Phytophthora root rot of chickpea: inoculum concentration and seasonally dependent success for qPCR based predictions of
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ORIGINAL PAPER
Phytophthora root rot of chickpea: inoculum concentration and seasonally dependent success for qPCR based predictions of disease and yield loss S.L. Bithell 1
&
K. Moore 1 & Herdina 2 & A. McKay 2 & S. Harden 1 & S. Simpfendorfer 1
Received: 3 May 2020 / Accepted: 12 October 2020 # Australasian Plant Pathology Society Inc. 2020
Abstract Phytophthora root rot (PRR) caused by Phytophthora medicaginis (Pm) is an important disease of chickpea in Australia. There are limited control options, with avoiding planting chickpeas in paddocks with a high PRR risk a key management strategy. Currently, risk assessment is based solely on paddock history of PRR, without any measure of Pm inoculum. We developed a qPCR test to quantify Pm inoculum concentrations in soil and evaluated its ability to predict, prior to planting, PRR disease and yield loss in chickpea. The qPCR test was specific to Pm and did not cross react with other Phytophthora species found in Australian agricultural systems and was sensitive, being able to detect 35.6) were observed with P. trifolii DNA. The Ct value difference (approx. 20) showed the assay was approximately 1,000,000 times more sensitive at detecting Pm than P. trifolii. The calibration curve prepared using synthetic DNA showed a linear relationship between the Ct value and the copy number over 7 fold magnitude (2 to 2 × 10^6 copies/ul). The efficiency of the assay was 97.9%.
S. L. Bithell et al.
Evaluation of Pm qPCR sensitivity in soil samples Experiment 1 – concentration series: Pm soil DNA concentrations as measured using the qPCR test had a strong correlation (r2 0.92, P < 0.001) with the number of oospores used to spike sand samples, although there was greater variation in Pm soil DNA levels detected at the highest oospore concentrations (Fig. 1). An average of 373 Pm copies/g media were detected at the lowest concentration of 0.25 oospores/g sand demonstrating the Pm soil DNA test maintained good sensitivity at low oospore densities. Experiment 2 – soil type and moisture content series: The Pm soil DNA test was found to have good sensitivity with the ability to reliably detect oospores in a 100 g sample of dry sand. There was no significant effect of moisture content on the detection of Pm using the qPCR test (data not shown). However, there was an interaction between soil type and oospore concentration (Table SM4). For the 1.25, 2.5, 5 and 20 oospore treatments, lower Pm soil DNA concentrations were detected in the sand than in the soil, with the sand:soil mix treatment providing intermediate values that did not differ from sand or soil with the exception of the 1.25 and 5 oospore treatments which provided lower values for sand than the sand:soil mix treatment. When comparing between oospore treatments there were significant increases in Pm soil DNA
Fig. 1 Experiment 1 - Linear regression of Phytophthora medicaginis (Pm) oosporemycelium concentrations in dry sand against Pm qPCR DNA copies/g media (y = 1258.5 (x) – 398, r2 0.924, df = 20, P < 0.001). Fitted regress
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