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.

Reduced Sensitivity to Tebuconazole in Botryosphaeria dothidea Isolates Collected from Major Apple Production Areas of China.

China has the largest acreage and is the greatest producer of apples in the world. Apple ring rot (ARR) caused by Botryosphaeria dothidea is one of the major diseases affecting apple fruit production. Tebuconazole, a sterol demethylation-inhibiting (DMI) fungicide, has been increasingly applied to manage ARR in China. Resistance to tebuconazole in field populations of B. dothidea may be selected and accumulate to higher degrees over time. Establishment of tebuconazole sensitivity monitoring programs is critically important for resistance management and effective ARR control. However, the resistance level of B. dothidea to tebuconazole in China remains largely unknown. In this study, in vitro mycelial growth assays of B. dothidea in media amended with tebuconazole were conducted, and the sensitivity of B. dothidea to tebuconazole was determined with a set of 390 isolates collected from the major apple production provinces in China between 2006 and 2014. Results showed that the 50% effective concentration (EC50) value ranged from 0.011 to 0.918, 0.040 to 1.621, and 0.052 to 1.925 µg ml-1 with a median value of 0.194, 0.386, and 0.782 µg ml-1 in the isolates collected in 2006, 2010, and 2014, respectively. The frequency distribution of EC50 for tebuconazole was a nonnormal distribution (P < 0.05), suggesting that subpopulations with reduced sensitivity to tebuconazole had emerged in these B. dothidea isolates. The frequency distribution of the B. dothidea isolates collected in 2006 fit a unimodal curve and could be regarded as the baseline sensitivity to tebuconazole. The resistance levels increased over time with the average occurrence frequency of 43.3% and resistance index of 0.38. Positive cross-resistance was observed between tebuconazole and metconazole, which is another DMI fungicide, but multiple resistance was not detected between tebuconazole and non-DMI fungicides. Our results demonstrated that regular long-term resistance monitoring combining with prudent fungicide use should be implemented to prolong the lifespan of tebuconazole in management of ARR in apples.

Population genetic diversity in Chinese pomegranate (Punica granatum L.) cultivars revealed by fluorescent-AFLP markers.

Eighty-five pomegranate (Punica granatum L.) cultivars from six geographical populations located at Shandong, Anhui, Shaanxi, Henan, Yunnan, and Xinjiang Provinces were studied for its population genetic diversity by means of fluorescent-AFLP markers. The results indicated that 135-185 polymorphic loci were amplified by eight pairs of primers at species level. An average of 158.25 polymorphic loci was amplified for each primer combination. The polymorphism percentage ranged from 62.5% to 86.11%, and the average polymorphism percentage was 73.26%. This indicated that there was plentiful genetic diversity in pomegranate cultivars. The genetic diversity at the species level was higher than that at the population level. The order of the genetic diversity was Henan population > Xinjiang population > Shaanxi population > Anhui population > Shandong population > Yunnan population. Variance analysis showed that there was significant difference between populations in genetic diversity. The genetic differentiation coefficient between populations (G(ST)) was 0.2018, which indicated that gene differentiation was mainly within the population, and between populations, it accounted for 20.18% of the total variation. Gene flow (Nm) between the populations measured was 1.9027 based on the genetic differentiation coefficient between populations, indicating that there was mild gene flow between populations. The UPGMA cluster analysis showed that most accessions from the same population were clustered together, but there was partly gene exchange. All genetic parameters indicated that there was plentiful genetic diversity in pomegranate cultivars in China, of which Henan population was significantly higher than the other populations, and it had wide application foreground in pomegranate breeding in China.