| Citrus Black Spot (CBS) caused by Guignardia citricarpa (Dothideales, Loculoascomycetes in Ascomycota) Kiely. The anamorph of this pathogen is Phyllosticta citricarpa (McAlpine) van der Aa, belongs to Sphaeropsidales, Coelomycetes in Fungi Imperfecti. CBS is a common disease in China, mainly causes diverse spots on the rind of Citrus fruit, results in the fruits become unsightly and unsuitable for the fresh fruit market. The pathogen of CBS P. citricarpa is an Al quarantine pathogen for countries of the European Union, and it is also regulated as a quarantine pest in the USA. The export of citrus fruits from China and other countries where CBS is present is heavily regulated. As one of the origin places, there are many cultivated Citrus species and varieties in China, and many Citrus species are susceptible to CBS. However, there is no research to demonstrate whether the pathogen on different Citrus species is belonged to the same species. The biological and molecular characteristics of the pathogen from China are remaining unknown. In the study, Phyllosticta strains were obtained from infected fruits and leaves of the majority of the cultivated Citrus species and varieties across the main citrus producing regions of China, and identified using the methods of morphological and molecular biology. A nested-multiple PCR technology for diagnosing the4Phyllosticta species from citrus was established. Genetic diversity of4Phyllosticta species were further studied, the results were described as follows.1. Establishment of identification system of P. citriasiana By comparing the differences of the internal transcribed spacer region of Phyllosticta spp., specific upstream primer Pca8for P. citriasiana was designed. By combining the downstream primer ITS4, an accurate, sensitive and fast identification system of P. citriasiana was established. By using the established PCR identification system, diagnostic fragments of488bp were consistently amplified from P. citriasiana, but were never amplified from P. citricarpa and endophytic P. capitalensis, nor from the common pathogens for citrus. This method is highly sensitive (12pg of genomic DNA) and rapid (with in3 hour) for detection of P. citriasiana.2. Clarification of Phyllosticta species associated with citrus diseases in China Samples of citrus fruits and leaves with black spot or black spot-like symptoms were collected from cultivated Citrus species including mandarins, oranges, pomeloes and lemons in the ten citrus-producing provinces of China during2007to2011. Four hundred and ninety six Phyllosticta strains were isolated from these samples. These isolates were divided into four morphological groups, and74representative strains of these four groups were selected for phylogenetic analysis. Analyses inferred from the sequences of the internal transcribed spacer region of nuclear ribosomal DNA (ITS), partial actin gene (ACT), partial translation elongation factor1-alpha (TEF1),β-tubulin (Tub) and Calmodulin (Cal) showed these representative Phyllosticta isolates clustered in four distinct clades corresponding to three known, and an undescribed species. Phylogenetic trees based on individual gene and combined genes resolved the same results. Clade-I corresponded to P. citricarpa, the pathogen of black spot (the species regulated by EU and USA). It was found on oranges, mandarins and lemons, but was not found on pomeloes; Clade-Ⅱ corresponded to P. citriasiana, the pathogen of pomeloe black spot (also named as tan spot), was only found from pomeloes, but not from the other Citrus; Clade-Ⅲ corresponded to P. capitalensis, the endophytic fungus with a wide host range, was found on all citrus fruits and leaves with black spot or black spot-like symptoms in this study; Clade-IV corresponded to Phyllosticta citrichinaensis X. H. Wang, K. D. Hyde&H.Y. Li, sp. nov., a novel species described in this study, obtained from all four Citrus species with atypical citrus black spot symptoms.Among four Phyllosticta species, P. citrichinaensis has the smallest conidia, longest mucoid apical appendage, largest spermatia and ascospores. P. citrichinaensis could be distinguished from P. citricarpa, P. citriasiana and P. capitalensis by producing colonies with multiple annular ridges on PDA, cornmeal agar (CMA), and malt extract agar (MEA), and by not producing yellow pigment in OA. On media using sucrose, fructose, galactose, mannitol, sorbitol, glucose or maltose (1%w/v) as a sole carbon source, P. citrichinaensis grew slower than the other three Phyllosticta species from Citrus. On media using urea, tartaric acid ammonium, aspartic acid, CH3COONH4,(NH4)2SO4, NH4NO3, NaNO3, tryptone, proline or glycine as a sole nitrogen source, P. citrichinaensis grew slowest than the other three Phyllosticta species from Citrus. P. citrichinaensis almost could not grow on media using urea, tartaric acid ammonium, aspartic acid, CH3COONH4,(NH4)2SO4or NH4NO3as sole nitrogen source. P. citrichinaensis could grow at pH value of3.0-6.0, but grew the best at pH4.0, followed by3.0. At pH value of3.0-5.0, P. citrichinaensis grew faster than P. citricarpa and P. citriasiana, but slower than P. capitalensis.3. Establishment of nested-multiple PCR identification system of four Phyllosticta species By comparing the sequence differences of the first internal transcribed spacer region and18s region of four Phyllosticta species associated with citrus, species-specific upstream primers Pccl, Pcl, Pct4and Pca8(above1) for P. citrichinaensis, P. citricarpa, P. capitalensis and P. citriasiana were designed. By combination with ITS4as downstream primer, an individual PCR diagnosis of each species was established and optimized. For diagnosis of a suspect Phyllosticta culture or a lesion that was suspected to be related to Phyllostica, the DNA of the culture or lesion was used as template, ITS4/ITS5was used to the first round amplification, the amplified product (diluted by50-100fold) was then used as template, and using mixture of primers Pccl/ITS4, Pcl/ITS4, Pct4/ITS4and Pca8/ITS4was used for the second round PCR amplification at annealing temperature of60℃. Meanwhile, the DNA of standard isolate belonging to P. citrichinaensis, P. citricarpa, P. capitalensis and P. citriasiana respectively was used as positive control. The exact Phyllosticta species was identified by comparison the diagnosis bands with standard isolate of each species after electrophoresis. The nested-multiple PCR could detect the fungal DNA as low as2ag and increased the detection sensitivity at least106-fold compared to the traditional PCR method. The established nested-multiple PCR system in the study could not only identify Phyllosticta spp. culture, but also could identify suspect Phyllosticta spp.-associated lesion on citrus rind.4. Genetic diversity of Phyllosticta associated with citrus By optimization the concentration of DNA templates, Mg2+, dNTPs, TaqDNA polymerase and primers, a suitable ISSR-PCR system for Phyllosticta was established.11primers amplified stable, clear, repeatable, high polymorphism productions were screened from100primers. Eleven primers and the optimized ISSR-PCR system were used to investigate the genetic diversity of110isolates of Phyllosticta, and197bands were amplified, of which194polymorphism bands. The polymorphism rate was98.5%, showing rich genetic diversity of Phyllosticta. NTSYS-pc2.10was used to build UPGMA dendrogram. It showed that the isolates of Phyllosticta were divided into four clades, corresponded to P. citricarpa, Pcitriasiana, P. capitalensis and P. citrichinaensis. Genetic differentiation of P. citricarpa was host-related, not geographical distance-related. Isolates collected from sweet oranges and lemons were distinct from isolates from mandarins, they gathered in different clades. Isolates collected from Citrus reticulata cv. Shatangju were different from isolates from other mandarins, and those isolates gathered in another clade. The genetic variation of P. citriasiana, P. capitalensis and P. citrichinaensis was not related to host and geographical distance. |
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