Fungal diseases are posing tremendous threats to global economy and food safety. Among them, Valsa canker, caused by fungi of Valsa and their Cytospora anamorphs, has been a serious threat to fruit and forest trees and is one of the most destructive diseases of apple in East Asia, particularly. Accurate and robust delimitation of pathogen species is not only essential for the development of effective disease control programs, but also will advance our understanding of the emergence of plant diseases. However, species delimitation is especially difficult in Valsa because of the high variability of morphological traits and in many cases the lack of the teleomorph. In this study, we delimitated species boundary for pathogens causing apple Valsa canker with a multifaceted approach. Based on three independent loci, the internal transcribed spacer (ITS), β-tubulin (Btu), and translation elongation factor-1 alpha (EF1α), we inferred gene trees with both maximum likelihood and Bayesian methods, estimated species tree with Bayesian multispecies coalescent approaches, and validated species tree with Bayesian species delimitation. Through divergence time estimation and ancestral host reconstruction, we tested the possible underlying mechanisms for fungal speciation and host-range change. Our results proved that two varieties of the former morphological species V. mali represented two distinct species, V. mali and V. pyri, which diverged about 5 million years ago, much later than the divergence of their preferred hosts, excluding a scenario of fungi–host co-speciation. The marked different thermal preferences and contrasting pathogenicity in cross-inoculation suggest ecological divergences between the two species. Apple was the most likely ancestral host for both V. mali and V. pyri. Host-range expansion led to the occurrence of V. pyri on both pear and apple. Our results also represent an example in which ITS data might underestimate species diversity.

A nested polymerase chain reaction (PCR) assay for detecting Valsa mali var. mali, the causal agent of apple tree Valsa canker, was developed. One pair of genus-specific primers was designed based on the ribosomal DNA internal transcribed spacer conservative sequence of the Valsa genus and one pair of species-specific primers was designed based on the specific sequence of V. mali var. mali. The specificity of the genus-specific and species-specific primers was evaluated against 10 V. mali var. mali isolates, 10 V. mali var. pyri isolates, 4 isolates from closely related Valsa spp., and 8 isolates from fungal species that are commonly isolated from naturally infected apple bark tissue. A distinct band of 348 bp in length was detected in all V. mali var. mali isolates but not in other tested species and the V. mali var. pyri variety. The sensitivity of this assay was evaluated by serial dilutions of DNA extracted from V. mali var. mali pure cultures and apple bark tissues with or without visible symptoms. The results showed that the assay was able to detect as little as 100 fg of DNA in mycelial samples and apple bark tissues with visible symptoms, whereas the lowest detectable concentration was 10 pg of DNA in symptomless apple bark tissues. The efficiency of the nested PCR assay was compared with that of fungal isolation assays. All symptomless and symptomatic samples from which the pathogen was successfully isolated yielded a PCR product of the expected size. The detection rate of nested PCR for symptomless samples was 64.7%, which was much higher than the detection rate of 20.6% by fungal isolation. The PCR analysis of different symptomless tissues showed that the incidence of V. mali var. mali was different in different tissues of apple trees. The average incidence of V. mali var. mali was 89% in terminal buds, 71% in internodes, and 48% in bud scale scars. Moreover, the incidence of V. mali var. mali in nonsymptomatic tissues was higher in orchards where more trees were infected. Taken together, the assay developed in this study can be used for rapid and reliable detection of V. mali var. mali in tissues of apple trees with or without symptoms and also for monitoring the presence of the pathogen at an early stage of disease development.

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