| Myxobacteria are a class of higher prokaryotes with social behavior.They have complex multicellular behavior and morphology,which are important for the research of cell differentiation,development and evolution.Myxobacteria have great research value in screening natural drugs because their metabolites have rich,diverse and novel biological activities.Potato is the fourth largest food crop in the world,following rice,wheat and maize,and is also the fourth main grain in our country.China is the largest producer of potatoes in the world.Potato is planted in the relatively cool areas such as Yunnan,Guizhou and Northeast China.The most serious disease of potato is potato late bright,and its pathogen is Phytophthora infestans.P.infestans mainly attacks leaves,stems and tubers.It is a destructive disease which can cause the death of potato stems and leaves and rot of tubers.Therefore,the development of highly effective and harmless pesticides against potato late blight is imminent.In this study,139 soil samples were collected from the central region of the Inner Mongolia Autonomous Region,and pH value and the content of water,organic matter,available potassium,hydrolyzed nitrogen,and available phosphorus of the soil samples were measured.The results showed that the soil samples in the central region of Inner Mongolia were basically neutral and alkaline,and the water content was very low,and the soil fertility status was generally poor.Myxobacteria were isolated from 139 soil samples by rabbit dung pellets inducing method,Escherichia coli inducing method and filter paper inducing method,and purified by directly picking fruiting bodies,antibiotic treatment and secondary induction.It was found that the dominant species of myxobacteria in the central region of Inner Mongolia included 6 genera,Myxococcus,Corallococcus,Pyxidicoccus,Cystobacter,Archangium and Melittangium.The most abundant resource of myxobacteria was found in Baotou of Inner Mongolia,while the myxobacterial resource of Wulanchabu was relatively poor.The population of myxobacteria in the samples from grassland and cultivated land was relatively abundant,while the population in the samples from woodland and unused land was less.The myxobacteria in the samples from Fluvo-aquic soils,Grey-cinnamon,Castanozems,and Bog soils showed a rich diversity,while the abundance of myxobacteria in the samples from Aeolian soils,Solonetzs,Skeletol soils,and Castano-cinnamon soils was poor.The correlation between the distribution of myxobacteria and the environmental parameters was analyzed and it was shown that the environmental parameters of the soil samples have no obvious correlation with the distribution of myxobacteria,which may be due to the fact that the number of isolated myxobacteria did not represent the actual number of myxobacteria in the soil samples or the influence of other undetected environmental factors.The strain HQK8-3 of Phytophthora infestans was used to dectect the antibiotic activity of the purified myxobacteria by the plate confrontation culture method.The results showed that 83% of the myxobacterium strains in central Inner Mongolia had the activity againt P.infestans,of which the proportion of the strains belonging to Myxococcus and Corallococcus was higher,the proportion of the strains belonging to Cystobacter and Melittangium was in the medium,and the proportion of the strains belonging to Pyxidicoccus and Archangium were lower than those of the cases.The antagonistic activity of the strain B25-I-1,B25-I-3 and X6-II-1 against P.infestans was significant.The strain B25-I-1,B25-I-3 and X6-II-1were further identified as Myxococcus xanthus,Myxococcus fulvus,and Myxococcus stipitatus by morphological characteristics,physiological and biochemical characteristics and 16 S rRNA gene sequence analysis.Antibacterial and antifungal activity analysis showed that strain B25-I-1 and B25-I-3 could kill and dissolve Escherichia coli,and showed a different degree of inhibition to Bacillus subtilis、Fusarium oxysporum、Rhizoctonia solanikuhn、Sclerotinia sclerotiorum and Verticillium dahliae,but did not exhibit inhibitory activity to Staphylococcus aureus and Saccharomyces cerevisiae.The strain X6-II-1 could also kill and dissolve the Escherichia coli,and showed the antibiotic activity against Bacillus subtilis and Fusarium oxysporum,but did not show inhibitory activity on the other indicator microorganism.The detection of growth promoting ability indicaed that the strain B25-I-1,B25-I-3 and X6-II-1 did not show the growth promoting effect on the production of ammonia,urease,indolo acetic acid,hydrogen cyanic acid and chitinase and the solubilization of phosphate,but all showed the ability of producing siderophores.The growth curves of the strain B25-I-1,B25-I-3 and X6-II-1 were measured by weighing method.It was shown that the biomass of the strain B25-I-1,B25-I-3 and X6-II-1 reached the maximum value respectively at sixth,seventh and seventh day of inoculation.The distribution of active substances produced by the strain B25-I-1,B25-I-3 and X6-II-1 was detected by filter paper disc mthod.The results showed that the antagonistic substances produced by strain B25-I-1,B25-I-3 and X6-II-1 distributed outside the cell.The fermentation parameters of three strains were studied by single factor analysis and orthogonal optimization.The results showed that the optimum fermentation conditions of strain B25-I-1 were as follows: temperature 32 ℃,incubation time 8 d and inoculation amount 10%;the optimum fermentation conditions of strain B25-I-3 were temperature 30 ℃,incubation time 7 d and inoculation amount 10%,and the optimum fermentation conditions of strain X6-II-1 were temperature 30 ℃,incubation time 7 d and inoculation amount 10%.The stability of the antibiotic activity of the active substances in the concentrated fermentation broth of strain B25-I-1 and B25-I-3 against P.infestans was tested by filter paper disc method.The results showed that the active substances produced by strain B25-I-1 and B25-I-3 were highly tolerant to temperature,the treatment of protease K,ultraviolet light and natural light,and were easy to be preserved for a long time under low temperature conditions.The antibacterial and antifungal activity analysis of the active substances showed that the secondary metabolites against P.infestans produced by the strain B25-I-1 and B25-I-3 exhibited different degrees of antagonism against Escherichia coli、Staphylococcus aureus、Bacillus subtilis、Saccharomyces cerevisiae、Fusarium oxysporum、Rhizoctonia solanikuhn、Sclerotinia sclerotiorum.The study of the biocontrol mechanism of the active substances showed that the active substances produced by two strains had strong inhibitory effect on the asexual reproduction and sexual reproduction of P.infestans.In addition,they could reduce the content of soluble proteins and the activity of the protective enzymes(PPO,POD,PAL,SOD)in P.infestans,and increase the oxidative damage and permeability of cell membrane.All of which may be the biocontrol mechanism of the active substances of strain B25-I-1 and B25-I-3 against P.infestans.The preventing diseases effects of active substances produced by strain B25-I-1 and B25-I-3 on detached potato leaves were detected.The results showed that the active substances produced by strain B25-I-1 and B25-I-3 could inhibit the infection of the detaced potato leaves by P.infestans significantly and had no damage to the leaves of different varieties of potato.The active substances produced by strain B25-I-1 and B25-I-3 were separated by TLC and HPLC,and the components with antibiotic activity were detected by HPLC-MS.It was found that the antagonistic components of strain B25-I-1 and B25-I-3 contained methyl(2R)-2-azido-3-hydroxyl-2-methylpropanoate and N-(3-Amino-2-hydroxypropyl)-N-methylsulfuric diamide.In this study,the diversity of myxobacteria resources in the central region of Inner Mongolia was studied for the first time,and on this basis,the strains with significant resistance to P.infestans were screened and their resistance to potato late blight pathogen was analyzed.The completion of this work may provide basic data for enriching our country’s myxobacteria resources and the research and development of biopesticides originated from myxobacteria resources against potato late blight and other Phytophthora diseases. |
