| Broccoli are nutritional vegetables,which are high in vitamin C,fiber and other nutritions,they also have health benefits,which are well received by consumers.Due to its top group is globular flower buds closely integrated,which are delicate structure,vulnerable to mechanical damage and pathogen infection destroying the appearance of broccoli as commodity and causing a lot of economic losses.The most serious fungal disease in broccoli is black spot disease caused by A.alternata.It can be a sign that in the early stage surface of broccoli florets appear black spots,and then quickly etiolate,rot,lead accelerated aging.In this research,focusing on the subject of broccoli,it was studied that the isolation and identification of A.alternata,the effect of storage temperature on its growth,the influence of quality of broccoli after the infection of A.alternata,the mechanisms of A.alternata pathogenicity were explored by optimization of cell wall degrading enzymes production process,analysis of pathogenic role of the enzyme on broccoli.Finally,it was studied that the inhibitory effect of cinnamaldehyde,cinnamic acid in vitro on A.alternata.The study could provide to provide theoretical reference for broccoli disease research,and propose a new idea for disease prevention and control by A.alternata.The results were as follows:1.The separation and identification of Alternaria spp.:It was obviously more damage after the infection of Alternaria spp.,on broccoli floret than pedicel,perhaps because of young bud tissues easily exposed to the air.Identification of black rot disease,there were two chain respectively Alternaria alternate(A.alternate)and Alternaria tenuissima(A.tenuissima),A.alternate shows stronger pathogenicity.2.Effects of different temperatures on growth of A.alternate:(1)The study was about the growth and pathogenicity of A.alternate in storage temperature include 4 ℃ 、 18 ℃ and 25 ℃.And A.alternate could grow and existpathogenicity on broccoli in the three temperatures.The optimum temperature was 25℃ for A.alternate.In the shelf life temperature of 18℃,the results showed A.alternate growed rapidly and had some damage on broccoli after infection.Clearly,In 4 ℃ it couldn’t completely prevent the mycelial growth and spore germination of A.alternate.(2)The treatment by heating 10 min under 55℃ and 85℃ with water bath could inhibit mycelial growth and spore germination of A.alternate.3.The effects of the infection of A.alternate on the broccoli’s quality:(1)After the infection of A.alternate,there were obvious variation on soluble protein,soluble sugar,vitamin C,chlorophyll,titratable acid and MDA both in floret and pedicel of broccoli.(2)The conclusion showed during A.alternate attack the treatment group of broccoli floret change greatly on soluble protein,and the broccoli floret became yellow quickly.It was speculated that after the infection of A.alternate,broccoli floret accelerated aging may be associated with increased soluble protein and chlorophyll degradation.4.The orthogonal optimization for production of cell wall degradation enzymes by A.alternate and its pathogenic effect on broccoli:(1)The results showed that the high-level cellulase activity(Cx)was produced by A.alternate at 30 ℃,pH 7,culture time 6 d,0.2% KNO3 and 0.5%carboxymethylcellulose as carbon and nitrogen sources,and reached at 110.53±1.02U/mL.(2)The activities of poly methyl galacturonase(PMG)exhibited their maximum activity were up to the maximum 128.75±0.96 U/mL at 30℃,0.2% KNO3 and 1%pectin,and culture time 6 d.(3)The conditions of producing β-glucosidase(β-GLU)by A.alternate was30 ℃,0.2% KNO3 and 1% glucose,and culture time 6 d,reached at 108.78±4.33U/mL.(4)In vivo experiments of broccoli three kinds of cell wall degradation enzyme activity produced by A.alternate was detected.The cell wall degradation enzymes activity were higher in broccoli floret than broccoli pedicel,and PMG showed thehighest activity among them.(5)In the study of treat broccoli floret with enzyme extracts directly include Cx,PMG,β-GLU,three kinds of enzyme extracts all could cause damage to broccoli floret,PMG showed most serious injury on broccoli floret;By observation of hydrolysis effect,PMG changed greatest on reducing sugar and conductivity,and Cx is higher than the β-GLU.5.The experiment was examined antifungal effect of three materials on A.alternate in vitro:(1)The results reflected when the concentration of 500 μg/mL,the inhibitory ratio of prochloraz,cinnamic acid and cinnamic aldehyde on growth of A.alternate were 100%,whereas chlorogenic acid wasn’t much evident(52.73±3.45%),the next test object were focused on prochloraz,cinnamic acid and cinnamic aldehyde.(2)After the addition of 50-250 μg/mL of three materials,they all showed inhibitory effect on A.alternate.When concentration of 200 μg/mL prochloraz was added,inhibition rate was up to 100%.Compared to control group-prochloraz,when the concentration of 250 μg/mL,the inhibition rate of cinnamic aldehyde reached100%,and cinnamic acid was 61.54±6.32%.It may conclude that the inhibitory effect cinnamic aldehyde is better than that of cinnamic acid.(3)The study indicated that prochloraz showed strongest inhibitory effect on A.alternate,the EC50 value was 61 μg/mL.Moreover,EC50 value of cinnamic acid and cinnam aldehyde were 0.200 and 0.145μg/mL,respectively. |
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