MECfiQfilQLOGICAL STUDIES ON THE
PHYfiZIOLOGECAL DESEASE OF

RICE EN YAIWAN

“rest: ‘or ”we Dogs-co of M. S.
MiCHlGAN STATE UNIVERSITY

Ming-huei Wu Shem:
19661

 

 

LIBRARY i”
Michigan State
University

   
      

 

 

MICROBIOLOGICAL STUDIES ON THE PHYSIOLOGICAL DISEASE

0F RICE IN TAIWAN

BY

Ming—huei Wu Sheng

A THESIS

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

MASTER OF SCIENCE

f

Department of Soil Science

1966

ABSTRACT

‘MICROBIOLOGICAL STUDIES ON THE PHYSIOLOGICAL DISEASE
OF RICE IN TAIWAN

by Ming—huei Wu Sheng

Studies were conducted on the microbial populations
in root environments of healthy rice and of rice exhibiting

symptoms of ”suffocation disease. a physiological disorder
associated with reductive soil conditions in eastern Taiwan.

NUmbers of total and anaerobic bacteria, fungi, and
aerobic and anaerobic cellulose decomposers reached seasonal
maxima during the tillering period. This was also the
period during which disease symptoms appeared and developed
to maximum intensity. These groups were much more numerous
on root surfaces than in the rhizosphere or edaphosphere,
and they responded to incorporated additions of straw or
green manure.

Actinomycetes, sulfate reducers and ammonifiers
were influenced to a much lesser degree by either organic
amendments or proximity to root surfaces.

Numbers of denitrifying bacteria showed relatively
little effect of organic amendments but appeared to be very
sensitive to plant influences. Extreme seasonal fluctuations

in numbers of denitrifiers, principally on root surfaces.

Ming—huei Wu Sheng

appeared to reflect changes in physiology of the rice
plant.l

Disease symptoms were successfully transmitted to
healthy rice seedlings in nutrient culture by root washings
from diseased plants, indicating the probable involvement
of micrdbial toxins. However, it appeared that a primary
factor affecting the nutrition and physiology of rice was
most likely the strongly reduced soil conditions observed:
Eh = -400 mv at tiller initiation. -250 mv at panicle initia-
tion and -150 to -100 mv at maturity.

- A review of the literature revealed numerous possi—
bilities for toxin production by denitrifying species.
Preliminary pure culture studies gave promise of useful
nutritional discrimination within this group for further

studies on rice rhizosphere physiology.

MICROBIOLOGICAL STUDIES ON THE PHYSIOLOGICAL DISEASE

OF RICE IN TAIWAN

BY

Ming—huei Wu Sheng

A THESIS

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

MASTER OF SCIENCE
Department of Soil Science

1966

ACKNOWLEDGEMENTS

The author wishes to eXpress grateful acknowledgement

to the International Rice Research Institute for financial
aid and thanks to Dr. I. C. MacRae for his suggestions
and assistance in carrying out this research. She would

like to thank Dr. R. L. Cook for generous encouragement
and for his assistance in making it possible to pursue

portions of the research with library and laboratory facili-

ties at Michigan State University. Also she would thank
Dr. A. R. Wolcott for guidance and advice during her stay

at East Lansing.

ii

TABLE OF CONTENTS

INTRODUCTION . . . . . . . . . . .
OBJECTIVES . . . . . . . . .

LITERATURE REVIEW . . . . . . . . .

Effects of Organic Matter
Rhizosphere Relationships

Denitrifying Bacteria

EXPERIMENTAL METHODS . . . . . .

Greenhouse Experiments
Inoculation Experiment
Field Locations

Microflora Studies
Nitrate Reducing Bacteria--Pure Culture Studies

RESULTS . . . . . . . . . .

Symptomology and Developmental Behavior of Rice

Inoculation Experiment
Microflora Studies
Pure Culture Studies

DISCUSSION . . . . . . . .

Seasonal Behavior of Microbial Groups
Relationships between Microflora and Disease

Symptoms
Nutrition of Denitrifiers

SUMMARY . . . . . . . . .
LITERATURE CITED . . . . . . . .

APPENDIX . . . . . . . . . . . .

iii

Page

l6
19

28
28
31
32
33
36
42
42
6O
61
73
84
84

87
92

93
96

111

10.

ll.

12.

LIST OF TABLES

Treatments in the first greenhouse
experiments . . . . . . . . .

Treatments in second greenhouse
experiments . . . . . . . . .

Nitrate reducing bacteria investigated
in pure culture . . . . .

o O O O O O 0

Experimental media . . . .

Height of rice plants in second greenhouse
trial (first crop rice, Lotung profile)

Number of tillers per hill in second
greenhouse trial (first crop rice, Lotung

profile) . . . . . . . . .

Weight of straw and number and weight of

rice grains per hill in second greenhouse
trial (first crop rice, Lotung profile)

Numbers of total bacteria and sulfate
reducers in nutrient solutions 10 days
after inoculation with root washings
from retreated and replanted rice
cultures in Lotung profiles from the
first greenhouse trial . . . . . . . .

Numbers of bacteria on rice roots in fall
crop (1964) and Spring crop (1965) . .

Numbers of actinomycetes on rice roots in
fall crop (1964) and spring crop (1965) .

Numbers of fungi on rice roots in fall crop
(1964) and spring crop (1965) . . . . .

Numbers of anaerobic bacteria on roots of
rice in fall crop (1964) and spring

crop (1965) . . . . . . . . . . . . .

iv

Page

29

31

38

39

45

45

46

6O

62

63

64

65

Table Page

13. Numbers of sulfate reducing bacteria on

rice roots in the spring crop (1965) . 66

Numbers of aerobic cellulose decomposers
on rice roots in fall crop (1964)

and spring crop (1965) . . . . .

14.
67

Numbers of anaerobic cellulose decomposers
on rice roots in fall crop (1964) and

spring crop (1965) . . . . . . . .

15.
68

NUmbers of denitrifying bacteria on rice
roots in fall crop (1964) and spring

crop (1965) . . . . . . . . . . . .

16.
69

NUmbers of ammonifiers on rice roots in
fall crop (1964) and spring crop

(1965) . . . . . . . . . . . . . . .
Aerobic growth of bacterial agar streak

cultures in relation to source of
nitrogen and growth factors . . . . .

17.
. . . 70

18.
75

Anaerobic growth of bacterial agar streak
cultures in relation to source of
nitrogen, growth factors and CO2 . . . . .

19.
76

20. Anaerobic growth of bacterial agar streak
cultures in relation to source of energy,

nitrate, growth factors and CO2 . . . . 77

Growth inhibition by Pseudomonas aeruginosa
ATCC 10145 under aerobic conditions
on cross-streaked agar plates . . . .

21.
. . 81

LIST OF FIGURES

Page

Figure

f rice tillers on Chungli (c).

) and Lotung (Ll) surface
Lotung profile (L2)

after transplanting.
greenhouse, 1964.) . . 49

1. Number 0
Pingtung (P
soils and on the
at various times

(Fall crop rice,

2. Weight of rice straw on Chungli (c).
Pingtung (P) and Lotung (Ll) surface
soils and on the Lotung (L2) profile.

(Fall crop rice, greenhouse. 1964.) . 49

3. Weight of rice grain on Chungli (c),
Pingtung (P) and Lotung (L1) surface

soils and on the Lotung profile (L2)
at various times after transplanting.
(Fall crop rice, greenhouse, 1964.) . 50

4. Number of rice grains on Chungli (c),
Pingtung (P) and Lotung (L1). surface
soils and on the Lotung (L2) profile.

(Fall crop rice, greenhouse, 1964.) . 50

5. Chungli surface 5011, fall crop rice, 1964,
10 days after transplanting. Left,

straw; right, green manure . . . .

51

fall crop rice. 1964,
Mont row:

ace soil,
fter transplanting.
right, green manure . .

fall crop rice. 1964, 10
transplanting. Left, straw;

O O O C

6. Pingtung surf
10 days a
left. StraW7

51

7. Lotung profile,
days after

right, green manure .

52

8. Lotung profile. fall crop rice. 1964, 10
Left, diseased

days after transplanting.

'plant; right, healthy plant . . 52

all crop rice, 1964, at
straw; right, green

9. Lotung profile, f
harvest. Left,

manure o . o . o

. . 53

vi

Figure

fall crop rice, 1964, at

From left to right:

Lotung profile,
(check), Pingtung

Chungli surface

10. Overall View,
flowering.
Lotung surface,
Pingtung surface
surface (manured),

SOil O O O O O O O O

O C O C O O O O O O C

1965: 15

ll. Lotung profile, spring crop rice.
days after transplanting . . . .

Spring crop rice, 1965, 15
days after transplanting. Amended 2 days
before transplanting with straw (left)
and green manure (right) . . . . . . .

13. Lotung profile, spring crop rice, 1965, 55
days after transplanting. Amended 2 days

before transplanting with green manure
(left) and straw (right) . . . . . . .

l4. Lotung profile, spring crop rice, 1965, 15
days after transplanting. Amended 2 weeks

before transplanting with straw (left)
and green manure (right). . . . . . . .
spring crop rice, 1965, 55
Amended 2 weeks
green manure

12. Lotung profile,

15. Lotung profile,
days after transplanting.

before transplanting with
(left) and straw (right) . . . . . . . .

spring crop rice, 1965, 15
days after transplanting. Straw incor-

porated into surface soil 2 days (left)
and 2 weeks (right) before transplanting .

16. Lotung profile.

17. Lotung profile, spring crop rice, 1965, 55 days
after transplanting. Straw incorporated

into surface soil 2 weeks (left) and
2 days (right) before transplanting. . . .

l8. Lotung profile, spring crop rice, 1965, 15
days after transplanting. Green manure
(Lupines) incorporated into surface soil
2 weeks (left) and 2 days (right) before

transplanting . . . . . . . . . . . . .
l9. Lotung profile, spring crop rice, 1965, 45
days after transplanting. Green manure

(Lupines) incorporated into surface soil
2 days (left) and 2 weeks (right) before

transplanting . . . . . . . .

vii

Page

53

54

55

55

56

56

57

57

58

58

Figure Page

20. Rice in nutrient solutions 25 days after
inoculation with root washings from
diseased rice grown in the Lotung
profile amended with straw (left)
and Lupines (right) . . . . . . . . . . . 59

viii

Table

23.

24.

25.

26.

27.

LIST OF APPENDIX TABLES
Page

Microbial numbers in the Lotung profile
samples at various stages of growth
of rice in the greenhouse. (First
trial, fall crop rice, 1964.) . . . . . . 112

Microbial numbers in Lotung soil at various
stages of growth of rice in the field.
(First trial, fall crop rice, 1964.) . . . 114

Microbial numbers in the Pingtung field
soil sample at various stages of growth
of rice (first trial, fall crop rice,
1964.) . . . . . . . . . . . . . . . . . . 116

Microbial numbers in the Lotung profile
sample at various stages of growth of
rice in the greenhouse (second trial,
spring crop rice, 1965.) . . . . . . . . . 118

Microbial numbers in Lotung soil at various

stages of growth of rice in the field
(second trial, spring crop rice, 1965.) . 120

ix

INTRODUCTION

Taiwan is a large semitropical island lying about
100 miles off the China mainland in the same latitude as
the Hawaiian Islands. It has a land area of 35,760 square
kilometers. Average January temperatures in lowlands range
from 15°C in the north to 20°C in the south. The summer
season lasts for six to seven months with temperatures
averaging 23—280C.

Rice is the most important crop of Taiwan. The
basic cropping pattern on Taiwan's paddy fields is to grow
two rice crops a year, one in the spring (the first crop
of rice) the other in the fall (the second crop rice).

In recent years, farmers of central and southern Taiwan
have gone into the growing of as many as four crops a year
on the same piece of land, because of year round warm
temperatures and especially good irrigation systems.

Organic matter content of tropical soils is generally
low. So is that of soils in Taiwan. It may be as high as
5% or as low as 1% but generally between 2-3%. Therefore,
maintenance of organic matter in soils, especially in paddy
fields, is considered essential. Unfortunately, the
application of green manure or straw to paddy soil aggra-

vates the seriousness of a major rice disorder which has

been known in eastern Taiwan for more than 30 years. until
recently this problem has been restricted to an area of
about 200 hectares in the eastern district of Ilan. How-
ever, during the present decade a similar unthrifty condition
of paddy rice has been recognized on extensive areas in
central and southern Taiwan. About 15,500 hectares were
affected in 1961, 25,635 hectares in 1962 and 3,570 hectares
in 1963 (162).

On the basis of field observations and other related
information, it is generally agreed that abnormal develop-
ment, poor growth and root rot observed in the problem area
in Ilan is a physiological disorder associated with poor
natural drainage and the resulting reductive soil condition.
Whether the same causes account for the newly arising rice
problem in the central and southern areas of Taiwan is more
controversial.

The apparent physiological disorder of rice in Ilan
is locally called "suffocation disease." It is one example
of a number of physiological diseases associated with poor
drainage. These may have different symptoms and are known
by various local names in other countries.

It is generally believed that the so-called "suffo—
cation disease" of rice is caused by chemically reduced
toxic products which interfere with nutrient uptake from
the soil. waever, the root zone is also the area of the

greatest activity of microorganisms. Any product of microbial

metabolism which is formed at the root surface, whether it
be harmful or beneficial, can have an immediate effect upon
the root cells. Conversely, changes in root physiology,
such as a decline or loss of oxygenating ability of rice
roots or changes in composition of root exudates or
sloughed off cellular debris, can drastically alter micro-
bial populations in the rhiZOSphere.

Since chemical methods seem to be unavailable to
define the physical and chemical status of the rhiZOSphere,
resort must be made to biological means of definition.
Changes in the microbial population in the root zone during
the growth of the plant will serve to indicate changes in
root physiology. Knowledge of the biological activities
of the microflora of root surfaces would be helpful in pre—

dicting the nature of possible toxic compounds.

OBJECTIVES

Microbial populations and activities in the rhizo—
sphere have been studied extensively for many upland crops.
very little is known in the case of rice, especially with
regard to harmful effects of microbial activities in poorly
drained paddy soil with the application of organic matter.
In order to reach a better understanding of the nature of
physiological disorders associated with these conditions
and to develop amelioration practices for rice fields made
unproductive by them, it seems an urgent matter to clarify
the relation between rice roots and the soil population.

A systematic study of rhizosphere physiology in its relation-
ship to development of disease symptoms is called for.

Such a study has been initiated in the investigation
reported here. The following objectives were undertaken:

1. To enumerate major taxonomic and physiological
groups of microorganisms in the rhizospheres of
healthy and diseased rice plants.

2. To correlate enumeration data with disease develop-
ment and with physiological age of rice.

3. To investigate effects of soil type and organic
amendments on disease development and microbial

numbers.

To demonstrate transfer of the disease by root
washings from diseased to healthy plants.

To develop media and procedures for more detailed
characterization of predominant physiological
groups found in the rhizosphere flora of diseased

rice.

LITERATURE REVIEW

Physiological disorders of rice occur in association
with flooding in many countries of Asia and America.
Symptoms and the stage of growth at which they occur are
different in different localities. In all cases, however,
an excessively reduced condition of the soil is an associated
phenomenon.

The corresponding diseased condition of rice is
called by different local names (131). In Burma, three
distinct patterns of symptomology are distinguished:
"Amiyit—po," "Miyit-po" and "yellow leaf." In Japan, the
names "Akagare," "Akiochi," "Aodachi," "Aogare" and
"Hideri—Aodachi" are applied to five distinctly different
sets of symptoms observed in rice under different soil,
management or climatic conditions conducive to stagnation
of impounded water in rice paddies. No distinction is made
in the name "Penyakit Merah" which is applied to two
distinct types of symptoms in Malaya. Names applied to
rice disorders associated with reductive soil conditions
in other countries include "Bronzing“in Ceylon, "Mentek"
in Indonesia, "Pansuk" in Pakistan, "Straight Head" in the
U.S.A. and "Suffocation Disease" in Taiwan.

Under tropical and subtropical climates, maintenance

of organic matter in soils, especially in paddy fields, is

6

considered essential. waever, the application of green
manures or straw in some soils aggravates the seriousness

of the physiological disease. The role of organic matter

in the growth of plants has been a subject of much investi—
gation and controversy since the sixteenth century. Adverse
effects on plant growth of straw and other mature plant
residues low in nitrogen were studied extensively prior to
1925 (31, 50). Since that time many workers (35, 50, 101,
115, 116, 136) have found that various crops suffer some
injury during the decomposition period of green manure.

Allison pointed out that the plant disease problem
cannot be viewed in only the narrow parasitic sense (6).
Associated saprophytic organisms may also reduce plant
vigor through the production of toxins or undesirable
products of organic matter decomposition, through competi-
tion for major and minor plant nutrients or through depletion
of the oxygen supply.

According to the discussion by Cochrane (30) there
are at least four general types of mechanisms which may be
responsible for adverse effects of readily decomposable
organic materials when they are added to soil:

1. Stimulated growth and activity of pathogenic
organisms.

2. Effects on the availability of nitrogen and phosphorus
in the soil.

3. An unfavorable microbiological balance in the soil
or the rhizosphere that leads eventually to plant

injury.

4. Chemical substances may cause root injury during
the microbial decomposition of the residue.
Research by many workers (10, 29, 30, 34, 44, 86,

87, 113, 116, 136, 137, 140, 151, 174) has served to focus
attention on several Specific effects of added organic
matter on the soil environment and the soil microbial popu-

lation. These are summarized in the following sections.
Effects of Organic Matter

Soil Aeration

It was assumed by many workers that the composition
of the soil air is changed unfavorably for the young plants
during the decomposition of organic amendments (13, 63,
101, 110, 113, 114, 168, 172). The incorporation of fresh
organic matter into the soil greatly affects aeration by
depressing the oxygen content and increasing the carbon
dioxide content. The extent of poor aeration may be
sufficient to reduce plant development (1) and crop yield
(34). Russell (113) indicated that green manuring will
increase the content of carbon dioxide and it may inhibit
germination or harm the very young root system of the
seedlings. Such results have also been reported by
others (2, 108, 113, 116, 139, 154). When oxygen content
falls below 1 per cent, there is a rapid and appreciable
decrease in potassium content of rice due to outward move-

ment of potassium from the roots (154). The injurious

effects on plants of low oxygen tension may be due more to
competition between the higher plants and the microorganisms
for the limited supply of oxygen (101) or nutrients

and oxygen (63) than any other single factor. Epstein and
Kohnke (34) suggested that planting of crops sensitive to

low oxygen and high carbon dioxide content should take place
about three weeks after the organic residues have been incor-
porated into the soil. In heavy, poorly drained soils, it

may be advisable to delay planting even longer.

Toxic Concentration of Gases

Ammonia (2), methane (4, 108, 115), hydrogen (108,
115) hydrogen sulphide (33, 104, 108, 111, 116, 154),
methyl mercaptan (133) may accumulate in concentrations
which are toxic to plants.

Takai, Koyama and Kamyra (134) have described the
following sequence of gas formation after soil amended with
fresh organic matter is flooded: oxygen is rapidly consumed,
accompanied by vigorous evolution of carbondioxide. Within
one day, hydrogen begins to evolve. The initially vigorous
production of carbon dioxide then decreases, with a recipro-
cal rapid increase in evolution of methane. Nitrate dis-
appears quickly, and ammonia is liberated as reduction
progresses. In the early stage of incubation, oxidation
reduction potential (Eh) drops rapidly, and ferric iron is
reduced. Active production of H28 and sulfides is not observed

until after a considerable period of incubation.

10

Accumulation of Active Ferrous
Iron and Reduced Manganese

The addition of organic matter is very effective in
promoting the formation of ferrous iron in soil (13, 25,
29, 33, 94, 108, 148, 154). Organic matter decomposition
and high moisture also favor the reduction of manganese
(108). Clark (29) pointed out that the concentrations of
these two elements in the soil solution have been observed
to change markedly with prolonged soil submergence and added
organic matter. Ponnamperuma, Bradfield and Peech (106)
suggested that the unthrifty growth of rice in poorly drained
or poorly aerated soils is due largely to the accumulation
of ferrous iron in the soil solution. According to Somers
and Shive (123), an unbalanced ratio of iron to manganese
produces specific types of chlorosis. Takai reported that
ferric iron is reduced vigorously in the early stages of
decomposition of organic matter in soil because of limited

oxygen supply fran the surface water (134).

Availability of Nitrogen and Phosphorus

Waksman (158) demonstrated that the decomposition of
younger plants results in liberation of some of the nitrogen
from protein as ammonia which may be reassimilated by the
microorganisms. In the presence of an excess of available
organic matter, the fungi, actinomycetes, and various
heterotrophic bacteria synthesize an extensive protoplasm.

For this purpose, they assimilate nitrate and ammonium

11

present in the soil and thus compete with higher plants.
According to Alexander (2), as carbon is assimilated for the
generation of new protoplasm by microflora, there is a con—
comitant uptake of nitrogen, phosphorus, potassium, and
'sulfur. Thus, in the presence of decomposable organic matter,
microorganisms reduce the quantity of nitrogen (8, 30, 42,

95, 141, 142, 174), phosphorus (30, 141) and other plant-
available nutrients in soil.

Phytotoxic By-products from Plants
or Microorganisms

Many studies have been made on phytotoxic by-
products from decomposition of plant residue (2, 10, 50,
75, 86, 94, 117, 118, 119, 137, 139, 162), living plants
(117) and microorganisms (2, 87).

Takijima (138) reported that the growth of paddy
roots was poor even at the earlier period of flooding due
to the harmful effects of major metabolites, such as carbon
dioxide, organic acids and unknown inhibitory substances.
He also pointed out that the physiological functions,
especially nutrient absorption and oxygenation of soil by
roots, therefore were retarded. He concluded that organic
acids and some organic metabolites harmful to plants should
play a key role in the occurrence of root damage in the ill—
drained paddy soil (135).

Takijima (139) found that organic acids (predominant-

ly formic, acetic, butyric, lactic, and succinic acids)

12

present in Japanese paddy fields where the soil was humic,
or ill-drained, or with heavy application of green manure,
exerted inhibitory effects on rice production.

Takijima and Sokuma (137) reported that water-logged
paddy soil treated with green manure--Chinese milk vetch--
caused the most extensive rice root injury and inhibited
the growth of rice. Growth inhibition due to some organic
acid in soil treated with vetch reached a maximum about
one week after water-logging and then decreased rapidly to
the same level as that in the controls.

A number of studies have been made on the physio—
logical basis of root rot which develops during plant
growth. The view that organic acids accumulating in rice
paddy soil may be one of the factors impeding the function
of roots has been expressed only recently in connection
with poorly productive paddy fields. Organic acids,
especially lower fatty acids present in soils of the paddy
fields in Japan, have been considered to be of great
importance in their inhibitory effects on rice production.
About ten fatty acids were found in paddy $011 (including
formic, acetic, butyric, lactic, oxalic, propionic and
succinic) by Takijima (139). Collison (31) and many
German scientists (10) have reported in the early days that
phenolic compounds (ferulic acid, p-coumaric acid, vanillic
acid and p-hydroxybenzoic acid) were liberated from plant

residues and affected or inhibited plant growth directly.

13

Schreiner and Reed (117) reported many organic compounds
other than organic acids, which are ordinarily regarded as
by—products of vegetable metabolism, to be highly toxic
to seedlings when present in sufficient quantities.
Recently, wang (160) found that lower aliphatic alcohols
which were formed in soils under waterlogged and anaerobic
condition may exert an influence upon plant growth on many
occasions in the soil. Studies on the effect of phenolic
acids upon the growth of paddy rice seedlings have also
been done by Wang. According to Wang's experiments (161)
the depressive effect of p—hydroxybenzoic, vanillic, and
syringic acids on the growth of paddy rice seedlings would
have become significant at 75 ppm, that of p—coumaric acid
at 50 ppm. Wang also found that paddy rice seedlings might
be more tolerant to lower fatty acids than young sugar cane.
Some microorganisms also produce many organic sub—
stances which are toxic to plant growth. Krassilnikov (66)
investigated more than 300 cultures of non-Sporefonning
bacteria for the production of plant growth inhibitors.
About 100 of the cultures suppressed plant growth and germi-
nation to some degree. Bacillus mesentericus, B. Subtilis,
.E- cereus, g. brevis, accounted for most of the inhibitory
Spore formers. MbCalla and Haskins (87) found that products

from certain strains of Pseudomonas fluorescens, g. pyocyanea,

 

and Bacterium spp. possess strongly herbicidal properties.
Alexander (2) noted that products of microbial metabolism

often have a detrimental effect upon higher plants, and

14

common non-pathogenic bacteria produce harmful effects
through the release of soluble toxic factors. He mentioned
that several antibiotics are assimilated by higher plants
through the root systems and thename translocated to above-
ground portions, that antibiotics are also knownto affect
the physiology of the plant entirely apart from any anti—
microbial action they might have. Livingston (74, 75, 178),
working on the harmful effect of bog soil, showed that
waters obtained by leaching a bog soil contain materials
which are toxic to the growth of numerous plant species.

It was noted that the substances were in some cases vokatile,

and in some cases apparently of colloidal nature.

Sulfate Reduction and Denitrification

Alexander (2) indicated that many bacteria, fungi,
and actinomycetes can convert sulfate and partially reduced
sulfur compounds to sulfide. In water-saturated soils
(under prolonged flooding) or in excessively compacted soils,
anaerobic decomposition of sulfates or proteins may result
in the formation of hydrogen sulfide which, even in low
concentrations, is toxic to plants (87). Vamos (152)
reported that one of the physiological rice diseases——
Brusone-—found in Hungary is caused by the toxic effect
of hydrogen sulfide. He also suggested that the disease
was more likely to occur on the heavier soils at lower
elevations and having higher contents of organic matter and

total nitrogen (150). As Takai, Koyama and Kamura (99)

15

explained, the hydrogen formed during the decomposition of
cellulose is used by the sulphate reducing bacteria
(Desulphovibrio desulfuricans) as a source of energy.
These bacteria obtain oxygen for utilization of the
hydrogen by reduction of the sulfates which are abundantly
present in the flood water and soil. vamos pointed out that
protein-decomposing bacteria also take part in sulphate
reduction. The application of organic matter to paddy
soil promotes production of toxic hydrogen sulfide, as
mentioned above. Hydrogen sulfide inhibits not only photo—
synthetic processes in the leaf, but also cytochrome oxidase
systems in the rice plant and nutrient uptake (20, 92).

It is widely known that denitrification requires a
goodly supply of readily oxidizable organic compounds (15,
156), high nitrate levels (167), low oxygen tension (5,
7, 20, 60, 99, 167) and poor drainage (3, 53, 99).
Bremner and Shaw (11) found that denitrification is much
more rapid in soil saturated with water than in soil at
lower moisture levels. The supply of oxygen in water-
saturated soil is not adequate to meet the requirement of
the soil microorganisms and, in consequence, the denitrify—
ing microorganisms utilize nitrate instead of oxygen as a
hydrogen accepter and so cause denitrification. The rate
of denitrification increases with the amount of straw
added. If at the same time the water content of the soil

is high, intensely anaerobic conditions may easily arise,

16

favouring denitrification as well as the reduction of nitrous
oxide to molecular nitrogen or the reduction of nitrate
to ammonia.

Nommik (99) reported that one of the many factors
that influence the rate and course of denitrification is the
content of easily oxidizable organic material to serve as
energy substrate. Supplying organic material as a rule
brings an increase in the microbial activity of the soil
which in turn means an increase in the consumption of oxygen.
High water content of the soil proved to be of decisive

importance to denitrification.

Rhizosphere Relationships

Investigations cited above provide ample evidence
that many inorganic or organic toxic compounds are associated
with microbiological activities in the rhizosphere. Numerous~
workers have reported on phenomena associated with activities
of microbes in the rhizospheres (28, 56, 64, 65, 127) of
upland crops.

Starkey (127, 129) reported that each kind of plant
has a typical rhizosphere population. He found that organisms
were many times more numerous on root surfaces than in the
soil close to the roots. The greater the distance from
the region of extensive root development the smaller the
number of bacterial inhabitants found in the soil. He also
pointed out that the greatest rhizosphere effect occurred

during periods of active plant development and the effect

17

disappeared promptly on death of the plant. He believed

that the rhiZOSphere effect is associated with normal growth.
The majority of the rhizosphere microorganisms are saprophytic
and convert both organic and inorganic materials in the
rhizosphere. The products of these transformations may be
beneficial or injurious to plants.

MacCalla (87) states that some of the organic
material produced by microbes in the rhizosphere is absorbed
by the plants, but the kind and amounts are not known.

Timonin (143) reported that different varieties of
plants affected the activities of the various groups of
soil microbes differently. Ishizawa gt 31. (51) noted that
the effect of organic substances upon microflora differed
according to the type of organic matter. Among micro-
organisms, the increase of actinomycetes was smallest, among
organic materials (glucose, alfalfa, rice straw and manure)
the effect of manure on the increase of microbes was
smallest. Bacteria were always higher than other micro-
organisms and did not show a rapid decrease for longer
periods. Ishizawa and Toyoda (52) found that total bacteria
and anaerobes were more and actinomycetes and fungi were
less abundant in paddy than in upland soil. Sulfate reducers
and denitrifiers were more numerous in paddy than other
soils in Japan, especially under conditions of abundant
supply of available organic matter. The latter two groups

of organisms were present not only in the surface but also

18

in the substratum. The authors observed that a high moisture
condition and a supply of available organic matter were
necessary for the maintenance of a high bacteria count.

Numerous rhizosphere studies indicate that the
abundance of microbial cells is affected by the kind of
plant and its stage of development and vigor. The maximal
number of bacteria is observed during the period of the most
active growth of the plant. Observations show that an
abundant growth of microorganisms takes place in the early
stages of plant growth, however the most vigorous growth of
microbes ensues during the period of flowering and in the
period directly preceding it (66). The abundance of mycelial
groups such as fungi and actinomycetes is greater on old
root surfaces (52).

It is suggested that the roots of plants may excrete
significant amounts of stimulative substances which can
promote bacterial growth (42, 164). Krassilnikov (79)

reported that small non—Sporing bacteria (B. denitrificans,

 

_§. fluorescens) were found to multiply extensively in rhizo—

 

spheres, presumably under the stimulation of organic root
excretions. It is generally accepted that the main factors
for the stimulation of microorganisms in the rhizosphere
are the excretion of organic substances and the sloughing
off of root hair and epidermal cells (112, 128). Mucoid
materials (59), nucleotides, flavones and other reducing
substances (81), pentose sugars or a closely allied

substance, D-xyloketone (l6), acetaldehyde (97) were found

19

by many workers in studying plant-microbial interactions in
the rhiZOSphere. Numbers of workers have found that root
excretions stimulated the maximum growth of various groups

of organisms, particularly these requiring“ amino acids

(58, 80, 109, 159, 165). Katznelson and Richardson (57)
found a higher production of bacteria stimulated by amino
acids in the rhizosphere of tomato plants than in the control
soil. The rhizosphere of diseased plants may contain

greater concentrations of microorganisms than that of healthy
plants (57).

There.is still very little information about the
kinds of microorganisms in the rhizosphere of different
plants and theil:significance in development of the plants.
Very little is known about rhizosphere microbial activities

associated with physiological disorders of paddy rice.

Denitrifying Bacteria

Denitrifying Species or denitrifying bacteria as a
group are frequently implicated in the above cited investi-
gations of rhizosphere phenomena and effects of inconpora-
ting organic matter in paddy soils of poor drainage. It
appears that this physiological group might be usefully
studied in greater detail in relation to physiological
disorders of rice.

All denitrifiers can produce nitrous oxide (96).
Mbst of the denitrifying bacteria reduce nitrate to nitrogen

gas and nitrous oxide in varying proportions,_§. nitroxus being

particularly active (158).

20

It was found that non-spore-

forming organisms of the genera Pseudomonas, Micrococcus

 

and Spirillum, aerobic spore formers (bacilli) and a number

of facultative anaerobes can reduce nitrate (170).

Mbre

than forty organisms have been reported as denitrifiers (88).

Denitrifying bacteria are summarized as follows:

 

 

 

 

 

 

 

 

 

 

 

 

 

Nitrate Reducers Denitrifiers Nitrate Reduction
Catalyzers(2)
E, coli (36) Spirillum Thiobacillus
A itersonii (88) denitrificans
Microcoocus
denitrificans *Microc. denitrifi- Chromabacterium
(36, 62) cans (170) mycoplana
Streptoccocus .B. denitrificans Serratia or Vibrio
faceaim (47, (88) species
69)

Clostridium spp.
(170)

Achromobacter
nephridii
(Corynebacterium
nephridii)(47)

*ffl, hartlebii (149)

‘A. arcticum (88)

A, liguefaciens
(153, 170)

*Bacillus cereus
(68)

*fg. licheniformis
(36)

‘B. proteus (88)

B. macerans (170)

.§._prodigiosus (88)

_B. licheniformis
(149)

{B. subtilis (96,
153, 170)

*Pseudomonas
stutzeri (88)

"U

. putida (88)

l2}? la? la? IIBI

. denitrificans
(88)

pg,
(107)

s. aeruginosa (71)

. indiqofera (89)

. fluorescens (88)

perfectomarinus

‘g§.,pseudiona11ei (124)

*Achromobacter

denitrificans (149)

21

Nitrate Reducers Denitrifiers Nitrate Reduction
Catalyzers (2)

 

 

KB. polymyxa (68)

_§. laterosporus (170)

 

.B. coagulans (170)

*Need amino acids

**Require ammonium

A comparison of the denitrifying population of grass—
land sods with and without nitrate addition was made by
Woldendorp (170). It revealed that under normal field
conditions without nitrate the population consisted mainly

of Bacillus species (B. cereus, B. circulans, g. macerans,

 

_§. coagulans, B. laterosporus) of which B. cereus was by

 

 

far the most numerous. These organisms did not give rise
to gaseous products but formed large quantities of nitrite

and some ammonia. Pseudomonas and Achromobacter were dominant

 

 

genera in soil to which nitrate fertilizer had been added.
valera and Alexander (149) reported, as did Woldendorp, that
the most rapid gas evolution was brought about by Pg.
aeruginosa. ‘

During the past decade, experiments have shown that
the producers of antibiotic substances are widespread in
soils. It has been determined that the plants absorb through
the roots different antibiotic substances produced by micro—
organisms in the Soil. It is known that mycerin, subtilin,

glyotoxin and others induce strong poisoning in plants by

22

comparatively small doses. Clavacin, suppresses the growth
of cereal roots at a dilution of l:l,000,000; subtilin
depresses the germination of wheat and pea seed at a dilution
of 1:100,000-1,000,000 (67).

Knight (68) listed a number of species of interest
as producers of antibiotics, including B. subtilis, B. brevis,
_§. polymyxa, B. licheniformis, B. circulans. ‘B. polymyxa
was originally detected as a producer of an antibiotic by
Stansly, Shepherd and White (125).

Pseudomonas pseudomallei is essentially an accidental
pathogen (124). .25. tabaci, can produce a true toxin which
is the cause of tobacco wildfire disease and causes several
leafspot necroses. The toxin appears to interfere with
the metabolism of a normal amino acid, methionine (124).

The most important denitrifying bacteria-figs.
aeruginosa—-is widely known for two distinctive properties:
one is production of the blue pigment, pyocyanin, the other
is its ability to lyse or to inhibit the growth of other
bacteria, especially Gram positive bacteria (176).

Pyocyanin is easily oxidized and reduced in cultures
and acts in conjunction with the cytochrome system of the
cells to increase reSpiration (37, 38). Chemically, pyocyanin
is a phenazine and is similar in structure to iodinine, a
pigment produced by Chromobacterium iodinum and to chloraphin,
a green pigment formed by ES. chlororaphis (43).

Some Ps. aeruginosa strains can produce a red

 

diffusible pigment--pyrorubrin--after 16 hours of incubation

23

at 37°C and an additional 24 hours at room temperature.
Strains producing pyrorubrin did not appear to produce
pyocyanin and vice versa. _Four types of diffusible pigment

are produced by EB: aeruginosa (157):

 

Pigment Percent of strains
Blue—green or brownish green 68.2%
Light-brown 10.5%

Red pyrorubrin 3.5%
No pigment 17.8%

Morihara reported recently that gs. aeruginosa can
produce proteinase on non-carbohydrate carbon sources (93).

The study of nutritive differences among microorganisms
found in the rhizosphere offers a means for increasing know-
ledge of the physiological activities of the root system
of plants. Many contributions have been made by numerous
investigators. They have found that nutrient requirements
are very Specific, not only for the growth of denitrifying
bacteria but also for the formation of pigments. Burton
found that phosphate, sulfate, magnesium, potassium and iron
ions were essential for growth of Bi- aeruginosa. Magnesium
promoted the synthesis of pyocyanin (43). Glycerol and
tartrate appeared to yield a greater population than
citrate (149). Glutamic acid appeared to be superior to
sugars or fatty acids as hydrogen donor in respiration (170)
and also for pyocyanin synthesis (43). Glucose over 1% may

inhibit the production of pyocyanin by BE- aeruginosa (176).

24

The presence of amino acids stimulated many denitrifying
bacteria (55, 176) as well as the entire denitrification
process (170). Nitrate served as an extremely effective
electron acceptor (124).

The reaction of media is also a critical factor.
Net only does the optimum pH range vary with the bacterium,
but the specific activity in gas evolution is greatly dif-
ferent at different pH's for several microorganisms.
Wijler and Delwiche (167) point out that total denitrification
rates were quite constant above pH 6.0, but the proportion
of nitrous oxide and nitrogen were pH dependent. Above
pH 7.0, nitrous oxide could be readily reduced to nitrogen.
Below pH 7.0, the reduction of nitrous oxide was strongly
inhibited. Below pH 6.0, nitrous oxide became predominant,
and the rate of denitrification was decreased. Karlsen
found that denitrification would occur at pH 5.8 to 9.2,
with an optimal range between pH 7.0 and 8.2. YCung (176)
reported that, in nutrient broth incubated two weeks at 370C,
only pyocyanin and a fluorescent pigment were formed.
Acid cultures of fig. aeruginosa produced no pigments, formed
no antibiotic substances of any kind (176).

Various synthetic and semisynthetic media have been

used in studies of pyocyanin production by gs. aeruginosa.

 

A synthetic medium consisting of_gL1-alanine or glycine at

0.4% concentration, combined with 0,8%_1-1eucine, 1.0%

glycerol and salt mixture has been Shown to be the most

25

suitable medium for pyocyanin production by five representa-
tive strains of fig. aeruginosa by Burton (19).
Liu (73) found that hemolysin, lecithinase and

protease of gs. aeruginosa were produced with cellophane

 

covered plates and completely synthetic agar of simple
composition containing_l-alanine, aspartic acid, glutamic
acid, glucose and salts. He also observed that glucose
and phosphate played a unique role in production of these
extracellular toxins. Glucose appeared to be more important
than just an energy source. It was suggested that products
of anaerobic metabolism of glucose are needed in the pro-
duction of these toxins. The most critical factor in the
production of these extracellular toxins appeared to be the
concentration of phosphate. The best medium for the pro-
duction of these toxins appeared to be 0.45%_l-alanine,
0.45% glutamic acid, 1.2% glucose and 0.003% phosphate
(KH2P04) .

Other pigments produced by Pseudomonas Species

 

include fluorescin and pyroverdin (163). Lenhoff found
that the oxygen tension in the growth medium influenced the
formation of pyroverdin. Large amounts of the pigment were
produced in well—aerated cultures with high oxygen tension
(71).
Knight and Proom (68) found the following nutritional

patterns to be characteristic of Bacillus spp.:

l. .B. subtilis, B. licheniformis and B. megatherium

grew with ammonia as sole nitrogen source and in the

26

absence of added growth factors.

_B. cereus and_§. brevis grew in the absence of added

growth factors but required mixtures of amino
acids instead of ammonia only as sources of

nitrogen.

_B. pumilus and B. macerans grew with ammonia only

in the presence of biotin and aneurin.

_B. alvei required amino acids and aneurin; B.

circulans and B. coagulans had more complex require—
ments.

Lochhead and Chase (78) described seven cultural

media, ranging from a simple basal medium to complex media,

for qualitative studies of rhizosphere bacteria in relation

to their amino acid nutrition:

Medium B Basal medium
" A " plus amino acid
" C " " growth factors
" A G " " amino acids plus growth factors
" Y " " yeast extract
" S " " soil extract
" Y S " " yeast extract and soil

extract

Rovira (111) found that,for some organisms, yeast

extract exhibited similar growth promoting properties as

did pea root exudate, but for others, the exudate stimulated

growth in the presence of yeast extract.

The root exudate

27

could not be completely replaced by glucose, soil extract,
vitamin-free casamino acids or a synthetic mixture of known
growth factors.

Wasserman used either tryptone—glucose-yeast extract
agar or Difco Pseudomonas Agar F to maintain stock cultures

of Pseudomonas spp. (163).

EXPERIMENTAL METHODS

Experiments were conducted during the fall of 1964,
which is the normal season for the second crop of rice in
Taiwan, and again in the spring of 1965, the season during
which the first crop is normally grown under a two-crop
system. Greenhouse experiments and field plantings were
used as sources of soil and rhizosphere samples for studying
micrdbial populations associated with diseased and healthy

rice plants.

Greenhouse Experiments

Greenhouse experiments were designed to investigate
effects of organic amendments on microbial populations and
disease development in rice growing on normal and problem

soils.

First Trial

In the first trial (fall, 1964), four soils were
used: (1) Lateritic paddy soil from Chungli, (2) alluvial
rice soil from Taichung, (3) soil from a problem area in
Pingtung and (4) Lotung silty clay loam from a problem area
in Ilan.

The Lotung soil was taken in two ways. In one case,

a profile sample, including top soil and subsoil, was taken

28

L31

29

without disturbance of its structure to insure the occurrence
of the disease. In the other, surface soil was composited,
as was done in the case of the other soils.

Organic amendments are outlined in Table 1. Freshly
chopped green Sesbania at the rate of 20 tons per hectare
was used for green manure. Chopped rice straw was used
at the rate of 5 tons per hectare. Organic amendments and
soils were mixed thoroughly before dispensing into four
replicate boxes of each treatment. In the case of the
Lotung profile, the organic materials were incorporated

only into the top soil, as in field practice.

Table 1. Treatments in the first greenhouse experiment.

 

 

 

 

 

 

 

 

 

 

 

 

 

Soil C T
Samples Chungli Taichung
Treatment No Green Straw No Green Straw
Manure Manure Manure Manure
Treatment C C C T T T
Code 0 g s o g 5
Soil P L
Samples Pintung Lotfing
No Green No Green
Treatment Manure Manure Straw Manure manure Straw
Treatment
Code P0 P9 PS L10 L19 L1S
Soil L2
Samples Lotung
(Undisturbed profile)
No Green
Treatment Manure Manure Straw
Treatment

Code L2 L2 L2
0 g

 

30

The boxes containing the Lotung profiles (L2)
were flooded 2 days before transplanting rice (not usually
done in the field). All other soils and treatments were
flooded 14 days before transplanting. The water level was
maintained at 4 to 10 mm above the soil surface continuously
until the 25th day after transplanting. At that time,
water was drained off to Observe effects on rice development
of improved soil aeration.

Seedlings of a susceptible strain of rice, Taichung
65, were grown on Taichung soil in the field to an age of
23 days before transplanting into the experimental boxes.
On August 6, 1964, five seedlings were planted per hill and
two hills per box (30 x 30 x 30 cm), except that four
hills per box (60 x 30 x 30 cm) were planted on the Lotung
profiles.

The rates of applied fertilizer were 80, 60, and 60
kg per hectare of N, P205 and K20,respective1y. Ammonium
sulfate, superphosphate and potassium chloride applications
were split, half two days before transplanting and half 30
days later.

All experimental units, except those involving
Taichung soil, were grown to maturity in the greenhouse.
The rice cultures on Taichung soil were grOWn outdoors on

the Chung-Hsing University farm.

Second Trial
In the first rrial, disease symptoms developed only

on the Lotung profile (L2). Accordingly, only Lotung

31

profile cultures (L3) were used in the second trial.
Organic amendments were incorporated at two different times,
two days and fourteen days before transplanting (Table 2).
Because Sesbania grows poorly during the winter months, it
was necessary to use lupines for green manure. The rate

of nitrogen fertilizer was increased from 80 to 120 kg

per hectare. The soil was flooded at the time of organic

matter addition and was not drained at any time.

Table 2. Treatments in the second greenhouse experiment.

 

 

 

 

Check Green manure Straw
Incorporated before transplanting
(No organic amendment) 2 days 2 weeks 2 days 2 weeks

 

Forty-one-day-old seedlings of the Taichung 65 rice
strain were transplanted on March 10, 1965. Five seedlings
per hill and four hills per box (60 x 30 x 30 cm) were
planted, as in the first trial, and the rice was grown to
maturity in the greenhouse.

Soil pH and oxidation-reduction potential (Eh)
were measured between plants at a depth of 10-15 cm daily
up to the time of maximum tillering and weekly in later

stages of growth (26, 105, 171).

Inoculation Experiment

An inoculation experiment was conducted to investi-

gate, in a preliminary manner, the etiology of disease symptoms

32

observed during the first greenhouse trial.

Healthy rice seedlings were grown in 1X Heagland's
solution (49) from seeds sterilized before germination by
immersion for one hour in 1:500 Uspulum solution. These
seedlings were germinated and grown in 100 ml bottles covered
with black paper.

Boxes of the Lotung profile which had produced
severe disease symptoms during the first greenhouse trial
were used to cultivate diseased rice again in the same way,
with incorporated rice straw and green manure. When severe
symptoms of the disease appeared about one week after trans-
planting rice to these boxes, soil leachates and root wash—
ings were collected from them.

Soil leachates and root washings from diseased rice
cultures were mixed with equal volumes of fresh 2X Hbagland's
solution in large bottles. Healthy seedlings grown on 1X
Hoagland's solution were then transferred to these bottles.

When plants inoculated in this way reproduced symptoms
of the disease, a 1 ml aliquot of the nutrient solution was
withdrawn from each bottle for enumeration of total bacteria
and sulfate reducers. Nutrient agar was used for bacteria,

and yeast extract-sodium sulfate agar for sulfate reducers.

Field Locations

Soil and rhizosphere samples of second crop rice
were taken in the fall of 1964 from farm paddies at two

locations where the disease was known to occur. One

33

location on Lotung soil in Ilan was chosen because the
disease occurs regularly in that area. The otherlocation
was in Pingtung where the development of disease symptoms
is less predictable.

With first crop rice in the spring of 1965, only
one field location was sampled. This was on Lotung soil

in Ilan.

Microflora Studies

Soil and rhizosphere samples for enumeration of
microbial populations were taken from both greenhouse experi—
ments and field location. Times of sampling were based on
the stage of physiological development of the rice:

(1) Seedling stage (prior to or at transplanting time)

(2) Tillering stage (when first detectable tiller
appeared)

(3) Time of maximum tiller number

(4) Panicle development (panicle primordium first
detectable by naked eye--usually 1—2 mm long)

(5) Anthesis (first visible anther)

The first symptoms of physiological derangement,
when observed, usually appeared at the tillering stage
and reached maximum intensity about the time of maximum
tiller number.

Samples of non-rhizosphere soils (S) were taken
mid-way between plants in the field or between hills in

the greenhouse box cultures. Core samples 10 cm in

34

diameter and 9 to 15 cm deep (depending on age of rice)
were taken.

Similar cores were taken for estimation of root (R)
and rhizosphere (Rh) populations. During the early stages
of development, these cores included the entire root system of
a single hill. In later stages of growth, only a part of
the root system would have been included. In the greenhouse
experiments, one hill per treatment was sacrificed for this
purpose on each sampling date. In treatments where no
disease symptoms developed, the sampled hill was selected
at random from one of the four replicate boxes of each
treatment. In treatments where disease symptoms appeared,
the most severely injured hill for that treatment was
taken. The corresponding non-rhizosphere (S) sample for
each treatment was taken from an inter—hill area adjacent
to the hill taken for the root and rhizosphere sample.

Samples were placed in individual plastic bags for
transport to the laboratory. Aliquots of non-rhizosphere
soil samples (S) were suspended immediately in physio—
logical saline solution and quantitative dilutions made for
inoculation of enumeration media. Root and rhizosphere cores
were immersed in water, with gentle kneading and agitation,
to remove the bulk of the soil. They were then trans-
ferred to physiological saline solution and agitated for
5 minutes on a flask shaker. Rhizosphere (Rh) populations
were estimated on serial dilutions of the suspension so

obtained and calculated on the basis of the dry weight of

35

soil remaining after evaporation of the saline solution.

After removal of the rhizosphere soil, the roots
were placed in fresh saline solution and shaken for 20
minutes on a flask shaker. Microbial numbers estimated on
serial dilutions of this suspension were calculated on the
basis of the oven dry weight of root tissue and are desig-
nated as root (R) populations (84, 110, 130).

Emphasis was placed upon enumerating physiological
rather than taxonomic groupings. Selective media were used
to differentiate between microorganisms with Specific bio-
chemical activities. However, counts were made also of the
major taxonomic groups to give further insight into the bio—
chemical capabilities of dominant microflora.

The following media were used:

(1) Sulfate reducers
Sodium lactate—asparagine (Van Delden, 9)
(2) Denitrifiers
Asparagine-nitrate-citrate broth (Timonin, 145)
(3) Ammonifiers
Urea broth (Viehoeven, 4)
(4) Cellulose digesters (aerobic)
Cellulose dextrin agar (Fuller and Nerman, 39)
(5) Cellulose digesters (anaerobic)
Cellulose dextrin agar (Fuller and Nerman, 39)
(Incubated in N2 atmosphere)

(6) Anaerobes

Sodium thioglycollate agar (Brewer, 12)

36

(7) Bacteria

Soil and yeast extract agar (Bunt and Rovira, l7)
(8) Actinomycetes

Dextrose-casein agar (Jensen, 54)
(9) Fungi

Rose Bengal agar (Smith and Dawson, 122)

After inoculation, all media were incubated for
appropriate periods at 280C.

With broth media and the sodium lactate—asparagine
agar for H28 production, numbers were estimated from a table
of most probable numbers based on a lO-fold dilution
series and 5 tubes per dilution (85). With agar media
numbers were calculated from the mean of 3 replicates of a

significant dilution in a dilution plate series.

Nitrate Reducing Bacteria—-Pure Culture Studies

In the microflora studies, it was found that numbers
of cellulose digesters, sulfate reducers, nitrate reducers
and ammonifiers were frequently more numerous in rhizo—
Spheres of diseased than of healthy rice. It appeared
that each of these groups might be studied in greater detail
in its relationship to development of disease symptoms.

To this end, a nutritional study of nitrate reducing bacteria
in pure culture was undertaken. The objective was to find
differences in nutritional requirements of representative
Species which might be used to develop solid media for

enumeration and isolation of more restricted physiological

37

types among the larger group of nitrate reducers.

Eighteen species and strains of facultatively anaero-
bic bacteria with known capability for reducing nitrate were
used (Table 3). StoCk cultures were maintained on nutrient
agar, with periodic transfer at 300C followed by storage
in the log phase at 10°C.

Eleven experimental media were employed (Table 4).
Media 103 through 112 are modifications of media described
by Woldendorp (170) and by Valera and Alexander (149).

Stock solutions listed in Table 4, when combined in the
proportions shown, give the following concentrations of

minerals in the final medium: 123 mg NazH P04, 724 mg

KH2P04, 89 mg MgSO4-7H20, 53 mg CaClz'ZHZO, 380 ug CuSO4'5HéO:
440 ug ZnSO4c7H20, 310 ug MnSO4- H20, 250 ug H3BO4, 5 ug
NaMoO '2H 0 and 1 mg Fe (as FeEDTA) per liter.

4 2
In preparing these media, buffer and mineral salts

solutions and agar were combined and sterilized by auto—
claving at 121°C for 15 minutes. Energy sources, amino
acids, vitamins and iron chelate were added through a
Millipore filter. The pH of the hot medium was adjusted
just before pouring the plates at 45 to 50°C.

Plates for anaerobic incubation were allowed to
solidify and then were placed immediately into an H2
atmosphere for storage until ready to use.

For aerobic incubation, plates were stored in
sealed glass jars containing a few ml of 10 per cent

glycerol to maintain humidity.

38

Table 3. Nitrate reducing bacteria investigated in pure
culture.

 

 

 

 

 

 

 

 

 

 

Laboratory . . . . .
number organlsm Source 1dent1f1cation
15 Achromobacter hartlebii Ach1 365
9 Bacillus cereus ATCC 6464
10 Bacillus cereus ATCC 14579
13 Bagillgs circulans ATCC 4513
.Bagillus,goagulans Woldendorp2 1963 II
5 .Bacillus.laterosnorus Woldendorp 468B
Bacillus licheniformis Woldendorp Pl
6 Bacillus licheniformis Woldendorp 430
14 Bacillus licheniformis ATCC 14580
11 Bacillus macerans ATCC 843
12 Bacillus macerans ATCC 8244
16 Micronecsns isnitrifigans ATCC 13543
Pseudomonas W ATCC 10145
.EfiandeQnAS.aaruginQSa Woldendorp
18 .Pseudomonas.denitrificans ATCC 13867
17 Pseudomonas fluorescens ATCC 11250
3 Pseudomonas stutzeri Woldendorp
Serratia marcescens MSU MPH3

 

 

1American Type Culture Collection, Rockville,
Maryland.

2J. W. weldendorp, Laboratory of Microbiology,
Agricultural University, Wageningen, Netherlands.

3Department of Microbiology, Michigan State
University.

39

Table 4. Experimental media.

 

 

Medium number

 

 

 

Component
103 104 105 106
Stock sol'n. A1 510 m1 510 m1 510 ml 510 ml
" " 32 90 ml 90 ml 90 ml 90 m1
" " c3 180 m1 180 m1 180 m1 180 ml
" " D4 10 ml 10 ml 10 ml 10 ml
" " E5 10 ml 10 ml 10 ml 10 m1
KNO3 2.5 g 2.5 g 2.5 g ---
Asparagine -—- --— -—- 2.5 g
Glucose 2.5 g 2.5 g 2.5 g 2.5 g
Vitamins6 --- --- 20 ml 20 ml
Amino acids7 --— 20 mi 20 ml 20 m1
Na thioglycollate --— --- --- --—
Yeast extract --- —-— -—— -—-
Na citrate —-— —-— --- ———
K H2P04 -—- --— --- ---
Mg SO4~7H20 --— --— --- ---
CaC12-6H20 —-- --- —-— -—-
FeC13-6H20 -—- --- --- -——
Agar 15 g 15 g 15 g 15 9
H20 to: 1 liter 1 liter 1 liter 1 liter
pH 7.3 7.3 7.3 7.3
lStodk sol'n. A: 1.42 g NaZHPO4 per liter (.01 M).
2Stock Sol’n. B: 1.36 g KH2P04 per liter (.01 M).
3Stock Sol'n. C: 493 mg MgSO4°7H20 (.002 M) plus
294 mg CaClz-ZHQO (.002 M) per liter.

4Stock Sol'n. D: 38 mg CuSO4-5H20, 44 mg ZnSO4-7H20,
31 mg MnSO4-H20, 25 mg H3803, and 5 mg na2M604-2H20 per liter.

5Stock Sol’n. E: 769 mg FeEDTA per liter.

Vitamin stock solution to provide 1 ug biotin,
2 ug vitamin 312' 2 ug folic acid, 100 ug riboflavin,
500 ug thiamine, 500 ug nicotinic acid, 500 ug pyridoxine —
HCl, 500 ug ca—pantothenate and 5 mg inositol per liter in
the final medium.

. 7Amino acid stock solution to provide 950 mg
V1tamin-free casamino acids, 50 mg tryptophane and 10 mg
Cysteine per liter of final medium.

4O

 

 

Medium number

 

 

107 108 109 110 111 112 113
510 ml 510 m1 510 m1 510 ml 510 m1 510 ml --—
90 ml 90 ml 90 ml 90 ml 90 ml 90 ml ---
180 m1 180 m1 180 ml 180 m1 180 ml 180 ml -—-
10 ml 10 ml 10 ml 10 ml 10 ml 10 ml ---
10 ml 10 ml 10 ml 10 ml 10 ml 10 m1 ---
2.5 g 2.5 g 2.5 g ——- 1.25 g 1.25 g 1.0 g
-—- -—- --- 2.5 g 1.0 g 1.0 g 1.0 g
2.5 g 2.5 g 2.5 g 2.5 g 2.5 g 2.5 g ---
--— —-- 20 ml 20 ml 20 ml --- ——-
-—- 20 ml 20 ml 20 ml 20 ml -—- ---
500 mg 500 mg 500 mg 500 mg 500 mg 500 mg 500 mg
--- --- --- --- -—- 10. g ---
--— --- --— --- --- -—- 8.5 g
--- --- --— --- --- --- 1.0 g
--- -—- --— --- -—- --- 1.0 g
--- —-- -—- --— —-- -—- 0.2 g
--- —-- --- --- --- --- 0.03 g
15 g 15 g 15 g 15 g 15 g 15 g 15 g
1 liter 1 liter 1 liter 1 liter 1 liter 1 liter 1 liter
7.3 7.3 7.3 7.3 7.3 7.3 7.1

 

41

Streak inoculations were made, using 12 to 18 hour
cultures in nutrient broth as inoculum. With slow growers,
34 to 42 hour cultures were found to be more reliable

(Achromobacter hartlebii, Bacillus macerans, g. laterosporus).

 

For anaerobic incubations, atmOSpheres of H2 and/or
CO2 were used in two cabinet type anaerobic incubators.

The cabinets were evacuated to 5 cm Hg and filled with H2
twice to exhaust O2 to very low concentrations. Palladium-
impregnated asbestos was used as a catalyst to reduce re-
maining traces of 02. Small tubes of methylene blue agar,
visible through the glass door panel, were used to indicate
the establishment of reducing conditions. Partial or
complete replacement of H2 with CO2 was effected only

after reducing conditions had been established.

Aerobic incubations were accomplished in loosely
covered glass jars containing a few m1 of 10 per cent glycerol
to maintain humidity.

Both aerobic and anaerobic incubations were carried
out at 28 to 30°C. After various incubation periods, growth
was estimated visually on a scale of 0 to 6 and pigmenta-
tion on a scale of 0 to 4. In some experiments, cross—

streak inoculations were made and appropriate notations for

synergism or antagonism were used.

RESULTS
Symptomology and Developmental Behavior of Rice

First Greenhouse Trial

Typical symptoms of suffocation disease were not
observed in rice growing in the Chungli, Pingtung or Lotung
(L1) surface soils (Figs. 5, 6).

Rice in the Lotung profile (L2) cultures with
Sesbania green manure started to wilt just 2 days after
transplanting. As the disease progressed, the leaves
rolled inwards, beginning with older, basal leaves.
Severely affected plants were severely stunted (Fig. 7).

At 10 days, lower leaves were yellowing from the margin

and apex toward the midrib and base. Roots of these plants
were poorly developed, coarse in texture, with short
lateral roots, very little lateral branching and no root
hairs (Fig. 8). The roots were dark brown to black in
color, and their lower portions were decaying, with a
putrid odor.

After maximum tillering, brown spots appeared on the
older leaves of both diseased and non—diseased plants in
check and amended cultures.

The first symptoms did not begin to appear until

5 days after transplanting in the Lotung profile cultures

42

43

where straw was incorporated. At 22 days, however, the
injury with the straw treatment was more severe than

with green manure. When the flood water was drained from
the boxes after 25 days, plants in the green manured profile
cultures gradually recovered their green color and grew
normally again, whereas the plants where straw had been

used recovered very little.

These symptoms are the same as are regularly observ-
ed in the field on Lotung soil (70). From the observed
production of gas bubbles on the surface of the flood water,
it appeared that the symptoms developed during the height
of decomposition of the added organic matter. With
reference to stage of development of the rice itself,
symptoms began to appear at about the same time as the first
tillers and became progressively more severe through the
tillering period. The severely affected plants were markedly
stunted, tillered poorly (Fig. l), headed late and produced
thin, narrow panicles with light weight, poorly filled
kernels (Fig. 4). Yields of straw and grain were severely
reduced (Figs. 2, 3).

Yields of straw and grain were severely reduced by
the straw amendment on the Pingtung and Lotung (Ll) surface
soils, even though no symptoms of disease were observed.

This was likely due to microbial competition for nitrogen.
This explanation is supported by the stimulating effect on

growth and yields of the Sesbania green manure. This

44

effect was expressed on all four soils. Even in the severely
injured Lotung profile (L2) cultures, rice following green
manure recovered after drainage to produce larger yields
of straw than the check and equivalent yields of grain.

Panicle development and maturity of rice were
delayed 20 days in all soils by amendment with either
straw or green manure, which suggests a delayed release of
nitrogen from soil sources in the presence of microbial
competition during earlier stages of decomposition of
these materials.

Since it appeared that nitrogen at the rate of 80 kg
per hectare was inadequate for maximum yields in the first
trial, the rate was increased to 120 kg in the second

trial.

Second Greenhouse Trial

From Tables 5 and 6 it can be seen that, in the
second trial, vegetative development was again retarded by
both straw and green manure in Lotung profile cultures of
rice, as it was in the first trial. The retardation was
greater when the amendments were incorporated two days
before transplanting than when they were allowed to de—
compose in the flooded soil for two weeks before trans—
planting (Figs. 12, 14, l6, 18). This difference gradually
diminished during later stages of growth (Figs. 13, 15, 17,
19). Final yields of grain and straw showed little benefit

from the earlier incorporation (Table 7). Yields of grain

45
and straw were drastically reduced by both organic amend-

ments, as compared with the unmanured check cultures.

Height of rice plants in second greenhouse trial

 

 

 

 

 

Table 5.
(first crop rice, Lotung profile).
Green Manure Straw
Age of rice «~—
plant Incorporated before planting
(days after No LSD0 5
transplanting) manure 2 days 2 weeks 2 days 2 weeks ’
cm. cm cm cm
25 34.9 26.4* 27.4* 23.8* 30.1* 3.01
50 65.1 56.1* 59.0* 51.7* 57.4* 7.58
75 73.4 69.5 70.0 65.2 65.6 6.59

 

*Significantly less than for no manure.

Number of tillers per hill in second greenhouse

 

 

 

 

 

Table 6.
trial (first crop rice, Lotung profile).
Green Manure Straw“?
Age of rice in
plant Incorporated before planting
(days after No LSDO 5
transplanting) manure 2 days 2 weeks 2 days 2 weeks ‘
cm cm cm cm
25 7.8 5.1* 5.2* 5.0* 5.7* 1.17
50 16.6 16.0 15.9 13.1 13.4 N.S.
75 19.2 18.6 16.9 13.7* 13.7* 4.39
Efficient
tillers _ 14.2 9.7* 10.1* 8.0* 9.0* 3.34

 

*Significantly less than for no manure.

46

Comparing yields in the second trial (Table 7)
with those in the first (Figs. 2, 3), it is apparent that
much more straw was produced in the second trial (for the
unmanured check, 83 gigs. 24 g) and less grain (9 g gs.

18 9, again for the check treatment). The dominantly
vegetative character of growth during the second trial may
have been partly a seasonal effect. Hewever, more nitrogen
was applied and the cultures were grown to maturity under
flooded conditions in the second trial, instead of being
drained at the maximum tiller stage as in the first trial.
It is likely that the continuously flooded condition was
responsible for the fact that rice in green manured cultures
failed to recover from the early retardation as completely
as in the first trial.

Table 7. Weight of straw and numberauxlweight of rice

grains per hill in second greenhouse trial
(first crop rice, Lotung profile).

 

 

Green Manure Straw

 

Incorporated before planting
No LSDO 5
Measurement Manure 2 days 2 weeks 2 days 2 weeks '

 

 

Grains per
hill 322.33 278.77* 197.69* 289.95* 210.20* 5.23

Grains (gm) 8.83 4.54* 4.77* 2.56* 3.07* 3.39
Straw (gm) 82.75 65.65 67.36 45.69* 52.08* 21.50

 

*Significantly less than for no manure.

47

In the second trial, the symptoms of young seedlings
were the same as occurred in the first trial and began to
appear 2 days after transplanting in lupine-amended soil
and 5 days after transplanting with straw. No symptoms
developed in check soils.

In amended cultures, root injury and rolling and
chlorosis of leaves became progressively worse through the
stage of maximum tiller number. After that, leaves regained
their green color and plants developed more or less normally,
except that they remained stunted in comparison with check
plants, and panicle formation, anthesis and maturity were
greatly delayed.

Brown spots began to appear on the older leaves,
spreading along the edges from the tip toward the base,
after 43 days after transplanting (maximum tiller number
stage). The brown spots gradually turned reddish and in-
creased greatly after anthesis (84 days after transplanting).
At this time, most of the older leaves were dead. The most
severely affected plants were in the straw-treated boxes.

Soil oxidation-reduction measurements failed to
show any differences between healthy unmanured cultures and
those which were injured by straw or lupine green manure
treatments. In the early stages of growth, the redox
potential was below -400 mv. It increased as the rice grew
older, reaching -250 mv at the time of panicle development
and -150 to -100 mv at maturity. These potentials are well
below the critical threshhold range of -6 to -100 mv found
in association with "Akagare," a Similar disorder of paddy

rice in Japan (131)-

FIGURES

Effects of Organic Amendments on Growth
Characteristics and Yields

49

b‘ufimro‘1]"mflg
I green. manure
I: check
straw

    

8

Number per In"

8

 

Fig. 1. Number of rice tillers on Chungli (c), Pingtung (P)
and Lotung (L ) surface soils and on the Lotung
profile (L ) at various times after transplanting.
(Fall crop rice, greenhouse, 1964.)

  

‘ I n mm
'1’ C21 Check ,
A
3 U
i u
u g
.0 /
i. g
'3 IO ;
a é
. /

 

  

 

 

Fig. 2. Weight of rice straw on Chungli (c), Pingtung (P)
and Lotung (L ) surface soils and on the Lotung
profile (L2). (Fall crop rice, greenhouse, 1964.)

Fig.

Fig.

50

 
     

 

' r-u ' . ‘
Wan. 6;;
’i “I {and )
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33' codes
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if» .L. ‘w
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. 0 ' , J;
' L1 L1
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3. Weight of rice grain on Chungli (c), Pingtung (P)
and Lotung (L1) surface soils and on the Lotung
profile (L ) at various times after transplanting.
(Fall crop rice, greenhouse,1964.)

.4

.“er oi Rice Gram .
( full for-ml )

- (l rec-n manure

Ll check

straw

 

Number For lull
-‘iiiliiii?

 

c r' Ll L,
SOIISUIPIu

if 7 ”L. __ _——_1

 

4. Number of rice grains on Chungli (c), Pingtung (P)
and Lotung (Ll), surface soils and on the Lotung
profile (L2). (Fall crop rice, greenhouse, 1964.)

 

Fig. 5. Chungli surface soil, fall crop rice, 1964, 10 days
after transplanting. Left, straw: right, green
manure; center check.

1

«(Vt I

‘11 m n

I

"M
i Tykx

 

 

 

Fig. 6. Pingtung surface soil, fall crop rice, 1964, 10
days after tranSplanting. Front row: left, straw;
right, green manure; center check.

52

 

Fig. 7. Lotung profile, fall crop rice, 1964, 10 days

after transplanting. Left, straw; right, green
manure.

 

 

Fig. 8. Lotung profile, fall crop rice, 1964, 10 days
_after tranSplanting. Left, d1seased plant:
right, healthy plant.

 

T's

.1.qu '13,, (Lof‘MS). (L'MS’A
- ——--9

“fi—

 

Fig. 9. Lotung profile, fall crop rice, 1964, at harvest.
Left, straw; right, green manure.

 

Fig. 10. Overall View, fall crop rice, 1964, at flowering.
From left to right: Lotung surface, Lotung
profile, Pingtung surface (check), Pingtung
surface (manured), Chungli surface soil.

54

 

 

Fig. 11. Lotung profile, spring crop rice, 1965, 15 days
after transplanting.

55

 

 

Fig. 12. Lotung profile, spring crop rice, 1965, 15 days
after transplanting. Amended 2 days before trans-
planting with straw (left) and green manure (right).
Unmanured check in center.

 

 

Fig- 13. Lotung profile, spring crop rice, 1965, 55 days
after transplanting. Amended 2 days before trans-
planting with green manure (left) and straw (r1ght).

Unmanured check in center.

56

 

Fig. 14. Lotung profile, spring crop rice, 1965, 15 days
after transplanting. Amended 2 weeks before trans-

lanting with straw (left) and green manure (right).
nmanured check in center.

 

 

Fig. 15. Lotung profile, spring crop rice, 1965, 55 days
after transplanting. Amended 2 weeks before
transplanting with green manure (left) and
straw (right). Unmanured check in center.

Fig. 16.

Fig. 17.

57

 

#4

Lotung profile, spring crop rice, 1965, 15 days
after transplanting. Straw incorporated into
surface soil 2 days (left) and 2 weeks (right)
before transplanting. Unmanured check in center.

 

Lotung profile, spring crop rice, 1965, 55 days
after transplanting. Straw incorporated into
surface soil 2 weeks (left) and 2 days (right)
before transplanting. ‘Unmanured check in center.

Fig. 18.

Fig. 19.

58

 

 

Lotung profile, spring crop rice, 1965, 15 days
after transplanting. Green manure (Lupines)
incorporated into surface soil 2 weeks (left)

and 2 da 3 (right) before transplanting.
Unmanure check in center.

 

 

Lotung profile, spring crop rice, 1965, 45 days
after transplanting. Green manure (Lupines)
incorporated into surface soil 2 days (left)
and 2 weeks (right) before transplanting.
Unmanured check in center.

Fig. 20.

59

 

Rice in nutrient solutions 25 days after
inoculation with root washings from diseased
rice grown in the Lotung profile amended with
straw (left) and Lupines (right). Center three
plants inoculated with root washings from healthy
rice grown in the unamended Lotung profile.

60

Inoculation Experiment

Similar symptoms of physiological disorder were
found when Lotung (L2) profiles which had been treated with
straw or Sesbania in the first trial were retreated with
straw or lupines, respectively. When root washings from
these retreated and replanted cultures were used to
inoculate rice plants growing in nutrient solution,
similar symptoms again developed.

Within one week after inoculation, the leaves began
to turn yellow. The height of the plants was markedly
reduced, in comparison with plants inoculated from replanted
cultures on the Lotung profile which had not received organic
amendments either in the fall or the spring. By the 22nd
day after inoculation the leaves of the affected plants were
completely yellow. The relative appearance of injured and
uninjured plants at this time can be judged from Fig. 20.

Numbers of total bacteria and of sulfate reducers
found in the inoculated nutrient solutions at the time
when distinct disease symptoms were apparent are shown in
Table 8.

Table 8. Numbers of total bacteria and sulfate reducers in
nutrient solutions 10 days after inoculation with
root washings from retreated and replanted rice

cultures in Lotung profiles from the first green—
house trial.

 

 

Source of inoculum

 

Microbial group

 

No Green Straw
__, Manure Manure
Total bacteria x 106 per ml 11.9 20.4 23.0

Sulfate reducers x 103 per ml 23.0 54.0 35.0

¥

61

Numbers of total bacteria were two-fold greater in
nutrient solutions inoculated with root washings from amended
cultures than from unamended checks. Sulfate reducers were
also greater where inocula from amended cultures were used,
but maximum numbers were associated with the green manure
treatment.

When soil leachates from these same replanted Lotung
profiles were used as inoculum, only mild symptoms developed
in nutrient solution cultures and no estimates of microbial

numbers were attempted.

Microflora Studies

Microbial numbers as determined for root surfaces
(R) and for non—rhizosphere (S) samples are recorded for the
two greenhouse trials and the three field locations in Tables
22 to 27 in the Appendix. Differences between edaphosphere
soils (S) counts and rhizosphere soil (Rh) counts were not
very great or very consistent. The rhizosphere counts (Rh)
have been recorded only for the two field locations on the
fall crop in 1964 (Tables 24, 25).

.Microbial numbers on root surfaces (R) were frequently
much greater than in either the rhizosphere or the edaphosphere.
Fluctuations in numbers on root surfaces were frequently
large and frequently appeared to be directly related to
organic amendments in the greenhouse or the diseased con-
dition of the plant in the field. For this reason, the root

counts have been summarized in Tables 9 through 17.

62

 

 

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mUHm mo ucmfimoam>mn Mo mmmum ucmfigmmne

 

 

.Amomav mono
Awomav mono Hamm Ga muoon moflu C0 mummomfioomv mmoHSHHmU UHQOHmmcm

mcflumm mam
mo mnmafisz

.mH mHQmB

69

 

 

 

m.N 0.0Nm o.ma @.H m.o mmmmmmfln
H.~ m.n m.m o.n «.0 msuammm wood namflm mcspmcflm
o.NH 0.0% II H.m m.m cmwmmmfln
m.m o.oa nu 5.H m.o msuammm moma
O.NN o.wN o.©H N.m II 00mmmwan
o.HH m.m N.m N.m H.o mauammm ¢oma namflm mcsuoq
m.m o.am b.m o.omH ¢.m Bmuum
0.0H o.@¢ o.oa o.oooa ¢.H .cmE .HU
>.m m.m H.HH o.w m.H Momzo moma
o.oooma o.mm m.m m.m II 3muum
o.ooooa o.oooma «.m o.HH o.@a .cmE .Hw mmsos maamonm
o.ooooa o.ooooa m.m ¢.m m.q xomao vmma lawmuw mmsuoq
moa x muoou mun m\mquE§Z
muausumz mammsucd GodumHUHCH mHmHHHE GodumauacH cod
. . . . . . . . . .9H :0 w
maoflamm Eseflxmz HMHHHB ucmwm 0 max coflpmooq HHom
no
moflm mo mmmum Hmpcwamoam>ma pcmEummHB

 

 

. mo
mCflHmm 0cm Awomav QOHU Hamm CH muoou moflu no aflumuomn maflmmauuacwwfimmmwwmQEMm

.wa mHQmE

70

 

 

 

o.ma 0.5 o.¢m N.m ¢.H nwmmmwfin
o.¢H m.o o.oa o.mm H.H maufimmm wood wamflm mcsumcflm
m.m H.m m.m m.m h.a Ummmmmfln
n.H F.H ¢.N m.m n.a mauammm moma
0.5H o.¢a o.¢ o.mm In wmmmomfla
¢.m o.¢m o.¢H o.ma 5.0 mauammm womfi namflm mcsuoq
0.5H o.mm o.mm o.mv m.h BMHum
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. . . . . Hamoum
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mananmm Esaflxmz Hmaafle “swam . a. m
no
moflm mo mmmum HmucmEQon>wQ ucprmmue

 

 

.Amomav mouu mcflnmm 0cm Awmmav mono Hamm CH muoou GUflH :0 mumHMHcoafim mo mHmQEsz .na maame

71
Major Taxonomic Groups

Bacteria and actinomycetes on root surfaces were
frequently high during early vegetative growth through maxi—
mum tiller number and then tended to decline to stable low
numbers (Tables 9, 10). In the greenhouse experiments,
numbers shortly after tranSplanting were usually distinctly
higher where energy materials had been added as green
manure or straw. In the field samples, there appeared to
be no consistent relationship to diseased or healthy plants.

Fungi also increased in responsetn organic amendments
during the period from tiller initiation to maximum tiller
number (Table 11). Later, numbers were consistently lower
in amended cultures than in the checks. In the field.
fungi were frequently more numerous on roots of healthy

than diseased plants.

Physiological Groups

1. Anaerobic bacteria

Variations in numbers of anaerobic bacteria (Table 12)
showed a similar tendency to decline from high numbers
early to lower numbers late in the season as was seen
With total bacteria (Table 9). However, in both fall and
spring crops at the Lotung field locations, numbers were
consistently higher on diseased roots. In the 1965 green—
house experiment. it appeared that the anaerobes may have

responded to the straw amendment at the time of the first

 

72

sampling. In general, however. the anaerobes did not re-
flect effects of added energy materials as clearly as the

three major aerdbic groups above.

2. Sulfate reducers

Sulfate reducers were counted only on the spring
crop (Table 13). In both the greenhouse and the field,
numbers were consistently higher on roots of plants showing
disease symptoms than of check or healthy plants. although

the differences were not great.

3. Cellulose decomposers

Both aerobic and anaerobic cellulose decomposers
(Tables 14, 15) responded to additions of green manure or
straw. Except for one field location (Lotung 1965).
numbers were rather consistently higher on roots of diseased
plants in the field and of amended cultures in the green-
house during the early vegetative period through maximum

tiller number or panicle initiation.

4. Denitrifying bacteria

Of all microbial groups studied, the denitrifiers
Showed the most extreme fluctuations in numbers. both on
root surfaces (Table 16) and in the rhizosphere (Tables
24. 25). The seasonal pattern was related inversely to
that observed in most groups considered in above sections.

Numbers were low in the beginning and reached maximum

73

numbers later, usually in the late stages of anthesis or
maturation.

In the 1964 greenhouse experiment. a dramatic
increase in numbers at the end of the season occurred later
with straw than either the checks or green manured cultures.
In the 1965 experiment and at all field locations, however,
this group was rather consistently higher on roots of
amended cultures and diseased plants from the late vege-
tative or early flowering period through the remainder

of the season.

5. Ammonifying bacteria

Root populations of ammonifiers (Table 17) showed
only moderate fluctuations during the season. There was a
tendency, as with the denitrifiers, to start from low
numbers and rise to a maximum at some later stage of develop-
ment. However, there was no consistent relationship to
organic amendment or condition of plant, except in the
1965 greenhouse experiment where larger numbers were
maintained in amended cultures from the maximum tiller stage

to maturity.

Pure Culture Studies

Woldendorp (170), using broth cultures, found that
his collection of denitrifying bacteria could be divided
into two groups on the basis of whether they could or could

not grow anaerobically in the absence of nitrate. Additional

74

distinctions could be made within each of these groups on
the basis of whether or not amino acids or vitamins were
required for growth and/or gas production. His observations
were. in general, consistent with those of valera and
Alexander (149).

Pure culture studies were undertaken with 18 species
and strains with a view to developing solid media for
differential enumeration and isolation of physiological
groups among the denitrifiers. The chemically defined
media of Woldendorp were used, with slight modifications
(see Table 4). Buffer concentration and the concentration
of Ca and Mg were reduced to avoid precipitation of phosphates
and iron. The vitamin component was augmented by addition

of inositol and Vitamin B as in media described by Chase

12
and Lochhead (78). Plates were streaked using loop
inoculations taken directly from heavy log-phase suspensions

in nutrient broth.

Aerobic growth

Visual estimates of growth under aerObic conditions
are shown in Table 18. In the absence of growth factors
(medium 103); growth of most organisms was restricted,
as compared with more complete media. On the basis of
Woldendorp's data, no growth would have been expected on
glucose with nitrate in the absence of growth factors in the
case of organisms 3, 5. 7, 9. 10, ll, 12. 13 or 15. The

fact that slight. slow growth did occur with a number of

75

.m on 0 mo mHmom m so >HHm5mH> pmumEHumm Comm um zuSOHwa

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

m N III III III N H sammH ooaa mamUHMHHustm .IF .wH
m m III III III m m omNHH 009m mcwommuosHm .mm. .bH
a m III III III a m mammH ooem mcmoHMHuuHcmo .m. .0H
N 0 III III III N\H o mom 0094 HHQmHuHmn .d .mH
a a III III III N H ommaH ooam mHsquHcmaoHH = .aH
N H III III III H o mHmv 008m mcmHSUHHU : .mH
N 0 III III III H O .VWNQ Gnu—Haw : : .NH
H 0 III III III N\H O mdm UUBfl mGMHmUmE : .HH
a w III III III m\H 0 mbme = = = .OH
an an III III III N\H O flmflw 0094 mSQHmU = .6
m m w m m m o ABVHm mHEHOMHcmnoHH = .m
a a a m m m m stHH mmmH mcmHsamoo = .s
m m I- I- l- H QH ESQ mHEoflawfiHH .... .a
m m a m m H «\H Hsvm was msuomwoumumH .m. .m
m w w m m m o mmzrbmz mcoommoume .m .w
a m a m m N o = meunsum = .m
m s d m m w m mucosmoHoz : Ih. .N
m w w m m w m s meOH UUB¢ mmochdnmm .mm .H
m>mo m m>m oaumm
hmo H mwmn h mmmp h m>m0 h mmm H Musouw
+ + + I I mcHEmuH>
+ + + + I moHom .Em Echmmuo
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+ I + + + mumwqu
+ + + + + mHmumcHz
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HHH mOH mOH vOH MOH “cocomaou
COHpHmOQEOU pcm HmQESQ ESHth
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mo mUHSOm ou GOHpmHmH CH mmHSuHso xmmuum ummm HmHumpomn mo ausowm UHQOHmfl .mH mHnt

76

.fi 0“ 0 mo mHmom m Co \HHHmCmHNV UmumEHumm 00mm um mmmp MH l m CH Cu3onwa

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

l I l l lH NIH SmmH 09$ mqmonHuuHcHu In. .3
Wlm mlm mWHIM mmHIM mWHIH olo ommHH 009d mCoommuosHm .mmm .bH
mlm mlm filfi fila film filfi mammH 00.3 mcmonHflHamt .HIH .3
olo olo olo Hlo olo m\HIo mom 009m HHQmHuHmC 4a. .mH
film mlm film film mlH mlm ommfiH 008.4. mHEHIanCwCoHH ._ .fiH
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Hlo mlo Hlo mlo Hlo mlo mfim 009R mCmumumE ._ .HH
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mlm mlm film film mlH HlH fimfio 009m mswnmo .. m
mlm mlm film film filH mlH :5va mHEomHamCUHH .. .m
mlm mlm mlfi filfi film mlm AEHH momH mCmHCmmoo .. .h
mlm mlm olm film NIH NIH ESQ mHsHochmaoHH h .a
mlH mlH film film fiIm\H fiIm\H Evm mmfi mswommomeBH .m .m
film filfi filfi filfi film film mszsz mCmommoumE .m .fi
HlH MIN mlN .Vlfi mlH mlH : Hummfifinwm .. .m
fil m fil m a la filfi fil m fil m 38330: .. In . m
mlm mlm filfi filfi film film mfiHoH ooem mmomwmsnmm .mm .H

a a a a a x.

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mCHQ mm mCHm mm wCHQ m IOEHE.
+ + I mCHEmpH> mEmHCmmCO
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+ + + mHmumCHS
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OHH mOH 50H prCOQEOU
HmQECC pCm COHuHmOQEOU ECHpmz
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mo monsom ou COHCMHmH CH meCUHCU Hmmuum Hmmm HMHHmuUmQ we Cusonm UHaowmmCm .mH mHQmB

77

.0 Cu 0 mo meom m Co mHHmsmH> omumEHumm .00mm um mmM© mH CH Cu30H0%*

.Noo ucmo Cmm OOH no NC ucmu Mme OOH*

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

N m o a m m m m NommH ooea mamonHHuHcmo .In .mH
o o o a o N m o omNHH 0094 mcmummnosHH .mm .NH
o a o a o m o m mfimNH noes mamoHHHuHHcmc .mfl .SH
0 o o N o o o o mom cues HHanuHma .a .mH
H N m m m a a m ommfiH ooaa mHsHochmaoHH = .fiH
o H m N N N m N mHma ooem mcmHsouHu = .mH
N N m N N N N m fifiNm cues = = .NH
I I I I I I I I mam cues mcmumoms = .HH
H N a a m m N N mamaH cues = = .OH
o N N m N m N N swam ooe< msmuwo = .m
o N a m N m a m Hsva mHsuomHamnoHH = .m
H N a m m m a m HonH momH mcmHsmmoo = .N
o N m m N m m N Havomfi mHsuoHHcmaoHH .F .a
o o N m H m H H Hsvmwoa msHommonmumH .m. .m
H N m m N m N H mszsz mcwommonms .m .a
H H N m o N o H = HHmNusum = .m
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N N a m m N m H mfiHOH ooem mmochsnmm .mm .H
«Ly «CH. ¥¥ «in urns. ink. wax. «in Q
N N N N N amuma m
00 oo 00 m oo NC Iospm
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.m00 UCm muouomw Cu3onm .mumuuHC .hmHma

.ON mHQmB

78

these probably reflects a carry-over of growth factors in the
inoculum.

The rather extensive growth of orgalism 7 is un-
explained. Physiological tests on milk and starch and for
production of indole and acetylmethylcarbinol (voSges—
Proskaur test) corresponded to the Bergey‘s Manual descrip-

tion of Bacillus coagulans.

Anaerobic growth

A major difficulty in anaerobic studies was the
complete elimination of traces of oxygen at the beginning
of incubation. Traces of oxygen can permit initiation
of growth and adaptation to anaerobic conditions in the
case of some facultative anaerobes (149). Media were

reduced chemically by storage in H2 prior to streaking.

Sodium thioglycollate was added to minimize oxidation

during the time that plates were necessarily exposed to

air while being streaked.

Data in Table 19 indicate that these precautions

were inadequate. In the absence of nitrate (medium 110),

no growth would have been expected for organisms 1, 2, 3,
16 or 18 (170). In the absence of growth factors

(medium 107). no growth would have been expected for

organisms 3, 5, 6, 7, 8. 9. 10, ll. 12. 13. 14. or 15.

Limited growth was observed in all of these, and rather

extensive growth in some, especially after 13 days. Plates

were exposed to air when the observations were made on the

79

third day, and oxygen absorbed during the observation period
may have stimulated growth during the following lO-day

incubation under anaerobic conditions. Rapid growth

 

of the two Pseudomonas aeruginosa strains was actually
observed during the 90-minute observation period.

As can be seen in Table 19, the introduction of
10 per cent CO2 into the H: atmosphere had little effect
on growth of most cultures. In the case of organism 6,
an apparent lysis of cells occurred with C02 on medium 109,
resulting in completely transparent zones after 13 days
where vigorous growth had been observed at 3 days.

In a subsequent experiment (Table 20), additional
precautions were taken to minimize the length of time
that media were exposed to air during inoculation. The
incubators were not opened during a 15-day incubation
period. HIn one incubator, after reducing conditions had

been established under H2, the H2 was completely replaced

With C02.

Under these conditions, complete suppression of

growth of several cultures was achieved. However, unexpected

responses to C02 were expressed.

The two Ps. aeruginosa strains (1 and 2) were
restricted in growth in the absence of nitrate (medium 110)

in the H2 atmosphere but grew profusely in C02. Wlth strain

2: this response to C02 was expressed also in the presence

of nitrate, without regard to source of energy or growth

factors (media 111, 112, 113). In the absence of nitrate,

80

growth of organisms 17 and 18 was also enhanced by C02.
Growth of Pg. stutzeri was completely inhibited by

C02 in the completely defined media 110 and 111, but not

when yeast extract was used as a source of growth factors

(medium 112). Nficrococcus denitrificans (# 16) was com-

 

pletely inhibited by C02 on all media. With gs. fluorescens

 

(# 17) a similar CO2 inhibition occurred only in media con-
taining nitrate (media 111 or 112).

Yeast extract was a better source of growth factors
for several organisms growing on glucose (medium 112)
than the defined vitamin and amino acid mixture (medium 111).
For most organisms, citrate supplied as energy source in

medium 113 was used less readily than glucose (medium 112).

Antagonistic relationships

Woldendorp (170) found that gs. aeruginosa strains
were the dominant denitrifying organisms in rhizospheres of
forage grasses. It appeared of interest to know whether
this might be due to antagonistic effects on other denitrify-
ing species. Accordingly, cross-streak inoculations were
made using organism # l as the test organism. Other
organisms were streaked at right angles to the test organism,
taking care to avoid direct contamination of the streaked
inocula.

Under aerobic conditions (Table 21), a number of
organisms were markedly inhibited in the vicinity of the

test strain when no growth factors were present (medium 103).

81

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 21. Growth inhibition by Pseudomonas aeruginosa
ATCC 10145 under aerobic conditions on cross-
streaked agar plates.
Medium composition and
number
Component 103 111
Glucose + +
Minerals + +
Nitrate + +
Asparagine - +
Organism Am. acids - +
Vitamins - +
mm* mm*
2. lgg. aeruginosa Woldendorp O 0
3. " stutzeri " 4 3
4. .§. marcescens MSU-MPH 8 0
5. lg. laterosporus 468B(W) 0 8
6. " licheniformis 430(W) 8 0
7. " coagulans 1963II(W) 8 0
8. “ licheniformis P1(W) 8 O
9. " cereus ATCC 6464 0 0
10. " " ATCC 14579 0 0
ll. " macerans ATCC 843 4 4
12. " " ATCC 8244 0 0
13. " circulans ATCC 4513 0 0
l4. " licheniformis ATCC 14580 8 O
15. g. hartlebii ATCC 365 0 0
16. .M. denitrificans ATCC 13543 6 2
17. ‘Pg. fluorescens ATCC 11250 2 2
l8. " denitrificans ATCC 13867 10 2

 

 

*Length of inhibition zone.

82

This suppression of growth was less frequently expressed

in the complete medium 111. In the case of g. laterosporus

 

(strain # 5), growth was inhibited only in the complete
medium.

Antagonistic growth inhibition by Pg, aeruginosa
under aerobic conditions was associated with the production
of a blue—green pigment in the absence of growth factors
and asparagine. When growth factors were present, a yellow-
green pigment was produced. The production of the yellow—
green pigment became apparent with the addition of amino
acids, was increased when vitamins were also added, and
was at a maximum in complete media containing asparagine
and growth factors supplied either as yeast extract or
as amino acid plus vitamin mixtures.

Under anaerobic conditions no pigments were produced

by Pg. aeruginosa strains. However, they appeared within

 

a few hours after cultures were removed from anaerobic
conditions into the air. This post—incubation development
of pigment was greater in media supplying glucose, rather
than citrate, as carbon source.

Under anaerobic conditions, Pg. aeruginosa (strain 1)
markedly inhibited the growth of g. macerans (strains 11 and
12) and of_M. denitrificans (strain 16). Uhder anaerobic
conditions, also, the ATCC strain (strain 1) showed a con-
sistent, though mild, dominance over Woldendorp‘s strain

(Strain 2). No other antagonisms were observed under

83

anaerobic conditions.
The characteristic bright red pigment of Serratia

marcescens was also produced only under aerobic conditions.

 

This was true also of a dull reddish brown pigment produced
by the g. licheniformis strains (numbers 6, 8, 14) on
nutritionally rich media. Neither of these genera, however,
showed any antagonistic effects on other strains when they

were used as test organisms, instead of Ps. aeruginosa.

DISCUSSION

The present investigations were undertaken on the
proposition that the study of rhizosphere microorganisms is
one useful means of approach in obtaining a better know—
ledge of the physiological activity of the root system of
rice plants and of factors concerned with such practical
problems as evaluating a given soil treatment.

Research by numerous investigators has developed
the view that, in a constant soil environment, a restricted
but dynamic equilibrium is established among components
of the microbial population. Any change in this environment—-
produced by soil treatments, season or growing crop--will

shift the microbial balance.

Seasonal Behavior of Microbial Groups

The present biological studies have shown that there
can be great divergences in seasonal behavior of different
microbial groups associated with the development of rice
and/or the decomposition of added organic materials.

Numbers of fungi and of total and anaerobic bacteria were
usually much higher on root surfaces than either the
rhizosphere or the edaphosphere during the vegetative and

early flowering stages of rice (Tables 22 to 27). This was

84

85

true also of both aerobic and anaerobic cellulose decomposers.
Numbers in these groups were usually enhanced by organic
amendments and showed a seasonal decline from large

numbers during the tillering and panicle initiation stages

to low numbers unrelated to organic addition at anthesis

and maturity (Tables 9, ll, 12, 13, 14).

Actinomycetes usually showed no relation to organic
amendments but did decline in numbers after maximum tiller-
ing or panicle initiation (Table 10). Only occasionally
were there any marked differences in numbers between root
surfaces and rhizosphere or edaphosphere (Tables 22 to 27).

The behavior of the above groups is consistent
with results reported by others for upland cr0ps (129, 144)
and for paddy rice (52, 116). The data indicate that the
size of the general microflora is largely determined by the
supply of readily available energy materials. These
energy sources may be in the form of residues from a
previous crop, amendments from extraneous sources, or from
exudates or abraded debris from roots of a growing crop.

It is of interest that both aerobic and anaerobic
types were able to respond to additions of organic
matter, even under flooded conditions and in the strongly
reductive environment observed in the greenhouse (Eh =—100
to -400 mv). It is likely that rice roots may have helped
to supply oxygen for growth of aerobes, particulary during

earlier stages of growth. This is supported by the fact

86

that aerobic cellulose decomposers declined sharply in
numbers after the tillering stage under continuous flooding
in the second greenhouse trial but not when soil was drained
at this time in the first greenhouse experiment (Table 13).

The distribution and seasonal behavior of the other
three microbial groups was less well defined. In the case
of sulfate reducers and ammonifiers,1 there appeared to be
no consistent relationship to their position in the root
zone, so that differences between root surface, rhizosphere
or edaphosphere were not great (Tables 22 to 27). Seasonal
variations in these two groups were also small. The
greatest numbers of sulfate reducers tended to come at the
beginning of the growth period, whereas larger numbers
of ammonifiers appeared at some later time.

With the denitrifiers, extreme seasonal fluctuations
in numbers were observed. These occurred mainly in the
root surface population or sometimes in the rhizosphere.
Variations in the edaphosphere from sampling to sampling
were relatively small.

Thus, the sulfate reducers, denitrifiers and ammoni-
fiers appeared to be much less influenced by organic amend—
ments than the other groups. The sulfate reducers and
ammonifiers appeared to be influenced relatively little by

nearness to the root surface. By contrast, the denitrifiers

‘

' 1A more Specific term for "ammonifiers" as estimated
in this study would be "urea hydrolyzers."

87

appeared to have been extremely sensitive to influences
arising from the growing rice plants by way of their root
systems. Among anaerobic groups, the denitrifiers were
usually less numerous than cellulose decomposers in earlier
samplings (Tables 12, 15). However, they were capable

of increasing dramatically to become the dominant anaerobic

group in later samplings.

Relationships between Microflora and Disease Symptoms

During the tillering period when disease symptoms
were developing to peak intensity, total and anaerobic
bacteria, fungi and cellulose decomposers were at their
highest numbers for the season. In the greenhouse experiments,
these groups were more numerous in the amended cultures.
These were also the cultures in which disease symptoms
developed on the Lotung profiles. At field locations
also, these microbial groups were, at times, higher on
diseased roots than healthy ones during the tillering
period.

It is difficult to assign a cause and effect
relationship to these associated phenomena. It is likely
that these saprophytic types were drawing their main
energy supplies from added organic materials and from
residues from the previous crop. Root exudates from the
growing rice would have stimulated growth of these
organisms by supplying growth factors, such as amino

acids. The fact that numbers were greater on diseased

88

roots in the field probably reflects more rapid release of
growth factors, as well as energy substrates, from roots
injured by other mechanisms associated with depletion of
oxygen and reduction of alternate electron acceptors, such
as ferric iron and manganic manganese.

There is the possibility that the above groups may
have included Species which produced toxins injurious to the
rice. However, it appears likely that their major role was
to deplete oxygen and bring about highly reduced soil
conditions. When redox potentials were measured in the
second greenhouse trial, Eh was less than -400 mv at the
beginning of the tillering period and had increased only
to —250 mv by panicle initiation. Bormer (101) and
Kononova (63) have expressed the opinion that competition
between higher plants and microorganisms for a limited
supply of oxygen may be more important than any other
factor in plant injury associated with organic manuring.

A second role of these main saprophytic groups
would have been to deplete nutrients, notably nitrogen
(31, 95). Nitrogen deficiency may have limited rice
Yields with the straw treatment in the first greenhouse
trial.

A more direct role in promoting disease symptoms
might be inferred for the sulfate reducers. These were
somewhat more numerous in amended soil during the tillering
Period and throughout the season in the second greenhouse

trial (Table 13). They were consistently slightly higher

89

on diseased than healthy roots in the field.

It is known that H S may inhibit cytochrome

2
oxidase systems and photosynthetic activity of plant

leaves (173). When Takahashi visited Taiwan in 1961, he
noted the similarity between "suffocation disease" and
"Akagare" which in Japan is found on soils in which there

is insufficient iron to precipitate sulfide. Ferrous iron
forms the complex nKZSO4°mFeSO4, thereby reducing the toxi—
city of H28 (22). waever,_the problem Lotung soil has been
examined by Takahashi (132) Ponnamperuma (106) and Chang (23)
and found to be high in ferrous iron. Thus, the small dif—
ferences observed in numbers of sulfate reducing bacteria
are probably of little significance as far as the associated
disease symptoms are concerned.

Of all the groups studied, the denitrifiers appeared
to be the most sensitive to the influence of the growing
plant, as evidenced by extreme seasonal fluctuations in
numbers on the root surface and/or rhizosphere, with
relatively minor fluctuations in the edaphosphere. In both
greenhouse experiments, this group was more numerous
during the tillering period on roots of the green manured
cultures where symptoms first appeared (Table 15). In
the second trial, without drainage, higher numbers were
maintained in amended cultures throughout the season.

At field locations denitrifiers were rather consistently

higher on diseased than healthy roots.

90

Again, cause and effect cannot be assigned to these
associations. It must be assumed that denitrifiers were
present in larger numbers on diseased roots because
nutritional conditions were more favorable. Whether they
contributed to the diseased condition of the plant by toxin
production or some other mechanism cannot be said.

It is known that a number of nitrate reducing
bacteria produce antibiotics and lytic enzymes. These may
have toxic effects on plants, as in the case of tobacco
"wildfire," which is caused by a toxin produced by
Pseudomonas tabaci (124).

In the nutritional studies reported here, it was
observed that the color of pigments produced by Pseudomonas
aeruginosa were different in media with and without growth
factors, and that the spectrum of antagonism was also
different. Thus, a change in composition of root exudates
associated with plant nutrition might bring about a change
from a toxic to a non—toxic product of the root surface
microflora.

The denitrifiers were enumerated on the basis of
gas production in aSparagine-nitrate-citrate broth. It
was assumed that the gas was N2 or N20, and that the
organisms responsible were capable of denitrification.

It cannot be assumed that, in the soil environment, these
organisms were actually maintained on a nitrate respiration.
However, if they were, a new problem arises as to where

the nitrate came from.

91

It has been shown that rice has a root structure
which permits oxygen to diffuse to the roots and to
oxygenate the environment in the immediate vicinity of the
roots (131). This would permit a nitrifying population to
remain active under flooded conditions on the root surface
or in the rhizosphere. An active nitrifying pOpulation would
be necessary to maintain an actively denitrifying population.
The nitrifying flora of flooded rice rhizospheres does not
appear to have been studied.

Although a specific microbial agent responsible for
the symptoms of the "suffocation disease" has not been
identified, the transfer of symptoms from diseased to
healthy rice by root washings was shown in the inoculation
experiment. This experiment makes it quite clear that
microbial agents areinvolved in some way.

Even if a microbial agent, or agents, are found,
it is unlikely that they will be primary agents in a
pathogenic sense. Rather, their presence will reflect
a degree of nutritional imbalance in the plant arising
from highly reductive soil conditions. According to
Yanagiswawa and Takahashi (173), low productivity in paddy
soils is frequently attributable to deficiencies of nitrogen,
potassium, phosphorus, or silica and, at later stages of
growth, to toxic substances produced in the soil. Chiu
(26) found that diseased rice on the problem Lotung soil in

eastern Taiwan contained more nitrogen and less potassium

92

and phosphorus than healthy plants. Lee (27) found

that the diseased rice on the same soil was higher in iron
and manganese, but that there was no difference between
soils taken from healthy and diseased areas in their content

of available iron and manganese.

Nutrition of Denitrifiers

It is of interest to note that counts of anaerobic
cellulose digesters and of denitrifiers frequently were
very much greater than the more general count for anaerobic
bacteria on Brewer's thioglycollate-dextrose—peptone agar.
This serves to emphasize the importance of medium composition
and cultural techniques in permitting significant groups of
organisms to grow in the laboratory.

The nutritional studies reported are preliminary
but show promise for developing procedures for enumeration,
isolation and nutritional characterization of anaerobic
organisms with the capability for reducing nitrate. The
microflora studies showed that this group is very sensitive
to influence by plant factors on root surfaces and in the
rhizosphere. It should be a useful group to work with
in further investigations of the rhiZOSphere physiology

of rice.

S UMMARY

The rhizosphere physiology of "suffocation disease"
of rice was investigated by following the seasonal distri-
bution of nine microbial groups in root environments of
healthy and diseased plants in two greenhouse experiments
and at three field locations in Taiwan. Fall and spring
crops of rice were studied.

In the greenhouse, disease symptoms similar to those
observed in the field were induced by amendments of straw
and green manure on only one of four soils tested~-an un-
disturbed Lotung profile from eastern Taiwan.

Early symptoms included chlorosis and rolling of
leaves, accompanied by darkening amd dying back of roots
and greatly reduced branching of secondary roots. These
symptoms became more severe as vegetative development of
tillers progressed. Some plants died before maximum
tillering.

After the tillering stage, surviving plants recovered
their green color but remained stunted and were delayed
in panicle initiation, anthesis and maturity as compared
With unmanured check cultures.

Recovery was more rapid and complete following
green manure treatment, even though symptoms appeared

earlier and were initially more intense with this treatment

93

94

than where straw was added.

Greater recovery occurred when soil was drained
after maximum tillering than with continuous flooding to
maturity. A two-week delay in transplanting after green
manure or straw were incorporated reduced injury and
hastened recovery.

Final yields of straw and grain were drastically
reduced by straw treatment, regardless of season or
drainage. Without drainage in the spring crop, yields
were also reduced by green manure, but not in the fall
crop where cultures were drained after maximum tillering.

Total and anaerobic bacteria, fungi, and aerobic
and anaerobic cellulose decomposers were most numerous
during the tillering period, responded to organic amendments,
and were much more numerous on root surfaces than in the
rhizosphere or edaphosphere. Actinomycetes were less
responsive to organic amendments or to the influence
of rice roots.

It appeared that demand for oxygen and other
electron acceptors by the above groups was responsible for
the strongly reduced soil conditions which were observed:
Eh = ~400 mv at tiller initiation, —250 mv at panicle initia-
tion and —150 to —lOO mv at maturity.

Sulfate reducers, denitrifiers and ammonifiers
(urea hydrolyzers) were affected relatively little by
organic amendments. Sulfate reducers and ammonifiers were

relatively insensitive to the proximity of the root, whereas

95

denitrifiers appeared to be extremely sensitive to plant
influences. Explosive increases in numbers of denitrifiers,
principally on root surfaces, appeared to reflect changes
in physiology of the rice plant.

Disease symptoms were successfully transmitted to
healthy rice seedlings in nutrient culture by root washings
from diseased plants, indicating the involvement of a
microbial agent.

Preliminary pure culture studies with denitrifying
bacteria gave promise of useful nutritional discrimination
within this group for further studies on rice rhizosphere
physiology. Numerous possibilities appeared for toxin

production by denitrifying Species and strains.

10.

ll.

12.

LITERATURE CITED

Alberda, T. 1953. Growth and root development of
lowland rice and its relation to oxygen supply.
Plant and Soil 5:1-28.

Alexander, M. 1961. Introduction to soil microbiology.
Wiley, New York, 472 p.

Allen, M. B., and Van Niel, C. B. 1952. Experiments
on bacterial denitrification. J. Bacteriol.
64:397-402.

Allen, 0. N. 1957. Experiments in soil bacteriology.
Burgess, Minneapolis.

.Allison, F. E., Carter, J. N., and Sterling, L. D.
1960. The effect of partial pressure of oxygen on
denitrification in soil. Soil Sci. Soc. Am. Proc.
24:283-285.

Allison, F. E. 1961. Twenty five years of soil
microbiology and a look to the future. Soil Sci.
Soc. Am. Proc. 25:432-439.

Allison, F. E., and Carter, J. N. 1960. Investigation
on denitrification in well aerated soil. Soil
Sci. 90:173-177.

Bartholomew, W. V., and Clark, R. F. 1950. Nitrogen
transformations in soil in relation to the rhizo-
Sphere microflora. Trans. Intern. Congr. Soil Sci.,
4th, Amsterdam. 2:112—113.

Bodily, H. L. 1944. The activity of microorganisms
in the transformation of plant materials in soil
under various conditions. Soil Sci. 57:341-349.

Bormer, H. 1960. Liberation of organic substances
from higher plants and their role in the soil sickness
problem. Botan. Rev. 26:393-424.

Bremner, J. M., and Shaw, K. 1958. Denitrification
in soil. II. J. Agri. Sci. 51:40-52.

Brewer, J. H. 1940. Clear liquid mediums for the

aerobic cultivation of anaerobes. J. Am. Med. Assoc.
115:598—600.

96

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

97

Brinhart, B. 1963. Soil air essential for good plant
growth. Horticulture 41:92.

Broadbent, F. E., and Stojonovic, B. E. 1952. The
effect of partial pressure of oxygen on some soil
nitrogen transformations. Soil Sci. Soc. Amer.
Proc. 16:359-362.

Broadbent, F. E. 1951. Denitrification in some
California soils. Soil Sci. 72:128-137.

Brown, R., Robinson, E., and Johnson, A. W. 1949.
The effects of D-xyloketose and certain root exudates

in extension growth. Proc. Roy. Soc. (London).
B, 136:577-591.

Bunt, J. S., and Rovira, A. D. 1955. Microbiological
studies of some subantarctic soils. J. Soil Sci.
6:119-128.

Burton, M. 0., Eagles, B. A., and Campbell, J. J. R.
1947. The amino acid requirements for pyocyanin
production. Can. J. Res., 25(C):121—128.

Burton, M. 0., Eagles, B. AH, and Campbell, J. J. R.
1948. The mineral requirement for pyocyanin pro-
duction. Can. J. Res. 26(C):15-22.

Cady, F. B., and Bartholomew, W. V. 1961.. Influence
of low pO on denitrification processes and products.
Soil Sci. Soc. Amer. Proc. 25:362-363.

Callahan, W. S., Beyerlein, B., and Mull, J. D. 1965.
Toxicity of Pseudomonas aeruginosa slime. J.
Bacteriol. 88:805-806.

Chang, S. C. 1960. The physiological disease of the
second crop of rice in Pingtung [in Chinese].
Newsletter of Soils and Fertilizers, Taiwan, No. 112.

Chang, s. c. 1962. My impressions on the physiological
disease of rice in Taiwan [in Chinese]. Newsletter
of Soils and Fertilizers, Taiwan, No. 139.

Chang, S. C. 1964. Evidence, significance and
research of suffocating disease of the second crop
of rice in southern Taiwan [in Chinese]. Joint
Committee on Rural Reconstruction, PID 287, March
23, 1964.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

98

Christensen, P. D., Toth, S. J., and Bear, F. E.
1950. The status of soil manganese as influenced,
by moisture, organic matter and pH. Soil Sci. Soc.
Am. Proc. 15:279-282.

Chiu, T. F., and Lee, L. D. 1962. Comparison by
pot culture experiments between Pingtung sick and
Taipei normal soil [in Chinese]. J. Agr. Assoc.
China. New Series No. 38.

Chiu, T. F., and Lee, L. D. 1956. Studies on
Chin-Sag-Tsan of Tung-San Ilan [in Chinese].
Natl. Taiwan Univ. Agr. Chem. Rep. No. 5.

C1afik.F. E. 1940. Notes on types of bacteria
associated with plant roots. Trans. Kansas Acad.
Sci. 43:75-84. '

Clark, F. E., Nearpass, D. D., and Specht, A. W.
1957. Influence of organic additions and flooding
on iron and manganese uptake by rice. Agron. J.
49:586-589.

Cochrane, V. W. 1949. Crop residues as causative
agents of root rot of vegetables. Connecticut
Agr. Exp. Sta. Bull. 526:3-31.

Collison, R. C., and Conn, H. J. 1925. The effect
of straw on plant growth. N.Y. Geneva Agr. Exp.
Sta. Tech. Bull. 114.

Dastur, J. F. 1937. "Pan-sukh" disease of rice in
the central provinces. Agr. and Livestock, India
7:509—511.

De, P. K., and Mandol, L. N. 1957. Physiological
diseases of rice. Soil Sci. 84:30-37.

Epstein, E., and Kohnke, H. 1957. Soil aeration as
affected by organic matter application. Soil Sci.
Soc. Am. Proc. 21:85-92.

Evenari, M. 1949. Germination inhibitors. Botan.
Rev. 15:153—194. '

Fewson, C. A., and Nicholas, D. J. C. 1960.
Utilization of nitric oxide by micro—organisms
and higher plants. Nature 188:794-796.

Friedheim, E. 1931. Pyocyanin, an accessory
respiratorywanzyme. J. Exptl. Med. 54:207—221.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

99

Friedheim, E., and Michaelis, L. 1931. Potentiometric
study of pyocyanin. J. Biol. Chem. 91:355-368.

Fuller, W. H., and NOrman, A. G. 1942. .A cellulose
dextrin medium for identifying cellulose organisms
in soil. Soil Sci. Soc. Am. Proc. 7:243-246.

Gato, Y., and Tai, K. 1956. On the differences of
oxidizing power of paddy rice seedling roots among
some varieties. Soil and Plant Food, Japan.
2:198-200.

Gerretsen, F. C. 1948. The influence of microorganisms
on the phosphate intake by the plant. Plant and
Soil 1:51-58.

Goring, C. A. A., and Clark, F. E. 1948. Influence
of crop growth on mineralization of nitrogen in
the soil. Soil Sci. Soc. Am. Proc. 13:261-266.

Grossowicz, N., Hayat, P., and Halpern, Y. S. 1957.
Pyocyanine biosynthesis by Pseudomonas aeruginosa.
J. Gen. Microbiol. 16:576—583.

Guenzi, W. D., and McCalla, T. M. 1962. Inhibition of
germination and seedling development by crop residues.
Soil Sci. Soc. Am. Proc. 26:456—458.

Gyllenberg, H. G. 1957. Seasonal variation in the
composition of the bacterial soil flora in relation
to plant development. Can. J. Microbiol. 3:131-134.

Halvorson, H. O., and Ziegler, N. R. 1933. A means
of determining bacterial populations by the dilution
method. J. Bacteriol. 25:101—121.

Hart, L. I., Larson, A. D., and McCloskey, C. S. 1963.
Denitrification by Corynebacterium nephridii. J. Bact.
89:1104.

Hays, E. E., and Wells, I. C. 1945. Antibiotic
substances produced by Pseudomonas aeruginosa. J.
Biol. Chem. 159:725—750.

Hoagland, D. R., and Arnon, D. I. 1938. The water-
culture method for growing plants without soil.
Calif. Agr. Exp. Sta. Circ. 347.

Huber, D. M., Watron, R. D., and Steiner, G. W. 1965.
Crop residue, nitrogen and plant disease. Soil
Sci. 100:5.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

100

Ishizawa, S., Suzuki, T., and Sato, T. K. 1958.
Studies on microorganisms and their activities
in soil. Natl. Inst. Agr. Sci. Tokyo, Bull. 8
(Ser. B).

Ishizawa, S., and Toyoda, H. 1964. Microflora of
Japanese soil. Natl. Inst. Agr. Sci., Tokyo.
Bull. 14 (Ser. B).

Janson, S. L., and Clark, F. E. 1952. Losses of N
during decomposition of plant material in the

presence of inorganic nitrogen. Soil Sci. Soc.
Am. Proc. 16:334.

Jensen, H. L. 1930. Actinomyces in Danish soils.
Soil Sci. 30:59—77.

Kopper, P. H. 1947. An atypical strain of Pseudomonas
aeruginosa. J. Bact. 54:359-362.

 

Katznelson, H. 1946. The rhizosphere effect of mangels
on certain groups of soil microorganisms. Soil
Sci. 62:343-354.

Katznelson, H., Lochhead, A. G., and Timonin, M. I.
1948. Soil microorganisms and the rhizosphere.
Botan. Rev. 14:9.

Katznelson, H., and Richardson, L. T. 1943. The
microflora of the rhizosphere of tomato
plants in relation to soil sterilization. Can.
J. Res. 21(C):249-255.

Katznelson, H., Rouatt, J. W., and Payne, T. M. B.
1956. The liberation of amino acids and reducing
compounds by plant roots. Plant and Soil 7:35—47.

Kefauver, M., and Allison, F. E. 1957. Nitrite re-
duction by Bacterium denitrificans in relation to
oxidation—reduction potential and oxygen tension.
J. Bacteriol. 73:8—14.

 

Kelley, W. P. 1912. The assimilation of nitrogen by
rice. Hawaii Agr. Exp. Sta. Bull. 24.

Kluyver, H. J., and Verhoeven, W. 1954. Studies
on true dissimilatory nitrate reduction. IV. An
adaptation in Micrococcus denitrificans. Antonie
Van Leeuwenhoek J. Microbiol. Serol. 20:337-358.

63.

64.

65.

66.

67.

68.

69.

70.

71.

72.

73.

74.

75.

101

Kononova, M M. 1961. Soil organic matter. Pergamon
Press. 450 p.

Krassilnikov, N. A. 1934. Influence of root secretion
on the development of Azotobacter and other soil
microbes. Microbiol. U. S. S. R. 3:343-359.

Krassilnikov, N. AH, Kriss, A. E., and Litvinov, M..A.
1936. The effect of the root system on the soil
microflora. Microbiol. U. S. S. R. 5:270-286.

Krassilnikov, N. A. 1958. Soil microorganisms and
higher plants. Office of Technical Services,
U. S. Dept. of Commerce, Washington, D.C.

Krassilnikov, N. A. 1960. The biological role of
microbes: Antagonistic producers of antibiotic
substances. Soil and Plant Food. V01. 5, No. 4.

Knight, B. C., and Proom, H. 1950. A comparative
survey of the nutrition and physiology of mesophilic
species in the genus Bacillus. J. Gen. Microbiol.
4:508—538.

 

Langston, C. W., and Williams, P. P. 1962. Reduction
of nitrate by Streptococci. J. Bacteriol. 84:603.

Lee, Y. C., and Lin, J. G. 1964. Rice [in Chinese].
In Crops and Fertilizer, Taiwan Fertilizer Corporation.

Lenhoff, H. 1963. An inverse relationship in the
effects of oxygen and iron on the production of
fluorescin and cytochrome by Pseudomonas fluorescens.
Nature 199:601-602.

Linford, M. B. 1942. Methods of observing soil
flora and fauna associated with roots. Soil Sci.
53:93-103.

Liu, P. V. 1964. Factors that influence toxigenicity
of Pseudomonas aeruginosa. J. Bacteriol. 88:1421—1427.

Livingston, B. E. 1923. Physiological aspects of
toxicity. J. Am. Soc. Agron. 15:313-323.

Livingston, B. E. 1907. Further studies on the properties
of unproductive soils. U.S. Dept. of Agr., Bur.
of Soils. Bull. 36.

76.

77.

78.

79.

80.

81.

82.

83.

84.

85.

86.

102

Lochhead, A. G., Timonin, M. I., and West, P. M.
1939. The microflora of the rhizosphere in relation
to resistance of plants to soil borne pathogens.
Sci. Agr. 20:414-418.

Lochhead, A. G., and West, P. M. 1940. The nutritional
requirements of soil bacteria--a basis for deter-
mining the bacterial equilibrium of soils. Soil
Sci. 50:409—420.

Lochhead, A. G., and Chase, F. E. 1943. Qualitative
studies of soil microorganisms. V. Nutritional
requirements of the predominant bacterial flora.
Soil Sci. 55:188—195.

Lochhead, A. G. 1946. Qualitative studies of soil
microorganisms. III. Influence of plant growth
on the character of the bacterial flora. Can. J.
Research 18(C):42—53.

Lochhead,A.G.,and Thexton, R. H. 1947. Qualitative
studies of soil microorganisms. VII. The
"rhizosphere effect" in relation to the amino acid
nutrition of bacteria. Can. J. Res. 25(C):20-26.

Loehwing, W. F. 1937. Root interactions of plants.
Botan. Rev. 3:195-239.

Lockett, J. L. 1937. Microbiological aspects of
decomposition of clover and rye plants at different
growth stages. Soil Sci. 44:425—439.

Loewenstein, H. 1957. Nitrogen loss in gaseous form
from soils as influenced by fertilizer and management.
Soil Sci. Soc. Am. Proc. 21:397—400.

Louw, H. A., and Webley, D. M. 1959. The bacteriology
of the root region of the oat plant grown under
controlled pot culture conditions. J. Appl.
Bacteriol. 22:216—226. I

Maloy, O.C., and Alexander, M. 1958. The most
probable number method for estimating populations
of plant pathogenic organisms in the soil.
Phytopathol. 48:126—128.

Martin, J. P., and Ervin, J. O. 1958. Greenhouse
studies on influence of other crops and of organic
materials on growth of orange seedlings in old
citrus soil. Soil Sci. 85:141.

103

87. MbCalla, T., and Haskins, F. A. 1964. Phytotoxic
substances from soil microorganisms and crop
residues. Bacteriol. Rev. 28:181-207.

88. MCElroy, W. D., and Glass, B. 1956. A symposium on
inorganic nitrogen metabolism. John Hopkins Press,
Baltimore. p. 233-259.

89. MCFadden, B. A., and Howes, W. V. 1961. Pseudomonas
indigofera. J. Bacteriol. 81:858-862.

 

90. MCGarity, J. W., Gilmour, C. M., and Bollen, W. B.
1958. Use of an electrolytic respirometer to study
denitrification in soil. Can J. Microbiol. 4:303-316.

91. .Meiklejohn, J. 1940. Aerobic denitrification. Ann.
Appl. Biol. 27:558—573.

92. .Mitsui, S., Aso, S., Kumarawa, K., Ishizawa, T. 1954.
The nutrient uptake of rice plants influenced by
hydrogen sulfide and butyric acid abundantly evolv-
ing under water-logged soil conditions. Trans.
Intern. Congr. Soil Sci., 5th., Leopoldville.
2:364—368.

93. Mbrihara, K. 1965. Production of proteinase on_non-
carbthdrate carbon sources by Pseudomonas aeruginosa.
Appl. Microbiol. B(2):793—797.

94. Mbtomura, S. 1962. Effect of organic matter on the
formation of ferrous iron in soils. Natl. Inst.
Agr. Sci. Tokyo. Soil Science and Plant Nutrition
Vol. 8, No. l.

95. MYers, H. E., and Anderson, K. L. 1954. Bromegrass
toxicity vs. nitrogen starvation. J. Am. Soc. Agron.
34:770.

96. Najjar, V. A., and Allen, M. B. 1954. Formation of
nitrogen, nitrous oxide and nitric oxide by extracts
of denitrifying bacteria. J. Biol. Chem. 206-214.

97. Nance, J., and Cunningham, L. W. 1951. Evolution of
acetaldehyde by excised wheat roots in solutions
of nitrate and nitrite salts. Am. J. Botany
38:604-609.

98- .Nielsen,.K. F., CUddy, T. F., and Woods, W. B. 1960.
The influence of the extract of some crops and soil
residues on germination and growth. Can. J. Plant
Sci. 40:188-197. 7

104

99. Nommik, H. 1956. Investigations on denitrification in
soil. Acta. Agr. Scand. 6:195-228.

100. Okajima, H., and Takagi, S. 1955. Physiological
behaviour of hydrogen sulphide in the rice plant.
II. Effects of HIS on the content of nutrients in

the rice plant. nst. Agr. Res. Tohoku Univ. Rep.
No. 6:89-99.

101. Page, J., and Bodman, G. B. 1953. The effect of soil
physical properties on availability of nutrients.

In Mineral nutrition of plants. The Univ. of Wisc.
Press.

102. Patrick, Z. A., and Koch, L. W. 1958. Inhibition of
respiration, germination and growth by substances
arising during the decomposition of certain plant
residues in the soil. Can. J. Botany 36:621-647.

103. PeCk, H. D. 1961. Symposium on metabolism of inorganic
compounds. V. Comparative metabolism of inorganic
sulfur compounds in microorganisms. Annual meeting

of the Amer. Soc. for Microbiology in Chicago, on
April 26: 1961.

104. Piper, C. S. 1950. Soil and Plant Analysis.

Univ.
of Adelaide, Adelaide, Australia.
105. Pearsall, W. A., and Marlimer, C. H. 1939. Oxidation
reduction potentials in water-logged soils, natural

water and muds. J. Ecol. 27:483-501.

106. Ponnamperuma, F. N., Bradfield, B., and Peech, M.
1955. Physiological disease of rice attributable
to iron toxicity. Nature 175:265.

107. Rhodes,.M., Best, A., and Payne, W. J. 1962. Electron

donors and factors for denitrification by Pseudomonas
,perfectomarinus. Can. J. Microbiol. 9:799-807.

 

108. Robinson, W. O. 1930. Some chemical phases of the
submerged soil condition. Soil Sci. 30:197.

109. Rouatt, J. W. and Atkinson, R. G. 1950. The effect
of the incorporation of certain cover crops on the
microbiological balance of potato scab infested
soil. can. J. Res. 28(C):140-152.

110. Rouatt, J} W;, and.Katznelson, H. 1961. A study of
the bacteria on the root surface and in the rhizosphere
soil of crop plants. J} Appl. Bacteriol. 24:164-171.

111.

112.

113.

114.

115.

116.

117.

118.

119.

120.

121.

122.

105

Rovira, A. D. 1953. Some quantitative and qualitative
aspects of the rhizosphere. Proc. Soil Sci. Conf.
Adelaide, June, 1953. -

Rovira, A. D. 1956. Plant root excretions in relation
to the rhiZOSphere effect. Plant and Soil 7:178-194.

Russell, E. J. and Russell, E. W. 1952. The soil
atmOSphere. £2 Soil conditions and plant growth.
8th Ed., Longmans, Green and Co., London, pp. 335—345.

Russell, E. W. 1961. Soil conditions and plant
growth. 9th Ed. Longmans, Green and Co., London.
688 p.

Russell, M. B. 1957. Soil aeration and plant growth.
In Soil physical conditions and plant growth.
Agronomy Monograph 2, Academic Press, New York.
pp. 253—301.

Sawada, Y., Nitta, K., and Igarashi, T. 1964. Injury
of young plants caused by the decomposition of green
manure. Soil Science and Plant Nutrition. Vol.

10, No. 4.

Schreiner, 0., and Reed, H. S. 1908. The toxic action
of certain organic plant constituents. Botan. Gaz.
45:73-102.

Schreiner, 0., and Shorey, E. C. 1909. The isolation
of harmful organic substances from soils. U.S.
Dept. of Agr., Bur. of Soils Bull. 53.

Schreiner, 0., and Shorey, E. C. 1911. Chemical
nature of soil organic matter. U.S. Dept. of Agr.
Bur. of Soils Bull. No. 74.

Schreiner, 0., and Lathrop, E. C. 1911. Examination
of soils for organic constituents, especially de-
hydroxystearic acid. U.S. Dept. of Agr. Bur. of
Soils Bull. No. 80. A

Skinner, F. A. 1960. The isolation of anaerdbic
cellulose decomposing bacteria from soil. J. Gen.
Microbiol. 22:539-554.

Smith, N. R., and Dawson. V. T. 1944. The
bacteriostatic action of rose bengal in media
used for plate counts of soil fungi. Soil Sci.
58:467-471.

123.

124.

125.

126.

127.

128.

129.

130.

131.

132.

133.

134.

106

Somers, I. 1., and Shive, J. W. 1942. The iron-manganese
relation in plant metabolism. Plant Physiol.
17:582-607.

Stanier, R. Y., Doudoroff, M., and Adelberg, E. A.
1963. The microbial world. 2nd Ed. Prentice-Hall,
Englewood Cliffs, N.J. 682 p.

Stansly, P. G., Shephard, R. G., and White H. J. 1947.
Polymyxin, a new chemotherapeutic agent. Johns
HDpkins Hosp. Bull. 81:43-55. '

Starkey, R. L. 1929. Some influence of the develop-
ment of higher plants upon the microorganisms in
the soil. I. Historical and introductory.

Soil Sci. 27:319-334. '

Starkey, R. L. 1931. Plant growth and microorganisms.
Soil Sci. 32:367-391.

Starkey, R. L. 1938. Some influences of the develop—
ment of higher plants upon the microorganisms in
the soil. VI. Microscopic examination of the
rhizosphere. Soil Sci. 45:207-249.

Starkey, R. L. 1958. Interrelations between micro-
organisms and plant roots in the rhiZOSphere.
Bacteriol. Rev. 22:154-172.

Suzuki, T., and Ishizawa, S. 1965. Soil micro-
organisms, their activities and soil fertility.
Natl. Inst. of Agr. Sci., Tokyo. Ser. B (Soil and
Fertilizers) No. 15:181-186.

Takahashi, J. 1960. Review of investigations into
physiological disease of rice. Intern. Rice
Commission Newsletter 9:1-6; 17-24.

Takahashi, J., and Chang, S. C. 1961. The physiological
disease of rice in Taiwan [in Chinese]. Newsletter
of Soils and Fertilizer, Taiwan, No. 125.

Takai, Y., and Asami, T. 1962. Formation of methyl
mercaptan in paddy soil. Natl. Inst. Agr. SCi.
Tkoyo. Soil Science and Plant NUtrition. V01. 8,

No. 3.
Takai, Y., Koyama, T., and Kamura, T. 1956.

Microbial metabolism in reduction process of paddy
soil. Part 1. Soil and Plant Food 2:63-66.

135.

136.

137.

138.

139.

140.

141.

142.

143.

107

Takai, Y. 1960. Essays of the 3rd. Congress of Isotope
Technology, Japan. p. 897.

Takijima, Y., Shiojima, M., and Konno, K. 1958.
Studies on soils of peaty paddy fields. I.
Geographical distribution, characteristics and
classification of soils in Miyagi prefecture.
Soil and Plant Food. Vol. 4, No. 2.

Takijima, Y., and Sakuma, H. 1961. Metabolism of
organic acids in soil of peaty paddy field and their
inhibitory effects on the growth of the rice plant.
Part II. Production of organic acids in water-
logged soil treated with green manure (Chinese milk
vetch) and its relation to growth inhibition of
rice seedlings. Natl. Inst. Agr. Sci. Tokyo.

Ser. B (Soil and Fertilizers) N0. 11:560-564.

Takijima, Y. 1963. Studies on behaviour of the
growth inhibiting substances in paddy soil with
special reference to the occurrence of root damage
in the peaty paddy fields. Natl. Inst. of Agr.
Sci., Tokyo. Ser. B (Soils and Fertilizers) No. 13:
118-245.

Takijima, Y. 1964. Studies on organic acids in paddy
field soil with reference to their inhibitory effects
on the growth of rice plants. Part I. Growth
inhibiting action of organic acids and absorption
and decomposition of them by soils. Soil Sci.
and Plant Nutrition. V01. 10, No. 5.

Takijima, Y. and Shiojima, M. 1958. Studies on soils
of peaty paddy fields. II. Soil characteristics
and metabolism under flooded conditions. Soil
and Plant Food, Vol. 4, No. 3.

Takijima, Y. and Shiojima, M. 1951. The influence
of plants on the mineralization of nitrogen and the
maintenance of organic matter in the soil. J.

Agr. Sci. 41:289-296.

Theron, J. J., and Haylett, D. G. 1953. The
regeneration of soil humus under a grass ley.

Emp. J. Exptl. Agr. 21:86-98.

Timonin, M. I. 1940. The interaction of higher plants
and soil microorganisms. I. Microbial populations
of rhizospheres of seedlings of certain cultivated
plants. Can. J. Res. 18(C):307-317.

144.

145.

146.

147.

148.

149.

150.

151.

152.

153.

154.

155.

156.

108

Timonin, M. I. 1940. The interaction of higher
plants and soil microorganisms. II. Study of the
microbial population of the rhizosphere in relation
to resistances of plants to soil borne disease.
Can. J. Res. 18(C):444-456.

Timonin, M. I. 1946. Microflora of the rhizosphere
in relation to the manganese deficiency disease
of oats. Soil Sci. Soc. Am. Proc. 11:284-292.

Totter, J. R., and Moseley, F. T. 1953. Influence
of the concentration of iron on the production of
fluorescin by Pseudomonas aeruginosa. J. Bacteriol.
65:45-47.

 

Tulecke, W. 1965. Isolation of an organism resembling
Achromobacter liguefaciens. J. Bact. 89:905-906.

 

Twyman, E. S. 1963. The iron manganese balance and
its effect on the growth and development of plants.
New Phytol. 45:92.

Valera, C. L., and Alexander, M. 1961. Nutrition
and physiology of denitrifying bacteria. Plant
and Soil 15:268—280.

Vamos, R. 1956. The role of excess nitrogen in the
"brusone‘l disease of rice. Acta. Biol. Szeged. 2:103—110.

Vamos, R. 1958. H S the cause of the brusone (akiochi)
disease of rice. Soil and Plant Food. V01. 4, No. 1:
37-40.

Vamos, R. 1959. "Brusone" disease of rice in Hungary.
Plant and Soil 11:65-67.

Vandecaveye, S. C., and Katznelson, H. 1938. Microbial
activities in soil. Soil Sci. 46:139-167.

Vlamis, J., and Davis, A. R. 1944. Effects of
oxygen tension on certain physiological responses
of rice, barley and tomato. Plant physiol.
19:33-51.

Voorhees, E. B. 1902. Studies in denitrification.
J. Am. Chem. Soc. 24:785-823.

Wagner, G. H., and Smith, G. E. 1957. Nitrogen
losses from soils fertilized with different nitrogen
carriers. Soil Sci. 85:125-129.

157.

158.

159.

160.

161.

162.

163.

164.

165.

166.

167.

168.

169.

109

Wahba, A. H. 1965. Pyrorubrin-producing Pseudomonas
aeruginosa. Appl. Microbiol. 13:291-292.

 

Waksman, S. A. 1952. Soil Microbiology. Wiley,
New Y0rk. pp. 183-189.

Wallace, T. H., and Lochhead, A. G. 1950. Qualitative
studies of soil microorganisms. IX. Amino acid
requirements of rhizosphere bacteria. Can. J.

Res. 28(C):1-6.

Wang, S. C., and Chuang, T. T. 1966. Soil alcohols,
their dynamics and their effects upon plant growth.
Soil Sci. (unpublished).

Wang, S. C. 1966. Dynamics of soil organic acids
(unpublished).

Wang, C. M., and Chang, S. C. 1964. Review of
studies on the physiological disease of rice in
Taiwan. Joint Commission on Rural Reconstruction.
PID-C-281, February, 1964.

Wasserman, A. E. 1965. Absorption and fluorescence
of water soluble pigments produced by four Species
of Pseudomonas. Appl. Microbiol. 13:175-180.

 

West, P. M. 1939. Excretion of thiamin and biotin by
the roots of higher plants. Nature 144:1050.

West, P M., Lochhead, A. G. 1940. Qualitative studies
of soil microorganisms. IV. The rhiZOSphere in
relation to the nutritive requirements of soil'
bacteria. Can. J. Res. 18(C):129-135.

Wheeler, B. E. J. 1963. The conversion of amino acids
in soils. II. Denitrification in percolated soil.
Plant and Soil 19:219-232.

Wijler, J., and Delwiche, C. C. 1954. Investigations
of the denitrifying process in soil. Plant and
Soil 5:155-169.

Williamson, R. E. 1964. The effect of root aeration
on growth. Soil Sci. Soc. Am. Proc. 28:86-90.

Woldendorp, J. W. 1962. The quantitative influence
of the rhizosphere on denitrification. Plant
and Soil 17:267-270.

170.

171.

172.

173.

174.

175.

176.

110

Woldendorp, J. W. 1963. The influence of living plants
on denitrification. Medeelingen van de Landbouwhoge-
schoolt12Wageningen, Nederland. 63(13):1-100.

Yamane, I. 1957. Nitrate reduction and denitrification
in flooded soils. Soil and Plant Food V01. 3:100-103.

Yamane, I. 1958. Metabolism in muck paddy soil.
II. Determination of gases evolved from paddy
field--estimation of decomposable organic matter.
Soil and Plant Food, V01. 4, No. 1.

Yanagiswawa, M., and Takahashi, J. 1964. Studies on
the factors related to the productivity of paddy
soil in Japan with special reference to the nutrition
of the rice plant. Bull. of the Natl. Inst. of
Agr. Sci. Japan. Ser. B (Soil and Fertilizers),
No. 14.

YOShida, T., and Sakai, H. 1963. Soil moisture
condition and organic matter decomposition-—micro-
biological studies on the soils of H0kkaido.
Hokkaido Natl. Agr. Exp. Sta. Present address,
Chugoku Natl. Agr. Exp. Sta. Bull. 6:197-202.

Yoshida, S. 1965. Chemical aspects of the role of
silicon in physiology of the rice plant. Bull.
Natl. Inst. Agr. Sci. Japan. Ser. B (Soil and
Fertilizer) No. 15:1-56.

Young, G. 1947. Pigment production and antibiotic
activity in cultures of Pseudonomas aeruginosa.
J. Bact. 54:109-117.

APPENDIX

Table 22. Sample codes used in Tables 23 through 27.

Soil sample source:

C = Chungli (field location)

Lotung (greenhouse experiments)

Lotung (field operation)

Pingtung (field operation)

Hot-‘0
II

Taichung (field operation)
Subscript g = green manure added in greenhouse
Subscript st = straw added in greenhouse

Subscript s = seedling before or at time of transplanting

Plant condition:

H

healthy

D

disease
Sampling zone:
R = root
Rh = rhizosphere soil
S = edaphOSphere soil
Examples:
TRs = Taichung soil, seedling root

DStR = Lotung soil, root count from diseased plant in
straw-amended pot

HRh = Lotung soil, rhizosphere count from healthy plants

112

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