Monitoring quantities of Phyllosticta citricarpa inoculum in sweet orange orchards using qPCR and young citrus plants as spore traps

Abstract

Monitoring of Phyllosticta citricarpa (causal agent of citrus black spot [CBS]) inoculum in orchards has previously been performed using volumetric spore traps. However, volumetric traps are nonspecific, and only capture ascospores (not pycnidiospores) of different Phyllosticta species. This research aimed to monitor and quantify the DNA of P. citricarpa using young citrus plants as a spore trap combined with quantitative PCR (qPCR), as well as quantify the number of ascospores and pycnidiospores, and to correlate results with weather variables. Citrus nursery plants were placed as traps under and outside tree canopies during fruit developmental stages (from October to July) in two seasons in commercial ‘Valencia’ sweet orange orchards. DNA was extracted from trap leaves to quantify P. citricarpa inoculum by qPCR of the ribosomal internal transcribed spacer 1 (ITS1 rRNA) region of P. citricarpa (Pc‐ITS). Correlations of Pc‐ITS to rainy days, leaf wetness and temperature were performed. Overall, the highest numbers of 400 Pc‐ITS/cm2 of leaf tissue, which represented up to 12 ascospores or pycnidiospores per cm2, were detected on leaves sampled from October to March, regardless of the trap position, season and orchard. Trap plants placed under canopies had up to 20‐fold more Pc‐ITS than those placed outside. Rainy days and leaf wetness were the variables most positively correlated with Pc‐ITS. Both results in the fluctuation of P. citricarpa inoculum in orchards and the most favourable weather variables associated with inoculum production contribute to better understanding of the critical periods for CBS management in citrus‐growing areas.