Phytopath. Z., 114, 3 1 - 4 0 (1985)© 1985 Verlag Paul Parey, Berlin und HamburgISSN 0031-9481 /InterCode: PHYZA3

Agricultural Research Department, Rts0 National Laboratory,

DK-4000 Rosktlde, Denmark

Instttute of Thallophytes University of Copenhagen,

0ster Fartmagsgade 2D, 1353 K, Denmark

Interactions between the vesicular-arhuscular mycorrhizalfungus Glomus hscicuhtum and Aphanomyces euteiches

root rot of peas

S. RoSENDAHL

With one figure

Received April 2, 1984

Abstract

Interactions between Glomus fasdculatum and Aphanomyces euteiches root rot of peas (Pisumsativum), were studied in pot experiments using irradiated soil.

Infections with the pathogen were suppressed by VAM when plants were challenge inoculatedafter two weeks. No reduction of the pathogen was detected when the plants were inoculated withboth fungi at the same time.

The suppression of the pathogen, obtained by preinoculation with G. fasciculatum, was notreduced when the inoculum level of the pathogen was increased thirty times.

The induced resistance to A. euteiches in VAM plants was partially a systemic effect. When rootsystems were split into two halves, one with mycorrhiza and one with A. euteiches, the oosporeproduction was reduced in both root systems. The infection with the pathogen was only suppressedwhen both fungi were present in the same pot. The background for the induced resistance is discussed.

Interaktionen zwischen dem vesikular-arbuskularen Mykorrhiza PilzGlomus fasciculatum und der Wurzelfaule der Erbsen, hervorgerufen

durch Aphanomyces euteiches

Wechselwirkungen zwischen Glomus fasciculatum und Aphanomyces euteiches Wurzelfaule derErbsen (Pisum sativum) wurde in Experimenten studiert, bei denen Erde verwendet wurde.

>?v enn I ilanzen nacri zwei iVocnen geimprt wurden, Konnten intektioneii durch den I atnogenmit VAM unterdruckt werden. Keine Verminderung des Pathogens wurde jedoch festgestellt, wenndie Pflanzen mit beiden Pilzen gleichzeitig geimpft wurden.

U.S. Copyright Clearance Center Code Stacemem: 0 0 3 1 - 9 4 8 1/85/1 401 "003 1 $ 0 2 . 5 0 / 0

-L̂ ie Unterdruckung des Pathogens, die durch die vorherige Impfung mit G-fasciculatumcrrcicht wurde, wurde nicht vermindert, wenn die Inokulumdichte des Pathogens 30maI erhoht

Die induzierte Resistenz gegenUber A. euteiches in VAM-Pflanzen war teilweise ein systemi-schcr Effekt. Wenn Wurzelsysteme in zwei Halften geteilt wurden, eine Halfte mit Mykorrhiza unddie andere Halfte mit A. euteiches, war die Oosporenproduktion in beiden Wurzelsystemen reduzien.Die Infektion durch den Pathogen war nur unterdriickt, wenn beide Pilze in demselben Topfvorhanucn waren. Die Orunulsge riir die mduzierte Resistenz wird besprochen.

Introduction

The significance of vesicular-arbuscular mycorrhiza (VAM) in the resistanceof plants to pathogens, has attracted great attention in the latest years (SCHENCKand KELLAM 1978, SCHENCK 1981, DEHNE 1982).

In most investigations VAM decreased the incidence of several root-pathogenic fungi (DEHNE und SCHONBECK 1975) and nematodes (KELLAM andSCHENCK 1980), whereas no clear effect was obtained in others (BAATH andHAYMANN 1983).

The resistance to pathogens, induced by VAM, seems to depened on thetime interval between the mycorrhizal inoculation and the challenge infectionwith the pathogen, hence a reduction of the disease is only obtained if themycorrhizal infection is well established at the time of challenge (BARTSCHI etal.1981).

The increased resistance in VAM plants is described by SCHONBECK (1979) asa strictly local effect occurring oniy in the mycorrhizal areas, whereas, otherauthors found increased resistance in the non-mycorrhizal parts of root systemsas well (DAVIS and MENGE 1980).

DEHNE (1982) describe the interactions between VAM and root-pathogensto be based on modifications of the disease since very virulent isolates or increasedinoculum levels of the pathogen may reduce or even eliminate the beneficialeffects of VAM.

The mechanisms behind the induced resistance in VAM plants remainunexplained, but many different mechanisms have been proposed (SCHONBECK1979).

Phosphorus may be important (DAVIS and MENGE 1980, GRAHAM and

MENGE 1982) and several mechanisms could be indirect effects of increasedphosphorus content in the VAM plants. Besides, the VAM plants are usually inbetter nutrional condition than the smaller non-VAM plants, and thereby lesssusceptible to the pathogens (DEHNE 1982). It is, therefore, important to workwith plants of the same size, either by using isolates of mycorrhizal fungi that donot give any growth response, or by adding phosphate to the non-VAM plants.

Aphanomyces euteiches DRESCHL. is described as the most destructive patho-gen on peas in several countries (PAPAVIZAS and AYERS 1974), and is regarded asthe principal limiting factor for pea production in several districts in USA(PFENDER and HAGEDORN 1983), Norway (SUNDHEIM and WIGGEN 1972) and the

southern part of Sweden (OLOFSSON 1967). The importance of the pathogen hasnot yet been investigated in Denmark.

Interactions between the vesicular-arbuscular 33

No resistant pea cultivars have been found, and chemical control seems to be

ineffective or very expensive. Losses can mainly be reduced by avoiding artas

heavily infested with the pathogen (PFENDER et al. 1981).

Few papers describe interactions between A. euteiches and other microor-

ganisms, and no antagonistic microorganisms have yet been found (PAPAVIZAS

and AYERS 1974).

The purpose of this study was to investigate the influence of Glomus

fasciculatum (THAXTER) GERDEMANN & TRAPPE, V—A mycorrhiza on

A. euteiches root of of peas, and to examine the nature of the mechanisms

involved in the induced resistance in VAM plants.

Material and Methods

2.1 Host plants and treatments

Peas were grown in a 2 • 1 mixture of sand and clay loam (pH 7) containing 12 ppm phosphorus(Olsen P). The mixture was irradiated with 0.8 Mrad (10 MeV electron beam) in order to killmycorrhizal propagules.

Two seeds of Pisum sativum L. (cv. Bodil), pregerminated on moist filterpaper for 48 hours andsurface sterilized m 70 % ethanol for two minutes, were planted in each 10 cm pot holding 1 kg of thegrowing medium. At planting, the pots were inoculated with 20 ml of a dense suspension ofRhizobium leguminosarum (RiS0 stram 1 a).

with A.eutetches, 4) inoculated with both G. fasciculatum and A. euteiches, were included in allexperiments except ror the split-root experiment (Section 3.3.2). The treatments m tnis experiment aregiven in table 3.

Six replicates of every treatment were tnade in all experiments. The pots were placed in agreenhouse at 20—25°C and watered as necessary.

2.2 Mycorrhizal inoculum

The G. fasaculatum inoculum was produced on maize grown in autoclaved, expanded clayLeca* for three months in 2000 cm' pots (H.-W. DEHNE pers. comm.). The inoculum containedinfected roots, spores and expanded clay particles with external mycelium, ihe VAJVi plants wereinoculated with approximately 10 g inoculum per pot at planting, and similar amount of uninfectedmaize roots and expanded clay were added to the non-VAM plants.

2.3 Aphanomyces euteiches inoculum

The A. euteiches isolate was obtained from L Sundheim, As, Norway. The culture wasmaintaineu on corn mesl aga.r at 4 C Zoospore inoculum was obtained according to the method oi

100ml maltose-peptone brotn medium (MPB). Ine medium was decantated aiter rive days, andreplaced with 100 ml sterile tap-water which was again replaced by 50 ml sterile distilled water aftertwo hours. After seventeen hours at 21 C the mycorrhizal mats were strained off and the zoospore

The plants were inoculated by pouring 10 ml of the zoospore suspension on the soil surface.Control plants received 10 ml distilled water.

2.4 Determination of dry weight, phosphorus content and degree of infection

Shoot and root dry weight were determined at harvest. The phosphorus concentration wasmeasured by the molybdate-blue method (MURPHY and RILEY 1962).

Representative root samples of each of the six replicates, was cleared in KOH and stained inTryphanblue according to PHILLIPS and HAYMAN (1970), and the infection with both mycorrhiza and

Phylopaih.Z.,Bd. 114, Hett 1 3

A. eutctchcs were estimated as percent mfected root length by the grid-line intersect method (GIOVAN-

Infection with A. euteiches was confirmed by occurence of oospores in the cortex, and theni\ corrhizal infection was identified by occurence of vesicles, arbuscles or chlamydospores.

The number of oospores per mm^ infected root was determined microscopically by measuringthe length and the diameter of the examined root piece. At every intersection between an infected rootand the grid-line, used for determination of infections, the number of oospores was counted in a250//m long root cylinder. For each of the six replicates, thirty such root cylinders were examined.

Experiments and Results

3.1 Influence of different challenge times

The influence of the time interval, between inoculation with the mycorrhizalfungus and the pathogen, on the interaction between the two fungi, was assessed.Three groups of plants, including all four treatments, were planted at 14 daysintervals and then challenge inoculated simultaneously 28 days after the firstplantmg (see Table 1).

All plants were harvested two weeks after challenge inoculation and dryweight of shoots and roots were determined along with the infection withG. fasciculatum and A. euteiches (Table 1).

VAM suppressed the infection with the pathogen when the challengeinoculation was made two and four weeks after planting (Treatment 8 and 12),whereas the pathogen was not influenced by VAM when the plants wereinoculated with both fungi at the same time (Treatment 4).

Aphanor

No.

nt Plan'

G.faT

tum. A:

t age (days) AT:. with:A. eut. harvest

T.

:inocula

Dry

Shoo

uble 1

ted wit

weigh,

(g)

h A. euteich

% Roo: VAM

itlength^ mjA. eut. V A M ' A.

md time ofl.fasacuU-

2M3A4M-(-A -5C 06M7A8M-t-A -9 C 0

lOMl l A12M-I-A -

P<0.05) , ' Vail

14 28-

-

28 42-

_

-

aes in each columr

0.160.120.100.550.570.530.551.661.671.561.50

1 followed by

0.190.110.140.310.310.370.300.560.600.600.61III

42.7a0

22.7b0

72.7c0

68.4c0

79.5c0

76.5c

-ac letters

052.lv50.lv

00

15.6X6.3y009.8yl.Oz

are no,

81.0a0

32.1b0

229.0c0

208.0c0

478.9d0

468.6d

; different (P

056.2x72.5x00

57.5x18.9y00

58.7X6.1z

<0.05

IS between the vesicular-arbusc 35

Estimated as percentage infection, VAM infection reached maximum .ihcronly two weeks. A reduction of the mycorrhizal infection was observed onlywhen plants were inoculated with both fungi at the same time (Treatment 4).

In spite of different values of percent infections with A. euteiches in the non-VAM plants, infections expressed as mg infected root were similar at all harvestsand thus independent of the root weight.

3.2 Effect of increased pathogen inoculum level

To determine the influence of increased inoculum, six different zoosporeconcentrations were used. The experiments were carried out at different times butunder similar conditions. The plants were inoculated with G. fasciculatum fourweeks before challenge inoculation and harvested two weeks after challenge.

The infection with A. euteiches were determined in the VAM and the non-VAM plants, and plotted against the zoospore concentration (Fig. 1).

The infection with the pathogen increased with increasing inoculum level inthe non-VAM plants, whereas the infection in the VAM plants was almostindependent of the zoospore concentration. The slope of the lines were signific-antly different (P<0.01) using the F-test.

3.3 Local or systemic effect of VAM

3.3.1 Infection pattern in non-split roots

It was examined whether the pathogen infected mycorrhizalh i l i h VAM l f h ifrequent

p g yn-mycorrhizal areas in the VAM plants of the experiment described

Fig. l . Relations between log zoo-spore concentration and the percentrootlength infected with A. euteichesin VAM and non-VAM plants (Sec-tion 3.2). VAM plants were challengeinoculated four weeks after planting

^ NON-VAM

Y=11.8LOGX-31.9

VAM

Y=3.1LOGX-7.9

X 10^ ZOOSPORES/POT

Porccntagcfre,,

zoosp./pot.

A.

no

infection types

euteiches infec

infc. ° M

T.

both futs VAM

al obserA

able2s of VAM plants

ngi). The right haand non-VAM r

vedM + A

challenge inoi

ootreq^ally

TotalObsv.

:ulated with differen

ts the hypothesis tha

^.^-Values'

' All values are significant (P > 0.05)

in Section 3.2. Roots of the plants inoculated with both fungi were examinedmicroscopically and the infection pattern was recorded for each intersectionbetween a root and the grid-line. The following categories were used: 1) noinfection, 2) infection with G.fasdculatum, 3) infection with A. euteiches,4) infection with both G.fascieulatum and A. euteiches (Table 2).

Significant (P > 0.05) x' values were obtained at all six inoculum levels ofthepathogen, when the hypothesis that the pathogen infected VAM and non-VAMroots of the same plant equally was tested.

3.3.2 Infection pattern in a split-root experiment

A split-root experiment was used in order to examine whether the mycorrhi-zal infection had any influence on the degree of pathogen infection when the twofungi were in separate pots.

Seeds were pregerminated for three days, whereafter the tip of the radicleswere severed in order to promote formation of side roots. After one week m

Table JDry weights and infections with G.fascieulatum and A. euteiches in a split-root experiment (Section3.3.2). (0: uninoculated control, M: inoculated with G.fascieulatum, A: inoculated with A. euteiches.

The plants were inoculated with G.fascieulatum four weeks before challenge

Tr(

Left

MOMM

:atment

Right

oAAM + A

Shoots

1.96a1.95a2.D6a2.26b

Dry

left

0.44vO.38x0.47v0.45V

weights (fRoots

right

0.26w0.29w0.24w0.42v

;)

total

0.70z0.67y0.71yz0.87Z

G.fasleft

88.0a0

85.7a85.9a

/o Rootl:cicul.right

000

83.9a

ength mfeA.e

left

0000

cted'uteiches

right

038.4x38.1x10.6y

Phosphor

Left

MOMM

Values in

CO

t

Rig

oAAM-

thes

ahte

ntrol, M: inoculat.uptakeed witl

PhosphorusShoots

;ht left

0.23a0.15b0.26a

ame column follow

the side roots

0.57x0.16y0.57X

ved by

Table

Rootsright

0.28z0.16y0.24z

F-test

separated \

4n exper

Shoot

4.50a2.84b5.24a

iment (Section 3.3.2) (0:

Phosphoi

left

0.58y2.66x

halves ai

•us uptake (mg/

°Mght

0.72z0.48y0.55y

Ltly different (P

ad placed in

un.no.uLacd

'plant)Total

7.663.548.45

<0.05)usmg

two 10 cmpots placed adjacent to each other.

Four treatments were included (see Table 3). Treatments with A. euteicheswere challenge inoculated with 10' zoospores per pot four weeks after inoculationwith G.fascieulatum. The plants were harvested after six weeks. For all plants,dry weights, infections with G.fascieulatum and A. euteiches, and phosphorusconcentrations in shoots and roots, were determined (Table 3 and 4).

ine mycorrnizai lntection was constant in all treatments, ine lniection witiithe pathogen was not influenced by the presence of VAM when the two fungiwere separated, but was significantly reduced when the two fungi were present inthe same pot. The phosphorus concentration (Table 4) was larger in the VAMroots than m the uninfected roots. A difference was also detected between thenon-mycorrhizal parts of the VAM plants and the roots of the non-VAM plants.

3.3.3 Influence of VAM on the oospore formation

The experiments described above was designed to test the influence of VAMupon the infection pattern. However, the sporulation ability of the pathogen isanother parameter which could be influenced.

The number of oospores per mm^ infected root was counted in the roots ofthe plants mentioned in Section 3.2, infected with 30 X 10* zoospores per pot, andin the plants from the split-root experiment (Section 3.3.2) (Table 5).

Nu

SpliNo

t-root (Sect. 3.3.2)t split (Sect. 3.2)

For each of the two e:( P < 0.05) using F-test.

. m m

') no

Tahle ii' infected root in VAM and non-VAM plants

four weeks after planting

Non-VAM plainon-VAM root

698a'748x

nents values followeiine of the root systen

us VAMs non-VAM roots

557xy

d by the same letters aiIS with VAM,') half of

inoc

plan,

thei

ulated Wl

VAMr(

479b'479y

t signific•oot systf

ah A.

,ots

antly

euteiches

differentth VAM,

38

No difference was detected in the oospore production, between mycorrhizaland non-mycorrhizal roots of VAM plants, although the oospore production wasreduced in the VAM plants, compared with the non-VAM plants.

Discussion

These experiments show that VAM infection can reduce root rot of peascaused by A. euteiches. This effect was increased with increasing time lapsebetween the mycorrhizal inoculation and the challenge inoculation with thepathogen (Section 3.1). This resuk is in agreement with BARTSCHI etal. (1981),who found that VAM did not protect roots of Chamaecyparis lawsoniana toPhytophthora cinnamomi unless the mycorrhizal infection was well estabhshed atthe time of challenge.

Percent infected rootlength was used as an estimate of the infection withA. euteiches, in order to avoid subjective estimates as e. g. "disease index" used inother experiments with root rot. However, a clear correlation was found hetweenpercent infected rootlength and the "disease index" in several pilot tests.

In the first experiment (Section 3.1) (Table 1), infected root weight was usedin order to compare infections in plants of different ages. By using this estimatethe problems in comparing infections in root systems of unequal size were met.However, both G. fasaculatum and A. euteiches will probably infect the finelateral roots more easily than the more hgnified main roots, and the obtainedvalues might therefore be over estimated.

A. euteiches was able to suppress the VAM infection in the plants inoculatedwith both fungi at the same time (Table 1, treatment 4). A similar effect wasreported by KRISHNA and BAGAYRAJ (1983) and BAATH and HAYMAN (1983) who

suggested that the infection was decreased because the pathogen reduced thephotosynthetic efficiency of the leaves and hence the root exudation rate.

Increased inoculum level of A. euteiches did not cause a correspondingincrease in infection in mycorrhizal plants (Fig. 1). This result contrasts withother reports deahng with Thielaviopsis hasicola (BALTRUSCHAT and SCHONBECK1975), and Fusarium oxysporum (DEHNE and SCHONBECK 1979).

The background for this discrepancy is not clear. The inoculum level of thepathogen used in the present experiment should be sufficient to cause maximuminfection in the plants (PAPAVIZAS and AYEKS 1974).

From Table 2 it appears that A. euteiches infected VAM and non-VAM rootsof mycorrhizal plants equally, though a general reduction in infections with thepathogen was evident in the plants preinoculated with G.fasdculatum. Thisindicates that the factor responsible for the increased resistance to infections withthe pathogen, was of systematic nature. However the split-root experiment(Section 3.3.2) showed that when the two fungi were in separate pots, VAM didnot suppress the infection with the pathogen (Table 3).

These apparently conflicting results might be explained by the presence of alimited systemic effect in the VAM plants, where the possible factor responsiblefor the induced resistance to infections could, if at all, only slowly be transported

upwards in the plant, and would therefore not easily be transmitted from ihemycorrhizal to the non-mycorrhizal half of the root system.

The difference in the amount of oospores per mm' infected root in non-VAM and VAM plants from the split-root experiment (Table 5) indicated thatVAM was able to reduce the oospore production of the pathogen, even when thetwo fungi were in separate pots. Further, it shows that the oospore productionwas not reduced in the VAM roots compared with the non-VAM roots of thesame plants. This supports the idea that the oospore production could besuppressed by a regular systemic effect and that this factor is different from thefactor responsible for the reduced infections in the VAM plants.

Increased resistance to pathogens in VAM plants may be due to increasedphosphorus concentrations in the roots (DAVIS and MENGE 1980, GRAHAM andMENGE 1982). In the split-root experiment (Table 3) A. euteiches infected non-mycorrhizal roots of V^AM plants and roots of non-VAM plants to the samedegree, in spite of larger phosphorus concentration in the mycorrhizal plants(Table 4). However, this enhanced phosphorus concentration was due to anindirect phosphorus supply from the mycorrhizal roots, and it is not knownwhen this increase did occur in relation to the challenge time. Increased phos-phorus concentration in the VAM roots, therefore, might still be responsible forthe inhibition of the infection with the pathogen.

No difference in the oospore production was detected between the mycor-rhizal and the non-mycorrhizal parts of the VAM plants in the split-rootexperiment (Table 5). Since a clear difference in the phosphorus concentrationwas evident between the two parts of the root (Table 4), phosphorus concentra-tion does not seem to govern the inhibition of the oospore production in theVAM plants.

In addition to increased phosphorus concentration, the increased resistancein the VAM plants has been explained as induction of compounds inhibitory tothe pathogen (BALTRUSCHAT and SCHONBECK 1975, KRISHNA and BAGAYRAJ

1983). Enhanced nutrional status of the host plant can not be excluded asresponsible for the induced resistance in the present experiment, but it is stillpossible that the mycorrhizal fungus induced one or several compounds inhibit-ory to A. euteiches. DAVIS (1963) found that the root nematode Meloidogyneincognita was able to induce resistance to A. euteiches in pea roots. This mightinclude the same mechanism as that obtained in the present experiment.

The present work indicates that early establishment of VAM is important, ifVAM is to be used to control A. euteiches in peas. However, additional experi-ments are needed to confirm if this also will be vahd under field conditions.

This work was part of a M. Sc. degree from University of Copenhagen, Denmark. I Wish tothank I.JAKOBSEN, Agricultural Research Department, Rise, and U. SOCHTING, Institute ofThallophytes, University of Copenhagen for advises and help-full discussions. M. ROSENDAHL,Institute of Datalog,y, University of Copenhagen, for statistical analysis, and J. HOCKENHULL,Agricultural University, Copenhagen and GLORIA ZAHKA for corrections of the manuscript.

40 ROSENDAHL

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Author's address: S. ROSENDAHL, Institute of Thallophytes, University of Copenhagen, 0ster

Farimagsgade 2 D, 1353 K. (Denmark).