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Phyllosticta citricarpa, Elsinoe fawcettii, Elsinoe australis, and Pseudocercospora angolensis are major pathogens of citrus crops worldwide and can cause non-characteristic symptoms that may lead to confusion regarding the causative agent. These fungi are subject to international phytosanitary regulations, and testing on fruits or leaves requires accurate and easy-to-use tools. New multiplex conventional PCR and real-time PCR assays were developed here to achieve highly accurate simultaneous detection of all four fungal pathogens in fruit tissues. We designed new oligonucleotide combinations for P. citricarpa, E. fawcettii, and E. australis and combined them with already available primers and hydrolysis probes to be used in either PCR assay. The limit of detection for multiplex conventional PCR was as low as 100 pg μL−1 for P. citricarpa, E. fawcettii, and E. australis and 10 pg μL−1 of target DNA per reaction tube for P. angolensis. The quadruplex real-time PCR assay successfully yielded repeatable positive results with as low as 242, 243, 241, and 242 plasmidic copies of target DNA of P. citricarpa, E. fawcettii, E. australis, and P. angolensis, respectively. Moreover, analysis of 60 naturally infected citrus samples yielded 100% concordant results by both assays. Our validation experiment revealed that the multiplex real-time PCR assay showed high specificity except a cross-reaction with P. paracitricarpa DNA. Sensitivity, repeatability, reproducibility, and robustness were verified, and the assay could be used following different DNA extraction procedures, supporting fitness for routine analysis. These new multiplex tools should be of great interest as cost-effective solutions for regulatory authorities and diagnostic laboratories, enabling testing for four important pathogens in single-tube reactions. • Development of new conventional PCR and qPCR assays for four citrus pathogens. • Very low limits of detection were found for multiplex conventional PCR. • qPCR had high specificity, sensitivity, repeatability, reproducibility, and robustness.

Sweet orange scab (SOS) is a fungal disease of citrus which is caused by Elsinoe australis. It affects the aesthetics of the fruit by forming wart-like protruded lesions on the fruit skin, and also affects the leaves which act as source of inoculum in the orchards. SOS is widespread in the different citrus species such as grapefruit (Citrus x paradisi Macfd.), sweet orange [C. sinensis (L.) Osb.], and mandarin (C. reticulata Blanco). In this study, we report the development of symptoms and changes in polyphenolic profile of ‘Rio Red’ grapefruit leaves in response to E. australis infection. Qualitative and quantitative analysis of polyphenols in ‘Rio Red’ grapefruit leaves inoculated with E. australis were conducted using high performance liquid chromatography (HPLC) at two different time points post inoculation. Development of SOS symptoms under light was recorded and categorized into four different morphological stages. Caffeic acid, luteolin-7-O glycoside, naringin, naringenin, apigenin-7-O glycoside and eriodictoyl were identified in healthy grapefruit leaves. The quantitative changes in the identified polyphenols were measured 9 days post-inoculation by comparing levels on the 9-day control versus the 9-day post-inoculated leaves. Total polyphenol levels decreased substantially in E. australis-infected ‘Rio Red’ grapefruit leaves 9 days post-inoculation. Levels of naringin, naringenin, apigenin glucoside and eriodictoyl decreased with E. australis infections indicating the pathogen’s ability to overcome some of plant’s defenses. Compared to 9-day controls, 9-day inoculated leaves had much less caffeic acid.