Molecular and morphological characteristics of Apiosporina morbosa, the causal agent of black knot in Prunus spp.

Abstract

Thirty isolates of Apiosporina morbosa, which were isolated from wild chokecherry (Prunus virginiana), an ornamental cultivar of chokecherry with purple foliage (P. virginiana ‘Shubert Select‘), and domestic plum (Prunus domestica), were compared for morphological and molecular characteristics, using culture techniques and data from internal transcribed spacers (ITS) I and II, restriction fragment length polymorphisms (RFLP), and sequence-related amplified polymorphism (SRAP) analyses. Strains of A. morbosa grew as olive-green colonies that changed in about 2 weeks to a dark-brown colour. Cultures produced single- or two-celled and cylindrical conidia with tapering ends. Conidia had a mean size of 4.51 μm × 10.97 μm. The overall colony and conidial morphology in cultures of the A. morbosa isolates was generally similar to that of an isolate of Cladosporium herbarum. Pseudothecia were globose and produced clavate asci with unequally two-celled and club-shaped ascospores having mean sizes of 6.68–7.37 μm × 16.42–16.78 μm. RFLP markers produced by endonucleases DdeI, Bst98I, and VspI in the ITS region differentiated the pathogen isolates from other gall-associated fungi, but no difference was observed among the A. morbosa isolates, using these markers. The sequences of the ITS region had >99% similarity among 30 isolates of A. morbosa, and a total of eight unique genotypes were found, based on ITS sequence alignment. The most common genotype was manifested in 21 of 30 A. morbosa isolates, based on the ITS sequences. Three A. morbosa isolates had an identical sequence to that of an isolate of C. herbarum. A comparison with an isolate of Cladosporium cladosporioides showed that A. morbosa differed from C. cladosporioides by 3%, based on ITS sequences. SRAP analysis detected 18 unique genotypes and a high genetic diversity (H = 0.256) among 25 isolates of A. morbosa. The pathogen population isolated from wild chokecherry produced more unique genotypes and higher genetic diversity (H = 0.283) than the population from ‘Shubert Select’ (H = 0.165). However, an exact test indicated no differentiation between these two populations (P = 0.334) with a gene flow rate of 4.28. Most of the isolates from ‘Shubert Select’ were clustered in a subgroup in a phylogenetic dendrogram. Phylogenetic analysis of ITS sequences of the A. morbosa isolates in our study and related taxa extracted from GenBank revealed that A. morbosa had the closest genetic distance with Cladosporium spp.