A phenotyping method to select lemon hybrids tolerant to mal secco disease: a resource for breeding and mapping

Mal secco, caused by Plenodomus tracheiphilus fungus, is a serious vascular disease that affects citrus, in particular lemons. Treatments can be applied only to prevent disease; therefore field tolerance or resistance are the major goals of breeders. As field phenotyping takes many years, we report a phenotyping method in greenhouse by artificial inoculation from P. tracheiphilus, with the aim of rapidly distinguishing resistant or tolerant individuals from those susceptible to the pathogen. Six-month plantlets of 53 Citrus latipes × Femminello ‘Siracusano 2KR’ hybrids plus the parentals, obtained from self-rooted cuttings, were stem-inoculated using a conidial suspension, and three biological replicates per hybrid were used. Two months after inoculation, the individuals, kept in the greenhouse, were monitored for symptoms appearance every two weeks, for a total of three surveys. The visual assessment of symptoms was coupled with real-time PCR pathogen quantification performed on DNAs from inoculated twigs. The same hybrids were evaluated for disease symptoms in open field, subjected to the natural pressure of the pathogen. Specifically, two replicates of each hybrid, grafted onto Carrizo citrange and planted in 2020 were phenotyped every month for 24 months. Results showed a significant correlation between real-time PCR quantification of P. tracheiphilus and phenotyping in greenhouse, and between field and greenhouse phenotyping (p<0.001). Analysis showed that around 70% of hybrids were phenotyped as susceptible both in the field and in the greenhouse. However, inoculated plantlet phenotyping led to an overestimation of the number of susceptible individuals. Based on our results, artificial inoculations of young, self-rooted plantlets could be used as a complementary approach to field evaluation to perform more rapid and reliable phenotyping of mapping populations, for the detection of loci potentially associated with disease resistance or susceptibility. The method could also be used to optimize lemon breeding programs for pathogen resistance.