Genetic Diversity of Populations of Monilinia fructicola (Fungi, Ascomycota, Helotiales) from China JIN-YAN FAN, a LI-YUN GUO, a JIAN-PING XU, b YONG LUO a,c and THEMIS J. MICHAILIDES c a Department of Plant Pathology, China Agricultural University, Beijing 100193, China, and b Department of Biology, McMaster University, Hamilton, ON, Canada L8S 4K1, and c Department of Plant Pathology, University of California-Davis, Kearney Agricultural Center, Parlier, California 93648, USA ABSTRACT. The genetic variation among 128 isolates of Monilinia fructicola (Fungi, Ascomycota, Helotiales) from China was an- alyzed using Inter-Simple Sequence Repeat (ISSR) markers and compared with those of samples from California, USA and New Zealand. A total of 72 reproducible DNA fragments were scored, of which 87.5% (63/72) were polymorphic. The Nei’s gene diversity and Shan- non’s diversity indices of three Chinese regional populations were very similar to that from California. However, several differences were observed among geographic populations of M. fructicola from both within China and between China and California. The analysis of molecular variance (AMOVA) of isolates from different geographic locations suggested that most of the observed genetic variation was found within populations. Results of this study are inconsistent with the hypothesis that the Chinese populations of M. fructicola were derived from a single or few recent migrants from other countries. Instead, our results suggest that M. fructicola has been in China long before its first official recording in 2003. Key Words. Brown rot, genetic variation, geographic distribution, ISSR. B ROWN rot is an important disease of stone and pome fruits worldwide. Monilinia fructicola (G. Winter) Honey. 1928, is one of the major causal pathogens of brown rot of stone fruits. This fungus can infect various parts of stone fruit trees and cause blossom blight, twig blight, and brown rot of green and mature fruits (Ogawa et al. 1995), resulting in severe losses during the growth season and in post-harvest storage. The fungal pathogen M. fructicola was first discovered in 1883 in eastern United States (Batra 1991), and has since been found mainly in America and Australia (Byrde and Willetts 1977). Because the distribution of this fungus in Europe was limited to only a few local areas, it has been listed as an A2 quarantine pathogen by the European Union (EPPO 2006). Peach and nectarine are considered important stone fruit crops and have been cultivated in China, where they originated, for 43,000 yr (Long 2000; Qu and Sun 2000). Since the late 1980s, both the production area and the total yield of these fruits have increased greatly in China. Unfortunately, brown rot caused by Monilinia spp. has been found also widely distributed in all the major stone fruit production areas in China (Zhu, Guo, and Chen 2008a). Xiang (1957) traced the earliest report of brown rot on stone fruits in China to the 1920s. By the 1950s, brown rot was found in many areas across China on a variety of stone fruits, in- cluding peach, plum, apricot, and mume (Dai, Xiang, and Zheng 1958; Xiang 1957). However, among the four pathogenic Mon- ilinia species (i.e. M. fructicola, Monilinia laxa, Monilinia fructi- gena, and Monilia polystroma) that have been reported to cause brown rot on pome and stone fruit (Byrde and Willetts 1977; van Leeuwen et al. 2002), only M. laxa and M. fructigena were doc- umented in China before 2003 (Wang et al. 1998; Xiang 1957; Zhu et al. 2005; Zhuang 1998). Monilinia fructicola was recently reported as a new pathogen in China (Zhu et al. 2005) and is currently listed as a quarantine pest of China (Bulletin of China Agricultural Ministry 2007). How- ever, during surveys carried out from 2003 to 2008, M. fructicola was found widely distributed in many areas in China, including Beijing, Shandong, Hebei, Zhejing, and Liaoning provinces. In- deed, it was the major pathogen causing brown rot on a diversity of stone fruits (Fan et al. 2007; L. Guo and X. Q. Zhu, unpubl. data; Zhong et al. 2008; Zhu, Guo, and Chen 2008b). In contrast, M. laxa, documented previously as the species widely distributed in China, was seldom found on the various hosts in these geo- graphic areas. Recently, up to 100% incidence of fruit rot has been reported in fruit packinghouses in Beijing (Chen et al. 2003). In addition, iso- lates of M. fructicola were collected from Shandong province in 2004 and from various locations in Shandong and Beijing in 2005. Some of these isolates were found highly resistant to the thiop- hanate-methyl fungicide (Fan, Fang, and Guo 2009). This fungi- cide is routinely sprayed in stone fruit orchards. The wide occurrence of M. fructicola determined right after this species was first reported in China led to our suspicion that this pathogen might not be a newly introduced fungus, but might have existed in China for a long time. If a population was established from one or a few newly introduced strains of a species, the genetic diversity in the population should be much lower than that of its source pop- ulation or a population of comparable size but which has had a much longer colonization history. Thus, the objectives of this study were to determine the genetic diversity of M. fructicola isolates from areas in China, using the PCR-based Inter-Simple Sequence Repeat (ISSR) technique (Zietkiewicz, Rafalski, and Labuda 1994), to compare them with isolates from California, USA, and New Zealand, and test the hypothesis that M. fructicola might have been in China for a long time before it was first reported in 2003. MATERIALS AND METHODS Isolates. A total of 164 isolates of M. fructicola were analyzed in this study. Among them, 128 isolates were from peach (n 5 96), nectarine (n 5 5), plum (n 5 19), pear (n 5 7), and ornamental peach (n 5 1). These isolates were collected from three provinces and the City of Beijing in China including Beijing (n 5 77), Hebei (n 5 5), Shandong (n 5 43), and Zhejiang (n 5 3). In addition, 28 isolates from the United States (California, n 5 27; North Caro- lina, n 5 1), 7 from New Zealand, and 1 from France were in- cluded for comparison (Table 1). All isolates were identified as M. fructicola based on both morphological characteristics (Lane 2002) and molecular features developed by Ioos and Frey (2000). In addition, one isolate of M. laxa was included as a reference. Mono-conidial cultures of each isolate were maintained on potato dextrose agar slants and stored at 4 1C. DNA extraction and quantification. The mycelia of each iso- late were obtained by culturing the fungus in pea broth for 4 d in the dark at 21 1C. The mycelia were vacuum-filtered through Whatman No. 1 filter paper. About 1 g of fresh mycelia was Corresponding Author: L. Y. Guo, Department of Plant Pathology, China Agricultural University, Beijing 100193, China—Telephone number: 186 10 6273 3027; FAX number: 186 10 6273 3027; e-mail: ppguo@cau.edu.cn 206 J. Eukaryot. Microbiol., 57(2), 2010 pp. 206–212 r 2010 The Author(s) Journal compilation r 2010 by the International Society of Protistologists DOI: 10.1111/j.1550-7408.2009.00467.x Published by the International Society of Protistologists Eukaryotic Microbiology The Journal of