Survey of Phyllosticta citricarpa mating type in the São Paulo citrus belt and optimization of in vitro ascospore production

Phyllosticta citricarpa, the citrus black spot causal agent, requires both mating types, designated as MAT1‐1 and MAT1‐2, to produce ascospores. Mating‐type ratio in some citrus‐growing areas has been characterized but remains little known in the São Paulo (SP) citrus belt, the largest sweet orange producer worldwide. A protocol for in vitro ascospore production was developed recently; however, there are few studies regarding the optimal conditions for their production. Thus, this study aimed to determine the mating‐type ratio in SP and quantify ascospores produced under different conditions in vitro. The mating types were identified by duplex PCR, and ascospore production was assessed in five culture media: potato dextrose agar (PDA), ½ PDA, malt extract agar (MEA), oatmeal agar (OMA) and citrus agar (CA), and at temperatures from 10 to 30°C. The ratio of MAT1‐1 and MAT1‐2 was similar to 1:1. Ascospore production reached peaks of 8.0, 7.0 and 3.0 × 103 ascospores/mL in MEA, PDA and ½ PDA, respectively. There were no ascospores observed in OMA and CA. P. citricarpa ascospores were observed only at 15, 20 and 25°C in PDA, with peaks of up to 2.0 × 103 ascospores/mL at 42, 35 and 21 days, respectively. There were no ascospores observed at 10 and 30°C. Both mating types are similarly distributed in the SP citrus belt, and in vitro ascospore production may be optimized by using MEA at 25°C. These data provide a better understanding of P. citricarpa sexual reproduction and a background for further studies with ascospores.