Identification, Distribution, Genetic Diversity And PCR Rapid Detection Of Botryosphaeria Spp. Causing Peach Tree Gummosis In China

Species in the genus Botryosphaeria are found on a wide range of monocotyledonous, dicotyledonous and gymnospermous plants as cosmopolitan endophytes, pathogens and saprophytes. Although well-known as pathogens causing canker, blight, dieback and gummosis disease in numerous woody perennial hosts, Botryosphaeria spp. have been overlooked as important causal agents of peach(Prunus persica L.) tree gummosis in China. Peach tree gummosis was thought to be associated with physiological disorder caused by pruning, extensive management or stresses. Disease symptoms are always associated with lenticels, wounds, and include gum formation on trunks, scaffold limbs, and braches, and necrotic lesions. The work presented in this thesis was initiated by the identification of Botryosphaeria spp. causing the disease in Hubei Province, which is one of the major peach producing areas in China, by analysis of conidial morphology, cultural characteristics, and nucleotide sequences of three genomic regions of the internal transcribed spacer region (ITS, ITS1-5.8S-ITS2), a partial sequence of β-tubulin gene, and the translation elongation factor1-α gene (EF1-α). And then, identification, pathogenicity and virulence of Botryosphaeria spp. in China, specific primer for PCR detection, and the fungal gummosis susceptibility of peach cultivars were also studied in this paper. The results were summarized as follows:1. Botryosphaeria spp. have been identified as causal agents of peach tree gummosis in Georgia, USA since1970s. To date, B. dothidea (Moug.) Ces.&De Not. has been reported as the only species associated with cankers on P. persica in China. Hubei Province is one of the most important peach producing areas in China. Fungal gummosis on trunks and branches has become a growing threat to the peach industry in this province and throughout Southern China. In order to determine the occurrence of gummosis and Botryosphaeria species in Hubei Province, a field survey was conducted of1,914trees in14peach orchards throughout the province. Gummosis was observed in1,899of1,914trees examined. A total of170samples of branches with gummosis were collected from orchards in Wuhan City, Xianning City, Xiaogan City, Yuan'an County (Yichang City), Suizhou City, Laohekou City, Shayang County (Jingmen City), Gong'an County (Jingzhou City), Zaoyang City, covering an area of300km by260km.. A total of116isolates of Botryosphaeria spp. were obtained. Culture characteristics, conidial morphology, pathogenicity, along with phylogenetics analysis of ITS, β-tubulin and EFl-a showed that at least three Botryosphaeria species occur on peach trees in Hubei Province, including B. dothidea (anamorph Fusicoccum aesculi Corda), B. rhodina (Berk.&M. A. Curtis)(anamorph Lasiodiplodia theobromae (Pat.) Griffon&Maubl.) and B. obtusa (Schwein.)(Diplodia seriata De Not.). Fusicoccum aesculi was isolated in all regions, but L. theobromae was found only in Shayang County in Jingmen City, and D. seriata only in Gong'an in Jingzhou City. Via artificial inoculation using mycelia on wounded twigs or branches, these three species were all found to be pathogenic, causing dark lesions and, sometimes, with gum exudation from diseased parts. Isolates of L. theobromae were the most virulent and caused most copious gum, and D. seriata had less gum than the other two species.2. The genetic diversity of F. aesculi isolated from different regions in China was studied based on conidial morphology, pathogenicity. phylogenetics and microsatellites analysis. The results showed that:in culture, isolates of F. aesculi had two types of conidia, namely fusiform, ellipsoid or ovoid, hyaline, aseptate conidia typical of the genus Fusicoccum, and fusiform, one septate conidia that are not typically observed. Five days after inoculation, vascular discoloration developed on peach twigs inoculated with each of the isolates of F. aesculi. There was significant difference in length of vascular discoloration among the18isolates ranging from5.8mm to40.1mm. Phylogenetic analysis combining three gene sequences of ITS, P-tubulin and EF1-α revealed the three Botryosphaeria species distributed in China, and differentiation existed in the isolates of F. aesculi. In the meantime, DNA fingerprinting produced by microsatellite primer M13, T3B, or (CTC)4RC were applied to analyze the difference among the isolates of F. aesculi. Seven groups were divided by Single Linkage in Cluster progress and Tree progress in SAS8.1software. Type one comprised of24isolates; type two comprised of6isolates. The other five types comprised of isolate WH-1822, TA-1, JM-20, CSJ-10and SH-5, respectively. The four aspects of conidial morphology, pathogenicity, phylogeny and microsatellite analysis confirmed the genetic diversity among isolates of F. aesculi in China.3. Fusicoccum aesculi is the most common pathogen of Botryosphaeria spp. associated with peach tree gummosis in China. In this study, a polymerase chain reaction (PCR)-based assay was developed for the specific detection of the fungal pathogen F. aesculi from peach trees. Variation among β-tubulin gene sequences of Botryosphaeria spp. was exploited to design primer pairs. Forward primer FaF, when used with the nonspecific reverse primer Bt2b, amplified a322bp DNA fragment for all20tested isolates of F. aseculi collected from peach trees. Specificity was also confirmed using those of closely related fungi including L. theobromae and D. seriata, and the other three fungal species isolated from peach or other hosts. The PCR assay using this pair of primers was sensitive enough to detect1pg/μl of genomic DNA of F. aesculi. A rapid DNA extraction procedure was applied to detect and identify F. aesculi from peach. These results indicate that this new primer is potentially useful for studies in areas or hosts in which the pathogen may be present, and prediction of inoculum production and disease development.4. Surveys in the field and experiments in doors were conducted to evaluate the peach cultivar susceptibility. A new grading standard for disease incidence was developed to score the severity of peach trees in the field. Seven peach orchards were surveyed, revealing that less severity of the same orchards compared with previous results, which was more effective and reliable for evaluation. Peach tree fungal gummosis, incited by Botryosphaeria spp., significantly depresses growth and yield on susceptible peach cultivars, on which little is known about the relative susceptibility in China. As F. aesculi is the most common species in peach trees,21cultivars were tested for susceptibility via artificial inoculation with F. aesculi isolate XNHG-241, which was identified as strong virulence in previous studies. Peach twigs inoculated with F. aesculi were placed in the plastic boxes in25℃for5days. Disease severity was evaluated by the length of vascular discoloration. Significant variation in disease susceptibility was detected across cultivars. Of the21cultivars tested, none of them was immune, and 'Shuguang' was the most susceptible and 'Zaofengwang' the least. The results of indoor tests were consistent with those in the field.