INTERRELATIONSHIPS BETWEEN MICROORGANISMS AND PLANTS IN SOIL doc

Developments
in
Soil Science
18
INTERRELATIONSHIPS BETWEEN
MICROORGANISMS AND PLANTS IN SOIL
Further Titles in this Series
1.
1.
VALETON
BAUXITES
2.
IAHR
FUNDAMENTALS OF TRANSPORT PHENOMENA
IN
POROUS hlEDI.4
3.
F.
E.
ALLISON
SOIL ORGANIC MATTER AND ITS ROLE
IN
CROP PRODUCTION
4.
R.
W.
SIMONSON
I
Editori
NON-AGRICULTURAL APPLICATIONS OF SOIL SURVEYS

5A.
G.
H. BOLT
cirict
M.
C.
M. BRL'GGENM%RT
/
Edirorsi
SOIL CHEMISTRY.
A.
BASIC ELEMENTS
5B.
G.
H. BOLT (Editor)
SOIL
CHEMISTRY.
B.
PHYSICO-CHEMICAL MODELS
6.
H.
E.
DREGNE
SOIL OF ARID REGIONS
7.
H. AUBERT
mil
M. PINTA
TRACE ELEMENTS
IN

SOIL
8.
M. SCHNITZER
unit
S.
U. KHAN (Editors)
SOIL ORGANIC MATTER
Y.
B.
K.
C.
THENG
FORMATION AND PROPERTIES OF CLAY-POLYMER COMPLEXES
10.
D. ZACHAR
SOIL EROSION
IIA. L.
P.
WILDING, N.
E.
SMECK
tirid
G.
F.
HALL (Editors/
PEDOGENESIS AND SOIL TAXONOMY.
I.
CONCEPTS AND INTERACTIONS
II.
L.

P.
WILDING. N.
E.
SMECKriritlG.
F.
HALL (Glitors)
PEDOGENESIS AND SOIL TAXONOMY.
11.
THE SOIL ORDERS
I?.
E.
B. A. BISDOM
iind
J.
DUCLOUX
f
Editor.?)
SUBMICROSCOPIC STUDIES OF SOILS
13.
P. KOOREVAAR.
G.
MENELIK
iiritl
C.
DIRKSEN
ELEMENTS OF SOIL PHYSICS
14.
G.
S.
CAMPBELL

SOIL PHYSICS WITH BASIC -TRANSPORT MODELS FOR SOIL-PLANT SYSTEMS
16.
G.
G.
C.
CLARIDGE
(itid
I.
B. CAMPBELL
ANTARCTICA; SOILS. WEATHERING PROCESSES AND ENVIRONMENT
15.
M. A. MULDERS
REMOTE SENSING
IN
SOIL SCIENCE
17.
K. KUMADA
CHEMISTRY OF SOIL ORGANIC MATTER
IN.
L'ANCL'RA
cirid
F.
KLJNC
I
Ectirors)
INTERRELATIONSHIPS BETWEEN MICROORGANISMS AND PLANTS
IN
SOIL
Developments
in

Soil Science
18
Interrelationships
between Microorganisms
and Plants in Soil
Proceedings
of
an International Symposium
Liblice. Czechoslovakia June
22-27.
1987
Edited by
Vlastimil Vaneura
Frantigek Kunc
Institute
of
Microbiology
of
the Czechoslovak Academy
of
Sciences,
Prague, Czechoslovakia
Organized by the Czechoslovak Society
for
Microbiology.
Czechoslovak Academy
of
Sciences, Prague
ELSEVIER
- Amsterdam - Oxford

-
New York
-
Tokyo
1989
Scientific Editor
PhMr. Vlastimil Vanfura, DrSc.
Scientific Adviser
RNDr. Josef Rusek, CSc.
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Interrelationships between microorganisms and plants in
soil.
(Developments in
soil
science
;
18)
Bibliography: p.
Includes index.
1.
Soil microbiology-Cong.
2.
Plants-Microbiology-Congresses.
3.
Rhizospherc-Congtrsscs.
4.
Phytopathogenic microorganisms-Congresses.
I.

Vanhra, Vlastimil,
1927-
111.
thkoslovensk6 spoldnost mikmbiologic~ pfi
hV.
IV.
Series.
QR111.IS7 1988 576’.5’09148 88447
ISBN
0-444-989226
(Vol.
18)
ISBN
0-44440882-7
(Series).
.
11.
Kunc, Frantikk,
1935-
0
Vlastimil Vanfura. FrantiSek Kunc
1989
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Preface
CONTENTS
1

Introductory lecture
LYNCH J.M.: Development and interaction between microbial
communities on the root surface
5
I. SYMBIOTIC MICROORGANISMS AND PLANTS
a. R h i
z
o
b i a
13
MISHUSTIN E.N., BONARTSEVA, G.A., MYSHKINA V.L.: Criteria of
root-nodule bacteria activity 15
ANTIPCHUK A.F., KANTSELYARUK R.M., RANGELOVA V.N., SKOCHINSKA-
YA N.N., TANTSYURENKO E.V.: New additional criteria for
estimation of grain legume-rhizobial symbiosis effectivity 23
culturasof root-nodule bacteria 29
and environmental factors on the efficiency of the
BONARTSEVA G.A., MYSHKINA V.L.: Nitrogen-fixation in pure
VOLKOVA T.N., CHERNOVA N.I.: Effects of rhizobia, host plant
legume-Rhizobium symbiosis 37
SIMAROV B.V., NOVIKOVA N.N., SHARYPOVA L.A., PROVOROV N.A.,
ARONSHTAM A.A., KUCHKO V.V.: Molecular-genetic basis for
Rhizobium selection
45
NASINEC V., CHLOUPEK
0.:
Preliminary results of a breeding
program for improving symbiotic nitrogen fixation in
alfalfa
51

COOPER J.E.: Nodulation
of
legumes by rhizobia in acid soils 57
NOVhK
K.:
Induction of phytoalexin in pea roots by rhizobia 63
b. Mycorrhizal funqi 67
as determinants for plant growth and survival 69
of ectomycorrhizal symbiosis in forestry 71
GIANINAZZI-PEARSON V.: Vesicular-arbuscular endomycorrhizae
CUDLIN P., MEJSTRfK V.: Ecological prospects of utilization
SCHONBECK
F.,
DEHNE
H W.:
VA-mycorrhiza and plant health 83
endomycorrhizal fungi 93
GIANINAZZI
S.:
Cellular relationships between plants and VA
SUNG
S.S.,
XU D.P., MUSTARDY L., KORMANIK P.P., BLACK C.C.:
Pyrophosphate dependent sugar metabolism in mycorrhizal
tree roots 99
HRSELOVh H., VEJSADOVh H., PRIKRYL
Z.,
VhCHOVh J., VANCURA
V.,
VfT A.: Effect of inoculation with vesicular-arbuscular

mycorrhizal fungi on growth of strawberries 109
VEJSADOVh H., HRsELOVh H., PRIKRYL
Z.,
VANEURA V.: Inter-
relationships between vesicular-arbuscular mycorrhizal
fungi, Rradyrhizobium japonicum and soybean plants
SCHENCK N.C., SIQUEIRA J.O., OLIVEIRA E.: Changes in the
incidence of VA mycorrhizal fungi with changes in eco-
systems
phosphorus fertility
of
arable soils and VAM infection
in different crop plants
OTTO G.: The appearance of endotrophic mycorrhiza in apple
seedlings from soils previously cropped with fruit trees
FERRER R.L., PRIKRYL
Z.,
GRYNDLER M., VANEURA V.: Natural
occurrence of vesicular-arhuscular fungi in grape vine
and apple trees
on sites disturbed by
SO,
emmissions and on strip-mine
spoil banks in Northern Bohemia
KORMANIK P.P.: Importance of first-order lateral roots
in the early development of forest tree seedlings
PEREZ Y., SCHENCK N.C.: The international culture collection
of VA mycorrhizal fungi (INVAM)
KROPhfEK
K.,

CUDLfN P.: Preparation of granulated mycor-
rhizal inoculum and its use in forest nurseries
11. ASSOCIATIVE MICROORGANISMS OF THE ROOT SYSTEM
VANEURA V.: Inoculation of plants with Pseudomonas putida
BURNS R.G., ALSTRbM
S.,
BURTON C.C., DARTNALL A.M.: Cyano-
genic microbes and phosphatase enzymes in the rhizo-
sphere: properties and prospects for manipulation
SOBIESZCZAfiSKI J., STEMPNIEWICZ R., KRZYSKO
T.:
Pseudo-
monas
sp.
as producer of plant growth regulators
CHRISTENSEN H.: Specific growth rate determination of rhizo-
sphere bacteria: evaluation of root-colonizing ability
based on the tritiated-thymidine method
STRZELCZYK E., POKOJSKA A., KAMPERT M., MICHALSKI L.,
KOWALSKI
S.:
Production of plant growth regulators by
non-mycorrhizal fungi associated with the roots
of forest trees
of bacterial population in the rhizosphere of inoculated
plants
DbBEREINER J.: Recent advances in associations of diazotrophs
with plant roots
HbFLICH G.: The use of rhizosphere microorganisms for sti-
mulating N, fixation and plant growth

RUPPEL
S.:
Isolation and characterization
of
dinitrogen-
-fixing bacteria from the rhizosphere of Triticum
aestivum and Ammophila arenaria
REDKINA T.V., MISHUSTIN E.N.: Nitrogen-fixing microorganisms
of the genus Azospirillum and their relations with higher
plants
tion of nitrogen fixed by soil diazotrophs by rice plants
LIPPMA” G., KEGLER G., WITTER
R.:
Relationship between
VOShTKA M.: VA mycorrhiza in stands of two hardwood species
PEREBITYUK A.N., PUCHKO V.N.: Survival and distribution
KALININSKAYA T.A., KRAVCHENKO I.K., MILLER Y.M.: Assimila-
115
125
131
137
141
149
157
171
177
183
185
191
20 1

207
213
223
229
243
253
263
269
KOZHEMYAKOV A.P.: Quantitative estimation of nitrogen
fixation by barley associative bacteria using tracer
technique
KRAVCHENKO L.V., MAKAROVA N.M.: Use of root exometabolites
by associative nitrogen-fixing microorganisms
KALININSKAYA T.A.: The influence of different forms of
combined nitrogen on nitrogen-fixing activity of azo-
spirilla in the rhizosphere of rice plants
JANDERA A., HANZLfKOVA A,, SOTOLOVA I.: Ecological function
of enzymes in the rhizosphere
HANZLfKOVA A., gOTOLOVh I., JANDERA A.: Chitinase in the
rhizosphere and on plant roots
SOTOLOVh I., JANDERA A., HANZLfKOVh A.: fl-1,3-glucanase
in the rhizosphere and on plant roots
TESAROVA
M., SIMEK
M.:
Rhizosphere microflora of managed
grasslands
LASrK
J., VANfURA V., HANZLrKOVh A., WURST
M.:

Polysaccha-
ANDREYUK E.I., IUTINSKAYA G.A.: Soil microorganisms
and transformation of bacterial polysaccharides in soil
KUNC F.
,
RYBAROVA
J.
:
Degradation of d4C-2
,
4-dichloro-
phenoxyacetic acid in artificial rhizosphere soil
SAT0
K.:
Effect of the herbicide, Benthiocarb (Thiobencarb)
on seasonal changes in microbial populations in paddy
soil and yield qf rice plants
pollution on the microorganisms from barley and field
pea rhizosphere
rhizosphere of rice and flooded soil in rice fields
ride compounds in the rhizosphere
BALICKA N., TEICHERT E., WEGRZYN T.: Effect of industrial
SIDORENKO O.D.: Role of sulphate-reducing bacteria in
111.
SOIL-BORNE PHYTOPATHOGENIC MICROORGANISMS
BOCHOW H.: Possibilities of protecting plant roots
against phytopathogens by biological means (biological
contro
1)
RUTHERFORD E., EPTON H.A.S., BENTON R.A.: Improvement

of propagation by use of fungicides
fATSKh V., SMRZ
J.:
Relationships between soil mites and
microorganisms in apple seedling rhizosphere
VESELP D.: The effectiveness in vitro of Pythium oligandrum
Drechsler mycoparasite against Phoma exigua Desm. var.
foveata inciting the gangrene in potato tubers
STEINBRENNER
K.:
Detrimental effects of Gaeumannomyces
graminis
THINGGAARD
K.:
Biological control
of
root pathogenic
DUSKOVA E., PROKINOVA E.: Interaction between growing sub-
PIETR
S.J.,
KEMPA R.: Cucumber rhizosphere pseudomonads as
NOVhK
K.,
STANEK M.: Production of phytoalexin in pea roots
fungi by Trichoderma
strate composition and Fusarium wilt of carnation
antagonists of Fusarium
273
277
283

287
293
30
1
307
315
323
329
335
343
349
355
357
371
377
383
389
395
403
411
419
IV. BIOLOGICAL PREPARATIONS STIMULATING GROWTH
AND IMPROVING HEALTH CONDITIONS OF PLANTS
HANCOCK
J.G., WEINHOLD A.R., VanGUNDY S.D., SCHROTH M.N.:
KHOTYANOVICH A.V.: Microbial formulations used in plant
VESELP D.: Biological control of damping-off pathogens by
Introduced microbes enhance root health and plant growth
production in the USSR
treating sugar-beet seed with a powdery preparation of the

mycoparasite Pythium oligandrum in large-scale field
trials
tion with the use of Bactoleg preparation under different
ecological conditions
DOMEY
S.,
LIPPMA" G.: Stimulation of plant growth
by phosphate solubilizing bacteria
CATSKh V.: Biological control of phytotoxic and phyto-
pathogenic microorganisms in the plant rhizosphere
VRANP J., DOBIAS
K.,
FIKER A.: Yields
of
potatoes and their
contamination
by
fusaria after inoculation with bacteria
and fungi in field experiments
on yield and stress resistance of crops
BAKONDI-ZmORY
E.,
KOVES-PECHY K.,
Sods
T., SZEGI J.: N-fixa-
BERGMA" H.: Effect of natural amines and lipid components
INDEX
OF
ORGANISMS
SUBJECT INDEX

425
427
439
445
451
457
463
469
415
481
487
PREFACE
On June
22-27,
1987, a stimulating gathering of rhizosphere micro-
biologists organized by the Czechoslovak Society for Microbiology took
place at the Liblice chateau near Prague (Czechoslovakia). Specialists
from 15 countries met to assess the advances in a field which has re-
cently attracted considerable interest and which is also important for
society at large. The study of the function of microorganisms in the
root system of crop plants and in'its immediate vicinity, the effect
of the plants themselves on this function, the interrelationships
among different microorganisms in the rhizosphere, the elucidation
of
the mechanisms of microbial action in the agroecosystom
-
all these
lines of research
are
intimately associated with the problems

of
soil
fertility and crop yields. These in turn have a direct hearing on the
nutrition of mankind, mobilization of natural resources, etc., and on
environmental protection and formation.
at the symposium; most of them are included in this volume. The pro-
blems to be studied have been divided into four topics. The first
deals with symbiotic microorganisms (rhizobia, mycorrhizal fungi), the
second focuses on associative microorganisms of the root system, the
third on soil-borne phytopathogenic and phytotoxic microorganisms,
while the fourth touches on preparations to stimulate growth and im-
prove plant health. The individual sections were headed
by
invited
lectures by outstanding specialists.
The creative atmosphere of the meeting was such as to deserve
a lengthier treatment than is possible here; let it be said in this
limited space only that the meeting documented the most up-to-date
knowledge of the interrelationships between microorganisms and plants
in the rhizosphere and the possibilities of utilizing these relation-
ships for improving plant growth, health and yields. The application
of biological preparations may in the. future partially replace the
use of agrochemicals and thus contribute to environmental improvement
and enhancement of the quality of soil, water and foodstuffs.
of the Department of Microbial Ecology and Department of Experimental
Mycology of the Institute of Microbiology, Czechoslovak Academy of
Sciences, who helped with the organization of the symposium. Our thanks
are
also due to our colleagues who kindly corrected the English text
in communications by non-English participants. Among these colleagues

were J.E.
Cooper,
R.G. Burns,
V.
Gianinazzi-Pearson, J.G. Hancock
and
J.M.
Lynch.
More than a hundred oral and poster communications were presented
It is our pleasant duty
to
express wsincere thanks to the staff
-1-
We hope that the book will provide interesting readinq and valu-
able information not only for rhizosphere microbiologists, but also
€or plant physiologists and pathologists, soil scientists, microbio-
logists, agronomists and scientists interested in environmental
protection.
V.
VanEura
F.
Kunc
-2-
INTRODUCTORY LECTURE
This Page Intentionally Left Blank
DEVELOPMENT AND INTERACTION BETWEEN MICROBIAL COMMUNITIES ON THE ROOT
SURFACE
Lynch M.J.
Microbiology Department,
AFRC Institute of Horticultural Research,

Littlehampton, West Sussex, BN17 6LP, UK
-ABSTRACT
Microbial biomass formation on root surfaces can be measured in
plants growing in solution with or without an inert solid support. Car-
bon flow to the biomass can be measured by growing plants in solution
or
soil on a continuous source of 14C02 and the expected biomass for-
mation predicted. The lack of correlation between measured and predic-
ted biomass can be explained by oligotrophic growth of the micro-orga-
nisms. Microbial species within communities on the root surface can
interact with each other, and a target for root inoculation is to
elev%te the PFOllation of beneficial organisms within the community.
RHIZOSPHERE ANATOMY
The rh.izosphere is today regarded as the zone of microbial proli-
feration in and around roots. A variety of light and electron micro-
scopic techniques have been used to observe bacteria and fungi around
roots (the ectorhizosphere), on the root surface (the rhizoplane) and
within the root (the endorhizosphere) (Lynch, 1982). Bacteria develop
as discrete colonies on the root surface, leaving large areas of the
root surface uncolonized (probably greater than 80
%).
There tends to
be
a
proliferation of colonization at the junctions between the inter-
cellular spaces of the epidermis. Bacterial colonization of the cortex
has also been found but it has not been reliably estimated.
The major interest in fungi of the rhizosphere has been focussed
on mycorrhizas. In some tree species ectomycorrhizas are formed
so

ex-
tensively that they can be separated from the roots and weighed. Endo-
mycorrhizal colonization however is usually assessed by staining with
lactophenol and trypan blue and examining the roots microscopically.
However root colonization following fungal inoculation of the rhizo-
sphere with nonsymbiotic fungi has not usually been assessed. Rather
the success of inoculated Organisms has been determined by measuring
-5-
the number of C0lOW-f~ propagules of the inoculant. This does not
necessarily relate to the fungal biomass present.
CARBON FLOW
TO
THE RHIZOSPHERE
Growth of micro-organisms in the rhizosphere is dependent on root
derived carbon which includes exudates (leaked from living roots),
secretions (actively pumped from the roots), lysates (passively rele-
ased from the roots during autolysis) and mucilage (giving rise to
mucigel which is of both plant and microbial origin). The
C:N
ratio
of these materials has not been measured with precision but it has been
estimated to be around 40:l (Lynch, 1986). Plants can be grown on a
source of uniformly-labelled 14C02 to assess the flow of carbon from
roots to the microbial population; this flow can account for up
to
40
%
of the plant photosynthate produced (Whipps and Lynch, 1985). It
is unclear however if carbon or nitrogen are the growth-limiting sub-
strates to specific components of the rhizosphere population. It can

be expected that the
C:N
ratio of bacterial cells will usually be
between 5:l and 1O:l (Barber and Lynch, 1977) but it
is
not known what
proportion of the total
N
available to roots is intercepted by rhizo-
sphere micro-organisms. The contribution of substrates exogenous to the
rhizosphere to the nutrition of rhizosphere organisms is also unclear.
For example, fungi may colonize plant residues and continue to use them
as substrates while hyphae spread to colonize roots. In natural systems
there may therefore be a two-way flow of carbon into the rhizosphere.
This consideration could be crucial in attempts deliberately to colo-
nize the rhizosphere by beneficial organisms, viz it may be necessary
to introduce the organism on a substrate which will give it a competi-
tive advantage over other organisms which are only present as slow-
-growing
or
dormant propagules.
RHIZOSPHERE NUTRITION
Table 1 defines some terms which have traditionally been used to
describe the nutrition of aoil organisms, and compares them with modern
ecological terminology.There is a similarity in meaning between auto-
chthonous and oligotrophic, and zymogenous and copiotrophic,
but
the
terms certainly do not equate. Traditionally rhizosphere organiams have
been regarded as zymogenous, and this would imply that they will disap-

pear from the rhizosphere when the substrate supply becomes exhausted.
In practice the truly successful rhizosphere inoculant would be ex-
pected to exhibit copiotrophic growth on the substrate base on which
-6-
Table 1 Nutrition of soil organisms
WINOGRADSKY
(1924)
Autochthonous
-
low but steady Zymogenous
-
rapid metabolism
level of activity on native soil of soil organic matter
MODERN ECOLOGICAL TERMINOLOGY
Oligotrophic
-
scavenging of Copiotrophic
-
growth on co-
scarcesupply of nutrients such pious nutrients such as fresh
as trace carbon compourds in
the soil atmosphere
organic manures
it is introduced to soil, it would be zymogenous on the carbon products
available from the substrate base and from the root-derived carbon, it
would become oligotrophic when the supply
of
these substrates is re-
duced and then it would remain in the soil in the autochthonous mode
until fresh substrate became available again. For organisms which might

pose some risk to the environment this latter mode would not be a de-
sirable trait.
MEASURED AND CALCULATED MICROBIAL
BIOMASS
By measuring the flow of carbon to the rhizosphere using the
14C method and assuming a growth yield
of
0.35
g
of bianass
per
g of car-
bon (glucose) substrate consumed (ignoring any carbon used in maintenan-
ce of the population), the rhizosphere biomass can be calculated. Furt-
her by counting the number of cells associated with the root using
a washing technique and by determining the mean cell weight of the
members of the population by growing them in luxuriant media, the bio-
mass of organisms actually present on the root can be determined.
Table
2
indicates that the biomass observed is usually greater than
would be expected than that calculated from the carbon flow measuye-
ments. This must mean that cellp which colonize the roots shrink under
the (natural) conditions of substrate limitation compared with their
growth on luxuriant nutrients
or
that the organisms colonizing roots
grow as oligotrophs and acquire a proportion of their carbon for growth
by utilizing trace carbon compounds.
a

dominant Gram-negative rod from the roots (Enterobacter cloacae
M0/1)
and comparing its growth on gnotobiotic wheat roots with
E.
cloacae
C2/4 which had been isolated as a colonist of straw (Chapman and Lynch,
This concept was tested by growing wheat roots in soil, isolating
-7-
Table 2 Calculated and measured substrate inputs to the rhizoaphere
Barber and Whipps and Lynch
Lynch (1977) (1984)
Barley Barley Wheat
Microbial biomass
Mean cell weight, 10-l’
g
1.9 3.2 3.2
Bacterial biomass, 9.mg-l dry root 2.6 2.0 5.4
Substrate input
Calculated, gC.mg-’ dry root 37.0 28.0 78.0
Measured, 9C.rng-l dry root 3.1 7.5 7.9
1985). The two bacterial strains were of similar size but in a popula-
tion of each strain there was
a
very large variation in cell length
(1-4
,um)
and this was independent of the substrate availability (nu-
trient broth or root-derived carbon). To account for the inadequacy
of the budgets with a mixed bacterial population on roots described
in

Table
2,the cell size during rhizosphere growth would have to de-
crease by
3
and 10 times on average compared with growth on nutrient
broth. Therefore if all rhizosphere bacteria behaved like
E.
cloacae,
it seems most likely that there is substantial oligotrophic growth
in the rhizosphere. The trace amounts of carbon for this growth could
be introduced from forced aeration of roots in experimental systems.
The results also indicate the difficulty in analysing soil population
biology generally even though not all bacteria may vary in cell size
to the extent of
E.
cloacae.
fate of soil organisms and it is frequently assumed that biomass can
be calculated from counts of viable cells. This could only be valid
if the mean cell size or weight under natural soil conditions is known.
It is increasingly common to use antibiotic marking to trace the
COMMUNITY INTERACTIONS
When two organisms come together in vitro or on the root surface
they can potentially interact in several ways depending on their phy-
siological characteristics. However, those characteristics are often
dependent on the substrate base on which the organism grows. For
example a potential antagonist may produce an antibiotic on a nutrient-
-rich agar contained in a Petri dish but the root itself may not pro-
vide the necessary substrates. Then again, even if one plant species
provides the substrate, another may not. Thus caution must be exercised
in extrapolating in vitro laboratory screens of potential biocontrol

agents
to
the microbial interaction occurring in the field.
Chemical
(a.g.
pH) and physical (e.g. temperature) factors will likely govern
any interaction and such factors can be investigated in the laboratory.
Whereas screening procedures for indentifying microbial antagonism in
the laboratory must be as quick and simple as possible, they should
consider wherever possible field factors which could influence the in-
teraction.
Enterobacter cloacae is a bacterium which is a natural colonist
Of
the endorhizosphere (Kleeberger et al., 1983) and has proved
to
be ef-
fective in controlling damping-off diseases of pea and cucumber (Hadar
et al., 1983). In vitro studies showed that the sugar composition of
the growth medium determined the inhibitory effect of
E.
cloacae on
Pythium ultimum and that growth inhibition was linked
to
binding of the
bacteria
to
the hyphae, thus indicating that a lectin-type interaction
is probably involved (Nelson et al., 1986). This interaction may not
however be the exclusive mode of action in the biological control.
In addition

to
lectin interactions, the following have been pro-
posed as modes of action which could be involved in biological control:
competition for available substrates, production of antibiotics, pro-
duction of cell walldegrading enzymes, physical restriction of patho-
gens
to
reduce site occupancy, ionophore production by antagonists
to
impede ion uptake
by
the pathogen and cross protection or induced re-
sistance in the host. Many of the investigations of practical bio-
control systems have paid little attention
to
the mode of action but
rather have concentrated on isolating antagonists from the soil by
either in vitro or in vivo study and then evaluating their field
effectivness (Cook and Baker, 1983). The search for potential antago-
nists might prove more rewarding if the modes of action are considered.
There is increasing evidence for the range of actions possible (Lynch,
1987a) but the truly successful biocontrol agent is unlikely
to
act in
a single mode.
or more antagonist actions, and then introduce the others by genetic
engineering with recombinant DNA or protoplasting and using somoclonal
variation.
A
problem that could arise from this approach is that the

genetic modification could reduce the ecological competitiveness and
rhizosphere colonization by the organism. Furthermore it is likely
that regulatory authorities will be far more stringent about the
release of such modified organisms into the enviroment. Therefore
at this stage it seems most reasonable
to
search for organisms with
It
should be possible
to
isolate antagonists with one
-9-
as many of desired traits as possible by isolating them from the en-
vironment.
Soil-borne diseases which appear to be good candidates for bio-
control include those caused by the sclerotial-forming pathogens, such
as Rhizoctonia and Sclerotinia. We have particularly considered Tricho-
derma spp., Gliocladium spp. and Coniothyrium minitans as potential bi-
control agents. Most studies
of
the mode of action have been on Tricho-
derma spp., which are not natural rhizosphere colonists and therefore
have to be introduced to soil on a substrate base. It is unclear how-
ever under these circumstances if the antagonist then becomes a rhizo-
sphere colonist.
#
Compared with other potential biocontrol fungi, Trichoderma
spp.
have a rapid growth rate on agar media and straw (Harper and Lynch,
1985; Lynch 198733). This efficiency of competitive substrate utiliza-

tion can be decreased at low
(5
'C) temperature (Lynch, 1987b) and
low
(-7.0
MPa) water potential (Magan and Lynch, 1986). The suppression
of one organism by another on agar is dependent on the relative ino-
culum
size of the antagonist and pathogen. From in vitro experiments
with Trichoderma versus Fusarium (Lynch, 1987b) and experiments in
soil with Trichoderma versus Rhizoctonia (C.J. Ridout, J.R. Coley-Smith
and J.M. Lynch, unpublished) it seems necessary to have the antagonist
present at an inoculum level which is an order of magnitude greater
that of the pathogen but at present this can be difficult to achieve
because it is difficult to determine the biomass of specific fungi in
nature. Trichoderma spp. can become predatory on pathogens (mycopara-
sitic) and whereas this can enhance the effectiveness of the action
of antibiotics or cell-wall degrading enzymes produced by the anta-
gonist, the mycoparasitic action per se may not be an essential requi-
site for biocontrol.
We have analysed the extracellular enzymes of a range of isolates
of
_T.
harzianum and
2.
viride using gel electrophoresis, isoelectric
focussing, chromatofocussing and fast protein liquid chromatography
(Ridout et al., 1986 and unpublished). Proteins produced by the various
isolates differ, and more are induced, by growing Trichoderma spp. on
the cell walls of the pathogen Rhizoctonia solani. In addition to glu-

can endo-1,3-~-glucosidase and chitinase, proteases are amongst the
major enzymes produced. Whereas all these enzymes may contribute to the
biocontrol action,the degree of contribution is unclear.
spp. and recently a volatile pyrone, dec-2,4-dien-5-olide possesing an-
tifungal properties has been isolated from
2.
harzianum (Claydon et al.,
1987). Both the organism and the antibiotic are effective against
Several antibiotic materials have been isolated from Trichoderma
-10-

solani and a range of other pathogens and this metabolic property
R.
of the antagonist could be important in its biocontrol action.
CONCLUSION
Microbial communities in the rhizosphere consist of some popula-
tions which have beneficial effects on plant growth and others such
as pathogens which are harmful. The community structure is governed
by environmental, and plant and microbial physiological factors. Pre-
sent knowledge of these factors and the quantitative analysis of the
populations and communities is fragmentary. It is likely that genetic
exchange will take place between members of the communities, for
example plasmids may be exchanged between bacteria, between bacteria
and fungi or even with the plant. This could have consequences in at-
tempts to introduce genetically modified organisms into the rhizo-
sphere but until there is a more complete understanding of natural
community structures, this will be difficult to assess. Biological
control of root diseases appears to be one of the most useful targets
to aim for in the manipulation of the rhizosphere and this might be
achieved with organisms which are not generally regarded as rhizosphere

organisms.
REFERENCES
BARBER, D.A., LYNCH, J.M.: Microbial growth in the rhizosphere. Soil
CHAPMAN, S.J., LYNCH, J.M.: Some properties of micro-organisms from
Biol. Biochem. 9: 306-308, 1977.
degraded straw. Enzyme Microb. Technol. 7: 161-163, 1985.
CLAYDON, N., ALLAN, M., HANSON, J.R., AVENT, A.G.: Antifungal alkyl
pyrones of Trichoderma harzianum. Trans. Brit. Mycol. Soc.,in
press, 1987.
Control of Plant Pathogens. American Phytopathological Society,
St. Paul 1983.
HADAR,
Y.,
HARMAN, G.E., TAYLOR, A.G., NORTON, J.M.: Effects of pre-
germination of pea and cucumber seeds of seed treatment with
Enterobacter cloacae on rots caused by Pythium spp. Phytopatho-
logy 71: 569-572, 1983.
COOK, R.J., BAKER, K.F.: The Nature and Practice of the Biological
HARPER, S.H.T., LYNCH, J.M.: Colonisation and decomposition of straw
KLEEBEIdC;ER, A., CASTORPH, H., KLINGMULLER,
W.:
The rhizosphere micro-
by fungi. Trans. Brit. Mycol. SOC. 85: 655-661, 1985.
flora of wheat and barley with
special
reference to Gram-negative
-11-
bacteria. Arch. Microbiol. 136: 306-311, 1983.
LYNCH, J.M.: Interactions between bacteria and plants in the root
environment. In: RHODES-ROBERTS, M.E., SKINNER, F.A. (Eds.):

Bacteria and Plants. pp. 1-23. Academic Press, London, 1982.
Agric. Hortic. 3: 143-152, 1986.
rhizosphere. In: FLETCHER, M., GRAY, T.R.G., JONES, J.G. (Eds,):
Ecology of Microbial Communities. pp. 55-82. Cambridge University
Press, 1987a.
a potential antagonist of plant pathogens. Curr. Microbiol.,in
press, 1987b.
fungi involved in decomposition of cereal residues.
J.
Gen. Micro-
biol. 132: 1181-1187, 1986.
Attachment of Enterobacter cloacae to Pythium ultimum hyphae:
possible role in the biological control of pre-emergence damping-
-off.
Phytopathology 76: 327-335, 1986.
electrophoretic profile of extracellular protein induced in Tricho-
derma spp. by cell walls of Rhizoctonia solani. J. Gen. Microbiol.
132: 2345-2352, 1986.
LYNCH, J.M.: Rhizosphere microbiology and its manipulation. Biol.
LYNCH, J.M.: Biological control within microbial communities of the
LYNCH, J.M.: In vitro identification of Trichoderma harzianum as
MAGAN,
N., LYNCH, J.M.: Water potential, growth and cellulolysis of
NELSON, E.B., CHAO, W L., NORTON, J.M., NASH, G.T., HARMAN, G.T.:
RIDOUT, C.J., COLEY-SMITH, J.R., LYNCH, J.M.: Enzyme activity and
WHIPPS, J.M., LYNCH, J.M.: Substrate flow and utilization in the rhizo-
sphere of cereals. New Phytol. 95: 605-623, 1984.
tion. Ann. Proc. Phytochem. SOC. Eur. 26: 59-71, 1985.
C.R. Acad. Sci. (Paris) D178, 1236-1239, 1924.
WHIPPS, J.M., LYNCH, J.M.: Energy losses by the plant in rhizodeposi-

WINOGRADSKY,
S.:
Sur la microflore autochthone de la terre arable.
-12-
1.
SYMBIOTIC MICROORGANISMS
a.
Rhlzobia
AND
PLANTS
This Page Intentionally Left Blank
CRITERIA OF
ROOT-NODULE
BACTERIA
ACTIVITY
Mishustin
E.N.,
Bonartseva
G.A.,
Myshkina
V.L.
Institute of Microbiology,
USSR
Academy of Sciences,
117 812
Moscow,
USSR
ABSTRACT
The
possibilities

of
the
Rhizobium active strains selection in
the pure culture and on
the
early
stages
of the legume-Rhizobium
symbiosis
formation
are
discussed.
It
is
now
100
years since
the
discovery of fixation of molecular
nitrogen by legumes. In 1886
a
short one-page
article
by
Hellriegel
appeared in "Landwirtschaftlichen Versuchstationen" asking "What kind
of nitrogen do plants need?".
Two
years
later

a
detailed 230-page
study
was
published
by
Hellriegel
and Wilfarth in
a
special
journal
on
beet-raising which
was
devoted to nitrogen utilization in
cereals
and legumes.
To commemorate this
day,
a
special
session was
held
by
the
British
Royal Society at the end of last year at
which
the
results of previous

and
prospects
for
future research into molecular nitrogen fixation
by
microorganisms
were
discussed.
A
special meeting devoted to
this
issue
has
been
held
in
MOSCOW.
Following the works of
Hellriegel
and Wilfarth, numerous observations
of nitrogen fixation
by
legumes have been
made
in different countries,
including Russia.
To-day
we
have abundant literature dealing
with

this
subject. Specifically, available evidence
shows
that
symbiosis
between
a legume and root-nodule bacteria
very
often turns out
to
be ineffect-
ive. This can
be
explained
by
the fact that,
as
a
result of crop rotat-
ion on cultivated soil, root-nodule bacteria have to spend much
of
their
lifetime without a host plant, which adversely affects their
activity. Hence,
the
necessity of artificial inoculation of bean
seeds
with
different
races

of root-nodule bacteria. In Russia, this has been
done
by
Budinov
at
the beginning of the century. Somewhat
later,
be-
fore World
War
11,
I
initiated
large-scale
complex work
to
inoculate
legumes in different soil and
climatic
regions of the
USSR.
This work
proved to be very effective.
It
is
interesting
to
note that positive
results
were

obtaQed not only when
a
new legume crop had to be intro-
duced but also on fields
where
particular legume crops had been grown
for
a
long
time.
This fact leads one
to
assume that prevailing root-
-nodule cultures
are
not
always
the
most
effective ones.
crop inoculations have been generalized
at
the Institute of Agricultu-
ral
Microbiology of VASKhNIL. In many
cases a
positive effect
was
observed.
It

is
clear
that
by
using this technique the development
of legume crops can
be
improved.
This
is
extremely important since
legumes (and particularly perennial ones) not only offer valuable har-
vests but also enrich soil in nitrogen. In addition, upon reaping,
considerable amounts of biomass remain in the fields, which
is
beneficial
for accumulation of soil humus. Most other crops,
especially
cultivated
ones,
are
know to
deplete
soil of humus. In planning efficient crop
rotation
this
fact needs to
be
taken into account.
Results of almost

3
thousands
tests
of the effectiveness of legume
As
€or fertilizers, numerous experimental studies show
that
these
when considering the effect of inoculation, one has to
admit
that
can
at
best
sustain
the
soil structure and never improve
it.
there
is
no reason for applying
it
to
all
areas under legume crops.
Thus in the Soviet Union
we
have an annual production of the "rhizotor-
fin" preparation sufficient for inoculating about
2

million hectares,
while
areas
under legume crops total roughly
26
million hectares.
So,
places
have to
be
identified
where
the application of this preparation
will
be
more beneficial. Obviously, these must
be
soils
containing ine-
ffective, root-nodule
bacteria.
We
believe that analytically
this
problem can
be
solved
by
analysing some physiological characteristics
of root-nodule

bacteria
isolated from the soils in question.
teria
to
be
used in soil-improvement preparations such
as
nitragin,
rhizotorfin,
e.
A
preliminary selection of active cultures can
be
realized on
the
basis
of the above-stated principle.
was
visiting a field station in Madison
(WI,
USA)
I
learned that
inoculation of soybean took
place
on
all
fields.
I
asked the manager

what the reason
was
for doing that.
He
pointed to
a
poster
on the
wall
and said:
"Very
good publicity.
It
is
cheap and
it
may
work".
Our
experimental study
is
concerned
with
determining the
time-
-variation of the number
of
root-nodule bacteria under
a
crop rotation

system
and analysing changes in their activity. The experiments
are
carried
out with different
types
of soil.
A
similar
problem
arises
in selecting cultures of root-nodule
bac-
Sometimes, however,
it
is
the publicity that takes over. When
I
On
the basis
of
the available data
it
can be concluded that there
-16-