Identification Of Five Fungal Pathogens Causing Citrus Diseases In China

This study was conducted from2009to2011. Some unknown diseases are threatening citrus industry in China. In this study, the causal agents of five new diseases were determined based systemic tracking survey of disease development, causal agent isolation, pathogenicity test, investigation of morphological and molecular characteristic after field investigation. The biological characteristics of an emerged pathogen were studied, and the effective fungicides against it were screened in vitro. The clarification of causal agents would lay the foundation for the effective control of the diseases. The research results are summarized as follows.1. Study of the Pathogen Causing Citrus Target Spot. Target spot of satsuma mandarin(Citrus unshiu Marcov.), as well as kumquat (Fortunella margarita(Lour.) Swingle) in Chenggu County, Shaanxi Province, China apparently began by2006or earlier and has caused substantial economic losses to the citrus producers. This new disease only prevailed in the late winter and early spring. A Cryptosporiopsis species was consistently recovered from the infected leaves, shoots and branches by tissue isolations and single spore isolation with capillary. Koch’s postulates were fulfilled by inoculating theconidial suspension of Cryptosporiopsis sp. onto the leaves of satsuma mandarin. Phylogenetic analysis based on LSU indicated that this taxon was clustered into the genus Cryptosporiopsis (teleomorph:Neofabraea, Dermateaceae). Phylogenetic analysis based on ITS, SSU and TUB indicated that the isolates of Cryptosporiopsis sp. constituted a distinct clade. Further study also demonstrated that this taxon was morphologically distinct from other species of Cryptosporiopsis, thus suggesting it might belong to an undescribed species. The name Cryptosporiopsis citricarpa sp. nov. was given to accommodate this fungal pathogen in this study. C. citricarpa showed very good growth on the media with sucrose, fructose, maltose, glucose and sorbitol as a sole carbon source, with ethanol being the worst. C. citricarpa could not utilize carbamide and even was killed by it with concentration of0.2%wt/vol. C. citricarpa showed better growth at pH value of4.0-5.0than the other pH condition. Difenoconazole and prochloraz showed high effectiveness to inhibite Cryptosporiopsis citricarpa from hypha growing and conidia germinating.2. Investigation of Botrytis-Induced Injury of Citrus Fruits. In this study, we investigated if Botrytis-mold of citrus petals was associated with defect or scars on fruit rinds by tracking survey, fungicide spraying and petal inoculation since (at) the flower-dropping stage. Results showed that Botrytis sp. infected citrus petals during the late stage of flowering, the infected petal was gray-molded. Some molded petals did not drop off, substituting they sticked to the fruitlets. Botrytis sp. extended from floral debris onto the fruit surface, caused spots with slight depressed innitialy, then conical or ridge-like outgrowths on the rind. The associated corky scars remained as blemishes on the fruit surface as the fruit enlarged and matured, the protruding parts became gradually flat, or even slightly depressed, roughness, cork-like (corky scars). Inoculation experiment indicated that Botrytis sp. was pathogenicity to citrus fruitlets, causing black, depressed spot, even fruitlet-dropping. Spraying fungicide procymidone at the stage of flower-dropping could decrease the ration of defect rind fruits. Thereby, we concluded that the Botrytis-mold of citrus petals was associated with defect or scars of fruit. The species of Botrytis sp. was indentified as Botrytis cinerea Pers. Ex. Fr. by morphology and phylogenies of ITS sequence.3. Identification of the Pathogen Causing Foot Rot of Fortunella margarita (Lour.) Swingle. Foot rot of kumquat(Fortunella margarita (Lour.) Swingle) is a big problem in Guilin, Guangxi Zhuang Autonomous Region, where kumquat is one of the main cash crops. The affected trees decline gradually and finally wilt as the lesion girding the trunk.The suspected pathogen isolates of Phytophthora citrophthora, which identified by morphology, were always obtained from affected trunks. The pathogenicity of those isolates were confirmed by inoculating the representative isolate GLC15on kumquat seedlings, shoots and branches. The identification was further confirmed by alignment the sequences of internal transcribed spacer (ITS) and translation elongation factor1alpha gene (TEF-1α), amplified by PCR. The sequences were deposited in Genbank with accession numbers GU133068(ITS) and HQ637486(TEF-1α), while the pathogen culture was deposited in Institute of Biotechnology, Zhejiang University.4. Identification of the Pathogen Causing Shoot Canker of Kumquat in China. Shoot and branch canker and tree decline was observed on kumquat(Fortunella margarita (Lour.) Swingle) cv. Guban during2008-2011in the orchards of Yangshuo County, Guilin City, Guangxi Zhuang Autonomous Region, China. Two species of Fusarium were consistently isolated from the infected shoots and branches. On the basis of morphological and cultural features, they were identified as Fusarium oxysporum and a new species within the Gibberella fujikuroi complex, respectively. The morpho-cultural identification were confirmed by the high sequence identity of the translation elongation factor1-alpha (TEF1) sequence of F. oxysporum and a new species within the Gibberella fujikuroi complex (F sp. GLB1) with those available in the GenBank databases. Koch’s postulates were established by pathogenicity testing on kumquat (cv. Guban).5. Study of the Pathogen and Disease Cycle of Greasy Yellow Spot on Changshanhuyou. Changshanhuyou (Citrus paradisi Mac. cv. Changshanhuyou) is one of the main cash crops and famous local fruits in Quzhou, Changshan, Zhejiang province. Greasy yellow spot is the most destructive diseases for Changshanhuyou. It causes premature leaf drop, and may leave the tree almost completely defoliated when the disease is not adequately controlled. In this study we identified the causing agent of greasy yellow spot as of Mycosphaerella citri by morphology and alignment of the sequences of internal transcribed spacer (ITS). It induced two types of symptoms, greasy spot and brown small round spot on leaves, while caused yellow spot with irregular shape on fruits. Conidia produced on the brown small round spot could infected the leaves, and induced both greasy spot and brown small round spot, thus inferred that conidia that produced in the canopy year around were the main source of infection for young tissues.