| Winter wheat-summer maize double cropping is the main cultivation method in north China.Since 1990,the technology of straw returning to field was popularized,and the area and quantity of the straw became more and more,with more serious occurrence of Wheat Sheath Blight(WSB)year by year.For a long time,the advantages and disadvantages of straw returning to the occurrence of WSB have been controversial.In this paper,after the separation and identification of the population structure of Rhizoctonia isolates in straw returning field with more than 15 years in Hebei,Shandong and Henan provinces,temporal and spatial distribution of the dominant pathogen,R.cerealis,in the system that winter wheat-summer maize double cropping were analyzed using real-time quantitative PCR technique.Then,field plot experiments were conducted to determine the composition of microbial communities in wheat rhizosphere soils with different straw returning years with 16S rRNA and ITS sequences used for deep pyrosequencing,and the correlation between microbial community structure and occurrence of WSB was also evaluated.The species and content of major organic compounds in wheat rhizosphere soil were detected by GC-MS technology.The allelopathy of compound with relatively higher content on the biological characteristics and virulence related factors of R.cerealis were further investigated by indoor bioassay tests.Meanwhile,the dynamic changes of major allelochemicals in wheat rhizosphere soils were monitored.The purpose of this study is to analyze the chemical ecological mechanism of the sharpening of WSB every year under straw returning conditions,and to provide reference for improving the comprehensive prevention and control technology system of the disease.The main results are as follows:1.284 strains Rhizoctonia isolates including dicaryon R.cerealis(247 strains,86.97%of the total)and multinuclear R.solani(37 strains,13.03%of the total)from 105 wheat fields under straw application(≥15 a)in 47 cities in Hebei,Shandong,Henan provinces were isolated,purified and identified.All the strains could be classified into four groups:AG-D,AG-B(0),AG-2 and AG-4,and the number of every group was 219,28,22 and 15,respectively,accounting for 77.11%,9.86%,7.75%and 5.28%,respectively.The percentage of the strong virulence strain,AG-D,decreased by more than 11%,compared with the previous reports.However,the relative proportion of R.solani increased significantly by 12.4%.2.284 Rhizoctonia isolates and 6 standard strains were divided to five pathogenic types including VT1,VT2,VT3,VT4,and VT5,according to the disease index of WSB caused by inoculating the all pathogens to Shixin 828(high susceptible),Jimai 22(medium susceptible)and Zhoumai 22(medium resistance).After inoculating the Rhizoctonia isolates in the rhizosphere soil,the average disease index of each pathogenicity type was 25.67,40.42,45.78,49.45,and 58.55,respectively,with the amount of 19,41,53,78,and 93,accounting for 6.69%,14.44%,18.66%,27.46,and 32.75%,respectively.The pathogenicity of the 284 strains for the three wheat varieties in Hebei,Shandong,and He’nan provinces was the strongest to Shixin 828,following with Jimai 22 and Zhoumai 22.The pathogenicity of the strain isolated in Henan province was the strongest,and the pathogenicity was the weakest in Hebei provinces.3.Under the condition of straw returning to field,the distribution of R.cerealis in the plant body,root system and rhizosphere soil at different growth stages of wheat was significantly different.The distribution of R.cerealis in the wheat plant showed a trend of increasing first and then decreasing.At the three-leaf stage,the DNA content of R.cerealis was the lowest,and the highest content was 3774.60 ng/g fresh tissues at the standing stage.At the heading stage,the DNA content of R.cerealis was reduced to 2518.93 ng/g fresh tissue.The distribution of R.cerealis in roots also increased firstly and then decreased with the highest content at the jointing stage.The content of R.cerealis in wheat rhizosphere soil and aboveground parts of maize plant body both showed a gradually increasing trend with the value of 310.90 ng/g fresh tissue and 1499.43 ng/g fresh tissues,respectively.The content of R.cerealis in maize rhizosphere soil showed a trend of decreasing firstly and then increasing.At the tasseling stage,the content was the lowest,with the value of 170.63 ng/g fresh tissues.4.The disease incidence and disease index of WSB were both gradually increased under the condition of straw returning to field for a long time.In the field that the straw returning for four years,the disease index of WSB increased by 2.3%and 8.9%respectively,than that in the field with no straw returning at the returning green stage and at jointing stage.In the field that the straw returning for twenty two years,the disease index of WSB increased by 11.5%and 20.8%respectively,at the mentioned the stages.The community structures of bacteria and fungi in the rhizosphere soil of wheat significantly changed,where the straw returning to field for 22 years.With the extension of straw returning period,the diversity of fungi increased significantly,while the bacteria diversity was significantly reduced.There was a negative correlation between the WSB occurrence and the abundance of bacteria,and a positive correlation with the abundance of fungi.The correlation indexes were 0.5576 and 0.9525,respectively.5.With the increase of straw returning period,the abundances of Acidobacteria,Firmicutes,Bacteroidetes,and Actinobacteria in the wheat rhizosphere soil were significantly reduced,but the abundance of Verrucomicrobia increased significantly.With the increase of straw returning period,the abundance of Ascomycota was increased gradually,while the abundance of Zygomycota gradually decreased.The relative abundance of potential biocontrol bacteria groups,such as Flavobacterium and acidophilus GP4,were negatively correlated with the years of straw returning to field,while the relative abundance of Rhizoctonia was positively correlated with the years of straw returning.The composition of microbial communities in the rhizosphere soil of wheat was changed under the condition of straw returning to field for a long time,which the transformation was conducive to occurrence of WSB.6.There were mainly 27 kinds of organic compounds in wheat rhizosphere soil,which contained organic acids,esters,amides,alkanes,alcohols,and aldehydes,with the relatively high content,which were account for 44.52%,15.98%,9.20%,2.39%,0.52%and 0.12%of the total compound content in the field with straw returning in Hebei,Shandong and Henan province.3-phenyl-2-propenoic acid,caproic acid,dibutyl phthalate,4-hydroxy-3,5-dimethoxybenzoic acid and o-hydroxy benzoic acid all had an obvious allelopathy on R,cerealis.3-phenyl-2-acrylic acid contributed to the mycelial growth,sclerotia number and fresh weight,cellulase activity of R.cerealis significantly at the concentration of 0.001 μg·mL-1-0.5μg·mL-1,and the contribution rate was from 1.4%to 45.9%.There was little effect on the activity of pectinase and xylanase after being treated by the 3-phenyl-2-acrylic acid at the higher concentration.The mycelial growth,sclerotia number and fresh weight of R.cerealis were promoted by the caproic acid at the lower concentration,with no effects on cellulase,pectinase and xylanase.The mycelial length,sclerotia number and fresh weight of R.cerealis were 5.6 cm,18.6 per dish,and 20.5 mg,respectively,after being treated by Dibutyl phthalate,which had little difference with those of the control,while the activities of cellulose and pectinase increased by 8.6%-25.8%and 13.5%-39.7%,respectively.Hydroxybenzoic acid promoted the mycelial growth,and showed a significant inhibitory effect on sclerotia number and fresh weight.The mycelial growth was inhibited by 4-hydroxy-3,5-dimethoxybenzoic acid,and the sclerotia number and fresh weight were promoted,significantly.The two allelochemicals had no significant effect on the three cell wall degradation enzymes.7.After pretreatment with 3-phenyl-2-propenoic acid and hexanoic acid,the mycelial infection rate of R.cerealis in the stem base of Liangxing 99 was significantly increased,and the occurrence of WSB was also significantly increased.Compared with the treatment of inoculating for 15 d,the mycelial infection rate in leaf sheath,middle column thin wall and vessel wall cells were increased by 13.4%,12.5%and 16.7%,respectively,after being treated by irrigating 3-pheny1-2-acrylic acid and inoculating together.At the same time,the mycelial infection rate increased by 8.1%,10.7%and 19.5%respectively,after being treated by irrigating caproic acid.The mycelial infection rate only in the cell mycelial tissue increased significantly after being treated by irrigating dibutyl phthalate for 15 days.But,the mycelial infection rate in the epidermis and the middle column parenchyma cells did not change significantly.The damage of cell wall tissue and vessel wall in the middle column was significantly aggravated.8.From the seedling stage to the maturity stage,the content of caproic acid,3-phenyl-2-acrylic acid,hydroxybenzoic acid and 4-hydroxy-3,5-dimethoxybenzoic acid were all increased at first and then decreased in wheat rhizosphere soil in straw returning field in Hebei,Shangdong and Henan provinces.The four allelochemicals reached the highest values at the tillering stage,with the value of 3.25 μg·mL-1,1.45 μg·mL-1,58.76 μg·mL-1 and 0.22 μg-mL-1,respectively.However,the dibutyl phthalate showed a downward trend,which values were 55.30μg·mL-1 and 22.45 μg·mL-1,respectively,at the seedling stage and at the maturity stage,with a decrease of 146.25%.In summary,the R.cerealis AG-D with strong pathogenicity,the increase diversity of fungi and the reduction diversity of bacteria in the wheat rhizosphere soil caused by the straw returning to field,and the promotion effects of allelochemicals to the pathogenicity of R.cerealis might be the main reasons for the occurrence of WSB in straw returning to field. |
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