Strain-specific late blight forecasting

Strain-specific late blight forecasting

Summary

Strains of Phytophthora infestans within the US are mostly clonal, and different clones are identified using specific DNA markers. Clones vary in traits such as fungicide sensitivity, aggressiveness, and the ability to overcome R genes. Knowledge about traits of P. infestans aids disease control. If DNA genotyping assigns strains to a lineage of known phenotype, recommendations can be made promptly and growers can make informed management decisions. In 2010 and 2011 we found 20 new and diverse genotypes of P. infestans concentrated in north central New York. The ratio of A1 to A2 mating types among these genotypes was close to the 50:50 ratio expected for sexual recombination. These genotypes were diverse at the glucose-6-phosphate isomerase locus, differed in their microsatellite profiles, showed different banding patterns in a restriction fragment length polymorphism assay using a moderately repetitive and highly polymorphic probe (RG57), were polymorphic for four different nuclear genes and differed in their sensitivity to the systemic fungicide mefenoxam. The null hypothesis of linkage equilibrium was not rejected, which suggests the population could be sexual. In the event that any of these new and diverse genotypes becomes prevalent in the upcoming years, we have determined their host preference, their susceptibility to the tomato resistance genes Ph2 and Ph3, their sensitivity to mefenoxam, the effect of temperature on germination and on mycelial growth. Having this information will allow us to provide field-specific recommendations to both conventional and organic farmers.

• Table 1. Phytophthora infestans isolates investigated in this study.

Objectives/Performance Targets

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

This objective has been completed. Differences in sensitivity to mefenoxam were seen among recent genotypes of P. infestans in the US (Figure 1).

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

I am currently in the process of determining host preference for recently emerged genotypes of P. infestans. I have completed at least two biological replicates of this assay for a subset of the recently emerged genotypes (Figure 2).

3. 3. 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.

I am currently in the process of determining the effect of temperature on the rate of germination for recently emerged genotypes of P. infestans. I have completed at least two biological replicates of this assay for a subset of these genotypes (Figure 3).  

There are significant differences in the rate of indirect germination at 4ºC among recent genotypes of P. infestans in the US. Genotypes that can germinate very rapidly might have a selective advantage in climates where leaf wetness occurs only for short periods of time.

4. 4. 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.

Because the purpose of this objective is to understand the effect of climate change on the development of the pathogen, an additional sub objective was included. I tested the effect of temperature on mycelial mass. This assay was done by exposing the different strains of P. infestans (growing on pea broth), to each of four different temperatures (10, 15, 20 and 25 ºC). After six days mycelial mass at each temperature was weighted. In general, mycelial growth at 20 and 25?C tended to be greater than growth at 15 ?C, and mycelial growth at 10?C was consistently less than growth at 15 ?C (Figure 4).

• Figure 3. Proportion of sporangia that had germinated at 30 and 120 min at 4ºC.
• Figure 2. Sporangia produced per infection site on potato and tomato for a subset of recently emerged genotypes of P. infestans in the US.
• Figure 4. Relative mycelial growth at different temperatures.
• Figure 1. Response of Phytophthora infestans isolates to mefenoxam.

Accomplishments/Milestones

Last year I have finished assessing the sensitivity to the systemic fungicide mefenoxam for diverse genotypes of Phytophthora infestans. These P. infestans isolates were detected near central and western New York State in 2010 and 2011. Mefenoxam sensitivity was assessed as described previously by Therrien et al., (1993){Therrien, 1993 #395;Therrien, 1993 #5}, except that mefenoxam was used in place of metalaxyl. Strains were grown on pea agar amended with Ridomil Gold EC (Syngenta, Greensboro, NC) such that the final concentrations of the active ingredient (mefenoxam) were 0, 5, or 100 ug 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 ug 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 (Figure 1). The isolates tested, differed in their sensitivity to mefenoxam ranging from sensitive to intermediate.

This past year I have begun to determine host preference as well as differences in rate of germination at 4ºC for a subset of genotypes. Host preference was determined by inoculating 20 µl of the same sporangial suspension on each of five potato and tomato leaflets. Inoculated leaflets were left for six days at 15ºC. After six days of incubation, each leaflet was washed with 3 mL of distilled water and sporangia counts were done using a haemocytometer. The experiment was repeated at least twice for each isolate. In general, all isolates tested were able to infect and sporulate on both potatoes and tomatoes (Figure 2).

To determine differences in rate of germination at 4ºC, two microscopic slides with three independent repetitions each (three circular water agar droplets that had each been inoculated with 20 µl of the same sporangial suspension) were assessed for each of the two time points studied (30 and 120 min). Percentage of total germination that was indirect was calculated for each of the two time points considered. The experiment was conducted at least twice for each isolate. Differences in the rate of indirect germination can be observed (Figure 3). For example, within 30 min at 4°C, more than 35% of the GDT-04 sporangia had liberated zoospores, whereas zoospore release from the other five genotypes tested ranged from about 5% to about 20%.

This past year, I have also finished assessing the effect of temperature on mycelial mass. This assay was done by exposing the different genotypes of P. infestans (growing on pea broth), to each of four different temperatures (10, 15, 20 and 25 ºC). After six days mycelia was lyophilized and dry weight at each temperature was determined. In general, mycelial mass at 20 and 25?C tended to be greater than growth at 15 ?C, and mycelial mass at 10?C was consistently less than growth at 15 ?C (Figure 4).

So far the project has progressed as planned. This upcoming year I am planning on finishing the host preference and differences in germination rates assays. Furthermore, I am planning on adding an experiment that will assess susceptibility of the recently emerged P. infestans genotypes to the tomato R genes Ph2 and Ph3. Preliminary results for this experiment are shown in Table 2.

Reference:

Therrien, C. D., Tooley, P. W., Spielman, l. J., Fry, W. E., Ritch, D. L., and Shelly, S. E. 1993. Nuclear DNA content, allozyme phenotypes and metalaxyl sensitivity of Phytophthora infestans from Japan. Mycological Research. 97:945-950.

• Table 2. Effect of tomato resistance genes, Ph2 and Ph3, on recently emerged genotypes of Phytophthora infestans in the United States.

Impacts and Contributions/Outcomes

The data I am gathering now, will enable rapid and appropriate disease management strategies against any of the recently emerged isolates of Phytophthora infestans in the northeastern US. The information that I will gather through this project will be useful in the event that any one of these genotypes might become a dominant strain in the future. The assessment for mefenoxam sensitivity, host preference, differences in germination rate at 4ºC, and the effect of temperature on mycelial mass indicated that these isolates are phenotypically diverse. Given that phenotypic analysis may take weeks to months, it will be important to learn the characteristics of each of these strains to be able in the future through rapid genotyping techniques to be able to assign strains to a lineage of known phenotype.

The ultimate goal of this project is to incorporate the information gathered for each of the isolates studied in a disease forecast hosted on the Cornell Decision Support System. This DSS is available to anyone in the Northeast at http://blight.eas.cornell.edu/blight_lab/.

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 8^th, 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., 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. Accepted in PLoS ONE.

Collaborators: