Identification and Fungicide Screening of Fungal Species Associated with Walnut Anthracnose in Shaanxi and Liaoning Provinces, China.

Walnut is cultivated around the world for its precious woody nut and edible oil. Recently, walnut infected by Colletotrichum spp. resulted in a great yield and quality loss. In August and September 2014, walnut fruits with anthracnose were sampled from two commercial orchards in Shaanxi and Liaoning provinces, and five representative isolates were used in this study. To identify the pathogen properly, four genes per region (internal transcribed spacer, glyceraldehyde-3-phosphate dehydrogenase, actin, and chitin synthase) were sequenced and used in phylogenetic studies. Based on multilocus phylogenetic analysis, five isolates clustered with Colletotrichum fioriniae, including its ex-type, with 100% bootstrap support. The results of multilocus phylogenetic analyses, morphology, and pathogenicity confirmed that C. fioriniae was one of the walnut anthracnose pathogens in China. All 13 fungicides tested inhibited mycelial growth and spore germination. Flusilazole, fluazinam, prochloraz, and pyraclostrobin showed the strongest suppressive effects on the mycelial growth than the others, the average EC50 values ranged from 0.09 to 0.40 µg/ml, and there was not any significant difference (P < 0.05). Pyraclostrobin, thiram, and azoxystrobin were the most effective fungicides on spore germination (P < 0.05), and the EC50 values ranged from 0.01 to 0.44 µg/ml. Pyraclostrobin, azoxystrobin, fluazinam, flusilazole, mancozeb, thiram, and prochloraz exhibited a good control effect on walnut anthracnose caused by C. fioriniae, and preventive activities were greater than curative activities. Pyraclostrobin at 250 a.i. µg/ml and fluazinam at 500 a.i. µg/ml provided the highest preventive and curative efficacy, and the values ranged from 81.3 to 82.2% and from 72.9 to 73.6%, respectively. As a consequence, mancozeb and thiram could be used at the preinfection stage, and pyraclostrobin, azoxystrobin, flusilazole, fluazinam, and prochloraz could be used at the early stage for effective prevention and control of walnut anthracnose caused by C. fioriniae. The results will provide more significant instructions for controlling the disease effectively in northern China.

Baseline Sensitivity and Control Efficacy of Pyraclostrobin Against Botryosphaeria dothidea Isolates in China.

Botryosphaeria dothidea is an important fungal pathogen that causes apple ring rot, which can significantly reduce apple yield. Fungicide applications are the main control measure of apple ring rot worldwide. Pyraclostrobin is a quinone outside inhibitor (QoI) fungicide that has yet to be registered for control of B. dothidea in China. Baseline sensitivity of B. dothidea to pyraclostrobin (EC50 of mycelial growth inhibition) was assessed for 97 isolates collected in Shandong Province. The EC50 values ranged from 0.7010 to 7.1378 µg/ml with the mean value of 3.0870 µg/ml and displayed a unimodal frequency distribution. After cultured on fungicide-free PDA medium or on apples for multiple generations, the B. dothidea-resistant isolates (RST) remained resistant to pyraclostrobin, but exhibited similar virulence as the susceptible isolates (ST). Cross-resistance investigation revealed that pyraclostrobin was not cross-resistant to tebuconazole, flusilazole, carbendazim, and iprodione. Field evolution showed that pyraclostrobin at 200 and 250 g a.i./ha provided greater than 80% control efficacy against apple ring rot disease when applied as a therapeutic or preventive fungicide. The efficacy was similar to fungicides that have been registered for apple.