Strain-specific late blight forecasting

Final Report for GNE13-056

Project Type: Graduate Student
Funds awarded in 2013: $15,000.00
Projected End Date: 12/31/2015
Grant Recipient: Cornell University
Region: Northeast
State: New York
Graduate Student:
Faculty Advisor:
Dr. William E. Fry
Cornell University
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Project Information

Summary:

An understanding of the phenotypic diversity within populations of Phytophthora infestans is important to select the most effective mitigation tactics. Here we systematically assessed five traits (mating type, pathogenicity on potato and tomato, sensitivity to mefenoxam, the effect of temperature on release of zoospores, and the effect of temperature on mycelial growth) of a diverse panel of P. infestans isolates. The panel consisted of i) the dominant clones in the US from the 1990s to 2013, ii) a recombinant population detected in northeastern US in 2010 and 2011, iii) a natural sexual population from Mexico, and iv) an isolate from the Netherlands. There were significant differences among isolates for growth rate and for growth rate as influenced by temperature, mefenoxam sensitivity, rate at which zoospores are released from sporangia, and for host preference.

Introduction:

Late blight caused by the pathogen Phytophthora infestans, has been a major threat to global food security ever since the Irish famine of the 1800's. In the US, late blight is potentially important on nearly all of the approximately 1.1 million acres of potato production (Agricultural Statistics Board, NASS, USDA). The worldwide cost of potato late blight alone exceeds $5 billion per year, including $1 billion spent on fungicides (Judelson, USAblight). On tomatoes, the disease can be and has been equally devastating. The most recent example occurred in 2009 when infected tomato transplants were distributed via national large retail stores who obtained transplants from a national supplier. The subsequent pandemic in the mid-Atlantic and northeast regions eliminated tomato plants in many organic farms and home gardens (Fry et al. 2013).

      Management of late blight mostly involves cultural procedures and fungicides designed to reduce the introduction, survival, or infection rate of P. infestans. Resistant cultivars are employed rarely in potato production due to the absence of durable resistance (R) genes. The use of fungicides has helped control late blight for more than a century, but the pathogen has proven capable of evolving resistance to certain highly effective fungicides. For example, the fungicide metalaxyl (now mefenoxam) was used with great success in the 1970s and 1980s, but resistant strains appeared that caused devastating losses (Fry and Goodwin 1997, Goodwin et al. 1996). Interestingly, due to year-to-year population shifts, chemicals that lose effectiveness one year may regain value. For example, the 2009 late blight pandemic in the eastern US, was caused by a mefenoxam-sensitive lineage. The fact that mefenoxam was effective was not known widely until late in the season, too late to aid growers. This shows that knowledge of such traits can provide management opportunities.

      Phenotypic analysis may take weeks to months, whereas certain molecular analyses of genotype can be accomplished in hours or days. If molecular tools can rapidly identify a genotype and if the epidemiologically important traits of that genotype are already known, genotypic analysis can inform management decisions.  However, it is also the case that we are only just beginning to understand the phenotypic diversity for several traits in P. infestans.  Thus, we initiated a study to investigate some of the phenotypic diversity among known different genotypes of P. infestans. 

      In this study, we assessed the mating type, pathogenicity on potato and tomato, sensitivity to mefenoxam, the effect of temperature on zoospore release from sporangia, and the effect of temperature on mycelial growth on a diverse panel of 66 P. infestans isolates. The panel consisted of i) the dominant clones in the US from the 1990s to 2013, ii) a recombinant population detected in northeastern US in 2010 and 2011, iii) a natural sexual population from Mexico, and iv) an isolate from the Netherlands. The population structure of this panel of isolates was assessed using SNP markers obtained through genotyping by sequencing (GBS).

Project Objectives:

My overall objective is to provide the data necessary to construct a strain-specific forecast for late blight of potato and tomato. This will be achieved through four specific sub objectives:

1. Determine host preference for new genotypes of Phytophthora infestans strains collected in the Northeastern US in the years 2010 and 2011.

2. Determine the effect of temperature on incubation period (time between inoculation and the appearance of typical late blight symptoms), latent period (time between inoculation and the start of the production of spores), sporulation capacity and lesion area.

3. Determine sensitivity to an important fungicide (mefenoxam) of P. infestans strains collected in the Northeastern US in the years 2010 and 2011.

4. Determine the effect of temperature on the rate of germination for new genotypes of P. infestans strains collected in the Northeastern US in the years 2010 and 2011.

Cooperators

Click linked name(s) to expand
  • Dr. William E. Fry

Research

Materials and methods:


Isolates

The isolates used in this study included six US clonal lineages (US-7, US-8, US-11, US-22, US-23, and US-24), 18 isolates that seem to have characteristics of a sexually reproducing population collected in and around west-central New York State in 2010 and 2011(from now on referred to as the NYS-2010/11population) (Danies et al. 2014), one individual from the Netherlands, and 36 isolates collected in Central Mexico where sexual reproduction is ubiquitous. For one of the six US clonal lineages (US-23), six individuals that showed differences in their microsatellite profiles were included. Isolates were maintained on pea agar (Jaime-Garcia et al. 2000) with antibiotics (ampicillin (100 µg ml-1), rifampicin (125  µg ml-1), and pentachloronitrobenzene (25  µg ml-1)) and on tomato and/or potato leaflets (depending on the isolate) at 20ºC.

Phenotypic assays

Mating type

Mating type was determined by pairing an unknown isolate with a known isolate of P. infestans, either A1 mating type (US970001 US-17 genotype) or A2 mating type (US040009, US-8 genotype), on rye B (Caten and Jinks 1968) or pea (Jaime-Garcia et al. 2000) agar media. Negative controls consisted of pairing the unknown isolate with itself. Petri plates were kept at 20°C for 10 to 14 days. The hyphal interface of the two colonies was investigated microscopically using 125X magnification. Isolates that formed oospores at the interface with the known A1 isolate were designated A2 and those that formed oospores with the known A2 isolate were designated A1. The known isolates (A1 and A2) were paired as positive controls, while negative controls consisted of pairing the known isolates with themselves (same mating type).

      Isolation of single zoospores was done for isolates that produced oospores in the presence of both the A1 and the A2 mating type testers as well as in the negative control (unknown isolate paired with itself). To do this, sporangia were washed with sterile distilled water from sporulating lesions on leaflets. The sporangial suspensions were adjusted to 8,000 sporangia per ml using a haemocytometer and maintained at 4ºC for 3 to 4 h to induce zoospore release. Subsequently, inoculations were carried out in 100 mm petri plates containing 20 ml of water agar (0.75%). Three independent petri plates were inoculated with 20, 40, or 100 µl of zoospore suspension of the same isolate. The zoospore suspension was spread using a sterile glass rod. Plates were then kept at 10ºC for 12 h to encourage zoospore germination and subsequently maintained at 15ºC for another 24 h. Individual germinated zoospores were then picked using a sterile scalpel and placed onto pea agar medium. Colonies formed from single zoospores were again tested for mating type as explained above.

Pathogenicity on potato and tomato

A sporangial suspension was used for inoculation of potato and tomato leaflets. Sporangia were washed from sporulating lesions on tomato or potato leaflets, which had been maintained in water-agar moist chambers at 20ºC for 6 to 8 days prior to inoculation. The sporangial suspension was adjusted to 10,000 sporangia per ml using a haemocytometer and maintained at 4ºC for 2 h.

      In order to determine differences in pathogenicity on potato and tomato, each isolate was inoculated onto both potato ‘Yukon Gold’ and tomato ‘Rutgers’ leaflets. Plants were grown in the greenhouse (ca 25°C daytime and 20°C nighttime) and when four to five weeks old, recently matured leaflets were harvested. Inoculations were carried out in 150 mm petri plates containing 75 ml of water agar (1.5%) in the smaller half – which served as the lid (top).  Leaflets were placed (abaxial side up) on the base of the moist chamber. Each moist chamber contained five potato or five tomato leaflets, abaxial side up.  All five leaflets were inoculated with 20 µL of a sporangial suspension (described above) of the same isolate, deposited on one side of the main vein of the leaflet. After the leaflets were inoculated, the petri plate was sealed with parafilm and incubated at 15°C with a 16-h light period. Two days after inoculation, inoculum droplets were dried with Kimwipes, subsequently sealed with parafilm and incubated at 20ºC. The experiment was conducted at least twice for each isolate.

      Lesion size and number of sporangia per lesion were measured at six days after inoculation. Lesion areas were estimated by taking two perpendicular measurements (length and width) starting from the widest diameter, using a ruler. Subsequently, the number of sporangia produced on each lesion was determined. Individual lesions were excised and placed into 14-ml disposable polypropylene culture tubes with 3 ml of preservative solution (0.04 M copper sulfate, 0.2 M sodium acetate, acetic acid, pH 5.4) (Spielman et al. 1991). The tubes were then vortexed for 10 seconds to dislodge and suspend sporangia, and aliquots counted with a haemocytometer. Haemocytometer counts were repeated at least twice.

Mefenoxam sensitivity

Mefenoxam sensitivity of isolates was assessed as described previously by Therrien et al. (1993), except that mefenoxam was used in place of metalaxyl. Isolates were grown on pea agar amended with Ridomil Gold SL (Syngenta, Greensboro, NC) such that the final concentrations of the active ingredient (mefenoxam) were 0, 5, or 100 μg ml-1. Mycelial plugs (8 mm diameter) were obtained from actively growing cultures, transferred to the test plates and incubated for approximately 10 to 12 days, or until growth on the control mefenoxam plate (0 μg ml-1) was approximately 75 to 90% of the diameter of the petri plate. Assessment of mefenoxam sensitivity was determined on the basis of radial growth of cultures grown on plates amended with mefenoxam (5 or 100 µg ml-1) compared to non-amended controls. Growth on mefenoxam-amended plates, 5 and 100 µg ml-1, was represented as a proportion of the growth on the non-amended control plates.

Rate of indirect germination at 4ºC

Sporangia were observed at 30 and 120 minutes after incubation at 4°C. Inoculation was performed as described above for the pathogenicity on potato and tomato assay except that the sporangial suspension was adjusted to 4,000 sporangia per ml and was immediately used to conduct the rate of indirect germination experiments. Independent measurements of total germination were carried out for each respective time point. That is, a unique slide with three circular water agar droplets that had each been inoculated with 20 µl of the same sporangial suspension was assessed for each time point. This was due to the difficulty of maintaining slides at 4°C while assessing germination microscopically. Percentage of total germination that was indirect was calculated for each of the time points considered. The experiment was conducted at least twice for each isolate.

      Effects of time and lineage on zoospore release (indirect germination) were analyzed using JMP Pro 11 (SAS Institute). A mixed effects model was conducted, where time, lineage, and their interaction were fixed effects, and trial and replicate nested within trial were considered random effects. Differences among lineages in mean indirect germination, within a time period (30 or 120 min), were identified using contrasts and Bonferroni corrected P-values.

Effect of temperature on mycelial growth

To determine the effect of temperature on mycelia growth, 1 cm diameter discs of hyphae growing on agar medium of each lineage were placed in a 100 x 15 mm petri plate containing 10 ml of vacuum filtered pea broth. Each replicate consisted of four plates, each incubated at one of four different temperatures (10, 15, 20, and 25ºC) for eight days. Mycelia were subsequently dried using vacuum filtration, frozen at -80ºC, lyophilized, and placed in a drying chamber until it was weighed using a Sartorius A120S analytical balance. Two to six independent replicates were conducted for each isolate.

      Effects of temperature and lineage on mycelial growth were analyzed using JMP Pro 11 (SAS Institute). A mixed effects model analysis was conducted, where temperature, lineage, and their interaction were fixed effects, and replicate nested within lineage was considered a random effect. Because growth at 10ºC for all isolates was significantly less than growth at 15, 20, or 25ºC, we eliminated this temperature from our analyses to detect lineages that exhibited significant growth differences as a function of temperature at 15, 20, and 25ºC. Growth differences among lineages as a function of temperature at 15, 20, and 25ºC were identified using contrasts and Bonferroni corrected P-values. These lineages were further investigated using an LSMeans Differences Student’s t test.

Population structure using SNP markers obtained through genotyping by sequencing (GBS)

Genomic DNA was isolated with a DNeasy® Plant Mini Kit (QIAGEN, Germany). Genotyping-by-Sequencing was performed as described by Elshire et al. (2011) at the Institute of Genomic Diversity (Cornell University). Briefly, genome complexity was reduced by digesting total genomic DNA from individual samples with the typeII restriction endonuclease ApeKI, which recognizes a degenerate 5 bp sequence (GCWGC, where W is A or T), and creates a 5’ overhang (3 bp). Digested products were then ligated to adaptor pairs with enzyme-compatible overhangs; one adapter contained the barcode sequence and a binding site Illumina sequencing primer. Samples were then pooled, purified, and amplified with primers compatible to the adapter sequences. GBS library fragment-size distributions were checked on a BioAnalyzer (Agilent Technologies, Inc., USA). The PCR products were quantified and diluted for sequencing on the Illumina HiSeq 2500 (Illumina Inc., USA).

      Samples sequenced in triplicates or duplicates (for five US-23 isolates, two Mexican isolates, and one isolate from Netherlands) served as technical replicates. Each of two 96-well plates, were multiplexed on a single Illumina flow cell lane. This comprised a total of 192 samples (including two blanks as controls). The GBS discovery pipeline for species with a reference genome, available in TASSEL (version 3.0.166 Date: April 17, 2014) (Bradbury et al. 2007), was used. Sequence reads were mapped against the P. infestans T30-4 draft genome sequence downloaded from the Broad Institute. After merging triplicates or duplicates we ended up with 66 P. infestans isolates and 570,192 SNPs. We subsequently filtered for proportion of missing data < 0.2 and for a minor allele frequency > 0.1. The resulting number of SNPs after filtering was 98,013. To estimate population structure in our panel, a Principal Coordinate Analysis (PCoA) was performed using filtered SNPs.

Research results and discussion:


Phenotypic assays

Mating type

Results for mating of each of the 66 isolates used are shown in Supplementary Table 1. In total there were 30 lineages of A1 mating type and 24 lineages of A2 mating type. For six of the 36 Mexican isolates assayed, oospores were observed in the negative controls (isolates paired against themselves) (Supplementary Table 1). Single-zoospore (uninucleate) isolates derived from ‘self-fertile’ strains paired against A1 and A2 mating type testers were also able to ‘self-fertilize’. Pea agar plates (where isolates were routinely maintained) of these six Mexican isolates were further assessed for oospore production. On these plates isolates were not paired against themselves or against other isolates. Yet, production of oospores was again observed.

Pathogenicity on potato and tomato

Pathogenicity on potato and tomato was assessed for a subset of isolates within our panel (three isolates representing dominant clones in the US from the 1990s to 2013, ten isolates from a recombinant population detected in northeastern US in 2010 and 2011, seven isolates from a natural sexual population from Mexico, and one isolate form the Netherlands). Differences in pathogenicity on potato and tomato were observed among the 21 different P. infestans genotypes studied. Clonal lineage US-24, 7 isolates from Mexico, and one isolate from the Netherlands, showed a strong preference for potatoes (Figure 1). In contrast, clonal lineage US-7, and all isolates from the NYS-2010/11 population seemed to grow equally well on both potatoes and tomatoes (Figure 1). Lesion size ranged from 14.74 cm2 (MX-02, on potato) to 0.49 cm2 (MX-04, on tomato). For all isolates studied lesion size was greater on potatoes than on tomatoes. For the seven Mexican isolates studied, lesion area on tomatoes was restricted to the place where the inoculum drop was deposited. Sporulation ranged from 53,389 sporangia per ml (MX-02, on potato) to 0 sporangia per ml (MX-01, MX-03, and MX-13, on tomato).

Mefenoxam sensitivity

Sensitivity to mefenoxam was assessed for 65 of the 66 isolates (isolate MX-36 was not included due to its slow growth rate). In general, the US standards used showed the response expected (Figure 2A and Supplementary Table 1). Isolates belonging to clonal lineages US-7 and US-11 were highly resistant; the isolate belonging to clonal lineage US-8 displayed an intermediate resistance, and isolates belonging to clonal lineages US-22, US-23, and US-24 where generally sensitive to mefenoxam. The NYS-2010/11 isolates were mostly sensitive to mefenoxam (Figure 2B and Supplementary Table 1). In contrast, isolates from Mexico showed a wide variety of response to mefenoxam (Figure 2C and Supplementary Table 1). Eight isolates were resistant to mefenoxam, nine displayed an intermediate phenotype, and 18 were sensitive. For two isolates, MX-23 and MX-27, mycelial growth seemed to be enhanced by the presence of mefenoxam.

Rate of indirect germination at 4ºC

The rate of indirect germination was assessed for a subset of isolates within our panel (13 isolates belonging to the NYS-2010/11 population and 13 isolates from Mexico). Varied responses were observed among the isolates studied (Figure 3). Sporangia of MX-3, MX-4, MX-19 and MX-26 released zoospores more rapidly than did sporangia of the other isolates. For example, within 30 min at 4ºC, approximately 75% of the MX-3 and 52% of the MX-4 sporangia had liberated zoospores, whereas the average percentage of sporangia that had released zoospores for all isolates studied was approximately 20%. Zoospore release among these isolates did not differ significantly (P > 0.05). For 14 isolates zoospore release was significantly slower (within the first 30 min of incubation at 4ºC) than that observed for isolates MX-3, MX-4, MX-19, and MX-26. These isolates were GDT-5, GDT-6, GDT-7, GDT-12, GDT-15, GDT-16, MX-1, MX-2, MX-8, MX-16, MX-17, and MX-23. 

Effect of temperature on mycelia growth

The effect of temperature on mycelia growth was assessed for 56 of the 66 isolates. Differences in mycelial growth in response to temperature were found (P < 0.0001). Mycelial growth at 10?C was consistently less than growth at 15, 20 or 25ºC for all isolates studied (Supplementary Table 2). We thus excluded this temperature from our analyses and proceeded to investigate differences in mycelial growth at 15, 20, and 25ºC. Differences in response to temperature within an isolate were analyzed. P values were adjusted for multiple testing using a Bonferroni correction. Six out of the 56 isolates evaluated (Supplementary Table 2) showed significant differences for growth between 15, 20 and 25ºC (P < 0.0009). Isolates MX-3 and MX-15 grew significantly less at 25ºC than at either 15 or 20ºC; Isolate MX-13 grew significantly better at 15ºC than at either 20 or 25ºC; Isolate MX-35 grew significantly less at 15ºC than at either 20 or 25ºC; and isolates MX-21 and MX-34 grew significantly better at 20ºC than at either 15 or 25ºC. With a less conservative P-value (α = 0.05) 12 additional isolates (US-8, US-22, US-23, GDT-05, GDT-16, MX-1, MX-2, MX-5, MX-9, MX-18, MX-28, and MX-23) also showed differences in mycelial growth between temperatures (15, 20, and/or 25ºC) (data not shown). Average dry weights six days after incubation for all isolates assessed are shown in Supplementary Table 2 and Supplementary Figure 1.

Population structure using SNP markers obtained through genotyping by sequencing (GBS)

The total number of tags before merging was 2,285,465. Out of the total number of tags, 1,163,220 (50.9%) were aligned to unique positions, 913,010 (39.9%) were aligned to multiple positions, and 209,235 (9.2%) could not be aligned to the reference genome. Triplicates or duplicates were merged and 570,192 SNPs. After filtering we ended up with 98,013 high quality SNP markers.

      A principal coordinate analysis was performed to provide spatial representation of the relative genetic distances among isolates of P. infestans included in this study (Figure 4). The first two principal coordinates explained 14.9 and 5.8 percent of the total variation, respectively. The first principal coordinate separated isolates belonging to the NYS-2010/11 population and clonal lineage US-22 from all other isolates. The Mexican isolates formed a single cluster that included clonal lineages US-7, US-8, US-11, and US-24. The second principal coordinate further separated isolates into three groups: 1) individuals belonging to clonal lineage US-23, 2) the isolate from the Netherlands, and 3) all other isolates.      

Discussion           

Obtaining precise phenotypic data is challenging due to the high variance observed in the assays conducted. Yet, the findings from this study show that statistically significant differences exist within the phenotypic traits studied among the panel of P. infestans isolates analyzed. Undoubtedly, the phenotypic diversity present in P. infestans is a key factor contributing to the pathogen’s success throughout time. The phenotypic traits studied were mating type, pathogenicity on potato and tomato, sensitivity to mefenoxam, the rate of indirect germination at 4ºC, and the effect of temperature on mycelial growth. Among this diverse panel of isolates, we identified individuals of the A1 and the A2 mating type. Interestingly, six of the 36 Mexican isolates assayed seemed to be ‘self-fertile’. There are two possible explanations for this: 1) the strain being assessed consisted of a mixture of two or more diverse genotypes of P. infestans, or 2) the isolate was able to ‘self-fertilize’ as has been previously reported (Goodwin and Drenth 1997, Savage et al. 1968, Smart et al. 1998). To test the first hypothesis, single-zoospore (uninucleate) isolates derived from ‘self-fertile’ strains were paired against A1 and A2 mating type testers. The derived single-zoospore isolates were also able to ‘self-fertilize’, thus a mixture of two or more genotypes cannot explain this phenomenon. To test for the second hypothesis, pea agar plates (where isolates were routinely maintained) were further assessed for oospore production. Production of oospores was again observed and thus the second hypothesis that these isolates are ‘self-fertile’ cannot be rejected.

      Pathogenicity on potato and tomato differed among the isolates evaluated. All isolates tested were able to produce symptoms and sporulate on potato leaflets but only a subset of the isolates was capable of sporulating on tomato leaflets. In general, genotypes US-8 and US-24, as well as the isolates from Mexico and the isolate from the Netherlands showed a strong preference for potato and were not at all aggressive on tomatoes. Genotypes US-7 and US-11 as well as the rare and diverse genotypes detected in the Northeast in 2010 and 2011 were pathogenic on both potato and tomato.

      A broad range of responses to sensitivity to mefenoxam was observed among the panel of isolates studied. The most remarkable differences were observed within the Mexican isolates, where sensitivity ranged from extremely sensitive (no growth in the presence of mefenoxam) to highly resistant (where mycelial growth was enhanced by the presence of mefenoxam). Isolates from the NYS-2010/11 population were in general sensitive to mefenoxam.

      The rate at which sporangia released zoospores differed among isolates. As had been previously reported by Danies et al. (2013), striking differences were observed in the percentage of sporangia capable of releasing zoospores within 30 minutes of incubation at 4ºC. For the panel of isolates included in this study, sporangia that had released zoospores within 30 minutes of incubation at 4ºC, ranged from approximately 2% to 80%.

      Differences in mycelial growth were observed at different temperatures. For all isolates studied, growth at 10ºC seemed to be greatly stunted. The vast majority of isolates (50) were not significantly different from each other in terms of mycelial growth at 15, 20 or 25ºC. However, six isolates were significantly different from the others.

      Up until today, the population structure of P. infestans in the US has been simple, composed mostly of a few clonal lineages (Fry et al. 2013). Therefore, in the US, it has been possible to phenotype individual clonal lineages (Danies et al. 2013), and provide information to farmers that would allow them to make informed management decisions. Yet, sexual reproduction events have been reported (Danies et al. 2014, Gavino et al. 2000), and sexual reproduction is certainly possible in the USA.  If sexual reproduction were to become common, the diversity of the pathogen would increase, thus complicating the task of linking phenotypic trait to genotype.

      The phenotypic data for diverse isolates of P. infestans along with the SNP markers generated through techniques such as genotyping-by-sequencing will enable a genome-wide association study to find SNP markers associated with traits of interest. This will hopefully form the basis for future research that would lead us to the development of a specific DNA-based method to identify phenotypic traits of interest. An understanding of the genetic basis of complex traits (most likely controlled by multiple genes) that are important to the pathogenicity or epidemiology of P. infestans would be of value in managing late blight because rapid analysis using molecular markers could inform the selection of the most effective mitigation tactics.

References

Bradbury PJ, Zhang Z, Kroon DE, Casstevens TM, Ramdoss Y, Buckler ES. 2007. TASSEL: software for association mapping of complex traits in diverse samples. Bioinformatics 23: 2633-2635.

Caten CE, Jinks JL. 1968. Spontaneous variability of single isolates of Phytophthora infestans. I. Cultural variation. Canadian Journal of Bototany 46: 329-348.

Danies G, Small IM, Myers K, Childers R, Fry WE. 2013. Phenotypic characterization of recent clonal lineages of Phytophthora infestans in the United States. Plant Disease 97: 873-881.

Danies G, Myers K, Mideros MF, Restrepo S, Martin FN, Cooke DEL, Smart CD, Ristaino JB, Seaman AJ, Gugino BK, Grünwald NJ, Fry WE. 2014. An ephemeral sexual population of Phytophthora infestans in the Northeastern United States and Canada. PLoS ONE 9: doi: 10.1371/journal.pone.0116354.

Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler ES, Mitchell SE. 2011. A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species. PLoS ONE 6.

Endelman JB, Jannink J. 2012. Shrinkage estimation of the realized relationship matrix. Genomic Selection 2: 1405-1413.

Fry WE, Goodwin SB. 1997. Re-emergence of potato and tomato late blight in the United States. Plant Disease 81: 1349-1357.

Fry WE, McGrath MT, Seaman A, Zitter TA, McLeod A, Danies G, Small IM, Myers K, Everts K, Gevans A, Gugino BK, Johnson S, Judelson H, Ristaino J, Roberts P, Secor G, Seebold K, Snover-Clift K, Wyenandt A, Grünwald NJ, Smart CD. 2013. The 2009 Late Blight Pandemic in Eastern United States. Plant Disease 97: 296-306.

Gavino PD, Smart CD, Sandrock RW, Miller JS, Hamm PB, Lee TY, Davis RM, Fry WE. 2000a. Implications of sexual reproduction for Phytophthora infestans in the United States: generation of an aggressive lineage. Plant Disease 84: 731-735.

Goodwin SB, Drenth A. 1997. Origin of the A2 mating type of Phytophthora infestans outside Mexico. Phytopathology 87: 992-999.

Goodwin SB, Sujkowski LS, Fry WE. 1996. Widespread distribution and probable origin of resistance to metalaxyl in clonal genotypes of Phytophthora infestans in the United States and Western Canada. Phytopathology 86: 793-799.

Goodwin SB, Spielman LJ, Matuszak JM, Bergeron SN, Fry WE. 1992. Clonal diversity and genetic differentiation of Phytophthora infestans populations in northern North America. Phytopathology 82: 955-961.

Gregorius HR. 1980. The probability of losing an allele when diploid genotypes are sampled. Biometrics 36: 643-652.

Grünwald NJ, Flier WG, Sturbaum AK, Garay-Serrano E, van den Bosch TB, Smart CD, Matuszak JM, Lozoya-Saldana H, Turkensteen LJ, Fry WE. 2001. Population structure of Phytophthora infestans in the Toluca Valley region of central Mexico. Phytopathology 91: 882-890.

Jaime-Garcia R, Trinidad-Correa R, Felix-Gastelum R, Orum TV, Wasmann CC, Nelson MR. 2000. Temporal and Spatial Patterns of Genetic Structure of Phytophthora infestans from Tomato and Potato in the Del Fuerte Valley. Phytopathology 90: 1188-1195.

Melhus IE. 1915. Germination and infection with the fungus of the late blight of potato (Phytophthora infestans). Agricultural Experiment Station of the University of Wisconsin. Research Bulletin 37: 1-64.

Mizubuti ESG, Fry WE. 1998. Temperature effects on developmental stages of isolates of three clonal lineages of Phytophthora infestans. Phytopathology 88: 837-843.

Sato N. 1994. Effect of sporulating temperature on the limit temperature in indirect germination of the sporangia of Phytophthora infestans. Annals of the Phytopathological Society of Japan 60: 60-65.

Savage EJ, Clayton CW, Hunter JH, Brenneman JA, Laviola C, Gallegly ME. 1968. Homothallism, heterothallism, and inter-specific hybridization in the genus Phytophthora. Phytopathology 58: 1004-1021.

Smart CD, Willmann MR, Mayton H, Mizubuti ESG, Sandrock RW, Muldoon AE, Fry WE. 1998. Self-fertility in two clonal lineages of Phytophthora infestans. Fungal Genetics and Biology 25: 134-142.

Spielman lJ, Drenth A, Davidse LC, Sujkowski LJ, Gu W, Tooley PW, Fry WE. 1991. A second world-wide migration and population displacement of Phytophthora infestans? Plant Pathology 40: 422-430.

Therrien CD, Tooley PW, Spielman lJ, Fry WE, Ritch DL, Shelly SE. 1993. Nuclear DNA content, allozyme phenotypes and  metalaxyl sensitivity of Phytophthora infestans from Japan. Mycological Research 97: 945-950.

Vleeshouwers VG, et al. 2011. Understanding and exploiting late blight resistance in the age of effectors. Annual Review of Phytopathology 49: 507-531.

Research conclusions:

The overall objective of this study was to investigate the phenotypic diversity of Phytophthora infestans. I have characterized phenotypes of the most recent and most prevalent strains of this pathogen in the US (US-8, US-22, US-23, and US-24), and growers throughout the Northeast have already used this information to make informed management decisions. For example, US-23 has been the dominant lineage in the US since 2011. Based on the data I have generated, growers have learned that if US-23 is in their fields, they must protect both potato and tomato crops. Furthermore, growers are now aware that they may use the fungicide mefenoxam to control late blight epidemics caused by this genotype. I have also investigated a novel set of rare and diverse genotypes of P. infestans detected in the Northeast in 2010 and 2011. The genetic characteristics of this population were consistent with a recombinant population. Greater diversity was detected in that region during each of 2010 and 2011 than had been observed in the entire United States in the previous ten years. The likelihood that many different migrations from diverse sources, or that many mutations caused the high degree of genotypic diversity found, seem low. Through parentage exclusion analyses using microsatellite markers and four nuclear gene sequences I found that clonal lineage US-22 could be a parent of some, but not all, of the new genotypes detected in 2010 and 2011. My best inference is that these isolates represent progeny that originated from at least two recombination events. The geographic location(s) of those recombination events remains unknown. The eventual impact of this recombination event cannot be predicted at this moment. The fact that individuals from this event were detected only in 2010 and 2011 and not in 2012 or 2013 suggests that these isolates were not as aggressive or as fit as subsequent dominant clonal lineages. However, the fact that there is now evidence for a second recombinant population of P. infestans detected in the US indicates that sexual recombination is certainly possible, and there is no reason to believe that such populations will not occur in the future. Diligence in monitoring populations might enable the location of a recombination to be identified so that proper mitigation techniques could be applied.

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Education/outreach description:

Refereed jouranl articles

Danies, G., Myers, K., Mideros, M. F., Restrepo, S., Martin, F. N., Cooke, D. E. L., Smart, C. D., Ristaino, J. B., Seaman, A. J., Gugino, B. K., Grünwald, N. J., Fry, W. E. 2014. An ephemeral sexual population of Phytophthora infestans in the northeastern United States and Canada. PLoS ONE 9: e116354. Doi:10.137/journal.pone.0116354

Conference presentations

Danies, G., Romero-Navarro, J. A., Gonzalez-Garcia, L. N. Myers, K., Bevels, E., Bond, M., Wu, Y., Restrepo, S., and Fry, W. E. Genetic architecture of complex traits of Phytophthora infestans determined through genome-wide association mapping. August 3rd, 2015. American Phytopathological Society Annual Meeting. Pasadena, CA, USA. (Oral presentation)

Danies, G., Myers, K., Bevels, E. J., Bond, M. and Fry, W. E. Phenotypic characterization of a sexual population of Phytophthora infestans in the northeastern United States and Canada. August 8th, 2014. North American Late Blight Symposium. Minneapolis, MN, USA. (Oral presentation)

Danies, G., Myers, K., Mideros, M. F., Restrepo, S., Martin, F. N., Cooke, D. E. L. Banks, E., Ristaino, J. B., Seaman, A. J., Gugino, B. K., Grünwald, N. J. and Fry, W. E. A novel recombinant population of Phytophthora infestans in northeastern USA. July 3rd, 2014. Oomycete Molecular Genetics Network Meeting. Norwich, UK. (Oral presentation)

Danies, G., Martin, F., Myers, K., Cooke, D. E. L., Smart, C., Seaman, A. and Fry, W. E. Population structure of Phytophthora infestans in central New York in 2011. August 14th, 2013. American Phytopathological Society Annual Meeting. Austin, TX, USA. (Oral presentation)

Project Outcomes

Project outcomes:

N/A

Farmer Adoption

N/A

Assessment of Project Approach and Areas of Further Study:

Areas needing additional study

Obtaining precise phenotypic data is challenging due to the high variance observed in the assays conducted. Yet, the findings from this study show that real statistical differences within the phenotypic traits studied exist within our panel of Phytophthora infestans isolates. Undoubtedly, the phenotypic diversity present in P. infestans is a key factor contributing to the pathogen’s success throughout time. Future efforts will be devoted to finalizing the phenotyping assays of the diverse panel of P. infestans isolates included in this study. The phenotypic data for diverse isolates of P. infestans along with the SNP markers generated through genotyping-by-sequencing hopefully will enable a genome-wide association study to find SNP markers associated with traits of interest. This will hopefully form the basis for future research that would lead us to the development of a specific DNA-based method to identify phenotypic traits of interest. An understanding of the genetic basis of complex traits important to the pathogenicity or epidemiology of P. infestans would be of value in managing late blight because rapid analysis using molecular markers could inform the selection of the most effective mitigation tactics.

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.