in vitro production and germination of Phyllosticta citricarpa ascospores and response of citrus genotypes after inoculating with ascospores and conidia

• Grant number: 24/01277-0
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: February 01, 2025
• End date: February 28, 2027
• Field of knowledge: Agronomical Sciences - Agronomy - Plant Health
• Principal Investigator: Geraldo José da Silva Junior
• Grantee: Leonardo Aparecido Brandão da Silva
• Host Institution: Escola Superior de Agricultura Luiz de Queiroz (ESALQ). Universidade de São Paulo (USP). Piracicaba , SP, Brazil

Abstract

The citrus black spot (CBS) is the third greater cause of sweet orange premature fruit drop in the São Paulo citrus belt, which represents the Brazilian larger area cultivated with sweet orange. CBS is caused by Phyllosticta citricarpa, a fungus that produces ascospores and conidia as inoculums. Protocols of ascospore production were developed in 2017, but the amount of ascospores produced in vitro remains low. The effect of temperature and wetness period over conidial germination was already reported; however, for ascospores these researches were not performed. In addition, the response of citrus genotypes inoculated with conidia has been studied, but for ascospores it remains little known. Therefore, this project will have as objectives: i) to estimate the minimum and maximum temperatures and to optimize ascospore production by using several photoperiods and crossing methods; ii) to assess the effect of temperature and wetness period over in vitro ascospore germination; iii) to assess the reaction of 'Valencia' fruit of diverse diameters when inoculated with ascospores in comparison to conidia; iv) to assess the penetration and colonization processes by ascospores in sweet orange fruit using fluorescence microscopy; v) to assess the resistance/susceptibility of citrus ancestors, commercial varieties and hybrids inoculated with both spores. Two P. citricarpa isolates from compatible mating types (MAT1-1 and MAT1-2) will be crossed and stored at temperatures from 10 to 30ºC to estimate the minimum and maximum temperatures to ascospore ejection. The ascospore production will also be assessed under five light periods (0, 8, 12, 16 and 24 hours) at 25ºC, as well as in five crossing methods. The ascospores will be collected and quantified weekly and up to 56 days after crossings. The ascospore germination will be assessed in temperatures from 10 to 40ºC, until 48 hours of wetness in suspension containing 2% sweet orange juice. Under greenhouse conditions, 'Valencia' fruits from 1.5 cm to 7.0 cm diameter will be inoculated with ascospores and conidia at 103 spores/mL, and the CBS symptoms will be assessed until the final fruit maturity. The infectious and colonization processes caused by conidia and ascospores will be compared through fluorescence microscopy. The assessments of citrus genotypes inoculated with both P. citricarpa spores will be performed in partnership with the Deparment of Agriculture & Fisheries in Queensland, Australia. The applicant will request a "Bolsa de Estágio de Pesquisa no Exterior (BEPE)" to conduct this last project stage. With the results obtained, it is hope to improve the ascospore production process, understand better the influence of environmental factros over P. citricarpa spore germination, generating mathematical models that may be used to develop a CBS predictive system, as well as to describe the resistance of citrus hybrids and species to CBS when caused by both inoculums.