Population Genetics of Phytopthora infestans

 Phytophthora infestans (Mont.) de Bary is a devastating pathogen of potatoes (Solanum tuberosum L.) and tomatoes (Solanum lycopersicum L.) worldwide, and causes a disease known as Late Blight.  Montagne first identified the pathogen as Botrytis infestans (Mont.) in 1845, but De Bary renamed it to P. infestans in 1876.  P. infestans is famous for causing the Irish famine in 1845. Almost 1 million people was died from starvation, malnutrition and disease and another 1 million was emigrated to avoid the famine in Ireland.  P. infestans continues today to be a devastating pathogen, capable of causing complete crop loss, and is estimated worldwide to cost producers approximately US$3 billion per year in yield losses and chemical control. Taxonomic Classification Kingdom: Chromista Phylum: Oomycota Class: Peronosporomycetidae Order: Pythiales Family: Pythiaceae Genus: Phytopthora Species: P. infestans The Centre of Origin & Migration Pathways– P. infestans  Two Centres of origin have been proposed for P. infestans are the central highlands of Mexico and the South American Andean region.  From different evidence, it is clear that Central highlands of Mexico is the true Centre of origin (Fry et al., 2009; Grünwald & Flier, 2005. Fig.: The series of migration events that likely led to the panglobal spread of a single clone of Phytophthora infestans (Goodwin 1997).  Until the 1980s, the A2 mating type was restricted to central Mexico, and only the A1 mating type was found to be distributed worldwide  In Korea, A2 mating s of P. infestans have been reported by Koh et al in 1994 and So and Lee in 1993 Fig.: Possible paths of the second global migration of P. infestans in the late 1970s and 1980s Biology of Phytophthora infestans  P. infestans has asexual (clonal) and sexual stages and is heterothallic.  Gametangia (male antheridium; female oogonium) are produced on separate thalli known as A1 and A2 compatibility (mating) types. Isolates of both mating types must be present for sexual reproduction to occur. A thick walled resting spore (oospores) develops within the fertilized oogonium.  The asexual cycle of P. infestans begins with the production of multinucleate sporangia. Sporangia can germinate either directly through the production of a germ tube, or indirectly through the production of zoospores. EPIDEMIOLOGY  Late blight development is favored by cool and moist conditions. The optimum temperatures for disease development are 16-21°C, with sporulation occurring at a relative humidity of around 90%.  Disease symptoms occur on the leaves, stems, fruits or tubers of potato and tomato plants. Characterization of P. infestans Isolates  Genetic and Biological (Phenotypic) markers can be used to characterize plant pathogen populations.  In P. infestans, given below genetic and phenotypic markers have been used study genetic structure.        Genotypic Markers Mitochondrial DNA haplotype (mtDNA) Allozyme Restriction Fragment Length Polymorphisms (RFLPs) fingerprinting using probe RG-57 Randomly Ampified Polymorphic DNA (RAPD) Amplified Fragment Length Polymorphisms (AFLPs) Simple Sequence Repeat Markers (SSRs)  Phenotypic Markers  Mating type  Fungicide resistance testing (Metalaxyl sensitivity) Mitochondrial DNA haplotype  Mitochondrial DNA (mtDNA) has been used to determine the population genetics and evolutionary biology of the pathogen among Phytophthora species.  P. infestans has a circular mtDNA molecule that is approximately 37914 bp long, depending on the specific haplotype  There are four known mtDNA haplotypes, namely Ia (37922 bp), Ib (37957), IIa (39870 bp) and IIb(39840 bp) Fig: A scheme of the mitochondrial genome of P. infestans illustrating the location of the various amplification products (shaded). Allozyme  Allozyme Alleles have been used to compare genetic diversity in populations from different locations.  Incase of P. infestans, 17 Allozyme was identified. Among them Glucose-6-phosphate isomerase (Gpi), Peptidase (Pep) and Malic enzyme loci have been widely used.  Gpi is an enzyme that catalyzes the isomerisation of glucose-6-phosphate to fructose-6phosphate while Pep is an enzyme that catalyzes the breakdown of short peptides into free amino acids.  The Allozyme genotype of P. infestans in korea was first reported by Koh et al. in 1994. Table: Differentiation of mating type, allozyme pattern, and mtDNA haplotype of P. infestans isolates collected in Korea from 2002 to 2004 Restriction Fragment Length Polymorphisms (RFLP) finger printing using probe RG-57  RFLP fingerprinting consists of first cutting genomic DNA into fragments using a restriction enzyme (s), followed by an agarose gel according to size.  Probe RG-57 hybridizes to approximately 25 fingerprint loci, and can be used to differentiate homozygous from heterozygous loci based on band intensity.  The advantages of probe RG-57 is that it is not prone to spontaneous mutation; has somatic stability. Fig.: DNA fingerprint patterns revealed by probe RG57. 568 is the A1 parent and 575 is the A2 parent. Band numbers are indicated to the left. Approximate fragment sizes are indicated to the right, in kilobases. Randomly Ampified Polymorphic DNA (RAPD)  The Randomly Ampified Polymorphic DNA (RAPD) Techniques has been used as an Auxiliary tools for the genetic analysis, classification or identification of plant pathogenic fungus.  Punja et al. described novel genotypes of P. infestans in British Columbia during 1990s by using RAPD analysis, and postulated that sexual reproduction was responsible for the variation. Fig. RAPD profiles of P. infestans genomic DNAs amplified by the primer OPC-5. About 0.6 kb of specific DNA fragments, indicated by arrows, was found only from A1 mating type isolates. Lanes M1 and M2 are 100 bp DNA ladder and 1 kb DNA ladder, respectively Amplified Fragment Length Polymorphisms (AFLPs)  AFLPs are based on the selective PCR amplification of a subset of genomic restriction fragments, using different primer and restriction enzyme combinations.  In P. infestans, AFLPs have only been used in a few population studies .  In Mexico and Europe, AFLPs were used to differentiate isolates. Simple Sequence Repeats (SSRs)  Simple Sequence Repeats (SSRs), also known as microsatellites, are short tandem repeats (2-6 bp) randomly distributed in a genome.  They are multiallelic, co-dominant, highly polymorphic.  The first SSRs for P. infestans were developed by Knapova et al. in 2001.  Initially, they developed six SSR markers (Pi4B, Pi4G, PiG11, Pi1D, Pi2D and Pi2H)  Lees et al. (2006) subsequently developed twelve other SSR markers (Pi02, Pi04, Pi16, Pi26, Pi33, Pi56, Pi63, Pi65, Pi66, Pi70, Pi89 and D13). Mating type determination  Mating type determination is conducted by pairing an unknown isolate of P. infestans with an isolate of a known mating type on synthetic medium.  The production of oospores can be observed by viewing the bottom of a paired Petri dish under a compound microscope. An isolate that produces oospores when paired with a known A2mating type, is designated an A1 mating type, and vice versa.  This method is considered reliable and is widely used but more recently, PCR–based techniques have been used for determining P. infestans mating types. Fig.: Mating culture established by growing an A2 isolate (left; Na1-2) together with an unknown isolate (right; YY-8). A and C: zoospores and mycelia produced; arrows/B: oospores formed. Korea 2001-2004 939 6.8 Metalaxyl sensitivity  Sensitivity to the fungicide Metalaxyl and its more recent isomer Mefenoxam has also been used to characterize P. infestans isolates.  Metalaxyl resistance is controlled by a dominant gene influenced by minor genes  Therefore, isolates are often broadly grouped as sensitive, intermediate resistant and resistant. Control 5 ppm Sensitive Intermediate resistant Resistant 10 ppm 100 ppm Table: Frequency of mating type occurrence and metalaxyl resistance of P. infestans isolate obtained from Korea in 2001-2004 Year Isolate Mating Type Response to metalaxyl A1 A2 S MR R 2001 94 84 10 5 73 16 2002 158 138 20 10 5 73 2003 465 437 28 11 248 206 2004 22 216 6 10 25 187 Total 939 875(93.2) 64(6.8) 31(3.3) 466(49.6) 442(47.1) Conclusion  The genetic and phenotypic diversity of P. infestans populations has subsequently been investigated in many countries.  New populations of P. infestans appear as a result of Mutation, Parasexual Recombination, Mitotic Recombination, Interspecific Hybridization, Migration into regions and / or sexual recombination within them.  A global marker database for P. infestans, containing information on Mitochondrial DNA (mtDNA) haplotypes, Allozyme genotypes, RFLPs obtained using the RG57 probe, RAPD markers , Mating types and sensitivity to Metalaxyl, other factors. Thanks to All…….