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.

Management of fire blight is complicated by limitations on use of antibiotics in agriculture, antibiotic resistance development, and limited efficacy of alternative control agents. Even though successful in control, preventive antibiotic sprays also affect non-target bacteria, aiding the selection for resistance which could ultimately be transferred to the pathogen Erwinia amylovora. Trunk injection is a target-precise pesticide delivery method that utilizes tree xylem to distribute injected compounds. Trunk injection could decrease antibiotic usage in the open environment and increase the effectiveness of compounds in fire blight control. In field experiments, after 1-2 apple tree injections of either streptomycin, potassium phosphites (PH) or acibenzolar-S-methyl (ASM), significant reduction of blossom and shoot blight symptoms was observed compared to water- or non-injected control trees. Overall disease suppression with streptomycin was lower than typically observed following spray applications to flowers. Trunk injection of oxytetracycline resulted in excellent control of shoot blight severity, suggesting that injection is a superior delivery method for this antibiotic. Injection of both ASM and PH resulted in the significant induction of PR-1, PR-2 and PR-8 protein genes in apple leaves indicating induction of systemic acquired resistance (SAR) under field conditions. The time separating SAR induction and fire blight symptom suppression indicated that various defensive compounds within the SAR response were synthesized and accumulated in the canopy. ASM and PH suppressed fire blight even after cessation of induced gene expression. With the development of injectable formulations and optimization of doses and injection schedules, the injection of protective compounds could serve as an effective option for fire blight control.

Fire blight is a devastating disease of apple and pear caused by the bacterium Erwinia amylovora. One of its main symptoms is canker formation on perennial tissues which may lead to the death of limbs and/or the entire tree. E. amylovora overwinters in cankers which play an important role in initiating fire blight epidemics. However, knowledge of pathogen biology in cankers is scarce, in part due to limitations of classical microbiology methods and the inability of most molecular techniques to distinguish live from dead cells. In this work, a viability digital PCR (v-dPCR) protocol using propidium monoazide (PMA) was developed, allowing for the first time the selective detection and absolute quantification of E. amylovora live cells in apple and pear cankers collected in two time periods. Some key factors affecting the v-dPCR performance were the maceration buffer composition, the target DNA amplicon length, the thermal cycle number and the use of sodium dodecyl sulfate or PMA enhancer for Gram-negative bacteria to improve the effect of PMA. In the future, this methodology could shed light on E. amylovora population dynamics in cankers and provide clues on the effect of management practices, host cultivar, host water/nutritional status, etc., on bacterial survival.