| Maize(Zea mays L.)seeds comprise micro-scale ecosystems that sustain fungal communities and are highly ubiquitous and abundant,which plays an integral role in plant growth and health.Some fungi that infect crop kernels also produce toxic secondary metabolites(mycotoxins),directly causing mildew,discoloration and low germination rate year by year.In addition,many breeding resources are also lost frequently because of the same problem.Pathogenic fungi are particularly problematic,as they are responsible for the re-emergence of past diseases,the movement of diseases across administrative borders,and the introduction of diseases into new areas,posing tremendous threats to maize production.However,the time dynamics of fungal communities(mycobiota)associated with maize seeds are poorly understood.In this study,we examined the inter-annual variability in fungal communities of maize seed samples(Zheng 58)with varying storage times ranging from 6 months to 12 years.To understand the pathogenicity,pathogenesis and genetic diversity of seed-borne Fusarium,dominant fungal Fusarium species from the time-series samples were examined.The results are as follows:1.We investigated the mycobiota of 11 maize seed samples with storage times ranging from 6 months to 12 years.Mycobiota were characterized by a culture-based approach,and fungal species were identified through r DNA-ITS sequence analyses.From a total of 169 pure fungal isolates obtained from both the seed surface and internal tissues,we identified 16 distinct species(belonging to 10 genera)associated with maize seeds,all but one of which were ascomycetes.Among these species,seven were exclusively isolated from internal tissues,two species were isolated only from the seed surface,and another six species were isolated from both the surface and internal tissues.Aspergillus niger was consistently found under all storage conditions and dominated fungal communities with a relative abundance of 36%–100%.Species of Fusarium(9%–40%)and Penicillium(9%–20%)were also frequently isolated,but other species appeared sporadically and were isolated from fewer than three seed stocks.According to our results,while the overall incidence of fungal infection generally declined with storage time,there was no consistent association between seed storage time and fungal species richness or relative abundance;furthermore,the composition of the mycobiota associated with maize seeds was highly variable among the samples.The detection of the four major mycotoxigenic fungal genera,specifically Aspergillus,Fusarium,Penicillium,and Alternaria,was alarming,and the isolation of a potential controlling agent as well as information about their temporal occurrence will contribute to the management of mycotoxins in the future.In addition,based on the analysis of correlation between the rate of seeds with endophytic fungi and seed vigor indexes,67% fungi effected negatively on seed vigor;and 11% endophytic fungi effected positively on seed vigor,which could promote germination and seedling growth of maize seed.2.We sampled 45 Fusarium strains associated with maize seeds both externally and internally,with storage times varying from six mouths to 12 years,to analyze the genetic differences and phylogenic relationship intra-or inter-species based on r DNA-ITS sequence data and inter-simple sequence repeats(ISSR).The results showed that 293 fragments were amplified from 45 isolates with 15 screened primers,among them,the number of polymorphic loci was 276,which accounted for 94.2% in the total amplified fragments.Each primer amplified a different number of fragments between 400~3000bp.The cluster analysis suggested that the genetic similarity coefficient of 45 isolates was from 0.55 to 0.93.All the test strains were divided into two ISSR groups at genetic similarity of 0.70.Group ? included 17 isolates in F1 by r DNA-ITS sequence analysis,and the group ? had 4 isolates in F2.Therefore,there were certain relativity between ISSR group partition and species identification.In the same ISSR group,a high genetic similarity existed in isolates from same part or storage time of seed,while the genetic similarities of isolates from different part or storage time of seed were low.The genetic diversity of Fusarium isolates were correlated obviously to their separation parts and storage time of maize seed.3.Fusarium spp.is one of main pathogenic fugus of maizeseeds mycobiota.However,the pathogenic activities of seed-bore Fusarium species with storage time of seeds are poorly understood.In the present report,we sampled 36 Fusarium spp.strains associated with maize seeds both externally and internally,with storage times ranging from six mouths to 12 years,to examine pathogenicity inter-or intra-species based on seed germination rate,average germination days,seedling growth in lab,as well as germination rate,average germination days,seedling blight,ear rot disease,economic characteristics of ear in the field.The main results are as follows:(1)According to our results,the germination rate of seed infected by Fusarium spp.in lab were significantly higher than that in field.And the germinatin rate of different treatments of the strains from 8~12 year were not obviously different from the control,but were distinctly higher than that from 0.5~5 year,which increased with the extension of storage time.Moreover,average germination time of seeds infected by the Fusarium strains from 0.5~5 year was 13.6~15.6d,which was longer 1~3d than control in field.However,average germination time of seeds infected by all Fusarium strains was only 3.7~4.0d in lab,and there was no evident differences between all stains and control.Except for individual strains,the height and fresh weight of seedling treated by the strains from 8~12 year were obviously higher than from 0.5~5 year,which increased with extention of storage time.While the main root length,root fresh weight and root crown rate treated by all strains generally declined with storage time,there was no consistent association between seed storage time and diameter of seedling or the number of fibril.(2)The incidence and disease index of seedlings caused by different Fusarium strains in lab as well as the incidence of seedling blight and ear rot caused by Fusarium strains in fieldmaintained high consistency,and they all generally decreased with storage time.(3)For the economic characteristics of the ear,the yield of different treatments were evidently different from each other,which increased with the storage time.Similarly,effect of all Fusarium strains on ear length,ear diameter,rows each ear,kernels each row and kernels each ear were the same as yield.However,there was no consistent association between seed storage time and weight of thousand kernels.(4)For pathogenicity of the Fusariumspeciesfrom same(or similar)storage time and same seperated part,from seed vigor index,incidence of seedling, seedling blight,ear rot disease,as well as economic charateritics,there were no significantly relationship between F.verticillioides and F.proliferatum strains.In addition,for the seperated part of Fusarium strainsfrom same(or similar)storage time,pathogenicity of most Fusarium strains from external surface of seeds by several indexeswere stronger than ones from internal tissues.4.To understand pathogenic mechanism of F.verticillioides from internal tissues of seeds with varying storage years,the main physiological and biochemical characteristic of seedling using sand culture method were determined by inoculating maize seeds with spore suspension.By comparing with the controls,increased relative permeability of plasma membrance,activity of POD,content of soluble sugar,MDA,proline and soluble protein were found in this study.And decreased activity of SOD,content of chlorophyll and reductive sugar were detected.In addition,the activity of SOD,the content of chlorophyll,MDA,proline and soluble protein were positively correlated with the storage time of the seeds with F.verticillioides.However,the activity of POD,relative permeability of plasma membrance,content of soluble sugar related negativly to the storage time of the seeds with F.verticillioides.Moreover,there was no obviously relativity between the content of reducing sugar of seedling and the storage time of the seeds with F.verticillioides. |
PDF Full Text Request