Journal of
Plant Breeding and Crop Science

  • Abbreviation: J. Plant Breed. Crop Sci.
  • Language: English
  • ISSN: 2006-9758
  • DOI: 10.5897/JPBCS
  • Start Year: 2009
  • Published Articles: 409

Full Length Research Paper

Effectiveness of pyramided genes in conferring resistance to anthracnose disease in common bean populations

Kiryowa M.
  • Kiryowa M.
  • Department of Agricultural Production, College of Agricultural and Environmental Sciences, Makerere University, P. O. Box 7062, Kampala, Uganda.
  • Google Scholar
Nkalubo S. T.
  • Nkalubo S. T.
  • National Crops Resources Research Institute (NaCRRI), NARO, P. O. Box 7064, Kampala, Uganda.
  • Google Scholar
Mukankusi C.
  • Mukankusi C.
  • Centro Internacional de Agricultura Tropical (CIAT), P. O. Box 6247, Kampala, Uganda.
  • Google Scholar
Male A
  • Male A
  • Centro Internacional de Agricultura Tropical (CIAT), P. O. Box 6247, Kampala, Uganda.
  • Google Scholar
Gibson P.
  • Gibson P.
  • Department of Plants, Soils, Agricultural Systems, Southern Illinois University, Carbondale, IL, USA.
  • Google Scholar
Tukamuhabwa P.
  • Tukamuhabwa P.
  • Department of Agricultural Production, College of Agricultural and Environmental Sciences, Makerere University, P. O. Box 7062, Kampala, Uganda.
  • Google Scholar
Rubaihayo P.
  • Rubaihayo P.
  • Department of Agricultural Production, College of Agricultural and Environmental Sciences, Makerere University, P. O. Box 7062, Kampala, Uganda.
  • Google Scholar


  •  Received: 01 March 2019
  •  Accepted: 01 May 2020
  •  Published: 31 January 2021

 ABSTRACT

Anthracnose disease (Colletotrichum lindemuthianum (Sacc. et. Magn) Lams. Scrib.) is one of the most devastating diseases that constrain common bean production in Uganda. A cascading pedigree pyramiding scheme was used to develop common bean populations to evaluate the effectiveness of pyramided and single resistance genes (Co-42, Co-43, Co-5, and Co-9) on disease development. Detached leaf trifoliates of F4:6 genotypes were screened with four C. lindemuthianum races (352, 713, 767 and 2047). Disease severity data were subjected to ANOVA. Races, genotypes and Race x Genotype interaction were significant. Genes Co-42 and Co-5 conferred resistance to the four races and the gene pyramids Co-42+Co-5+Co-9 and Co-42+Co-5 had the lowest severity scores. Gene Co-43 conferred resistance to race 352 and weak resistance to race 713; whereas, gene Co-9 conferred resistance to race 352.  Co-43+Co-9 gene pyramid showed resistance only to race 352. The Co-42 and Co-5 genes conferred resistance to all the four races 352, 713, 767 and 2047. The single gene Co-42 was not significantly different from the pyramids Co-42+Co-5+Co-9 and Co-42+Co-5 (P<0.01). Similarly, the Co-5 gene was not significantly different from Co-42+Co-5, Co-42+Co-9 and Co-5+Co-9 pyramids. The Co-9 gene showed antagonism in all pyramids. These results indicate that pyramiding of resistance genes would be effective for disease management in Uganda, but pyramids with Co-9 gene would be less effective.

Key words: SCAR markers, Colletotrichum lindemuthianum, broad-spectrum resistance.


 INTRODUCTION

Colletotrichum lindemuthianum (Sacc. et. Magn) Lams. Scrib., the pathogen that causes anthracnose of common beans  (Phaseolus  vulgaris  (L.))  has  a  high  degree  of pathogenic and genetic variability (Mahuku and Riascos, 2004). New races of the pathogen continually emerge, which has made  single  resistance gene deployment less effective as a control strategy. The pathogen is reported to have a reproductive/ telemorphic phase, Glomerella lindemuthianum which is partly responsible for its genetic and physiologic variability through genetic recombination (De Silva et al., 2017). This enables it to adapt to new sources of host resistance and easily breakdown host resistance (Melotto et al., 2000). New races continually emerge based on the gene-for-gene hypothesis; this has posed a risk of rendering single resistance gene deployment less effective against the disease. Studies in Uganda have indicated that C. lindemuthianum has a high pathogenic variability found to be highest in the Eastern and South Western highland regions (Mwesigwa, 2008; Kiryowa et al., 2016); 24  new physiological races have been reported (Kiryowa et al., 2016).

Resistance to bean anthracnose is conditioned by thirteen (13) major genes Co-1 to Co-13 (Lacanallo et al., 2010) with only co-8 being recessive. Loci Co-9/Co-33 and Co-7/Co-3 are allelic (Méndez-Vigo et al., 2005) and multiple alleles exist at the Co-1, Co-3, Co-4, Co-5 loci. Genes Co-1, Co-12 and Co-13 are Andean in origin while the rest are Mesoamerican (Kelly and Vallejo, 2004). Resistance gene pyramiding, the combination of two or more resistance genes in a cultivar (Ye and Smith, 2008) is recommended as a strategy to increase broad-spectrum resistance against C. lindemuthianum pathogen. But the conventional gene pyramiding approach is costly (Joshi and Nayak, 2010) requiring extensive phenotyping with several races of the pathogen over many generations. DNA markers have enormous potential to improve the efficiency and precision of conventional plant breeding through marker-assisted selection (MAS), which is proven to speed up breeding through laboratory based selection of individual plants with the desired trait(s).

Marker assisted selection (MAS) has been extensively used in common bean breeding programs (Miklas et al., 2006) and the availability of molecular markers linked to the major anthracnose (Co-) genes provides an opportunity to pyramid multiple disease resistance genes (Kelly and Vallejo, 2004). Kelly et al. (1994) recommended pyramiding resistance genes Co-1 Andean and Co-2 Mesoamerican to confer resistance to all known C. lindemuthianum races in North America. Young and Kelly (1996) suggested pyramiding major resistance genes Co-6 and Co-5 in combination with Co-1 for durable resistance. Ragagnin et al. (2009) used RAPDs and SCAR markers to pyramid genes Co-4, Co-6, Co-10 with Phg-1 angular leaf spot and Ur-ON rust resistance genes in a susceptible ‘carioca’ market class cultivar in Brazil. Genchev et al. (2010) used RAPD and SCAR markers to pyramid Co-1 and Co-4 genes to confer effective resistance against C. lindemuthianum races in Bulgaria. Ferreira et al. (2012) used SCAR, CAPs and RAPD markers to successfully pyramid Co-2 and Co-3/9 genes, along with, I and bc-3 common mosaic virus resistance genes to develop a market class bean genotype. For successful resistance gene pyramiding, there is need to evaluate the different pyramid combinations under a diverse C. lindemuthianum population. The purpose of this study therefore is to assess the effectiveness of single and pyramided resistance genes against bean anthracnose disease.


 MATERIALS AND METHODS

Parent materials and Ugandan breeding locations

The cultivars used as parents were obtained from the Legumes Program, National Crops Resources Research Institute (NaCRRI), Namulonge, located 00 32'' N of the Equator and 320 37'' E, 27Km North of Kampala and elevated at 1,150 meters above sea level. The parents and their traits are presented in Table 1. All crosses and advancement from F1 to F4 generations were conducted under screen house conditions at NaCRRI. Marker-assisted selection (MAS) during gene pyramiding for fixation of alleles and phenotypic screening of advanced lines were conducted at the International Center for Tropical Agriculture (CIAT), based at the National Agricultural Research Laboratories (NARL), Kawanda, located at 0° 24’ 38.15” N and 32o 32’ 14.06” E and elevated at 1,147 m above sea level.

Development of populations

A  cascading  pedigree  gene  pyramiding  scheme  (Servin   et   al., 2004) was used to develop populations. In this breeding scheme, only one cross was made at each generation beginning with two founding parents and followed by an intermediate genotype and one founding parent. The root genotype (Servin et al., 2004) combining all the desired resistance genes in heterozygous state was obtained and fixation of these genes was achieved through self-pollination.

Selection scheme

Marker-assisted selection (MAS) was used to select individuals with the desired single and pyramided genes during the pedigree and fixation steps of the breeding scheme.

Pyramiding scheme

The donor parents G2333 and PI207262 for anthracnose resistance were crossed in a screen house to combine the four anthracnose resistance alleles in F1a plants (Figure 1). The F1a plants were crossed with RWR719 to produce F1b plants. The SCAR markers SAS13, SBB14, SAB3 and SB12 run on extracted DNA of 105 F1b plants to identify  plants  that  possessed  the  target genes. 35  F1b plants showed the fragments associated with resistance. Six of these plants selected and crossed with the susceptible varieties K132, NABE4, NABE13 and NABE14. F1c seeds of the crosses were harvested and planted as individual plants under screen house conditions. Markers SBB14, SAB3, SB12 were run on DNA extracted from 46 F1c plants. Three plants possessing genes in heterozygous state became the root genotype.

Fixation steps of the pyramiding scheme

F2 seed from the root genotype was planted and DNA extracted from 69 plants. MAS was conducted to identify plants that inherited 0, 1, 2, 3 target resistance genes. Selfing continued up to F6 generation to ensure that the genes were fully fixed in homozygous state, but MAS was conducted up to F4 generation of the fixation scheme. The F6 advanced lines were classified into 10 groups according to the alleles inherited as indicated in Table 4.

Molecular markers used in MAS

The  target  genes,  linked  DNA  markers  used  to  tag  the   genes amplified fragment sizes, orientation and primer sequences are presented in Table 2. Sequence characterized amplified regions (SCAR) markers were used to tag the resistance genes. The primers were obtained from the Department of Molecular and Cellular Biology, University of Cape Town, Randebosch, South Africa. Later batches in premix form were obtained from Bioneer Corporation, Munpyeong-dong, Daejeon, South Korea. A 25/100 base pairs (bps) mixed DNA molecular weight marker, specifically designed for determining the size of double strand DNA from 25 to 2,000 bps was used to estimate fragment sizes.

DNA extraction

Leaf samples were collected from 14-day old plants raised in a screen    house.     Genomic    DNA    was    extracted    using    the Cetyltrimethylammoniumbromide (CTAB) method adapted from Doyle and Doyle (1987).

DNA amplification, gel electrophoresis and imaging

To prepare a polymerase chain reaction (PCR) master mix using PCR reagents, double distilled water (ddH2O), deoxy nucleoside triphosphates (dNTPs including dATP, dGTP, dCTP and dTTP), forward and reverse primers, MgCl2, PCR buffer and Taq polymerase enzyme were mixed based on the concentrations in Table 3. Nineteen microliters (19 μl) of the master mix were pipetted into individual PCR tubes and 1.0 μl of 40 ng plant DNA was added to each tube to make a total PCR reaction volume of 20 μl. When the PCR Bioneer Premix (Bioneer Inc, Korea) was used instead of the PCR reagents,  the PCR master mix was prepared by adding ddH2O to a 1.5ml Eppendorf tube, followed by the forward and reverse primers according to the working concentrations in Table 3. Amplification of DNA was carried out in a Bioneer Thermal cycler (Bioneer Inc, Korea) with an initial denaturation step at 95°C for 5 min and 35 cycles each of a denaturation step at 94°C for 20 s, an annealing step at 64°C (SBB14), 68°C (SAS13), 65oC (SAB3), 65oC (SB12), 64oC (SH18) and 63°C (PYAA19800) for 40 s; an extension step at 72oC for one (1) min followed by a final extension for 10 min at 72oC. The amplicons were resolved on 1.5% agarose gels in 1X TBE (0.045 M Tris–borate and 1 mM EDTA, pH 8.2) at 100V for 90 min and stained with 0.5 µg/ml ethidium bromide for 10 min. Gel images were captured using the SynGene G: BOX gel documentation system (Syngene, Frederick, MD, USA).

Phenotypic screening

Seeds of F6 lines were sown in five liter plastic pots filled with top soil mixed with sand and sterilized manure in the ratio of 5:3:2 respectively. Diammonium Phosphate (DAP) fertilizer was applied prior to sowing and watering was done daily. Four C. lindemuthianum races 352, 713,767 and 2047 were cultured on Potato Dextrose Agar (PDA) media and incubated in darkness at 22 - 24°C for four days before sub-culturing onto modified Mathur’s Agar media (500 g) (Champion et al., 1973). Inoculum was prepared by scrapping germinated conidia off the growth media into a jar with small amounts of distilled water to form a suspension. A hemocytometer was used to adjust the concentration to 1.2 x 106 conidia ml-1 (Inglis et al., 1988) and 0.1% Tween 20 was added as a surfactant. The detached leaf technique (Tu, 1986) was used to differentiate the bean families. Leaf trifoliates were detached 14 days after planting and immersed in the suspension containing C. lindemuthianum spores. The inoculated leaf trifoliates were placed in transparent plastic containers with moistened  paper  towels  and covered with transparent covers to maintain humid conditions (Figure 2). The containers were placed on wooden shelves fitted with PhillipsR TLT 18-20W/75RS Fluorescents tubes that supplied approximately 50 µmoles m-2s-1 of light to enable prolonged physiological processes of the detached leaves up to 28 days. A 12 h day light and 12 h night regime, and room temperatures were maintained between 22 and 25°C, which is recommended for successful infection of C. lindemuthianum (Awale et al., 2007) 

Disease symptoms were scored after a seven day incubation period using a modified 1 – 9 scale (Balardin et al., 1997) where; 1 = no symptoms (resistant), 2 – 3 = very small lesions mostly on primary leaves (resistant), 4 – 9 = numerous enlarged lesions or sunken cankers on the lower side of the leaves (susceptible). This experiment was designed in a Randomized Complete Block Design (RCBD) with three replicates.

Data analysis

Disease severity data were subjected to analysis of variance (ANOVA) using GenStat Discovery, 12th Edition (Anonymous, 2009). To determine whether pyramid group means were significantly different with respect to anthracnose resistance levels, a Tukey’s Honest Significant Difference (HSD) test was carried out to test the null hypothesis that all gene group means are equal; Tukey Test statistic; HSD = q√MSE/nc. Where; q = value from studentized range table, MSE = Mean Square for Error from ANOVA table, nc = number of replicates per treatment. Standard error of pyramid group means (SEM) was computed using the formula; SEM = s/√n, where; s = sample standard deviation and n = sample size. Sample standard deviation (s) was computed using the formula; s = √1/N-1 ∑Ni=1(xi – x)2, where; x1 …., xN = the sample data set, x = mean value of the data set, N = size of sample data set.


 RESULTS

F4:6 populations with pyramided and single resistance genes

Sixty nine F4:6 lines were obtained after the fixation steps (Table 5). Nine lines inherited three anthracnose resistance genes, 17 inherited two alleles, 28 inherited a single allele; while 12 did not inherit any of the target alleles. Twenty seven possessed Co-42 allele, eight possessed Co-43 allele, 33 possessed Co-5 allele, while 21 possessed Co-9 allele. Images of the amplified DNA fragments for the four dominant SCAR markers SAS13, SAB3, SB12 and one codominant marker SBB14 are presented on Figure 3.

Evaluation of families for resistance to anthracnose

Five lines that possessed alleles Co-42, Co-5 and Co-9 were resistant to the four races. Three of the four lines possessing Co-42 and Co-5 alleles were also resistant to the four races. Four of the five lines possessing allele Co-42 were resistant to the four races. Two families that possessed allele Co-9 and the family that possessed Co-43+Co-9 pyramid showed resistance to only race 352. Six families that did not inherit any resistance alleles were susceptible to the four races. Three of four families possessing Co-5 allele were resistant to the four races. Presence of the Co-9 gene was always associated with symptoms except in the three-gene pyramid.

Effectiveness of single and pyramided genes in conferring resistance to diverse C. lindemuthianum races

Data from the analysis of variance for anthracnose disease severity are presented in Table 6. Gene-groups, races  and  interaction  between gene-groups  and  races were all highly significant (P < 0.01) with gene-groups contributing highest total variation followed by races.

Severity scores of gene groups inoculated with diverse C. lindemuthianum races are presented in Table 7. Race 2047 had the highest overall mean score followed by races 767, 713 and 352. The mean scores for pyramid and single-gene groups respectively were not significantly different from each other (Lsd 0.05). Among the two-gene pyramid groups, the Co-43+Co-9 group had the highest overall mean severity score across the four races, followed by Co-5+Co-9 and Co-42+Co-9, both of which succumbed to race 2047. The pyramid group Co-42+Co-5 had the lowest severity score and did not succumb to any race.

The mean severity score for the three-gene pyramid Co-42+ Co-5+Co-9 was significantly lower than the two-gene pyramid groups. The mean score for Co-42+Co-5 pyramid group was significantly lower than scores for the Co-5+Co-9 pyramid group, but was not significantly different from the Co-42+Co-9 pyramid group. The three-gene pyramid Co-43+Co-5+Co-9 was not developed for comparison. Overall severity scores of single-gene groups were significantly different from each other and from the no-gene group. The Co-42 gene group had the lowest severity score followed by Co-5, Co-43 and Co-9 gene groups. The mean severity score of Co-9 gene-group was significantly higher than mean scores of all the single-gene groups and was the only group with severity score falling in the susceptible range.

The mean score of the Co-42 single-gene group was not significantly different from the mean scores of the Co-42+Co-5+Co-9 and Co-42+Co-5 pyramid groups, but was significantly lower than the mean scores of the Co-42+Co-9, Co-43+Co-5 and Co-5+Co-9 pyramid groups. The mean score of the Co-5 single-gene group was not significantly different from mean scores of the Co-42+Co-5, Co-42+Co-9 and Co-5+Co-9 pyramid groups, but was significantly higher than the score for the three-gene pyramid Co-42+Co-5+Co-9. The mean score of the Co- 43 single-gene  group  was  significantly  less  than  the  Co-43+Co-9 pyramid group. The mean score of the Co-9 gene group was significantly higher than scores for all the pyramid gene groups.


 DISCUSSION

The mean severity score for the three-gene pyramid Co-42+ Co-5+Co-9 was significantly lower than the two-gene pyramid groups. The mean score for Co-42+Co-5 pyramid group was significantly lower than scores for the Co-5+Co-9 pyramid group, but was not significantly different from the Co-42+Co-9 pyramid group. The three-gene pyramid Co-43+Co-5+Co-9 was not developed for comparison. Overall severity scores of single-gene groups were significantly different from each other and from the no-gene group. The Co-42 gene group had the lowest severity score followed by Co-5, Co-43 and Co-9 gene groups. The mean severity score of Co-9 gene-group was significantly higher than mean scores of all the single-gene groups and was the only group with severity score falling in the susceptible range.

The mean score of the Co-42 single-gene group was not significantly different from the mean scores of the Co-42+Co-5+Co-9 and Co-42+Co-5 pyramid groups, but was significantly lower than the mean scores of the Co-42+Co-9, Co-43+Co-5 and Co-5+Co-9 pyramid groups. The mean score of the Co-5 single-gene group was not significantly different from mean scores of the Co-42+Co-5, Co-42+Co-9 and Co-5+Co-9 pyramid groups, but was significantly higher than the score for the three-gene pyramid Co-42+Co-5+Co-9. The mean score of the Co- 43 single-gene  group  was  significantly  less  than  the  Co-43+Co-9 pyramid group. The mean score of the Co-9 gene group was significantly higher than scores for all the pyramid gene groups.

The Co-9 gene was the least effective against the pathogen and was also associated with increased severity when combined with other genes implying it was antagonistic when combined with other genes and therefore should be avoided in gene pyramiding programs. Its presence in the three gene pyramid Co-42+Co-5+Co-9, however, was not antagonistic probably because of the combined effectiveness of the Co-42 and Co-5 genes. Kelly and Vallejo (2004) reported the Co-9 gene to possess a very specific breeding value against Andean races of C. lindemuthianum. Alzate-Marin et al. (2003) reported the Co-9 gene to be susceptible to even the weak Mesoamerican races 65 and 69. The cultivar PI207262 possessing the Co-9 gene was reported to be overcome  by  many   anthracnose   races   (Kelly,  2004)  implying the ineffectiveness of the Co-9 gene as a resistance source. This explains the poor resistance spectrum observed with the Co-9 gene in this study. Kelly and Vallejo (2004) recommended its use only to diversify resistance in gene pyramids because of its independence and potential value in controlling Andean races. 

In this study, the single gene Co-42 was found to be as effective as the best pyramid combinations Co-42+Co-5 and Co-42+Co-5+Co-9 and the single gene Co-5 was as effective as pyramid combinations Co-42+Co-5 and Co-42+Co-9. This implies that the two single genes possess factors that promote broad-spectrum resistance and that these factors are higher in Co-42 gene. These two genes would confer effective and broad-spectrum resistance in single deployment against a diverse C. lindemuthianum population. The Co-5 gene was reported to be among the most effective genes in Central America and Mexico but with limited use by bean breeders (Kelly and Vallejo, 2004). It was reported to possess a wide resistance spectrum conferring resistance to 31 races (Balardin et al., 1997). The Co-42 gene was reported to exhibit the most broad-spectrum resistance against C. lindemuthianum in common beans (Young and Kelly, 1996; Balardin and Kelly, 1998; Awale and Kelly, 2001) and controls of up to 97% of all currently identified races of C. lindemuthianum (Melloto et al., 2000). Young et al. (1998) reported the Co-4 locus to be a complex gene family with three anthracnose resistance alleles residing at the locus namely Co-42 in cultivars G2333 and SEL 1308, Co-4 in cultivar TO (Young et al., 1998) and Co-43 in cultivar PI207262 (Alzarte-Marin et al., 2007). Melotto and Kelly (2001) fine mapped the complex Co-4 locus to reveal an open reading frame designated as  COK-4  that encodes for protein kinase and was strongly associated with anthracnose resistance. The predicted amino-acid sequence of COK-4 had a high degree of similarity with expressed sequences generated by resistance genes in other crops such as Pto in Lycopersicon pimpinellifolium and L. esculentum; and Xa21 in O. Sativa. Further genetic analysis by Melotto and Kelly (2001) revealed that other genes may be tightly clustered with Co-42, although segregation data indicated a single gene. These tightly clustered genes could be responsible for the broad spectrum resistance associated with the Co-4 locus.

In this study, the Co-43 allele showed a mildly susceptible reaction to races 767 and 2047, however, it is still a highly beneficial allele to specific races and has the potential to add value if combined with other compatible single genes in pyramid. The Co-43 allele was, however, reported to possess a narrower anthracnose resistance spectrum than the Co-4 allele in cultivar TO and the Co-42 allele in cultivar G2333 (Alzate-Marin et al., 2007).

The Co-42+Co-5+Co-9 and Co-42+Co-5 pyramid had the lowest severity scores and did not succumb to any of the races implying that these pyramids are highly effective in conferring broad-spectrum resistance to C. lindemuthianum in Uganda. Kelly (2004) proposed combination of Co-42, Co-5 and Co-6 genes in North America and Co-12 and Co-42 gene pair for Central America. The Co-12 gene confers resistance against Andean races while Co-42 confers resistance against Mesoamerican races. Resistance gene pyramids whichincorporate at least two unique modes of action are reported to delay the evolution of virulent pathotypes (Roush, 1998). Results further revealed that gene pyramids, depending  on  number  and the specific single genes combined were not consistent in conferring broad-spectrum resistance. It was observed that a higher number of genes in a  pyramid  significantly  reduced  the severity of symptoms on the host implying that gene pyramiding has the potential of increasing the potency of resistance.  According  to  Wheeler  and  Diachun (1983); Schafer and Roelfs (1985); and Kolmer et al. (1991), pyramids with higher resistance gene numbers are most likely to remain effective over long periods of time before breaking down. Their argument is based on the probability hypothesis (Mundt, 1991) which states that “cultivars possessing multiple race-specific resistance genes (pyramided genes) owe their durable resistance to a low probability of the pathogen independently mutating to virulence at multiple avirulent loci corresponding to the host resistance genes”. According to this hypothesis the pyramids Co-42+Co-5+Co-9 and Co-42+Co-5, which were the most effective in conferring resistance, are likely to remain effective over a longer period of time. However, the mechanisms by which gene pyramids increase durability is still unknown and there is no strong evidence for gene number as the dominant mechanism for the durability of pyramids (Mundt, 1991).


 CONCLUSIONS

Resistance gene pyramiding was effective in conferring broad-spectrum resistance in some cases and was ineffective in other cases. Effectiveness of pyramided resistance genes heavily depended on the fitness of individual resistance genes combined. It is therefore crucial that plant breeders identify and use favorable resistance gene combinations as opposed to mere accumulation of resistance genes in a cultivar in gene pyramiding programs. The gene combinations Co-42+Co-5+Co-9 and Co-42+Co-5 and the single genes Co-42 and Co-5 were the most effective resistance options against C. lindemuthianum and are recommended for control of the bean anthracnose disease in Uganda through development of resistant varieties. The races 2047 and 767 are recommended for use in screening for resistance to bean anthracnose disease. Further investigations would be beneficial in identifying new effective gene combinations for against C. lindemuthianum based on other available resistance genes.


 CONFLICT OF INTERESTS

There are no competing interests.


 ACKNOWLEDGMENTS

The authors appreciate the Uganda National Council of Science and Technology (UNCST) which funded the study through the Millennium Science Initiative (MSI) project. Additional funding was provided by the National Agricultural Research Organization (NARO) ATAAS project and CIAT-Uganda/ ECABREN.



 REFERENCES

Alzate-Marin AL, de Morais MA, de Oliveira EJ, Moreira MA, de Barros EG (2003). Identification of the second anthracnose resistance gene present in the common bean cultivar PI207262. Annual report. Bean Improvement Cooperation 46:177-178.

 

Alzate-Marin AL, de Souza KA, Silva MGM, Oliveira EJ, Moreira MA, Barros EG (2007). Genetic characterization of anthracnose resistance genes Co-43 and Co-9 in common bean cultivar Tlalnepantla 64 (PI 207262). Euphytica 154:1-8.
Crossref

 

Anonymous (2009). GenStat Discovery, 12th Edition. Lawes Agricultural Trust.

 

Awale HE, Kelly JD (2001). Development of SCAR markers linked to Co-42 gene in common bean. Annual Report. Bean Improvement Cooperative 44:119-120.

 

Awale H, Falconi E, Villatoro JC, Kelly JD (2007). Control and characterization of Colletotrichum lindemuthianum Isolates from Ecuador and Guatemala. Annual Report. Bean Improvement Cooperative 50:85:86.

 

Balardin RS, Kelly JD (1998). Interaction among races of Colletotrichum lindemuthianum and diversity in Phaseolus vulgaris. Journal of the American Society for Horticultural Science 123:1038-1047.
Crossref

 

Balardin RS, Jarosz AM, Kelly JD (1997). Virulence and molecular diversity in Colletotrichum lindemuthianum from South, Central and North America. Phytopathology 87:1184-1191.
Crossref

 

Campa A, Rodriguez-Suarez C, Paneda A, Giraldez R, Ferreira J (2005). The bean anthracnose resistance gene Co-5 is located in linkage group B7. Annual report Bean improvement Co-operative 48(48):68-69.

 

Champion MR, Brunet D, Maudit ML, Ilami R (1973). Method of controlling the resistance of bean varieties to anthracnose (Colletotrichum lindemuthianum (Sacc & Magn.) Briosi et Cav. C. R. Hebd). Seances Academy of Agriculture France 59:951-958.

 

Darben LM, Gonela A, Elias HT, Pastre HH, Goncalves-Vidigal MC (2017). Common bean germplasm resistant to races 73 and 2047 of Colletotrichum lindemuthianum. African Journal of Biotechnology 16(19):1142-1149.
Crossref

 

Davide LMC, Souza EA (2009). Pathogenic variability within race 65 of Colletotrichum lindemuthianum and its implications for common bean breeding. Crop Breeding and Applied Biotechnology 9:23-30.
Crossref

 

De Silva DD, Crous PW, Ades PK, Hyde KD, Taylor PWJ (2017). Life styles of Colletotrichum species and implications for plant biosecurity. Elsevier, Fungal Biology Reviews 31:155-168.
Crossref

 

Doyle JJ, Doyle JL (1987). A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 19:11-15.

 

Ferreira JJ, Campa A, Perez-Vega E, Rodriguez-Suarez C, Giraldez R (2012). Introgression and pyramiding into common bean market fabada of genes conferring resistance to anthracnose. Theoretical and Applied Genetics 124:777-788.
Crossref

 

Genchev D, Christova P, Kiryakov I, Beleva M, Batchvarova R (2010). Breeding of common bean for resistance to the physiological races of anthracnose identified in Bulgaria. Biotechnology and Biotechnological Equipment 24(2):1814-1823.
Crossref

 

Inglis DA, Hagedorn DJ, Rand RE (1988). Use of dry inoculum to evaluate beans for resistance to anthracnose and angular leaf spot. Plant Disease 72:771-774.
Crossref

 

Joshi KR, Nayak S (2010). Gene pyramiding-A broad spectrum technique for developing durable stress resistance in crops. Biotechnology and Molecular Biology Review 5(3):51-60.

 

Kelly JD (2004). Advances in common bean improvement: some case histories with broader applications. In: Proceedings XXVI IHC-Advances in Vegetable Breeding. Eds. J.D McCreight and E.J. Ryder.
Crossref

 

Kelly JD, Afanador L, Cameroon LS (1994). New races of Colletotrichum lindemuthianum in Michigan and implications in dry bean resistance breeding. Plant Disease 8:892-894.
Crossref

 

Kelly JD, Vallejo VA (2004). A comprehensive review of the major genes conditioning resistance to anthracnose in common bean. HortScience 39:1196-1207.
Crossref

 

Kiryowa JM, Ebinu A, Kyaligonza V, Nkalubo ST, Paparu P, Mukankusi C, Tukamuhabwa P (2016). Pathogenic variation of Colletotrichum lindemuthianum causing anthracnose of beans (Phaseolus vulgaris) in Uganda. International Journal of Phytopathology 5:89-98.
Crossref

 

Kolmer JA, Dyck PL, Roelfs AP (1991). An appraisal of stem and leaf rust resistance in North American hard red spring wheats and the probability of multiple mutations to virulence in populations of cereal rust fungi. Phytopathology 81(3):237-239.

 

Lacanallo GF, Gonçalves-Vidigal MC, Vidigal Filho PS, Kami J, Gonela A (2010). Mapping of an Andean gene resistant to anthracnose in the land race Jalo Listras Pretas. Annual Report of Bean Improvement Cooperative 53:96-97.

 

Mahuku SG, Riascos JJ (2004). Virulence and molecular diversity within Colletotrichum lindemuthianum isolates from Andean and Mesoamerican bean varieties and regions. European Journal of Plant Pathology 110:253-263.
Crossref

 

Mahuku SG, Buruchara R, Navia M, Otsyula, R (2007). Development of PCR markers tightly linked to Pyult1, a gene that confers Pythium root rot resistance in the common bean genotype AND1062. Phytopathology 97:69-79.

 

Melotto M, Balardin RS, Kelly JD (2000). Host-pathogen interaction and variability of Colletotrichum lindemuthianum. In: Prusky D, Freeman S and Dickman MB (eds) Colletotrichum Host Specificity, Pathology, and Host-Pathogen Interaction (pp 346-361) APS Press.

 

Melotto M, Kelly JD (2001). Fine mapping of the Co-4 locus of common bean reveals a resistance gene candidate, COK-4, that encodes for a protein kinase. Theoretical and Applied Genetics 103:508-517.
Crossref

 

Mendez de Vigo B, Rodriguez C, Paneda A, Giraldez R, Ferreira JJ (2002). Development of a SCAR marker linked to Co-9 in common bean. Annual Report. Bean Improvement Cooperative 45:116-117.

 

Méndez-Vigo B, Rodríguez-Suárez C, Pañeda A, Ferreira JJ, Giraldez R (2005). Molecular markers and allelic relationships of anthracnose resistance gene cluster B4 in common bean. Euphytica 141:237-245.
Crossref

 

Miklas PN, Kelly JD, Beebe SE, Blair MW (2006). Common bean breeding for resistance against biotic and abiotic stresses: from classical to MAS breeding. Euphytica 147:105-131.
Crossref

 

Mundt CC (1991). Probability of mutation to multiple virulence and durability of resistance gene pyramids. Phytopathology 80(3):221-223.
Crossref

 

Mwesigwa JB (2008). Diversity of Colletotrichum lindemuthianum and reaction of common bean germplasm to anthracnose disease. MSc. Thesis. Makerere University. Uganda.

 

Nkalubo ST (2006). Physiological races of dry bean anthracnose (Colletotricum lindemuthianum) in Uganda. PhD thesis. University of KwaZulu Natal, South Africa.

 

Ragagnin VA, De Souza TLPO, Sanglard DA, Arruda KMA, Costa MR, Alzate-Marin AL, Carneiro S, Moreira MA, De Barrios EG (2009). Development and agronomic performance of common bean lines simultaneously resistant to anthracnose, angular leaf spot and rust. Plant Breeding 128:156-163.
Crossref

 

Roush RT (1998). Two-toxin strategies for management of insecticidal transgenic crops: can pyramiding succeed where pesticide mixtures have not? Philosophical Transactions of the Royal Society, London 353:1777-1786. 
Crossref

 

Schafer JF, Roelfs AP (1985). Estimated relation between numbers of urediniospores of Puccinia graminis f.sp. tritici and rates of occurrence of virulence. Phytopathology 75:749-750.
Crossref

 

Servin B, Martin CO, Mezard M, Hospital F (2004). Toward a theory of marker-assisted gene pyramiding. Genetics 168:513-523.
Crossref

 

Tu JC (1986). A detached leaf technique for screening beans (Phaseolus vulgaris) in vitro against anthracnose (Colletotrichum lindemuthianum). Canadian Journal of Plant Science 66:805-810.
Crossref

 

Wheeler H, Diachun S (1983). Mechanisms of pathogenesis. pp. 324-333 in: Challenging problems in plant health. T. Kommedahl and P.H. Williams, eds. American Phytopathological Society, St. Paul, MN.

 

Ye G, Smith FK (2008). Marker-assisted gene pyramiding for inbred line development: basic principles and practical guidelines. International Journal of Plant Breeding 2(1):1-10.

 

Young RA, Melotto M, Nodari RO, Kelly JD (1998). Marker assisted dissection of oligogenic anthracnose resistance in the common bean cultivar G2333. Theoretical and Applied Genetics 96:87-94.
Crossref

 

Young RA, Kelly JD (1996). Characterization of the genetic resistance to Colletotrichum lindemuthianum in common bean differential cultivars. Plant Disease 80:650-654.
Crossref

 




          */?>