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Background  Phytophthora blight caused by  Phytophthora cajani  is an emerging disease of pigeonpea ( Cajanus cajan  L.) affecting the crop irrespective of cropping system, cultivar grown and soil types. Current detection and identification methods for  Phytophthora  species rely primarily on cultural and morphological characteristics, the assessment of which is time-consuming and not always suitable. Sensitive and reliable methods for isolation, identification, zoospore production and estimating infection severity are therefore desirable in case of Phytophthora blight of pigeonpea.    Results  In this study, protocols for isolation and identification of Phytophthora blight of pigeonpea were standardized. Also the method for zoospore production and  in planta  infection of  P. cajani  was developed. Quantification of fungal colonization by  P. cajani  using real-time PCR was further standardized.  Phytophthora  species infecting pigeonpea was identified based on mycological characters such as growth pattern, mycelium structure and sporangial morphology of the isolates and confirmed through molecular characterization (sequence deposited in GenBank). For Phytophthora disease development, zoospore suspension of 1 × 10 5  zoospores per ml was found optimum.  Phytophthora  specific real-time PCR assay was developed using specific primers based on internal transcribed spacer (ITS) 1 and 2. Use of real-time PCR allowed the quantitative estimation of fungal biomass in plant tissues. Detection sensitivities were within the range of 0.001 pg fungal DNA. A study to see the effect of elevated CO 2  on Phytophthora blight incidence was also conducted which indicated no significant difference in disease incidence, but incubation period delayed under elevated CO 2  as compared to ambient level.    Conclusion  The zoospore infection method for Phytophthora blight of pigeonpea will facilitate the small and large scale inoculation experiments and thus devise a platform for rapid and reliable screening against Phytophthora blight disease of pigeonpea. qPCR allowed a reliable detection and quantification of  P. cajani  in samples with low pathogen densities. This can be useful in early warning systems prior to potential devastating outbreak of the disease.

bmc plant biology

An efficient method for zoospore production, infection and real-time quantification of Phytophthora cajani causing Phytophthora blight disease in pigeonpea under elevated atmospheric CO2