements

Bioassay

Preparation of Inoculum

Tests for Aseptic Conditions

Statistical Analysis

Gas Chromatography and Mass Spectrosc0py

RESULTS AND DISCUSSION . . . . . . . . .

Mist Chamber
Content of Exudates

Ethanol
TMSi Derivatives

Plant Morphology
Plant Disease
Bioassay
CONCLUSIONS . . . . . . . . . . . . . .

LITERATURE CITED 0 O O O O C O O C 0

LA)

«>wa

11
12
14
16
19
19
20
20
21
21
22
22

25

25
25

25
37

54
61
68
74

76

LIST OF TABLES
Table Page

1. The effects of time on germination,
tissue injury, and sterilization of
pea seeds soaked in 0.2% HgC12 plus
Tween 80 . . . . . . . . . . . . . . . . . . 15

2. The effects of gas composition on the net
accumulation of ethanol in the root
exudates of peas treated for 6 days . . . . 27

3. Ethanol production in the exudates from
roots of four pea plants treated with
N2 containing 30% C02 . . . . . . . . . . . 32

4. Ethanol production in the exudates from roots
of four pea plants treated with 30% C02
and air . . . . . . . . . . . . . . . . . . 32

5. Ethanol production in the exudates from
roots of four pea plants treated with
air for 48 hours, then N2 without CO
for 48 hours, then air for 48 hours at

high light intensity . . . . . . . . . . . . 33
6. Ethanol production in the exudates from

roots of four pea plants treated with

N2 Without C02 0 o o o o o o o o o o o o o o 33
7. Ethanol production in the exudates from

roots of four pea plants treated with
air for 48 hours, then N2 without CO
for 48 hours, then air for 48 hours . . . . 34

8. Effects of the gas composition of the
rhizosphere on the amino acid and
carbohydrate contents in root exudates
of peas grown in an aseptic mist
chamber . . . . . . . . . . . . . . . . . . 46

9. Carbon dioxide evolution by the roots of
four pea plants treated with air . . . . . . 55

10. Carbon dioxide evolution by the roots of four
pea plants treated with N2 without C02 . . . 55

Table

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

Carbon dioxide evolution by the roots of
four pea plants treated with air for
48 hours, then N without C02 for
48 hours, then air for 48 hours under
low light . . . . . . . . . . . . . .

Carbon dioxide evolution by the roots of
four pea plants treated with air for
48 hours, then N without C02 for
48 hours, then air for 48 hours under
high light . . . . . . . . . . . . .

Carbon dioxide evolution by the roots of
four pea plants treated with air and
N2 containing 30% C02 (anaerobic) and
infected by Fusarium . . . . . . . .

Effects of gaseous composition of root
environment upon the morphological
characteristics of pea plants treated
for 6 days . . . . . . . . . . . . .

Effects of gaseous composition of root
environment upon the dry weight of
pea plants treated for 6 days . . . .

The effects of time and gaseous atmosphere

upon the germination and growth of g.
solani f. pisi macroconidia in the
rhizosphere of pea roots . . . . . .

Effects of severe anaerobiosis on the
intensity of root rot disease and
growth of Fusarium in the rhiZOSphere
of peas . . . . . . . . . . . . . . .

The effects of anaerobiosis upon the
morphology of plants infected with
Fusarium root rot . . . . . . . . . .

Effects of gas composition on the
accumulation of carbohydrates and
amino acids in the rhiZOSphere of
peas grown in an aseptic mist chamber
and inoculated with Fusarium . . . .

The effects of ethanol concentration and
gaseous composition upon the
germination and initial growth of g.
solani f. pisi macroconidia . . . .

Page

56

56

57

58

59

64

65

67

69

71

Table Page

21. Effects of ethanol concentration and gaseous
composition upon the germination and
growth of E. solani f. pisi in solution
containing 20/lg alanine . . . . . . . . . . 73

LIST OF FIGURES

Figure Page
1. Diagram of pyrex glass mist chamber system . . 12
2. Diagram of apparatus used to mount seedling

into mist chamber . . . . . . . . . . . . 17

3. Time required to remove oxygen from mist
chamber by flushing the system with
N2 gas at various flow rates . . . . . . . 26

4. Effect of gas composition on ethanol
production by the roots of aseptic
peas grown under low light conditions . . 28

5. Effects of flooding period on ethanol
production by aseptic roots under low
light conditions . . . . . . . . . . . . . 29

6. Effects of light intensity on ethanol
production by aseptic pea roots
subjected to N2 containing 30% CO2 . . . . 3O

7. Effects of light intensity on the ethanol
production by aseptic pea roots
treated with air for 48 hours, then
N2 without C02 for 48 hours, then air
for 48 hours . . . . . . . . . . . . . . . 31

8. Gas chromatogram of TMSi derivatives of

exudates from roots of 12 pea plants

treated with air for 6 days . . . . . . . 38
9. Gas chromatogram of TMSi derivatives of

exudates from roots of 12 pea plants
treated With N2 for 6 days 0 o o o o o o o 39

10. Gas chromatogram of TMSi derivatives of
exudates from the roots of 12 pea
plants treated with air for 48 hours,
then N without C02 for 48 hours,
then air for 48 hours under low light . . 40

Figure

11.

12.

13.

14.

15.

16.

Gas

Gas

Gas

Gas

Gas

The

Page

chromatogram of TMSi derivatives of

exudates from roots of 12 plants

treated with N2 containing 30% C02

for 6 days . . . . . . . . . . . . . . . . 41

chromatogram of TMSi derivatives of

exudates from the roots of 12 pea

plants treated with air containing

30% CO2 for 6 days . . . . . . . . . . . . 42

chromatogram of TMSi derivatives of

exudates from roots of 12 pea plants

treated with air for 48 hours, then

N2 without C02 for 48 hours, then air

for 48 hours under high light . . . . . . 43

chromatogram of TMSi derivatives of
exudates from roots of 8 pea plants
treated with air for 17 days. . . . . . . 44

chromatogram of TMSi derivatives of

exudates from roots of 8 pea plants

treated with N containing 30% C02

for 8.3 days, Eben air for 8.7 days . . . 45

effects of anaerobiosis and the
presence of Fusarium upon ethanol
accumulation in the rhiZOSphere . . . . . 63

INTRODUCTION

Maximum plant growth requires ample quantities of
oxygen available to the root system. Peas, tomatoes, and
other vegetable crops are quite sensitive to oxygen stress.
Short periods of soil flooding frequently result in root
diseases reducing plant growth and yield.

Many of the fine textured soils frequently become
saturated after a heavy rainfall or when located in low
areas of a field. As the water‘content of soil approaches
the saturation point, soil pores normally occupied by air
become filled with water, greatly reducing the supply of
oxygen to plant roots.

Root diseases are increased by high soil moisture.
Walker (1942) reported that root diseases of peas grown in
Wisconsin were invariably greater in wet years and on wet
soils. Often soil anaerobiosis inflicts damage to the
roots within a few hours after the soil is saturated. The
resultant root injury, esPecially to young seedlings, af-
fects plant growth throughout the growing season. Prolonged
soil flooding may also reduce the resistance of plant roots
to the surrounding biota.

Most studies have related oxygen stress to nutrient

uptake, plant growth, and alterations in plant metabolism.

2

No effort has been directed toward delineating the role of
soil flooding or soil anaerobiosis in root diseases. Since
most of the vegetables in this stage are grown on soils
which frequently become saturated during the seedling stage,
a study should determine whether the majoreffect of
anaerobiosis is on the host, making it more or less sus-
ceptible to disease, or on the pathogen making it more or
less virulent.

This investigation was designed to determine the
effects of soil anaerobiosis on root exudates and the
inoculum potential of Fusarium root rot of peas. Knowledge
of this interrelationship should lead to a clearer under-
standing of the role exudates have in host pathogen
interactions.

The objectives of this study were: 1) to determine
the effects of anaerobiosis on the exudation and respiration
of sterile pea roots, 2) to determine the role of anaerobic
metabolites and gases on Fusarium spore germination and
growth, and 3) to determine the role of anaerobiosis on

infection by Fusarium.

 

LITERATURE REVIEW

Root Exudates

 

There is much evidence that intact cells of plant roots
exude sufficient quantities of organic compounds to support
large p0pulations of microorganisms in the root zone or
rhizosphere; Schroth and Hildebrand (1964) and Rovira (1962
and 1969). Compounds identified by Rovira (1969) in the
exudates included: amino acids, sugars, peptides, organic
acids, enzymes, vitamins, nucleotides, and many others which
attract, stimulate, or inhibit soil microflora. The quantity
of root exudates tended to control the number of micro—
organisms in the rhizosphere, whereas their quality controlled
the specificity of the microorganisms. Many factors of the
environment affected the quality and quantity of root
exudates. These factors included: 1) plant species, age,
and nutrition, 2) temperature and light, 3) presence or
absence of microorganisms, 4) supporting medium, 5) moisture
content, and 6) chemical and physical damage.

Pearson and Parkinson (1961) and Schroth and Snyder
(1961) designed experiments and reported that amino acids
are exuded from the zone immediately behind the root tip of
beans while most of the sugars are exuded from the hypocotyl.
By subjecting older plants to 14C02 and measuring the

3

4

radioactivity at the roots, McDougall and Rovira (1969)
showed the greatest amount of metabolites was exuded at the
tips of lateral roots.

The mechanisms involved in root exudation are essen-
tially unknown. Evidence that exudation is a metabolic
process involving cell permeability was demonstrated by
Hiatt and Lowe (1967) and McDougall and Rovira (1960).

They reported that potassium cyanide and 2,4 denitrOphenol,
inhibitors of oxidative phOSphorylation, increased both the
permeability of membranes and the production of root
exudates. However, since low concentrations of oxygen have
been reported to increase exudation by Brown and Kennedy
(1966), root exudation may be primarily controlled by the

energy processes within the cell.

Root Physiology

 

The oxygen supply in flooded soils is greatly reduced
to that dissolved in water. Erickson and van Doren (1960)
have shown that short periods of flooding caused a decrease
in the diffusion of oxygen to plant roots reducing plant
growth and yield. In addition to less growth, the quantity
of mineral elements in peas is reduced by soil flooding;
Cline and Erickson (1959).

Crawford (1966) reported soil flooding increased the
rate of glycolysis in the roots of species susceptible to
saturated soil conditions while there was no change in

the glycolitic rate of tolerant species. Flooding also

5

increased alcohol dehydrogenase activity in the susceptible
plants producing ethanol; Crawford (1967). After studying
the rates of other enzymes, Crawford concluded that the
activity of alcohol dehydrogenase was an enzyme induction
process which could be controlled by soil flooding.

Ethanol accumulation in plants has been determined as
an index of anaerobic soil conditions. Kenefick (1962)
showed that the ethanol content of sugar beets increased
under anaerobic conditions. Fulton and Erickson (1964)
reported the concentration of ethanol in the xylem exudates
of tomatoes increased during short periods of soil flooding.
Bolton and Erickson (1970) reported a lOO-fold increase in
the ethanol content of root exudates of tomato plants dur-
ing anaerobiosis. A concurrent five-fold increase in the
ethanol content of the roots suggests that most of the
ethanol produced during anaerobiosis is exuded at the site
of production.

Cossins and Turner (1959 and 1963) and Lin gt a1.
(1965) presented evidence that a functional alcohol
dehydrogenase complex is active in pea roots. They reported
that during anaerobiosis ethanol was produced and exuded
during germination. When the seedlings were aerated they
oxidized exogenous ethanol to acetaldehyde.

Most of the amino acids and two monosaccharides have
been identified in the exudates of pea roots. Rovira
(1956) and Boulter gt 31. (1966) reported 21 amino acids

were exuded into the culture medium of l4-day-old pea roots.

 

6

Paper and column chromatography revealed that 26.1 mg of
amino acids were exuded per g of dry root during the first
14 days. During the first seven days, glucose and fructose
were the primary sugar exudates. Twenty-five micrograms of
each monosaccharide was exuded from each plant; Rovira
(1956). Information on the effects of anaerobiosis upon the
qualitative or quantitative exudation of amino acids and
sugars is unavailable. There is, however, contradictory
evidence concerning the role of oxygen in root exudation.
Ayers and Thornton (1968) reported more material reactive to
ninhydrin was released from roots of wheat and peas when
grown in solution culture saturated with air as opposed to
a solution culture saturated with gas containing 2% 02. In
contrast, Brown and Kennedy (1966) reported increased pro-
duction of sucrose, fructose, galactose, glucose, and
substances reactive with ninhydrin occurred in exudates
collected from soybean seedlings germinated at low oxygen
concentrations. They also reported bacterial contamination
favored increased sugar production in exudates collected at
all oxygen concentrations.

The evolution of volatile organic compounds such as
ethylene has been noted in diseased, senescing, and wounded
tissue by Burg (1962). Other volatile organics in the soil

atmosphere have been neglected.

7

Fusarium Physiology

The need of macroconidia of Fusarium species for
exogenous nutrients in germination has been established for
several species and may be fairly general; Sisler and Cox
(1954) and Marchant and White (1966). Cochrane gt gt.
(1963) found that macroconidia of E. solani f. phaseoli
required exogenous carbon and nitrogen and a growth factor
present in yeast extract, which later could be replaced
completely by ethanol or acetoin and partly by acetaldehyde
or one of several amino acids.

Nutrient requirements for infection are higher than
those for spore germination and so the nutrients supplied
by root exudate are generally essential for infection;
Garrett (1970). Toussoun, Nash, and Snyder (1960) reported
that glucose was essential for the penetration stage of
infection of bean hypocotyl by macroconidia of E. solani
f. phaseoli, but glucose without a nitrogen source delayed
host penetration and the onset of pathogenesis. Nitrogen-
ous compounds did favor early penetration and pathogenesis
with the organic forms being more effective than the in—
organic forms.

Root exudates provided the necessary energy to increase
the inoculum potential, Garrett (1970), of selected root
saprophytes making them pathogenic. In turn, adverse soil
conditions, such as soil flooding, degraded the integrity

of root cells making them more susceptible to infection.

Fusarium solani f. Etgt, an important component of the
pea root rot complex, is a good example of the conversion
of a soil saprOphyte to a pathogen by root exudates. In a
seedling test, Lockwood (1962) reported that the root rot
of peas increased significantly when the sand was saturated
immediately after inoculating with E. solani f. Etgt. Payne
gt gt. (1966) designed experiments and reported that the
permeability of root cells was altered by anaerobiosis as
more amino acids leaked from the roots of peas growing in
saturated sand. These and others, Geisler (1965) and Cook
and Flentje (1967), suggested that soil anaerobiosis played
an important role in the root rot complex of peas.

Soil flooding also affects the survival of soil fungi.
Newcombe (1960) demonstrated the survival of chlamydospores
was dependent upon soil aeration. More recently, Griffin
(1968) designed an experiment which demonstrated the gas-
eous requirements for the production of spores by E. roseum
f. cerealis. He showed less air was required for the
production of macroconidia than for the production of
chalmydospores. Macroconidia were produced within the water
film while chlamydospores were produced only in the air
space of a simulated soil matrix. The report by Cochrane
gt gt. (1963) agrees with Griffin as they demonstrated
macroconidia germinated best at oxygen concentrations of
3-4%. However, there was essentially no germination of

macroconidia under zero oxygen concentrations.

9

Greater partial pressures of C02 in flooded soils fre-
quently approach levels which promote growth of certain
fungi. Durbin (1955) reported the growth of fungi and
certain other microorganisms was directly related to CO2
concentration even at levels toxic to plant roots. In
contrast, Cochrane gt gt, (1963) reported the germination
of macroconidia accelerated when metabolic C02 was removed
from the atmosphere. The report by Papavizas and Davey

(1962) agrees with Durbin in that Fusarium growth increased

 

prOportionally to C02 concentrations up to 30%.

Cook and Flentje (1967) reviewed earlier studies and
presented evidence that exudates from flooded seeds and not
water alone influenced germination and subsequent growth of
chlamydOSpores of E. solani f. Etgt in soils contiguous to
germinating peas. They also reported mycelial growth near
the host was greatest in soil with an intermediate water
content. This evidence implies that during anaerobiosis
the nutrient content in pea root exudate approached the
critical level required for germination and exceeded the
level for fungal survival; Cochrane gt gt. (1963) and Cook

and Snyder (1965).

Exudate Measurements

Gas liquid chromatography (GLC) has attained great
SOphistication with great Speed, sensitivity, and versa—
tility. GLC methods now encompass a wide range of

analytical applications; examples include analytical

10

methods for carbohydrates and related polyhydroxy compounds,
amino acids, and amino sugars; Sweeley gt gt. (1963), Zumwalt
(1971), and Karkkainen and Vihko (1960). The analysis of
these compounds by GLC techniques has been advanced through
extensive studies involving numerous derivatization methods
and chromatographic systems; McBride gt gt. (1968) and
Pierce (1970). GLC analysis of trimethylsilylated (TMSi)
derivatives of the above compounds is the preferred method.
This method is simple in its formation, quantitative in
yield (95-100%), and forms sufficiently volatile compounds
that give good resolution in chromatography without losses

during sample concentration by evaporation.

MATERIALS AND METHODS

Description gt Plant Growth Chamber

 

 

A Sherer growth chamber model CEL 25-7 was used for
controlling the environmental conditions. The unit was
equipped with temperature and photoperiod controls. The
relative humidity was controlled by evaporation of free
water.

The unit was modified to provide a range in quality
and intensity of light. Low light conditions of 0.63 g
cal cm"2 min‘l (Langleys) were achieved by using 6
fluorescent bulbs, 4-25 watt, and 5-75 watt incandescent
bulbs. High light conditions of 1.1 Langleys were achieved
by 6 fluorescent bulbs, 4-100 watt, 4-150 watt, and 1-200
watt incandescent bulbs. The energy emitted for each light
intensity was measured by a Beckman and Whitley thermal
radiometer and an inline Sargent recorder.

Temperatures were maintained at 18.31.5 and 23.3i.5 C
for the low and high light intensities. These air tempera-
tures simulated the temperature of field conditions in
Michigan during the spring growing season. Temperatures
inside the mist chamber were l8.0i.5 C. The conditioned
air was filtered through eight layers of moist cheesecloth

and circulated through the growth chamber.

11

12

Description gt the Root Chamber System

 

 

A pyrex glass mist chamber was designed and constructed
to produce fine water drOplets which continuously bathed the
plant roots. Figure 1 shows the details of the root chamber.
The chamber was equipped with a variable flow Masterflex
pump* model 7545-15 which recirculated the sterile nutrient
solution. The inline pressure was 1515 psi producing a flow
of 25 ml min‘1 at the orifice of the nozzle. The circulating
solution was filtered by an inline cellulose ester filter+
which had a mean pore size of 0.22p. The fine droplets of
spray were produced by a small brass nozzle with an orifice
of 0.5 mm which formed a full cone spray. The brass nozzle
was secured to the glass access tube with epoxy cement,
Figure l.

The gas exhaust apparatus was designed to remove water
from the escaping gases without resisting flow. The dry
barrier prevented contamination of the system by micro-
organisms. A U-shaped gas exhaust tube collected and
returned the condensate to the solution reservoir via the
condensate return, Figure 1. Gas flowing into the chamber
via the gas inlet was sterilized by filtration. Filtration
was achieved by flowing the gas through a Millipore filter
having a mean pore size of 0.22A, Treatment gases flowed

through the chamber at the rate of 2515 ml min'l.

 

*Obtained from the Cole Parmer Co., Chicago, Ill.

+Obtained from the Millipore Corp., Bedford, Mass.

l3

 
 
 
  

CaClZ drying tube

Condensate separator

Water cooled condenser-——f

I

 

 
 

 

 

.3)
Plant port ’g:
.3
"K
‘2.)
1’.” '
Gas exhaust tube ‘kw
§ 19/38

Condensate return

 

 

 

Mist chamber

Spray nozzle ——————-————\\\+
Gas inlet ——————————————~___+

1—10.2 cm—d

 

 

 

Access tube

 

Solution return

 

Solution reservoir

 

(200 m1)

 

Access port <—""

 

 

 

Figure 1. Diagram of pyrex glass mist chamber system.

20.3 cm

 

14

A glass reservoir was connected to the root chamber with
a ground glass joint (S 19/38), Figure 1. This connection
functioned as the drain to the mist chamber through which the
nutrient solution flowed, by gravity, to the reservoir. The
reservoir was equipped with an access port that was fitted
with a silicone rubber septum.

It is believed the sterile mist chamber removed essen-
tially all the biochemical, microbiological, and physical
factors which obscure the true quality and quantity of actual i

root exudates. This system is especially suited for studying

 

root reSponse to the composition of rhizosphere gases. This
system provided a method of manipulating the gas composition
of the rhiZOSphere without changing the thickness of the
water film on the roots. Rapid changes in the gaseous com-
position of the rhizosphere are also possible with this
apparatus. The advantage of this system is that the entire
apparatus can be sterilized by autoclaving and aseptic con-

ditions maintained for prolonged periods of time.

Preparation gt Seedlings

 

 

Peas (Pisum sativum L.; variety, Miragreen*) were used

 

throughout this investigation. Mature seeds were surface

sterilized by immersing in 50% ethanol for 30 seconds fol-
lowed by six washings with distilled water. Then the seeds
were immersed in 100 ml of 0.2% HgClz containing two drOps

of "Tween 80". After five minutes the seeds were washed

 

*Obtained from the Ferry-Morse Seed Co., Buffalo, N.Y.

15

free of HgClz with six washings of sterile redistilled water

 

and soaked in redistilled water for 12 hours. Sterilization
time was determined by the experiment reported in Table l.
The seeds, imbibed with water, were transferred to 500 ml
gas diffusion vessels where they germinated in the dark in

sterile redistilled water.

Table l. The effects of time on germination, tissue injury,
and sterilization of pea seeds soaked in 0.2%
HgC12 plus Tween 80.

 

 

 

Aseptic
Soaking Germination Lesions seeds
time - min % per seed %
5 100 6.0 100
10 100 8.0 100
15 84 8.0 100
20 90 7.8 100

 

The water was vigorously aerated by compressed air
scrubbed by acid, base, and water baths and sterilized by
filtration. After 72 hours, 48 of the most uniform seedlings
were transferred to sterile glass storage vessels 80 x 100 mm.
Approximately 35 ml of a 50% modified Hoagland's solution
were added to the storage vessels and sterilized by auto-
claving. The radicle of each seed was placed in the nutrient
solution and the seedlings were held in position by placing
them between two glass rods. These vessels were placed in
the dark for 48 hours, then placed in the growth chamber for

48 hours.

16

Four uniform seedlings were transferred to each root
chamber. The green plumule of each plant measured 2515 mm
and the primary root 3515 mm in length. During this trans-
fer the seedcoat was removed and the seed and root were
washed with a sterilized nutrient solution. Methods of
sterile technique were executed during all transfers in-
cluding the use of sterile gloves while installing seedlings
in the plant holder, Figure 2. The plant port and holder
were constructed from the lower half of a female and male
ground glass joint, reSpectively. The base of the male was
partially closed forming a small opening 6 mm in diameter.
The plumule of the pea seedling was inserted through the
bottom of the plant holder to the seed and secured to the
holder with sterile parawax (a mixture, by weight, of l 9
hard filtered paraffin : 8 g petroleum jelly). This seal
proved to be a barrier to gas, water, and microorganisms,
yet was plastic, enabling the plant to expand during growth.
Plant leaves were protected from desiccation by a poly-
ethylene bag removed 12 hours after the root chamber was
installed in the plant growth chamber. The root chambers
were wrapped with aluminum foil simulating the dark condi-

tions of the soil.

Treatment gt Seedlings

 

 

Seedlings were transplanted 7 days after seed sterili-
zation. Seedlings were treated for the following 6 days and

harvested. Preliminary studies indicated this stage of

 

 

17

Plant holder

‘- 1'! 1') .‘L1 '1

Parawax barrier

 

 

Plant port

 

 

sf Mist chamber

Figure 2. Diagram of apparatus used to mount seedling into
mist chamber.

18

plant growth was the most dynamic for pea roots. During

this period the secondary root initials emerge rupturing

the primary root, eXposing the young root tip. It is

believed this phase of root development is most sensitive

to the gas composition of the rhizosphere.

The treatments of this investigation were:

1)
2)

3)

4)
5)

6)

7)
8)

Experiments 1-5 were run under low light conditions and
experiments 6-8 were conducted under high light conditions.

Treatments 1-6 were run for 6 days.

Air control,

N2 without C02,

Air for 48 hours, then N2 without C02 for

48 hours, then air for 48 hours--low light,
70% N2 with 30% C02,

70% air with 30% C02,

Air for 48 hours, then N2 without C02 for

48 hours, then air for 48 hours-—high light,
Air plus inoculum,

70% N2 with 30% C02 plus inoculum for 10 days,

then air for 7 days.

moved from the N2 gas by flowing the treatment gas through

a 35 x 2 cm plexiglass cylinder filled with ascarite.*

pH of the circulating nutrient solution was 6.5 at the

beginning of each eXperiment and changed very little during

each experiment except in those treated with 30% C02 where

 

*Registered trademark for C02 absorber from Arthur H.
Thomas Co., Philadelphia, Pa.

 

 

Carbon dioxide was re-

The

19

the pH drOpped to 5.5I.3, 12 hours after the initiation of
the experiment.
Seedlings in experiments 7 and 8 were treated for

17 days. Those in eXperiment 8 were treated with 30% C02 +

 

70% N2 for 10 days then switched to air (352 ppm C02) for the
remaining 7 days. Experiment 7 was treated with air for the

duration of the eXperiment. Macroconidia of Fusarium solani W:

 

f. pisi were injected into the nutrient solutions of these

two eXperiments 5 days after the onset of the experiments.

 

The Spore concentration of the circulating nutrient solution a

was 1.0 x 106 macroconidia ml"1 for both experiments.

Collection gt Root Exudates

 

 

Aliquots, 0.5 ml per chamber, were extracted from the
reservoir with a hypodermic needle and syringe via the
rubber septum. Samples were taken at 4, 6, 12, or 24 hour
intervals and analyzed for ethanol. At the end of each
experiment the nutrient solution containing the root exudates
was measured and frozen. Water was removed from the solution
by lypholization. The lypholized samples were weighed, dried
further at 105 C for 60 min, derivatized and analyzed for

amino acid and sugar content.

Collection and Measurement gt Root Gases

 

 

Gases evolved by pea roots were determined by analyzing
the gas composition before and after passing it through the

chamber. Oxygen and C02 were monitored by a magnetic moment

20

Beckman oxygen analyzer and a Beckman model 215A infrared C02
analyzer. Organic volatiles were collected on an organic com-
pound trap and removed, by heating, onto a gas chromatograph
column for analysis. The trap was constructed of two QD
plastic connectors joined together by a tygon tube 5 x 1.27 cm.

The trap was filled with 0.5 g of Porapak type 0* having a

 

 

mesh size of 50-80 with essentially no resistance to gas flow.

Plant Measurements

 

 

The shoot, root, and seed of each plant were separated, a
measured, dried, and weighed at the end of each experiment.
The mean value of 12 plants was considered representative of
that component.
All debris from the roots of treated plants was deter-
mined by taking the difference between the dry weight of the

inline prefilter before and after each experiment.

Bioassay

In this experiment the germination of Fusarium macro-
conidia in ethanol was tested in the presence of five gaseous
atmOSpheres. Sterile redistilled water containing 0, 10,
100, 1000 ppm of ethanol, by weight, was saturated with air,
30% C02 + 70% N2, 30% CO2 + 70% air, N2 without C02, and N2
passed through a heated column filled with elemental COpper
to remove residual oxygen. The dissolved oxygen concentra-

tions of these solutions were 8.23, 0.13, 4.71, 0.18, and

 

*Obtained from Waters Associates Inc., Farmington, Mass.

21

0.41 ppm, respectively. Analysis of the dissolved oxygen
content was by the Winkler method.

Stoppered Erlenmeyer flasks (125 ml) were used for this
experiment. Each vessel contained 100 m1 of the treatment
solution. The pathogen concentration used was 3.0 x 106
Spores, mostly macroconidia, per m1. Aliquots, 1 ml per

sample, were removed periodically for spore germination and

ethanol consumption analysis.

Prgparation gt Inoculum

 

 

Fusarium solani f. pisi, obtained from Dr. Lockwood's

 

laboratory, was the pathogen used in this investigation. The
pathogen was cultured on slants of potato dextrose agar (PDA).
Spores were obtained from lS-day agar cultures incubated in
the dark at 22 C.

Spores were washed from the agar surface, centrifuged,
and resuspended four times in sterile redistilled water. The
concentration of spores, mostly macroconidia, were standard-
ized by the hemacytometer method. Germination was determined
by counting the germ tubes of 100 spores from each replicate.
The criterion for germination was the formation of a micro-
sc0pically visible germ tube. Length of germ tubes was

measured by an ocular micrometer.

Tests for Asgptic Conditions

 

Samples from all eXperiments were cultured on PDA to

detect contamination. The pH of PDA was adjusted to 7.0

22

with 40 ml of 0.1 E KZHPO4 1"l PDA to provide a medium con-
ducive to fungal and bacterial growth. Inline filters from
the root chamber were also plated and cultured for 2 weeks,

then examined. All contaminated replications were discarded.

Statistical Analysis

 

Analysis of variance was used to determine the statis-
tical differences among the treatments in this investigation.
The hierarchal or nested design was used for all experiments
involving the root chamber. The average of each root chamber
was used to calculate the sums of squares in the analysis of
variance. A randomized block design was used for the bio-

assay eXperiments. The average value of each replication was

used to calculate sums of squares in the analysis of variance.

Significant differences among the data were determined by the

least significant difference (LSD) method.
Gas Chromatogrgphy and Mass Spectroscopy

Ethanol was identified and measured by injecting the
samples, taken from the reservoir, directly into the gas
chromatograph. One microliter samples were injected into a
Beckman GC-ZA gas chromatograph equipped with a hydrogen
flame detector and a 2 m x 3.2 mm stainless steel column
packed with Porapak 08 (100/120 mesh). Oven temperature was
isothermal at 175 C with the flow rate of the helium carrier
gas at 80 m1 min'l.

The nutrient solution and exudates from three chambers

were lypholized to dryness. Exudates were silylated in the

.u—L‘

 

23

presence of the remaining salts before analysis by gas
chromatography and mass spectroscopy.

The silylated derivatives (TMSi) of the root exudates
were formed by substituting a silyl group for an active hy-

drogen and/or replacing the metal component of a salt; Pierce

 

(1970) and personal communication with the Biochemistry

Department, M.S.U. A sixty molar excess of N,O-Bis (trimethyl-

 

silyl) trifluoroacetamide (BSTFA)* containing 1% of the

catalyst trimethylchlorosilane (TMCS) was added to the dried

 

sample and sonicated for five hours at 70 C in an ultrasonic é,
cleaning vessel filled with water. The ultrasonic treatment

physically dismantled the salt-exudate complex enhancing silyl-

ation. During silylation the solvent properties of BSTFA and

its reaction products were used to dissolve the amino acids

and carbohydrates in the exudates. The dissolved TMSi samples

were centrifuged and concentrated by evaporation. The sample

was redissolved in SQ/Ll of BSTFA containing 1% TMCS and sgfill
dimethylformamide. The above reactions were carried out in a

Kimax vial (100 x 13 mm) covered with a teflon-lined cap.

The TMSi derivatives were quantitatively and qualita-
tively analyzed by using a Perkin-Elmer 900 gas chromatograph
and an LKB 9000 gas chromatograph-mass spectrometer, respec-
tively. Quantitative analysis of the TMSi derivatives was
performed by injecting 5/ul samples into the gas chromatograph
equipped with a hydrogen flame detector. The column used was

a 3.3 m x 3.2 mm stainless steel tube packed with 3% SE-30

 

*Obtained from the Regis Chemical Co., Chicago, Ill.

24

ultraphase on chromosorb W (HP)* (80/100 mesh). The oven
temperature was programed from 90-200 C at 2° min'l. The
injector and manifold temperatures were 250 C. Flow rate of
the helium carrier gas was 40 ml min'l. Standards of glucose,
fructose, ribose, and sucrose were weighed, then silylated by
the same procedure used for the exudates.

Mass spectrometric analysis was performed by eluting the F“

samples through the SE-30 column used above. The separated

compounds flowed from the column, whose temperature was pro-

 

gramed from 90-200 C at 2 C min-l, into the ionization E
chamber of the LKB single-focusing mass spectrometer. The
ionization voltage was 70 ev while the temperature of the ion
source was 290 C. TMSi derivatives were volatilized at 250 C
in the flash heater at the head of the column. Helium car-
rier gas flowed through the system at 40 ml min—1.

Conditions for the gas chromatography-mass spectrometric
analysis of the exhaust gases from the rhizosphere were
similar to those listed above. The changes for this analysis
included a 3.3 m x 3.2 mm stainless steel column packed with
3% 0V 101 on chromosorb W (HP) (80/100 mesh). A 15.2 cm x
6.35 mm stainless steel pre-column was packed with the 0.50 g
Porapak Q. The unknown compounds in the pre—column were vola-
tilized at 275 C and eluted into the main column which was at
room temperature. Then the main column was programed at 6°
min‘1 from 50-150 c and 2° min-1 from 150-250 c. All com-
pounds were identified by studying the important fragmented

ions recorded on a bar graph spectrogram.

 

*Obtained from the Pierce Chemical Co., Rockford, Ill.

RESULTS AND DISCUSSION

Mist Chamber

 

The mist chamber proved to be an excellent system for TT
studying the effects of gas composition on plant roots. By

simply altering the flow rate of gases entering the mist

 

chamber, the gaseous composition could be changed very ‘
rapidly for determining the short-term effects on plant roots E’
or very slowly simulating natural field conditions, Figure 3.
The system also provided a means for manipulating the gas
environment, extracting root exudates, observing morphologi-
cal changes, and inoculating roots without contaminating the
root zone. Sterile conditions were maintained for 88% of the
experiments. The present design should also maintain sterile
conditions for prolonged periods when aseptic plant roots are
transferred to the system.
Additional uses of this system might include measure-
ments of water and nutrient uptake, evaluations of disease
resistance, the energy requirements of roots in the presence
and absence of soil microflora and the effects of rhizosphere

gases on these reactions.
Content gt Exudates

Ethanol: Roots of intact pea seedlings were tested for

loss of ethanol into the rhizosphere during anaerobiosis.

25

Flow rate-ml min-1

300*

250‘

200‘

150‘

100-

50‘

 

26

\\\\\“‘N“‘*~———————o

 

Figure 3.

1

50 100 150 200 250
Time-min

Time required to remove oxygen from mist
chamber by flushing the system with N2 gas
at various flow rates.

 

 

27

Data obtained from the treatments with different gases under
high and low intensities of light are presented in Figures
4-7 and Tables 2-7. The accumulation of ethanol in root
exudates was quite different depending upon the gas
composition of the rhizosphere and the light intensity.
Ethanol was absent from the root exudates of peas grown

in a sterile aerobic rhizosPhere. In contrast, large

 

quantities of ethanol accumulated in root exudates under

anaerobic conditions, Table 2. Similar results have been

 

found by others; Kenefick (1962) and Bolton and Erickson i;
(1970). However, the accumulation of ethanol in the root
exudates of their experiments was greatly reduced as the

roots were not under aseptic conditions.

Table 2. The effects of gas composition on the net
accumulation of ethanol in the root exudates of
peas treated for 6 days.

 

 

Ethanol
Treatment mg/g dry root
Air 0.0a
Air containing 30% CO2 134.3
N2 WithOth C02 146.2
N2 containing 30% C02 269.5

 

aEach value represents the average of three replications.

Ethanol mg 9'1 dry weight

300

 

28

 

 

 

7
0—0 Air
o——-o N2 without C02
Or——fl Air containing 30% C02
250-
m———a N2 containing 30% C02
200‘
150-
100q
50-
0‘ ——9 *9 ‘ikgi Ae<}—e ssg
I l l I 1 I
24 48 72 96 120 144
Time-hours
jFigure 4. Effect of gas composition on ethanol production by

the roots of aseptic peas grown under low light
conditions. (Each point represents the average
value of three replications.)

 

3'53: I n

 

 

 

 

90—
80a
70‘
1:“
m 60‘
-H
m
3
a
H
'U 50-
,..|
I
m
m
E
'4 40
o
a
m
.c
4.)
m
30j
20.
10'
Figure 5.

29

o—-o N2 without C02 for 96 hours

D———o .Air for 48 hours, then N2 for
48 hours

 

1%. air-J. -
I 2‘

 

 

I
24 48 72 96

Time-hours

Effects of flooding period on ethanol production
by aseptic roots under low light conditions.
(Each point represents the average value of
three replications.)

 

1200T

1000-

,J 800-
.c
U)
-H
(D
3
>1
H
vs

T

m 600
m,
E.
H
o
r:
(U
.C:
4..)

m 400

200‘

Figure 6.

3O

 

o—-o High light

o—o Low light

 

 

1 l j

1
72 96 120 144

Time-hours

48

Effects of light intensity on ethanol production
by aseptic pea roots subjected to N containing
30% C02. (Each point represents the average value
of three replications.)

 

300

250

E 200
m
«4
m
3
w
5..
to

'7' 150
Us
m
E
H
o
c
m
3‘3

m 100

50

Figure 7.

31

o—-o High light

D————O Low light

 

 

N2 .
: Air
’9/ 4*»
I T —l
24 48 72 96 120 144

Time-hours

Effects of light intensity on the ethanol
production by aseptic pea roots treated with air
for 48 hours, then N2 without CO for 48 hours,
then air for 48 hours. (Each vaIue represents
the average of three replications.)

32

Table 3. Ethanol production in the exudates from roots of
four pea plants treated with N2 containing 30% C02.

 

 

 

Dmafionof Ethanol Remaining volume
anammm concentration of nutrient solution
hours ppm ml
17 6a 174
24 9 171
35 14 . 168
43 11 164
48 14 160
60 33 156
66 37 152
72 36 148
87 43 144
96 51 140
108 51 136
113 53 132
120 53 128
132 56 124
140 65 120
144 62 116

 

aEach value represents the average of three replications.

Table 4. Ethanol production in the exudates from roots of
four pea plants treated with 30% C02 and air.

 

 

Duration of Ethanol Remaining volume
treatment concentration of nutrient solution

hours 42pm ml
6 la 173
24 2 165
48 2 152
72 2 140
96 5 126
120 12 111
126 16 107

 

aEach value represents the average of three replications.

33

Table 5. Ethanol production in the exudates from roots of
four pea plants treated with air for 48 hours,
then N2 without CO for 48 hours, then air for
48 hours at high light intensity.

 

 

 

 

 

Duration of Ethanol Remaining volume
treatment concentration of nutrient solution

hours ppm ml
48 2a 173
56 3 166
58 8 162
70 15 158
74 30 154
80 36 150
86 50 146 (gm
96 62 142 ;'
120 57 127 ii

 

aEach value represents the average of three replications.

Table 6. Ethanol production in the exudates from roots of
four pea plants treated with N2 without C02.

 

 

Duration of Ethanol Remaining volume
treatment concentration of nutrient solution
hours gppm ml
12 1a 172
24 2 168
48 6 156
72 7 140
132 37 104

 

aEachvalue represents the average of three replications.

34

Tdfle7. Ethanol production in the exudates from roots of
four pea plants treated with air for 48 hours,
then N2 without C02 for 48 hours, then air for

 

 

 

48 hours.
.muafionof Ethanol Remaining volume
nemmam concentration of nutrient solution
hours ppm ml
12 1a 175 m
24 l 170 rfik
54 l 160
66 4 152
72 6 147
79 7 142
90 12 136
92 14 130 '
138 10 120 t;
142 8 114
149 10 110

 

aEach value represents the average of three replications.

The possibility that C02 might affect root exudation of
ethanol during soil flooding was also considered. The fact
that gases taken from an anaerobic soil at a depth of two
meters contained 15.5% C02 (Baver, 1956) suggests that very

high concentrations of C02 may occur in the rhizosphere of

respiring roots when flooded. Values of 20% C02 have also

been reported to accumulate in very heavy soils containing

decomposing organic matter; Kidd (1914). To study this
possibility, air containing 30% C02 and a mixture of 70% N2
and 30% C02 were obtained from the Matheson Gas Products
Company. These gases were circulated through the mist chamber
for six days and the ethanol content of the circulating

mineral nutrient solution was monitored. Data is presented

35

in Figures4 and 6. The addition of 30% C02 to 70% N2
caused a 200% increase in ethanol accumulation. With 30%
C02 and 70% air, ethanol accumulated at a rate similar to
that of N2. This is illustrated by the nearly parallel
curves for these two treatments in Figure 4.

These results indicate that high concentrations of CO2
affect not only the permeability of the root membranes but
also the metabolism of plant roots.

The low pH (5.5) created by saturating the mineral
nutrient solution with 30% C02 may have increased the

permeability of membranes in stressed roots. This phenomenon
explains in part the greater accumulation of ethanol in the
exudates of roots subjected to N2 with 30% C02. However,
the formation of ethanol in roots treated with air contain-
ing 30% C02 suggests the high partial pressures of C02,
occurring in cells subjected to high concentrations of C02,
inhibit the normal cycling of the tricarboxylic acid (TCA)
cycle, White gt gt. (1968). Just as in the case of oxygen
stress, when reactions in the TCA cycle are inhibited by
their accumulated products (i.e., reduced pyridine nucleo-
tides) high concentrations of CO2 could inhibit the
decarboxylation reactions, reducing the rate of the cycle.
Assuming this occurs, pyruvate is then reduced to ethanol
forming an alternate electron sink even in the presence of
oxygen. In any case, whether or not the forementioned
alternate pathways occur, the results present concrete
evidence that metabolic pathways of aerated roots are

altered by high concentrations of C02.

 

 

36

Ethanol production during anaerobiosis was also affected
by light intensity. Figures 6 and 7 show more ethanol ac-
cumulated in the root exudates of plants subjected to high
light regardless of the gas used in the rhizosphere. Bolton
and Erickson (1970) reported similar results by showing higher
ethanol concentrations accumulated in xylem exudates of tomato

plants grown under high light conditions. It is believed this
phenomenon occurs as a result of higher photosynthetic rates
during the higher light and temperature conditions. Ethanol
was absent from the exudates of aerobic roots under high light.
The rate at which ethanol accumulates during anaerobiosis
appeared to be unaffected by the concentration of ethanol in
the rhiZOSphere; Figure 5. However, when the atmOSphere of
the root zone is made aerobic, the ethanol content of
anaerobic root exudates declined as shown in Figure 7. These
results indicate that exuded ethanol was reabsorbed and/or
oxidized by the roots. Similar results were reported by
Cossins and Turner (1962) . They reported that an active
alcohol dehydrogenase in the cotyledons is active in peas dur—
ing germination but the activity declines soon after
germination. In this study, ethanol disappeared from the
exudate of 11-day—old pea roots indicating that an active
alcohol dehydrogenase enzyme may be present in the roots.
’hese data provide evidence that the reaction in equation (1)

ccurs in the roots of peas:

 

CH 3-COH ,_a1cohol dehydrogenase i CH3CHZOH (1)
acetaldehyde + ethanol
DPNH DPN

biphosphOpyridine nucleotide (DPNH) is the reduced pyridine

 

 

 

J;

 

37

nucleotide and DPN+ is the oxidized pyridine nucleotide.
Since the direction of equation (1) in peas is controlled by
the presence or absence of oxygen it appears that initially

the reaction is driven to the right during anaerobiosis by

the abundant supply of substrate and DPNH.
Root exudates of intact pea seed-

TMSi derivatives:
Data

lings were analyzed for amino acids and carbohydrates.

obtained from chromatographing their TMSi derivatives are
presented in Figures 8-15 and Table 8. The exudation

patterns were quite different depending upon the length and

degree of anaerobiosis and the presence or absence of

Fusarium.
The column used in this study gave excellent separation

of the TMSitderivatives of root exudates as long as they

were in small quantities. Alanine, valine, leucine,

glycine, proline, serine, threonine, and glutamic acid were
Good separation of

eluted before the pentoses and hexoses.
exudate carbohydrates also occurred on the SE-30 column.

At the onset of this investigation attempts were made
to silylate root exudates using BSA in pyridine. This

procedure was soon abandoned as most of the amino acids were
masked by the tailing pyridine peak. Attempts were made to

remove the pyridine by partitioning the TMSi derivatives in
hexane and water as described by Wood gt gt. (1965) . This

procedure is excellent for TMSi sugars as hexane is in—

soluble in water yet retains the carbohydrates, but only

compounds the TMSi amino acid-pyridine problem.

 

 

38

 

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47

As reported by Stalling gt gt. (1968) TMSi alanine and
TMSi glycine cannot be separated from mono (trimethylsilyl)
acetamide, a reaction product of BSA. BSTFA and its reaction
product mono (trimethylsilyl) trifluoroacetamide are more
volatile than BSA and do not coincide with the elution peaks
of alanine or glycine. The success in silylating amino acids
and sugars in the presence of high salt concentrations with F“
BSTFA holds considerable promise in identifying additional
root exudates of plants as the derivatives can be prepared in

a single step and in a relatively short time.

 

Attempts were also made to separate the mineral salts in P;
the nutrient solution from root exudates using an ion ex-
change resin, Rollins gt gt. (1962). This, as well as organic
methods of separating salts and amino acids, did not effi-
ciently separate the small quantities of amino acids from the
mineral salts.

Exudates were quantitatively analyzed by the methods
outlined by Sawardeker and Sloneker (1965). The minimum
quantity of TMSi sugars detected by the hydrogen flame was
0.25/ug and 1.0/«g for the amino acids.

Mass spectrometric analyses showed the silylation pro-
cedure used in this study formed the silylamine or
silylester of essentially all the amino or carboxyl and/or
hydroxy groups, respectively. The general reaction is
recorded in equations (2) and (3). Fully silylated amino
acids were previously reported by Ruhlman (1961) and others

cited there. The above conditions for derivatization

48

 

o
n
2R-CH-COOH + 3CF3-C-N-Si(CH3)3 gmcs g, (2)
l I 70°C, 5 hrs'
amino acid BSTFA

O
H
2R-CH-C-OSi(CH ) O
I 3 3 + 3CF3-E—NH2
(CH3)3Si-N-Si(CH3)3 A
trifluoroacetamide
TMSi amino acid

 

R O
H
H-con + nCH3-C—N-Si(CH3)3 Eggs L (3)
l l 70°C, 5 hrs’
R? Si(CH3)3

polyhydroxy sugar

B n
H-cOSi(CH3)3 + nCF3-C-NH-Si(CH3)3
RI

poly TMSi sugar mono (trimethylsilyl) trifluoroacetamide

were selected on the basis of the dissolution and silylation
of the amino acid and carbohydrate standards. One milligram
of each dried standard was silylated in 4SQ/rl BSTFA and
50/41 DMF. Samples, 5;.1, were injected into the gas
chromatograph. Anomerization of some carbohydrates may have
occurred during this treatment; Sweeley gt gt. (1963). Con-
sequently, the anomeric peaks, if they were identified, were
combined during the quantitative measurements of the carbo-
hydrates.

Considering the amino acid content of pea root exudates,
Figures 8-15 show that 4 or possibly 7 amino acids were

exuded by the roots of 6-day-old pea seedlings. This is

 

49

fewer than the 21 identified by Rovira (1956) in the root

exudates of l4-day-old peas grown in sand. Apparently

additional amino acids leaked from the roots during the

removal of sand by washing. Their methods of removing the

mineral salts from the exudates and forming the hydrochloride

salts of the amino acids may have hydrolized the enzymes

(Chang and Bandurski, 1964) and other proteins in the root ra‘

exudates.

 

Table 8 shows oxygen stress and/or high partial pres-

sures of C02 definitely promoted root exudation of alanine.

 

Alanine was the predominant amino acid in the exudate of all
gas treatments. This finding coincides with that of Cossins
(1964) who reported large amounts of alanine metabolized via
the transamination of pyruvate in young pea seedlings. In
this study the largest concentrations of alanine accumulated
in the exudates of plants treated with N2 and/or C02. This
phenomenon is evidence that only a portion of the pyruvate

is reduced to ethanol during anaerobiosis. More alanine
accumulated in the exudates of plants subjected to air, then
N2, then back to air at intervals of 48 hours than when sub-
jected to N2 for 6 days. There was also more alanine in the
exudate of plants treated with the same gases but under low
light. The tremendous quantity of alanine produced in the
air treatment containing 30% C02 is evidence for a carbon
dioxide stimulated process in the metabolism of alanine under
aerobic conditions. It should be mentioned that the attenua-
tion of the alanine peaks in Figures 8-13 was too high (160-320)

to be included in these chromatograms.

50

The accumulation of leucine and aspartate appeared to
be a function of high C02 and high light, as 8.4 and 0.9
mg/g dry roots were excreted into the rhizospheres treated
with no oxygen and high C02. Additional light also increased
the accumulation of these two amino acids in the rhizosphere
treated with an intermittent aerobic-anaerobic atmosphere.
Glutamate accumulated only in the rhizOSpheres treated with rm
gases containing oxygen, except the control.

Dubinina (1961) reported oxygen deficiencies of pumpkin,

tomato, and willow roots led to increases in endogenous free

 

amino acids. The fact that more amino acids were exuded by l
stressed pea roots in the present study is evidence that
greater quantities of free amino acids are produced in
anaerobic pea roots.

The greatest loss of identified carbohydrates by plant
roots occurred during periods of oxygen stress. Low oxygen
compounded by high concentrations of C02 caused up to 4.6 mg
of carbohydrates to be exuded per gram of root, Table 8.

Pea roots treated with air exuded trace amounts of carbo-
hydrates. With the addition of 30% C02, carbohydrate
exudation increased to at least 0.3 mg per gram of dry root.
Treatment with N2 caused very little leakage of carbohydrates.
The addition of 30% C02 to N2 caused 4.6 mg of the measured
carbohydrates to leak into the rhiZOSphere. Roots treated
with an intermittent aerobic-anaerobic atmOSphere also

showed an increase in the carbohydrate content when compared

to the control.

51

Among the carbohydrates listed in Table 8, glucose is
the most consistent exudate. For the air treatment root
exudates contained a trace of glucose while 0.3 mg/g was
exuded when 30% C02 was added. A nitrogen environment
caused 9/Lg of glucose to accumulate in the rhizOSphere.
By adding 30% CO2 to the NZ,
to l3/xg. The treatments with 48-hour intervals of aerobic- ‘e

the glucose content increased

anaerobic atmospheres contained 5 and 69419/9 of root for
low and high light, reSpectively.

Except for the air control, ribose was found only in

 

the N2 treatment containing 30% C02. Besides the control, flJ
fructose occurred in the exudates from the air-Nz-air (low
light) and N2 with 30% C02 treatments. Sucrose was
identified in the air-NZ-air (low light), and the air with
30% C02 and N2 with 30% C02 treatments. They contained
212, 59, and 69Q/Ag/g dry root, reSpectively.

These results demonstrate the adverse effects
anaerobic rhizosphere conditions have upon root exudation.
That anaerobic conditions simulated by an N2 atmosphere are
intensified by adding 30% C02 is indicated by the larger
quantity of exudates. These results are similar to those
of Hiatt and Lowe (1967) and others. These authors suggest
that anaerobiosis causes the cytOplasm to derange and become
less dense than that of aerobic roots. Similar changes of
the cytOplasm result from treatment of roots at a pH of 4.4.
This phenomenon may have contributed to the increased exuda-
tion of roots subjected to 30% C02 as the pH of the circulating

nutrient solution drOpped to 5.5.

52

Several peaks were eluted from the column at tempera-
tures greater than 165 C. Of these only sucrose was
positively identified. Many of these peaks had mass
spectrograms very similar to those characteristic of TMSi
carbohydrates. For example, the spectrogram of the peak at
168 C in Figure 11 contained fragmented ions whose masses
were 204 and 217 which were 10 and 40% of the parent peak F.L
and are characteristic of carbohydrate fragments. It also
contained fragments with a mass of 305 which was 30% the

height of the parent peak. The peak is often found in the

 

mass Spectrograms of closed ring carbohydrates. '

Glycerol phOSphate was also identified in the exudates
of all treatments by mass spectroscopy. No quantitative
measurements of this compound were made during this
investigation.

The possibility that volatile organic compounds evolved
from the roots of stressed plants was also considered. To
study this possibility a porapak organic compound trap was
attached to the gas outlet of the mist chamber for 48 hours.
Porapak containing the trapped gases was emptied into the
precolumn of the gas chromatograph-mass spectrometer.
Qualitative measurements of the unknowns was possible by
loading the column at room temperature and eluting these
compounds into the mass spectrometer by programing the
temperature of the column. Background components of this
procedure were determined by applying the above procedures

to Porapak which filtered the gases prior to flushing the

53
rhizosphere. The ethylene and/or acetylene content of the
rhizosphere was determined by the direct injection of 10 m1
gas samples into the gas chromatograph.

Ethanol, acetaldehyde, and water were trapped and
identified from the exhaust gases of stressed plants. No
organics were detected in the exhaust gases of the rhizo-
Sphere treated with air. Quantitative evaluation of these F5
organics was impossible by these methods as the samples were
eXpended during mass Spectrosc0py. However, the relative

height of the parent peaks of acetaldehyde to ethanol was

 

4:5 in the bar graph spectrogram. E

Volatilization of ethanol, accumulated during anaerobic
germination of peas, was also reported by Cossins and Turner'
(1962). They showed 5% of the ethanol produced was lost by
evaporation. The above results are interpreted as evidence
that the alcohol dehydrogenase complex of peas was Operative
during aerobic and anaerobic conditions when the ethanol
substrate was present. Since no acetaldehyde was detected
in samples chromatographed for ethanol, it is suggested that
most of the acetaldehyde produced by metabolism in the
rhizosphere is lost into the gaseous atmosphere of the soil.
Microbial contamination as a possible explanation for the
observed production of acetaldehyde has been excluded by
tests which indicated strict asepsis was maintained.

Carbon dioxide evolution by the treated roots was
determined by measuring the differential C02 content of the

inlet and exhaust gases. Data from these measurements are

54

presented in Tables 9-13. Generally, less CO2 was produced
by roots treated with air than those treated with N2 con-
taining no C02. Evolution of C02 by plant roots subjected
to air-Nz-air at 48-hour intervals was less during the N2
treatment under low light conditions but increased when
treated with high light, Tables 11 and 12. As eXpected,
respiration also fluctuated with the time of day.

The increase in root respiration immediately after the
rhizosphere was converted from aerobic to anaerobic condi-
tions without C02, as shown in Table 11, provides additional
evidence of the over-response mechanisms characteristic of
plants. Infected roots pretreated with a mixture of 70% N2
and 30% C02, then changed to air, produced more C02 than

infected roots grown in an aerobic atmOSphere, Table 13.

Plant Morphology

 

After six days of treatment, plants were removed from
the mist chamber and separated into roots, shoot, and seed.
The component parts were measured, dried, and weighed. The
data are presented in Tables 14 and 15.

Growth of the primary root was reduced very little by
the gas treatments. Secondary root development was greatly
reduced by oxygen stress and/or high C02 concentration.

As shown in Table 14, more secondary roots developed and
grew in the treatment containing 21% oxygen. When the 02
content decreased to 14.7% and the C02 content increased to

30%, essentially no secondary roots formed. N2 gas resulted

 

Table 9.

pea plants treated with air.

Carbon dioxide evolution by the roots of four

 

 

 

[€02]
Date Time Gas ppm mg/g/hr
3/5 830 Air sea 2.20a
3/5 1015 Air 105 2.62
3/5 1100 Air 76 1.90
3/5 1400 Air 88 2.20
3/5 1500 Air 97 2.42
3/5 1600 Air 94 2.35
3/6 1430 Air 101 2.52
3/6 1600 Air 110 2.75
3/6 1750 Air 103 2.57
3/6 1900 Air 95 2.73
3/7 600 Air 93 2.32
3/7 1230 Air 104 2.60

 

aEach value represents the average of three replications.

 

 

 

 

Table 10. Carbon dioxide evolution by the roots of four
pea plants treated with N2 without C02.
[C02]

Date Time Gas ppm mg/g/hr
3/11 2230 N2 1613 15.45a
3/12 830 N2 245 23.51
3/13 1600 N2 112 10.75
3/14 1030 N2 107 10.27
3/14 1230 N2 108 10.37
3/14 1630 N2 102 9.79
3/14 2200 N2 156 14.97
3/15 900 N2 112 10.75
3/15 1030 N2 113 10.84

 

aEach value represents the average of three replications.

 

 

56

 

 

 

 

Table 11. Carbon dioxide evolution by the roots of four

pea plants treated with air for 48 hours, then

N2 without C02 for 48 hours, then air for 48

hours under low light.

[C02]

Date Time Gas ppm mg/g/hr
3/16 1830 Air 122a 7.90a
3/16 2000 Air 135 8.74 H
3/17 1000 Air 117 7.58
3/17 1730 Air 101 6.54
3/17 2015 Air 102 6.60
3/17 2200 Air 101 6.54
3/18 2230 Air 91 5.89
3/18 2300 N2 added -— --
3/18 2315 N2 339 21.95 ,
3/19 900 N2 63 4.08 E
3/19 2130 N2 52 3.37
3/21 2100 Air 74 4.79
3/22 915 Air 108 6.99
3/22 1400 Air 102 6.60
3/22 2115 Air 95 6.15

 

aEach value represents the average of three replications.

 

 

 

 

Table 12. Carbon dioxide evolution by the roots of four
pea plants treated with air for 48 hours, then
N2 without C02 for 48 hours, then air for 48
hours under high light.
[C02]
Date Time Gas ppm mg/g/hr
4/10 1130 Air 88a 7.39a
4/10 2000 Air 95 7.98
4/11 900 Air 105 8.82
4/11 1700 N2 134 11.26
4/12 830 N2 133 11.17
4/12 1530 N2 133 11.17
4/14 1430 Air 105 8.82

 

aEach value represents the average of three replications.

 

57

 

 

 

 

 

 

Table 13. Carbon dioxide evolution by the roots of four pea
plants treated with air and N2 containing 30% C02
(anaerobic) and infected by Fusarium.
[C02]
Aerobic Anaerobic
Date Time Appm mgKg/hr ppm mg/g/hr
4/19 1130 107b 5.14b -- --a
4/20- 910 118 5.67 -- --
4/20 1900 133 6.39 -- -- F“
4/21 1600 113 5.43 -- --
---------- Addition of pathogen ---------
4/21 1845 120 5.77 -- --
4/21 2230 84 4.04 -- --
4/21 6380 64 3.08 -- --
4/22 6324 63 3.03 -- --
4/22 1030 102 4.90 -- -- ,
4/22 1440 112 5.38 -- -- F
4/22 2045 105 5.05 -- --
4/24 2315 93 4.47 -- --
4/26 1015 103 4.95 -- --
- Changed to Air 5
4/27 1400 139 6.68 341b 59.48
4/27 1545 133 6.39 218 38.03
4/27 1915 114 4.81 181 31.57
4/27 2130 115 5.53 218 38.03
4/28 2000 114 4.81 213 37.16
4/29 1100 97 4.66 141 24.42
4/29 2130 169 8.12 187 32.62

 

aDashed lines indicate C02 content beyond range of

instrument.

bEach value represents the average of three replications.

58

 

 

 

 

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mo.o m.m v Hm.s so. oms
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mo sumcoq mo rumcoq mumpcooom mo spasms .

 

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.mmmp o How topmouu mucmsm.mmm mo mowumwumuomumso
moms ucoEcoss>co uoou mo cesusmomEoo msoommm mo muoommm .va manna

59

 

 

 

 

rs; 1| .1...er
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mo usmsoz mum on» com: ucoEcous>co uoou mo sowusmomEoo msoomom mo muoommm .ms osome

60

in the reduction of the number of secondary root initials
nearly 50%. When 30% CO2 was added to the N2 the number was
reduced an additional 50%. Air, then N2, then air at 48-
hour intervals did not reduce secondary root development nor
growth unless plants were grown under high light. Shoot and
root growth was best when the roots were treated with air.
In contrast, significant reductions in the length of shoots

occurred among plants whose roots were treated with an

 

atmosphere low in 02 and high in C02.

Roots subjected to anaerobic conditions for 2 or 6 days

 

were darker than aerated roots. However, new but thinner F.
secondary root tips, which were white, continued to grow or

elongate after the 2-day stress. Roots subjected to high
concentrations of C02 were considerably thicker than those

grown in the presence or absence of 02.

Leaves of plants treated with N2 and N2 with 30% CO2
became chlorotic after 24-48 hours. Treatments containing
30% C02 brought on chlorosis more rapidly than N2 alone.
Plants treated with N2 for 48 hours became chlorotic 1-2
days after treatment. The leaves of all plants subjected
to treatments containing 30% C02 appeared to have thicker
cuticles than those treated with air or N2 alone.

The above results compare favorably with those reported
by Williamson and Splinter (1968) and others. The results
indicate that either a lack of 02 or excessive C02 causes
injury to the root and with time causes physiological

changes in the leaves.

61

The dry weight of plants treated in this investigation
are reported in Table 15. These values are closely related
to the morphological data presented above. Root weights were
significantly less in those plants treated with mixtures of
gases low in 02 and/or high in C02. These gases also in-
creased the sloughing off of cell debris. The quantity of
debris accumulated on the nutrient solution filter was: 3.6, r-
2.3, 1.1 and 0.4 mg for 8.5, 7.0, 0.3, and 0.9% of the total
root weight of treatments; N2 containing 30% C02, air contain-

ing 30% C02, N2 without C02 and intermittent air-NZ-air at

 

48—hour intervals, respectively. The hydrolytic products of i;
this debris provide substantial quantities of soluble material

in the soil environment. However, in the sterile mist chamber

any hydrolytic enzymes secreted into the circulating mist

media by the root would be diluted, hence inactive. Con-

sequently, it is believed the exudates reported in this study

are truly the products of excretion and not autolysis.

Plant Disease

 

Roots of intact pea seedlings were tested for their
increased susceptibility to Fusarium root rot during severe
anaerobiosis. The gas treatment containing N2 and 30% CO2
was chosen for its adverse effects upon root exudation and
morphology and its suggested stimulative effects at this CO2
concentration upon Fusarium growth; Papavizas and Davey
(1962). Macroconidia of E. solani f. ptgt, cultured and

prepared by the methods described earlier, were injected into

the sterile nutrient solution 5 days after the onset of the

62

experiment. Samples were removed from the reservoir via the
access port and analyzed for spore germination and ethanol
content.

Table 16 shows a definite increase in the germination
and growth of Fusarium in the rhizosphere of anaerobic
plants. Two hours after inoculation, spore germination was
nearly 300% greater in the root zone of stressed plants. At
18 hours the percent germination was the same but growth
(i.e., germ tubes) of the pathogen was greater in the
stressed treatment. After 29 hours germination had doubled
and germ tube growth was 27-fold greater in the anaerobic
than in the aerobic rhizosphere. By 94 hours germination
had peaked at 91% in the anaerobic rhizosphere with con-
current germ.tube growth of 400/iincluding the branches.
Many of the macroconidia in the rhizosphere containing air
had constricted forming chlamydOSpores whose contents were
granular having the same appearance as those cultured in
distilled water. Six days after inoculation the atmOSphere
of the stressed plants was changed to air until the experi-
ment was terminated.

The presence of Fusarium in the anaerobic rhiZOSphere
decreased the rate of ethanol accumulation as shown in
Figure 16. The point of inflection in the ethanol curve
12 hours after inoculation was concurrent to the rapid
growth of the inoculum, Table 16. The fact that the
decrease in the rate of ethanol accumulation was simul-
taneous to pathogen growth provides additional evidence that

ethanol was metabolized by the pathogen. The ethanol

 

 

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bosom comm

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com: ESsHMmsm mo oocomonm osu poo msmOsoosomcm mo muoommo one .os ossmsm
musoslofise
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64

Table 16. The effects of time and gaseous atmosphere upon
the germination and growth of F. solani f. pisi

macroconidia in the rhizOSpherE of pea roots.

 

 

 

 

 

Treatments
Time
hrs Aerobic Anaerobic
Length of Length of
Germination germ tube Germination germ tube
% xu- % /‘L
0 0a 0a 0a 0a
3 13 0 36 12
8 l6 0 41 12
18 39 0 39 56
30 43 4 82 108
42 __ —- 92 300
96 26 16 91 400

 

aEach value represents the average of two replications.

content declined very rapidly soon after the rhizOSphere was

aerated, Figure 16. This rate far exceeded that of auto—
oxidation in a sterile rhiZOSphere, Figure 7, suggesting
that ethanol was oxidized by the pathogen. These results
support those of Cochrane gt gt. (1963) who showed the
exogenous carbon requirements of germinating macroconidia
could be fully replaced by ethanol.

Anaerobic conditions caused a 5-fold increase in the
growth of Fusarium with a c0ncomitant 4-fold increase in
infection and disease, Table 17. Roots grown in the mist

chamber having an atmOSphere of air were infected at the

broken walls of the primary root where the secondary roots

had emerged. Roots grown in a mixture of 70% N2 and 30%

CO2 showed black lesions on both the roots and epicotyl.

 

65

Table 17. Effects of severe anaerobiosis on the intensity of
root rot disease and growth of Fusarium in the
rhizosphere of peas.

 

 

Treatment Disease indexa Dry weightb - mg
Air 1.9C 8.1C
70% N2, 30% C02 7.6 42.7

 

aDisease index is rated 0-12, with 12 assigned to dead plants.

Fusarium mycelia were removed from nutrient solution by

bDry weight of pathogen growth in rhizosphere for 12 days.
filtration. This weight includes the cell debris. F-

cValues are the average of two replications.

The tips of roots treated with N2 containing 30% CO2 were com-

 

pletely black while the aerobic root tips were not infected. *3
This phenomenon suggests that more nutrients were exuded at

the root tips or the integrity of this area of the root was

affected more by anaerobiosis or a combination of both re-

sulted in greater disease. This data agrees with that of

Lockwood (1962) who showed 24% more disease occurred in pea

roots grown in saturated conditions. Since the plants in his

study were not grown under aseptic conditions, the smaller

increase in the disease of saturated plants may have resulted

from the competitive absorption of root exudates.

Pea roots became necrotic 8 days after inoculation. A
brown exudate was noted from the anaerobic treatment 4 days
after inoculation while it was not observed in the exudate of
the aerobic treatment until 8 days after inoculation. A
similar exudate containing 10 to 20 phenolic compounds was
reported by Sherrod and Domsch (1970). They showed the re-
lease of phenols was an integral part of the pathogenicity

mechanism of pea root disease.

66

As in previous treatments, the length of the primary
root was unaffected by anaerobiosis. However, secondary root
growth and deve10pment was severely restricted by the
anaerobic conditions as is shown in Table 18. Total dry
weight of roots grown in the air treatment was 15.4 mg while
4.2 mg of dry weight was produced by each plant treated with
N2 containing 30% C02. Shoot growth was also reduced by
anaerobiosis. The length of stems and leaves was reduced
40% while the dry weight of the shoots was reduced by nearly
80%. These results are interpreted as evidence supporting
the work of Kende (1964) and others. They reported that an
auxin or kinetin-like substance synthesized in the root is
essential for prOper growth and development of the shoot.
The author contends the synthesis and transport of these
substances is inhibited by anaerobiosis.

As reported earlier in Table 13, greater quantities of
C02 were produced by infected roots in the anaerobic treat-
ment when it was changed to air than by infected plants
treated aerobically. The 4-9 fold increase in C02, eXpressed
on the basis of root weight, may be explained by the greater
growth of the pathogen in this treatment as shown in
Table 17.

Fewer carbohydrates accumulated in the rhiZOSphere of
anaerobically treated plants after they were inoculated by
Fusarium. Table 19 shows a 70% decrease in the measured
carbohydrate content of exudates in the treatment involving

N2 which contained 30% C02 as compared to the aerobic

 

 

 

 

 

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.uos uoou Edsummsm
sus3 pouooMCs mucosa mo macsosmuoe osu coma msmOsoouooco mo muoommo one .ms osooe

68

treatment. Both fructose and sucrose were absent from the
exudates of diseased and anaerobic plants. Ribose was absent
from the exudates of both treatments. In contrast, glucose
was more abundant in the exudates of the anaerobic than in
the aerobic treatment.

Fewer amino acids were identified in the anaerobic
exudates of diseased plants, Table 19 and Figure 15. There
was a 60-fold increase in glutamate production in the
exudates of the anaerobic as compared to the aerobic treat-
ment. The conSpicuous absence of alanine, present in the
exudates of sterile plants of both treatments (Table 8) and
the absence of aspartate and leucine identified in the
sterile exudate of the mixture of 70% N2 and 30% C02 suggest
these three amino acids were metabolized by Fusarium.

By comparing the total carbohydrate and amino acid
contents of the sterile roots, Table 8, and inoculated roots,
Table 19, it is apparent that the presence of Fusarium may
have increased root exudation. This phenomenon may in part
be the result of plant age. But, as Rovira (1956) points
out, there is just a 20% increase in root exudates of peas
from day 10-21. Since the increase in exudates exceeds this
value, even when a growing pathogen is present, it is
believed this increase results from the presence of the

pathogen.

Bioassay

From the results obtained in the mist chamber several

factors were Operative, causing the increased disease in

 

69

 

 

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ocsEo one moumsomsoosoo mo c0suMsoEooom osu so COsusmomEoo new no muoommm .ms osome

70

the anaerobic rhizosphere. These factors could possibly be
separated by isolating the microorganism and subjecting it
to various concentrations of sterile gases and ethanol.

Gases sterilized by filtration were bubbled through
100 m1 of sterile redistilled water containing 0, 10, 100,
and 1000 ppm of ethanol. Macroconidia of E. solani f. ptgt
were added to the solution and incubated at 25 C for 24 hours. ma‘
Data are presented in Table 20.

Germination was low in all the treatments. Ethanol

concentration alone had essentially no effect upon germina—

 

tion regardless of the treatment gas. Those gases containing E5
very low concentrations (less than 0.41 ppm) of oxygen in-
hibited germination and develOpment of the germ tube. The
air treatment containing 30% C02 showed some stimulation of
germination by ethanol. After 24 hours 33% of the spores had
germinated in the solution containing 1000 ppm ethanol with
34, 16, and 3% germinating in 100, 10 and 0 ppm of ethanol,
respectively. Approximately 50% germination occurred in
those solutions diffused with air. Again there was no
effect of ethanol concentration on germination.

The second bioassay was similar to the first except
for the addition of a nitrogen source in the form of 20/ig
of alanine. In this eXperiment ethanol content was deter—
mined before and after the treatment period and adjustments
were made for volatilization. When the ethanol standards
were autoclaved and cooled approximately 30% of the ethanol

was lost by volatilization.

 

71

 

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0 o o o o o ooa No usosunz N2
0 o m o o 0 OH NO usonufl3 NZ
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0 o o o o o ooa Noo usonunz «z
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0 o o o o o 9 N00 usonuwz Nz
ma m 0 mm m NH oooa Noo wom mancnmucoo “Ha
mm NH 0 am a 0 cos Noo wom mcflcflmucoo “Ha
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ma ma o m H o o N00 wom mcflcwmucoo Ham
0 m m o H H oooa NOU wom mewcwmpcoo NZ
0 o o o o o ooa NOU mom mowcwmucoo NZ
0 0 NH 0 o H OH Noo mom mcHCHmucoo Nz
o o o o o o o Noo mom mancnmucoo Nz
ma mm mm Hm «v om OOOH HH<
m NH o m m o 00H HH<
mm mm ma om mm ov 0H HH¢
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MSNonsu EH

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wwIGOHumaHEHmO

mucwEumoHB

 

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wnu com: coaufimomEoo msommmm cam cofiumnucwocoo Hocmnum mo muomwmm one .om canoe

72

Germination and growth of Fusarium occurred only in
treatments diffused with air and air containing 30% C02.
When an exogenous source of nitrogen was present greater
growth resulted in those treatments containing ethanol. As
before, germination and growth of the macroconidia appeared
to be reduced by increasing the concentration of ethanol,
Table 21. However, after 72 hours fungal growth showed a
significant increase in the highest ethanol concentration
used.

These results are interpreted to mean that ethanol
alone has very little effect upon germination and growth of
Fusarium. Conversely, when an exogenous source of nitrogen
is added, ethanol does promote the growth of Fusarium. The
greater germination in those treatments containing only
alanine (i.e., 0 ppm ethanol) is evidence that nitrogen
enhances spore germination. But with growth the endogenous
reserves of carbon are eXpended and the requirements for
exogenous carbon increase. These results agree with
Cochrane (1963) and indicate the carbohydrate supply of the

culture medium of these spores was more than adequate.

 

73

 

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.mnoon Nb um coma mucmEmusmmmzn

.musoc am no wows mucwEmusmmmzm

 

 

 

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vu.o m.NH m.mm In mo. qu
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N.N Nm mm mm ooa
m.N ov an N OH
N.N mN mm II o
Ema Hocmnum
IINOU wom mcwcwmucoo Hߢ
m.m ON mm mma oooa
m.N mm mm mm ooa
o.N am mm m 0H
v.N no cam I: o
o o Ema Hocmcumnnnfld
mE 4“ 5mm,
noomocumm mo monsp Euwm moowumcfifinmm cofiumfismcoo mucwfiummus
ucmflmz who mo numcmq usooumm Hocmsum

 

.mcwcmam m<\dN mewcflmucoo coHuDHOm cfi Hmwm .m HcmHOm .M «o nuBoum can
coflumcfiaumm may coma cowuwmomEoo moommmm paw cowumuucoocoo Hocmcum mo muommmm .HN manna

i
a ' . h ' 32,9754

L;

CONCLUSIONS

1) A mist chamber was designed to maintain sterile
conditions in the rhiZOSphere. The gaseous atmOSphere could
be changed at will with continuous monitoring of the F**
exudates.

2) Pea root exudation was increased by anaerobiosis.

Exudation of ethanol, amino acids, and carbohydrates was

 

increased by both oxygen stress and high C02 partial pres- E;
sure. These effects were additive as more root metabolites
were lost when the rhiZOSphere was treated with a mixture of
70% N2 and 30% C02.

3) Both ethanol and acetaldehyde volatilized from the
rhizosphere.

4) Greater respiration occurred in the rhizosphere of
roots pretreated with N2 and 30% C02 than from roots treated
with air.

5) Oxygen stress and high concentrations of C02
reduced the growth and development of secondary roots.

Shoot growth was also reduced by anaerobiosis.

6) Pathogen germination and growth as well as plant
disease were increased by anaerobic conditions in the
rhizosphere.

7) No germination of E. solani f. pisi occurred in

absence of oxygen.

74

75

8) Ethanol content increased Fusarium growth only in
the presence of an exogenous source of nitrogen.

These results provide evidence that anaerobiosis pro-
motes the severity of Fusarium root rot of peas. Low
concentrations of oxygen combined with 30% C02 reduced
plant growth and development. Under these circumstances,
the integrity of the root declines resulting in the loss of [-
additional metabolites which in turn promote fungal germina-
tion and growth. Increased growth of the pathogen intensifies

the inoculum potential of the fungus. The reduced integrity

 

1‘"

of the cells lowers the resistance of the root to the F;
surrounding biota. The result of these combined factors was

increased plant infection. Once the plant actively responds

to the presence of the pathogen the ensuing metabolic

reSponses are both complex and varied.

LITERATURE CITED

 

 

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