| With the increase of the world's population,potato has become the fourth largest food crop,and its output has been listed as an important part of the national economy.The potato late blight caused by Phytophthora infestans has been widely recognized as the world's largest crop disease.Nowadays,the prevention and control methods for potato late blight in production mainly include chemical control,agricultural control,and disease resistance breeding.Because agricultural control is time-consuming and labor-intensive,and the cost of disease-resistant breeding is high.And chemical control of disease is fast and effective,but excessive utilization of chemicals has caused chemicals-resistance of pathogen and environmental pollution.The advantages of biological control due to environmental protection and pollution-free are widely concerned.At this stage,many researches reported the use of bacteria,fungi,actinomycetes and their metabolites to inhibit P.infestans,but successfully practical application of antagonists for control late blight in potato production is still not present.The one of the most important reasons might be that the suppression mechanism of antagonists on P.infestans is not clear.The HT-6 strain was isolated by our lab from Linzhi city in the Zhuang Autonomous Region of Tibet.It is the endophytic bacterium from the leaves of susceptible walnut trees and had very strong inhibition for mycelia growth and sporangia germination of P.infestans.And a series of investigations have been carried out on its restraint to P infestans.The stability was tested,and the fermentation conditions of the bacterial liquid were optimized,and the classification status was studied,but the cell free fermentation broth did not inhibit the P.infestans.In order to further understand the potential of antagonistic HT-6 in inhibiting P.infestans and its physiological and biochemistry mechanism,this experiment detected the persistent restriction effect of HT-6 strain and cultured bacterial stock solution on P.infestans,in prolonging dual culture time.In addition,disease prevention effect of HT-6 on in vitro,the morphological and partial physiological changes of P.infestans prohibited by HT-6 as well as the effect of blocking HT-6 partial biochemical process on its constraint to P.infestans were investigated.The main findings are as follows:1.The inhibition of HT-6 strain and bacterial liquid on mycelial growth of P.infestans could keep for 15 days,inhibition rate of the former was 84.00%,and that of the latter was67.71%;the inhibitory effect of the living strain is stronger than that of the bacterial liquid cultured at certain conditions.2.In the process of preventing disease of potato in vitro tissue,when the concentration of HT-6 bacterium was 2.88×10~8?/mL,the disease prevention effect was the best.HT-6 could cause the morphological aberration of P.infestans sporangia and mycelia and their rates were44.13%and 37.80%,respectively.The mycelial morphological changes included excessive mycelia branching,serious invagination of cell membrane,bulge of mycelia surface,mycelial contents accumulation,mycelial distortion etc;And the sporangia morphological changes were from lemon to baseball,fan-shaped,uneven distribution of contents and leakage,or sporangia wall thickening and other distortion phenomena,and various distortions.3.After being inhibited by antagonistic HT-6,content of the soluble sugar,protein and starch in the mycelium of P.infestans were significantly different from that of the control as the culture time.In which,content of the soluble sugar and starch in inhibited mycelia increased with the dual culture time prolongation as control,but their contents inhibited mycelia were far below that of control.However,the content of soluble protein in inhibited mycelia decreased with culture time prolongation,indicating that that HT-6 slowed down the synthesis of soluble sugar and starch,while accelerated the decomposition of proteins in the mycelium.4.The activities of the resistance enzymes PAL,POD and PPO increased in P.infestans mycelia inhibited by HT-6 compared with control as the prolongation of culture time.At 12days,the three enzymes activities increased and reached the highest;Furthermore,the antagonistic HT-6 could also enhance the electrical conductivity of the P.infestans mycelium,and raise the relative permeability of the mycelium membrane.These changes of P.infestans caused by HT-6 not only damaged the mycelia but depressed the respiration.The degree of damage and inhibition rate of respiratory were 32.58%and 15.99%,respectively.5.After blocking the biochemical process of HT-6 by Glucosamine hydrochloride,ATP and citric acid,the effect of its inhibiting P.infestans was investigated.The results showed that although the concentration gradients of three chemicals were set low in the preliminary experiment,it could also be found that interference with chemicals on the glycolysis process of HT-6 could actually decrease the inhibition effect of HT-6 on mycelia growth of P.infestans.Moreover,the best effect was achieved by mixing the chemicals with HT-6 for 12h.6.In the subsequent experiments,the experimental conditions were optimized,and increasing the concentration of ATP may inhibit the activity of pyruvate kinase,and the inhibitory strength of HT-6 against P.infestans decreased with the increase of the concentration of the reagent,It was found that raising the compounding concentration of ATP and citric acid,could inhibit phosphofructokinase(PFK)activity,and with the increase of compounding concentration,the inhibition effect of HT-6 on P.infestans was evidently weakened.Similarly,complex of ATP and potassium dihydrogen phosphate as well as ammonium sulfate at a concentration of 0.3 g/mL had significant effect for the inhibition of HT-6 against P.infestans compared with the absence of the reagent.But as the concentration of the reagent increased,the effect on HT-6 was not strong,and there were no significant difference between treatment and control. |
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