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Title: Plasmolysis and vital staining reveal viable oospores of Peronospora effusa in spinach seed lots

item KUNJETI, SRIDHARA - University Of California
item Anchieta, Amy
item SUBBARAO, KRISHNA - University Of California
item KOIKE, STEVEN - University Of California - Cooperative Extension Service
item Klosterman, Steven

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/16/2015
Publication Date: 1/12/2016
Citation: Kunjeti, S.G., Anchieta, A.G., Subbarao, K.V., Koike, S.T., Klosterman, S.J. 2016. Plasmolysis and vital staining reveal viable oospores of Peronospora effusa in spinach seed lots. Plant Disease. 100:59-65.

Interpretive Summary: Downy mildew disease of spinach, caused by the plant pathogenic microorganism Peronospora effusa, is a major disease constraint on spinach worldwide, and in the U.S. This microorganism produces long-term survival structures, known as oospores, through sexual reproduction. Spinach seed lots were confirmed positive for the presence of oospores over three decades ago, yet the extent of oospore infestation in modern commercial spinach seed lots was not known. This study was undertaken to assess the level of oospores present in commercial seed lots, which may transmit the pathogen. One thousand seeds of each lot, or 500 seeds in some cases, were washed with water and the sediment from this wash-off was examined by microscopy for oospores. The results indicate that 16 percent of the 82 spinach lots tested are infested with oospores of P. effusa. Also, 95 percent of the seed lots tested were positive for detection of DNA of P. effusa using a previously developed P. effusa-specific DNA-based test. Thus the number of spinach seed lots positive for inoculum of P. effusa is probably underestimated by examination of the sediment from windows of only 1000 seeds. Knowledge acquired in this study is an important first step to provide awareness of the amount of infestation of spinach seeds with P. effusa, and to devise strategies, such as seed treatments or other means to eliminate the pathogen from seed production fields, to curtail the spread of P. effusa on spinach seed.

Technical Abstract: Production of oospores by Peronospora effusa, the causal agent of downy mildew on spinach (Spinacia oleracea), was reported on spinach seed over three decades ago. In view of the rapid proliferation of new races of P. effusa worldwide, seed borne transmission has been suspected but methods to test the viability of seedborne oospores have not been available. Eighty-two seed lots of contemporary spinach cultivars were evaluated for the presence of P. effusa using a seed-wash method and the sediment examined by microscopy. Sixteen percent of the analyzed seed lots were positive for oospores and 6% for sporangiophores characteristic of P. effusa. Peronospora effusa-specific quantitative PCR (qPCR) assay showed that 95% of the 59 tested seed lots were positive for P. effusa. The viability of oospores from five seed lots was tested using two independent methods, one involving plasmolysis and the other by trypan blue staining. The oospores plasmolysed in 4 M sodium chloride (NaCl) but were deplasmolysed in water, demonstrating an active and viable cell membrane. Similarly, viable oospores failed to take up the trypan blue stain. Overall, 59% of the oospores were viable in the plasmolysis test, and 45% with the trypan blue test. These results indicate the presence of P. effusa oospores in contemporary spinach seed lots, and transmission of viable oospores of P. effusa in spinach seed is a common occurrence. Elimination of the pathogen on seed, in addition to other management approaches, will be useful to prevent the pathogen spread on spinach seed.