Use of Troyer citrange seedlings as a model system to study the temperature influence in citrus black spot caused by Phyllosticta citricarpa ascospores and conidia

• Grant number: 25/05785-3
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: October 01, 2025
• End date: June 30, 2026
• Field of knowledge: Agronomical Sciences - Agronomy - Plant Health
• Principal Investigator: Geraldo José da Silva Junior
• Grantee: Leonardo Aparecido Brandão da Silva
• Supervisor: Andre Drenth
• Host Institution: Escola Superior de Agricultura Luiz de Queiroz (ESALQ). Universidade de São Paulo (USP). Piracicaba , SP, Brazil
• Institution abroad: University of Queensland, Brisbane (UQ), Australia
• Associated to the scholarship: 24/01277-0 - in vitro production and germination of Phyllosticta citricarpa ascospores and response of citrus genotypes after inoculating with ascospores and conidia, BP.DR

Abstract

Citrus black spot (CBS) is the third greater cause of premature fruit drop in São Paulo citrus belt, which represents the largest area cultivated with sweet orange worldwide. CBS is caused by Phyllosticta citricarpa, a fungus that produces ascospores and conidia as inoculums. Despite the importance of ascospores in this disease spread and introduction in CBS-free areas, few studies have been performed regarding this inoculum. Ascospore production protocol development in 2017 by Australian researchers has been changing this scenario. Our research group studied the effect of different temperatures and wetness durations on the in vitro P. citricarpa ascospore and conidial germination, observing that both spores showed the same minimal (~10ºC) and maximum (~40ºC) temperatures to germinate and form appressorium, but the optimal temperature for ascospores (~30ºC) was higher than that for conidia (~24 ºC). This study was performed with Brazilian isolates; however, to validate and use these results to develop a CBS predictive system, it is necessary to use isolates from other countries and perform in vivo studies. In addition, the long CBS incubation period in sweet orange hampers these in vivo studies. Thus, using other citrus species with a shorter incubation period may be an alternative after proper validation, such as Troyer citrange (Citrus sinensis x Poncirus trifoliata). This project will aim: i) to assess the in vitro P. citricarpa ascospore and conidial germination at five different temperatures using Australian isolates; ii) to assess CBS incidence and severity in Troyer citrange leaves inoculated with ascospores and conidia under different temperatures; iii) to assess CBS intensity in sweet orange fruit inoculated with ascospores and conidia in semiarid conditions, and use Troyer citrange seedling to monitor P. citricarpa inoculum. Four P. citricarpa Australian isolates from compatible mating types (MAT1-1 and MAT1-2) will be crossed to obtain the ascospores, whereas the conidia will be collected from two of these isolates. Both spore suspensions will be deposited on polystyrene dishes and kept at 10, 25, 30, 35 and 40ºC, and spore germination will be assessed after 48 hours of wetness. Leaves of Troyer citrange seedlings will be inoculated with ascospore and conidial suspensions and stored at 10, 25, 30, 35 and 40ºC in growing chambers. After symptom onset, CBS incidence and severity will be assessed biweekly up to 120 days after inoculations (DAI). Final incidence and severity, as well as AUDPC will be compared among the temperatures and both spores. In an orchard located in a semiarid region, sweet orange fruit will be inoculated with ascospores and conidia and CBS intensity assessed over time. In another sweet orange orchard under subtropical climate, Troyer citrange seedlings will be placed under canopy and some leaves will be used to assess CBS symptoms and other to extract DNA and quantify P. citricarpa inoculum through qPCR. The results obtained will be compared with those from our study with Brazilian isolates in order to validate using Troyer citrange seedlings in studies regarding weather variable effects on CBS intensity, as well as developing a CBS predictive system.