Apple bitter rot caused by Colletotrichum species is a growing problem worldwide. Colletotrichum spp. are economically important but taxonomically un-resolved. Identification of Colletotrichum spp. is critical due to potential species-level differences in pathogenicity-related characteristics. A 400-isolate collection from New York apple orchards were morphologically assorted to two groups, C. acutatum species complex (CASC) and C. gloeosporioides species complex (CGSC). A sub-sample of 44 representative isolates, spanning the geographical distribution and apple varieties, were assigned to species based on multi-locus phylogenetic analyses of nrITS, GAPDH and TUB2 for CASC, and ITS, GAPDH, CAL, ACT, TUB2, APN2, ApMat and GS genes for CGSC. The dominant species was C. fioriniae, followed by C. chrysophilum and a novel species, C. noveboracense, described in this study. This study represents the first report of C. chrysophilum and C. noveboracense as pathogens of apple. We assessed the enzyme activity and fungicide sensitivity for isolates identified in New York. All isolates showed amylolytic, cellulolytic and lipolytic, but not proteolytic activity. C. chrysophilum showed the highest cellulase and the lowest lipase activity, while C. noveboracense had the highest amylase activity. Fungicide assays showed that C. fioriniae was sensitive to benzovindiflupyr and thiabendazole, while C. chrysophilum and C. noveboracense were sensitive to fludioxonil, pyraclostrobin and difenoconazole. All species were pathogenic on apple fruit with varying lesion sizes. Our findings of differing pathogenicity-related characteristics among the three species demonstrate the importance of accurate species identification for any downstream investigations of Colletotrichum spp. in major apple growing regions.

Managed colonies of European honey bees (Apis mellifera) are under threat from Varroa destructor mite infestation and infection with viruses vectored by mites. In particular, deformed wing virus (DWV) is a common viral pathogen infecting honey bees worldwide that has been shown to induce behavioral changes including precocious foraging and reduced associative learning. We investigated how DWV infection of bees affects the transcriptomic response of the brain. The transcriptomes of individual brains were analyzed using RNA-Seq after experimental infection of newly emerged adult bees with DWV. Two analytical methods were used to identify differentially expressed genes from the ~15,000 genes in the Apis mellifera genome. The 269 genes that had increased expression in DWV infected brains included genes involved in innate immunity such as antimicrobial peptides (AMPs), Ago2, and Dicer. Single bee brain NMR metabolomics methodology was developed for this work and indicates that proline is strongly elevated in DWV infected brains, consistent with the increased presence of the AMPs abaecin and apidaecin. The 1361 genes with reduced expression levels includes genes involved in cellular communication including G-protein coupled, tyrosine kinase, and ion-channel regulated signaling pathways. The number and function of the downregulated genes suggest that DWV has a major impact on neuron signaling that could explain DWV related behavioral changes.

Colletotrichum fioriniae of the C. acutatum species complex is an important hemi-biotrophic pathogen of vegetables and fruits in temperate regions world-wide. In apple, it is one of the primary species responsible for bitter rot disease. Understanding the disease cycle is complicated because many broadleaf plants can be hosts of C. fioriniae. By detecting and quantifying rain-splashed C. acutatum species complex conidia in over 500 samples from heavily bitter-rot-infected apple orchards and nearby forested woodlots over two summers we show that conidial quantities were higher in the woodlots than in the orchards. Testing of over 1,000 surface disinfected leaves of apple and 24 different forest plant species showed that overall C. fioriniae was an abundant leaf endophyte, with high variation in leaf colonization area. Endophytic isolates from leaves were pathogenic on apples, and multi-locus sequence analysis showed 100% identity between most isolates from leaves and diseased fruits. Apple leaves endophytically infected with C. fioriniae were present in a conventionally managed orchard and abundant in an untreated orchard. These lines of evidence, in the context of previously published research, lead us to hypothesize that the main ecological role of C. fioriniae is that of a leaf endophyte, which we present as a generalized C. fioriniae infection cycle that provides an updated framework for its integrated management in agricultural systems.

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Apple growers in the Mid-Atlantic region of the United States have reported increased losses to bitter rot of apple. We tested the hypothesis that this increase is because the Colletotrichum population has developed resistance to commonly used single-mode-of-action (single-MoA) fungicides. We screened 220 Colletotrichum isolates obtained from 38 apple orchards in the Mid-Atlantic region for resistance to 11 fungicides in FRAC (Fungicide Resistance Action Committee) groups 1, 7, 9, 11, 12, and 29. Eleven (5%) of these isolates were resistant to FRAC group 1 with confirmed beta-tubulin E198A mutations, and two (< 1%) were also resistant to FRAC group 11 with confirmed cytochrome-b G143A mutations. Such low frequencies of resistant isolates indicate that fungicide resistance is unlikely to be the cause of any regional increase in bitter rot. A subsample of isolates was subsequently tested in vitro for sensitivity to every single-MoA fungicide registered for apple in the Mid-Atlantic US (22 fungicides; FRAC groups 1, 3, 7, 9, 11, 12, and 29), and thirteen fungicides were tested in field trials. These fungicides varied widely in efficacy both within and between FRAC groups. Comparisons of results from our in vitro tests with results from our field trials and other field trials conducted across the Eastern US suggested that EC₂₅ values (concentrations that reduce growth by 25%) are better predictors of fungicide efficacy in normal field conditions than EC₅₀ values. We present these results as a guideline for choosing single-MoA fungicides for bitter rot control in the Mid-Atlantic US.