Analysis Of Anti-oomycetes Substances And Disease Prevention Of HT-6 Strain Induced By Phytophthora Infestans

As an important food crop,potato?Solanum tuberosum L.?is widely used in the world because of its cold-resistance,drought-resistance,easy storage,rich nutritional value,and prevention of hypertension and diabetes.However,the prevalence and frequent occurrence of potato late blight has become an important obstacle to potato production.The potato late blight caused by Phytophthora infestans brings up to 7 billion dollars in global losses each year,and the losses of late blight in China also reach about 1.7 billion dollars.At present,chemical agents such as metalaxyl or the selection of disease-resistant varieties are mainly used to prevent late blight in production.However,due to environmental problems caused by chemical pesticides and long breeding time,many scholars gradually realized the importance of biological control.Current studies have found that bacteria,fungi,and actinomycetes can all have a strong inhibitory effect on the growth of P.infestans.Different types of antagonistic bacteria produce different anti-oomycetes substances.Even if the same class of antagonistic bacteria,different strains may produce different anti-oomycetes substances.For example,the bacilysin produced by Bacillus pumilus and the lipopeptide substances Iturin A and Surfactin produced by Bacillus subtilis WL2 have strong anti-oomycetes effects on the growth of P.infestans.Antagonistic bacterial can not only produce anti-oomycetes substances to inhibit the growth of P.infestans,but also enhance the activity of resistant enzymes in potato plants,enhance potato disease resistance and not easily become infected with P.infestans.In recent years,our laboratory has screened many antagonistic strains that have a strong inhibitory effect on P.infestans.Among them,an antagonistic bacteria HT-6,selected from the leaves of the leaf spot of walnut in Tibet by Li Liyan,has a strong inhibitory effect on P.infestans.This strain was identified as Bacillus subtilis by Zhang Congying in our laboratory.However,it is not clear what kind of substance was used to inhibit P.infestans.This test analyzes the anti-oomycetes substances produced in HT-6 induced by P.infestans,and discusses its in vitro disease prevention effect,which lays a foundation for further isolation and purification of anti-oomycetes substances.The research results are as follows:1.Compared the effects of HT-6 on P.infestans mycelia growth between HT-6 cultured in LB liquid medium and rye liquid medium,the anti-oomycetes effect of HT-6 was not significant difference.The inhibition rate was 89.61%and 86.58%,respectively.Various types of aberrations occurred in the mycelia of P.infestans inhibited by HT-6,and the total distortion rate was as high as 40.33%.2.The conditions and blocking methods for HT-6 to produce anti-oomycetes substances were investigated.The free cell fermentation broth after culturing alone had no anti-oomycetes effect,and intracellular extracts and cell wall extracts of HT-6 strain also showed no inhibitory effect.Three plate isolation tests demonstrated that the anti-oomycetes substance is not a volatile substances.However,the free cell agar blocks in the anti-oomycetes zone had a significant inhibitory effect,and the anti-oomycetes effect of the agar blocks gradually increased with the incubation time or the number of agar blocks.Comparing the blocking effect of HT-6's anti-oomycetes effect after adding copper sulfate,SDS and chitosan,it was found that chitosan was the best.3.Distilled water and different organic solvents were used to extract anti-oomyceres substances from HT-6.It was found that methanol extract?74.03%?had the strongest inhibitory effect on mycelium growth,followed by ethyl acetate extract?69.26%?and water extract?53.25%?.Methanol extract was the best inhibitor on the direct germination and zoospore release of P.infestans.The direct germination rate and zoospore release rate of sporangia were 10.21%and 9.59%,respectively.4.The effects of different solvent extracts on potato tissues and disease control were compared.The results showed that,methanol extract and water extract were better than other extracts in the potato tuber disease prevention and treatment,and the disease prevention effects were 73.77 and 67.44,respectively.In the prevention of potato leaves in vitro,the disease prevention and treatment effects of methanol extract were the best,and the prevention effect?66.78?in advance was significantly better than the cure treatment effect?63.34?.5.The changes of defensive related enzymes?PPO,PAL,POD?and metabolites?soluble protein?in potato tubers were measured after treated with methanol extract.It was found that several enzyme activities and protein contents increased significantly compared with the control,and the methanol extract and P.infestans synergistically increased the contents of resistant enzymes and protein,also.6.The environmental stability of methanol extract was clarified.The suitable pH value was 4 to 10,and the anti-oomycetes rate was the strongest?74.46%?in a neutral environment?pH7?.The anti-oomycetes activity was stable at a temperature of 40?¹00?,and the anti-oomycetes rate was above 50%,but the tolerance decreased significantly when reaching121?.The anti-oomycetes rate still reached 71.00%when irradiated under UV light for 10hours.Mg²⁺and Ca²⁺could partially offset the inhibitory effect of methanol extract on the pathogenic P.infestans?the anti-oomycetes rates were 70.13%and 69.26%respectively?,And Zn²⁺and Fe³⁺could enhance the inhibitory effect of the extract,especially the anti-oomycetes rate of the extract after Fe³⁺treatment could reach 74.89%.