Virulence and isozyme variations within fungi causing Ascochyta blight of Phaseolus vulgaris.

Fungal isolates obtained from

foliar

samples taken from bean

with

symptoms of Asenchgta Étight

ir,

various regions

of

East Africa and Colombia were

all

related

to

P.

esigluL

var,

il;iuersisporo based

on the

symptoms produced

on

inoculated bean and isozyme analysis. Isozyme banding patterns revealed genetic variations between African and American strains

of

P.

erigao

var. diuersisporo and

within

the African isolates of this fungus.

815

Med. Fac. l-andbouww. Rijksuriv. Gent, 55(3a), 1990

VIRULENCE

AND

ISOZYME VARIATIONS

WITHIN

FUNGI

CAUSING

Ascochyta

BLIGHT

OF

Phaseolus vulgaris

L.

Obando*

**,

J.

Kummert*, P. Lepoivre*

&

J'P'

Baudoin*

'

Llnitâ de Phytopathologie

**

Unité de Phytotechnie des Bégions chaudes, Facuké des Sciences Agrcnomiques de Gembloux

2, Passage des Oéportés, B-580o Gembloux, Belgique

INTRODUCTION

The literature

repofts

several species

of fungi

which

are

considered

to

be responsible for Ascochgta

blill.t.

Phonza erignta Desm.

var.

d;iaersisporo @ub.) Boerema

.urr". t"u"."

damage

in

bean (Phnseohts uulgr:'ris ^L-) and cowpea (Vigna' ungnti'allata

(L.)

Walp.)

in

many

areas

of l,atin

America,

the

United States, Europe,

and

Africa

(Bo"r"*"

et

ol.,

1981); leaves infected

with

this fungus bear large necrotic spots, greyish brown

in

color, often

with

concentric rings of pycnidia. Phoma erigua Desm. var. erigua" formerly known as Ascochyto, phaseol.orum sacc.,

is

another agent of Ascochuta blicht; spore characteristics of this organism are similar

to

those of P. erigrua var. d:iaerispoto,

but

symptoms are different, consisting

of

small specks and spots

on

older

leaves and pods. A

ihird

agunt causing Ascochgta blight, Stagonosporopsr.s hartensis (Sacc.

&

Malbr') Èetr., formerly

inown

u" Ascochgta boltshauseri Sacc., is readily distinguished from both

var.

of

Phonza erigil..a.

by the

occurrence

of

large, frequently multi-septated conidia (Boerema et

aI.,

1981).

s. htrtelsis

induces small reddish brorvn spots

on

leaves, pods, and stems.

Traditionally,

the

classification

of

the

fungi

causing Ascochgta

blight

was based primarily on morphological characters, leading to tâxonomic confusion mainly between P.

érigra

vur. diuersispora and P. erigua var. erigrua, thus hampering the rational selection of resistant bean genorypes; therofore, other methods of identification are needed.

The possible taxonomic interest

of

the technique

of

gel electrophoresis

to

solve problems of genetic variability has been recognized (Burdon and Marshall, 1983). Enryme banding patterns were used

to

study the variation

within

formae speciales and beËween

species

of

a wide range

of

fun$

(Burdon and Marshall, 1981).

At

the physiological race lèvel, however, few differences were detected (Burdon et oÙ., 1981). The purpose

of

our study was

to

characterize the pathogenicity of various fungal isolates of different origins obtained

from foliar

material showing symptoms of Ascochgta blight, and

to

study the correlative variations

of

the electrophoretic banding pattern of selected enzymes within these isolates.

MATERIEL AND METHODS

Fungal material

Dr. W.lvt. Loerakker,

"Plantenziektenkundige

Dienst'

(Wageningen, The Netherlands),

provided

reference

strains

PD

79161

(=strain

Rl) of P.

erigrua

var. d,iuersispora

originating

from

Kenya,

and PD

79/269 _1=s1çuin

R2) of S.

hprtsnsis originating from The Netherlands.

Dr.

E.S, _{Van Reenen-Hoekstra, "Centraal Bureau} voor Schimmelcultures" (Baarn, The Netherlands) gave

us

strain

CBS ?29.68 (=strain R^3)

of P.

erig:ua

van

eîigua, originating from Brazil.

Dr. M. Gerlagh, "instituut voor Plantenziektenkundig Onderzoek" (Wageningen, The Netherlands), donated strain 4.45 (=strain R4) of P. erigao var. diuersispora originating from Rwanda, strain 50987 (=s1çuin R5) of P. eri4:u.c var. diuersi.spora originating from Guatemala,

strain

40987

(=strain

R6) of P. erigua var. erigiln

originating

from Guatemala, and strain COL 5 (=strain R7) of S. hortzlltjts originating from Colombia.

Eighteen isolates were obtained from leaves of P. auLgaris

et

Vigma sp., showing severe symptoms of Ascocàgfa blight collected

in

difTerent locations in l,atin America and East

Africa (table

l).

Foliar

lesions rvere treated 20-30 seconds

in

AgNO.{

and

then incubated on oal meal agar (OMA) at 26'C, 16 h day/8 h night to allow the Éathogen to gro\.v out for isolation. Fungal strains were maintained on OS'{A medium.

Inoculation and incubation of the plants

Plantlets of P. uulgaris (cv. Ica Llano Grande obtained from CIAT, Coiombia, and Karama Demi obtained from ISABU, Burundi) were inoculated at the

first

trifgliolate leaf stage by run-off spraying of leaves with an aqueous spore suspension (3

x

10o spores/ml containing 0,1% Tween 20) prepared from 10 day-old colonies on OMA.

Inoculated plants, arranged

in a

randomized complete block design (using three planis per fungal sirain), were maintained

for

3

hr

under a relative humidity

of

about 70%, and lvere then transferred and maintained

for

15 days

in

a cabinet

at

20-22'C

with

a mist-type vaporizer operating continuously.

Disease assessment

was

made

during

15

days

following

inoculation

by

daily evaluation of the percentage infected leaf area per plant.

Extraction and electrophoresis of enzymes

Isolates were grown on potato dextrose broth (PDB) in flask seeded each

with

t!!'o plugs excisecl from a fungal coiony grorving on OMA, and incubated 6 days

in

the dark ât 24"C. The cultures then were filtered through mulsin and rinsed with distilled water. Five g of mycelium were ground

in

a motar, with addition of dry ice,

in

5 ml O.2M Tris buffer

pH

8.5

containing sucrose

(lM)

ancf mercaptoethânol (0.056M). Homogenization was obtained

in

a French press at 7 kg/cm'. The crude extract was centrifuged at 30,000 g for

t

h and supernatânt maintained at -20"C.

Disc electrophoresis on polyacrylamide gel was performed according to Wetter and Dyck (1983). The gels were assayed for a range of enzymes (table 2) by immersion

in

the reaction mixtures containing appropriate substance

of

the

different enzymes (Vallejos, 1983).

Electrophoretic patterns

were

interpreted according

to the

methods

of

"the

principal

components analysis" (PCA)

and "the

hierarchical cluster analysis" (HCA) (Dagnelie,1975).

817

Ig!lg-!: Fungai isolates from diseæed leaves of HÆeoL16

sp. ùd Vigm sp. ællæted in South America of Eæt Africa

S5

o

s7 lsolates sl S2 S3 s4 S8 s9 s10

slt

sl2 sl3 s14 sl5 sl6 st7 s18 Altitude 1600m 1250m 1250m 900m I 250m 800m 900m i470m 2300m I 600m 1700m I 700m 2000m 2600m I 700m 2700m 1635m I ?Ocm Host plmt P. Dubark P. wl4aris V. globrescw V. rodiaLa V. inutgo V. ulLgtuielato V. unguiwbta P. wvLris P. twlgot* P. utLlgat* P. uulgaris P. wl4ot* P. elgaris P. Luat6 P. wLgaris P, !-ùLgoris P. ut|garis P. tttLgorts I lAauons-Rutagma BDI trlosso BDI trlosso BDI J\{probo BDI Mosso BDI BrrjumbuË BDI 1\lpambo BDI Murogwe BDI ljenda BDI lheta BDI Rutegma BDI Popaym COI Sydundoy Col Rio Negro COL Popa!'e cOL Quito EQU Bukaw Zil I{ulungu Zd Collætor Yed Perreaux-lSABUZ 1985 Perreaux-ISABU 1986 Perreaux-ISABU 1986 Peneaux-ISABU 1986 Perreaux-ISABU 1986 Perredx-ISABU 1986 Peneau-ISABU 1986 Perreaux-ISABU 1986 Peûeaux-ISABU 1986 Perreau-ISABU 1986 Perreaux-ISABU i986 Baudoin-FSAGx3 1986 Baudoin-FSAGX 1985 schnit-clAT4 1986 Raùdoin-FSAGx lgAT Brodoin-FSÂGX 1987 Perreau-ISABU 1986 Pemeaux-ISÂBU 1986

ci'

I _BDI

= Burundi, COL = Colombia. EQU = Equator, L\R = Z'a\re 2 tsaBU = Institut des Sciences

^Sronomiques

du Burundi, Burundi 3 _FsaG"

= Faculté des Sciences agrcnomiques de Gemblou, Iklgium 4 _ctat

&ble2:

Enzymes studied after protein electrophoresis I I I I I I I I I I Enryme Acid phosPhatase Alcohol dehYdrogenase Alkaline PhosPhatase Catalase Esterase Galactose dehYdrogenase Glutamate dehYdrogenase Malate dehYdrogenase Peroxydase Shikimic dehYdrogenase Enzyme Commission 3.1.3.2. 1.1.1.1. 3.1.3.

l.

1. I 1.1.6. 3.1.1.1. 1.4.1.3. 1.1.1.37. 1.1 1.1.7. 1.1.1.25. Abbreviation ACP ADH AKP CAT EST GaDH GDH MDH PER SDH RBSULTS

Symptoms induced by the fungal strains

Symptoms of P.

etigao

var' diaersisporo (reference strains

Rl'

R4'

or

R5) on P'

aulgaris

(Karama

d"*i ;t"i";

Llano Grande)' appeared 2-3 days iollo&il1c inoculation' Rings

of

pycnidia formed àn the surface of the necrotic spots, and leaves collapsed when

the

necrotic

l""ion,

rua"h"d

aUout

half

theii

surface

(ilgure

1)'- AIso' necrotic specks formed along stems

".,i-

on

nodes, which turned black approximately 10 days aiter inoculation

AfterinoculationwithS'hmtensis(referencestrainRTorR2)'orP'etigru'avar'

erignra (reference strains R3

or

R6)' the percentage

of

infected leaf remained l€ss then

10*

with

no symptoms on stems or nodes'

Symptoms

inaucei

lï-ittitt""

S.1

to

S18 originating from South America

or

East

Africa,alllookedsimila.,o"ti,o."ofthereference-strains(R1,R.1,orR5)ofP.erigla

vat.

d'irersispora.

Enzymes analYsis

No activiry for

catalase'

malate

dehydrogenase' peroxydase'

or

shikimic dehydrogenase were detected

for

the

isolates

of f'îgi tttt"à Atid

phosphatase and

alkaline

phosphatase

*.."-inr"ii""t

for all

funeal

lsolates

examined.

The

banding patterns for alcohol

a"nyîrog"n*", "'t"'ut"'

galacto-se dehydrogenase' glutamate

dehydrogenase, variec.l Ji,f.,in"rf.r" eighteen furigal isolates and the seven reference strains (table 3).

;!? ? _-x ;9 '-9o2 oàÉ d é4

(D

F9 >t <O ! I e € o D a

l,

t:

lo

t.

l9 l?, l9

l"

l-o I ô

f:

à !l I J È_d H

Principal

component analysis

(PCA)

showed

that the 31

enzy-matic bands, reflecting

the

enzymatic polymorphism

of the

25 fungal isolates

or

strains, could be grouped into eight main components, responsible for 89% of the total variability. The first ihreé axis of PCA explained 57%

of

the total variability (24.2%

lor

axis

Zl,

l8'5%

for

axis 22 and 14.6%

for

axis Z3). Figures 2 and 3 indicate the position of the 25 fungal isolates or strains

in

plans 27-22 and

zl-23,

and. their association into six different groups.

No fungal isolates originating from South America

or

East Airica were associated

with

groups

Gl

or

G2, corresponding to refer€nce strains of S. hortercis (R2 and R7) or reference strains of P. eri.gnto var. erigru'a (R3 and R6)'

All

south-American isolates (s12, S13,

Sl4,

S15 and s16) were associated into group G3, together

with

the reference strain R5 (P. eriguo var. d.iuersisPoro) originating

irorn

Guatemala.

All

African isolates (S1

to

Sl1)

belonged

to

groups G4a, G4b

or

G4c.

Group G4c contained

the

reference strains R1 and R4

of

P.

etigaa var'

diaersispora ,

originating from Africa.

_-

Figure

4

represents

the

dendogram obtained

after

hierarchical cluster analysis

(HCA),

*ith ."u".,

groups explaining 76%

of the variability of the

enzymatic polymorphism. Both HÔA and CPA led to a similar classification of the 25 fungal isolates àr itrains, except that group G4b, as defined

with

cPA, split into rwo groups (5 and 6) in the HCA method.

Eigure l. Evolution of the

Percentage

of infected leaf

area on

D rnildar;s

(Karama Demi)

inoculated with

Phona exigua

L -

vvL9e11-var. drversispora (Rl:#*

), s- hortensis

(R2:

æ

)'

P.

exig'ua

vat, exigua

(R3:

.-

_).

@

50 40 L *Jo 3 o e ll) o

*10

67891011r213

Time after inoculation (daYs)

O

ç

/--\

o,'x\

9 ol

9 " :l /

(rv

aff

^\g

^v-Rô

.*Po

_oai

o-,^.\c

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_(uso

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L \I\ P CD .\U

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O OS.I_

5sâ

hqi

c) v _^. Ftl! 0)IP Lt+

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- ':vJ _< v)_\U

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cf, ôt

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ro (o o) u)

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Tq9ïç

s(r)ôlo

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Group

I

R2 R7 o< Group

2

"" o? 5 rl s15

'

si4 Grouo .Kf,

3

^-s16 clâ R4 S5 Group

4

55 Group 5 S7 R1 S2 (o S8 s11 s1 S4 Grcuo

_'

6 s10 s18 ç? Group

7

'-s17

Figure 4.

Dendogram

resulting

froûl

fron the electrophoretic

and

reference

strâins of

hierarchical cluster

analysis

banding

paÈtern

cf the isolates

Ascochgta

blight

agents.

DISCUSSION

Fungal isolates obtained from

foliar

samptes taken

in

the

field from

beans with symptoms of Ascochyta

blight

in

various regions

of

Africa and South America were all relaied

to

the species P. erigftlo var. ùiuetsisporo on the basis of symptoms produced on inoculated bean.

These results confirm

that P.

erigtua var. diuersi^sporo

is

highly

pathogenic on beans, whereas P. erigtravat. eîigaa and s. horterasis are moderately pathogenic.

Isozyme analysis

is a

versatile and inexpensive technique

to

be used

to

reveal genetic variations among organisms,

for

the

clarification

of

fungal ta-xa,

and

for

the identification of fungal cultures to the species or subspecies level.

This technique has been used

successfully

to distinguish

species

of Peronosclerosporo (Micales

et

ol.,

1988), Endothia and Cryphonectria (Micales

et

aI.,

1987).

in our

study,

the high

degree

of

dissimilarities between reference strains

of

S. hnrlsnsis,

P. erigua var. erigua

and

P.

erîgztd,

var.

di.uercispota agrees

with

their traditional separation into different entities according to classical taxonomic criteria. This

point is parti;ulary

important to distinguich P.

erigÆ

var. diuersisporo from P. erigu.a

iar.

erigpo, the morphological characteristics of both species being very similar.

in

accordance

with

this

symptomatological study, the electrophoretic pattern

ol

the

isolates

originating

from

East

Africa or south

America

were linked to

the corresponding reference strains of P. eri'gua var. ùiuersisporo '

The study

of

the

population genetics

of

specific fungi

is

another area

in

which isoryme analysis can be used.

Difference

in cultural

characteristics beflveen

the

isolates

of P.

erig:ua

var. d,iaersi,spora from Colombia, and those from Western Europe, has already been observed

(Gerlach, personal

communication). According

to

electrophor€tic

patterns, it

is notetvorthy

that all

isolates collected

from

South America were associated

within

a

specific group, including the Am€ricain reference strain of P. erigrua var' di'uersi.sporo .

Che anaiysis

of

the

status

of

the

African isolates seems more complex: these isolates (tcgether

with

the

reference strains originating from Africa) were associated

into

three àitferent groups (according

to

PCA method) or four groups (according

to

HCA method), independently of host plant, altitude or area of origin.

Such intraspecific differences between isorymes

from

isolates

in the

Eastern hemisphere,

or of the

Americas,

has

been observed

with the

soybean

rust

fungi Phakospora parhErhizi @onde e,

ot.,

1988). The low frequency of shared putative alleles in<licatàs

thal

two populations of P.

porlllrhizi

are involved

in

causing rust on soybean: one

in

Asia and Australia, and another one in America (Brazil and Puerto Rico).

However,

such

intraspecific differences

are rarely

related

to

differences in pathogenicity. One exception was

the

correlation,

in

asexual populations

oî

Pu.ccinia giromini.s

_f.

sp.

tritici

between isozyme phenotypes and nine mqior virulence groups

*itnin tn"

populations

of this

fungus

in the

United

States.

Within

each virulence grouping,

all

individual isolates had exactly the same isozyme banding patterns (Burdon àna Roàlfs, 1985b). These authors also used isozyme patterns oT Puccinia recon'dita to trace independent introductions

of

this

pathogen

into

the United States (Burdon and Roelfs, 1985a).

In the

case

of

Ascochyta

blight of

bean,

it is difficult to

speculate

on

the significance

of

the differences

in

isoryme banding patterns belween Africa and American

stiains

of P. erigta

var. diuerstsporo and

within

the African isolates

of

this

fungus. Nevertheless,

su"h

differ"tces

in

isoryme banding patterns between Ascochgta

blight

agents

could

be connected

to

the

fact

that Phaseolu.s species show different levels of ràsistance

to

given African

or

American isolates of P. erigua var. ùiuersispoto (Obando et

ol.,

1988).

Nothing

is

known about the genetics of P.

erigan

var. diuetsispora, so

that

the analysis

of the

isozyme

pattern cannot

be

related

to

phenorypical markers. The inteipretation

of our

electrophoretic data is thus restricted

to

a simple nband counting" whicfr provides

us

a

static view

of

the

genetic population structure

of the

pathogen. Isoryme analysis, when combined

with

genetic knowlegde

of the

pathogen, allows to

!!i '{i

-t:

825

control plant pathogens.

For Ascochyta blight of Phnseolw, isozyme analysis appeared to be a powerful tool to measur€ the genetic variability within and between incipient fungal species. The technique proved useful

to

characterize fungal

taxa and

to

identify

genetic variation among P.

eri4ta

var. diuersisporo, which is of uppermost importance

for

the selection of resistant genotypes of Phaseolus sp.

REFERENCES

BOEREMA, G.H., 1972. Ascochyta phaseolorum synonymous with Phoma erigpa. Neth. J' Pl. Path. 78, 113-115.

BOEREMA. G.H., G. GRÛGER, M. GERLAGH and H. NIRENBERG' 1981. Phomn erigua var.

O

tîr"Iîî-"

and related fungi

on

Phaseolus

beans. Zeitschirft Pflanzenkrankh. 88,

BONDE, MR., G.L. PETERSON, W.M. DOWLER and

B.

MAY, 1984. lsozvme analysis to

differentiate

species

of

Peronnsclerosporo causing

downy mildews

of

maize. Phytopathologlr 7 4, 127 8-1283.

BONDE, M.R., G.L. PETERSON and W.\{. DOWLER, 1988.

A

comparison

of

isozymes of Phakospora

panhyrhizi

from the

eastern hemisphere

and the

nerv

world. Phytopathologr 78, 1491-1494.

tsirBDON, J.J. and D.R. MARSHALL, 1981. Isozyme variation betr'r'een species and formae speciales of the g€nus Pucctnin Pers. Can. J. Bot. 59, 2628-2634.

BLADON, J.J., D.R. I'{ARSHALL and N.H. LUING, 1981. Isozyme analysis indicates

that

a

virulent ceieal rust pathogen is a somatic hybrid. Nature 293, 565-566.

BURDON, J.J., D.R. MARSHALL, 1983. The use

of

isorymes

in

plant

disease research. 13: Isozymes

in

Plant Genetics and Breeding, Part

A.

S.D. Tanksley

and

T'J. Orton (Eds), Elsevier Science Publishers B.V. Amsterdam, 401-412.

BLRDON, J.J., and A.P. ROELFS, 1985a. Isozlme and virulence

variation

in

asexually reproducing populations of wheat ieaf and stem rust. Phytopatholo$r 75, 907-913. BURDON, J.J., and A.P. ROELFS, 1985b, The eifect of sexual and asexual reproduction on

the

isozyme structure

of

populations

af

Puncini,a g,raminis. Phytopathologr 75,

r 068- 1 073.

DAGI{ELIE, P., 1975. Anall'se statistique à plusieurs variables. Les Presses Agronorniques de Gembloux, 362 p.

MICALES, J.4., M.R. BONDE and G.L. PETERSON, i9E6. The use

of

isozyme analysis in fungal taxonomy and genetics. Mycotaxon 27,405-449.

MICALES,

J.4.,

R.J. STIPES and M.R. BONDE, 1987. On

the

conspecificify

of

End,cthia

e*"'s,'r3'"

o

"*,

r*;îffi"

r:i:î:fi

"Ëii:'

ig';'*É

J"'L J

?,

u. i u

tu n.

"

t

o

p ho ma

erigua

in

the secondary

gene

pool

of

Pinseolus nulgarts. Annual Report

of

the Bean Improvement Cooperative il1, 117-118.

VALLOJOS,

C.8.,

19S2. Enzyme

activiti

straining.

In:

Isorymes

in plant

genetics and breedind. Part A. Tanksley S.D. & Orton T.J. (Eds). Elsevier Science Publishers B.V.

Amsterdam.

WETTER, L. and J. DYCI(, 1983. Iscenzyne analJ'sis of cultured cells and somatic hybrics.

In:

Handbook

of

Plant Cell

Culture - vol I:

Technique

for

Propagation and Breeding. A.D. Evans, R.Vr'. Sharp, P.V. Arnmerato and Y. Yamada (Eds). Macmillan Pubiishing Co, 607 -627.

We tank Prof. J. Semal and Dr. Gerlach for their support during this work and for many helplul discussions. Grateful acknowledgment are due to Prof. J.J. Claustriâux and B. Saint Ghislain (Unité de Statistique et Informatique, FSAGx)

for

statistical processing of the data. L. Obando is indebted to the "Administration Générale de

la

Coopération au Développement, Brussels, Belgium"

for

research grants. We thank A.M.

Pollart

for

able technical assistance.