Abstract
In recent years, a devastating fungal disease characterized by small black spots (<1 mm) on sandy pear (Pyrus pyrifolia Nakai) leaves has occurred in sudden outbreaks in southern China, resulting in severe defoliation and a loss of fruit quality and yield. To identify the etiology of the disease, 147 fungal isolates from sandy pear leaves showing typical black spots were collected from 30 orchards; 10 fungal species were included among the isolates, and the prevailing 124 Colletotrichum isolates were subjected to morphological and molecular characterization. Based on differences in colony morphology, the isolates were separated into five groups (I to V); the groups were specifically separated according to a combination of colony color, pigment, sporulation, perfect stage, and spore size. In addition to their morphological features, the five groups were assessed for their molecular and taxon statuses based on ITS sequences, multilocus regions (ITS, ACT, TUB, CHS-1, GAPDH), and the Apn2/MAT locus, which provided molecular proof for the identification of these isolates as Colletotrichum fructicola Prihastuti. The result also indicated different resolution of the three loci in delimiting the species. Koch’s postulates were fulfilled by inoculating conidium suspensions of the representative isolates on attached and detached leaves of sandy pear cvs. Cuiguan, Xueqing and Huali No. 2 with analogous conditions (under saturated humidity and with tiny wounds) to those that appeared in the field, and similar symptoms were elicited by the isolates, suggesting that C. fructicola was causing the disease. To our knowledge, this is the first report of C. fructicola causing leaf black spot of pear. The etiological clarification and characterization of the C. fructicola isolates provide useful information to aid in the understanding of leaf black spot disease in pears and for designing management strategies to control this economically significant disease.
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Acknowledgments
This study was financially supported by the earmarked fund for Pear Modern Agro-industry Technology Research System (CARS-29-10) and the Chinese Ministry of Agriculture, Industry Technology Research Project (200903004–05). The authors would like to thank Professor Yangdou Wei University of Saskatchewan, Canada, and Chaoxi Luo, Huazhong Agricultural University, China, for critical revisions of the manuscript.
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The authors declare no conflict of interest. This research work does not include any animal studies.
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Fig. S1
Phylogenetic analysis of Colletotrichum fructicola isolates and other species belonging to the C. gloeosporioides species complex based on the ITS sequences using the MEGA 5.2.2 software with 1,000 bootstrap replicates and other default parameters. Type strains are marked with symbol “*”. A and B, Phylogenetic tree constructed using the UPGMA and ML methods, respectively. The isolates of C. boninense and C. hippeastri are designated as outgroups. Bootstrap support values of more than 60 % are shown at the nodes. The isolates obtained in this study are indicated by a triangle (▲). Of these isolates, the 117 isolates showing identical ITS sequences are represented by FJ-1 and are indicated as “FJ-1 and other isolates”. (JPEG 642 kb)
Fig. S2
Phylogenetic tree based on the ITS sequences of the 25 Colletotrichum fructicola isolates obtained in this study and those of other reported isolates. The isolates of C. boninense, C. siamense, and C. gloeosporioides are shown as outgroups. Bootstrap support values greater than 60 % are shown at the nodes. The isolates obtained in this study are indicated by a triangle (▲). Out of these isolates, the 20 isolates showing identical ITS sequences are represented by FJ-1 and are indicated as “FJ-1 and other isolates.” The accession number, host and original country of each isolate are successively presented in the phylogenetic position of each C. fructicola isolate. An isolate obtained from Pyrus bretschneideri fruit in China is indicated by a blank triangle (Δ). (JPEG 655 kb)
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Zhang, P.F., Zhai, L.F., Zhang, X.K. et al. Characterization of Colletotrichum fructicola, a new causal agent of leaf black spot disease of sandy pear (Pyrus pyrifolia). Eur J Plant Pathol 143, 651–662 (2015). https://doi.org/10.1007/s10658-015-0715-7
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DOI: https://doi.org/10.1007/s10658-015-0715-7