Evaluation of Pinto Genotypes of Common Bean for Resistance to Anthracnose

Common bean (Phaseolus vulgaris L.) is an important crop grown for household revenue, food, and nutrition security in many parts of the world, especially in Africa and Latin America. Anthracnose caused by Colletotrichum lindemuthianum is a major disease of common bean globally. The objective of this study was to determine the response of selected pinto bean genotypes to seven races of C. lindemuthianum the causative fungus for anthracnose. A total of 56 pinto bean genotypes and three checks were evaluated for resistance to C. lindemuthianum races 51, 65, 73, 247, 253, 263, and 1085. Significant differences were observed among the 56 pinto genotypes in their reaction to the seven races, which was generally skewed towards susceptibility except for races 51 and 73. There was no genotype that was resistant to all seven races. In general, the genotypes that showed resistance to most of the races were those that carried Co‐42, which highlighted the importance of this locus to anthracnose resistance in pinto beans. Three genotypes—NDZ14006‐4, NDZ14110‐4, and NDZ14043—showed superior resistance (resistant to six of the seven races).


| Introduction
Common bean (Phaseolus vulgaris L.) is an important grain legume for direct human consumption (Broughton et al. 2003).It plays a major role in household food and nutrition security in Latin America and Eastern and Southern Africa (Uebersax et al. 2023).It is a source of protein and the essential micronutrients iron and zinc, which tend to be deficient in most African diets.Furthermore, common bean is an important source of dietary fiber and carbohydrates (Wortmann 1998).
Several biotic and abiotic stress factors limit the production of common bean (Binagwa et al. 2021;Hamabwe et al. 2023;Njobvu et al. 2020;Portilla et al. 2022;Siamasonta et al. 2021).Anthracnose, caused by Colletotrichum lindemuthianum, is a major disease of common bean and can cause yield losses of up to 100% depending on varietal susceptibility and environmental conditions (Pastor-Corrales and Tu 1989).High humidity coupled with cooler ambient temperature enhances the spread of anthracnose.Anthracnose affects all aerial parts of the plant including leaves, stems, and pods (Schwartz and Pastor-Corrales 2005).It is a seed-borne disease, whose primary mode of transmission is through the planting of infected seed (Ferreira, Campa, and Kelly 2013).The informal seed system practice of sharing farm-saved seed, which is prevalent in many African countries, makes control and management of anthracnose challenging.Though anthracnose can be effectively controlled by use of fungicides, small-scale farmers cannot afford them, and there are health risks associated with their use.Therefore, the development and use of anthracnose-resistant varieties is the most cost-effective and environmentally friendly management strategy.
The development of common bean varieties with durable resistance to anthracnose remains challenging because of the extensive genetic variability for C. lindemuthianum.To date, over 298 races have been characterized globally (Nunes et al. 2021) using a set of 12 race differential cultivars (Pastor-Corrales 1991).Extensive genetic variation for C. lindemuthianum has also been reported in many African countries (Mungalu et al. 2020;Sansala et al. 2023).The extensive genetic variability and rapid evolution of new races makes it challenging to develop varieties with durable resistance to anthracnose.C. lindemuthianum has coevolved with the Andean and Middle American gene pools, resulting in Andean races that are virulent mostly on Andean genotypes and Middle American races that are virulent mostly on Middle American genotypes (Geffroy, Sicard, et al. 1999).
Resistance to C. lindemuthianum is oligogenically controlled involving mainly major-effect resistance loci (Kuwabo et al. 2023;Mungalu et al. 2020;Zuiderveen et al. 2016).To date, about 13 major-effect loci (Co-1 to Co-18) have been reported (Lateef et al. 2024).The resistance provided by these major-effect loci follows gene-for-gene action, that is, conferring resistance to specific races (Flor 1955).These major-effect loci have been classified into Andean and Middle American loci.The clusters of resistance (R) genes underlie some of the resistance loci such as Co-3 (Campa, Garcia-Fernandez, and Ferreira 2020;Oblessuc, Francisco, and Melotto 2015).Though there is no single gene that confers resistance to all races as indicated by previous work in Tanzania where races 3068 and 3264 overcame the three resistance genes  in the differential cultivar G2333 (Mpeguzi et al. 2020), the locus Co-4 2 confers resistance to multiple races of C. lindemuthianum.
The pinto bean class, which belongs to the Durango race of the Middle American gene pool, is a major market class in countries such as the United States.Anecdotal evidence suggests that there is a growing interest in pinto beans in some African countries such as Tanzania and Zambia.Anthracnose may hinder expansion of pinto bean production in Africa.There is no previous study to determine the response of pinto beans to specific races of C. lindemuthianum in Africa.Therefore, identification of pinto bean germplasm resistant to C. lindemuthianum is important for meeting future varietal needs of farmers in Zambia.The objective of this study was to evaluate the response of 56 pinto genotypes to C. lindemuthianum races 51, 65, 73, 247, 253, 263, and 1085.

| Plant Materials
A pinto bean collection comprising 56 pinto varieties and breeding lines was used (Data S1).These varieties and breeding lines were sourced from public bean breeding programs in the United States.Dr. Phillip Miklas of the USDA-ARS, Prosser, Washington, USA, assembled the collection.The varietal names or breeding line codes are provided in Table S1.Two genotypes, Kabulangeti and Lusaka, which are Andean landraces from Zambia, were used as susceptible checks (Mungalu et al. 2020;Nalupya et al. 2021), whereas the Middle American landrace "Colorado de Teopisca" designated as G2333 was used as a resistant check.

| Inoculation and Anthracnose Severity Assessment
The 56 pinto genotypes and checks were inoculated with previously characterized races of C. lindemuthianum, namely, races 51, 65, 73, 247, 253, 263, and1085 (Sansala et al. 2023).These races were characterized from isolates collected from Zambia (Sansala et al. 2023) and were selected for use in this study to obtain a wide virulence range and representation of Andean, Middle American, and mixed races of C. lindemuthianum.
The 56 genotypes and checks were planted in a Styrofoam tray, which had 200 wells with each well measuring 3 cm long, 3 cm wide, and 5 cm deep.The experiment was set up in a completely randomized block design with three replications.Each replication had two seedlings; therefore, a total of six seedlings per genotype were evaluated.Seedlings in Styrofoam trays were grown to the unifoliate (fully expanded primary leaf) stage and then inoculated with races 51, 65, 73, 247, 253, 263, and 1085.Inoculation was conducted for a single race at time.Inoculated seedlings were transferred to the high-humidity (> 90°C) growth chamber where they were maintained for 72 h.At the end of 72 h, the materials were transferred to the green house where they were maintained at room temperature and humidity for 5-7 days to allow for anthracnose disease development.
The 1-9 CIAT scale (Balardin and Kelly 1998) was used to score anthracnose severity.Plants with no visible symptoms or a few small lesions mostly on primary leaf veins were classified as resistant (scales 1-3), seedlings with small lesions mostly on leaves and stems seedlings were considered moderately resistant (scales 4-6), and seedlings with numerous or enlarged lesions, with sunken cankers on leaves and seedling stem, or dead plants were classified as susceptible (scales 7-9).

| Data Analysis
The analysis of variance (ANOVA) on anthracnose reaction severity scores was conducted using PROC MIXED in SAS 9.3 (SAS Institute 2011) based on the statistical model: where anthracnose severity score for genotype i in replication k, α i was the fixed effect of genotype, γ was the random variable effect of a replication, and £ was the random error associated with replication k for genotype i.

| Results
The Middle American resistant check G2333 showed a consistent resistant reaction (< 2.0) to all seven races (Table 1).As for the susceptible checks, the reaction to the seven races was variable.The first susceptible check-Kabulangeti-was highly susceptible to three races (51, 65, and 247), moderately resistant to two races (253 and 1085), and resistant to two races (73 and 263).The second susceptible check-Lusaka-was highly susceptible to three races (51, 73, and 247), moderately resistant to three races (253, 263, and 1085), and resistant to one race (65).

| Race 247
The disease scores of the 56 genotypes for reaction to race 247 were skewed towards susceptible (Figure 4).The results revealed that 5 (8.3%) genotypes were resistant, 7 (11.7%)were moderately resistant, and 48 (80%) were susceptible to race 247.The local susceptible checks Kabulangeti and Lusaka were both susceptible to race 247, whereas the anthracnose resistant check G2333 was resistant.The range of scores for reaction to race 247 was 1-9, with an average score of 7.5.Three genotypes (NDZ14086, NDZ14006-4, and G2333) had score of 1.

| Race 253
The disease scores of the 56 genotypes for reaction to Middle American race 253 were skewed towards susceptible (Figure 5).

| Race 263
The disease scores of the 56 genotypes for reaction to race 263 were skewed towards susceptible (Figure 6).The results revealed that five (8.3%) genotypes were resistant, one (1.7%) was moderately resistant, and 50 (90%) were susceptible to race 263.The local susceptible checks Kabulangeti and Lusaka were both moderately resistant to race 263, whereas the anthracnose resistant check G2333 was resistant.The range of scores for reaction to race 263 was 1-9, with an average score of 7.8.Three genotypes (G2333, Kabulangeti, and NE2-18-12) had score of 1.

| Race 1085
The disease scores of the 56 genotypes for reaction to race 1085 were skewed towards (Figure 7).Of the 56 evaluated, 7 (13.3%)were resistant, 11 (18.3%) were moderately resistant, and 41 (68.3%) were susceptible to race 1085.The local susceptible check Kabulangeti was resistant, whereas the susceptible check Lusaka was susceptible.The resistant check G2333 was resistant.The range of scores for reaction to race 1085 was 1-9, with an average score 7.3.
In addition to resistant loci Co-4 2 , the anthracnose resistant genotype G2333 carries the Co-5 and Co-7 resistant loci.

| Discussion
Of the 56 pinto genotypes evaluated in this study, nine had the Co-4 2 anthracnose resistance allele.The average severity score (2.4) for these nine genotypes was significantly lower than the average score (6.2) for the 47 genotypes that did not have the Co-4 2 .The nine genotypes with Co-4 2 provided variable levels of resistance to the seven races, suggesting that other host resistance genes could have been involved the resistance they showed.It is important to note, however, that the resistance allele Co-4 2 was not effective against all seven races.All nine genotypes with Co-4 2 allele were susceptible to races 251 and 1085 except genotypes NDZ14110-4 and NDZ14006-4, which were moderately (severity score of 3) resistant to races 251 and 1085, respectively.The resistant check G2333, which also carries Co-4 2 , was the only one among genotypes with Co-4 2 that was resistant to all seven races used in the current study.This broad-spectrum resistance in G2333 could be attributed to additional resistance loci Co-5 and Co-7 that it carries.The susceptibility to races 263 and 1085 of some genotypes that carry Co-4 2 underscores the need for pyramiding additional resistance loci such as Co-5 to confer broad-spectrum resistance to C. lindemuthianum similar to the one observed in the resistant check G2333.Race 263 was highly virulent on all the 56 pintos except NE2-18-12 and the resistant check (G2333).Surprisingly, the susceptible check (Kabulangeti) was resistant.The genotype NE2-18-12, therefore, would be a valuable source of resistance for genetic improvement of pinto beans in geographic areas that have race 263.However, in areas that have races 51, 65, 247, and 1085, NE2-18-12 would not be an effective source of resistance.
The genotype BelDakMi-RR-5 was interesting because it was resistant to five races including the highly virulent race 1085, and yet, it does not carry Co-4 2 or any other known major anthracnose loci.Future studies aimed at understanding the genetic basis of this observed resistance would be worthwhile.
A total of nine pinto genotypes were highly resistant to race 65.These nine genotypes can be used as sources of resistance to race 65 not only in Zambia where race 65 is widespread but also in other countries such as Brazil where it is an important race because of its high frequency of occurrence and wide physiological variability (Paulino et al. 2022;Ribeiro et al. 2016).Six pinto genotypes were highly resistant to the Middle American race 73, which is present in Zambia, but also an important race to other countries including Brazil and the United States, where Middle American genotypes are predominantly grown (Balardin, Jarosz, and Kelly 1997;Kelly, Awale, and Wiersma 2020).These six highly resistant genotypes would be valuable sources of resistance in these countries.

| Conclusions
The genotypes presented variable reaction to the seven races.The most virulent race was 263, whereas the least virulent was 51.Except for races 51 and 73, the reaction of the 56 pinto genotypes was skewed towards susceptibility, which highlighted the lack of resistance in most of the pinto beans to the races used in the current study.In general, the genotypes that showed resistance to most of the races were those that carried Co-4 2 , thereby highlighting the importance of this locus.The genotypes NDZ14006-4, NDZ14110-4, and NDZ14043 showed superior resistance among the 56 pinto genotypes used in the current study as each was resistant to six of the seven races used in the current study.These three genotypes, which all carry Co-4 2 , can be used for genetic improvement of pinto beans for anthracnose resistance.None of the 56 pinto genotype that showed resistance to all seven races of C. lindemuthianum similar to the resistant check G2333.This result underscored the importance of pyramiding loci Co-4 2 and Co-5.It would also be useful to pyramid the major resistance locus Co-1 (Andean locus), with Co-4 2 and Co-5 loci in pinto genotypes to provide them with durable resistance to multiple races of C. lindemuthianum found in Zambia.This pyramid would be necessary because of the presence of both Andean and Middle American races in the beangrowing regions of Zambia (Sansala et al. 2023).

FIGURE 1 |
FIGURE 1 | Frequency distribution of severity scores for race 51 of Colletotrichum lindemuthianum inoculated on 56 pinto genotypes.

FIGURE 2 |
FIGURE 2 | Frequency distribution of severity scores for race 65 of Colletotrichum lindemuthianum inoculated on 56 pinto genotypes.

FIGURE 7 |
FIGURE 7 | Frequency distribution of severity scores for race 1085 of Colletotrichum lindemuthianum inoculated on 56 pinto genotypes.

TABLE 1 |
Means and ranges for anthracnose severity scores measured on 56 pinto genotypes and checks inoculated with seven races of Colletotrichum lindemuthianum in the green house at University of Zambia.