Strains

30 isolates of P. capsici were used, obtained from plants with symptoms of ‘wilt’, in 12 commercial production lots of different varieties and hybrids of five types of chili peppers (poblano, güero, serrano, jalapeño and chilaca), located in the most important municipalities in the production of this crop in Guanajuato: Dolores Hidalgo, San Luis de la Paz, Juventino Rosas, Silao, Salvatierra and Cortázar (Table 1). These isolates were identified using the keys of ^{Erwin and Ribeiro (1996)}. In addition, two strains PCT17 and PCC6, donated by Dr. Sylvia Patricia Fernández Pavía of the Plant Pathology Laboratory-UMSNH.

From all the isolates, including the reference strains, monozoosporic cultures were made according to the protocol of ^{Mitchell and Kannwischer-Mitchell (1995)}; ^{Erwin and Ribeiro (1996)}. Cultures were kept in V8C solid medium (160 ml of V8 juice, 3 g CaCO3 and 840 ml of deionized water), transferring them every six weeks.

Genetic diversity

The DNA of the 30 isolates and the two reference strains was obtained from mycelium of monozoosporic cultures, using the method proposed by ^{Doyle and Doyle (1987)}. It was quantified with the help of a spectrophotometer (NanoDrop 8000) and the dilutions were standardized at a concentration of 120 ng µl^-1 of DNA.

Amplified fragment length polymorphisms (AFLP) were detected based on the protocol described by ^{Vos et al. (1995)}, using a NEN Global IR2 sequencer, brand LI-COR, which generates and allows the automatic capture of the digital image. The adapter-restriction site primers Eco R1 (E+2) used in this work were: E+AT, E+AG y E+AC; the adapter- restriction site primers Tru 91 (T+2) were T+CC, T+CA, and T+CG. With these primers, the six combinations used for the analysis were formed: E+AT/T+CC, E+AT/T+CA, E+AG/T+CC, E+AG/T+CA, E+AG/T+CG, y E+AC/T+CG.

The images generated were analyzed using the program ‘Cross Checker 2.91’, which encodes the bands using a binary code, where each band is represented as present (1) or absent (0), it was considered that a fragment of DNA is monomorphic if 99% of the population has it and polymorphic to those that differ from this percentage (^{Cavalli-Sforza and Bodmer; 1981}). The binary data were analyzed with the program NTSYS-pc version 2.1 (Numerical Taxonomy Multivariate Analysis System) (^{Rohlf, 2005}) to generate a similarity matrix based on the Dice coefficient (^{Núñez-Colín and Valadez-Moctezuma, 2010}), which considers only the coincidences to determine the similarity between two isolates. A Bootstrap procedure was carried out with 2 000 resamples with replacement, from the average similarity matrix, the genetic relationships between the genotypes studied were determined, generating a consensus dendrogram applying the Unweighted Pair Group Method using Arithmetic Average (UPGMA) (^{Sneath and Sokal, 1973}).

Only well-defined and medium- to high-intensity bands were considered for analysis (Figure 1). A total of 807 bands were obtained, of which 78.81% were polymorphic. The percentage of band polymorphism is similar to that reported by ^{Kersey et al. (2005)}, who, when analyzing 31 isolates of P. capsici from New Mexico USA, found that 80% of 1 155 bands were polymorphic. The level of polymorphism was higher than that obtained by ^{Lamour and Hausbeck (2001a)} for P. capsici isolates from Michigan USA, who reported an average percentage of polymorphisms of 43% for 94 bands.

Figure 1 Amplified fragment length polymorphisms (AFLP), genetic profiles generated by the combination of E+AT/T+CA primers in 30 P. capsici isolates from the state of Guanajuato and 2 reference strains, showing the definition and intensity of the amplified DNA fragments (bands). 

The similarity matrix based on the Dice coefficient shows values ranging from 76 to 95% similarity with an average of 86%, these genetic relationships are reflected in the consensus dendrogram obtained in this work (Figure 2). In it, one can see the formation of four well-defined groups, group I formed with two isolates from Cortázar and one from Silao, which have the least similarity with the rest; the groups with the largest number of isolates are group II, formed only by isolates from Dolores Hidalgo, and group III, formed by a mixture of isolates from Silao and Dolores Hidalgo, group IV is formed by six subgroups, highlighting that five of them are formed only with isolates from the same municipality, these are the subgroups formed only with isolates from Silao, Dolores Hidalgo, San Luis de la Paz, Juventino Rosas and the subgroup formed by the two reference strains, which, following the observed trend could, come from the same locality.

Figure 2 Consensus dendrogram of 30 P. capsici isolates from the state of Guanajuato and 2 reference strains. The numbers on the nodes are the percentage of dendrograms above 70% that were supported by bootstrap analysis. 

In this group only a subgroup formed, which mixes two strains originating from Salvatierra with one from Cortázar. These results seem to indicate a tendency of the isolates to group according to their municipality of origin. Except for the monozoosporic cultures originating from Silao and Cortázar, which grouped with isolates from different localities, which is indicative of a high genetic diversity.

The isolation site-based association is consistent with the study of variability of P. capsici isolates originating from four different regions of Michigan USA reported by ^{Lamour and Hausbeck (2001a)}, in which the grouping found was based on the collection site. Likewise, in what was reported by ^{Parada-Rojas and Quesada-Ocampo (2018)}, who, using microsatellites to analyze the genetic relationships of 50 P. capsici isolates originating in several USA states, find a population structure based on geographical origin. In Mexico, ^{Castro-Rocha et al. (2016)}, analyzing the diversity of 80 isolates originating in the north and center of this country, using SNP-type markers, point out that the isolates from Chihuahua form two groups and the rest form three closely related groups, composed mainly of isolates from Aguascalientes, Guanajuato and Michoacán, finding a separation by regions of isolation.

If one considers what was pointed out by ^{Brasier and Hansen (1992)}, that the genus Phytophthora has a strong selection for climate, so most of its species are found in areas of cool subtropical or tropical temperatures, and that humidity, nutrient content, pH and native microorganisms are factors that influence the adaptation of this pathogen. So, it seems that the differences between the environmental conditions of the regions of Guanajuato where the collections were carried out are the main factors that influenced the formation of the groups in this study.

The municipalities of the north of the state, San Luis de la Paz and Dolores Hidalgo, have a semi-dry climate, with an annual rainfall of 387.5 mm and an average annual temperature of 16 °C; Silao, Juventino Rosas and Cortázar, which are located in the center of the state, have a subtropical-subhumid climate with an annual rainfall of 688 mm and average annual temperature of 19.4 °C, very similar to the climatic conditions of Salvatierra, which is in the south of the state, with a predominantly humid temperate climate with an annual rainfall of 730 mm and average annual temperature of 18.1 °C (^{INEGI, 2013}). Inconsistencies in the grouping by isolation site could be explained by sampling effects, genetic drift or crossing in small populations, which can lead to changes in genetic diversity (^{Goodwin, 1997}) and consequently changes in the groupings.

All the isolates of P. capsici from Guanajuato were unique genotypes, this was expected by the results obtained in the genome sequencing project, where they report that the density and diversity of the variants of a single nucleotide between genomes of P. capsici is notably higher than in other eukaryotic genomes, which has confirmed that the isolates of P. capsici have a large genetic variation in the form of SNPs, which can occur as frequently as 1 in 40 bp (^{Lamour et al., 2012}).

When including in the analysis their morphological and physiological characteristics described in a previous work (^{Pons et al., 2020}), no clear association was found between the genetic groups with morphology, type of compatibility, sensitivity to mefenoxam, the type of chili pepper or the degree of virulence. This absence of correspondence was found in other Phytophthora spp. (^{Lebreton and Andrivon, 1998}; ^{Mahuku et al., 2000}; ^{Abu-El Samen et al., 2003}), where no correlation was found between the groups defined by molecular markers and the virulence, type of mating or response to metalaxyl of the isolates analyzed.

Despite the absence of these correlations, the results of this work indicate that the population of Phytophthora capsici from Guanajuato maintain its characteristic of high levels of diversity, as has been reported in other populations of this pathogen by several authors (^{Lamour and Hausbeck 2001a}; ^{Hausbeck and Lamour 2004}; ^{Gevens et al., 2008}; ^{Hurtado-Gonzáles et al., 2008}; ^{Meitz et al., 2010}; ^{Lamour et al., 2011}; ^{Gobena et al., 2012}). As the two types of mating were found in several of the collection municipalities (^{Pons et al., 2020}), it is likely that a part of this diversity results from sexual recombination and another from mutations that, according to that reported by ^{Goodwin (1997)}, is the primary source of the new genetic diversity in oomycetes. Regardless of its origin, the presence of genetic diversity in the population of P. capsici from Guanajuato indicates that there is a genetic potential in the population of pathogens for the development of resistance to fungicides and to overcome the defenses of the host, so it is necessary to monitor it continuously and consider it for the design of strategies for its control.

Conclusions

There is genotypic variability among P. capsici isolates from Guanajuato. All isolates of P. capsici from Guanajuato are unique genotypes since no clonal individuals were found. The genetic groups that form are based on the place of origin of the isolates. No clear association was found between genetic groups and virulence, mating type or response to metalaxyl in the isolates analyzed.