The Study Of Isolation And Identification Of Major Fungal Diseases Of Blueberry And Disease Control

Blueberry (Vaccinium spp.) is a kind of perennial shrub fruit tree belongs to Ericaceae family. The fruit contains rich nutrients, which contributes to its biological function such as cardiac and anti-cancer etc. It has been listed as one of the five major health fruits. With the rapid development of blueberry market, its planting area expands continuously, but the blueberry disease has gradually become one of the major factors hindering the development of blueberry industry. There are researches on blueberry diseases at home and abroad, but the lack of systematic classification of pathogens, especially the fungal pathogens. This study will clear the main fungal pathogens and control methods of the southern highbush blueberry in Zhejiang Province, and provide the theory basis and the technical support for the control of fungal diseases.The research mainly included four parts. Firstly, typical samples with leaves and fruits and stems of major fungal diseases were collected. Pathogens isolate and purification were obtained by tissue isolation method and single spore isolation method, respectively. The pathogenicities of pathogens were proved by Koch’s postulates, and the combination of routine morphological and molecular biology were used to identify pathogens. Secondly, the biological characteristics of the six kinds of pathogens were tested with a single variable. Then by using mycelium growth rate method, variations of mycelium growth of pathogenic bacteria were determinated under the different temperature, pH value, illumination and culture medium. Lethal temperature were tested too.Thirdly, study on chemical control of 6 kinds of pathogenic. The effective preventing fungicide was confirmed by inhibition of mycelium growth, calculated the toxicity regression equations and the effective inhibition of the concentration. Finally, to carry out the preliminary biological control research, the antagonistic strains of pathogenic were isolated by the methods of flat growth restraint.The main results were summarized as following:1.6 kinds of the main pathogenic fungi isolated from blueberry were identified: gray mold pathogen X1-1(Botrytis cinerea), stem canker pathogen JA4 (Phomopsis sp.) and JB2(Botryosphaeria dothidea), stem blight pathogen ZA1 (Pestalotiopsis clavispora), leaf spot pathogenYB-1(Pestalotiopsis sp.) and YB-2(Colletotrichum gloeosporioides).2. The optimum growth temperature of XI-1 was 20℃, and the optimum pH value was 6. The optimum medium of mycelium growth medium were PDA and BPY, lethal temperature was 40℃,10 min. The optimum growth temperature and medium of stem canker pathogen JA4 were 25℃ and PDA, pH 4-8, respectively. The optimum medium of sporulation was MEA+blueberry stems culture, and lethal temperature was 45℃,10 min. The optimum growth temperature of stem canker pathogen JB2 was 25～30℃, under the condition of weak acid mycelium growth rate was the fastest. Light was beneficial for mycelium growth. The optimum medium of mycelium growth and sporulation were MEA+blueberry stems, and lethal temperature was 55℃,10 min. The optimum growth temperature of stem blight pathogen ZA1 was 25℃, the optimum pH value was 4, and the nutrient requirement was lower, PDA medium was the best, and its lethal temperature was 55℃,10 min. The optimum growth temperature of leaf spot disease pathogen YB-1 was 25-30℃, the optimum pH value was 4-7, under the condition of light mycelium growth rate was the fastest, the applicable range of the medium was widely. The optimum growth temperature of leaf spot disease pathogen YB-2 was 25℃, and the optimum pH value was 5, under the condition of light mycelium growth rate was the fastest, the optimum medium of mycelium growth medium were PDA、PMA、PSA、LB、BPY、MEA. Lethal temperature of YB-1 and YB-2 were 45℃,10 min.3. The hyphae growth inhibition rates of 6 kinds of pathogen were tested by fungicides of Thiophanate methyl, Mancozeb, Difenoconazole, Azoxystrobin, Iprodione, Pyrimethanil or Leaf Kuzuo. It was found that Thiophanate methyl and Iprodione could significantly inhibit mycelium growth of XI-1, Thiophanate methyl, Difenoconazole and Iprodione could significantly inhibit mycelium growth of JA4, JB2x YB-K YB-2, Difenoconazole could significantly inhibit mycelium growth of ZA1, Azoxystrobin on inhibitory effects of six kinds of pathogenic were the worst.4. Antagonistic bacterium BL-01 was isolated from the healthy leaves of blueberry by preliminary screening. The sequence was amplified by 16S rDNA universal primers (27F/1492R), finding that BL-01 was belonged to the genus Bacillus. Then amplified sequences by 16S rDNA specific primers, rpoB gene primers and gyrA gene primers, finding that BL-01 was Bacillus pumilus. Morphological performance, the physiological and biochemical characteristics of antibiotic bacteria were observed:BL-01 was mostly thin rod, the size of 0.4-0.7×2.2-3.1μm, gram-positive, central spore, lateral flagella, capsule, the utilization of glucose acid but no gas produced. This antagonistic bacteria has antagonistic effect on 6 kinds of pathogenic bacteria, the best was for JA4. The inhibition of fermentation supernatant was 53.19%, better than the mycelium and fermentation liquid.