INTERRELATIONSHIPS BETWEEN MICROORGANISMS AND PLANTS IN SOIL doc

: 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.:

Developments in Soil Science 18

INTERRELATIONSHIPS BETWEEN MICROORGANISMS AND PLANTS IN SOIL

Further Titles in this Series

NON-AGRICULTURAL APPLICATIONS O F SOIL SURVEYS

5 A G H B O L T cirict M C M BRL'GGENM%RT / Edirorsi

SOIL CHEMISTRY A BASIC ELEMENTS

I? E B A B I S D O M iind J D U C L O U X f Editor.?)

SUBMICROSCOPIC STUDIES OF SOILS

CHEMISTRY O F SOIL ORGANIC MATTER

IN L'ANCL'RA cirid F KLJNC I Ectirors)

INTERRELATIONSHIPS BETWEEN MICROORGANISMS AND PLANTS I N SOIL

Developments in Soil Science 18

Interrelationships

between Microorganisms and Plants in Soil

Proceedings of an International Symposium

Liblice Czechoslovakia June 22-27 1987

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

Published in co-edition with

ACADEMIA, Publishing House of the Czechoslovak Academy of Sciences Prague

Czechoslovakia

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Library of Congress Cataloging-in-Publication Data

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

0 Vlastimil Vanfura FrantiSek Kunc 1989

All rights reserved No part of this publication may be reproduced stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying recording or otherwise, without the prior written permission of the copyright owners

Printed in Czechoslovakia

P r e f a c e

C O N T E N T S

1 Introductory lecture

I SYMBIOTIC MICROORGANISMS AND PLANTS

MISHUSTIN E.N., BONARTSEVA, G.A., MYSHKINA V.L.: Criteria o f

and environmental factors on the efficiency of the

BONARTSEVA G.A., MYSHKINA V.L.: Nitrogen-fixation in pure

program for improving symbiotic nitrogen fixation in

CUDLIN P., MEJSTRfK V.: Ecological prospects of utilization

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

HRSELOVh H., VEJSADOVh H., PRIKRYL Z., VhCHOVh J., VANCURA V ,

VfT A.: Effect of inoculation with vesicular-arbuscular

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 s p 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-

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

ANDREYUK E.I., IUTINSKAYA G.A.: Soil microorganisms

and transformation of bacterial polysaccharides in soil

KUNC F , RYBAROVA J : Degradation of d 4 C - 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

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

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

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

and J.M Lynch

More than a hundred oral and poster communications were presented

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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

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INTRODUCTORY LECTURE

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DEVELOPMENT AND INTERACTION BETWEEN MICROBIAL COMMUNITIES ON THE ROOT SURFACE

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

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

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the number of C 0 l O W - 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

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

is unclear however if carbon or nitrogen are the growth-limiting sub- strates to specific components of the rhizosphere population It can

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-

RHIZOSPHERE NUTRITION

Table 1 defines some terms which have traditionally been used t o describe the nutrition of aoil organisms, and compares them with modern ecological terminology.There is a similarity in meaning between auto-

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

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Table 1 Nutrition of soil organisms

MODERN ECOLOGICAL TERMINOLOGY

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

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-

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

grow as oligotrophs and acquire a proportion of their carbon for growth

by utilizing trace carbon compounds

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

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Table 2 Calculated and measured substrate inputs to the rhizoaphere

Microbial biomass

Substrate input

1985) The two bacterial strains were of similar size but in a popula-

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

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

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

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

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

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

It should be possible to isolate antagonists with one

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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-

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

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-

1987) Both the organism and the antibiotic are effective against Several antibiotic materials have been isolated from Trichoderma

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- - R solani and a range of other pathogens and this metabolic property

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,

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 s p e c i a l reference to Gram-negative

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bacteria Arch Microbiol 136: 306-311, 1983

Bacteria and Plants pp 1-23 Academic Press, London, 1982

Agric Hortic 3: 143-152, 1986

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-

- o f f 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

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.:

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

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1 SYMBIOTIC MICROORGANISMS

a Rhlzobia

AND PLANTS

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CRITERIA OF ROOT-NODULE BACTERIA A C T I V I T Y

M i s h u s t i n E.N., Bonartseva G.A., Myshkina V.L

I n s t i t u t e of Microbiology, USSR Academy of S c i e n c e s ,

of n i t r o g e n do p l a n t s need?" Two years l a t e r a d e t a i l e d 230-page

s t u d y w a s p u b l i s h e d by H e l l r i e g e l and W i l f a r t h i n a special j o u r n a l

on b e e t - r a i s i n g which w a s d e v o t e d t o n i t r o g e n u t i l i z a t i o n i n cereals and legumes

To commemorate t h i s d a y , a special s e s s i o n was h e l d b y t h e B r i t i s h Royal S o c i e t y a t t h e end of l a s t y e a r a t which t h e r e s u l t s of p r e v i o u s and prospects for f u t u r e r e s e a r c h i n t o m o l e c u l a r n i t r o g e n f i x a t i o n b y microorganisms were d i s c u s s e d

A s p e c i a l meeting d e v o t e d t o t h i s i s s u e has been h e l d i n MOSCOW Following t h e works of H e l l r i e g e l and W i l f a r t h , numerous o b s e r v a t i o n s

of n i t r o g e n f i x a t i o n b y legumes have been made i n d i f f e r e n t c o u n t r i e s ,

i n c l u d i n g R u s s i a To-day w e have a b u n d a n t l i t e r a t u r e d e a l i n g w i t h t h i s

s u b j e c t S p e c i f i c a l l y , a v a i l a b l e e v i d e n c e shows t h a t symbiosis between

a legume and r o o t - n o d u l e b a c t e r i a very o f t e n t u r n s o u t t o b e i n e f f e c t -

f o r e World War 11, I i n i t i a t e d large-scale complex work t o i n o c u l a t e legumes i n d i f f e r e n t s o i l and climatic r e g i o n s o f t h e USSR T h i s work proved t o b e very e f f e c t i v e I t is i n t e r e s t i n g t o n o t e t h a t p o s i t i v e

r e s u l t s were o b t a Q e d n o t only when a new legume c r o p had t o b e i n t r o - duced b u t a l s o on f i e l d s where p a r t i c u l a r legume c r o p s had been grown

f o r a long t i m e T h i s f a c t l e a d s one t o assume t h a t p r e v a i l i n g r o o t - -nodule c u l t u r e s are n o t a l w a y s t h e most e f f e c t i v e ones

c r o p i n o c u l a t i o n s have been g e n e r a l i z e d a t t h e I n s t i t u t e of A g r i c u l t u -

r a l Microbiology of VASKhNIL I n many cases a p o s i t i v e e f f e c t was

observed It is clear t h a t b y u s i n g t h i s technique t h e development

of legume c r o p s can be improved T h i s is extremely i m p o r t a n t s i n c e

legumes (and p a r t i c u l a r l y p e r e n n i a l o n e s ) n o t only o f f e r v a l u a b l e h a r -

v e s t s b u t a l s o e n r i c h s o i l i n n i t r o g e n I n a d d i t i o n , upon r e a p i n g ,

c o n s i d e r a b l e amounts of biomass remain i n t h e f i e l d s , which is b e n e f i c i a l

f o r accumulation of s o i l humus Most o t h e r c r o p s , especially c u l t i v a t e d ones, are know t o d e p l e t e s o i l of humus In planning e f f i c i e n t c r o p

r o t a t i o n this f a c t needs t o be taken i n t o account

R e s u l t s of almost 3 thousands t e s t s of t h e e f f e c t i v e n e s s of legume

A s € o r f e r t i l i z e r s , numerous experimental s t u d i e s show t h a t t h e s e

when c o n s i d e r i n g t h e e f f e c t of i n o c u l a t i o n , one h a s t o admit t h a t can a t best s u s t a i n t h e s o i l s t r u c t u r e and never improve it

t h e r e is no reason f o r applying i t t o a l l a r e a s under legume c r o p s Thus i n t h e S o v i e t Union w e have a n annual production of t h e " r h i z o t o r -

f i n " p r e p a r a t i o n s u f f i c i e n t f o r i n o c u l a t i n g about 2 m i l l i o n h e c t a r e s , while areas under legume c r o p s t o t a l roughly 26 m i l l i o n h e c t a r e s So,

places have t o be i d e n t i f i e d where t h e a p p l i c a t i o n of t h i s p r e p a r a t i o n

w i l l be more b e n e f i c i a l Obviously, t h e s e must be soils c o n t a i n i n g i n e -

f f e c t i v e , root-nodule bacteria W e b e l i e v e t h a t a n a l y t i c a l l y this

problem can be solved by a n a l y s i n g some p h y s i o l o g i c a l c h a r a c t e r i s t i c s

of root-nodule bacteria i s o l a t e d from t h e s o i l s i n q u e s t i o n

t e r i a t o be used i n soil-improvement p r e p a r a t i o n s such a s n i t r a g i n ,

r h i z o t o r f i n , e A p r e l i m i n a r y s e l e c t i o n of a c t i v e c u l t u r e s can be

r e a l i z e d on t h e basis of t h e above-stated p r i n c i p l e

was v i s i t i n g a f i e l d s t a t i o n i n Madison (WI, USA) I l e a r n e d t h a t

i n o c u l a t i o n of soybean took place on a l l f i e l d s I asked t h e manager what t h e reason was f o r doing t h a t H e pointed t o a poster on t h e w a l l and s a i d : "Very good p u b l i c i t y I t is cheap and it may work"

Our experimental study is concerned with determining t h e t i m e -

- v a r i a t i o n of t h e number of root-nodule b a c t e r i a under a c r o p r o t a t i o n

s y s t e m and a n a l y s i n g changes i n t h e i r a c t i v i t y The experiments are carried o u t w i t h d i f f e r e n t t y p e s of s o i l

A similar problem arises i n s e l e c t i n g c u l t u r e s of root-nodule bac-

Sometimes, however, i t is t h e p u b l i c i t y t h a t t a k e s over When I

On t h e b a s i s of t h e a v a i l a b l e d a t a it can be concluded t h a t t h e r e

-16-

i s a drop i n r h i z o b i a l p o p u l a t i o n s u n d e r non-legume crops, w i t h t h e i r

a c t i v i t y r e d u ced These processes proceed d i f f e r e n t l y f o r d i f f e r e n t

t y p e s of s o i l

ency of r o o t - n o d u l e b a c t e r i a i s o l a t e d from s o i l b y v e g e t a t i o n e x p e r i - ments W i t h t h a t i n mind, w e h av e made a s t u d y of a c o r r e l a t i o n t h a t

mi gh t e x i s t between biochemical p r o p e r t i e s of r h i z o b i a and t h e i r n i t r o -

g e n - f i x i n g e f f i c i e n c y A l a r g e c o l l e c t i o n o f Rhizobium c u l t u r e s h a s been compiled, t h e a c t i v i t i e s o f which were d e t e r m i n e d i n v e g e t a t i o n

The development o f c r i t e r i a r e f l e c t i n g t h e e f f i c i e n c y of root-nodu-

l e b a c t e r i a is t h e r e f o r e one o f t h e main i s s u e s i n symbiotic n i t r o g e n

f i x a t i o n Two d i f f e r e n t approaches t o t h i s problem have be e n u n d e r t a k e n The f i r s t o n e i s s e e k i n g i n d i r e c t a c t i v i t y c r i t e r i a c o r r e l a t i n g w i t h

t h e n i t r o g e n - f i x i n g capacity of r h i z o b i a i n t h e symbiosis The s e c o n d approach i s b a s e d o n e s t i m a t i n g t h e a c t i v i t y e x e r t e d b y n i t r o g e n a s e a t

t h e e a r l y stages of t h e s y m b i o s i s u s i n g t h e a c e t y l e n e r e d u c t i o n method Research aimed a t f i n d i n g c i r c u m s t a n t i a l c r i t e r i a f o r t h e a c t i v i t y

of r o o t - n o d u l e b a c t e r i a i n p u r e c u l t u r e s h a s a l o n g h i s t o r y Numerous

p h y s i o l o g i c a l and b i o c h e m i c a l c h a r a c t e r i s t i c s of r h i z o b i a have bee n

a n a l y s e d i n t h i s c o n t e x t Y e t , t h e problem r e m a i n s t o p i c a l a nd w e s t i l l have no r e l i a b l e p r a c t i c a l c r i t e r i a f o r i d e n t i f y i n g a c t i v e s t r a i n s

I n this work a n a t t e m p t i s made t o e s t a b l i s h a c o r r e l a t i o n between several b i o c h e m i c a l parameters of n o d u l e bacteria a n d t h e i r n i t r o g e n -

- f i x i n g e f f i c i e n c y i n t h e symbiosis For t h i s purpose w e had a t o u r

d i s p o s a l a c o l l e c t i o n of r o o t - n o d u l e c u l t u r e s t h e a c t i v i t y of which had been e s t a b l i s h e d i n g r e e n h o u s e e x p e r i m e n t s

I t h a s been proposed t h a t a c o r r e l a t i o n m i g h t e x i s t between t h e

a c t i v i t y l e v e l of Rhizobium c u l t u r e s and t h e i r p r o d u c t i o n of v i t a m i n s

W e have d e t e r m i n e d t h e a b i l i t y of r o o t - n o d u l e b a c t e r i a c h a r a c t e r i z e d

b y d i f f e r e n t n i t r o g e n - f i x i n g e f f i c i e n c i e s t o produce v a r i o u s v i t a m i n s ( t h i a m i n , r i b o f l a v i n , p a n t o t h e n i c and n i c o t i n i c a c i d s , b i o t i n , i n o s i -

n e , c o b a l a m i n ) The o b t a i n e d d a t a show t h a t i t i s o n l y t h e r i b o E l a v i n ( v i t a m i n B2) and cobalamin ( v i t a m i n B12) c o n t e n t of c e l l s of p u r e

Rhizobium c u l t u r e s t h a t e x i b i t a c o r r e l a t i o n w i t h t h e e f f i c i e n c y of

a c u l t u r e i n symbiosis w i t h legumes I t c a n be s e e n t h a t t h e c e l l ATP

c o n t e n t f o r h i g h l y a c t i v e s t r a i n s o f E p h a s e o l i , 5 m e l i l o t i and

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- R

s t r a i n s

leguminosarum is more t h a n twice as h i g h a s t h a t i n l o w - e f f i c i e n c y

I n this work a n a t t e m p t w a s made t o measure hydrogena6e a c t i v i t y

n o t h i g h ( B o n a r t s e v a e t a l , 1 9 8 3 ) The b e s t r e s u l t s were o b t a i n e d b y

t h e s p e c t r o p h o t o m e t r y method, i n terms of b o t h t h e enzyme a c t i v i t y

ma gn i t u d e a n d the d i f f e r e n t i a t i n g power it o f f e r s between a c t i v e a n d low-active s t r a i n s

T a b l e 1 Hydroqenase a c t i v i t y i n Rhizobium m e l i l o t i and g p h a s e o l i

a

I n t h e l a s t f e w years there h a v e been p u b l i c a t i o n s d e a l i n g w i t h

a c c u m u l a t i o n of b i o p o l y m e r s i n c e l l s of r h i z o b i a h a v i n g d i f f e r e n t

e f f i c i e n c y An i n c r e a s e d l e v e l of g l y c o g e n , DNA and l i p i d s w a s reported

i n a c t i v e Rhizobium s t r a i n s ( B o n a r t s e v a and Myshkina, 1 9 8 5 ) Q u a n t i t a -

t i v e l y , t h e s e b i o p o l y m e r s c a n b e e s t i m a t e d b y c h e m i c a l methods u s i n g

f l u o r e s c e n c e microscopy and IR- spectr ophotome tr y o f whole b a c t e r i a l

c e l l s ( F i g 1 )

-18-

of i n a c t i v e s t r a i n s w i t h l a r g e c o n t e n t of p o l y - 8 - o x y b u t y r a t e w i l l

f l u o r e s c e n c e i n b r i g h t g r e e n l i g h t

-19-

Having t h e n e c e s s a r y i n s t r u m e n t ( f l u o r e s c e n c e s p e c t r o p h o t o m e t e r

o r f l u o r i m e t e r ) , one c a n proceed f r om a q u a l i t a t i v e d e t e c t i o n o f t h e

p o l y - 6 - o x y b u t y r a t e c o n t e n t t o q u a n t i t a t i v e measurements

To do t h i s , aq ue ou s s u s p e n s i o n s w i t h d i f f e r e n t c o n c e n t r a t i o n s of bacteria grown i n t h e p r e s e n c e o f 3R p hos ph ine were prepared Low-ac-

t i v i t y s t r a i n s qive a s t r o n g f l u o r e s c e n c e peak a t 470 nm; s t r a i n s

w i t h i n t e r m e d i a t e a c t i v i t y y i e l d a smaller p e a k ; a n d e f f e c t i v e l y no

f l u o r e s c e n c e a t t h e g i v e n w a ve l e ng t h c a n be d e t e c t e d w i t h a c t i v e s t r a i n s ( F i g 2 )

i n d i v i d u a l p h y s i o l o g i c a l and b i o c h e m i c a l parameters of p u r e r h i z o b i a l

c u l t u r e s , e x h i b i t i n g a c l e a r c u t c o r r e l a t i o n w i t h t h e i r s y m b i o t i c

n i t r o g e n - f i x i n g a c t i v i t y

s h o u l d b e , i f p o s s i b l e , i n t e r r e l a t e d Thus o n e c a n be c o n c u r r e n t l y measuring t h e a c t i v i t y of t h e s u b s t r a t e - i n d e p e n d e n t d e h y d r o g e n a s e , t h e This s e l e c t i o n scheme must be easy t o implement a n d measure ments

-20-

hydrogenase, and t h e a c t i v i t y of some s p e c i f i c dehydrogenase F o r

example, u s e of t h e IR-spectrophotometry t e c h n i q u e a l l o w s s i m u l t a n e o u s

d e t e r m i n a t i o n of t h e c o n t e n t of such s t o r e d compounds a s p o l y - B-oxy-

b u t y r a t e and glycogen W e would n o t have had such a mixed p i c t u r e of

e x p e r i m e n t a l e v i d e n c e r e l a t i n g t o i n d i r e c t a c t i v i t y c r i t e r i a f o r r o o t - -nodule b a c t e r i a , had t h e a u t h o r s been closer t o some s t a n d a r d b o t h i n

took 2 weeks f o r the d i f f e r e n c e i n n i t r o g e n - f i x i n g a c t i v i t y t o be

r e l i a b l y e s t a b l i s h e d f o r small bean p l a n t s and 3 weeks from t h e s t a r t

of t h e e x p e r i m e n t s f o r l a r g e bean p l a n t s This e x p e r i m e n t d u r a t i o n

seems t o be q u i t e r e a s o n a b l e f o r an a c c u r a t e d e t e r m i n a t i o n o f the

e f f i c i e n c y of r h i z o b i a l s t r a i n s Moreover, w e b e l i e v e t h a t by a d j u s t i n g some e x p e r i m e n t a l c o n d i t i o n s ( i n c r e a s i n g C 0 2 c o n c e n t r a t i o n , supplemen-

t i n g t h e a g a r i z e d medium w i t h p h o s p h a t e s and growth f a c t o r s , e.)

REFERENCES

BONARTSEVA G A : T e s t i n g t h e e f f i c i e n c y o f r o o t - n o d u l e b a c t e r i a i n

terms o f a c c u m u l a t e d p o l y - 8 - o x y b u t y r a t e by v i t a l s t a i n i n g o f

t h e i r colonies w i t h 3R p h o s p h i n e ( I n R u s s i a n ) M i k r o b i o l o g i y a 54: 461-464, 1 9 8 5

BONARTSEVA G.A., MYSHKINA V.L.: F l u o r e s c e n c e i n t e n s i t y f r o m d i f f e r e n t -

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NEW ADDITIONAL CRITERIA FOR ESTIMATION OF GRAIN LEGUME-RHIZOBIAL SYMBIOSIS EFFECTIVITY

Antipchuk A.F., Kantselyaruk R.M., Rangelova V.N., Skochinskaya N.N., Tantsyurenko E.V

Zabolotny Institute of Microbiology and Virology, Ukrainian Academy

of Sciences, 2 5 2 143 Kiev, USSR

INTRODUCTION

Obtaining strains capable of forming an effective symbiosis with various species of host-plant is the main task of nodule-for- ming bacteria selection

scientists are paying attention to specific aspects of response of grain legumes It was shown (Mathysse et al., 1978) that adhesion

of microorganisms to plant surfaces was controlled by genotype of both organisms In the authors' opinion, based on examination of a range of rhizobial strains on various soya and lupin varieties (Volkova et al., 19851, the influence of host-plant on yield can be greater than that of nodule-forming bacteria The mechanism of va- rietal specifity is not yet studied, but its influence on the effec- tivity of legume-rhizobial symbiosis is apparent even when inocula- tion is performed with competitive rhizobial strains

From the literature it appears that an increasing number of

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MATERIALS AND METHODS

The estimation o f symbiotic effectiveness is often made with

a restricted amount of indices: the number and mass of nodules on roots, their acetylene-reducing activity, the yield quantity and its protein content Since it is known that intensity of symbiotic ni- trogen-fixation depends on plant.photosynthesis and transfer of assimilates into roots, we have scope for widening the range o f ta- ken indices

The properties characterizing the physiological state of the host-plant were used: the size of assimilating surface and chloro-

was determined in alcohol extracts from homogenized leaves with a

se of budding and the beginning of flowering

gume varieties

tests

The work was conducted over 5 years with a large number of le-

Effectivity of rhizobial strains was estimated in vegetation

RESULTS AND DISCUSSION

cesses connected with soya plant photosynthesis it was established that in 15 variants it lead to an increase (more than 5%) increase

The coincidence of such indices increases with crop capacity enlargement in the most sensitive varieties (Belosnezka and Tere- sinskaya-24) Strains 69 and 1 facilitating effective symbiosis with most of the examined varieties, were the best ones Similar results were obtained in analogous tests with soya varieties

Kievskaya, Teresinskaya-24 and Peremoga

rization greatly changed plant photoassimilating properties: the enlargement of the assimilating surface was noticed in 30 variants

In experiments with six pea varieties it was found that bacte-

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Table 1 The influence of bacterization of photoassimilating characte-

ristics and soya yield

content in leaves occurred together with

Varieties Albatros, Uladovsky, Streletsky were the most sensitive The characteristic feature of variety hialinovka was that following

protein in green mass increased rather than leaf surface and yield

On variety Tson bacterization produced an increase in yield and

protein content without noticeable changes in photoassimilating

properties

The majority o f strains showed high effectivity on at least four

sitive varieties

Study of lupins was performed on plants of two varieties: white (Stuart, Olezka, Soyus, Solnechniy) and yellow (Kopilovsky, Martin-2)

It was established that varieties of white lupin were more sensitive

to inoculation Yellow lupin differed by exhibiting a lower crop ca- pacity and a more prolonged vegetation period In plants of this

-2 5-

ristics and pea yield

138 116 119 146' 104 122 151' 106 103

see Table 1

Table 3 The influence o f bacterization on photoassimilating chara-

cteristic and yield of white lupin

species, the assimilating surface was mainly enlarged in response to inoculation (Table 3 ) This work permitted a comparigon of specific and varietal responses of the mentioned bean cultures to inoculation and the selection of strains showing high effectivity in the majority

of the examined varieties

REFERENCES

MATHYSSE, A.G., WYMAN, P.M., HOLMES, K.V.: Plasmid-dependent attach- ment of Agrobacterium tumefaciens to plant tissue culture cells Infect Immun 22: 516-522, 1978

VOLKOVA, T.N., ENKINA, O.V., MYAKUSHKO, Y.P., BARANOV, V.F., TCHERNOVA, N.I., GORELOVA, O.P.: The ratio of the role of plant and microbial components in the effectivity of bean-rhizobial symbiosis

(In Russian) Mikrobiologiya 54: 857-859, 1985

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NITROGEN-FIXATION I N PURE CULTURES OF ROOT-NODULE BACTERIA

Bonartseva G.A., Myshkina V.L

I n s t i t u t e of Microbiology, USSR Academy of S c i e n c e s ,

117 812 Moscow, USSR

ABSTRACT

F i x a t i o n of n i t r o g e n by Rhizobium i n p u r e c u l t u r e s i n t h e

p r e s e n c e of n i t r a t e under a n a e r o b i c c o n d i t i o n s h a s been e s t a b l i s h e d Both slowly-growing and fast-growing r h i z o b i a were i n v e s t i g a t e d

INTRODUCTION

The purpose of t h i s work w a s to reveal n i t r o g e n f i x a t i o n i n p u r e

c u l t u r e s of n o d u l a r bacteria i n t h e p r e s e n c e of n i t r a t e a n a e r o b i c a l l y Both e f f e c t i v e and i n e f f e c t i v e - s t r a i n s of Bradyrhizobium japonicum, Vigna rhizobium, Rhizobium lpeliloti, 5 p h a s e o l i , E leguminosarum, and 5 t r i f o l i i were u s e d i n experiments Bacteria were grown a n a e r o b i -

r a t e s a n a e r o b i c growth by a f a c t o r o f 3 to 4 , which c a n a t t a i n t h e

r a t e o f growth by means o f oxygen r e s p i r a t i o n Even b e f o r e t h e end of

t h e i r loq-qrowth phase b o t h s t r a i n s were c a p a b l e of r e d u c i n g a c e t y l e n e

Fig 1 Anaerobic growth of Bradyrhizobium japonicum 646 (A) and

(3,4) of nitrates Arabinose (1,3) or glucose (2,4) were present

in the medium

O.D., optical density

is effective in both cultures already on the first day of their growth From the very start anaerobic development of cultures under study

is intimately related to the process of denitrification

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T a b l e 1 Growth and n i t r o g e n a s e a c t i v i t y of Bradyrhizobium japonicum

646 and Vigna rhizobium 164 under a e r o b i c and a n a e r o b i c

To check t h i s , an e x p e r i m e n t w a s c a r r i e d o u t under t h e same c o n d i t i o n s

b u t u s i n g two d i f f e r e n t media: CS-7, which f a c i l i t a t e s nitrogenaseinduct-

i o n and a media c o n t a i n i n g p e a - e x t r a c t , which had n e v e r p r e v i o u s l y been observed t o g i v e r i s e t o n i t r o g e n a s e a c t i v i t y

Reduction of a c e t y l e n e t o e t h y l e n e c o u l d b e o b s e r v e d o n l y on t h e

CS-7 medium, which s t i m u l a t e s n i t r o g e n a s e i n d u c t i o n On t h e p e a - e x t r a c t medium no such p r o c e s s w a s d e t e c t e d and a t t h e same t i m e d e n i t r i f i c a t i o n was p o s i t i v e l y proceeding (Tab 2 )

A l l t e s t e d s t r a i n s from t h e c o l l e c t i o n of slow-growing c u l t u r e s from soybean and vigna proved t o be a b l e t o f i x n i t r o g e n u n d e r a n a e r o -

b i c c o n d i t i o n s , r e d u c i n g n i t r a t e s t o N2 w i t h NO; and N 2 0 a s i n t e r m e d i a t e

p r o d u c t s However, d i f f e r e n t s t r a i n s had d i f f e r e n t d e n i t r i f y i n g e f f i c - iency A c o r r e l a t i o n was o b s e r v e d between c u l t u r e development, N 2 0

f o r m a t i o n and e t h y l e n e f o r m a t i o n (Tab 3 )

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