Genetic resistance of common bean cultivar Beija Flor to Colletotrichum lindemuthianum

Anthracnose, which is caused by the fungus Colletotrichum lindemuthianum, is one of the most widespread and important diseases of the common bean (Phaseolus vulgaris L.) in the world. The objective of the present study was to characterize the genetic resistance of the Beija Flor cultivar by inheritance and to conduct allelism tests. The inheritance test was conducted in the F2 population derived from the Beija Flor (resistant) x TU (susceptible) cross inoculated with race 2047 of C. lindemuthianum. Furthermore, allelism tests exhibited a fitted segregation ratio of 15R:1S, thereby indicating the independence of the Beija Flor gene from the following previously characterized genes: Co-1, Co-2, Co-4, Co-42, Co-6, Co-12, Co-14, Co-15, and Co-Pe. Based on the aforementioned results, we are proposing the symbol Co-Bf to designate the new anthracnose resistance gene in the Brazilian Andean common bean cultivar Beija Flor. This cultivar is an important source of resistance to C. lindemuthianum that should provide a valuable contribution to the common bean breeding program for anthracnose resistance.


Introduction
The common bean (Phaseolus vulgaris L.) is an important protein source, especially in developing countries, for instance, in Latin America and Africa (Broughton et al., 2003). The most productive areas are among these main consuming regions; currently, Latin America shows the largest common bean production in the world followed by Africa (CGIAR, 2015).
The common bean is a crop with broad edaphoclimatic adaptation; therefore, it is affected by many diseases, which depreciate the quality of the product and decrease the crop productivity. These diseases can be fungal, bacterial or viral in origin (Pastor-Corrales & Schwartz, 2005). Anthracnose, whose causal agent is Colletotrichum lindemuthianum, is among the major fungal diseases that present symptoms in the aerial part of the common bean.
This disease stands out as one of the most important because it causes losses of up to 100% of the production and because it causes stains in the grains, depreciating the product (Rey, Balardin, & Pierobom, 2005). Temperatures above 25°C or below 18°C are limiting for both infection and pathogen development. In addition, sporulation is abundant in the pods at temperatures between 14 and 18°C (Pastor-Corrales & Schwartz, 2005).
Several of these Andean cultivars have been reported as sources of new anthracnose resistance genes (Gonçalves-Vidigal et al., 2008;Gonçalves-Vidigal et al., 2011;Gonçalves-Vidigal et al., 2012;Sousa et al., 2015;Castro et al., 2017). Among these Andean cultivars, the Beija Flor (BF) was collected in the state of Paraná, has a type I growth habit, medium-size seeds (36.02 g per 100 seeds), and morphological characteristics of the Andean gene pool (Singh, Gepts, & Debouck, 1991). The phaseolin seed protein test, as described by Kami, Velásquez, Debouck, and Gepts (1995) and conducted on seeds of Beija Flor, showed the presence of phaseolin 'T', which is characteristic of common beans from the Andean gene pool. Previous studies of anthracnose resistance showed that Beija Flor is resistant to the Andean race 7 and to the Mesoamerican races 9, 64, 65, 73, 89, 453, and 2047 of C. lindemuthianum. Due to its good resistance spectrum and resistance to one of the most virulent races, the race 2047, for which only one of the 12 differentiating cultivars is resistant (Pastor-Corrales, Erazo, Estrada, & Singh, 1994), the Beija Flor cultivar is of great value for the common bean breeding programmes. The search for new sources of Andean resistance to C. lindemuthianum is extremely important for common bean breeding programmes in tropical and subtropical regions.
Therefore, the objective of this study was to characterize the genetic resistance and to conduct allelism tests in the Beija Flor cultivar to Colletotrichum lindemuthianum.

Material and methods
Beija Flor (BF), which is studied herein, belongs to the Andean gene pool and is one of 26 Andean and Mesoamerican common bean landraces collected in the Northern and North-western regions of the state of Paraná in Southern Brazil (Vidigal Filho, Gonçalves-Vidigal, Kelly, & Kirk, 2007). The present work was conducted from March 2015 to October 2016 under greenhouse conditions and at the Common Bean Breeding and Molecular Biology Laboratory of the Núcleo de Pesquisa Aplicada à Agricultura (Nupagri), Universidade Estadual de Maringá, Paraná State, Brazil.

Population development
The inheritance resistance studies were conducted on the F 2 population derived from the Beija Flor (R) × TU (S) cross. The resulting segregating F 2 population was inoculated with race 2047 of C. lindemuthianum. TU is susceptible to race 2047, whereas Beija Flor is resistant. To determine the independence of the anthracnose resistance allele present in Beija Flor (BF) from other previously characterized anthracnose resistance alleles, BF was crossed with Andean bean cultivars Michigan Dark Red Kidney (Co-1), Jalo Vermelho (Co-12), Pitanga (Co-14), Corinthiano (Co-15), and Perla (Co-Pe), and with the Mesoamerican cultivars Cornell 49-242 (Co-2), TO (Co-4), AB 136 (Co-6), and G 2333 (Co-4 2 /Co-5 2 /Co-3 5 ). In all cases, BF was used as the female parent. F 1 seeds were planted and self-pollinated to obtain the corresponding F 2 seeds.

C. lindemuthianum races used in the study
The C. lindemuthianum races used in this work were 65 and 2047, which were obtained from the mycology collection of the Nupagri, and the monosporic cultures were prepared at the Common Bean Breeding and Molecular Biology Laboratory. The Mesoamerican race 2047 was chosen in this study due to its high virulence and importance in common bean breeding programs for anthracnose resistance, as well as the resistance response shown by BeijaFlor against this race. In the inheritance test, race 2047 was inoculated into the F 2 generation of the cross (R × S) Beija Flor × TU, and in the allelism tests with the F 2 generation of the crosses (R x R) between Beija Flor and resistant cultivars. The identification of the races was confirmed by inoculation of the isolates on a set of 12 common bean anthracnose differential cultivars (Pastor-Corrales, 1992).

Inoculation and disease evaluation
Monosporic cultures of each race of C. lindemuthianum used in this study were prepared in young green common bean pod medium and incubated at 25°C for 14 days. The inoculation of the parents and the F 1 and F 2 populations from each cross was carried out separately. Seedlings were grown under natural light in greenhouses supplemented by 400-w, high-pressure, sodium lamps, yielding total light intensity of 115 μmoles m -2 s -1 for 7 to 10 days until the seedlings had reached the first trifoliate leaf stage. Twelve parental and F 1 seedlings from each cross were inoculated, and the number of F 2 seedlings inoculated varied by cross, as shown in Table 1. Seedlings with fully developed first trifoliate leaves of common bean cultivars and the F 1 and F 2 populations were inoculated with spores of selected races of C. lindemuthianum. Spore suspensions containing 1.2 x 10 6 spores mL -1 were spray-inoculated on seedlings using a De Vilbiss, Model 15 Atomizer powered by an electric air compressor. In each cross, the F 2 seeds and those of the respective parents and one susceptible control (Michelite, Mexico 222) were used. After inoculation, the plants were maintained at >95% relative humidity at 21-23°C, with a 16-h day length (light intensity of 300 micromoles m -2 s -1 at 1 m height) in a mist chamber for 2 days. After this period, the plants were removed from the mist chamber and transferred to benches under suitable environment at 22°C with artificial light (12-h day length at 25°C) for seven days. Anthracnose disease reactions were rated visually using a scale from 1 to 9 (Pastor-Corrales et al., 1995).

Statistical analysis of data
Individual plants with disease reaction scores between 1 and 3 were considered resistant, whereas plants that scored 4-9 were considered susceptible. Segregation analysis of the disease reaction of 100 F 2 plants from the cross Beija Flor × TU were performed by the Chi-square ( 2 ) test, according to the Mendelian segregation hypothesis of a 3:1 (resistant to susceptible) ratio. Similarly, the disease reaction of the 829 F 2 plants derived from the crosses Beija Flor and the cultivars Michigan Dark Red Kidney, Jalo Vermelho, Pitanga, Corinthiano, Perla, Cornell 49-242, TO, AB 136, and G 2333 were tested for a goodness of fit of 15:1 (resistant to susceptible) ratio.

Inheritance of anthracnose resistance
Beija Flor and Pitanga (Co-14), Corinthiano (Co-15), Perla (Co-Pa), and G 2333 (Co-3 5 , Co-4 2 , Co-5 2 ) were resistant to Mesoamerican races 65 and 2047 of C. lindemuthianum used in this study. However, the Jalo Vermelho (Co-12), TU (Co-5), Michigan Dark Red Kidney (Co-1), Cornell 49-242 (Co-2), TO (Co-4), and AB 136 (Co-6) included in this study were susceptible to race 2047 and resistant to race 65 (Table 1). The differential cultivar TU, which was crossed with BF to study the inheritance of resistance, was susceptible to race 2047. This race was chosen because is one of the most virulent and only the G 2333 of the 12 differentiating is resistant to it. The Mesoamerican differential cultivar G 2333, which has three anthracnose resistance genes, has been evaluated as resistant to several hundred races of the anthracnose pathogen worldwide, including race 2047 Padder et al., 2017). Eleven different cultivars were susceptible to race 2047. Previous studies had revealed that the Beija Flor is an important source of resistance against races 7,9,64,65,73,89,453, and 2047 of C. lindemuthianum. Thus, the results of this study underscored the comprehensive resistance of BF to a broad diversity of Mesoamerican and Andean races of C. lindemuthianum.
All F 1 plants from the Beija Flor × TU cross were resistant to race 2047, suggesting that a single dominant gene conditioned anthracnose resistance in Beija Flor. Segregation for resistance in the F 2 populations was consistent with a 3 resistant:1 susceptible ratio for Beija Flor × TU ( 2 = 0.00; p = 1.0). These results support the hypothesis that a single dominant gene in the Andean common bean cultivar Beija Flor provides anthracnose resistance to race 2047 of C. lindemuthianum; thus, the anthracnose resistance of BF is monogenic and dominant.
In this test, the segregation at the F 2 population occurred at a ratio of 3R:1S ( 2 = 0.00; p = 1.0), showing that the action of only one dominant gene, present in the Beija Flor cultivar, gives resistance to race 2047 (Table 1). When Gonçalves-Vidigal et al. (2012) tested the F 2 population derived from a Pitanga × AB 136 cross, the authors obtained segregation of 3 resistant:1 susceptible with ( 2 = 0.012; p = 0.91), providing evidence that the resistance to race 2047 is conferred by a dominant gene present in the Pitanga cultivar. In 2015, Sousa and collaborators reported such results ( 2 = 0.002; p = 0.97) when they used race 2047 in F 2 population from the cross of Corinthiano × Cornell 49-242. According to the Chi-square test applied to the data obtained in the resistance inheritance test, dominant monogenic inheritance was hypothesized to be present in the Beija Flor cultivar in response to race 2047.

Allelism tests
The allelism tests evaluated the independence of the gene present in the Beija Flor cultivar relative to nine genes previously characterized and described in the literature. The allelism tests were conducted in the F 2 populations, where both cultivars involved showed resistance. Allelism tests were carried out with nine F 2 populations from crosses between the BF and Andean (Michigan Dark Red Kidney, Jalo Vermelho, Pitanga, Corinthiano and Perla) and Mesoamerican (TO, Cornell 49-242, AB 136, and G 2333) cultivars (Table 1). Segregation for resistance in the nine crosses fitted a 15 resistant:1 susceptible ratio.
These nine F 2 populations were from crosses between BF and the Andean cultivars Michigan Dark Red Kidney ( 2 = 0.609; p = 0.43), Jalo Vermelho ( 2 = 0.266; p = 0.60), Pitanga ( 2 = 0.170; p = 0.68), Corinthiano ( 2 = 0.006; p = 0.93), and Perla ( 2 = 0.011; p = 0.92), and Mesoamerican cultivars Cornell 49-242 ( 2 = 0.011; p = 0.92), TO ( 2 = 0.001; p = 0.98), AB 136 ( 2 = 0.011; p = 0.92), and G 2333 ( 2 = 0.606; p = 0.80). These results support the hypothesis that two independent dominant genes confer resistance to C. lindemuthianum in these F 2 populations; one anthracnose resistance gene present in the Andean bean BF was used as the female parent and the other gene was from the male parent. The segregation results for anthracnose resistance in the F 2 population from crosses between BF and the other Andean bean cultivars indicated that the anthracnose resistance dominant gene in BF is independent of Andean anthracnose resistance genes Co-1,  Similarly, the segregation for anthracnose resistance in the F 2 populations derived from the crosses between BF and the Mesoamerican cultivars Cornell 49-242, TO, AB 136, and G 2333 indicate that the dominant anthracnose resistance gene in BF is also independent of Mesoamerican anthracnose resistance genes Co-2,  .
The F 2 population of the cross between Beija Flor × Cornell 49-242, when inoculated with race 65 the segregation ratio, fitted a 15R:1S ratio and showed statistical results of the  2 = 0.011 and p = 0.92. Similar results were found in 2006 by Gonçalves-Vidigal and Kelly (2006) when they inoculated race 65 in a population from a Widusa cultivar × MDRK cross (p = 0.79).
The F 2 population derived from the crosses Beija Flor × TO ( 2 = 0.001; p = 0.98) and Beija Flor × AB 136 ( 2 = 0.011; p = 0.92), inoculated with race 65, showed segregation of 15R:1S, demonstrating the presence of two dominant genes. This indicates that the gene which confers resistance to the race 65 present at Beija Flor is independent of the genes Co-1, Co-2, Co-4 and Co-6.
The allelism test using the differentiating cultivar G 2333 was made with race 2047 of C. lindemuthianum because the cultivar has one anthracnose resistant allele, Co-4 2 , which confers resistance to that race. The result obtained from the F 2 population from the Beija Flor × G 2333 cross (15R:1S) shows the action of two independent and dominant genes, with one of them being the Co-4 2 present in the cultivar G 2333 and the other being a gene present in the Beija Flor cultivar. This segregation could be observed because, although cultivar G 2333 has three resistance genes, only Co-4 2 allele confers resistance to race 2047.
In summary, the current study on anthracnose resistance inheritance revealed that the resistance in BF is monogenic and dominant. Similarly, the allelism tests carried out in nine F 2 populations revealed that the anthracnose resistance gene in the Andean common bean landrace Beija Flor is independent from the Andean Co-1, , and Co-Pe, and from the Mesoamerican Co-2, Co-4, Co-6, Co-4 2 genes.
Previous work conducted by our group showed that the Beija Flor cultivar has morphological characteristics similar to bean cultivars from the Nueva Granada race of the Andean gene pool (Singh et al., 1991). The Beija Flor cultivar, which was collected in the state of Paraná, has a type I growth habit, mediumsize seeds (36.02 g per 100 seeds), and morphological characteristics of the Andean gene pool (Singh et al., 1991). The phaseolin seed protein test, conducted on seeds of BF, as described by Kami et al. (1995), showed that these seeds had phaseolin 'T', which is characteristic of common beans from the Andean gene pool. The initial anthracnose resistance studies of BF showed that BF was resistant to Andean race 7 and to the Mesoamerican races 9, 64, 65, 73, 89, 453, and 2047 of C. lindemuthianum. Due to its good resistance spectrum and resistance to one of the most virulent races, the race 2047, and because it is only one of the 12 different cultivars resistant to that race , the cultivar Beija Flor is of great value for the common bean breeding programmes.
The combined results of the monogenic inheritance and the allelism tests support the hypothesis that only a single dominant locus confers resistance to races 65 and 2047 of C. lindemuthianum in the Andean common bean landrace Beija Flor and that this cultivar is independent from other Andean and Mesoamerican bean anthracnose resistance loci previously reported. The authors propose that this single dominant anthracnose resistance locus in the Beija Flor cultivar provisionally be named as Co-Bf.
These results are particularly relevant to bean breeding programmes that wish to broaden the genetic base of the bean crop and the pyramid of Andean and Mesoamerican genes by conferring resistance to distinct races of C. lindemuthianum. Gene pyramiding results in bean cultivars with durable anthracnose resistance (Young & Kelly, 1996). The data obtained in the present research provides breeders with an anthracnose resistant gene of Andean origin that can be deployed in commercial cultivars, thereby providing an opportunity to improve the effectiveness of the anthracnose resistance gene pyramiding in common bean breeding programmes while also broadening the germplasm base of the common bean.

Conclusion
The inheritance and allelism tests revealed that Beija Flor has a dominant gene for resistance to C. lindemuthianum, being independent of previously characterized genes, which include the following: Co-1,  The results indicate that the Beija Flor cultivar is an important source of anthracnose resistance because it presents a new gene that can be used as a potential source of resistance. This gene can contribute to future common bean breeding programmes and commercial cultivars, with the purpose of increasing the spectrum of resistance to anthracnose.