ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2002 Vol. 45, Št. 1: 3 - 7 
Identification of physiological races of Colletotrichum lindemuthianum 
occuring in Slovenia 
Identifikacija fizioloških ras glive Colletotrichum lindemuthianum v 
Sloveniji 
Alenka MUNDA, Metka ŽERJAV, Jelka ŠUŠTAR - VOZLIČ 
Agricultural institute of Slovenia, Hacquetova 17, SI-1001 Ljubljana, Slovenia 
fax: + 386 (O) 1 280 52 55, e-mail: a1enka.munda@kis-h2.si 
Abstract. Bean anthracnose, the most important fungal disease of common bean in 
Slovenia, is caused by a mitosporic fungus Colletotrichum lindemuthianum (Sacc. et 
Magnus) Briosi et Cav. The most appropriate measure to control the disease is growing of 
resistant cultivars. Breeding for resistance is limited by the existence of severa! physiological 
races of the pathogen and continuous development and introduction of new ones. It is 
necessary to constantly monitor the occurrence and dynamic of physiological races existing 
in certain area. With this aim, 47 isolates were obtained from severa! bean accessions in 
different bean growing areas in Slovenia. The fungus was isolated from infected pods and 
leaves. A set of 12 internationally accepted differential bean cultivars was inoculated with 
spore suspension obtained from single spore isolates. Plant reaction was screened following 
1 - 10 severity scale. Four physiological races ofthe pathogen were identified. Using the 
binary nomenclature system they were assigned to races 23, 55, 103 and 131. The most 
frequent and widespread were races 55 and 23, named also lambda and delta. The research 
will continue by screening the autochthonous bean germplasm for susceptibility towards 
the existing races. 
Key words: bean anthracnose, Colletotrichum lindemuthianum, common bean, 
physiological races 
Izvleček. Fižolov ožig, kije pri nas najpomembnejša glivična bolezen fižola, povzroča 
gliva Colletotrichum lindemuthianum (Sacc. et Magnus) Briosi et Cav. Bolezen preprečujemo 
predvsem z gojenjem odpornih sort fižola. Žlahtnjenje na odpornost proti tej bolezni je 
težavno zaradi velikega števila fizioloških ras ter nenehnega razvoja in introdukcije novih 
ras. Zato moramo stalno spremljati pojav in dinamiko fizioloških ras na določenem območju. 
S tem namenom smo zbrali 47 primerkov glive C. lindemuthianum z različnih območij 
pridelovanja fižola v Sloveniji. Glivo smo izolirali iz okuženih fižolovih strokov in listov. 
S pridobljenimi izolati smo okužili 12 diferencialnih sort fižola, ki so mednarodno priznane 
za identifikacijo fizioloških ras te glive. Testne rastline fižola smo okužili s suspenzijo 
trosov, pridobljeno iz enotrosnih izolatov. Jakost okužbe smo ocenili po desetstopenjski 
skali. Odkrili smo štiri fiziološke rase glive in jih na podlagi binarnega nomenklaturega 
sistema identificirali kot rase št. 23, 55, 103 in 13 l. Najbolj pogosti in razširjeni sta rasi št. 
55 in 23, imenovani tudi lambda in delta. V nadaljnjih raziskavah bomo proučili odpornost 
domačih akcesij fižola na okužbo s slovenskimi rasami glive C. lindemuthianum. 
Ključne besede: Colletotrichum lindemuthianum, fiziološke rase, fižol, fižolov ožig 
4 Acta Biologica Slovenica, 45 (!), 2002 
Introduction 
Bean anthracnose is one of the most destructive diseases in bean growing areas world-wide as well 
as in Slovenia. It is caused by a mitosporic fungus C. lindemuthianum (Sacc. et Magnus) Briosi et Cav. 
The anamorphic state Glomerella cingulata (Stonem.) Spauld et v. Schrenk. f. sp. phaseoli can be 
produced in cul ture in certain pairings of conidial isolates, but has not yet been found in field conditions 
(BRYSON & al. 1992). C. lindemuthianum has a world wide distribution. It has been recorded on 
various legumes belonging to the genera Phaseolus, Vicia, Vigna, Glycine, Pisum and many others 
(LENNE 1992). Among them, P. vulgaris is the most important host, followed by P. acutifolius var. 
latifolius and Vigna unguiculata. Other legumes are only slightly susceptible (TU 1992). 
C. lindemuthianum shows an extensive intraspecific variability and exists in the form of severa) 
physiological races. Pathogenic variability among isolates of C. lindemuthianum was first observed 
by BARRUS (1911). He distinguished two races of the fungus (alpha and beta) by testing their 
pathogenicity towards 139 bean cultivars. This was the first description of physiological races in a 
plant pathogenic fungus. Later on extensive variation in the fungus has been found and severa) new 
races were described in the local populations of C. lindemuthianum throughout the world. Race 
identification was traditionally based on the nomenclature system using letters of the Greek alphabet. 
Many other nomenclature systems using local codes were also used, thus rendering the possibility of 
comparing the research results very difficult. Altogether 13 races, named by letters of Greek alphabet, 
were described till 1994 and many more were classified using local codes (MELOTTO & al. 2000). 
Bean anthracnose affects ali the above ground parts of beans causing dark brown depressed 
lesions. Affected leaves and stems bear brown lesions that can rapidly spread and girdle the plant. 
Symptoms are the most conspicuous on pods: rusty brown spots occur first, then sunken dark brown, 
eye-shaped lesions with brown to reddish edges develop. Black specks (acervuli) occur on the lesions 
and pink spore masses ooze from them in humid conditions. The disease is transmitted and spread 
mostly by infected seed. Seed treatment and production of clean seed, crop rotation and sanitation can 
significantly reduce the extent of the disease. Nevertheless, outbreaks of anthracnose stili occur, 
especially in areas with an extensive use of susceptible local bean accessions. The intensity of the 
disease depends on weather conditions and can reach the extent of an epidemic in the years with high 
relative humidity, frequent precipitation and moderate temperature. 
The most appropriate measure to control the disease is growing of resistant cultivars. Breeding for 
resistance against C. lindemuthianum has been practised since the beginning of the last century, when 
disease resistance was demonstrated in certain bean cultivars (BARRUS 1911, 1918). Eight independent 
dominant resi stance genes ( Co-1 to Co-8) were described in common bean (ALZATE-MARIN & al. 
1997, YOUNG & al. 1998, GEFFROY & al. 2000). The use of specific resistance genes has not 
always provided a durable resistance due to the continuous development of new physiological races 
which are capable of overcoming the resistant germplasm. When selecting for resistance in a particular 
region, the breeder should carefully choose a gene pair that would confer resistance to ali races known 
in that region (KELLY & MIKLAS 1999). 
The aim of our study was to investigate the pathogenic variability of the pathogen and identify the 
physiological races existing in the bean growing areas in Slovenia. 
Materials and methods 
Fungal isolates 
47 samples were collected from severa) bean accessions in different bean growing areas in Slovenia. 
The fungus was isolated from infected pods and leaves on potato dextrose agar (PDA). Sporulation 
was induced by growing isolates on sterilised bean pods. Single spore isolates were obtained by 
A. Munda, M. Žer jav, J. Šuštar-Vozlič: Identification of physiological races of ... 5 
spreading spore suspension on PDA plates and isolating individual germinating spores. To prevent a 
degeneration of single spore isolates during storage, spore suspension of single spore isolates was 
fixed on sterile filter papers, desiccated on sterilised silica-gel and stored at - 20 °C (FERREIRA & 
FUEYO 2001). 
Differential bean cultivars 
Physiological races of the fungus were identified by screening the disease severity (resistant or 
susceptible reaction) ona selected group of bean varieties - differentials. The reaction of differential 
cultivars strongly depends on infection techniques, experimental conditions, on host genotype and 
pathogenicity of the fungal isolate. Different bean cultivars, mostly local ones, were used as differentials 
and different reaction schemes were applied for assessing the disease severity. Severa! attempts have 
been made to select an intemational set of differential cultivars and to uniform the classification of 
races. Differential cultivars should be genetically uniform with clear resistant or susceptible reaction to 
ali isolates of the fungus (CHARRIER & BANNEROT 1970, DRIJFHOUT & DAVIS 1989). In 
1991, a set of 12 differential bean cultivars was intemationally accepted and a binary nomenclature 
system for identification of C. lindemuthianum races was proposed (PASTOR - CORRALES 1991 ). 
Using this system, each race is identified by a number, obtained by summing the binary value of ali 
differential cultivars showing a susceptible reaction. Among differential cultivars, four represent the 
Andean germ plasm (Perry Marrow, Michigan Dark Red Kidney, Kaboon and Widusa) and the others 
- the Middle American germ plasm (Michelite, Cornell 49242, Mexico 222, PI 207262, To, Tu, Ab 136 
and G2333). 
The seeds of differential bean cultivars used in our study were obtained from CIAT, Columbia. The 
seeds were surface sterilised with 1 % sodi um hypochlorite, left to germinate on moist filter paper and 
planted in sterilised sand (four seeds per pot). They were kept in greenhouse for two weeks until the 
development of primary leaves. 
Infection trials 
A single spore colony of each isolate was grown on PDA for I 4 days prior to inoculation. Spores 
were then scrapped from the plates and used for the preparati on of spore suspension. Spore concentration 
was adjusted to 106 - 107 per ml prior to inoculation of test plants. Plants with fully expanded primary 
leaves were infected. Both sides of leaves and stems were inoculated by brushing following the 
method ofTU & A YLESWORTH ( I 980). Four plants ofeach cul ti var were infected with each isolate 
and the whole experiment was repeated two to three times. Altogether I 690 inoculations were made. 
The plants were kept in a growth chamber at the temperature 20 °C and 14 h of light. During the first 
two days of incubation they were covered with transparent plastic bags to maintain a 100 % relative 
humidity. The disease expression was scored 8 days after the inoculation. The plant reaction was 
evaluated qualitatively (resistant or not) and quantitatively using ten- point severity scale (O- 9). Plants 
showing no infection or small necrotic lesions originating from hypersensitive reaction, were assigned 
resistant (score O -3), the rest were considered susceptible (score 4 - 9). 
Results and discussion 
Isolates of C. lindemuthianum from Phaseolus vulgaris grown in Slovenia were assigned to four 
physiological races based on their pathogenicity towards 12 differential bean cultivars (Table 1). Most 
of the isolates were pathogenic to differential cultivars originating from Andean gene pool. Among 
them, the cultivar Michigan Dark Red Kidney was the most liable to infection and showed a highly 
susceptible reaction to ali isolates. By contrast differential cultivars originating from MiddleAmerican 
gene pool were relatively resistant with the exception of the cultivar Michelite, which showed a 
moderately susceptible reaction to most of the isolates. 
Using the binary nomenclature system the races of C. lindemuthianum from Slovenia can be 
identified as races 23, 55, 103 and 131. The majority of isolates (25) represented race 55, 16 isolates 
represented race 23, and only 6 isolates represented races 103 and 131. 
6 Acta Biologica Slovenica, 45 (l ), 2002 
Table 1: Reaction of differential bean cultivars to races of C. lindemuthianum from Slovenia 
Tabela 1: Reakcija diferencialnih kultivarjev fižola pri okužbi s slovenskimi rasami glive C. lindemuthianum 
Race Differential bean cultivars 
2 4 8 16 32 64 128 256 512 1024 2048 
131 S 5-7 s. Ro Ro R , R, R, s 4-5 R, Ro R , Ro 
103 RIS 
4 s9 s 9 Ro R 3 s 8 RIS 4 R, R, Ro R , Ro 
23 s 7 s 9 s . Ro s 8 R ,_, R , R, R3 R o R , Ro 
55 s7 s. s . Ro s 8 S 5-7 R , R, R , Ro R, Ro 
Legend: Differential bean cultivars: 1- Michelite, 2 - Michigan Dark Red Kidney, 4 - Perry Marrow, 
8 - Cornell 49242, 16 - Widusa, 32 - Kaboon, 64 - Mexico 222, 128 - PI 207262, 256 -TO, 512 -
TU, 1024 - AB 136, 2048 - G 2333; R - resistant reaction, S - susceptible reaction, numbers 
indicate severity of symptoms (ten- point severity scale). 
Legenda: Diferencialni kultivarji fižola: 1- Michelite, 2 - Michigan Dark Red Kidney, 4 - Perry 
Marrow, 8 - Cornell 49242, 16 - Widusa, 32- Kaboon, 64 - Mexico 222, 128 - PI 207262, 256 -TO, 
512 -TU, 1024 - AB 136, 2048 - G 2333; R - odporen, S - občutljiv, številke kažejo jakost okužbe po 
desetstopenjski skali 
As has already been mentioned, many different systems of race classification are used in the studies of 
pathogenic variability in C. lindemuthianum. The results are difficult to compare since different bean 
cultivars are used as differentials. Nevertheless, comparing our results with other studies of C. lindemuthianum 
race composition in Europe in the past decades (CHARRIER & BANNEROT 1970, DRUFHOUT & 
DAVIS 1989, GOTH & ZAUMEYER 1965, KRUGER & al. 1977, YERKES & ORTIZ 1956) we can 
conclude that races 55 and 23 fully correspond with the races lambda and delta. The race lambda has been 
first reported in Europe in 1974 from the Netherlands and the race delta in 1953 from Germany (HUBBELING 
1974, FRANDSEN 1953). Both races are very virulent, showing high leve! of pathogenicity towards many 
bean cultivars. The minority of isolates tested in our study belonged to races 103 and 131 . They partly 
correspond with the races gamma and beta. Their reaction to differential cultivars, especially to the cul ti var 
Michelite, slightly differs from the reaction described in other studies; Michelite was moderate to susceptible 
in our study, while it was resistant in most other studies (ALAM & RUDOLPH 1993, FERNANDEZ & 
AL. 2000, GOTH & ZAUMEYER 1965, YERKES & ORTIZ 1956). 
The existence of a large number of C. lindemuthianum races and a continuous emergence of the new 
ones are very important aspects of the control of bean anthracnose. It is necessary to constantly 
monitor the race composition in a certain area and screen the local bean germplasm for susceptibility 
towards the existing races. With this aim we intend to infect 27 local bean accessions with isolates 
representing ali the four races of the pathogen occurring in Slovenia. Preliminary results of infection 
with three isolates of the delta race revealed that only 4 local accessions were resistant to the anthracnose 
pathogen, the rest were susceptible with a leve! of infection ranging from 5 to 8. 
Acknowledgements 
This work was supported by research grant L4-3284-0401-01 from the Ministry of education, science 
and sport and Ministry of agriculture, forestry and food of the Republic of Slovenia. 
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