A novel, multiplexed, probe-based quantitative PCR assay for the soybean root- and stem-rot pathogen, Phytophthora sojae, utilizes its transposable element

Authors: Haudenshield, James; SONG, JEONG - Chungnam National University; Hartman, Glen

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/20/2017
Publication Date: 5/25/2017
Citation: Haudenshield, J.S., Song, J.Y., Hartman, G.L. 2017. A novel, multiplexed, probe-based quantitative PCR assay for the soybean root- and stem-rot pathogen, Phytophthora sojae, utilizes its transposable element. PLoS One. doi.org/10.1371/journal.pone.0176567.

Interpretive Summary: Phytophthora sojae is a soil-borne, water-mold that can grow as mycelium, like a fungus, but can also form overwintering spores and swimming spores in moist and water-logged soils, rapidly spreading disease. It causes seed rot, pre- and post-emergence damping-off, and sometimes foliar blight in soybean, and crop loss may approach 100% with susceptible cultivars. It is one of several organisms that cause similar diseases in soybean, and can be a challenge to identify. Soybean is a major crop in numerous U.S. states, with a large economic impact. The causal organism is commonly found in field soils where ever soybean is grown. Molecular methods—called assays—can help farmers determine the presence and amount of this damaging organism in their fields, so that they can select resistant varieties of soybean, or use other disease control strategies. This report describes a new assay for detecting and quantifying even very small traces of the genetic material of the causal organism without inadvertently detecting related or unrelated microbes that might cause different soybean diseases. The assay differs from others, in both the precise genetic locus being detected, and in its biochemistry, such that it can be combined with other assays simultaneously, and have improved reliability. By using this assay, technicians can rapidly and reliably help farmers determine what organisms threaten their crops. It will also be useful to scientists who are studying this disease, to find improved resistance to the disease, and to evaluate the spread of the organism in the environment.

Technical Abstract: Phytophthora root rot of soybean (Glycine max Merr.) is caused by the oomycete Phytophthora sojae (Kaufm. and Gerd.). P. sojae has a narrow host range, consisting primarily of soybean, and it is a serious pathogen worldwide. It exists in root and stem tissues as mycelium, wherein it can form oospores which subsequently germinate to release motile, infectious zoospores. Molecular assays detecting DNA of P. sojae are useful in disease diagnostics, and for determining the presence of the organism in host tissues, soils, and runoff or ponded water from potentially infested fields. Such assays as published have utilized ITS sequences from the nuclear ribosomal RNA genes in conventional PCR or dye-binding quantitative PCR (Q-PCR) but are not amenible to multiplexing, and some did not utilize control strategies for type I or type II errors. In this study, we describe primers and a bifunctional probe with specificity to a gypsy-like retroelement in the P. sojae genome to create a fluorogenic 5’-exonuclease linear hydrolysis assay, with a multiplexed internal control reaction detecting an exogenous target to validate negative calls, and with uracil-deglycosylase-mediated protection against carryover contamination. The assay specifically detected 13 different P. sojae isolates, and excluded 17 other Phytophthora species along with 13 fungal species pathogenic on soybean. A diagnostic limit of detection of 34 fg total P. sojae DNA was observed in serial dilutions, equivalent to 0.5 genome, and a practical detection sensitivity of four zoospores per sample was achieved, despite losses during DNA extraction. The assay was used to specifically quantify P. sojae from infested soil, seed, and plant tissues.