Effects of Temperature on Spore Germination and Mycelial Growth of Calosphaeria pulchella, Cytospora sorbicola, and Eutypa lata Isolates Associated with Sweet Cherry Canker Diseases.

Although fungal canker diseases constitute a limiting factor to orchard productivity and longevity, little is known about the effects of temperature on spore germination and mycelial growth of the fungal causal agents. Accordingly, the germination of spores and colony growth of Calosphaeria pulchella, Cytospora sorbicola, and Eutypa lata were evaluated after incubation on 2% water agar and 4% potato dextrose agar, respectively, at 5, 10, 15, 20, 25, 30, 35, and 40°C. Temperature optima for spore germination and mycelial growth were derived from nonlinear models fitted to germination rates and colony diameter data. The optimal temperatures for spore germination of Cal. pulchella were 28.5°C for ascospores and 29.2°C for conidia. The optimal temperatures for Cyt. sorbicola conidia and E. lata ascospore germination were 25.8 and 23.1°C, respectively. The germination of ascospores and conidia of Cal. pulchella at temperatures below 15°C required an incubation time of at least 72 h. Ascospores of E. lata and conidia of Cyt. sorbicola germinated at 10°C after 36 h. The optimal temperature for colony growth of Cal. pulchella was 24.6°C, whereas it was 21.7°C for both Cyt. sorbicola and E. lata. Our study indicates that temperature requirements for basic biological functions are higher for Cal. pulchella than for Cyt. sorbicola and E. lata. The overall higher temperatures of California relative to other cherry-producing regions in the United States or worldwide could explain the prevalence of Calosphaeria canker in the state. Conversely, Cyt. sorbicola and E. lata appear better adapted to cooler temperatures.

Development of PCR-Based Assays for Rapid and Reliable Detection and Identification of Canker-Causing Pathogens from Symptomatic Almond Trees.

Trunk and scaffold canker diseases (TSCDs) of almond cause significant yield and tree losses and reduce the lifespan of orchards. In California, several pathogens cause TSCDs, including Botryosphaeriaceae, Ceratocystis destructans, Eutypa lata, Collophorina hispanica, Pallidophorina paarla, Cytospora, Diaporthe, and Phytophthora spp. Field diagnosis of TSCDs is challenging because symptom delineation among the diseases is not clear. Accurate diagnosis of the causal species requires detailed examination of symptoms and subsequent isolation on medium and identification using morphological criteria and subsequent confirmation using molecular tools. The process is time-consuming and difficult, particularly as morphological characteristics are variable and overlap among species. To facilitate diagnosis of TSCD, we developed PCR assays using 23 species-specific primers designed by exploiting sequence differences in the translation elongation factor, ß-tubulin, or internal transcribed spacer gene. Using genomic DNA from pure cultures of each fungal and oomycete species, each primer pair successfully amplified a single DNA fragment from the target pathogen but not from selected nontarget pathogens or common endophytes. Although 10-fold serial dilution of fungal DNA extracted from either pure cultures or infected wood samples detected as little as 0.1 pg of DNA sample, consistent detection required 10 ng of pathogen DNA from mycelial samples or from wood chips or drill shavings from artificially or naturally infected almond wood samples with visible symptoms. The new PCR assay represents an improved tool for diagnostic laboratories and will be critical to implement effective disease surveillance and control measures.