Pathogen Identification,Screening Of Prevention And Control Agents And Preliminary Study On Pathogenic Mechanism Of Brasenia Schreberi Leaf Rot

In recent years,diseases have become the main reason for limiting the development of Brasenia schreberi industry in Lichuan,Hubei.In this study,during the disease survey of Brasenia schreberi lactuca production area in Lichuan from2020 to 2021,it was found that the disease of Brasenia schreberi lactuca in Lichuan production area was mainly leaf rot with incidence rate mostly above 60%and some fields with incidence rate up to 100%,which seriously restricted the development of Brasenia schreberi lactuca industry in Lichuan.At present,there are very few reports on Brasenia schreberi disease at home and abroad,and there is no systematic naming of its disease species,while the species of Brasenia schreberi disease pathogens and their pathogenic mechanisms are not clear.Lack of scientific and reasonable control measures when diseases appear in the field.Therefore,in this paper,we isolated and identified Brasenia schreberi leaf rot pathogenic leaves,determined their biological properties,screened control agents by indoor virulence assay,and investigated the role of cell wall degrading enzymes in the pathogenic process of Brasenia schreberi leaf rot pathogens.The main results are as follows:（1）Pathogen identification and biological characterization:The pathogenic fungus was isolated from the leaf of Brasenia schreberi lactuca in Lichuan,Hubei,and was identified as strain YB-2 by pathogenicity assay,and was identified as Phytopythium helicoides by morphological identification and internal transcribed spacer（ITS）gene sequence analysis.The mycelial growth rate method was used to determine the biological characteristics of Brasenia schreberi leaf rot,and it was clear that different media,p H,temperature,light conditions,carbon and nitrogen sources had significant differences on the mycelial growth of the fungus.The optimum medium is V8 medium;the mycelial growth rate reaches its peak at p H=6,the optimum p H range for mycelial growth is 5-7;the growth temperature is 35°C,the mycelial growth rate reaches its peak,the optimum growth temperature for mycelia is 30-35°C,the optimum light condition is 12 h alternating light and dark,the optimum carbon source is sucrose,the optimum nitrogen source is glycine.（2）Screening of agents for indoor control of Brasenia schreberi leaf rot:determination of the inhibitory effect of six fungicides on the mycelial growth of Brasenia schreberi leaf rot using a toxic media-containing method.The results showed that 25%pyrimethanil suspension had the strongest inhibitory effect on the mycelial growth of Brasenia schreberi leaf rot with an EC₅₀value of 7.69μg/m L,followed by 500 g/L mildew emulsion with an EC₅₀value of 89.64μg/m L.（3）Analysis of the pathogenesis of Brasenia schreberi spp.leaf rot:The reducing sugars released by the enzymatic reaction were determined by DNS colorimetric method and the activities of four cell wall degrading enzymes,carboxymethyl cellulase（Cx）,β-glucosidase,polygalacturonase（PG）and polymethylgalacturonase（PMG）,measured experimentally were calculated.The results of in vitro induction of cell wall degrading enzymes activity by different inducing substrates showed that all the four cell wall degrading enzymes mentioned above could be produced by Brasenia schreberi leaf rot under the culture conditions containing different inducing substrates.Different induction substrates led to differences in the activities of cell wall degrading enzymes produced by Brasenia schreberi leaf rot.PMG activity was highest among the four cell wall degrading enzymes produced by Brasenia schreberi leaf rot when pectin,orange peel and Brasenia schreberi leaves were used as induction substrates;Cx activity produced by Brasenia schreberi leaf rot was highest when filter paper was used as induction substrate;and PMG activity produced by Brasenia schreberi leaf rot was highest when Brasenia schreberi leaves were used as induction substrate.Therefore,it can be predicted that PMG plays a major role in the process of Brasenia schreberi leaf rot infestation of Brasenia schreberi leaves.The leaves were artificially inoculated with Brasenia schreberi leaf rot and the changes of four cell wall degrading enzymes activities in the leaves were measured using DNS colorimetric method for 1d,3d and 5d in healthy leaves inoculated with Brasenia schreberi leaf rot,respectively.The results showed that four cell wall degradation enzymes induced in vitro could be produced after infection of Brasenia schreberi leaf rot bacteria.During the whole infection process,the activity of PMG on the diseased leaves of Brasenia schreberi was much higher than that of Cx,β-glucosidase and PG,and its activity increased with the extension of the infection time of Brasenia schreberi leaf rot bacteria.During the infection of PG and Cx,the enzyme activity first increased and then decreased.The activity ofβ-glucosidase and PMG gradually increased with the increase of infection time of Brasenia schreberi leaf rot.The results showed that cell wall degrading enzyme was the causative agent of Brasenia schreberi leaf rot bacteria infecting Brasenia schreberi leaves,and the type of cell wall degradation enzyme that played a key role in the infection of Brasenia schreberi leaf rot by Brasenia schreberi leaf rot was PMG.