Peronospora tabacina is a pathogen that causes the disease known as blue mold or tobacco mildew. In the past, it caused significant economic losses in US crops, with estimated losses of $250 million (^{Lucas, 1980}). This oomycete infects primarily the aerial parts of plants, such as leaves. However, under favorable environmental conditions, it can infect any stage of the crop and cause systemic infections (^{Milholland et al., 1981}; ^{Spurr and Todd, 1982}; ^{Caiazzo et al., 2006}). Its most common reproductive structures are asexual, known as sporangiophores or sporangia, containing multiple diploid nuclei. These sporangia are produced massively and are easily dispersed by wind, being the main means of reproduction and spread of this pathogen (^{Hall, 1989}; ^{Spring et al., 2018}). Under optimal environmental conditions, this pathogen can produce over 10⁵ sporangia/cm² in a single lesion (^{Cohen, 1976}).

Despite its importance, few studies have investigated the biology and population genetics of this pathogen. This may be because it is an obligate parasite, which makes it difficult to characterize and obtain a sufficient number of isolates (^{Derevnina et al., 2015}; ^{Nowicki et al., 2022}).

Genetic variation studies in plant pathogen populations have become increasingly important due to the availability of several molecular markers. These studies have applications in detection, diagnosis, taxonomy, epidemiology, and population structure, each requiring different sampling, genetic markers, and analyses (^{Milgroom, 1997}). Moreover, genotypic diversity measurements and patterns within populations can infer clonality or recombination (Milgroom, 1996).

DNA markers are widely used for analyzing plant pathogen population dynamics due to their high precision (^{Milgroom and Peever, 2003}). Microsatellites, also known as Simple Sequence Repeats (SSRs), are one of the available molecular markers that offer significant advantages. They consist of short DNA sequences of 1 to 6 nucleotides, repeated a certain number of times in tandem, and are abundant in the genomes of most eukaryotic organisms (^{Gupta et al., 1996}). Microsatellite analysis uses the PCR technique, requires small amounts of DNA, and its codominant nature makes microsatellites one of the most preferred markers for marker-assisted selection programs and genetic mapping and diversity studies (^{Gupta et al., 1996}; ^{Jarne and Lagoda, 1996}). Microsatellites are ideal for obtaining the genetic identification and fingerprinting of many organisms, including fungi and oomycetes, that show high polymorphism.

Several studies have aimed to characterize microsatellites of Peronospora tabacina. One such study was conducted by ^{Trigiano et al. (2012}), in which 10 microsatellite loci were characterized in 44 isolates of this pathogen collected from various regions of the world. The microsatellite loci were found to be polymorphic. Polymorphism is the genetic variation through time in populations, resulting from some type of mutation. The amplification of these microsatellites allows visualizing or indicating the presence of allelic variants, which is essential for distinguishing groups, populations, isolates, species, or higher taxonomic groups, identifying the source of populations, estimating population divergences, and identifying the gene flow between natural banks or seedbeds. Furthermore, seven of the ten microsatellites characterized in the study by ^{Trigiano et al. (2012}) were evaluated by ^{Nowicki et al. (2022}), who added two additional microsatellites to their analysis to assess the genetic diversity in 122 P. tabacina isolates. Thus, the objective of the present study is to identify and characterize molecular microsatellites in isolates of Peronospora tabacina collected from tobacco fields distributed across three producing states in Mexico, using 12 microsatellites.

Leaf samples with blue mold symptoms and pathogen signs were collected from commercial tobacco fields in Nayarit, Chiapas, and Veracruz, Mexico, between 2018 and 2021. Samples were taken to the Phytopathology Laboratory of the Research Center for Food and Development Culiacán Unit, where they were air-dried daily and stored between newspapers.

DNA extraction from each P. tabacina isolate was performed using the CTAB method according to the method reported by ^{Voigt et al. (1999}). The quantification of the obtained DNA was carried out using a Nanodrop One (Thermo Scientific, USA). A polymerase chain reaction (PCR) was initially performed for genotyping and confirmation of the genus and species of P. tabacina using the specific oligonucleotides PTAB and ITS4 under specific conditions described by ^{Ristaino et al. (2007}). Subsequently, the amplification and genotyping of 12 microsatellites were performed using the method proposed by ^{Trigiano et al. (2012}) and ^{Nowicki et al. (2022}). The PCR was carried out in a 15 µL reaction volume using 7.5 µL of Master Mix, 1 µL of each oligonucleotide, 4.5 µL of water, and 1 µL of DNA (15 ng µL^-1). The amplification conditions were as described by Trigiano et al. (2012). The amplified products were separated in 2% agarose gels stained with Gel Red and run in an electrophoresis chamber (BioRad, USA) at 80 V for 60 min. The expected amplicons were visualized using a Gel Doc ^TM XR + Imaging System photodocumentor (BioRad, USA). The purification of the amplicons was performed using the Wizard® SV Gel and PCR Clean-Up System kit (Promega, USA) following the manufacturer’s instructions.

The purified DNA products were sent for sequencing to the National Laboratory of Agricultural, Medical and Environmental Biotechnology located in San Luis Potosí, S.L.P. The obtained DNA sequences were manually aligned and edited using BioEdit Sequence Alignment Editor Software Version 7.2.5.0 (^{Hall, 2011}). Subsequently, the consensus sequences obtained were compared with the sequences deposited in the GenBank Overview NCBI database.

A total of 20 isolates of Peronospora tabacina were collected from different tobacco fields in Nayarit, Veracruz, and Chiapas (Table 1). The PCR technique was used to process the 20 isolates using the specific oligonucleotide pairs PTAB and ITS4 for P. tabacina, resulting in a 764 bp fragment in each isolate, which confirmed the identity of the oomycete under study.

According to the analysis of the amplification of the 12 microsatellites evaluated, 19 isolates showed 100% amplification for all microsatellites evaluated. For isolate Pt14Ta from Tantoyuca, Veracruz, amplification was not observed for two of the 12 oligonucleotide pairs evaluated (Table 2). The isolate was thus considered a partially clonal strain.

To confirm the results, the 12 microsatellite amplicons were sequenced for isolates Pt7SA and Pt16Na, and the consensus sequences obtained were compared with sequences deposited in GenBank.

The consensus sequences showed identity percentages ranging from 95.83 to 100% (Table 3) compared to the sequences of the P. tabacina isolates from the study by ^{Trigiano et al. (2012}). It should be mentioned that for the oligonucleotide pairs of the microsatellites PT034, PT041, and PT056, poor quality was observed in the obtained sequences even though they were performed in triplicate, so it can be considered that there is some problem with their design. In all the evaluated isolates in this study, the sequences (100%) of the microsatellites comprised dinucleotide regions (Table 4), mostly corresponding to repeated motifs or structures (GT)n or variations (TG)n. Motifs (AC)n, (CA)n, (AT)n, and (AG)n were also visualized, and these were perfect repetitions since the sequences were not interrupted by non-repeated nucleotides.

With the obtained data, it was determined that the P. tabacina isolates present in the tobacco fields of the main producing states in Mexico are genetically homogeneous since the amplification of the reference microsatellites was observed in the 20 isolates evaluated in this study. Likewise, in a study by ^{Edreva et al. (1998}), it was observed that P. tabacina isolates collected in Europe (France and Bulgaria) between 1978 and 1992 were genetically stable. These results were supported by the observation of a high similarity of the isoenzyme patterns of natural populations of the pathogen and the non-significant changes in these patterns. Similarly, ^{Zipper et al. (2009}) also reported genetic uniformity in European isolates of P. tabacina.

Oomycetes are diploid organisms with both asexual and sexual reproduction in their life cycle. Asexual reproduction tends to exhibit high clonality, whereas sexual reproduction generally has a higher degree of genotypic diversity (^{Chen and McDonald, 1996}). Populations that reproduce sexually produce offspring with a high level of genetic diversity, while the variation of asexual populations is limited by mutations that can occur within populations (^{McDonald, 1997}). Notably, P. tabacina is a pathogen that mainly reproduces asexually through sporangia and sporangiophores, while oospores, the sexual reproductive structures, are rarely observed (^{Blanco-Meneses et al., 2017}; ^{Nowicki et al., 2022}).

These results differ from those reported by ^{Nowicki et al. (2022}), who observed high genetic diversity and gene flow using nine microsatellite molecular markers evaluated in 122 P. tabacina isolates collected on three continents (Central, Southern, and Western Europe, the Middle East, Central and North America, and Australia). However, they reported the presence of partially clonal subpopulations among the isolates they evaluated. Additionally, ^{Nowicki et al. (2022}) mentioned that the high genetic variation and population structure observed among the evaluated isolates could be explained by continuous gene flow across continents and by the exchange of infected plant material and/or the dispersal of P. tabacina sporangia over long distances through wind (^{LaMondia and Aylor, 2001}).

The present study determined that the Peronospora tabacina isolates causing the disease known as blue mold of tobacco in the main tobacco-producing states in Mexico are genetically homogeneous.