Genotyping of South American clinical isolates of Pythium insidiosum based on single nucleotide polymorphism-based multiplex PCR

We aimed to genotype the South American clinical isolates of Pythium insidiosum using the single nucleotide polymorphisms (SNP) of the ribosomal DNA sequences (rDNA). Previously, an SNP-based multiplex-PCR was able to distinguish three different clades of P. insidiosum isolates. Thus, we used this assay to evaluate South American clinical isolates of P. insidiosum (n=32), standard strains from Costa Rica (n=4), Thailand (n=3), Japan (n=1), and India (n=1), a standard strain of Pythium aphanidermatum, and Brazilian environmental isolates of Pythium torulosum, Pythium rhizo-oryzae and Pythium pachycaule voucher (n=3). It was possible to allocate each American P. insidiosum isolate to clade I, the isolates of India, Japan, and Thailand to clade II, and the Thai isolate to clade III. P. aphanidermatum, P.torulosum, P.rhizo-oryzae and P.pachycaule voucher isolates were not amplified. For the first time, a P. insidiosum isolate from Uruguay, South America, was included in molecular analyzes. By SNP-based multiplex-PCR, it was possible to perform the identification and genotyping of the South American isolates of P. insidiosum, demonstrating similar genetic characteristics of these isolates.


INTRODUCTION
Pythium genus is an ecofriendly oomycete found in a varied ecosystem.Most species are saprobic or pathogens of plants, algae, fishes, insects, and mammals (ADHIKARI et al., 2013).Pythium insidiosum causes pythiosis, a relevant infectious disease in human and animals that is widely distributed throughout the world (GAASTRA et al., 2010).In Brazil, this oomycete is present predominantly in the swampy areas of Pantanal Mato-Grossense and Rio Grande do Sul ABSTRACT: We aimed to genotype the South American clinical isolates of Pythium insidiosum using the single nucleotide polymorphisms (SNP) of the ribosomal DNA sequences (rDNA).Previously, an SNP-based multiplex-PCR was able to distinguish three different clades of P. insidiosum isolates.Thus, we used this assay to evaluate South American clinical isolates of P. insidiosum (n=32), standard strains from Costa Rica (n=4), Thailand (n=3), Japan (n=1), and India (n=1), a standard strain of Pythium aphanidermatum, and Brazilian environmental isolates of Pythium torulosum, Pythium rhizo-oryzae and Pythium pachycaule voucher (n=3).It was possible to allocate each American P. insidiosum isolate to clade I, the isolates of India, Japan, and Thailand to clade II, and the Thai isolate to clade III.P. aphanidermatum, P.torulosum, P.rhizo-oryzae and P.pachycaule voucher isolates were not amplified.For the first time, a P. insidiosum isolate from Uruguay, South America, was included in molecular analyzes.By SNP-based multiplex-PCR, it was possible to perform the identification and genotyping of the South American isolates of P. insidiosum, demonstrating similar genetic characteristics of these isolates.Key words: Pythium insidiosum, Pythiosis, molecular detection, genotype, single nucleotide polimorphisms.

MICROBIOLOGY
State (RS) (SANTOS et al., 2014;WEIBLEN et al., 2016).Little is known about the presence of pythiosis in some countries of South America, such as Uruguay, where the first case in an equine was recently reported in Costas del Tacuarí, Departamento de Treinta y Tres (LABORATORIO REGIONAL ESTE DE DILAVE, 2012).
Due to the difficulty of diagnosing pythiosis and the high costs for laboratory identification of P. insidiosum, as well as the similarity to other agents, especially other oomycetes and filamentous fungi, there is a need for diagnostic tools that can identify rapidly this relevant microorganism.VILELA et al. (2015) proposed a biochemical assay for identification of oomycetes; however, this technique should still be used carefully for the evaluation of P. insidiosum isolates (KRAJAEJUN et al., 2018).
Molecular biology tools have been successfully employed for diagnosing pythiosis, mainly using polymerase chain reaction (PCR) targeting the P. insidiosum internal transcribed spacer (ITS) of the rRNA locus, i.e., the ribosomal DNA (rDNA region) that consists of 18S rRNA, internal transcribed spacer 1 (ITS1), 5.8S rRNA, internal transcribed spacer 2 (ITS2), and 28S rRNA (GROOTERS & GEE, 2002).Phylogenetic studies of P. insidiosum have already been used with different genetic markers to elucidate aspects related to epidemiology, pathogenesis, and hosts (SCHURKO et al., 2003a,b;KAMMARNJESADAKUL et al., 2011;AZEVEDO et al., 2012;RIBEIRO et al., 2017).The first phylogenetic analyses grouped P. insidiosum in three clusters: cluster I North, Central, and South America; cluster II Australia, North America, Southeast Asia, and Thailand; and cluster III North America and isolates from Thailand (SCHURKO et al., 2003a,b).However, these analyses have limitations owing to the high costs, time required for DNA sequencing, and the delay in obtaining results.
Simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers have been used for studies of diversity and relationship in different microorganisms, including P. insidiosum (SUPABANDHU et al., 2007;RUJIRAWAT et al., 2017).The purpose of our study was genotyping American clinical isolates of P. insidiosum using a fast, simple, and low-cost tool based on SNP multiplex PCR.
The PCR products of rDNA region presenting a single band with the desired length (approximately 500-800pb) were purified with PureLink PCR Purification Kit (Invitrogen), and the DNA was sequenced in an automatic sequencer (ABI-Prism 3500 Genetic Analyzer) using the primers ITS1 and ITS4 (WHITE, 1990).Phylogenetic analysis for rDNA region was conducted by Neighbor-joining (NJ) method with 10,000 bootstrap replicates.All rDNA sequences from P. insidiosum clinical isolates from South America and standard strains of Costa Rica, India, Japan and Thailand, as well as P. aphanidermatum, P.torulosum, P.rhizo-oryzae and P.pachycaule voucher and Phytopythium vexans (outgroup) were used to construct the phylogenetic tree.

RESULTS AND DISCUSSION
Based on the multiplex PCR targeting the three SNPs identified in the rDNA region, all the thirty-six South and Central American isolates of P. insidiosum and the five standard strains from Thailand, India and Japan were grouped in their respective clades, as suggested by RUJIRAWAT et al. (2017) (Table 1, Figure 1).We observed that the American clinical isolates, grouped in clade I, generated amplicons of approximately 490 and 660bp when using the primers ITS1/R1 and ITS1/R2, respectively.P. aphanidermatum, P.torulosum, P.rhizo-oryzae and P.pachycaule voucher were not amplified since these isolates do not belong to any P. insidiosum clade.
The multiplex PCR targeting the three SNPs identified in the rDNA region was developed by RUJIRAWAT et al. ( 2017) and has many advantages, such as 100% of sensitivity, and specificity, rapid and cost-effective identification, and genotyping of P. insidiosum.As these authors evaluated only one Brazilian isolate of P. insidiosum in their study, we proposed to evaluate an expressive number of P. insidiosum clinical isolates from South America using this technique.
The molecular phylogeny obtained for the rDNA region showed P. insidiosum as paraphyletic in relation to other Pythium species.However, it was observed that South and Central American P. insidiosum isolates were grouped together, forming a monophyletic group.In addition, isolates from other countries formed a basal-positioning group in relation to the American isolates (Figure 2).These results were consistent with AZEVEDO et al. ( 2012) and RIBEIRO et al (2017) that used rDNA (ITS) and cytochrome c oxidase subunit II as molecular markers and exo-1,3-β glucanase gene in phylogenetic analyses of Brazilian P. insidiosum isolates, respectively.Moreover, all isolates of P. insidiosum from India, Japan and Thailand were grouped in different clades as proposed by SCHURKO et al. (2003 a,b) and lately supported by SUPABANDHU et al. (2008).
According to RUJIRAWAT et al. ( 2017) multiplex PCR targeting the three SNPs identified in the rDNA (ITS) region were able to allocate P. insidiosum to clade-I provided two amplicons (approximately 490 and 660bp), whereas the clades-II and -III showed only one amplicon (approximately 660 and 800bp, respectively).The same results were obtained in this study, allowing to assign each American P. insidiosum isolates to clade I, isolates from India, Japan, and Thailand to clade II, and one Thai isolate to clade III.In addition, P. aphanidermatum, P.torulosum, P.rhizo-oryzae and P.pachycaule voucher were not amplified.Thus, these results evidenced that this molecular biology methodology is specific and sensitive for identification and genotyping of P. insidiosum, in agreement with RUJIRAWAT et al. (2017).
The genome sequences of P. insidiosum recently available can be a useful genetic resource for exploring aspects related the biology and evolution Ciência Rural, v.49, n.1, 2019.
Weiblen et al.P.insidiosum and other oomycetes since independently assessed genes may not provide much information when compared to genomes.However, genome analyses are still recent, expensive and laborious when compared to the available molecular analyses (RUJIRAWAT et al., 2015;TANGPHATSORNRUANGA et al., 2016).
For the first time, a P. insidiosum isolate from Uruguay were included in phylogenetic analysis.
A single case of equine pythiosis has been reported by the Laboratorio Regional Este de DILAVE (2012).It is of note there is still little knowledge about pythiosis in Uruguay.However, we are aware of other cases of equine pythiosis in that country (unpublished data).Additionally, MACHADO et al. (2018) suggested that P. insidiosum is a generalist pathogen that has the potential to move between the borders of southern Brazil, e.g., RS (the southernmost state in Brazil)  and Uruguay.P. insidiosum isolate from Uruguay was grouped in clade I, together with isolates from Brazil.This was evidenced by both multiplex PCR and phylogenetic analysis, proving that the South American isolates are grouped in the same clade, as previously suggested (SCHURKO et al., 2003a,b;KAMMARNJESADAKUL et al., 2011;AZEVEDO et al., 2012;RIBEIRO et al., 2017).

CONCLUSION
The SNP-based multiplex-PCR methodology has benefits (i.e., fast, simple, and lowcost) and was possible to carry out the identification and genotyping of the South American isolates of P. insidiosum.For the first time a P. insidiosum isolate from equine in Uruguay was identified and genotyped.Furthermore, the American P. insidiosum isolates evaluated showed similar genetic characteristics.

Figure 2 -
Figure 2 -Neighbor-joining tree based on sequence analysis of the rDNA ITS showing relationships among clinical isolates of P. insidiosum from South America (Brazil (BR) and Uruguay (Uy) and standard strains of Costa Rica (CR), India (IN), Japan (JP) and Thailand (TH), as well as P. aphanidermatum and environmental Pythium spp.(P.pachycaule voucher, P. rhizo oryzae and P. torulosum isolates).Bootstrap values expressed in percentages based on 10,000 replicates are present at their corresponding clades.

Table 1 -
Isolates of Pythium.insidiosum(n=36)and other species of Pythium (n=4) used for evaluation of the multiplex PCR assay and their information of GenBank acession number of rDNA sequence, isolate source, geographic origin and phylogenetic clade.
and no amplification)=No P. insidiosum genotype.=This isolate does not belong to P. insidiosum clade.* Municipality of Brazil.