Pythium Species and Population Identification Using DNA Markers PROGRESS REPORT – June 2001
AMERICAN FLORAL ENDOWMENT June 2001 – PROGRESS REPORT and APPLICATION for CONTINUED FUNDING
TITLE:
Pythium species and population identification using DNA markers
PRINCIPAL INVESTIGATOR:
Gary W. Moorman, Professor of Plant Pathology, Department of Plant Pathology, The Pennsylvania State University, 111 Buckhout Laboratory, University Park, PA 16802-4506. Phone: 814-863-7401. Fax: 814-863-7212. E-mail: gmoorman@psu.edu
CO-INVESTIGATORS:
Seogchan Kang and David Geiser, Assistant Professors of Plant Pathology, Penn State University, and Seong Hwan Kim, Chief Plant Pathologist, Pennsylvania Dept. of Agriculture, Bureau of Plant Industry, Harrisburg, PA.
REVIEW OF INDUSTRY NEEDS:
Knowing the identity of a pathogen and from where it came is crucial to formulating an effective management strategy for that pathogen. The research addresses this in relation to Pythium. The identification of Pythium species is important because while some species found associated with greenhouse crop roots are aggressive plant pathogens, others are weak pathogens, non-pathogens or may even be beneficial because they attack other parasites. It is possible that a pathogenic Pythium causing crop losses originated from a resident population within the greenhouse or was brought in on infected plant material from a distant population. Knowing its source is important so that control measures can be targeted to eliminate or manage the pathogen at its source. The proposed research will continue work initiated in 1999 to identify genetic markers that are characteristic of Pythium species and populations within species known to infect greenhouse crops.
PROGRESS:
Objective 1.
Determine DNA sequences of Pythium that can be used to identify species within the genus.
The polymerase chain reaction (PCR) and automated DNA sequence analysis were used to continue to characterize the DNA sequences of the ITS-1, 5.8s, and ITS-2 regions of nuclear ribosomal DNA from many additional isolates of three important species found in greenhouses. The sequences of Pythium aphanidermatum exhibit essentially no variation among the isolates examined despite the fact that some isolates had been obtained from other parts of the world for comparison to U.S. isolates. The sequences of P. ultimum and P. irregulare isolates show some variations but are uniform enough to identify each species. It was found that sequences could not be obtained for approximately one third of the P. irregulare isolates that were identified as P. irregulare using microscopy. Experiments are underway to determine why the DNA of these isolates cannot be sequenced and to compare these with sequenced isolates. Our data indicate that there are two major types of P. irregulare for which sequences were obtained. This ‘species’ may actually contain closely related organisms. Additional work is needed to understand P. irregulare because it is the most commonly isolated species from greenhouse crops. It is found in more different crops than P. ultimum or P. aphanidermatum..
Work continued on the ras-related protein gene sequence which had never before been determined for any Pythium. We have obtained the sequences for Pythium aphanidermatum, P. arrhenomanes, P. catenulatum, P. cylindrosporum, P. deliense, P. graminicola, P. myriotylum, P. torulosum, P. vanterpooli as well as Phytophthora infestan, P. cinnamomi, P. citricola,and P. cryptogea . Because the ras sequence does not clearly identify Pythium species (for example, P. aphanidermatum, P. arrhenomanes, and P. deliense cannot be differentiated from one another) no further work on this DNA segment will be conducted.
Developing a PCR/RFLP method for rapid species identification using DNA but that does not require sequencing equipment continues. Modifications are being made in the electrophoresis gel and the endonucleases employed. Once the method is proven, identification of Pythium species will be simpler than using microscopy and should greatly assist clinicians in determining the exact species causing crop losses.
Of high priority is the preparation of a publication on the sequences of the rDNA and the ras-related protein gene DNA of the Pythium species examined. We will submit these sequences to the world database, BLAST.
Objective 2.
Generate DNA fingerprints, using AFLP methods, for identifying populations within species with emphasis on P. ultimum, P. irregulare, and P. aphanidermatum, the most common species that cause significant crop losses in commercial floriculture.
It was found that AFLP was very effective for identifying P. ultimum, P. irregulare, and P. aphanidermatum (see attached example of fingerprints). It may be possible to identify species using AFLP rather than sequencing, AFLP will be applied to other species we currently have in our collection to verify this possibility.
While isolates within each species from well separated geographic locationscould be differentiated from one another using AFLP fingerprints, we feel that the our current protocol did not yield enough electrophoresis bands to reliably differentiate all isolates. Continued work is needed to obtain more bands and thus a more complex fingerprint to allow us to clearly resolve differences among isolates. In fact, changing one of the primers may result in twice as many bands. We have also found that the positioning of tubes in the thermocycler can have an influence on the number and intensity of bands. If small modifications fail to provide the information needed, additional primers will be tested. Failing that, a completely different technique will be sought. The identification of populations within species is the single most important aspect of this research. If the origin of the pathogen in a greenhouse can be determined, control measures can be directed at the source whether it is irrigation water, commercial potting mix, cuttings purchased elsewhere, or from the soil within the particular greenhouse. Using the current method, the AFLP patterns among P. aphanidermatum isolates exhibited almost no variation. This is similar to what was found with the rDNA sequences and may indicate that this species has little DNA variation. Because P. aphanidermatum is a very aggressive pathogen, additional AFLP work needs to be done to determine exactly from where it is entering the production system. We have made extensive collections at each of three commercial greenhouses with histories of Pythium problems, have microscopically identified the species present, and are in the process of using the AFLP technique to characterize isolates from infected plants, new and used potting mix, irrigation water, and soil from floors and walkways. We hope to determine whether isolates within one operation can be differentiated from each other. We are optimistic that it is possible because our AFLP analyses differentiate metalaxyl/mefenoxam (Subdue 2E/Subdue Maxx) sensitive P. irregulare strains from resistant ones all obtained in Pennsylvania even though these strains differ very little in other characteristics. This work should be complete by the end of 2001, contingent upon successful modification of the AFLP method. A graduate student who has worked for almost two years with AFLP on this project as part of a Master of Science degree is expanding her work into a Ph.D. project.
We are consistently finding P. aphanidermatum associated with poinsettia roots and almost no other crop or location within greenhouses. This is true even in greenhouses where P. irregulare is causes root rot in many crops other than poinsettias and is in the water supply. Research is required to determine whether poinsettias are particularly susceptible to P. aphanidermatum as compared to P. irregulare or whether the cultural practices or growing conditions for poinsettias makes them especially vulnerable to P. aphanidermatum. A second graduate student, beginning in August 2001, will work specifically on this aspect.
Objective 3.
Data from objectives 1 and 2 will be used to help establish an interactive database linking the genotypic data to phenotype, host plant, and geographic location information on the Pythium populations characterized in this study.
We are storing DNA sequences, AFLP fingerprint data, microscopic characteristic data, host plant information, and original geographic location for each isolate on the computer for analysis and for future use in the database. Following the completion of analysis of the ITS and ras DNA sequences obtained from the representative isolates, we will submit them to the World Wide Web database, BLAST. We will also deposit certain isolates with the American Type Culture Collection so that other researchers have access to them. This will be done by July 2001. It is important to develop a database of Pythium populations and continually add to it so that unknowns can be compared to those that have been well characterized. It then may be possible to trace a particular pathogen to its likely source using the database.
PUBLICATIONS:
Moorman, G. W. and Daughtrey, M. L. Pythium problems are escalating. We need to learn why. This was accepted for publication by GrowerTalks (Chris Beytes, editor; beytes@growertalks.com) in December 2000. He assures us that it will be published soon. AFE funding is noted in the article. (Enclosed)
Yoneyama, M., Lease, R., Geiser, D., Kang, S., Moorman, G., and Kim, S. 2000. Comparison of the ras-related protein gene sequence among species of Pythium and Phytophthora. Phytopathology 90:S87.
This is an abstract of a poster presented at the national American Phytopathological Society meeting in New Orleans, LA in August 2000. The poster prominently noted AFE funding. Ms. Yoneyama also presented this poster at the Undergraduate Research Exhibition at Penn State in March 2000.
Presentations were made by Moorman at the following locations and included results from this work and recognition of AFE funding:
2001 Mid-Atlantic Fruit and Vegetable Convention. Hershey, PA. 1/31/01.
Greenhouse crop disease scouting and identification IPM workshop.
Allentown, PA. 3/28/01; Landisville, PA. 3/29/01; Greensburg, PA. 4/2/01;
Middleburg, PA. 4/4/01.
Carla Garzon (graduate student) presented a poster at the Penn State Graduate Research Exhibition detailing her AFLP work on Pythium and acknowledged AFE funding. April, 2001.
She also presented a seminar to the Department of Plant Pathology faculty, staff, and students on this topic. March 2001.
ANTICIPATED IMPACTS:
The identification of Pythium species causing greenhouse crop losses will be simplified so that the species name will be routinely reported. It will be possible to trace the isolate causing crop losses to it’s point of origin within a greenhouse. Metalaxyl/mefenoxam-resistant populations will be more readily identifiable based on DNA characteristics rather than culture tests.
A database of population information will be established and available to everyone working with Pythium so that we can begin to trace populations obtained from anywhere in the greenhouse industry to their origin.
Growers will be better able to target Pythium control measures and manage this important pathogen at its source, before crop losses occur, based on information that can be provided by clinicians and researchers.
PLANS FOR FURTHER DISSEMINATION OF RESULTS:
Carla Garzon (graduate student) will present a poster at the national American Phytopathological Society meetings, acknowledging AFE funding, at Salt Lake City, UT in August 2001.
Moorman will continue to present information on the research at Extension meetings in Pennsylvania. Some of the information will also be presented at the Southeast Greenhouse Conference and Trade Show in Greenville, SC, June 2001.
A trade journal article will be prepared based on the AFLP work and submitted for publication late in 2001 or early 2002. Scientific journal article(s) will be prepared and submitted to Phytopathology, Plant Disease, Mycologia, or the Canadian Journal of Botany in 2001. Pathogenicity test results will be submitted to Biological and Cultural Control Tests in 2001.
An oral or poster presentation of final results will be made at the national American Phytopathological Society meetings in Milwaukee, WI in July 2002. Based on this work, Carla Garzon will prepare a trade journal article. It is anticipated that the second graduate student, Paola Ramon, will also present results of pathogenicity tests at this meeting.
LIST OF SUPPLES AND MATERIALS:
DNA extraction chemicals including DNAzol, alcohol, and buffer salts.
DNA restriction enzymes, DNA ligase enzymes, DNA size and mass standards and primers for sequencing and AFLP work
Disposable pipet tips, disposable nitrile (non-latex gloves), and electrophoresis agarose and reagents.
Agar, antibiotics and other reagents to isolate, purify and store Pythium isolates
DNA analysis software
