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Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing
Published on: March 31, 2017
First Report of Powdery Mildew on Potato Caused by Golovinomyces cichoracearum in California
M K Romberg1, J J Nuñez2, J J Farrar3
1University of California, Davis.
Abstract:
In October 2003, potato plants in three fields (cv. Desiree, Satina, Midas, and Mondial) in Lancaster, California exhibited symptoms and signs of powdery mildew. Disease symptoms were most severe on cvs. Desiree and Santina. Disease expression was greater along sprinkler lines and in localized areas from which the disease spread to surrounding plants. Severely affected plants began collapsing just prior to water cutoff. Early symptoms comprise small dark areas on the adaxial surface of leaves, along the veins, and at the petioles. Dark lesions consisting of mycelia and conidiophores were also visible on the main stems of affected plants. As the disease progressed, leaves were covered by a gray powdery fungal mass, and older leaves became necrotic. Conidial chains arising from the hyaline, epiphytic mycelia consisted of two to eight conidia. The cylindric to doliform conidia measured 16.8 to 22.8 μm wide (mean = 19.2, standard error = 0.36, N = 30) × 28.8 to 45.6 μm long (mean = 32.4, standard error = 0.75, N = 30). No cleistothecia were observed. Identification of the causal agent as Golovinomyces cichoracearum (synonyms G. orontii and Erysiphe cichoracearum) based on morphology was confirmed by internal transcribed spacer (ITS)-polymerase chain reaction (PCR). Conidia were washed off the affected leaves, concentrated by filtration and centrifugation, and sonicated to release genomic DNA. PCR was performed on the sonicated conidia with primers ITS4 and ITS5 (2), and the resulting amplicon was purified and sequenced. BLAST analysis of the ITS sequence revealed a 99% homology to E. cichoracearum from an Ambrosia sp. (GenBank Accession No. AF011292). Pathogenicity was confirmed on potato seedlings cv. Red La Soda. Inoculations were performed twice on six plants (three pots) each time. A sterile brush was used to transfer conidia from the affected leaves to seedlings consisting of two to three fully expanded leaves. A plastic bag was placed around each pot containing two seedlings for 1 to 2 days and then removed. Noninoculated controls were stroked with a sterile brush, placed in a plastic bag for 1 to 2 days, and kept in the greenhouse on a separate bench. Two control plants were included for each inoculation. Plants were maintained in a greenhouse at approximately 25 to 28°C and 40 to 60% relative humidity. After 7 days, dark spots were visible on the leaves of all inoculated plants, and conidiophores with conidia identical to those of the isolate used as the inoculum source were apparent after 10 days. The controls showed no disease symptoms or signs. To our knowledge, this is the first report of powdery mildew caused by G. cichoracearum on potato in California. The first field report of the disease was from Washington in 1950 (1), with subsequent reports from Utah and Ohio. References: (1) J. D. Menzies. Plant Dis. Rep. 34:140, 1950. (2) T. J. White et al. PCR Protocols. Academic Press, New York, 1990.
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