Study On The Mechanism Of Cell Wall Degrading Enzymes And Toxins In The Process Of Infecting Jujube Fruit By Alternaria Alternata

Alternaria alternata infects jujube fruit and causes jujube black spot disease.It forces a series of changes in the physiological,tissue and morphology of jujube fruit.It is responsible for a significant decline in yield and quality.It not only causes loss in production,but also causes postpartum loss,thus causes huge economic losses to the production and storage of the jujube industry.In this paper,we intend to research black spot disease caused by Alternaria alternata.The role of Alternaria alternata cell wall degrading enzymes and toxins in infections in jujube fruit was determined.The biochemical mechanism and morphological changes of Alternaria alternata infecting jujube fruit were elucidated.This study provides a theoretical basis for the prevention and control of black spot disease caused by Alternaria alternata infection.Our main findings are as follows:1.Isolation and Morphological Identification of Black Spot PathogenThe pathological samples were separated by tissue separation method.We obtained 50 strains of microorganisms in which 78% was Alternaria alternata,8% was Penicillium,4% was Fusarium,and 10%was Aspergillus bacteria.The isolated strain was returned to the healthy jujube.Hence,the pathogenicity test was carried out.It was found that only Alternaria alternata could cause jujube fruit disease with an incidence rate of 54%.After a second isolation and inoculation,it was found that the pathogenic Alternaria alternata coincided that in the first isolation.The isolated Alternaria alternata was identified as the main pathogen of jujube black spot.According to the morphological characteristics of the strain,it was initially identified as Alternaria alternata.The isolated A3 group of single spore strains has been proven to have strong pathogenicity.They can also be used for subsequent studies on Alternaria alternata infestation.2.Analysis of Cell Wall Degrading Enzyme Activity during the Inoculation of Jujube Fruit by Alternaria alternataIn order to determine the reducing sugar released by the enzyme reaction and calculate the cell wall degrading enzyme activity,the activity of the enzyme that degrades Alternaria alternata cell walls was measured by using 3,5-dinitrosalicylic acid(DNS)method and Coomassie Brilliant blue(Bradford)method.Alternaria alternata produced six kinds of cell-wall-degrading enzymes under the induction of different carbon sources outside the host.The enzymes were Polygalacturonase,carboxymethylcellulase,?-glucosidase,xylanase,polymethylgalacturonase,and pectin methyl trans-eliminase.However,there were some differences in the activity of the six cell-wall-degrading enzymes.The activity of ?-glucosidase produced by Alternaria alternata was significantly higher than that of the other five enzymes when using jujube pulp as an exogenous inducer.It was indicated that ?-glucosidase played an important role in the pathogenesis of Alternaria alternata.The activity of ?-glucosidase produced by filter paper as inducer was also higher than that of other inducers,reaching 10.104 U/mg.The activity of cell-wall-degrading enzyme produced by Alternaria alternata after the infection was same as results induced by different carbon sources in vitro.Six cell wall degrading enzymes were also produced.The activity of ?-glucosidase was higher than the other five enzymes,and the activity of ?-glucosidase at the junction of disease and health is significantly higher than that of the infected site and the uninfected site.The results showed that the cell-wall-degrading enzyme which plays a key role in the pathogenesis of Alternaria alternata is?-glucosidase.The activity at the border of disease is the highest in the process of infection.The optimal carbon source for inducing ?-glucosidase outside the host is the filter paper inducer.3.The stress of toxin on the peel of jujube during the process of infecting jujube with Alternaria alternataThe jujube peel tissue was treated with Alternaria alternata toxin extract.Then,the cell membrane permeability was measured.The malondialdehyde content,the activities of peroxidase,ascorbate peroxidase and catalase were also determined.The pathogenic mechanism of the toxin was studied.The results showed that the toxin produced by Alternaria alternata could inhibit the activity of active oxygen scavenging enzyme in cell tissues.The activity of catalase decreased by 67.06% in a short time,resulting an accumulation of high concentration of reactive oxygen species in the cells.It disturbed the normal metabolism of the tissue and caused the loss of normal physiological functions in cells.Due to the damage of the cell membrane,electrolyte leakage occured,and the electrical conductivity increased.The maximum relative conductivity difference was 33%.The malondialdehyde content was on an increasing trend which peaked at 36.6nmol/g.The degree of membrane lipid peroxidation was deepened.Therefore,the content of malondialdehyde can also be used as a marker of the degree of cell damage.After the toxin treatment,the degree of cell damage was deepened,which eventually caused the death of the host.The result showed that the toxin of Alternaria alternata had a stress effect on Junzao.It also reflected the relationship between pathogenicity and toxin.4.Morphological changes during the process of infecting jujube with Alternaria alternataIn order to investigate the changes of morphological structure of Alternaria alternata infecting jujube fruit,labeled the strain with the green fluorescent protein gene(GFP).The pathogen infection process was studied by molecular transformation system.After 6 hours of inoculation of GFP-labeled Alternaria alternata in jujube fruit,spore germination was observed on the surface of jujube fruits.After 12 hours of inoculation,the germ tubes formed by germination were gradually elongated.After 24 hours,sparse single hyphae formed and invaded into epidermal cells.After 48 hours,the jujube fruit tissue was filled with dense mycelium and grew in different directions.After the jujube fruit was infested,the black lesions gradually expanded,the center of the lesion was partially depressed,fruit shrinked and dryed,the diseased jujube tissue cells invaded by Alternaria alternata were damaged,and there were a large number of hyphae in the intercellular space and in the cells,tissue cell shape changes.From the perspective of histopathology,the process in which GFP-labeled Alternaria alternata infected jujube fruits was observed.The most direct evidence for the Alternaria alternata interaction is provided.