J.
Phytopathology 149, 403408 (2001)
2001 Blackwell Wissenschafts-Verlag, Berlin
ISSN 0931-1785
Laboratory of Phytopathology, Ghent University, Belgium
Bean Anthracnose: Inoculation Methods and Inuence of Plant Stage
on Resistance of Phaseolus vulgaris Cultivars
J. BIGIRIMANA and M. HO FTE
OFTE
Authors' address: Laboratory of Phytopathology, Ghent University, Coupure Links 653, B-9000 Gent, Belgium
(correspondence to M. Hofte. E-mail: monica.hofte@rug.ac.be)
With 3 gures
Received August 9, 2000; accepted March 9, 2001
Keywords: Colletotrichum lindemuthianum, Phaseolus vulgaris
Abstract
Bean anthracnose, caused by Colletotrichum lindemu-
thianum, severely reduces bean production world-wide
and rapid methods are required to investigate bean
C. lindemuthianum interactions. In this study, bean cvs
Boterkoning, Saxa and Admires were inoculated with
C. lindemuthianum race 69 using three dierent methods:
germinated seed inoculation, seedling inoculation and
detached leaf inoculation. Although the methods gave
similar results, inoculation of detached leaves was the
best method. In order to compare the susceptibility of
bean at dierent developmental stages, cvs Prelude,
Boterkoning, Saxa and Admires were used. Leaves
harvested from 6-, 8-, 10-, 12- and 14-day-old plants
were inoculated with C. lindemuthianum races 9, 69, 385,
and 401. Race 9 was virulent to the four bean cultivars
at all the tested stages; cvs Admires and Saxa were
resistant to races 69, 385 and 401, whereas cv. Boterk-
oning was susceptible. Plants of cv. Prelude were
sensitive at 8 days of age, but resistant when 12 and
14 days old. The present results suggest that the
resistance of certain bean cultivars is not equally
expressed at all the developmental stages.
Introduction
Common bean (Phaseolus vulgaris L.) is included in the
Phaseolae tribe of the Leguminosae (Debouck, 1991). It
is a very important crop which provides cheap protein
for many people in Latin America, Asia and Africa.
Field-grown crops face both abiotic and biotic con-
straints. Bean anthracnose, caused by Colletotrichum
lindemuthianum, is one of the most important bean
diseases world-wide. This pathogen can infect leaves,
pods and stems and causes yield losses of 90100%
(Pastor-Corrales and Tu, 1989; Lenne, 1992). The bean
C. lindemuthianum interaction seems to conform to the
gene-for-gene model (Beebe and Pastor-Corrales, 1991;
Neema et al., 1994), in which for each gene that confers
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avirulence to the pathogen there is a corresponding gene
that confers resistance to the host, and vice versa (Flor,
1942). The most important limiting factor for bean
anthracnose control is the existence of a large number of
C. lindemuthianum races (dierent pathotypes, with
dierent types or number of avirulence genes). In
addition, there is a continuous emergence of new races
that overcome existing bean resistance genes. It is
therefore important to characterize C. lindemuthianum
races in bean growing zones and to screen bean
germplasm in order to obtain resistant cultivars for
use in farms or in bean breeding programmes. Collet-
otrichum lindemuthianum races are determined using a
set of 12 bean cultivars (dierentials) internationally
used for the purpose (Pastor-Corrales, 1991). Dieren-
tials are inoculated to characterize isolates, and races are
designated according to susceptible dierentials. Inocu-
lation of dierentials has to be repeated for each isolate,
which requires many infection tests. In addition, in
order to identify sources of resistance, characterized
races need to be tested on local and imported germ-
plasm, which increases the number of infection tests
required. Consequently it is important to use an easy,
reliable and rapid inoculation method to study the
beanC. lindemuthianum interaction.
Several inoculation methods have been used to study
this interaction. The dierence between methods is
based on the plant organ to be inoculated and the
method of transmitting spores from a spore suspension
to the host. Methods include dipping germinated seeds
in a spore suspension (Kruger et al., 1977), spraying
spores on seedlings (Mastenbroek, 1960; Schwartz et al.,
1982), brushing spores onto seedlings (Cardenas et al.,
1964) or onto detached leaves (Tu, 1986), and injecting
inoculum into stems (Schwartz et al., 1982). The choice
of inoculation method is dependent on the technical
facilities that are available. A comparative study of these
dierent methods is essential to check if they give
www.blackwell.de/synergy
404
reproducible results under the same conditions. It is also
necessary to simplify or to nd more reliable methods.
Furthermore, tests with dierent methods are often
performed at dierent bean developmental stages, which
may inuence results. It is important therefore to nd
the best method to use and the developmental stage of
bean cultivars to be inoculated. We here compare results
from three dierent inoculation methods and discuss the
advantages and disadvantages of each. In addition, the
inuence of bean developmental stages on resistance to
anthracnose is discussed and relevant recommendations
are formulated.
Materials and Methods
Colletotrichum lindemuthianum isolates and inoculum
preparation
Infection tests were performed using four dierent races
of C. lindemuthianum: 9, 69, 385 and 401 (Bigirimana
et al., 2000), from bean elds in Burundi, Central
Africa. Their origin is shown in Table 1. The fungal
isolates were grown in Petri plates at 25C, on Mathur
medium (Mathur et al., 1950) composed of: glucose
(2.8 g/l); MgSO4 (1.23 g/l); agar (20 g/l); neopeptone
(2 g/l); KH2PO4(2.72 g/l) and H2O (1 l). After sporula-
tion (10-day-old cultures), the plates were ooded with
10 ml of demineralized water and the spores were
scraped from the plates using a brush. The spore
suspension was ltered through four layers of
0.2 mm 0.2 mm Nylon mesh, the number of spores
was estimated with a haemacytometer and the inoculum
concentration adjusted to 105 spores/ml before inocula-
tion.
Bean cultivars, inoculation methods and experimental design
Four bean cultivars of P. vulgaris were used: cvs.
Prelude, Boterkoning, Admires and Saxa (Royal Sluis
Enkhuizen, the Netherlands). The material to be inocu-
lated was prepared according to the infection methods
used.
Seed inoculation
For germination, 50 seeds of each cultivar were placed
in humid plates ( 92% relative humidity), at 25C. The
plates were covered with plastic sheets to maintain high
humidity. After 3 days the bean seeds were at the V0
stage: emergence of the radicle, and transformation into
the primary root (CIAT, 1992). Germinated seeds
(24 per cultivar), were soaked for 5 min in 200 ml of
inoculum containing 105 spores/ml. A fresh spore
suspension was used for each bean cultivar. Inoculated
Isolate no.a Race no.b Province
06/038 385 Gitega
12/090 9 Muyinga
13/098 401 Ngozi
15/112 69 Ruyigi
a
Isolate number: identication number of the isolates in the local collection; bRace designation according
to the international binary system (Pastor-Corrales, 1991).
BIGIRIMANA and HO FTE
OFTE
seeds were replaced in humid plates, incubated in a dark
room at 1921C and after 2 days, seedlings at the
emergence stage (CIAT, 1992) were transferred to trays,
covered by a thin layer of compost (Klassmann-
Deilman, Geeste, Germany) and incubated at 1921C
in a moist chamber under at least 92% relative humid-
ity. Two moist chambers were used each containing four
trays with two germinated seeds of each bean cultivar.
These seeds were randomly placed in four lines.
Seedling inoculation
Bean seeds were sown in a potting compost (Klassmann-
Deilman) in the greenhouse at 28 3C under a
16 h light : 8 h dark photoperiod. Thirty seeds were
used for each cultivar, and the trays containing pots
were covered with plastic sheets to maintain the relative
humidity that was high enough for bean germination.
Primary leaves of 10-day-old plants were inoculated by
spraying the lower and upper surfaces of the leaves
until run-o with the spore suspension (105 spore/ml).
Eight plants (16 leaves) were inoculated per cultivar in
each replicate and the three cultivars required approxi-
mately 300 ml inoculum. Seedlings were thereafter
randomly placed in two moist chambers containing
four plants of each cultivar. Inoculated plants were
incubated at 1921C for 2 days in darkness followed by
a 12 h light : 12 h dark photoperiod.
Detached leaf inoculation
Beans were grown in conditions described in the
previous method and 10-day-old plants were used.
Detached leaves (four per cultivar) were placed in trays,
upper surface down on a humid surface; four trays were
used per experiment. The leaves were sprayed (approxi-
mately 150 ml per experiment) with the spore suspen-
sion until the lower surfaces were covered by the
inoculum. The trays were covered with transparent
plastic sheets to maintain a minimum relative humidity
of 92% and placed in a room at 20 1C for 2 days in
darkness, followed by a 12 h light : 12 h dark photo-
period.
Bean developmental stages
CIAT (1992) has dened 10 developmental stages of
P. vulgaris, divided into two phases: vegetative (V0 to
V4) and reproductive (R5 to R9). As the dierent stages
are never sharply xed in natural growth, substages can
be dened. In this study we were interested in seedlings
between emergence V1 and rst trifoliate leaf V3.
Cultivars Admires, Boterkoning, Prelude and Saxa were
Table 1
Commune (district) Hill Origin in Burundi of
C. lindemuthianum isolates used to
Giheta Nyamugari infect beans. (Data from:
Muyinga Mukoni Bigirimana et al., 2000)
Nyamurenza Kagoma
Nyabitsinda Nyabitsinda
Inoculation Methods and Resistance of Phaseolus vulgaris cvs to Bean Anthracnose 405

grown in the conditions described in the seedling

inoculation method above and leaves were harvested
6, 8, 10, 12 and 14 days after sowing. The ve bean
developmental stages considered were:
6-day-old plants: primary leaves not completely opened;
8-day-old plants: primary leaves opened, terminal buds
1 cm from the leaf petioles;
10-day-old plants: primary leaves totally opened, the
bud of the rst trifoliate leaf visible;
12-day-old plants: primary leaves totally opened, rst
trifoliate visible but not opened;
14-day-old plants: the rst trifoliate leaf opens, the
second trifoliate leaf appears.
The detached leaf method was used in this test. In
each experiment, four trays were used per cultivar, each
containing 20 leaves: four leaves per developmental
stage. Dierent stages of cvs. Boterkoning, Prelude and
Saxa were compared, using races 385 and 401. In an
additional screening experiment, cv. Admires was inclu-
ded and 8- and 14-day-old plants of the four cultivars
were tested for resistance to races 9, 69, 385, and 401.

Disease evaluation and data analysis

Seven days after seed or leaf inoculation, anthracnose
symptoms were evaluated on stems and leaves. A 19
severity scale was used, based on the percentage covered
by necrotic lesions on the plant organ: 1, no visible
anthracnose symptoms; 3, leaf or stems with a few
isolated small lesions, covering approximately 1% of the
total area; 5, larger lesions covering approxi-
mately 5% of the leaf or stem area; 7, large lesions
covering approximately 10% of the area; 9, large lesions
covering 25% or more of the leaf or stem area.
Experiments were repeated at least twice and data were
statistically analysed using the KruskalWallis multiple
comparison test completed by the MannWhitney
comparison test. For all techniques, the a 0.05 was
used. Score ratings were distributed into two classes:
leaves or stems scored as 13 were considered resistant
whereas scores 49 represented a sensitive reaction.

Results Fig. 1 Anthracnose symptoms on bean cultivars Boterkoning (Bot-

Dierent inoculation methods
The cvs Admires, Boterkoning and Saxa were inoculated
with race 69 of C. lindemuthianum using dierent
methods. Inoculated germinated seeds of cvs Saxa and
Admires displayed a resistant reaction, with resistant
stems of 84 and 93%, respectively (Fig. 1a). Cultivar
Boterkoning was susceptible to race 69 with only 5%
resistant stems. Race 69 was also tested on cvs Admires,
Boterkoning and Saxa by the seedling inoculation
method; cvs Saxa and Admires gave a resistant reaction,
displaying a few or no anthracnose lesions: 3 and 20%
of their leaves were sensitive, respectively. The percent-
age of sensitive cv. Boterkoning leaves was very high
(97%), which conferred a sensitive reaction to this
cultivar (Fig. 1b). When detached leaves of the three
bean cultivars were inoculated with race 69, similar
results were obtained: cv. Boterkoning was sensitive,
whereas cvs Saxa and Admires were resistant. The
erk.), Saxa and Admires (Adm.). Symptoms were evaluated 7 days
after inoculation with a spore suspension 105 spores/ml of
C. lindemuthiamum race. A severity scale of 19 was used. Data
represent results of two experiments and scores were grouped in two
classes: resistant (white bars) and susceptible (black bars) with scores
of 13 and 49, respectively. Bars with the same letter are not
signicantly dierent using the MannWhitney comparison test at
a 0.05, n 32. (a) Germinated-seed inoculation; (b), spray inocula-
tion on seedlings; (c) spray inoculation on detached leaves
percentage of resistant detached leaves were 0, 90, and
90%, respectively (Fig. 1c).
Inuence of plant stage on resistance
In a preliminary experiment, seedlings and detached
leaves of cv. Prelude were inoculated with C. lindemu-
thianum races 385, 401 and 69. Cultivar Prelude
displayed inconsistent results in dierent replicates
(results not shown), the inuence of bean developmental
406 BIGIRIMANA and HO FTE
OFTE

sown, followed by a intermediate stage that preceded

a completely resistant reaction observed on 12- and
14-day-old plants.
A screening test provided additional elements to the
investigation of this phenomenon: cvs Admires, Boterk-
oning, Prelude and Saxa were inoculated with the two
tested races (385 and 401) and with C. lindemuthianum
races 9 and 69. Race 385 (Fig. 3a) was virulent on

Fig. 2 Inuence of developmental stages on bean resistance to

anthracnose. Leaves of cvs Boterkoning (black diamond), Prelude
(j) and Saxa (m), were excised from 6-, 8-, 10-, 12- or 14-day-old
plants and inoculated with 105 spores/ml of C. lindemuthianum.
Symptoms were evaluated 7 days after inoculation using a 19 severity
scale. The number of resistant leaves (scores 13) are presented. Data
represents results of two experiments with n 32. (a) Inoculations with
race 385; (b) inoculations with race 401

stages possibly aecting resistance. In order to study this

phenomenon, races 385 and 401 were inoculated to cvs
Boterkoning, Prelude and Saxa, using the detached leaf
inoculation method. Leaves were harvested from 6-, 8-,
10-, 12- and 14-day-old plants.
Cultivar Boterkoning inoculated with race 385 was
susceptible at all the developmental stages. The number
of resistant leaves was always less than 20% (more than
80% of inoculated leaves were susceptible). The oppos-
ite case was observed for cv. Saxa, which was resistant at
all the stages tested (Fig. 2a). However, cv. Prelude
displayed another resistance trend: 37% of the leaves
were resistant when tested 6 days after the seeds were
sown, but this percentage decreased considerably on
8-day-old plants (0% resistant leaves), and later
increased again (49% of leaves from 10-day-old plants
were resistant and almost all the leaves were resistant
when harvested from 12- or 14-day-old plants). Cultivar
Prelude thus showed a sensitive reaction on 8-day-old
plants and a resistant one on older plants. The two
stages were separated by an intermediate reaction,
observed on 10-day-old plants.
Similar results were obtained when C. lindemuthianum
race 401 was inoculated on the same three bean cultivars
Fig. 3 Anthracnose symptoms on detached leaves of four bean
(Fig. 2b). The cvs Boterkoning and Saxa displayed a cultivars Boterkoning (Boterk.), Prelude (Prel.), Saxa and Admires
consistent reaction, being sensitive and resistant, (Adm.). Leaves were excised from (white bars) 8- and (black bars) 14-
respectively. On the contrary, cv. Prelude again showed day-old plants and inoculated with 105 spores/ml of C. lindemuthia-
dierent results according to developmental stage: 50% num. Symptoms were evaluated 7 days after inoculation using a 19
severity scale. The number of resistant leaves (scores 13) are
of the leaves from 6-day-old plants were resistant; a presented. Data represent the results of two experiments with n 32.
sensitive stage was observed 8 days after the seeds were (a), Race 385; (b), race 401; (c), race 69; (d), race 9
Inoculation Methods and Resistance of Phaseolus vulgaris cvs to Bean Anthracnose
cv. Boterkoning and avirulent on cvs Saxa and Admires.
Its virulence on the three bean cultivars was identical on
8- and 14-day-old plants. On cv. Prelude, race 385 was
virulent on 8-day-old-plants but failed to infect 14-day-
old plants. Races 401 (Fig. 3b) and 69 (Fig. 3c) gave
similar results on the four bean cultivars. Race 9 had a
dierent virulence capacity (Fig. 3d): it was virulent on
all four cultivars, on young and old plants aged 8 and
14 days, respectively.
Discussion
The seed inoculation method was rst used by Kruger
et al. (1977) during their investigation on C. lindemuthia-
num race kappa that broke down the commonly used Are
resistance gene. As only 3 days are needed to obtain plant
material to inoculate, germinated-seed inoculation is
rapid, and the plant material is homogeneously inocula-
ted, ensuring uniform inoculation. Unfortunately, much
plant material is needed for this method because a large
number of bean seeds rot in the germination phase and
a large quantity of inoculum is required, especially
when many bean cultivars are tested. It is dicult to
perform large experiments with the germinated-seed
method because moist chambers are space-consuming
and, in order to avoid mixing races, every isolate needs a
separate moist chamber. Therefore, the germinated-seed
method is not recommended for experiments involving
large numbers of C. lindemuthianum races and bean
cultivars.
In comparison with the seed-inoculation method,
spraying on seedlings requires less plant material but is
time consuming and space demanding. In addition, the
inoculum tends to reach the upper surface better than
the lower face on which the anthracnose start to develop
in natural infection (Tu, 1992). In the present study, this
limitation was solved by spraying each plant separately,
which was time consuming and demanding in inoculum
quantity. Moreover, the inoculum may run o from the
leaves and the uniformity of the inoculation decreases
consequently, negatively inuencing the results.
The detached leaf method solves most of the limita-
tions of spraying C. lindemuthianum spore suspension on
bean seedlings. It was introduced by Tu (1986) and
inoculation was carried out by brushing the inoculum
on leaves detached from bean plants, which is a tedious
task. In the present study, inoculation was carried out
by spraying the inoculum on the lower surfaces of bean
leaves placed on a horizontal surface in humid trays. In
this detached leaf method, uniform distribution of the
inoculum on leaves can be controlled when spraying.
Advantages of genetic importance were recognized for
the detached leaf inoculation (Tu, 1986): one plant can
be assayed several times and only excised leaves are
inoculated, which means that plants used in infection
tests are still able to produce healthy seeds. In compar-
ison with seedling inoculation, the detached leaf method
is equally demanding in plant material but needs less
inoculum and much less space and time. In conclusion,
inoculation on detached leaves appears to be the best
method to investigate bean anthracnose. This method
407
was used to study the inuence of bean developmental
stages on anthracnose severity.
Cultivars Admires, Boterkoning, Prelude and Saxa
were inoculated at dierent stages between emergence
V1 and rst trifoliate leaf V3, using C. lindemuthianum
races 9, 69, 385 and 401. Race 9 can break the resistance
present in the four bean cultivars used and was virulent
to them at all the tested stages. Towards races 69, 385
and 401, the four bean cultivars tested displayed three
dierent kinds of reaction: cvs Admires and Saxa were
resistant to the three isolates at all the developmental
stages; cv. Boterkoning was always susceptible;
cv. Prelude displayed a susceptible reaction on 8-day-
old seedlings, but was resistant when tested on 12- and
14-day-old plants. The susceptible and the resistant
phases are separated by an intermediate reaction that
was observed on Prelude 10 days after the seeds were
sown. However, this phenomenon probably depends on
the pathogen race used because cv. Prelude was
persistently susceptible to race 9 at all of the bean
stages tested. In their study on resistance mechanisms
to C. lindemuthianum, Pastor-Corrales et al. (1985)
suggested four dierent mechanisms because four
dierent groups of cultivars were identied: cultivars
resistant at all stages of growth, cultivars for which
seedlings are highly susceptible but adult plants are
resistant, cultivars moderately resistant and cultivars
moderately susceptible at all stages of growth (Pastor-
Corrales et al., 1985). However, the type and number
of isolates used, and plant stages that were tested are
unfortunately unknown, which makes it dicult to
compare these results to the results of the present
study.
For two cultivars that were, respectively, susceptible
and resistant to the delta race of C. lindemuthianum,
inoculation on primary, third and fth trifoliate bean
leaves resulted in the same virulence level of the
pathogen (Tu, 1986). The results suggest that at least
for certain bean cultivars that are resistant after the
primary leaf stage (e.g. Prelude), there is a sensitive stage
in very young seedlings before the stages tested by Tu
(1986). Some resistance genes may not be expressed at
early developmental stages such as V0 (germination),
and V1 (emergence). This lack of resistance has been
demonstrated in other plant disease systems: resistance
to bacterial blight on rice, mediated by the resistance
gene Xa21, was not expressed in early stages of
development (Mazzola et al., 1994), and only observed
at 21 days post-inoculation. Moreover, it was proved
that in the tomatoCladosporium fulvum interaction, cell
death was developmentally regulated in a tomato line
containing the Cf-9 resistance gene (Hammond-Kosack
et al., 1994).
The inuence of bean stages on resistance to
C. lindemuthianum is to be studied in detail. In the
study of gene-for-gene interaction where race charac-
terization is a very useful tool, cultivars such as Prelude
should not be used as dierentials because dierent
developmental stages may provide dierent results and
lead to wrong conclusions. In addition, seed inoculation
408
is not recommended for screening as it may have a
negative inuence on conclusions and recommendations
to be addressed to farmers and breeders. The detached
leaf inoculation is the best method to use especially
when large numbers of pathogen races and bean
cultivars are to be tested. Valid screening for practical
application should be carried out at the most sensitive
stage: on 8-day-old plants in greenhouse conditions
(28 3C). As it appears that bean plants are more
susceptible at seedling stage, farmers should avoid, when
possible, plant infection at this developmental stage in
order to help plants escape the disease.
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