Quantification of allele E198A in beta-tubulin conferring benzimidazole resistance in Monilinia fructicola using real-time PCR

BACKGROUND: Thiophanate-methyl, a member of the benzimidazole class of fungicides, is used in California to control brown rot of stone fruit caused by Monilinia fructicola (G. Wint.) Honey. The goal of this study was to develop a real-time polymerase chain reaction (PCR) assay as an efficient method to quantify the E198A allele of beta-tubulin that confers benzimidazole resistance. RESULTS: Using the real-time PCR assay, the frequency of allele E198A (FEA) in a population was determined from the quantities of DNA amplified with the E198A allele-specific primer pair HRF/HRR and the M. fructicola-specific primer pair MfF6/MfR6. The average proportions of highly resistant isolates determined with the conventional fungicide sensitivity method were within the range of average FEA values determined with the real-time PCR assay. We also determined the FEAs of M. fructicola populations sampled from 21 stone fruit orchards in California. Only one orchard showed a high FEA over 0.20, seven orchards had values between 0.01 and 0.1, and 13 orchards had values less than 0.01. CONCLUSION: The real-time PCR assay developed in this study provides a potentially useful tool to efficiently quantify benzimidazole resistance for large M. fructicola populations.     

Phoma diseases: Epidemiology and control

Phoma is the most widely distributed and omnipresent genus of the order Pleosporales and the largest genus with some 3,000 taxa described so far. Of these, approximately 110 species are pathogenic and occupy varied ecological niches. The genus Phoma is polyphyletic and is not really delimited, with unclear species boundaries that make it a taxonomically controversial genus. Fungi belonging to Phoma commonly occur on crop plants that are economically important, where they cause devastating plant diseases. Pathogenic members of Phoma sensu lato species attack crop plants with symptoms ranging from leaf blight to root rot, and even wilting of the plant. In infected crop residues and field stubbles, the pathogen produces abundant pycnidia and pseudothecia that serve as the source of primary inoculum, whilst repeated crops of conidia produced inside pycnidia are the main source of secondary infection during the same growing season. After successful infection, the pathogen produces various phytotoxins that alter photosynthetic efficiency and actin cytoskeleton-based functions, and cause electrolyte leakage from cells. Controlling the diseases caused by members of Phoma sensu lato is challenging and efforts have been made to identify resistant varieties that can be used in various plant breeding programmes. Studies have also been conducted to devise cultural and biological control measures as well as to evaluate the efficacy of fungicides against members of Phoma sensu lato. In this review we aim to discuss the disease epidemiology and control measures that can be practised to protect crops from Phoma diseases.     

Control of Hymenoscyphus fraxineus,the causal agent of ash dieback,using composting

Viability of Hymenoscyphus fraxineus inocula following temperature treatments for different exposure times was examined in vitro and in aerated flask‐ and large‐scale composting tests using green waste. After an exposure for up to 10 days at 20°C, 97.3% of H. fraxineus mycelium and pseudosclerotia plate cultures remained viable. No viability was detected following a 3‐day exposure to 40°C or a 1‐day exposure to 45°C although pseudosclerotia were more tolerant than mycelium to an exposure to 35°C. Primordial apothecia of H. fraxineus emerged from 62%–100% of infected ash rachises collected from two infected sites and stored at 4°C for 0–5 months; exposure to compost for up to 10 days at 20°C did not affect this emergence. No emergence of H. fraxineus apothecia was observed from ash rachises that were exposed to compost at 45°C for 1 day or at 35°C or 40°C for 3 days in flasks or at 40°C for 1 day or at 30°C for 5 days in a large‐scale composting system. Based on a fitted model, estimates of the survival of H. fraxineus inoculum in infected ash rachises exposed to compost at 50°C for 1 day were 0.081% of that in the untreated H. fraxineus ash rachis inoculum. Increasing loss in viability of H. fraxineus inoculum in infected ash rachises during longer and warmer exposures to compost at 35°C–45°C corresponded with a reduced concentration of pathogen DNA detected in the rachises using real‐time PCR. However, exposure of rachises to compost at >53°C resulted in a smaller reduction in pathogen DNA detected than exposure to compost at lower temperatures, possibly due to the inhibition of enzymatic degradation of DNA at elevated temperatures.