Study On Control Technology And Mechanism Of Chrysanthemum Continuous Cropping Obstacles

Chrysanthemum(C.morifolium Ramat.)is a perennial root herb belonging to the Chrysanthemum genus in the Asteraceae family.Chrysanthemum is native to China and is popular in worldwide,with strong cultural heritages and displays a wide range of variability among its cultivars due to a wide range of colors,size and flower patterns.Chrysanthemum is an important ornamental plant,ranked as one of the traditional ornamental flowers in China and one of top four for cut flowers in the world.However,sustained monoculture often leads to the inhibition of plant growth,the decrease of the soil microbial diversity and changes in soil microbial community composition,particularly to the accumulation of soil-borne pathogens,which,in turn,results in declines in chrysanthemum growth and more frequent incidences of Fusarium wilt disease,causes significant decrease in the yield and quality of chrysanthemum and serious economic losses to the chrysanthemum growers.Due to the lack of scientific guidance,facilities often applied excessive agro-chemical products in the prevention of chrysanthemum Fusarium wilt disease and improvement of the continuous cropping soil.As a result,the abuse of agro-chemical products inevitably increased the cost of chrysanthemum cultivation and brought about the pollution of soil ecological environment.In this study,the chrysanthemum ’Jinba’ was used as the plant material,and a series of field experiments were conducted in the site which had been cropped continuously with chrysanthemum for more than 5 years and was heavily suffered with Fusarium wilt disease,to screen the integrated optimization of control technology about chrysanthemum continuous cropping obstacles and Fusarium wilt disease,and to study the control mechanism.In addition,the function of CmWRKY1,CmWRKY6 and Cm WRKY8 responding to infection by F.oxysporum in chrysanthemum were preliminarily verified.The main contents and conclusions are as follows:1.Twelve contrasting greenhouse experiments were conducted,which were as follows:different depth of soil tillage,30 cm(30dp)and 40 cm(40dp)respectively;Bio-organic fertilizer treatments with different dosage were 0.75 kg/m2(BOF1),1.50 kg/m2(BOF2),2.25 kg/m2(BOF3)and 3.00 kg/m2(BOF4);Organic fertilizer treatments with different application amounts were 0.75 kg/m2(OF1),1.50 kg/m2(OF2),2.25 kg/m2(OF3)and 3.00 kg/m2(OF4);Different chemical fungicide treatments,namely ’Dazomet’(DAZ)and’Carbendazim’(MBC).Through these field experiments,compared the different treatments on the growth index and incidence of Fusarium wilt disease of chrysanthemum and on the value of soil enzyme activities and the contents of available nitrogen,available potassium and organic matter in continuous cropping soil.The results indicate that,among all different treatments,the DAZ application had the most significant promotion effect on the growth of cut chrysanthemum,and has the lowest incidence of the Fusarium wilt disease.However,compared with control,the contents of available nitrogen,available potassium and organic matter after DAZ treatment decreased by 0.99%,22.25%and 2.77%,respectively.And the catalase activity was also significantly decreased after DAZ treatment.Among different amounts of bio-organic fertilizer or organic fertilizer treatments,BOF2(1.50 kg/m2)and OF2(1.50 kg/m2)treatment can not only promote the growth of cut chrysanthemum,reduce the incidence Fusarium wilt disease of chrysanthemum,but also improve the nutrient content and soil enzyme activities in continuous cropping soil.The improvement effect of BOF2 on the continuous cropping soil was better than that of OF2.In different soil tillage treatments,the incidence of Fusarium wilt in 30dp and 40dp decreased by 18.70%and 42.18%,respectively.Soil tillage had a positive effect on the growth of chrysanthemum,soil nutrients content and enzyme activities in continuous cropping soil.Moreover,soil tillage of 40 cm(40dp)was better than that of continuous cropping soil of 30 cm(30dp).Our findings preliminary screening for three chrysanthemum continuous cropping soil improvement treatments with better effect,namely,DAZ,BOF2 and 40dp,from the commonly used continuous cropping soil improvement measures.It provides a basis for rational application of organic fertilizer and microbial organic fertilizer in soil improvement of continuous cropping of cut chrysanthemum.Meanwhile,it also provides guidances for the control of chrysanthemum Fusarium wilt disease.2.A series of greenhouse experiments were conducted to investigate the practical effects of application of bio-organic fertilizer in the amount of 1.50 kg/m2(B),application of Dazomet in the amount of 30 g/m2(D),application of Dazomet 30 m2 in combination with bio-organic fertilizer 1.50 kg/m2(DB)and tilling the soil down to a depth of 40 cm in combination with application of Dazomet 30 g/m2 and the application of bio-organic fertilizer 1.50 kg/m2(DB+40dp)the on chrysanthemum growth,with a focus on the potential mechanisms underlying the responses of the soil microbiome.The results showed that DB+40dp treatment significantly(P<0.05)increased the plant height,dry weight of plant,dry weight of root,diameter of flower,and ray floret number when compared with other treatments.And compared with the control,the incidence of Fusarium wilt disease was decreased by 91.68%in DB+40dp treatment.And the DB+40dp treatment enhanced the activities of catalase,urease,sucrase,and β-D-glucosidase,and significantly increased the levels of available nitrogen,phosphorus,and potassium in the soil.The weighted and unweighted UniFrac distances in the principal co-ordinates analysis(PCoA)revealed that soil bacterial and fungal community compositions were separated according to the treatments.The abundance of genera potentially expressing growth-promotion,such as Pseudomonas and Bacillus,was increased in the DB+40dp treatment.The redundancy analysis(RDA)implied that the composition of the microbiome was correlated to soil enzymatic activities and soil potassium availability in the rhizosphere soil of chrysanthemum plants.Our findings suggest that the DB+40dp treatment is a better strategy for improving chrysanthemum growth and regulating the rhizosphere microbiome in monoculture soils than the methods presently employed by commercial chrysanthemum producers.3.A strain of bacteria was isolated from the rhizosphere soil of a healthy ’Jinba’ by gradient dilution coating,and it was named K.Through the morphological observation and the sequence analysis of 16S rDNA,the strain was preliminarily identified as Bacillus subtilis,and was further named as B.subtilis-K.The strain B.subtilis-K was co-cultured with pathogenic Fusarium oxysporum-B2 on PDA plate,and the results showed that B.subtilis-K had obvious inhibitory effect on the growth of F.oxysporum-B2.The strain B.subtilis-K was labeled with GFP to further study the inhibitory mechanism of B.subtilis-K on the growth of F.oxysporum,and the strain was named as B.subtilis-K-GFP.The plant height of chrysanthemum were significantly promoted(P<0.05)after B.subtilis-K-GFP inoculation using root-drenching method.The colonization of B.subtilis-K-GFP on the root tips of cut chrysanthemum was observed on the 7th and 14th days after inoculation.And the colonization of B.subtilis-K-GFP in the rhizosphere soil was stable at 104 cfu/g on the 14th day after inoculation.The results of this study provided reference for the biological control of chrysanthemum Fusarium wilt disease and improved the understanding of the colonization dynamics of antagonists in rhizosphere soil.In addition,it provides a basis for further evaluating the potential of the strain to promote plant growth and disease control under field conditions,as well as its application as biofertilizer and biological control agent.4.Based on the full-length cDNA sequence of CmWRKY1,CmWRKY6 and CmWRKY8 previously obtained by our laboratory,the gene fragments of CmWRKY1,CmWRKY6 and CmWRKY8 were cloned(Each gene fragment was 400bp),and the pYL156-GFP-CmWRKY1/6/8 silencing vector were constructed.Solution(OD=1.5)of Agrobacterium tumefaciens transformed with pYL156-GFP-CmWRKY1/6/8 and pYL156-GFP(control)plasmids were mixed with TRV1 in a ratio of 1:1,then the Chrysanthemum nankingense was transformed by vacuum osmotic method.The results showed that the green fluorescence signal of GFP could be observed in the newly appeared leaves of C.nankingense 10 days after infection and the expression of CmWRKY1,CmWRKY6 and CmWRKY8 gene in leaves with green fluorescent signals were 0.31,0.21 and 0.16 times that of the control,respectively,which indicated that the CmWRKY1,CmWRKY6 and CmWRKY8 gene were successfully silenced.When compared with pYL-GFP(control),the Fusarium wilt disease symptoms of pYL156-GFP-CmWRKY6 and pYL156-GFP-CmWRKY8 were obvious severe after infection of F.oxysporum-B2,which indicated that Cm WRKY6 and CmWRKY8 gene played an important role in the infection of the F.oxysporum.The results of this study provided a new idea for the control of Fusarium wilt disease in chrysanthemum and preliminary identified the candidate genes for molecular breeding of continuous cut chrysanthemum resistant varieties.