Pathogenicity differentiation of Colletotrichum fructicola causing precocious defoliation in pear in southern China

Abstract:【Objective】In recent years, precocious defoliation of pear, mainly caused by Colletotrichum fruticola in southern China has become serious and resulted in large losses of fruit production in these areas. The aim of this study is to determine the pathogenicity differentiation of C. fruticola and establish the laboratory methods determining virulence of this pathogen.【Methods】The strain PAFQ32 of C. fru- ticola, which has strong virulence, was isolated from diseased leaves of Pryus pyrifolia cv. Cuiguan in Fujian. Mycelial discs (5 mm in diameter) were taken from the colony margins of 5-d-old cultures on PDA medium, and fresh PDA discs were used as controls. Isolates were cultured on SNA (synthetic nu- trient-poor agar) medium for 3 days at 28 °C. Conidia were harvested and put into a 2.0 mL sterilized Eppendorf tube, and the conidial suspension was diluted with sterile water to a final concentration of 1× 106 conidia per mL. The strong pathogenic strain PAFQ32 of C. fructicola was inoculated from the twigs (P. pyrifolia‘Cuiguan’), leaves (P. pyrifolia‘Cuiguan’), and fruit (P. bretschneideri‘Huang- guan’) wounded with various methods. Inoculation of detached twigs was performed by using 1-year- old twigs (10.0 cm in length) with mycelial plugs on wounds and incubated at 25 °C in plastic contain-ers covered with plastic film. The lesion lengths were recorded at 9 dpi. Inoculation of detached mature pear fruit was performed by placing mycelium plugs or dropping an aliquot of 6.0 μL conidial suspen- sion (106 conidia per mL) on wounds. Fresh PDA discs (5 mm in diameter) and sterile water were used as the controls, respectively. The lesion lengths were recorded at 7 dpi. Inoculation of detached leaves was performed by placing mycelium plugs or dropping a 6.0 μL aliquot of conidial suspension on the left side of a leaf after wounding. The lesion lengths were recorded at 4 dpi. The results of each treat- ment were compared, and then chose a laboratory method to determine the pathogenicity of 111 strains of C. fruticola that were isolated from the leaves and fruits of P. pyrifolia and P. bretschneideri in 7 or- chards in Anhui, Fujian, Guangxi, Hubei, Jiangsu, Jiangxi, and Zhejiang. The pathogenicity of strains from different sources were observed and divided into different pathogenic types, and the relationship between the pathogenicity differentiation of the strains and their sources were analyzed【. Results】Labo- ratory determination of virulence showed that, on inoculated twigs, the incidence rate was 100% by scald holing and holing, 80% by girdling, 40% by puncturing with 10 needles, and 20% by puncturing with 3 needles after three days of inoculation. The average diameters of lesions treated with scald hol- ing were significantly bigger than the latter four wounding treatments, but it varied greatly. On inoculat- ed fruit, the average diameters of lesions produced by inoculating with mycelium plugs were larger than by inoculating with conidial suspensions under the same wound treatment, and there was no significant difference among the average diameters of lesions produced by inoculating mycelial plugs with differ- ent wounding treatments. On inoculated leaves, the average diameters of lesions produced by punctur- ing with 3 needles were significantly larger than with 1 needle on the right and the back side of the leaves. Under the same wound treatment, the expansion of the lesions produced by inoculating with my- celial plugs was faster than that by inoculating with conidial suspensions. The results showed that the method of inoculating with mycelial plugs on punctured pear leaves had good material uniformity and significant effect, and took less time, and thus was better than the other inoculation treatments. The pathogenicity of 111 strains were determined by this treatment. The results showed that there were significant differences in pathogenicity among C. fructicola strains from different provinces in southern China. According to cluster analysis, the strains could be divided into three types including 17 strains with strong pathogenicity (15.3%), 89 strains with middle pathogenicity (80.2%) and 5 strains with weak pathogenicity (4.5% ). There was a significant difference in the proportions of the pathogenic types of the strains from different areas. This difference was related to climate differences.【Conclusion】It was suggested that rapid laboratory determining the virulence of C. fruticola could be carried out by inoculating with mycelial plugs on leaves wounded by puncturing. There was significant pathogenicity differentiation in C. fructicola causing leaf early defoliation in pear in southern China, and the strains with middle pathogenicity were the dominant group.