Identification of Colletotrichum species associated with postbloom fruit drop in Brazil through GAPDH sequencing analysis and multiplex PCR

The genus Colletotrichum comprises a group of important fungal pathogens that can infect a wide variety of host plants worldwide. Postbloom fruit drop (PFD) of citrus plants is responsible for extensive crop losses annually, and is particularly detrimental to Brazilian citrus production. The disease was first associated with Colletotrichum gloeosporioides and subsequently linked to Colletotrichum acutatum. However, a new species, C. abscissum, was described in 2015 as the causative agent of PFD in Brazil. The species description used a small number of strains and the distribution of the pathogen remains unclear. The proportion of PFD caused by this species is also unclear, because both C. abscissum and C. gloeosporioides are associated with the disease as well. By analyzing sequences of the GAPDH intron region, we identified 227 isolates of Colletotrichum associated with PFD in orchards of So Paulo state, 172 isolates were identified as C. abscissum and 55 as C. gloeosporioides. Morphological characters and multilocus sequencing confirmed species C. abscissum was the only species in the C. acutatum complex associated with PFD disease in Brazil. Although described as sensitive to benzimidazole-based fungicides, 20% of C. gloeosporioides isolates were found in regions with high use of this class of fungicide. Evaluated strains exhibited resistance to this fungicide in vitro. Because previously described primers differentiate between C. acutatum and C. gloeosporioides complexes, but not the particular species associated with PFD, we proposed and validated primers for a single multiplex PCR that specifically distinguished the C. abscissum and C. gloeosporioides sensu stricto.