Studies On The Infection Pathway And Pathogenic Difference Of Fusarium From Stalk Rot And Ear Rot On Maize

Both stalk rot and ear rot on maize are the important diseases,and they can occur in each maize growing area in China.With the promoting of straw returned policy in China,the pathogen in straw can survive into the soil that cause the increase of pathogen in soil,which led to the disease of stalk rot and ear rot happen heavily year by year.Stalk rot on maize can cause plants susceptible to lodging that influence the grain weight,causing yield loss;ear rot on maize can not only decrease the yield loss,but also affect maize quality by the toxins,the infected kernels can threaten the health of human and animal.Both stalk rot and ear rot on maize can be caused by Fusarium graminearum and Fusarium verticillioides,and the same Fusarium between these two diseases can be affected with each other.At present,there have been few studies on the relationship between the infection cycle of stalk rot and ear rot.In this study,we accordingly aimed to clarify the genetic relationship,pathogenicity and infection cycle of these diseases in maize systematacially.We studied the infection cycle of Fusarium in maize by using EGFP expressing strains that infect maize seeds,survey the infection status through the whole growing season,to clarify the relationship between the diseases caused by the same Fusarium,which provides a basis for control the diseases and disease-resistant breeding that makes great significance.1.Population structure and genetic diversity of Fusarium from maize stalk and ear rot strains in northeast ChinaA total of 295 Fusarium were obtained from samples of stalk rot and ear rot in maize,which collected from 42 areas in Liaoning Province,Heilongjiang Province,the Inner Mongolia Autonomous Region in northeast of China.Molecular identification of Fusarium isolates were using EF-1?sequencing that based on morphological characters which showed that the isolates belong to 7 species:F.graminearum,F.verticillioides,F.subglutinans,F.proliferatum,F.equisetu,F.temperatu,and F.incarnatum.Among all the species,F.graminearum was the most prevalent species of stalk rot in maize,and F.verticillioides was the most prevalent species of ear rot in maize.ISSR molecular marker technology was able to distinguish the Fusarium graminearum species complex at the subspecies level,however,it did not distinguish isolates that caused different diseases;to analyze the F.verticillioides isolates,ISSR was also unable to discriminating between isolates of these two diseases.2.The same specie Fusarim from stalk rot and ear rot showed similarities and differences in pathogenicityPathogenicity assays were conducted in three groups:corn stalk rot at adult stage and corn ears at milk-stage,the same specie Fusarim from different diseases showed similarities and differences in pathogenicity.In the corn stalk rot at seedling stage assay,the disease severity index of seedlings inoculated with stalk F.graminearum isolates was slightly higher than that of seedlings inoculated with F.graminearum isolates from infected ears,whereas the pathogenicity of F.verticillioides isolates were nearly identical;in the corn stalk rot at adult stage assay,pathogenicity of F.graminearum isolates from ear rot was higher than F.graminearum isolates from stalk rot,and F.verticillioides from these two diseases showed little difference;in the corn ears at milk-stage assay,pathogenicity of F.graminearum isolates from these two diseases showed slight difference,while pathogenicity of F.verticillioides isolates from ear rot was higher than it from stalk rot.3.The EGFP gene expression vector constructed and transformSeamless cloning technology was used in constructing the veator,which contained hygromycin B resistant marker.The plasmid containing the EGFP gene was then transformed into F.graminearum and F.verticillioides from corn stalk rot and ear rot using the PEG-CaCl₂ mediated transformation method,electroporation and agrobacterium mediated transformation.The transformants showed stable resistance against hygromycin B and fluoresced under blue light after continuous culturing for five generations.4.The wild-type strains and their transformants showed similarities and differences in biological phenotypeF.graminearum and F.verticillioides wild-type strains and their transformants showed no significant differences growth situation in different pH PDA culture medium.The conidiation of F.graminearum wild-type strains was higher than the transformants,while the conidiation of F.verticillioides transformants FV-C79 was little difference to the wild-type strains,and the conidiation of transformants FV-D108 was higher than the wild-type strains.The activity of cell wall degradation enzymes showed similarities and differences,among all the transformants,the cellulose and pectinase activity was higher than the wild-type strains;the polymethyl galacturonase activity of F.graminearum transformants was higher than the wild-type strains,while the polymethyl galacturonase activity of F.verticillioides transformants was lower than the wild-type strains.Pathogenicity between wild-type strains and their transformants showed no significant differences.5.Observing the infection process of Fusarium transformants with EGFP gene expressed in maizeBy observing under a fluorescence microscope,inoculation after 6-12 h,the conidium began to germination and growth,non-invaded into the seeds,after 24 h,the conidium were almost fully germination,and after 48 h,mycelium formed reticulate structure.During early days of corn seeds germination,mycelium formed specific organization to break the seeds epidermis that invaded into the host,and colonization into the corn with continuously extended.For the overall level of the infection cycle,F.verticillioides can infect corn seeds and spread to the kernel,causing ear rot disease,so do the F.graminearum obtained from ear rot,whereas F.graminearum obtained from stalk rot cannot completely spread to the kernel cause ear rot disease.Re-isolate and purified the isolates from fluorescent kernels,and inoculated to corn seeds,the results indicated that the re-isolated isolates can cause a second infection and thereby initiating an infection cycle.