Nitrogen Diagnosis Of Oilseed Rape At Different Growth Stages And Leaf Positions Using Chlorophyll Fluorescence Techniques

Nitrogen(N)is the mineral element required by plants in the greatest quantity.Both Nlimited or N-excessive conditions directly influence the photosynthesis,and play vital roles in yield formation.Oilseed rape(Brassica napus L.),as a major crop in Zhejiang province,and is in large demand of N fertilizer during the growth.So it is important to monitor N status strictly for fertilizer management in different growth stages.Chlorophyll fluorescence(Chl F),as a probe of photosynthesis in vivo,provides rich information and has been widely used in the researches of photosynthetic physiology,abiotic and biotic stress,nutrient diagnosis and so on.However,due to the differences of the detection mode and the detection principle in different Chl F measurements,the obtained signals are diverse.Meanwhile,the accumulation and translocation of N element usually change in different growth periods,which also result in the variation of the sensitivity of Chl F signals to N stress.Therefore,this study collected mulsessilele fluorescence signals from oilseed rape plants respectively using OJIP transient by plant efficiency analyzer(PEA),pulse amplitude modulated chlorophyll fluorescence(PAM-Chl F)imaging and multicolor fluorescence(MCF)imaging in different growth stages and leaf positions,aimed to analyze the effects of N fertilizer rate on leaf photosynthesis,the effects of growth stages and leaf positions on leaf photosynthesis.Then finally established an optimal N diagnostic method in the whole growth.The contents and conclusions of this study are listed as follows:Firstly,the effects of N fertilizer rate on the photosynthetic apparatus in photosystem II(PSII)in oilseed rape leaves were studied.The effects of N fertilizer rate on photosynthesis were analyzed by the integrated application of PEA,PAM-Chl F imaging and MCF imaging.The results showed that N deficiency caused damage to the oxygen evolving complex(OEC)at the PSII donor site,as well as the reaction center and the acceptor side.Meanwhile,the electron transfer efficiency in the electron transport chain and the actual photochemical efficiency in rape leaves decreased.The absorbed light energy was dissipated by heat.While the contribution of N excess on the electron transport and the improvement of the actual photochemical efficiency only appeared in the bolting stage based on the long-petiole leaves.Secondly,the photosynthetic performance in rape leaves under different N levels was studied in different growth stages and leaf positions.The results showed that different growth stages made impacts on the amplitude of fluorescence curves,as the amplitude slightly decreased in the seeding stage but obviously increased in the bolting stage.Meanwhile,the damage on photosynthesis caused by N deficiency aggravated during the growth,but the restoration of N excess worked until the bolting stage.The photosynthesis also changed in different leaf positions.The short-petiole and long-petiole leaves responsed earlier to N stress especially the long-petiole ones,the sessile leaves responsed slower due to the apical dominance under N stress.In addition,the photosynthetic capacity of the short-petiole and long-petiole leaves was relatively weak,and the absorbed light energy was mainly used for photochemical reaction and fluorescence quenching.The photosynthetic capacity of the sessile leaves was relatively high,and the absorbed light energy was mainly used for the demand of leaf growth.Thirdly,N diagnostic based on PEA,PAM-Chl F imaging and MCF imaging in different growth stages were compared.By combining principal component analysis(PCA)with linear discriminant model,the results showed that the diagnosis accuracies were different in three fluorescent techniques.For PEA,the classification accuracies of the linear discriminant model in the early seeding stage and bolting stage were respectively 86.96% and 79.17%,which indicated a relatively high diagnostic capability of N deficiency,N normal and N excess.Moreover,the N diagnosis based on the sensitive fluorescence parameters was able to differentiate plants under N deficiency,N normal and N excess based on the long-petiole leaves.While for PAM-Chl F imaging,the linear discriminant model based on the data of the slow chlorophyll fluorescence curves showed higher classification accuracies in the late seeding stage and bolting stage which were both 83.30%.In each growth stage,all the N deficient plants were almost identified,except two misjudged ones in the late seeding stage.The model mistook some N normal plants as N excessive ones.In general,the combination of PAM-Chl F imaging with machine learning was superior in the identification of N deficient plant in the whole growth stage.For OJIP measurement,N diagnosis could be realized in the bolting stage by the parameter sensitivity analysis based on the long-petiole leaves.For MCF imaging,N diagnosis based on the parameters was applicable in the seeding stage,as the parameter RF/Ir F in the sessile leaves was able to identify oilseed rape plant under different N levels since the early seeding stage.Fourly,the photosynthesis in the rape pods was explored by PEA instrument,and the relationship between the photosynthesis and seed yield and quality was further explored.The results showed that excessive N application was helpful to improve the photosynthetic performance of rape pods,which was in accordance with the existing researches.While the photosynthetic performance of the high-quality rape plants which were low in both erucic and glucosinolates was relatively weak.The pod samples under different N levels and varieties could be identified by the optimal fluorescence parameters obtained from the parameter selection method named random forest.The samples under N deficiency,N normal and N excess and those from varieties respectively named ZD630,ZS758 and GY605 could be classified by fisher discriminant analysis,which could make it earlier in the fertilizer management and the breeding work in the agricultural production.What's more,high correlations were found between fluorescence parameters and seed yield and quality,and this indicated that the nondestructively quantitative prediction of seed yield and quality in the early podding stage could be realized by PEA,and this would definitely make the seed yield statistic and quality analysis much easier.Only one technique of the OJIP transient was used due to the advantages of small size,hand-held and fast detetion in the instrument.In comparison,the instruments of PAM-Chl F imaging and MCF imaging was large in size,and have strict requirements in the detection such as detection distance and detection time.