Field trials

In trials established in 2013, the reaction to natural infection by Psph was rated on a scale of 1 to 9 (^{Shoonhoven and Pastor-Corrales, 1987}), where 1 to 3 is considered resistant, 4 to 6 intermediate reaction, and 7 to 9 susceptible. Field visits to assess the incidence of Psph were carried out in the reproductive stage. Crosses between susceptible and resistant varieties were made to obtain Fs1 (varieties of the Nueva Granada race as females and resistant plants of other races as a source of resistance). Parents and F1 plants were inoculated in the primary leaves as described above.

Identification of molecular markers associated with resistance

From the plants of the sown differential varieties, tissue from the youngest trifoliate leaf was collected in the vegetative stage for DNA extraction, following the CTAB procedure described by ^{Doyle and Doyle (1987}). For the quantification and determination of DNA purity, a NanoDrop 8000^® spectrophotometer was used, placing 2 μl of DNA on the pedestal and configuring the program for an optical density of 30 nanograms per microliter and absorbance at 260 nm.

Six sequence-tagged site (STS) markers associated with three genes of halo blight resistance described by ^{Fourie et al. (2004}); ^{Miklas et al. (2009}, ²⁰¹¹) were used. To determine the usefulness of these markers, 10 seeds of each differential variety were sown, and a mixture equivalent in weight of leaf tissue was made for the extraction of DNA from the seedlings of each one. DNA was used to amplify by PCR the six markers by following the indications of ^{Fourie et al. (2004)}; ^{Miklas et al. (2009}, ²⁰¹¹). The amplification products were separated by 1.5% agarose gel electrophoresis with TBE buffer. The gels were left to run 2 h at 120 V, revealed with ethidium bromide, and documented by photograph on a UV transilluminator.

Identification of resistant genotypes in the greenhouse

Inoculation with races 2 and 6 of Psph showed reaction heterogeneity in the symptoms of seedlings of some varieties; that is, in the same material, there were plants with susceptible and resistant reactions. There were also uniform resistant genotypes. Genotypes of intermediate reaction were also identified according to the scale used ( 0 to 1, resistant; 2 to 3, intermediate, and 4, susceptible) (^{Innes et al., 1984}). Due to the diversity of resistant genotypes of different types, both races seem to be widely distributed in Mexico. The genotypes resistant to race 6 were: Pinto 168, Bayo 66, and Bayo 161 of the Jalisco race; Azufrado Tapatío, Lagunero 80, and Pinto Saltillo of the Durango race; and Bayo Azteca of the Mesoamericana race. In this group, Azufrado-type varieties were included, and all were susceptible.

Through inoculation with two isolates of Psph (Races 2 and 6), five varieties resistant to race 2 and eight to race 6 were identified, none of the Azufrado type (Table 1).

^{Guising et al. (2014}, ²⁰¹⁶) characterized the reaction of 283 bean accessions for their resistance to race 6 and identified five resistant ones, suggesting their use as sources of resistance, including a bean native to Puebla, Mexico. In Guanajuato, only the varieties Flor de Mayo M38 and Pinto Saltillo have been used in crosses; the rest of the resistant materials have not been used due to their undesirable agronomic characteristics, such as a type of grain without commercial value, long cycle, and climbing growth habit. Likewise, for selection in segregating populations, this should be done through inoculation with the races present in the region for which the varieties are being developed and not only based on field reaction, where escapes may occur (^{Ghising et al., 2014}).

Reaction of Azufrado genotypes established in the field

In the field, the 10 Azufrado-type materials established in June showed a severe attack by halo blight (data not shown). In this trial, precipitation was high in the vegetative phase during July, and temperatures were favorable for the development of halo blight (Figure 1). Due to the splashing of rain from infected plants onto healthy plants, Psph infection was uniform. It is also likely that Psph was present in the soil from previous bean sowings at the site.

Figure 1 Maximum and minimum temperature and decennial precipitation from January to November 2018 in Celaya, Guanajuato. 1 and 2 indicate the sowing dates. 

In the trial with 16 materials established in July, the incidence of the disease was lower; this was proven because the control varieties were the same in the two trials, JANASA, Azufrasin, and Azufrado Higuera, which showed lower incidence in the second trial (Table 2). This indicates that environmental conditions influence the incidence of the disease since the sowing date of late July and a drought period in mid-August did not favor the early development of halo blight; for the controls of both trials, the same seed source was used.

In the trial of 16 genotypes (Table 2), there were two outstanding ones with incidence < 3.0, both derived from the same cross: (G17421/AZ 26// FME)-1-8-2 and (G17421/AZ 26// FME)-1-8-1. The cross and selection in the segregating population from which these lines were derived were carried out in the test locality; these genotypes should be inoculated under controlled conditions to verify their resistance. Currently, the production of Azufrado Higuera in Sinaloa is carried out under irrigation and with certified seed, and chemical protection for plants is used against pathogens (^{Félix-Gastelum et al., 2016}).

As for the crosses made to incorporate resistance to Azufrado-type varieties, F₁ and F₂ seed was obtained from the crosses: Aluyori x Flor de Mayo Dolores, Azufrasin x Minimi, Azufrasin x Flor de Mayo Dolores, Azufrasin x Junio León, San Rafael x Janasa and Raramuri x Janasa. These populations were incorporated into the local improvement program to continue their assessment.

Use of markers associated with Psph resistance genes

In the differential varieties, except for Tepari 1072, the presence of the six markers available in the literature was determined (^{Fourie et al., 2004}; ^{Miklas et al., 2009}; ^{Miklas et al., 2011}) and of these, only three produced the expected amplicons, ST8, B10 and SW13 (Table 3), while R13, SAE 15 and H11 presented different amplicons, which may be due to differences in the genetic background of the loci that these markers label. In fact, two of the last three markers, R13 and H11, mark the same gene that ST8 and its developers (^{Miklas et al., 2009}) indicate that they only work on Andean material and the black and red types of the Mesoamericana race.

The resistance reported in the differential varieties corresponds to the genes Pse-1 and Pse-3; these do not have resistance to races 2, 6, and 8. That is, the markers do not work in the selection against those races; therefore, their usefulness is limited to races 1, 3, 4, 5, 7, and 9 (^{Miklas et al., 2009}). The three markers that showed the expected response allow the identification of bean genotypes resistant to races 1, 3, 4, 5, 7, and 9 (^{Miklas et al., 2009}), which will be useful to incorporate resistance to the varieties of greater demand of the Azufrado and Cacahuate Bola types, but not against races 2 and 6 that have been isolated in Guanajuato and Sinaloa.

Recent studies of ^{Trabanco et al. (2014}); ^{González et al. (2016)}; ^{Took et al. (2017}) indicate the presence of different genotypes resistant to race 6, with non-race-specific resistance, which, when combined with race-specific alleles, can result in lasting resistance. In the case of the resistant pinto-type genotype US14HBR6, resistance against race 6 was determined to be monogenic (^{Duncan et al., 2014}). Of the materials studied, Flor de Mayo M38, Pinto Saltillo, and Pinto Lagunero 80 with resistance reaction to race 6 do not have the three markers; it is inferred that they have some other resistance gene.

These results suggest the need to develop new markers to be able to use assisted selection efficiently (^{Miklas et al., 2009}) or base it on inoculation under controlled conditions. Recently, ^{Took et al. (2017}) pointed out that resistance to race 6 includes both alleles with non-race-specific (quantitative effects) and race-specific effects (qualitative effect), and that the combination of both types of alleles will provide lasting resistance.

Regarding crosses between resistant and susceptible varieties, it was observed that the B10 marker is present in all (resistant and susceptible); that is, it will not be useful for assisted selection in this group of parents (Table 4). The susceptible variety Azufrasin presented two of the three markers; in this case, none of the three markers will be useful for the selection in crosses with this variety. The results of the parents show that markers, especially T8 and SW13, appear heterogeneously in some; that is, in different proportions (Table 4).

This suggests their use to homogenize the presence of these in those genotypes. Likewise, the absence of the corresponding marker in F1 plants of the Aluyori by Flor de Mayo Dolores cross indicates the presence of self-fertilization. Recently, several authors have conducted studies to identify sources of quantitative resistance, considering that this, in combination with race-specific resistance, may be more durable (^{Trabanco et al., 2014}; ^{González et al., 2016}). ^{Took et al. (2017}) mapped alleles of resistance to Psph race 6 from different sources and showed evidence of durable resistance by combining non-race-specific alleles with race-specific alleles. They suggest the use of molecular markers for the improvement of resistance, especially of the non-race-specific allele.

The varieties Flor de Mayo M38 and Pinto Saltillo have been used extensively in crosses with similar materials; they should be used in crosses with materials of the Cacahuate Bola and Azufrado types. Selection in segregating populations should be made through inoculation with the races present in the region for which the varieties are being developed. In the case of the Bajío region, a region with consecutive rainfed and irrigated cultivation cycles, the possibility of maintaining healthy seeds of susceptible varieties, such as Azufrado and Cacahuate Bola, is limited and is compromised by the reuse of grain as seed by producers.