Phylogeny and Fungal Community Structures of Helotiales Associated with Sclerotial Disease of Mulberry Fruits in China

Abstract

Mulberry fruit sclerotiniose is a prevalent disease caused by the fungal species Ciboria shiraiana, Ciboria carunculoides, and Scleromitrula shiraiana of the order Helotiales, and severely affects the production of mulberry. However, these species have only been identified using morphological and rDNA-ITS sequence analyses, and their genetic variation is unclear. To address this, morphological and two-locus phylogenetic (ITS and RPB2) analyses were conducted using culture-dependent and independent methods for 49 samples from 31 orchards across four provinces in China. Illumina MiSeq sequencing was used to assess the fungal communities collected from fruits varying in disease severity and color from an orchard in Wuhan. Conidial suspensions of C. shiraiana and C. carunculoides isolated from diseased fruits, disease fruits affected with hypertrophy and pellet sorosis sclerotiniose, and mycelia of Sclerotinia sclerotiorum were determined to be pathogenic to the mulberry cultivar ‘YSD10’. However, fruits inoculated with S. sclerotiorum mycelia exhibited non-typical disease symptoms, and mycelia and conidia obtained from C. carunculoides and S. shiraiana strains were not pathogenic. Maximum parsimony and Bayesian analyses using the sequences of the assessed loci indicated species variability with no evidence of geographic specialization. Metagenomic analysis revealed that the diversity of fungal communities was reduced with disease progression. Furthermore, within a single fruit, the presence of two Ciboria spp. was detected. These results provide novel insights into Ciboria spp., revealing the secondary infections caused by conidia in diseased fruit, genetic variations of the pathogens, and the occurrence of coinfection. This improved understanding of fungal pathogens will aid in developing effective disease control strategies.