Detection of the sensitivity of Colletotrichum gloeosporioides to three fungicides

Abstract：【Objective】Colletotrichum gloeosporioides (anamorph of Glomerella cingulata) , a ubiquitous fungal pathogen, is the agent causing Glomerella leaf spot (GLS) on apples. Under favorable conditions, GLS can result in seventy-five percent defoliation by harvest, weakening trees and reducing yield.In recent years, an epidemic of GLSA broke out in most apple planting areas in China, because of an indefinite occurrence period and resulitn in a severe impairment. The disease is initiated when conidia attaches to the plants surfaces via wind or rain splash dispersal, where trhe conidia germinates and differentiates into a specialized infection structure called appressorium. After invasion, C. gloeosporioides grows biotrophically, proliferates into neighboring cells, turns into a necrotrophic development and eventually results in lesions on the plants. Planting a resistance cultivar is considered to be a key strategy and one of the most efficient methods of controlling plant diseases. Many quality cultivars derived from the“Delicious”group, including'Gala''Gold Delicious', were widely planted. Some biocontrol products are applied to plants for protection and achieve good benefits of social and economic, but the commercial biocontrol product showed no effect on GLS in leaves and fruits. Therefore, this disease is primarily managed with chemical controls, such as benzimidazole fungicides, sterol demethylation in-hibitors (DMI) and imidazole. The severity of GLS can be reduced in the orchard by applying some fungicides beginning about 6 weeks after petal fall and continuing every 2 weeks until 2 to 3 weeks before harvest. Combinations of chelated zinc materials and dithiocarbamate fungicides improve control. However, while frequent applications are effective, they also increase production costs and can have damaging effects on the environment and human health. Moreover, plant pathogens can become resistant to fungicides and it can then fail as a control. For investigating the sensitivity of C. gloeosporioides to benzimidazole, DMI and imidazole, diseased leaves were collected from different apple gardens, and the obtained pathogen isolates were tested to see if they were resistance to benzimidazole, DMI and imidazole. This study could provide a theoretical basis for effective scientific controlling of GLSA.【Methods】One hundred and seventeen C. gloeosporioides strains were isolated from their diseased leaves, which were collected from different apple cultivars including'Gala''Golden Delicious' 'Jonagold''Jinxiuhong''Jinhong'and'K12'in different apple growing areas, such as Anyang, Henan; Zhengzhou, Henan; Zhaoyuan, Shandong; Qufu, Shandong; Yixian, Hebei; Xianyang, Shanxi and Dangshan, Anhui.The obtained isolates were separated using the single-spore method. The cultural characteristics of all the different isolates were studied on 6 cm diameter PLA plates. The morphology of the colony and conidia were studied after the plates were incubated for 10 days at 25 ℃ under a light microscope. For infection assays, a droplet (2 μL) of freshly harvested conidial suspensions was placed onto the obverse of the apple leaves ('Gold Delicious') , while 2 μL conidial suspensions were also inoculated into the wounded leaves by stabbing with a toothpick. Inoculated leaves were placed on plates containing water agar (12%) . Disease lesions were examined at 4 days postinoculation. Each test was repeated three times with three repetitions for each time. In all, we obtained one hundred and seventeen C. gloeosporioides strains from five hundred and thirty-five isolates tested. To evaluate the resistance of C. gloeosporioides against thiophanate-methyl, prochloraz and tebuconazole, we tested the pathogen growth by using distinguishing dosages and the hyphae growth inhibition method. C. gloeosporioides strains were cultured on the PDA plates for seven days in the dark, then transferred into fresh PDA plates with different dosages, and the diameter of the colony was investigated on the seventh day. The effective inhibiting concentration (EC50) and resistance frequency were calculated by using the DPS software. For investigating the mechanism of C. gloeosporioides resistance against thiophanate-methyl, β-tubulin gene sequences of 28 strains were amplified and sequenced. The fungal genomic DNA of the twenty-eight strains was isolated from the fungal vegetative hyphae using the cetyltrimethylammonium bromide (CTAB) procedure combined with RNase A and proteinase K treatment. PCR was conducted with pfu DNA polymerase, and PCR products were purified and sequenced by a bio-company. The partial amino acid sequences of β-tubulin proteins were aligned and analyzed by using the Bioedit software.【Results】The resistance frequency of the 117 isolates to thiophanate-methyl was 100%, which can be attributed to high level resistance. The β-tubulin gene sequence alignment of 27 of the selected 28 isolates indicated that GAA at codon 198 mutated into GCA, which resulted in a substitution of glutamic acid for alanine at codon 198. In addition, phenylalanine acid of the isolate Dj1-1-6 mutated into tyrosine acid at codon 200. The effective inhibiting concentrations (EC50) of hyphen radial growth on tebuconazole ranged from 0 to 0.843 0 mg·L-1 with a mean value of 0.155 5 mg · L-1. The sensitive assay showed that 75.21% of the 117 isolates had a low resistance to tebuconazole, and the hyphen inhibition rate was 92.72% at 5 mg·L-1. All 117 isolates were highly sensitive to prochloraz with a mean value of 0.011 9 mg·L-1, and the average inhibition rate of the colony growth was 95.02% at 0.5 mg·L-1.【Conclution】This study shows that C. gloeosporioides has a high resistance to thiophanate-methyl, a low resistance to tebuconazole and a high sensitivity to prochloraz.