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fillifllmlfllflflil'l

 

 

 

 

 

 

 

NLlllllHlflfl

uses QM

LIBRARY
Michigan State
University

 

 

 

This is to certify that the l

thesis entitled

Races of Bean Anthracnose (Colletotrichum Lindemuthianum)

From Malawi, Germplasm Screening and the Genetics of

Resistance in Selected Bean (Phaseolus Vulgaris L.)
Genotypes

presented by
James Morris Bokosi

has been accepted towards fulfillment
of the requirements for

M.S.

dPgflvin Crop & Soil Science

c7/
\:;;%7f;zg/ f «(1;374<fl--

Major professor

 

 

c.7539 MSUisnn Arr-mm» ‘ ' m m" 'Jlnstitution

 

 

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RACES OF BEAN ANTHRACNOSE (COLLETOTRICHUM
LINDEMUTHIANUM) FROM MALAWI, GERMPLASM
SCREENING AND THE GENETICS OF RESISTANCE IN

SELECTED BEAN (PHASEOLUS VULGARIS L.)

 

GENOTYPES

By

James Morris Bokosi

A THESIS

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

MASTER OF SCIENCE

Department of Crop and Soil Sciences

1986

 

 

 

ABSTRACT

RACES OF BEAN ANTHRACNOSE (COLLETOTRICHUM
LINDEMUTHIANUM) FROM MALAWI, GERMPLASM
SCREENING AND THE GENETICS OF RESISTANCE IN
SELECTED BEAN (PHASEOLUS VULGARIS L.)
GENOTYPES

 

By

James Morris Bokosi

Bean anthracnose caused by Colletotrichum linde—

 

muthianum is a major fungal disease of beans in Malawi
and is found in all bean growing areas. Studies of 16
isolates showed more pathogenic variation. Alpha-Brazil
and lambda were the only known races identified. Many
of the Malawian bean landraces were susceptible to a
mixture of7 two Malawian isolates. Screening of other
bean germplasm identified additional sources of resist-
ance. Studies on genetics of resistance in selected
bean genotypes to two Malawian isolates, BA3 and BAH, of
g; lindemuthianum indicated that resistance to 8A3 was

mainly governed by a single pair of genes while that of

 

 

BAA was by two pairs of interacting genes. No linkage
was detected in factors conditioning resistance to the

two isolates.

 

In memory of my father

ii

 

ACKNOWLEDGMENT

I am grateful to the following:

Dr. M. w. Adams, my major professor, for
collecting the Malawian isolates, his sup—
port, patience, guidance throughout my study
and editing this manuscript.

Dr. A. w. Saettler, a member of the guidance
committee, for his invaluable suggestions,

understanding, review of the manuscript, and
allowing me to use his laboratory facilities.

Dr. J. D. Kelly, a member of the guidance
committee, for collecting the other isolates
and his critical review of this manuscript.

F. Correa for sharing with me his expertise on
isolation and inoculation techniques, and all
my friends that helped me in various ways.

 

My mother for her understanding.
My wife for her patience.

My daughter Mphatso, born during my absence
and deprived of fatherly love for 2 years.

F.A.O. of the United Nations for granting the
scholarship, and the Bean/Cowpea CRSP for
additional support.

TABLE OF CONTENTS

3353
List of Tables. . . . . . . . . . . . v
List of Figures . . . . . . . . . . . vii
INTRODUCTION . . . . . . . . . . . . 1
LITERATURE REVIEW. . . . . . . . . . . 3
GENERAL MATERIALS AND METHODS. . . . . . . 26
RACE IDENTIFICATION . . . . . . . . . . 31
CROSS PROTECTION VERIFICATION. . . . . . . Al
GERMPLASM SCREENING . . . . . . . . . . AA
GENETIC STUDY . . . . . . . . . . . . 56
DISCUSSION . . . . . . . . . . . . . 68

 

LITERATURE CITED . . . . . . . . . g. . 8M

 

iv

 

LIST OF TABLES

TABLE Page
I Isolates of bean anthracnose (Colletotri-
chum lindemuthianum), from Malawi. . . 32
2 Isolates of bean anthracnose (Colletotri-
chum lindemuthianum), from Tanzania and
Kenya . . . . . . . . . . . . . 33
3 Proposed standard range of differentials

for the identification of races of
Colletotrichum lindemuthianum, accord-
ing to E. Drijfhout, IVT, Holland. . . . 35

 

4 Reaction of standard anthracnose differ-
entials to isolates of Colletotrichum
lindemuthianum from Malawi . . . . . . 38

5 Reaction of standard anthracnose differ-
entials to six isolates of Colletotri-
chum lindemuthianum from Tanzania and

 

 

one from Kenya. . . . . . . . . . 39
6 Selected varieties and their reaction to

two isolates. . . . . . . . . . . A2
7 Bean landrace germplasm identified as

resistant to a mixture of two isolates
of Colletotrichum lindemuthianum from

 

 

 

Malawi. . . . . . . . . . . . . A7
8 Malawian bean landraces identified as

resistant to three isolates of

Colletotrichum lindemuthianum. . . . . M9
9 Reaction of some Malawian bean varie-

ties of Phaseolus vulgaris L. to
three isolates of Colletotrichum
lindemuthianum. . . . . . . . . . 51

 

10 Reaction of other bean varieties of
Phaseolus vulgaris L. to two isolates
of Colletotrichum lindemuthianum. . . . 54

 

 

 

TABLE

ll

l2

13

Page

Malawian bean landraces identified
as resistant to a Tanzanian isolate
of Colletotrichum lindemuthianum. . . . 55

F2 reactions to two isolates of

Colletotrichum lindemuthianum

and expected ratios of resistant

(R), and susceptible (S) plants . . . . 61

Observed and expected frequencies
in reaction of F2 populations
of two crosses segregating simul-
taneously for reaction to two
isolates of Colletotrichum linde-

 

muthianum from Malawi . . . . . . . 66

vi

 

Figure

1

LIST OF FIGURES

Lower side of a bean leaf showing typical
symptom of Colletotrichum lindemuthianum

 

An aerosol containing can (Spra-tool No.
8011 Soil Power Pak, Industrial Products
Co.). . . . . . . . . . . . .

A Sample of Malawian bean landraces used
in screening for sources of resistance

Reaction of some Malawian bean germplasm
to isolate BAA . . .

Trifoliate leaf of F2 from a Ruddy x
1212 D cross showing a resistant reac—
tion to BA3 (right) but susceptible to
BAA (left). . .

Page

14

36

45

52

6O

 

 

INTRODUCTION

Beans (Phaseolus vulgaris L.) play a very

 

important nutritional role in the diets of people in
Malawi. Compared to other legumes, beans are second
only to groundnuts in total production. But groundnuts
are generally grown for cash and beans are grown
essentially for food. Beans are high in protein content
and provide a balanced diet when eaten with maize, rice,
or cassava, which is quite common in Malawi. Thus, they
serve as an economic source of protein for the majority
of the population, who cannot afford expensive protein
sources like beef, fish or poultry. When immature pods
or green tender leaves are eaten, as vegetables, they
provide vitamins. This practice is quite common.

Despite the importance, bean production has
lagged behind demand and yields are consistently low.
The main constraints are many but diseases are probably
the single most important single factor limiting yields.
Among these diseases, bean anthracnose, caused by

Colletotrichum lindemuthianum (Sacc and Magn.) Scrib. is

 

one of the most widespread and destructive. Efforts to
control this disease are complicated by the pathogenic
variability that exists for this organism.

l

 

This study was conducted with the following
objectives:

1. To identify pathogenic variation in Malawian
isolates of bean anthracnose.

2. To identify sources of resistance in the
Malawian bean germplasm pool.

3. To identify sources of resistance in foreign
bean germplasm pool.

A. To determine the inheritance of genetic
resistance involving a Malawian cultivar and

Malawian isolates of the disease.

 

 

 

 

 

 

LITERATURE REVIEW

The Pathogen

Bean anthracnose is incited by the fungus
Colletotrichum lindemuthianum (Sacc. and Magn.)
Scribner. It has been common for most workers to cite

the fungus to Colletotrichum lindemuthianum (Sacc. and

 

Magn.) Briosi and Cavara. Earlier publications classi-

fied it as Gleosysorum lindemuthianum (Sacc. and Magn.).

 

The pathogen is a member of the Fungi Imper-

fecti. The perfect stage of the fungus has been
reported, and was originally called Glomerella
lindemuthianum. Kimati and Gali (40), renamed it Q;

cingullata.

g; lindemuthianum produces a septate, branched
mycelium which changes color from hyaline to nearly
black upon maturity. It produces hyaline unicellular
conidia (18) measuring 14 to 5 by 22 M which usually
contain a clear vacuole-like body near the center. The
shape of the conidia may be oblong, cylindrical, kidney-
like with rounded or slightly pointed ends. The conidia

are born in acervuli on unbranched conidiophores.

 

 

In cultures optimum growth of the fungus occurs
at 22—2500 (18) while optimum conidial production takes
place from 14-180C. It has been reported by Edgerton
(25) that temperatures above 30°C limit or prevent
conidial production. Sporulation is best at pH 5.2-6.5
and virtually stops below pH 3 but is unaffected by
aeration or ultraviolet light (48).

Cultures of C; lindemuthianum are frequently
grown on bean pod agar (BPA) medium; sterilized pods,
potato—dextrose agar (PDA), and Czapek medium (67). But
Mather et a1. (MB) found excellent Sporulation on a
medium containing glucose, mineral salts and Neopeptone.
Chaves (18) reported that isolates may lose viability
and pathogenicity when repeatedly transferred in cul-
ture, unless occasionally re-isolated from inoculated
plants or stored under low temperatures. In evaluating
the cryo—preservation and pathogenicity of some fungi,
Hwang et al, (38) found that neither freezing nor
ultra-low temperature storage (in liquid nitrogen--
-1500C to -l96°C) altered the pathogenicity or damaged
the viability of the C; lindenmuthianum isolates, and no
morphological changes were observed in subcultures after

four years of storage in the liquid nitrogen.

 

The Host
In most cases, C; lindemuthianum occurs on
common bean (Phaseolus vulgaris L.). But, while the

 

disease is more severe on P; vulgaris, it has also been
reported on other hosts including lima beans (3;
limensis Macf.), scarlet runner bean (E; coccineus
Willd), tepary bean (P; acutifolious Gray var.

latifolius Freeman), Cowpea (Vigna unguiculata Walp),

 

kudzu bean (Dolichos biflorus L.) and broad bean (Vicia

faba L.).

The Disease

Bean anthracnose is known wherever §;_ vulgaris
is cultivated. The disease has been reported in Africa,
Asia, Australia and Oceania, Europe, North America,
Central 'America, West Indies, and South America. The
wide geographic distribution has been well illustrated
by the Commonwealth Mycological Institute (22).

Although the pathogen was actually collected by
mycologists as early as 18H3 (65) the first description
of bean anthracnose in 1875 was based on specimens
originating in Bonn, Germany. The disease is of high
economic importance. Crop losses can approach 100% when
infected seed is planted under conditions favorable for

disease development (67).

 

 

 

From about 1912 to 1920 bean anthracnose was con-
sidered the most serious bean disease in the United
States, losses in Michigan in 1914 were estimated (67)
at about $1.5 million and double that amount in 1915.
However, since about 1920 the importance of the disease
in the United States has declined because of use of
disease-free seed and development of resistant varie—
ties. But the disease remains one of the most destruc-
tive bean diseases in other parts of the world, espe-
cially in East Africa and Latin America.

Compared to other bean diseases, 9; lindem-
uthianum is ranked first as a priority disease in
practically all of Eastern Africa (the major bean
production area in Africa) where it causes substantial
yield losses.

In Malawi, where the common bean P; vulgaris L.
is one of the most important grain legumes, second only
to groundnuts (27) diseases are probably the most
important single factor limiting bean yields. Bean
anthracnose occurs in practically all bean growing
districts and most bean varieties are susceptible.
Because of its widespread and destructive nature, it is
ranked as the number one fungal bean disease and the

objective is to breed for resistance to it.

 

 

 

Epidemiology

Humidity and Temperature

 

Bean anthracnose is favored by humid or rainy
weather combined with comparatively low temperatures.
Lauritzen (A3) found that high humidity, greater than
92% or free moisture, and temperatures between 13° to
26°C were required for infection. The lowest limit for
infection during a 24 hour period of incubation was
IAOC. No infection occurred above 270C and was
prevented or inhibited at 13°C. Abundant infection
occurred at 170C.

Rahe and KuE (53) have reported limited infec—
tion and development of the fungus when temperatures are
greater than 300C. However, the pathogen is capable of
tolerating temperatures as low as -150 to -200C, for

several days.

Spread

The build up of an epidemic requires moderate
rainfall at frequent intervals (18). This condition is
essential for local dissemination of conidia. Cultiva—
tion of bean fields while rain or dew is present on the
foliage provides for dissemination of the disease
through contact of spore masses with man, insects,

animals and implements (65, 67).

 

 

Work done in Canada (Southern Ontario) on Fleet-
wood, a white bean cultivar, demonstrated that periods
of heavy rain are responsible for the spread of C;
lindemuthianum in the field (62). The direction of
spread from an initial infection focus followed the
direction of the prevailing wind. Field temperature of
Southern Ontario did not appear to be a decisive factor
in the infection and spread of the disease. The spread
was limited to the dispersal distance of splashing
raindrops. Disease severity was highest at or near the
axis of the sector nearest the initial infection focus
and decreased gradually toward the periphery of the
sector.

For long distance spread, the seed is the
principle means of dissemination. C; lindemuthianum is
a seed borne disease and will survive from one season to
the next as dormant mycelium within the seedcoat or the
cells of the cotyledon or as spores between cotyledon or

elsewhere in the seed.

Longevity

The survivability of Q; lindemuthianum varies
greatly depending on environmental conditions. Tu (63)
found that moisture had a profound effect. The fungus
survived at least 5 years in infected pods and seeds

that were air dried and kept in storage at 4°C. Dry

 

infected plant material had similar longevity when left
in the field in sealed polyethylene envelopes and having
no contact with water. An alternating wet-dry cycle was
detrimental to the survival of the fungus. Infected pod
segments, under laboratory conditions, lost viability
after three cycles of 72 hour wet and 72 hour dry

periods.

Control

Cultural Practices

 

One method of limiting the spread of bean
anthracnose is by the use of disease-free seed. This
has been done in various regions of the world (67).

Heat treatment has been used in some cases but
leads to reduced seed viability.

Walker (65) and Zaumeyer (67) recommended the
practice of crop rotation of three years duration. In
Kenya, Van Rheenen et al. (64) reported that beans grown
in association with maize showed less incidence of bean
anthracnose. Mixed cropping effected a kind of cultural
control of the pathogen especially in the wetter areas
but yield was considerably less under mixed cropping
than in monoculture.

It is highly recommended that people refrain
from working in the field when conditions favor the

spread of the disease. Above all, quarantine and legal

 

10

requirements for bean seed exchange and/or marketing

between and among countries need to be adhered to.

Chemical Control

Various chemicals have been evaluated and used
to control bean anthracnose (18, 67) either through seed
treatment or foliage sprays. An effective seed treat-
ment must be able to destroy the mycelium and spores in
the seed without greatly impairing germination.

In Tanzania, Shao (59) reported that benomyl
(Benlate, 0.55 g/l) gave effective control of bean
anthracnose as well as angular leaf spot. Yield losses
by anthracnose in unsprayed treatments were 46%.

In Malawi, Edje and Mughogho (26) evaluated six
fungicides and found that triphenyltin hydroxide
(Du-Ter)- was the most effective in controlling the
disease. Sprays with Daconil 75% w’p at 3.5 g/liter or
Zineb 70% w p at 3.7 g/liter at bi-weekly intervals have
been found effective (27).

Pyeregrine (51) recommended the use of fentin
hydroxide at the rate of 1.5 oz. a.i./150 gallons of

water per acre for good quality seed production.

Use of Resistant Varieties
The observation that bean varieties differ in

their reaction to anthracnose came as early as 1883 (65)

 

11

but the first organized screening for resistance and
susceptibility of different varieties to different
isolates of the fungus was by Barrus (10). A bean
selection from Red Kidney showed resistance to the alpha
and beta races of C; lindemuthianum and was named Wells'
Red Kidney. A few years later, Burkholder (14) produced
a resistant white marrow bean variety resulting from a
cross between Well's Red Kidney and a white marrow bean.
McRostie (46) utilized the same Well's Red Kidney in a
cross with a selection (p.B. No. 1986) of Michigan
Robust to develop a navy bean resistant to the disease.
Search for resistance to bean anthracnose has been
expanded into many bean producing areas and regions (18,
67). Goth and Zaumeyer (33) tested fifty-four bean
varieties for their reaction to four races of
anthracnose and found that snap bean varieties resistant
to alpha, beta, and gamma races could be developed by
recombining resistance in existing snap bean varieties
while resistance to the delta race could be derived from
the dry bean, PI304110.

In Malawi, varietal improvement is aimed at
producing disease resistant varieties (27). Earlier
screening identified some sources of resistance from the
land races. In addition to identifying resistance in

the land races, attempts are being made to use exotic

 

12

cultivars in crosses with Malawian gemplasm, to develop

new resistance sources.

Plant Infection and Symptomology

 

Under favorable conditions conidia of C; lindemu-
thianum may germinate in six to nine hours (23) to form
a germ tube and appressorium which are attached to the
host cuticle by 21 gelatinous layer. Penetration occurs
by means of an infection peg which forms on the side of
the appressorium in contact with the host and penetrates
the cuticle and epidermis by mechanical pressure applied
by the appressorium and infective hyphae which develop
from it (23, 65). The infective hyphae (18, 67) con-
tinue to grow and enlarge between the cell wall and
protoplast for two to frnu' days without apparent damage
to tflua host cells. Later, the cells are degraded and
the protoplast dies and water soaked lessions appear.

The symptoms appear on all above-ground parts of
the plant. The term 'anthracnose' is descriptive of the
appearance (fl? the symptoms in the form of 'burnt black'
lesions (16). The fungus primarily attacks young organs
of the plant (stems, leaf petioles, leaf veins, and the
fruit). The lesions are characteristically dark brown
to black. In moist weather, pinkish spore masses may be
present on the surface. Lesions on leaves appear mostly

on the veins and (N1 the lower side of the leaf (Figure

 

 

 

13

1). Petioles and stems may also bear lesions. In the
hypocotyl they often extend to deep cankers. The most
striking symptoms (65) appear on the immature pod.
Lesions develop from small brown spots which enlarge
quite rapidly. They become dark brown to black in the
center and light to pinkish at the border. As the pod
matures, the colors lighten and the edges of the lesions
are raised. When the pods are opened the lesions are
often found to have penetrated through the seed coat
into the seed.

Affected seeds bear lesions colored in various
shades of brown. Plants which grow from such seeds show
blackened cankers on the cotyledon. Badly infected
plants may form lesions several inches long from
coalesced spots especially on the stem and young stems

may collapse due to cracking and rotting of the tissues.

Host Pathogen Interactions

 

Disease resistance of higher plants usually
depend on metabolic responses of host cells to the
pathogen, and successful pathogens overcome all of the

defense mechanisms of their hosts.

Cell Wall Degradation

 

Plant infection tut microbial pathogens usually

involves degradation of the cell walls of the plant.

 

 

Figure 1. Lower side of a bean leaf showing typical
symptoms of Colletotrichum lindemuthianum.

 

 

15

The importance of pectic degrading enzymes is indicated
by the ability of these enzymes to macerate plant tissue
and to cause cell death. Anderson and Albersheim (2)
indicated that the pectic degrading enzymes secreted by
the pathogens and the proteins present in plants which
inhibit them can play a critical role in the infection
process. Among the enzymes secreted by plant pathogens
to degrade the polysaccharides of plant cells, poly-
galacturonases are considered of great importance in the

infection process (28).

Phytoalexins

Phytoalexins are "low molecular weight antimicro-
bial compounds that are both synthesized by and
accumulated in plants after exposure to microorganisms"
(24). Many plants respond to invasion by a pathogen or
non-pathogenic microorganism by accumulating phyto-
alexins (l, 7, 8, 34, 35, 55, 66). Considerable
evidence support the view that accumulation of phyto—
alexins at the sight of infection is one mechanism by
which plants resist disease (21). It cannot be said
that all plants have the ability to synthesize phyto-
alexins but the ability to produce them is a charac-
teristic of plants that is widespread (1). Indeed
several types of resistance responses to attempted

infection apart from phytoalexins have been reported.

 

 

16

Plants can entrap invading microbes by secreting extra-
cellular' materials composed, in different host-microbe
interactions, of lignin-like compounds, callose,
silicon, cellulose or unidentified materials.

Many pathogens are capable of detoxifying the
phytoalexins produced by their hosts. There often
appears to be competition between plants' ability to
accumulate inhibitory concentration cfi‘ phytoalexins and
the ability of the microbes to detoxify phytoalexins.
Successful pathogens are able to tip the balance in
their favor. They are able to evolve a mechanism of
avoiding toxic effects of phytoalexins.

One mechanism might be the ability of an
infective strain to grow away from the areas in which
the plant is accumulating toxic levels of phytoalexins.
A pathogen might secrete a toxin which kills the plant
cells in the region of the pathogen before those cells
are capable of synthesizing enzymes needed for produc-
tion of phytoalexins. Some pathogens can become tolerant
1x3 high concentration cfi‘ phytoalexins. Pathogens may
secrete molecules that specifically inhibit synthesis of
phytoalexins such as secretion of an effector molecule
that activates a repressor of a gene encoding an enzyme
required to synthesize the phytoalexins or by secretion

of molecules that inhibit a required enzyme (21).

 

17

Further still, some pathogens may penetrate their hosts
so efficiently that they invade the host tissue before
the tissue can accumulate enough quantities of phyto-
alexins to stop the pathogen. Other pathogens can be so
well adapted to their hosts that they grow through the
plant tissue biotrophically, without causing apparent
damage to host cells and without triggering host
defenses (9).

The amount of phytoalexins accumulated in chal-
lenged plants is highly variable (21) and is likely to
depend on such things as the physiological state of the
plant (temperature, water potential, nutrition), time of
day, eliciting microbe, and the genetic background of
the cultivar. The mechanisms by which phytoalexins
accumulate in plant tissues are not well understood.

In studying the relationship between symptom
expression and phytoalexin concentration in P; vulgaris

infected with C; lindemuthianum, Bailey (6) demonstrated

 

that four antifungal compounds-~phaseollin, phaseol-
lidin, phaseollenoflavin and kieveton--accumulated in
all infected tissues. Skipp et a1. (61) showed that
phaseollin caused drastic changes in 21 range cfi‘ fungal

species. Spores of the bean pathogen C; lindemuthianum

 

were killed within two minutes of exposure to a solution

containing 10 mg phaseollin per ml. Phaseollin was also

18

shown in) kill plant cells. But Darvill and Albersheim
(21) have reported that phytoalexins are ineffective
antibiotics, requiring millimolar concentration to
inhibit microbes. Thus they are less effective, on a
molar basis, as compared to commercial pesticides but
have the advantage of being concentrated where they
would be most effective. They are also indiscriminate
in that they affect procaryotic and eucaryotic cells at
similar concentration and appear to be toxic to higher

plant cells and mammalian cells in addition to microbes.

Elicitors

 

The molecules that signal plants to begin the
process of phytoalexin synthesis are called elicitors.
Elicitors can be solubilized from plant cell walls both
by enzymes secreted by infecting microbes and by enzymes
in the plant that are thought ‘to be activated during
infection. Many biotic and abiotic elicitors have been

identified (21). Most work with elicitors has centered

 

 

 

 

 

on two experimental systems: Colletotrichum linde-
muthianum, Phaseolus vulgaris, and Phytophtora
megasperma var. Sojae--Glycine max soybean. In each
case, researchers were investigating race--specific
monogenic resistance. Evidence indicates that these

elicitors do not induce differential phytoalexin

accumulation in host varieties to explain the striking

 

 

l9

race—-variety specificity characteristic of natural
systems (1). An elicitor was extracted from P; vulgaris
hypocotyl sections and cell suspension cultures follow-
ing a treatment with denatured ribonuclease A, a known
abiotic elicitor (24). The elicitor was capable of
passing through a dialysis membrane and inducing phyto-
alexin accumulation in healthy tissue on the other side

of the membrane.

Induced Resistance

 

Inoculations with a non-pathogenic race of g;
lindemuthianum has been shown (12, 29, 52, 60) to induce
resistance against a pathogenic race. The recognition
of the organism by the host and by the organism early in
the development of the interaction appears to be essen-
tial for the activation of a disease resistance mecha-
nism.

Induced resistance has also been reported with
heat treatment of infected plants (54). Andebrhan et
al. (3) was able to induce resistance by irradiating
susceptible bean hypocotyls with ultraviolent light.
Resistance was accompanied by accumulation of phyto-
alexins.

There is also gradual acquisition of resistance,

apparently with age. As the bean plant ages, tissue

 

20

that is susceptible when young gradually acquires resist-

ance to Q; lindemuthianum.

 

Pathogenic Variation

 

Physiological specialization or races of C.

 

lindemuthianum were first observed by Barrus (10), who

 

noted that 'varieties resistant 1K) repeated inoculation
made with an isolate obtained in one locality' became
seriously infected when inoculated with an isolate from
another locality. The results demonstrated that at
least two physiological races were involved. The races
were later designated alpha and beta.

In 1922, Burkholder (15) isolated 21 third race
and designated it gamma. Many more races have since
been reported. These include delta (4), epsilon (l3),
kappa (42) which was originally called ebnet (57),
alpha-brazil (30), lambda (36), which was originally
called alpha SN, jota (37), and lambda—mutant (32).
C-236 is another race added to this list.

The race nomenclature system used by most
workers is based upon Greek letters but in other
instances (18) researchers have used different codes
such as Groups I-III in Mexico, 1-8 in Australia, A-X in
Germany and Races PV6, D10, Egb, In, and L5 in France.

In Kenya, the occurrence of alpha race has been

reported (41). Leakey and Simbwa Bunnya (44) have

 

21

indicated the existence of a number of races in Uganda,
and reported that some isolates with affinites to the
alpha and beta managed partially to overcome the
immunity of the cultivar Cornell 49—242. Shao (59) has
reported the existence of alpha, beta, gamma, epsilon
and kappa races in Tanzania. In Malawi, Ayonoadu (5)
reported the presence of beta, gamma, and delta and

three new races.

Sources of Resistance

 

The detection of varietal resistance to C.

 

lindemuthianum can be traced back to Barrus (10) who
later released a resistant selection known as Wells Red
Kidney (11). Since then, considerable effort has been
devoted to screening for broader and more stable
resistance to the disease.

Sources of resistance to alpha race has been
found in Charlevoix, Manitou, Montcalm and Mecosta red
kidney varieties (56). Sanilac and Fleetwood varieties
are resistant to alpha, beta, and gamma races but
susceptible to delta. Tuscola confers resistance to
alpha and beta but is susceptible to gamma and delta
races. The Venezuelan accession P1326418 (Cornell
49-242), which contains the ARE gene, is resistant to
alpha, beta, gamma, delta, epsilon and lambda raes (18),

but is susceptible to C-236, kappa, and jota races.

 

22

Resistance to alpha, beta, and kappa has been identified
in Coco ala creme, Kievit Koekork, Bo-22 and Evolute,
while PI1501414, Titan and Metorex confer moderate
resistance to kappa (42). A black seeded Mexican bean,
P1165422 and 21 small light tan Colombian bean, P1207264
(37), show resistance to kappa and jota races. A
variety Nep-2 has resistance to delta race and breeding
lines with favorable plant architecture as well as delta
race resistance are IMMJ available (39). Fouilloux (31)
reported Mexico 222 and Mexico 227 as sources of
resistance 1x) kappa zuui Brazilian-alpha races. Bo-22,
Kaboon, To (Mexique 2 gene) and Tu (Mexico 3 gene) are
reported 1x3 give resistance to the same two races (18).
Other varieties, A8136, BAT841, and INT7420, appear to

be effective against most races of g; lindemuthianum.

 

Ayonoadu (5) showed that some cultivars from Malawi gave
differential reactions to alpha, beta, gamma, and delta
races. Two cultivars (Acc. Nos. 2 and 118/2) were able
to resist all four races.

Workers at Centro Internacional de Agricultura
Tropical (CIAT), in screening for sources of resistance
to bean .anthracnose ‘have obtained some important
results. More than 13,000 bean accessions from the CIAT
germplasm bank were screened during 1978-1981 (19).
Among these, 162 were resistant or intermediate to the

Popayan isolates of C; lindemuthianum, 27 accessions

 

 

 

23

were resistant to all known and available races from
Europe, Colombia and other parts of Latin America. Many
of the resistant sources originated from Mexico and
Central America and showed great diversity for geo-
graphic origin, plant type, seed color and seed size.
Thus, :U: is considered possible to identify genotypes
with resistance effective against a wide range of
pathogen races from within and across different bean
production regions. The stability and lengevity of
anthracnose resistant varieties is dependent upon the

pathogenic variation displayed by C; lindemuthianum

(58).

 

Genetics of Resistance

 

The genetic control cfi‘ resistance 1x3 different
races of bean anthracnose has been a subject of consider-
able study. Burkholder (14) crossed Well's Red Kidney
with 21 white marrow bean and showed almost an exact 3:1
ratio in tune F2 segregating population, indicating a
single factor difference between the resistant and
susceptible plants with respect to one race of bean
anthracnose involved in the cross. Resistance was found
to be dominant. McRostie (46) obtained similar results.
In another cross by McRostie (47) between selection B,
resistant to alpha and beta races, and Germany wax,

susceptible to both races, a segregation ratio of 9R:7S

 

I
L

24

in the F2 was obtained with a mixed inoculum indicating
that a single dominant gene governed resistance to each
race.

The first evidence of greater complexity in the
genetic reaction to a particular race of C; linde-
muthianum, race beta, was by Andrus and Wade in 1942
(4).

Mastenbroek (45) described the existence of the
dominant ARE gene found in Cornell 49—242 and used it in
a breeding program. There were no undesirable linkages
noted in the back crossing program that was used.

Cardenas et a1. (17) evaluated nine varieties of
beans and their crosses for their genetic resistance to
alpha, beta and gamma races. The study showed that
genetic resistance to alpha was by dominant alleles at
each of two loci, unlinked but behaving as duplicate
factor loci. A similar system governed reaction to beta
and gamma races with evidence favoring multiple allelism
and dominance of alleles conferring susceptibility in
some cases for beta. Distinct systems of complementary
factors and genetic linkages were also observed but
genes belonging to members of the set(s) affecting any
one race alone showed independence with regard to
linkage.

Another study by Muhalet et a1. (50), involving

five cultivars and one breeding line of P; vulgaris,

 

25

showed that independent and complementary gene action at
1 or 2 loci accounted for a resistant reaction to beta,
gamma, and delta races. Resistance to the beta race was
further controlled by an allemorphic series of three

alleles.

 

GENERAL MATERIALS AND METHODS

Media Preparation

Potato dextrose agar (PDA) and bean pod—agar
bean (BPAB) were the two media used in this study. PDA
plates were prepared by dissolving 39 g of commercial
PDA (Difco) in 1 litre (1,000 ml) of glass—distilled
water. The suspension was steamed for about 20 minutes,
transferred (in 250 m1 lots) to prescription bottles and
autoclaved for 20 minutes at 2500F and 15 psi. The
contents were allowed to cool and then poured in small
lots of 25 ml to petri dishes. The plates were checked
for contamination after two days and any contaminated
plates were discarded.

BPAB plates were prepared by steaming 10 g of
navy bean seeds in 100 ml of glass distilled water for
60 minutes; the beans were then ground in a mortar and
pestle. 20 g grams of agar (Difco) and 20 g of ground
bean pods were then dissolved in 900 ml of distilled
water and steamed for 30 minutes. The previously
prepared 100 m1 + 10 g ground navy bean suspension was
then added to the 900 ml agar/ground pod mixture by
sieving it through a double layer cheese cloth, and

thoroughly stirred. The contents were autoclaved for 20

26

 

J
I
1

27

minutes at 250°F and 15 PSI, allowed to cool, and
poured in 25 ml lots to petri dishes, and checked for

contamination after 2 days.

Isolation of the Pathogen

For infected plant tissue that contained
sporulating lesions a drop or two of sterilized
distilled water was put on the lesion and a fine needle,
previously flamed, was used to dislodge the spores into
a suspension. A sterile pipette was then used to suck
the spore masses and transfer them to 1 ml sterile
distilled water in a test tube. The suspension was
diluted three or four times to reduce the spore
concentration to facilitate single spore isolations
later.

The spore suspensions from the 1 ml sterile dis-
tilled water were then poured onto the surface of petri
dishes containing potato dextrose agar (PDA) made from
39 g of Difco PDA, and 1,000 ml distilled water.
Inoculated plates were incubated at 19-210C, for 24 to
48 hours, during which time germinating spores could be
seen under the stereo-microscope. When the germinating
spores became visible, a fine needle was used to trans-
fer 10 to 12 of the spores plus a small amount of media
to two fresh PDA plates. These spores developed into

colonies and the colony showing maximum Sporulation and

 

28

freedom from contamination under iflue stereo-microscope
was selected, transferred and maintained on PDA.

For materials that showed little or no
sporulating lesions, the diseased tissue was excised
frmn the infected tissue and sterilized with 10% sodium
hypochlorite for two minutes and rinsed twice with
sterile glass-distilled water. The pieces were then
transferred to PDA plates and incubated at l9-210C.
During incubation, spores were produced and the same
procedure as described for sporulating lesions was used

to obtain single spore isolates.

Maintenance of Isolates and Inoculation Preparation

 

Cultures were maintained (”1 PDA and transferred
at 8-10 day intervals. Small discs of PDA from an aging
culture (8-10 days) were transferred to new PDA plates.
When greater Sporulation was needed for inoculum prepar~
ation, BPAB was the medium used. Spores for inoculation
were obtained by scraping them from plates that had been
incubated at 210C for 55 tr> 7 days. Glass sterile-
distilled. water of :25 ml containing 0.5 v/v Tween 80
(polyoxythelene sorbitan monooleate), a wetting agent,
was added to the spore—containing plates and spores were
dislodged with a sterile microscope slide. The suspen-

sion was filtered through a double layer of cheese

 

29

cloth. Spore concentration was adjusted to about 106
spores/ml by the use of a haemocytometer.

For a preparation. of mixed race inoculum the
same procedure was used except that the spore concentra-
tion of each race before the mixing was 2.0 x 106
instead of 1.0 x 106 spores/ml to take care of the
dilution effect when mixing. This ensured a concentra-
ion of 1.0 x 106 spores/ml for each of the two isolates

in the mixture.

Disease Assessment

 

Plants were rated at "+" for susceptible and "-"
for resistant, with no visible symptoms of infection. A
1-5 scale was adapted where resistance corresponded to
1-2 while susceptibility corresponded to 3-5, rating as
follows:

1. Clean, no visible symptoms of infection.

2. A few scattered, small lesions on the midrib
and occasionally on the main veins. Lesions
corky in appearance.

3. Many' small lesions scattered cni the Inidrib
and veins with collapse of the tissue.

4. Few large lesions scattered over the leaf
blade <n‘ many large lesions over the leaf

blade.

 

30

5. Many large coalesced lesions accompanied by
tissue breakdown and sometimes leaves became

chlorotic and abscised.

 

RACE IDENTIFICATION

The pathogenic variation of p; lindemuthianum
has been well documented (4, 10, 13, 15, 30, 32, 36, 37,
42). Ayonoadu (5) has reported this variation in bean
anthracnose from Malawi and identified the presence of
beta, gamma, delta and three new races. But the number
of race groups appeared to depend on number of differen-
tial varieties used and the system of classification was
unsatisfactory because of lack of standardization. This
study was aimed at using a proposed standard set of

differentials.

Materials and Methods

 

Collection of Isolates

 

Sixteen bean anthracnose isolates (Table 1 and
2) were collected for race identification. Nine of
these came from Malawi and were brought by Dr. M. W.
Adams in 1984 and 1985. Six came from Tanzania and one
from Kenya collected by Dr. J. D. Kelly during a trip to
Tanzania and Kenya in 1985. All isolates came from bean
plants that were naturally infected in the field. The
infected tissues were preserved by air drying upon their
collection and put under refrigeration (60C) when they

arrived in Michigan.

31

32

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.Hw\ome .ow\m>ma mcommmm MCflzoLm Lsom song cmxmp HHBLCHML Hacommom ommcm><o

.mw\mmma new
.mm\awma .Hm\owma .ow\o>ma mcowmom mafisocm Loom :H :mxmp omcmc ccsumcoaeoen

.omocomcsuc¢ :mom com A<mv oopmfl>ocnnm one mopmaowHw

 

 

 

limo? - coma Essie L84 mam
m.oaoa omnoa wona manna mam; w<m
m.oHoH omloa Nora mwcom mmmq ><m
m.mam mNIHN ONQH mzmcoaflq mama 04m
m.oaoa owloa soza «some when m<m
0.3m :muom cg 39:: eon is
N.H:m I omma mzoo mam; m<m
m.oaoa omuoa Nona swoon new; m<m
N.H:m I omma mzoo mama H<m
Aco<upoov Aomcwmv Aev ooflm socm mmumaomH
0:55 988 $332 US$03
Hawucflmm ocsumeoq809 Scam
Hzmamz

 

seem AessmfinuseooCHH EBLOHLpouoHHoov mmocomccucm smog mo moumaomH .H m4m<e

 

33

TABLE 2. Isolates of bean anthracnose(Colletotrichum
lindemuthianum) from Tanzania and Kenya.a

 

 

Isolateb Part Isolated From Site
BAlO Leaf Magamba
BAll Leaf Same
BA12 Leaf Lyamungu
BA13 Pod Kabete
BA14 Leaf Mlingano
BA15 Leaf Same
BA16 Leaf Magamba

 

aIsolate BA13 came from Kenya.

bIsolates are abbreviated (BA) for Bean Anthracnose.

 

34

Seed Source

 

The bean cultivars used as standard differen-
tials (Table 3) for race identification were from stocks
maintained by Dr. A. W. Saettler at Michigan State

University.

Plant Management

 

The twelve differential varieties were grown in
plastic flats containing 21 pasteurized. 3:1 mixture of
soil and vermiculite. Water was provided ens needed.
Overwatering was avoided especially during the first
week of pdanting which led to better germination. Each
flat had twelve inserts and each insert was planted to
six seeds, one differential variety for each insert.
The greenhouse temperature fluctuated between 24 and

300C.

Inoculation of Plants

 

Plants were inoculated using 21 spraying techni-
que. In this method the inoculum was sprayed as a fine
mist onto upper and lower leaf surfaces and the entire
stem. An aerosol containing can (Spra—tool No. 8011
power Ixnc, Crown Industrial Products Co.) was used as a
sprayer (Figure 2). Care was taken to avoid contamina-
tion between isolates more than one isolate was used.
Physical barriers were put in the chamber to separate

inoculations with different isolates. Inoculations were

 

35

 

 

I I I I I I I I I I oma m< NA
I + I I I I I I I I Nmm oonmz an
+ I + I I I I I I I oHB=Ho>m 0H
+ I + I + I I I .t I NamIa: nauseoo a
+ + I I I I + I I I omamoHHm w
I + I + I I I I + + momwoAHm F
+ I + + I + I I + I cameo mam 0000 o
I I + + + + I + I I zoLLm: 5.5m m
+ I + + + I I + + I omaficmm :
I I + + + + I + + I .¥.m.o .aonz m
+ + + + + I + + + + aco> oaaasms< N
I + + + + + + + + + sausages: H
ammo Hansen msoH menses «same meeao eosamam magma msmm means asentm> agate
mnaa< Hmflucococmfia wocmpmamom

 

.ecmaaoz .e>H .saOSLnHLo .m as mcaeeooom .eacmncssemecns
eszoflcpoumaaoo no moomc no :oHumoHeHucoUH on» so; mamflococoeeflc mo owcmc ocmccmom oomoqocm .m m4m<e

 

36

 

Figure 2. An aerosol containing can (Spra-tool No.
8011 Soil Power Pak; Crown, Industrial
Products Co.).

 

37

done 12 to 14 days after planting. Plants were put in
mist chambers maintained at near 100% relative humidity
and ambient greenhouse temperature. The high humidity
inside the chambers was maintained by using humidifiers
(Model 500 Hermidifier, Hermidifier Co. Inc.). The
humidifiers were supplied with tap water and made to run
continuously. Plants were kept in the chambers for
about a week. Symptoms usually appeared between 6 to 8
days after inoculations. Disease counts were done twice
for each inoculation, and every inoculation was

repeated.

Results

Isolates BA1, BA2, and BA3 (Table 4) were the same
as Alpha-Brazil. BA4, BA5, BA6 were unique isolates.
BA7 behaved as BA8 and BA9 and was different from all
known races. The Tanzanian isolates, (Table 5) BA10,
BA12, and BA14 differed from all known races. BA16 was
the same as lambda. BAll and BA15 were the same and
represented a unique isolate. The Kenyan isolate, BA13,

behaved differently from all known races.
All the Malawian isolates infected Auguille
Vert. Michelite was another poor source of resistance to
these isolates showing resistance only to BA6. Cornell
49-242, Evolutie and A8136 were the only differential

varieties that were resistant to all the isolates.

38

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.ucmpmflmom n I ”manapaoomsm u +m
I I I I I I I I I oma m<
I I I I I I + + + NNN coaxoz
I I I I I I I I I mausao>m

I I I I I I I I mzmIm: Hameeoo

 

 

I I I I + + + + + omzmoaHm
I I I I + I + .+ + oommoaHm
+ + + + + + I I I cameo mam 0000
+ + + + I + I I I gonna: xenon
I I I I I + I I I omaflcmm
+ + + + I + I I I .M.m.o cmmneoflz
+ + + + + + + + + .uLm> oaaflsms<
+ + + I + + + + + mpflaozoflz

m<m w<m ><m m<m m<m z<m mam m<m H<m >uoflem>

nmopmaomH Hafipcoeomhaa

 

 

.Hzmam: Eoem sscmHLQSEmUCHH ESQOHLquoHHoo
mo moumaomfl o» mamaucoLoMMHo chocoML5pcw ocmvcmom mo chHpomom .: mqm<e

 

39

.AomocomL5pc< :momv <m noumfl>ocnnm one moumaomHo
.mflcmucmw sore omen on» .mmcom seem ma ma<m opmaomHn
.DCMpmHmom u I “sandpaoomsm n +m

 

I I I I I I I 92 ma
I I I I I I I mmm coda:
I I I I I I I @330;

I I I I I I I szlm: Haocnoo

 

 

I I I + + I + om:moHHm
+ I + I + I I mmmwoaHm
+ + + + + + + oeoco mam 0000
+ + + I + + + ZOLLmz zonmm
+ I + I I I I omHHcmm
+ + I + + + + .M.m.o cmeSOHz
+ + + + + + + .peo> oaaflsmz<
+ I I I I I + ouHH650flz
oa<m mH<m na<m oma<m NH<m Haam oa<m >poflcm>

omopmaomH Hmfiococomufla

 

 

.nm>com song ozo ucm mflcmmcme soup azcmflnQSEoBCHH ESSOHLpOBoHHoo mo
woumHomH xflm on mamflpcocoumao omocomcspcm cemocmpm mo «coapomom .m m4m<e

 

no

All Tanzanian (non—Malawian) isolates attacked
the differential varieties Auguille vert. and Coco ala
creme, but Cornell 49—242, Evolutie, Mexico 222 and AB

136 showed resistance to all seven isolates.

 

 

CROSS PROTECTION VERIFICATION

Before the screening of the Malawi bean land
races was conducted a small study was done to evaluate
the independence of reaction of the two individual
isolates, BA3 and BA4, that were to be used as a
mixture. Various researchers, Berald et a1. (12),
Elliston et a1. (29), Rahe et al. (52), Skipp and
Daverall (60) have reported induced resistance by a non-
pathogenic race against a pathogenic one. This study
was done to verify any interference or non-interference

by the two isolates when used in the screening program.

Materials and Methods

 

Eight varieties (Table 6) from the standard
differentials were chosen based on their reaction to
each of the two individual Malawian isolates, BA3 and
BA4. These varieties were planted in plastic flats and
all treatments remained the same as those used for race
identification. In the preparation of the inoculum,
each isolate was prepared separately, adjusted to a
concentration of 2.0 x 106 and then mixed in equal
volumes. Inoculation procedures were the same as for

race identification.

41

 

42

TABLE 6. Selected varieties and their reactiona to two

 

 

 

isolates.
Individual Reaction Mixture
Variety BA3 BA4 Reaction
Auguille Vert. S S S
Michigan D.R.K. R S S
Coco ala creme R S S
P1167399 S R S
P1165425 S S S
Cornell 49-242 R R R
Mexico 222 S R S

AB 136 R R ‘ R

 

aS = Susceptible, R = Resistant.

 

43

Results
Assuming no interference between the two
isolates the expected are susceptible reaction for all S
+ S, R + S, and S + R combinations and resistance only
to the R + R combination. Any deviation from these, for
instance, getting a resistant from S + S, R + S, or S +
R combinations, would be indicative of some cross

protection or induced resistance.
The results presented in Table 6 show no

evidence of cross protection.

 

GERMPLASM SCREENING

Breeding for disease resistance to C; lipgg:
muthiaum depends on identifying bean genotypes that show
resistance to the races of the pathogen. Malawi has one
of the most diverse bean germplasm pools in Eastern
Africa. The germplasm collection at Bunda College
probably contains the largest collection of African bean
landraces (27). The genetic variability of this germ-
plasm pool should inevitably be reflected in disease
resistance. This study was conducted in order to iden-
tify sources of resistance to two of the isolates of C;
lipdemuthianum from Malawi. Some of the bean germplasm
pool was also tested against a Tanzanian isolate to
assess its potential as a source of resistance to other

C; lindemuthianum races.

Materials and Methods

 

Seed Source

The Malawian bean landraces were obtained
through the Bean Cowpea Collaborative Research Support
Program (CRSP), and supplied by Dr. M. W. Adams. These
landraces were collected from the central and northern

regions of Malawi and were quite diverse (Figure 3).

44

45

 

A Sample of Malawian bean landraces used in
screening for sources of resistance.

Figure 3.

 

46

The Malawian bean varieties were from the stocks
of the Bean Research Program of Bunda College of Agri-
culture in Malawi. The rest of the bean varieties came
from stocks maintained by Dr. Adams and Dr. A. W.

Saettler at Michigan State University.

Plant Management

The landrace lines were grown in plastic flats
containing a pasteurized 3:1 mixture of soil and vermicu-
lite, water was provided as needed and all management
remained the same as those used for differentials in
race identification. The same plant management was

applied to varietal screening.

Inoculation of Plants

 

Plants were inoculated using a spraying method.
All the procedures were as those used for race identi-
fication. The landraces were screened using the mixture
of BA3 and BA4 while varieties were screened with the

same isolates separately.

Results
When more than 220 bean landrace lines were
screened with a mixture of BA3 and BA4 isolates, 19
landraces (Table 7) were resistant to both isolates.

Only about 8.6 percent of the lines tested were

 

47

TABLE 7. Bean landrace germplasm identified as resist-
ant to a mixture of two isolates of Colleto—
trichum lindemuthianum from Malawi.

 

 

 

 

Seed Isolates
Accessiona g/100 seeds Color Resistant to
M86 35.0 Black BA3 + BA4
M817 21.3 Red "
M825 36.7 Brown "
M828 39.5 Brown "
M870 32.7 Brown "
M890 34.3 Tan "
M892 44.5 Red Tan "
MB97 28.5 White "
M899 28.9 White "
M8108 23.9 Mottled "
M8112 29.2 White "
M8113 25.9 White "
MB114 27.4 White "
M8119 42.0 Red "
M8124 40.0 Brown "
M8157 41.7 Red "
M8191 30.8 Black "
M8198 34.5 Red "
M8205 28.2 Red "

 

aM8 = Malawian Bean.

 

48

identified as sources of resistance to both isolates.
Although resistance to each isolate individually may be
higher than 8.6 percent, the results show a remarkably
low level of resistance to bean anthracnose in the
Malawian bean landrace germplasm. However, it is
unlikely that the 8.6 percent could have been much lower
had all the 9 Malawian isolates been used for screening,
since the two isolates chosen for the mixture (BA3 and
BA4) represented almost all the total possible
combinations of reaction of the 9 isolates to the
differentials used for race identification.

Screening using mixtures proved to be an effi-
cient way of searching for resistance when dealing with
large populations or limited seed where fewer repeats
are necessary. The system saves time and space.

There were some lines that showed resistance to
more than the two Malawian isolates when they were
inoculated with a third isolate, BA16 from Tanzania.
Out of 70 Malawian bean landrace lines screened to all
three isolates, six (Table 8) were shown to confer
multiple resistance. This represents about 8.5% of the
lines tested.

Reaction of Malawian Bean Varieties to
Three Anthracnose Isolates

 

 

Some of the Malawian bean varieties are resis-

tant to the individual isolates BA3, 8A4, and BA16. Six

 

 

49

TABLE 8. Malawian bean landraces identified as resistant
to three isolatesa of Colletotrichum linde-

 

 

 

 

muthianum.

Seed Isolates
Accessionb g/100 seeds Color Resistant to
MB99 28.9 White BA3+BA4+BA16
MB108 23.9 Mottled "
M8112 29.2 White "
MBll3 25.9 White "
MB198 34.5 Red "
MB205 28.2 Red "

 

aIsolates are abbreviated BA (Bean Anthracnose). BA3
and BA4 are from Malawi and BA16 is from Tanzania.

bM8 = Malawian Bean.

 

 

50

of the varieties conferred resistance to isolate BA3
(Table 9). However, there was very low frequency of
resistance to isolates BA4 and BA16. Only two varieties
gave resistance to isolate BA4 and one to BA16.
Although no variety was able to give resistance to all
three isolates, the Malawian variety 1212 D gave an
immune reaction to the two Malawian isolates (8A3 and
BA4). Variety 336 was also resistant to the Malawian
isolate BA4 and the Tanzanian isolate BA16. These two
isolates attacked many of the differentials and
represented the most virulent isolates. Figure 4 shows
the reaction of some Malawian bean germplasm to BA4.
Only one variety, 1768 D, succumbed to all three
isolates.

There was an appearance of complementarity
between the two Malawian isolates, 8A3 and BA4. Except
for the reaction of 1768 D, all the varieties attacked
by one isolate gave resistance to the other. The
Tanzanian isolate BA16 behaved similarly to the BA4
isolate, although its reaction to 1212 D was not tested.

Reaction of Other Bean Varieties of

P. vulgaris to Two Isolates of
C. lindemuthianum From Malawi

 

 

 

When other bean varieties were inoculated with
the two Malawian isolates, 8A3 and BA4, some resistance

to either isolate was identified. Six varieties (Table

 

 

 

51

TABLE 9. Reactiona of some Malawian bean varieties of
Phaseolus vulgaris L. to three isolates of
Colletotrichum lindemuthianum.

 

 

 

 

 

Seed Isolateb
Variety g/lOO seeds Color BA3 BA4 ' BA1645
336 26.9 Red S R R
lO39D 46.8 Mottled R S S
l212D 15.7 White R R NT
1251D 48.2 Mottled R S NT
1768D 41.3 Red S S S
2482D 36.6 Yellow R S S
2539D 41.2 Mottled R S S
Nasaka 44.9 Tan R S NT

 

as = Susceptible; R : Resistant; NT = Not Tested.

bIsolates are abbreviated BA (Bean Anthracnose).
BA3 and BAu are from Malawi and BA15 is from
Tanzania.

 

 

52

 

Figure 4.

Reaction of some Malawian bean germplasm to
isolate BA4.

 

 

53

10) gave resistance to isolate BA3 and 17 gave a
susceptible reaction. Approximately 1/3 of the varie—
ties tested were resistant to this isolate. Isolate
BA4, which represents the most virulent of the Malawian
isolates, managed to infect only 8 of the 24 varieties
tested. This indicates a higher frequency of resistant
varieties to susceptibles for this isolate. But this
was just the opposite for isolate BA3 which showed lower
frequency for resistance to susceptibility in the same
varieties. Although resistance to either isolate was
identified in quite a number of varieties, only three
varieties, Charlevoix, Montcalm, and Seafarer, gave
resistance to both isolates. Five varieties, 70-700 (an
experimental line), Isabella, Pompadour—Checa, Redkloud,
and Ruddy showed susceptibility to both isolates.

From the 70 bean landrace components tested
against the Tanzanian isolate, BA16, only 13 lines
(Table 11) showed a resistant reaction.

This indicates the potential role the Malawi
bean landraces have as sources of resistance to foreign
isolates of p; lindemuthianum. And since the BA16
isolate represented the more virulent, in terms of
number of differentials it attacked, it is entirely
possible that more resistant lines could be identified

for less virulent isolates that come from Tanzania.

 

54

TABLE 10. Reaction3 of other bean varieties of Phaseolus vulgaris L. to two
isolates of Colletotrichum lindemuthianum.

 

 

 

Seed Isolatesb

Variety g/lOO seeds Color BA3 BA4
Aurora 14.7 White S R
Bat 41 20.0 Dark Red 8 R
Bat 332 16.0 Cream S R
C-15 18.9 White S R
C-20 19.7 White S

70-700 52.9 Light Red S

Charlevoix 53.5 Dark Red R R
Fleetwood 18.9 White NT R
G-04534 62.4 Mottled R S
Isabella 53.5 Light Red 3 S
Jamapa 21.4 Black S R
Manitou 53.5 Light Red R s
Mecosta 53.5 Light Red R S
Midnight 16.8 Black S R
Montcalm 53.5 Dark Red R R
Pinto VI-lll 40.7 Pinto S R
Pompadour (Checa) 43.6 Mottled S S
Porrillo 22.0 Black S R
Puebla 13.0 Black s R
Redkloud 53.5 Light Red 3 s
Ruddy 50.5 Light Red S S
Seafarer 18.9 White R R
Swan Valley 18.2 White S R
T 39 16.8 Black S R

 

as = Susceptible,

R = Resistant, NT = Not Tested.

bIsolate are abbreviated BA (Bean Anthracnose).

BA3 and BA4 are from Malawi.

 

 

55

TABLE 11. Malawian bean landraces identified as resistant to a Tanzanian isolate of
Colletotrichum lindemuthianum.

 

 

 

Accession g/loo seeds Seed Color Isolate Resistant to
MB 12 23.5 Brown BA15
MB 14 18.2 Red "
MB 22 34.3 Black "
MB 23 24.9 Black "
MB 23 24.9 Black "
MB 99 28.9 White "
MB 108 23.9 Mottled "
MB 112 29.2 White “
MB 113 25.9 White "
MB 169 18.5 Dark Brown “
MB 198 ' 34.5 Red "
MB 205 28.2 Red "
MB 210 26.3 Cream . "

 

SAGIsolates are abbreviated BA (Bean Anthracnose).

 

 

 

GENETIC STUDY

The genetics of resistance to Q; lindemuthianum
has been studied in various bean genotypes using differ-
ent races (4, l4, 17, 45, 46, 47, 50). However, no such
study is documented for Malawi. Many of the bean
anthracnose studies have concentrated on screening for
resistance and not on genetics of resistance. Knowledge
of the genetics of resistance enables a plant breeder to
anticipate possible interactions and linkages in
selected crosses and devise the type of breeding
strategy and selection procedures to use. This study
was formulated to determine the inheritance of resis-
tance in five selected genotypes one of which was from
Malawi, and in which two bean anthracnose isolates from
Malawi were used.

The joint segregation of the F2 populations to
simultaneous reaction to two isolates of the pathogen
was tested against the theoretical joint segregation
ratios for the two isolates. This was obtained by
multiplying together the segregational ratios to each of
the isolates. Plants were classified as resistant to
both isolates, resistant to the first isolate but

susceptible to the second, susceptible to the first

56

 

 

 

57

isolate but resistant to the second isolate and
susceptible to both races, and denoted as R R, R S, S R,
and S S, respectively. The observed and expected
numbers of plants in their respective categories were

compared by using a X2 (Chi-Square) test.

Materials and Methods

 

Seed Source

Parental and F2 seeds used in the genetic study
were supplied by Dr. J. D. Kelly. The choice of the
parents of the selected crosses was based on their reac-
tion to two bean anthracnose isolates from Malawi (Table

6).

Plant Management
Plants were planted in 10 cm clay pots (1 plant

per pot). Soil/vermiculite mixture, water requirements
and greenhouse temperature remained the same as the
growth conditions for differentials used in race

identification.

Inoculation of Plants

 

Parental plants were screened using a spraying
technique for inoculation. The F2 plants were inocu-
lated with BA3 and BA4 using a brushing technique.
Under this method, a brush (No. 4 camel's hair brush)

was used to apply the inoculum. The inoculum was

 

 

58

deposited on the under side of each of the lateral
leaflets of the first trifoliate leaf.

Each of the lateral leaflets was inoculated with
one of the two isolates; two people using two separate
brushes, one for each isolate, performed the inocula-
tions. Plants were usually inoculated 14 to 18 days
after planting when the first trifoliate leaf was fully
expanded. Chamber and greenhouse environments remained

as for differentials.

Data Analysis
The segregating plants in the F2 populations for

resistance and susceptibility were tested against the
theoretical ratios of resistance to susceptible plants

by using a X2 (Chi—Square) test.

Results

Reaction of Parent Bean Cultivars to Each of the Two
Malawian Isolates

 

The Malawian navy bean variety 1212 D showed an
immune reaction to both isolates. This variety was the
only white seeded type used in the study. The rest were
all red seeded. BAT 41 gave a resistant reaction when
inoculated with BA4 isolate but was susceptible to BA3
isolate. Isabella showed a susceptible reaction to BA3
and was moderately susceptible to BA4. Montcalm gave

resistance to both isolates, however, this resistance

 

 

 

59

differed from that of 1212 D. Very tiny flecks occasion-
ally appeared on Montcalm as opposed to a complete immu-
nity shown in 1212 D. Ruddy was the only variety that
showed complete susceptibility to both isolates. The
reaction results and seed characteristics of these bean
varieties are presented in Table 6.

Reactions of F2 plants were observed in

susceptible leaflets (Figure 5).

BA3 Isolate

Four F2 populations were tested against the BA3
isolate. The observed segregation of resistant and
susceptible plants to this isolate is presented in Table
12. In crosses between Ruddy X 1212 D the proportion of
resistant to susceptible plants fit a 3:1 ratio. This
indicates that a single dominant gene conferred resist—
ance to this isolate. This gene came from the resistant
parent 1212 D. The same segregation ratio was observed
in the cross between Isabella and 1212 0, indicating a
similar gene system. In crosses between Montcalm X 1212
D the F2 plants showed no segregation. All plants gave
a resistant reaction to BA3. This may mean that the
genes conferring resistance from both parents are iden-
tical or are closely linked. It would seem unlikely
that the two parents have the same gene or genes for

resistance because of the previous differences observed

 

 

60

 

Figure 5. Trifoliate leaf of F2 plant from a Ruddy x
1212 D cross showing a resistant reaction to
BA3 (right) susceptible to BA4 (left).

 

 

61

rem on coauomcoc Lou scan Lozoa we» use m<m cu cofipommc

.cmemoo coaumasaoa ocean me no cones: Hmuoe-

Loo muasmoc on» mo>Hw magma o

.ucmumflmom AH< n m<n

.moumaomfl
no no acme Loam: scam

 

 

 

m». I m. H ma m.: H.m> z :5 mp z x m o NHNH x maamnmmH

I m< o o.mma o 00H mma m x m a NANA x aamoucoz

me. I m. a 0 :.mm e.m: mm m: am e x m a: Hem x sense

mm. I co. m ma m.:m m.o:a :m oma :ma m x m Q NANA x >uvsm

mp. I m. H m m.mH m.wm mm om me x x m G mama x mafiwnmmH

I m< o 0.00H o mma mwa m x m a mama x Eamoucoz

mNo. I Ho. MH m m.mo N.ma pm :N an m x m a: H<m x zoosm

om. I mN. H m 0.0: o.wma om :MH zwa m x m a mama x zvozm

com3pon m m m m m m 2 coauommm mmOLo
howaflnmnocm oHumm nooomaxm vooooaxm um>ewmgo aducotmm

 

«.mocmaa Amy oHnHoQoomsm cam
.Amv ocmumfimoc no mofipme eouooaxo ecm escmflguseoucfla EscoHLSOSBHHoo mo mopmaomfl oz» 08 macapomoe mm .ma mnmee

 

62

in the degree of resistance, as 1212 D showed complete
immunity, though the difference may be due to different
genetic backgrounds or modifying genes.

In the cross Ruddy x BAT 41 a 3:13 ratio was
observed although both parents are susceptible. I
postulate that one parental variety has a resistant gene
pair but is unable to express it, because of an inhibit-
ing factor. If the inhibiting factor is denoted by I
and the resistant factor by R, one variety has proposed
genotype IIRR and the other has genotype iirr. In the
F2 population resulting from a cross between the two
susceptible parents, 3/16 of the progeny would show
resistance and 13/16 susceptibility. Although this
ratio is not like the other two—factor ratios, it may be
explained in the same way with the additional assumption
that the inhibiting factor I is epistatic to the effect
of the resistant factor R.

In determining the appearance of the F2 plants,
it should be remembered that, wherever I is present,
resistance is inhibited, so that plants which inherit I
are susceptible whatever other factors they may receive
such that IIRR, IIRr, IiRR, IiRr, IIrr, Iirr and iirr
genotypes would show a susceptible reaction while iiRR
and iiRr would be resistant.

The above postulation fit the 3:13 ratio at a

very low probability (P between 0.01—0.025) and gives a

 

 

 

63

weak support to the inhibitory hypothesis. A ratio of
1:3 would have given a better fit (P between 0.25—0.5)
and this would mean that a single recessive gene confers
resistance to BA3. But both parents, Ruddy and Bat 41.
are susceptible to BA3 and this cannot be explained by a
single gene hypothesis. It would, therefore, seem
appropriate not to discard the 3:13 ratio on account of
its poor fit.

The results for reaction to the BA4 isolate show
a completely different mode of inheritance as compared
to the BA3 isolate. In crosses between Ruddy and 1212 D
the observed segregation of resistant and susceptible
plants fit a 13:3 ratio. Apparently two independent by
segregating loci are involved in conferring resistance
to the BA4 isolate in this particular cross. One of
these gene pairs segregates into a 3:1 and the other
into a 1:3 ratio. Both genes would have come from
variety 1212 D which was resistant to both isolates.
The single dominant gene responsible for the 3:1 segre-
gation may have been the same one that conferred resist-
ance to the BA3 isolate. The two genes segregate inde-
pendently and either one is capable of producing a
resistant reaction. Denoting the Ruddy x 1212 D cross
as aaBB x AAbb, the small b is epistatic resistant over
small a and large A epistatic resistant over large B

such that AABB, AABb, AaBB, AaBb, AAbb and aabb

 

64

genotypes would show a resistant reaction but aaBB and
aaBb genotypes would be susceptible.

In crosses between Ruddy x BAT 41 the observed
segregation of resistant to susceptible F2 plants fit a
9:7 ratio. This segregation pattern suggests that there
were two pairs of complementary genes conferring resist-
ance to this isolate. These genes may have come from
BAT 41 which was the resistant parent in the cross.

The F2 plants from the Montcalm x 1212 D cross
did not segregate for susceptibility. All the plants
gave a resistant reaction. This may mean that the genes
conferring resistance in the two parents are closely
linked or possibly the same genes since the difference
in the resistance reaction may be a result of differ-
ences in genetic background or results of effects of
modifying genes. The same findings were true for the
BA3 isolate. The segregation of resistant and suscep-
tible F2 plants in the cross between Isabella x 1212 D
showed a good fit to a theoretical ratio of 15:1. The
data indicate that two dominant duplicate gene pairs
confer resistance. One pair may have come from 1212 D
and the other from Isabella. However, Isabella is
moderately susceptible to the 8A4 isolate. It is
therefore more conceivable that both pairs of duplicate

genes could have come from 1212 D.

 

 

I
I. _l

65

Evaluation of the four F2 populations with
regard to their reaction to the BA4 isolate indicates
that resistance is controlled by more than one pair of
genes compared to that of BA3 which showed simple

genetic control--most1y by single dominant genes.

Joint Segregation in F2 Populations

 

There were three F2 populations that were
evaluated for joint segregation for reaction to the BA3
and 8A4 isolates. This was done in order to ascertain a
degree of association in factors conditioning reaction
to the two isolates. The results are shown in Table 13.

In the cross Ruddy x 1212 D the segregation
ratio of resistant and susceptible F2 plants was 3:1 for
the 8A3 isolate and 13:3 for BA4. The theoretical joint
segregation computed as the product of the two ratios
was 39:9:l3z3. The observed number of plants classified
in the reaction classes fit the theoretical ratio. This
means that there was independent association in factors
conditioning reaction to the 8A3 and BA4 isolates.

The observed joint segregation of F2 plants in
the cross between Ruddy x BAT 41 was computed but gave
inconclusive results. The joint segregation observed in
the cross between Isabella x 1212 D did not deviate from
the theoretical ratio of 45:3:15zl. It fit the expected

ratio at the probability level of 0.5-0.75.

 

 

 

66

 

 

 

 

 

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35 mm; m a m m 2.3113
910 mN.mA 0N ma z m o NANA
m>.o oo.m N m m z x
Ho.o =m.:m :m m: m m 933.33
mN.IH. -.m o.=m~ :wH :o nausea
m:.N mo.m : m m m Am”MAVAH"mv
om.a mm.~m 0: ma z m a NANA
oo.o mm.mN om o m m x
36 «TN: :3 mm m z >322
m Nx .nxm .nno noaumz z<m m<m Nm
nucmam ho Lenszz amoauococzp nnmao :oHuomoz new nmocu

I .azflmz a9... 3%

aszounuOUwHHou ho nonmaona oz» cc coaoomoe Lou mamzoocmumgzaqm wcaummohmon
monnoco oz» no macaumflsnon mm «o ceauomoe ca noaocwscccc cocooaxm new cw>eonno .mH mgmep

 

 

67

In all, the three F2 populations studied for
joint segregation for reaction to the BA3 and BA4

isolates showed independent association.

 

DISCUSSION

Based on the reaction of the nine Malawian

Colletotrichum lindemuthianum isolates to the standard

 

set of differentials for the identification of races of
p; lindemuthianum, according to T. Drijfhout, IVT,
Holland, an alpha Brazil race has been identified.
Although previous studies have reported the existence in
Malawi of beta, gamma, and delta races (5) this is the
first time that the existence of alpha Brazil is
revealed in Malawi. The other isolates tested show some
differences to the known races. The lack of correspon-
dence between Malawian isolates and the identified races
on the standard differentials is not surprising. Leakey
and Simbwa—Bunnya, (44), reported similar findings with
Ugandan and European isolates. It was reported from
CIAT (18) that when several isolates from different
areas of Colombia were tested against the European
differentials (the same as those used in this study)
they appeared to be different from all races of C;
lindemuthianum previously reported. All this reinforces
the view of the previously known world diversity of

races and sub-races of this organism.

68

 

 

 

 

 

69

A similar lack of conformity is evident for the
other isolates (six from Tanzania and one from Kenya).
Only one isolate, BA16, gave a reaction that clearly
identified it as lambda.

It is worth noting that as the number of stan—
dard differentials used increases, so does the likeli-
hood of separating isolates that might have been
previously lumped together in one group, based on their
reaction to fewer differentials.

A comparison between and among the Malawian and
the other isolates show some notable similarities and
differences. None of the Malawian isolates broke down
the resistance of Cornell 49-242, Evolutie and A8136.
Auguille Vert. was the most vulnerable differential,
being attacked by all the isolates. Another similarity
is evident when the most virulent Malawian isolate (8A4)
is compared to BA16, another most virulent isolate from
Tanzania. Both isolates attacked seven differentials
and gave a similar pattern of reaction except that BA4
attacked P1165426 instead of P1167399.

Some isolates found both in Malawi and Tanzania
were identical. For instance, isolate 8A6 from
Lilongwe-Malawi, was identical to BAll and BA15 from
Tanzania. Some of the Malawian bean anthracnose
isolates occurred in more than one location. Isolate

8A1 and BA3, both of which were from Dowa, in the

 

70

central region, were identical to 8A2 from Dedza, in the

southern region. These isolates were the same as alpha-
Brazil. Dedza was one location that showed more varia-

tion of isolates. This may be due to the greater number

of isolates collected from the district, which was far

more than from any other location/site.

When the results on race identification in this
study are compared to those of Ayonoadu (5), some
notable differences arise. This comparison, which is
based on five differentials, Michelite, Dark Red Kidney,
Perry Marrow, Sanilac and Cornell 49-242, show that the
16 isolates (8A1 to BA16) used in the current study
would have been classified into alpha, delta, gamma and
one unidentified group. BA1, BA2, BA3, and 8A5 would
have been identified as alpha. Isolate BA4 would have
been classified as delta. BA6 would have been called
gamma while BA7, BA8 and BA9 would have been determined
as a new race. This means that more known races and few
unique ones would have been identified. The current
study has identified only one known race, alpha-Brazil,
and many unique types from the Malawian isolates. It is
important to note that in the previous study, gamma was
found to be the most abundant race, comprising more than
half of the isolates. The present study found no such
domination by any single isolate although alpha-Brazil

(8A1, 8A2, and BA3) and the unique isolate represented

 

 

 

71

by BA7, BA8, and BA9, were both found three times. The
lack of domination by any single race can be explained
by the genetic diversity found in both host and
pathogen. Plants affect their own environments and thus
the environments of their pathogen. The nature, disper-
sion, and genetics of crops affect not only disease
development in local populations but the spread of
disease among populations, from field to field, and
region to region, and the genetic adaptation of fungal
parasites.

Traditional landraces and cultivars have
advantages to the limited resource farmer. They repre-
sent centuries of continuous selection for local perfor-
mance. The farmers' strategy was not so much for
greater performance in the best years as for adequate
performance in the worst. These landraces lag behind in
yield but may excel in adaptability, in resistance to
diseases, and other extreme conditions. Such qualities
are enhanced by genetic diversity. Modern agriculture
demands uniformity much to the detriment of yield stabi—
lity.

The co-evolution of host and parasite has a
strong equilibrium component, laid down in the genetic
make-up of both host and pathogen. The result is a
balance between the amount of host and the amount of

pathogen. The pathogen needs the host and is influenced

 

 

72

by it, where as the host does not need the parasite but
is nevertheless influenced by it. The balance results
from a dynamic equilibrium. Actual amounts of host and
pathogen vary between and within years. The more
diverse the genotypes, the more diverse the pathogen.
It is, therefore, less surprising that more unique
isolates were identified.

Another notable finding is that all the isolates
classified as 'new races' in the previous study by
Ayonoadu (5) had been from Bunda. It may not be surpris-
ing to find that the new races could have been either
alpha-Brazil (n: a unique isolate corresponding to BA6.
This isolate was found both at Bunda and in Tanzania by
the current study. It may have been transmitted through
bean seed exchange between Bunda-Malawi and Tanzania.

The importance of using a. standardized set of
differentials for race identification is further sup-
ported in this study. Proper race identification is
crucial for breeding for bean anthracnose and seems to
depend on the number and type of differentials. A plant
breeder may identify alpha, beta and gamma as the exist-
ing races in an area and may request seed, from else—
where, that is resistant to the three races. However,
if some unique races exist but were not revealed by the

differentials used, the requested ”supposedly resistant"

variety may show susceptibility. Plant breeders need to

 

 

73

test any given variety against indigenous known races.
This would act as a check for any unique races that may
have been incorrectly identified on account of the
number and type of differentials used.

Among the differences was the reaction of Mexico
222 to the Malawian and the other isolates. The three
Malawian isolates belonging to alpha-Brazil overcame the
resistance of Mexico 222 while none of the non- Malawian
isolates could do so. Michelite was resistant to many
of the other isolates but succumbed to all but one
isolate from Malawi. Coco ala creme gave an opposite
reaction to that of Michelite, showing susceptibility to
all other isolates but resistance to some of the
Malawian isolates.

The screening of the Malawian bean landrace
lines revealed a low level of resistance to the Malawian
C; lindemuthianum isolates. Only 8.6 percent of the
more than 220 bean landrace lines gave resistance to the
two isolates used in the mixed-isolate screening pro—
gram. This is rather puzzling because one would expect
that the diverse nature and persistence of these
landraces over the years would be reflected in their
ability to maintain adequate gene frequencies for
adverse environmental factors, disease resistance being
one of them, especially so for resistance to bean

anthracnose, a disease found in practically all bean

 

74

growing areas of Malawi and ranked as the most destruc-
tive. The only plausible explanation may be that under
natural conditions these environmental factors exert
varying selective pressures from season to season, field
to field and year to year. Because of this inconsistent
selective pressure, gene frequencies for various traits
shift from time to time depending on what pressures are
present. In a season when bean anthracnose becomes more
prevelant gene frequencies for resistance to the disease
may be selected for but if the disease becomes less
prominent in the next season, there may be selection
towards another trait. This kind of natural selection,
although assuring stable performance over numerous
adverse environmental conditions, reduces gene frequency
levels in the landrace populations for any single trait,
and this could be true not only for disease resistance
factors but for other characters as well.

The screening of the existing Malawi bean varie-
ties, although based upon far less numbers (only 8) in
comparison to the landraces (over 220), showed the
presence of some good sources for resistance.

With respect to diseases in natural populations,
where host and pathogens have had a long interdependent
association, disease levels may be expected to be low
and to vary only moderately over time. A change in

cultural practices may produce unexpectedly strong

 

75

changes in disease occurrence in a crop. The genetic
diversity found in the Malawian bean landraces
inevitably encourages more pathogenic variation but acts
as a buffer in ensuring that no single bean anthracnose
race dominates as this may destabilize the delicate
balance that exists, and thus threaten the survival of
certain genotypes. Any selection towards more resistant
genotypes is checked by increase in corresponding
virulent races and any build up of more virulent races
is minimized by an increase hi corresponding resistant
host genotypes. This lies at the crux of the dynamic
equilibrium referred to above, and explains the
diversity of the bean anthracnose races that exist in
Malawi.

In the quest for more economic crop production,
new management systems for specific crops are con-
tinuously developed. Some of these are counter-
productive in that they establish conditions favoring
higher disease risk. One trend in resistance breeding
is towards incorporation of vertical resistance genes.
This may lead to reduction in cultivars more vulnerable
to new physiological races. The introduction of another
cultivar with another resistant gene may cause it to be
overcome by another virulence factor in a different
race. The pathosystem enters a vicious cycle, the

boom-and-bust cycle, to the detriment of both farmers

 

 

 

76

and plant breeders. The pathogenic variability of bean
anthracnose in Malawi, inevitably, underscores the
importance of the need to understand the dynamics of the
landraces before attempting to improve them. Some
agricultural practices disturb natural stabilizing
mechanisms. Epidemics of plant diseases are often a
result of that disturbance.

Other bean varieties, (not from Malawi) have
also been identified as sources of resistance. Charle-
voix, Montcalm, and Seafarer were resistant to the two
Malawian isolates.

The differential varieties used for race identi-
fication have revealed some good sources of resistance
to some isolates used in this study and could be useful
in breeding programs. Of great interest are varieties
Cornell u9-2u2, Evolutie and A8136 which proved to be
resistant to all isolates. The performance of Cornell
49-242 reinforces the usefulness of the 'ARE' gene in
breeding for resistance to g; lindemuthianum in areas
from which the isolates tested in this study came.

The use of racial mixtures in screening for
resistance to g; lindemuthianum has been shown to be a
very efficient procedure. There is need, however, to
identify the reaction of each pure isolate, if only a

few need to be mixed, so that selections can be made for

 

 

 

 

77

the best combinations that represent the closest total
reaction if all the isolates were combined.

In the genetic study, there is evidence for a
simple inheritance mechanism controlling resistance to
BA3 isolate, while a more complicated system, involving
two genes, operates for the other isolate, BA4.

The Malawian variety 1212 D seems to possess a
single dominant gene for resistance to BA3 but possibly
two genes for resistance to the BA4 isolate. These
results indicate that 1212 I) could be a. good source of
resistance to the two isolates. The single dominant
inheritance observed for BA3 in the crosses involving
1212 D suggest that this gene system could easily be
incorporated in breeding for resistance to this isolate.
Resistance could be incorporated and fixed within a
short time. However, selections in the -early genera-
tions (F2 and F3) may be less efficient because all
heterozygotes would show resistance alongside the true
breeding dominant homozygotes.

Breeding for resistance to the BAR isolate can
also be achieved by using 1212 D as the resistant
parent. Although the mechanism of inheritance for BAH
is not as simple as for BA3, the independent segregation
shown in this study would facilitate a less complicated

breeding strategy.

 

78

The results of this genetic study have also
shown that there is independent association in factors
conditioning resistance to BA3 and BA“ isolates. All
the joint segregation for reaction to the two isolates
in two crosses studied fit the theoretical ratios
assuming independence. No unusually high frequencies of
parental phenotypes were observed in any of the crosses.
This means that the genes controlling resistance to BA3
and BAH isolates segregate independently.

This is important because it shows that breeding
for resistance to the two isolates can be less compli—
cated than would have been the case had joint segrega-
tion shown linkage.

Variety 1212 D has also been observed to confer

some resistance to halo blight (Pseudomonas syringe Pv.

 

Phaseolicola) and angular leaf spot (Isariopsis
griseola) (20, 49). This makes the variety a poten-
tially useful parent for disease resistant breeding in
Malawi. One disadvantage of 1212 D is that it is a navy
bean and may not appeal to many of the Malawian farmers
and consumers who prefer the red kidneys. The size and
color problems may limit using it in its pure form and
lead to using it as a parent in crosses. Being a
Malawian variety it holds adaptational advantages to

certain geographical and climatalogical areas of Malawi.

 

 

79

In order to increase bean production, all
factors that limit production need to receive careful
evaluation. It becomes apparent, however, that not all
factors can be dealt with at one time. In most cases,
there is inadequate financial, institutional and per-
sonnel support to deal with all problems of production;
at the same time such an approach may slow progress.
This inevitably leads to an approach that is based on
well defined priorities depending on what factors limit
production more significantly and how best to solve
them. In Malawi, bean diseases are the single most
important factor limiting production and C; lindemu-
thianum is one of the most widespread and destructive
among them. Control methods that involve the use of
chemicals are less attractive because of the expense
involved as well as their associated environmental
problems.

The use of disease-free seed and other cultural
practices, though helpful, have a prospect of limited
success because farmers keep their own seeds and they
may not follow sanitary practices. The ultimate solu-
tion to controlling this seed-borne disease is to grow
resistant varieties. This study has identified some
sources of resistance in the landraces. The few
resistant landrace lines offer some opportunities in

breeding for resistance. The results of screening of

 

80

eight Malawian bean varieties show additional sources of
resistance and comprise another useful focal point for
breeding against the disease.

This study has also reinforced the existence of
pathogenic variability of g;_ lindemuthianum in Malawi.
This means that any proposed breeding program should aim
at producing varieties that offer broad resistance.
Some of the differentials have shown such resistance.
Cornell H9-2M2, Evolutie and A8136 were resistant to all
the tested isolates. The use of any variety will, how—
ever, depend on other acceptable characteristics. It
has to be agronomically suitable and acceptable to the
consumer.

Obviously these resistant sources differ in
their performance for various traits. Those that excel
in most of the desired traits will have to be selected
for further evaluation, selection and/or hybridization.
For instance, the variety Montcalm, which offers resis-
tance to two of the Malawian isolate and is reportedly
resistant to some Malawian halo blight pathotypes, may
be selected over Cornell 49-242 on account of its seed
coat color (red), since consumers generally prefer large
red to small black seeds.

Consideration should also be given to how much
improvement a given resistant variety needs. For

instance, an existing variety may be chosen over a land-

 

81

race line on the basis that a landrace line may require
more improvements in other traits before becoming useful
as a variety even though both might be resistant to bean
anthracnose.

The breeding strategy will be based on selection
and hybridization. The selection step is now possible
because this study has identified landrace lines and
varieties that offer good sources of resistance to bean
anthracnose. The foreign varieties will have to be
grown under Malawian conditions in order to determine
their agronomic performance in that environment.
Further selections will then need to be made. In some
cases, some selections may perform well enough to be
'released as varieties. In situations where no single
variety performs well in all major traits, crosses will
have to be made to combine the desired traits and
develop superior recombinants through selection.

The presence of pathogenic variation in the
isolates tested suggests that screening for races of
anthracnose will have to be a continuous process. These
isolates came from just a few of the bean growing areas
and it would not be surprising to find additional varia—
tion in some of the areas where no isolates were
collected and even within those from where the current
tested isolates came. The search for resistance sources

will have to be an ongoing process.

 

 

The ultimate success of any bean improvement
program in Malawi will depend on an understanding of the
genetic, agronomic and socio-cultural factors that limit
bean production. This is a complex phenomenon and will

require continued research in order to reach a synthesis

of understanding.

 

 

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