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ARS Home » Northeast Area » Ithaca, New York » Robert W. Holley Center for Agriculture & Health » Emerging Pests and Pathogens Research » Research » Research Project #435528

Research Project: Monitoring and Molecular Characterization of Pythium species in Floricultural Greenhouses of NY and OK

Location: Emerging Pests and Pathogens Research

Project Number: 8062-22410-007-015-S
Project Type: Non-Assistance Cooperative Agreement

Start Date: Sep 1, 2018
End Date: Aug 31, 2021

1. Assess the incidence and diversity of pathogenic Pythium species in floricultural crops grown outdoor (chrysanthemum) and indoor (poinsettia) on the same season. 2. Monitor the movement of inoculum between outdoor and indoor crops grown simultaneously in the same floricultural operations.

Materials and Methods: Isolates. Mycelium of Pythium isolates from several hosts and other sources collected in floricultural greenhouses in Long Island, New York, will be provided by Margery Daughtrey (Cornell University, Long Island Horticultural Research & Extension Center), the Entomology and Plant Pathology Plant Disease and Insect Diagnostic Laboratory (Oklahoma State University), or collected by members of the research team. Upon arrival mycelia will be lyophilized and stored at -20°C until used. Isolates will be tentatively identified based on morphology and species identification will be confirmed by analysis of DNA sequences of the internal transcribed spacer (ITS) region. DNA extraction. DNA extractions will be performed from 30 µg of dehydrated mycelium using DNeasy Plant Mini Kits (Qiagen, Valencia CA), according to the manufacturer instructions. DNA quality and concentration will be measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Fragment analysis. P. aphanidermatum, P. cryptoirregulare and P. irregulare simple sequence repeats (SSR) markers developed by Lee and Moorman (6) will be used to characterize pathogenic Pythium populations by location (greenhouse) and host (poinsettia and chrysanthemum). Labeled SSR forward primers will be used (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. PCR amplifications will be carried out in a 20 µL reactions using 50 ng of DNA, 2 µl of 10× PCR buffer, 1.2 µl of dNTP mixture, 0.15 µl of Taq DNA (Takara Bio Inc, Japan), and 1 µl of each 5 µM primer. PCR temperature profiles: 94ºC (2 min), followed by 35 cycles of 94ºC (30 sec), annealing temperatures ranging from 55°C to 60°C depending on the specific primer set (30 sec), extension at 72°C (30 sec), and final extension at 72°C (10 min). PCR products will be resolved by capillary electrophoresis on an ABI 3730 DNA Analyser (Applied Biosystems) by loading 1 µL of the diluted PCR, 9µL Hi-Di™ formamide, and 0.5 µL LIZ 600 size standard (Applied Biosystems). Electropherograms will be analyzed using GeneMapper software version 3.7 (Applied Biosystems, Foster City, CA). Statistical analysis. The genotypic matrix will be constructed based on the sizes of microsatellite alleles. Groups formed by genetic relatedness, host of origin, geographical distribution and year of collection will be used to define populations. The genetic relationships between isolates will be analyzed using three different methods: Bayesian admixture, principle coordinate analysis (PCoA) and minimum spanning tree (MST) using an algorithm concept based on the haplotype frequencies and the distance among different haplotypes. Data formatting and population structure analyses will be conducted using GenAlEx 6.5 (Peakall and Smouse 2012) and R 3.4.3 (R Core Team 2018).