Application Of Electrophysiological Information In Prediction And Control Of Sclerotinia In Brassica Napus And Orychophragmus Violaceus

Sclerotinia caused by Sclerotinia sclerotiorum(Lib.)de Bary which has a wide host range and strong destructiveness is the most serious fungal disease in Brassica napus L.and Orychophragmus violaceus.Pathogenic Organics from plant and fungal are important pathogenic factors in the pathogenic process of Sclerotinia.At present,it is still difficult to quantify the source and contribution of pathogenic organics and the infection ability of S.sclerotiorum.Dimethachlon[N-(3,5-dichlorophenyl)-succinimid]is the first choice fungicide for controlling Sclerotinia in the production of B.napus and O.violaceus,however,its widespread and inappropriate application pose a potential risk to the ecological environment,wild animals and human beings.Therefore,it is of great significance for the efficient,safe and ecological prevention and control of sclerotinia in B.napus and O.violaceus to establish a rapid and accurate early identification and prediction method of sclerotinia,resolve the pathogenic role of pathogenic organics and the infection ability of S.sclerotiorum,as well as develop an efficient degradation technology of dimethachlon residues.In this study,the role of pathogenic organics of sclerotinia in B.napus and O.violaceus and the application of electrophysiological information in its prediction and control were studied.The main conclusions are:(1)The responses of cell membrane permeability,membrane lipid oxidation and antioxidant enzyme activity and oxalic acid in seedling B.napus,flowering B.napus and flowering O.violaceus infected by S.sclerotiorum and S.sclerotiorum conbined with an important pathogenic organic matter(oxalic acid)were investigated,and the physiological and biochemical basis of plants'electrophysiological information applied to the early prediction of Sclerotinia was clarified.Seedling B.napus,flowering B.napus and flowering O.violaceus were were used as the research objects,the physiological and biochemical parameters of B.napus and O.violaceus at different infection times were measured by inoculation of S.sclerotiorum and compound inoculation of S.sclerotiorum and OA.The results indicated that S.sclerotiorum infection significantly(p?0.01)increased electroconductibility(EC),ion extravasation percentage,superoxide anion production rate,proline(pro)content,malondialdehyde(MDA)content and OA content in the leaves of seedling B.napus,flowering B.napus and flowering O.violaceus,and their the increasing range gradually increased with the increase of the inoculation amount of S.sclerotiorum.On the whole,S.sclerotiorum infection significantly(p?0.01)decreased the activities of superoxide dismutase(SOD),catalase(CAT),peroxidase(POD)and polyphenol oxidase(PPO)in the leaves of seedling B.napus and flowering B.napus,and significantly(p?0.01)decreased the activities of SOD,POD and PPO and increased the activity of CAT in the leaves of flowering O.violaceus.0.10 m M OA promoted the change responses of cell membrane permeability,membrane lipid oxidation and antioxidant enzyme activity and oxalic acid in the leaves of seedling B.napus,flowering B.napus and flowering O.violaceus after S.sclerotiorum infection.The physiological and electrophysiological responses of seedling B.napus,flowering B.napus and flowering O.violaceus infected by S.sclerotiorum were obvious,which inevitably led to the obvious responses of their electrophysiological information,and well presented the feasibility of plants'electrophysiological information applied to the early prediction of Sclerotinia in B.napus and O.violaceus.(2)The acquisition technologies of intrinsic electrophysiological information,intracellular water metabolism information and nutrient transport information of B.napus and O.violaceus were established.The theoretical relationships between the clamping force(F)and leaf capacitance(C),and resistance(R),impedance(Z),capacitive reactance(Xc)or inductive reactance(XL)were revealed as linear model C=y₀+k₀F and 3-parameter exponential decay model R,Z,Xc or XL=y based on based on the Gibbs free energy and the Nernst equation for the first time,respectively.The intrinsic C,R,Z,Xc and XL in plant leaves were successfully monitored using the above-mentioned relationships.Subsequently,according to the intrinsic electrophysiological information of plants,the intracellular water use parameters(Intracellular water-holding capacity(IWHC),water use efficiency(IWUE),water-holding time(IWHT),water transfer rate(WTR))and nutrient transport parameters(Nutrient flux per unit area(UNF),the active transport flow of nutrient(NAF),nutrient transfer rate(NTR),nutrient transport capacity(NTC),nutrient active transport capacity(UAC))were defined to evaluate the physiological activities such as electrophysiological information,intracellular water metabolism and nutrition transport of different plants.The results demonstrated that plant's intrinsic electrophysiological information C,R,Z,XC and XL could accurately characterize the physiological states of B.napus,O.violaceus and other plants,and IWHC,IWUE,WTR,UNF,UAF,NTR,NTC and UAC could accurately reveal and characterize their intracellular water utilization strategies and nutrient transport strategies.It provided a technical support for plants'electrophysiological information applied to the early prediction of Sclerotinia in B.napus and O.violaceus.(3)The effects of S.sclerotiorum and S.sclerotiorum conbined with OA infection on electrophysiological information,intracellular water metabolism and nutrient transport of seedling B.napus,flowering B.napus and flowering O.violaceus were clarified,and the health threshold of Sclerotinia based on electrophysiological information was proposed.Seedling B.napus,flowering B.napus and flowering O.violaceus were were used as the research objects,the electrophysiological parameters of B.napus and O.violaceus at different infection times were measured by inoculation of S.sclerotiorum and compound inoculation of S.sclerotiorum and oxalic acid(OA).The results shown that S.sclerotiorum infection significantly(p?0.01)decreased C,IWHC,WTR(or NTR),UAC and NTC of seedling B.napus,flowering B.napus and flowering O.violaceus,and significantly(p?0.01)increased their Z,R,XC and XL.With the increase of the inoculation amount of S.sclerotiorum,the decreasing range of C,IWHC,WTR(or NTR),UAC and NTC and the increasing range of Z,R,XC and XL gradually increased.0.10 m M OA promoted the infection of S.sclerotiorum to rape at seedling B.napus,flowering B.napus and flowering O.violaceus.The significant changes of electrophysiological information,intracellular water metabolism information and nutrient transport information after S.sclerotiorum infected seedling B.napus,flowering B.napus and flowering O.violaceus were earlier at least 2?4 days than the symptoms of Sclerotinia.The C,Z,R,Xc,XL,IWHC,WTR(or NTR),UAC and NTC(160.71?249.04 p F,,0.16?0.19 M?,0.17?0.29 M?,0.22?0.34 M?,0.31?0.49 M?,2050.54?3952.76,72.48?92.22,42.31?71.04 and 86.47?149.27,and 165.37?199.97 p F,0.12?0.18 M?,0.13?0.18 M?,0.22?0.32 M?,0.30?0.48 M?,2126.61?2820.36,82.59?119.13,57.48?94.40 and 63.64?125.22,as well as 489.36?606.53 p F,0.07?0.08M?,0.05?0.11 M?,0.09?0.11 M?,0.06?0.18 M?,10833.99?14718.28,294.57?407.94,171.19?203.37 and 445.12?577.23)of the control groups in seedling B.napus,flowering B.napus and flowering O.violaceus could be used as their health thresholds or disease indicators,combine with the initial symptoms of Sclerotinia and predict it of B.napus and O.violaceus.(4)The role of plant pathogenic organics in the pathogenic process of S.sclerotiorum and the infection ability of S.sclerotiorum to B.napus and O.violaceus were resolved,and well proved the feasibility of plants'electrophysiological information applied to the early prediction of their Sclerotinia.B.napus and O.violaceus were used as the research objects,a two-way isotope tracer culture system was established by using sugar beet sucrose and sugar cane sucrose with large difference in stable carbon isotope values.S.sclerotiorum cultured by sugar cane sucrose and sugar beet sucrose alternately infected the tested plants cultured by sugar beet sucrose and sugar cane sucrose.And then The infection system was cultured at high?¹³C value(30‰)sucrose medium.The results indicated that the?¹³C value,stable carbon isotope fractionation value and S.sclerotiorum proliferation coefficient in infection system of S.sclerotiorum-B.napus(or O.violaceus)increased gradually with the extension of infection time(within 72 hours),indicating that the pathogenic role of plant pathogenic organics increased gradually with the extension of infection time.The disease progression(Si)based on stable carbon isotope fractionation value and S.sclerotiorum proliferation coefficient could well characterize the infection ability of S.sclerotiorum to B.napus and O.violaceus.The infection of S.sclerotiorum to B.napus was consistented with the two parameter single rectangular hyperbolic model S_i=1.9495 t/(8.8600+t),R~2=0.9997,p<0.0001,while the infection of S.sclerotiorum to O.violaceus.was consistented with the exponential growth model Si=0.1920 e ^{0.0391 t},R~2=0.9851,p=0.0030.These results indicated that B.napus and O.violaceus had different characteristics,and O.violaceus was more susceptible to S.sclerotinia than B.napus.It was consistent with the result that the electrophysiological information changes of O.violaceus infected with S.sclerotiorum were significantly faster than that of B.napus infected with S.sclerotiorum,which well proved the feasibility of plants'electrophysiological information applied to the early prediction of their Sclerotinia.(5)The prediction method of sclerotinia based on electrophysiological information and the prediction,and the prediction and control effect of sclerotinia in B.napus and O.violaceus were evaluated.The disease index,control effect and dimethachlon dosage of sclerotinia of B.napus and O.violaceus in the predictive and conventional control areas were investigated.The results indicated that the C,IWHC,WTR(or NTR),UAC and NTC of B.napus(or O.violaceus)were significantly(p?0.01)decreased,and its Z,R,XC and XL were significantly(p?0.01)rised after spraying S.sclerotiorum solution for2 days(or 1 day),as compared with the electrophysiological information,intracellular water metabolism information and nutrient transport information before spraying or the above mentioned health thresholds;which sugestting S.sclerotiorum had begun to infect B.napus and O.violaceus at this time,and then the control of sclerotinia could carry out by spraying dimethachlon.This was also consistent with the result that O.violaceus was more susceptible to S.sclerotiorum than B.napus based on the union characterization of stable carbon isotope fractionation value and S.sclerotiorum proliferation.Moreover,the significantly changes of the electrophysiological information,intracellular water metabolism information and nutrient transport information of B.napus and O.violaceus were earlier 3?4 days and 2?3 days than the apparent symptoms,respectively.After spraying dimethachlon for 7 days,the control effects of sclerotinia of B.napus and O.violaceus in the predictive and conventional control areas were 91.07%and 68.42%,87.73%and 69.27%,respectively.And the dimethachlon dosage in the predictive control area was half of that in the conventional control area.(6)The degradability of dimethachlon by the mixed bacterial agent of Providencia stuartii JD and Brevundimonas naejangsanensis J3 was evaluated,and the biodegradability of free JD-J3 bacterial agent and immobilized JD-J3 bacterial agent on dimethachlon in B.napus,O.violaceus and soil was studied.The results showed that the free mixed bacterial agent degraded 85.56%of 50 mg/L dimethachlon in liquid culture within 5 days,which efficiency was 1.23 and 1.26 times of strains JD and J3,respectively.The optimal condition for dimethachlon degradation was p H 7.0,30?,6.5×10~8 cfu/m L of the JD-J3 mixed bacterial agent,50 mg/L of dimethachlon.Free JD-J3mixed bacteria metabolized dimethachlon through the typical pathway,in which dimethachlon was firstly transformed into 3,5-dichloroanilin and succinic acid,and the former was then converted to phenol,which was subsequently degraded to muconic acid,and muconic acid and succinic acid further subjected to the mineralization.Moreover,the JD-J3 mixed bacteria immobilized in a charcoal-alginate-chitosan carrier obviously surpassed free JD-J3 mixed bacteria in terms of degradability,stability and reusability.The results of the degradation performance evaluation test found that the free JD-J3 mixed bacteria could effectively remove the high dimethachlon residues in B.napus and O.violaceus,it effectively reduced the half-lives of dimethachlon residues in B.napus and O.violaceus by 2.22 folds and 2.42 folds compared to natural dissipation,respectively.Compared to free JD-J3 mixed bacteria and natural dissipation,immobilized JD-J3 mixed bacteria significantly enhanced the degradation of the high dimethachlon residues in soils.The study provided feasible,efficient and rapid products for bioremediating dimethachlon-contaminated plants and soils in-situ.(7)The biodegradation of dimethachlon residues in B.napus,O.violaceus and soils by free and immobilized JD-J3 mixed bacteria was clarified.B.napus,O.violaceus and their planting soils were used as the research objects,the biodegradation effests of dimethachlon residues in B.napus,O.violaceus and their planting soils in the predictive and conventional control areas by free and immobilized JD-J3 mixed bacteria were investigated.The results demonstrated that after spraying free JD-J3 mixed bacteria for 60 hours in the predictive and conventional control areas,100%and 98.56%of dimethachlon residues in B.napus plants were removed,almost all dimethachlon residues in O.violaceus plants were removed.Compared to natural dissipation,free JD-J3 mixed bacteria could effectively reduced the half-lives of dimethachlon residues in B.napus and O.violaceus plants in the predictive and conventional control areas by4.40 folds and 3.20 folds,4.60 folds and 3.34 folds,respectively.After applying immobilized JD-J3 mixed bacteria for 60 hours in the predictive and conventional control areas,99.66%and 98.33%of dimethachlon residues in B.napus planting soils were removed,98.96%and 98.03%of dimethachlon residues in O.violaceus planting soils were removed.Compared to natural dissipation,immobilized JD-J3 mixed bacteria could effectively reduced the half-lives of dimethachlon residues in B.napus and O.violaceus planting soils in the predictive and conventional control areas by 3.44 folds and 2.78 folds,3.35 folds and 2.27 folds,respectively.This study provided a reference for the efficient,safe and ecological control system of sclerotinia of B.napus and O.violaceus,and this technical system main including the acquisition of electrophysiological information,the establishment of prediction method of sclerotinia based on plants'electrophysiology,the prediction and control of sclerotinia in B.napus and O.violaceus,the preparation and evaluation of the high-efficiency degradation bacteria agent of dimethachlon,as well as the biodegradation of dimethachlon residues in B.napus,O.violaceus and their planting soils.