Identification Of Pathogens On Walnut (Juglans Regia) In Shandong Province

Walnut (Juglans regia) is important "woody grain" ecological species. As the continuous improvement of intensive culture and cultivated area expansion of walnut, disease hazard becomes mote and more serious, and has became a serious problem of the walnut industry. In this study, based on the survey of disease prevalence in walnut producing areas and the pathogens of walnut major diseases were separated; and after identification of morphological and DNA molecules integration inoculation experiment, identify the major disease pathogens of walnut.. The main results are as follows.1. Walnut major diseases in Shandong Province: By the sentinel survey to walnut growing areas in Shandong Agricultural University the Forestry Experimental Station, Qiu store Luyuan forest Research Institute, Wenshang Golden Tan Walnut Institute, Linyi City, peony-growing areas of Walnut in 2009-2010, found that: the main disease of walnut severity followed by walnut anthracnose, walnut blight, walnut canker, walnut shoot dieback and Walnut brown apical necrosis.2. Walnut anthracnose: Walnut anthracnose mainly against fruit, but also against leaves and young shoots. Selected 17 strains from isolated as the test strains. On PDA, characteristic of the tested strains show that, oil ball within the mycelium, conidia, single-cell and colorless, cylindrical, oil ball internal, 10.4-15.0μm×4.6-6.4μm; Formations of appressorium are nearly round, trilobed, irregularly shaped, light brown or dark brown. The average size of 10.2μm×7.3μm. Obtained Genbank accession numbers (HQ845100-HQ845107, HM016792-HM016798, GU597322, HQ828096), the result of phylogenetic analysis show that all tested strains is divided into three groups, distribution by location.The three groups all together with C. gloeosporioides on a branch. Selection strains of'w-2','M9','wsg2'do pathogenicity test, June inoculation fruit average incidence of 100%, leaves the average incidence of 100%, the average incidence of 86% of the branches. In July, average incidence of 62% of fruit, the average incidence of 100% of the leaf, the average incidence of 83% of the branches. So through morphology and ITS molecular identification integrate pathogenic experiment were clear that Walnut anthracnose pathogen is C. gloeosporioides.3. Walnut blight and brown apical necrosis: Tow type diseases have different symptoms. brown apical necrosis disease symptoms began to appear after flowering, lesion at the base of the pistilOr apical of fruit, Walnut blight late onset of brown apical necrosis and mainly against the fruit peel, leaf and shoot. Alternaria spp., Pantoea agglomerans, Xanthomonas arboricola pv. juglandis can be found in the Ministry of separation of two diseases , So they are complex infectious diseases.Selected 34 fungal and 4 bacterial strains from isolated as the test strains .On PCA ,characteristics of 34 strains were divided intothree groups, conidial morphology and sporulation phenotype are two types. According to morphological characteristics will be 33 strains identified as Alternaria alternata and one strain identified as Alternaria tenuissima . Molecular identification using ITS (Genbank accession numbers: JF835805-JF835834, JF835903-JF835906) and RAPD random primers OPA2-1 (JF835834-JF835868), OPA1-3 (JF835869-JF835899) DNA sequence for phylogenetic analysis. These sequences have the distinction of Alternaria, but can not use these sequences for effective classification of microspore Alternaria species on the species level, Alternaria fungi are quite different in genetically.Inoculated with on behalf strain of (A. alternata)'6hao','10-39'and on behalf strain (A. tenuissima) of'10-19'with stab wounds, These strains can be pathogenic were pathogenic, pathogens isolated can Re-inoculation from the onset position.Pantoea agglomerans: colonies round, smooth, yellow wax, cell growth was gathered, Straight shape, 0.9-3.0μm×0.6-1.2μm, around with 4-6 flagella. The ITS sequence HM016799 of HXJ have 99% homology with ITS sequence HQ242740,GU477762,GQ494018 of (Pantoea agglomerans). Therefore, it was identified as Pantoea agglomerans .Inoculation proved the Pantoea agglomerans main cause endogenous to the apical fruit necrosis, and caused branches diseased and leaf spot disease and involved in the process of blight disease.Xanthomonas arboricola: colonies round, smooth, yellow-green, short rod-shaped, 0. 3-1.8μm×0.3-0.8μm, a flagellum-side students.Both ITS sequence JF835909 of lxj1 and sequence JF835910 of lxj2 have 99% homology with sequence AB558555of Xanthomonas arboricola pv.juglandis. Therefore, it was identified as Xanthomonas arboricola pv.juglandis. Inoculation proved the Xanthomonas arboricola mainly caused blight spot on fruit, leaves and shoots, also induced necrosis of young fruit on the apical.4. Walnut canker: Selected 4 strains from isolated as the test strains. The tested strains cultured in PDA medium characterized by the same microscope.The beginning show white mycelium, gradually turns gray or black, well-developed aerial mycelium, conidia, fusiform, base obtuse or slightly, transparent, droplet-like inclusions, 23-27μm×4-8μm, average of 24.2μm×4.8μm. The ITS sequence JF835907 of 3hao strain has homology with Botryosphaeria dothidea up to 99% .So it was identified as Botryosphaeria dothidea. Field observations and inoculation experiments showed that the bacteria can cause canker on the base of the trunk and main lateral branch, infection young shoots dieback symptoms appear serious disease caused tree death.5. Walnut dieback: B. dothidea and Phomopsis sp. can Separable from the Ministry of the disease .Phomopsis sp.can produce alpha and beta two types of conidia, alpha conidia ,fusiform or oval-shaped, colorless, single cell, containing oil ball; beta conidia, slender such as lines, curved ,sometimes hooked at one end, colorless, single cell.ITS sequence JF835912 of Z3-19Y5 strain have 99% homology with sequence AF103001of Phomopsis amygdali and EU520050 of Phomopsis vaccinii. But the morphological characteristics of Z3-19Y5 strain with the Phomopsis amygdali are quite different, with P. vaccinii device in the conidia, conidial morphology of influenza A are significantly different. Therefore, it was identified as Phomopsis sp.. Selected Phomopsis strains Z3-19Y5 for inoculation, pathogenic rates 75%. So that bacteria can pathogenic alone, but also caused dieback complex with B. dothidea. For taxonomic status of Phomopsis sp. needs further study.