To investigate the effect of applying different gradients of nitrogen reduction on leaf production capacity of Korla fragrant pear,no fertilizer treatment(CK),no nitrogen fertilizer treatment(N0),conventional fertilizer treatment(N),and three nitrogen fertilizer reduction gradients(N1,N2 and N3,which reduced nitrogen use by 10%,20%and 30%,respectively,compared with conventional fertilizer)were set up in a total of six treatments.Leaf nutrient content,net photosynthetic rate,chlorophyll fluorescence,chlorophyll content,leaf area index and yield were compared under different fertilizer application methods based on multi-year fertilizer trials.Nitrogen fertilization significantly increased leaf and branch nutrient content,leaf chlorophyll content,leaf area index,net photosynthetic rate,chlorophyll fluorescence and yield,and soluble solids and VC content in fruit.Nitrogen reduction of 10% had no significant effect on leaf and branch nutrient content,chlorophyll fluorescence,net photosynthetic rate,chlorophyll content,leaf area index and fruit soluble solids,VC,stone cells and total acid content compared to complete N application,and N reduction of 10% to 20% had no significant effect on the orchard yield and was able to maintain it at the normal range level.Net photosynthetic rate,chlorophyll fluorescence parameters,and yield were significantly and positively correlated with N,P,K,Fe,Mn,Cu,and Zn contents in leaves and branches.According to the experimental results and analysis,the recommended range of nitrogen fertilizer reduction for Korla fragrant pear of 10—12 years old is 10%—20%(240—270 kg/ha)on the basis of fully applied nitrogen(300 kg/ha)as the optimal amount of nitrogen fertilizer reduction in fragrant pear orchards.

In order to explore the potential of melatonin to maintain the fruit quality of postharvest pear fruits,providing references for the research and development of storage and fresh preservation technologies of postharvest fruits,we carried out this research.In which Xinli No.7 pear fruits harvested at mature stage were selected as materials,and they were soaked in 0(control),0.02,0.20 and 1.00 mmol/L melatonin solutions for 2,4,8,12 and 24 h,respectively.Then we sampled and determined the effects of melatonin treatments on the internal qualities of pear,the activities of cell wall lysis related enzymes,the activities of antioxidant related enzymes,and the content of endogenous melatonin and salicylic acid in fruit peel and sarcocarp.The results showed that the hardness of the pear fruits soaked with 0.02,0.20 and 1.00 mmol/L melatonin for 8 h was 38.51,42.79,and 40.95 N/cm²,respectively,and the ethylene release rate decreased by 66%,70% and 71% respectively compared with the control.The soluble solid content(SSC)of pear fruits soaked with 0.2 mmol/L melatonin for 8 h was 94 g/L,which was significantly higher than that of the control(80.2 g/L)and was 1.17 times of the control.And the titrable acid(TAC)of pears of the same treatment was 1.013 g/L,which was also significantly higher than that of the control(0.90 g/L)and was 1.13 times of the control.In addition,treating pear fruits with exogenous melatonin could significantly increase the enzyme activities of superoxide dismutase(SOD),peroxidase(POD)and catalase(CAT),reduce the enzyme activities of polygalacturonase(PG),pectin methylesterase(PME)and Lipoxygenase(LOX),and could increase the endogenous melatonin content in fruit peel and the endogenous salicylic acid content in fruit(peel and sarcocarp).To sum up,soaking pear fruits with 0.2 mmol/L melatonin solution for 8 h had the maximum potential to maintain the fruit quality of postharvest pears,which could keep better fruit quality,keep lower cell wall lysis related enzyme activities and higher antioxidant enzyme activities,and could make the pear fruits having higher level of endogenous melatonin and salicylic acid content.

In order to explore the effect of applying biochar on the improvement of soil physical and chemical properties in pear orchards and the continuous impact on the fruit quality of Huangguan pear,the positioning experiments for four consecutive years were conducted to provide theories and technologies support for rational and efficient fertilization in Huangguan pear orchards in Hebei Province.The experiment was carried out in the Huangguan pear experimental base in Jinzhou City,Hebei Province.Taking the treatment without biochar as the control(CK),the application of 0.45(B1),0.90(B2),1.35(B3),1.80 kg/m²(B4)4 treatments were used to study the effects of different amounts of biochar on soil organic matter,nitrate nitrogen,available phosphorus,available potassium,electrical conductivity and pH,and explored the effects on fruit quality(such as total soluble sugar,titratable acid,firmness,soluble solids,and single fruit weight)of Huangguan pear during ripening.The application of biochar for four consecutive years significantly reduced the soil nitrate nitrogen content,and increased the contents of organic matter,available phosphorus,available potassium and pH in the surface layer(0-20 cm),and the effect on the surface layer(0-20 cm)was greater than that in the 20-40 cm soil layer.B3 treatment had the best improvement effect on soil nutrients.The influence of soluble solid content on the fruit quality of Huangguan pear showed an increasing trend,and titrable acid showed a decreasing trend.There was no significant difference between B2 and B3 treatments on the quality of Huangguan pear.Considering the long-term cumulative effect of soil and the economic cost of biochar,0.90-1.35 kg/m² biochar application rate was recommended as the appropriate application rate.Combined with the analysis of the effects of the amount of biochar on the soil physicochemical properties and pear quality,under this test condition the recommended amount of biochar was 0.90 kg/m².

In order to clarify the biological function of gibberellin receptor GID1 in Pyrus betulifolia,and provide a good foundation for future development of P.betulifolia dwarf rootstocks using CRISPR/Cas9 gene editing technology.Pyrus betulifolia was used as the test material,and the PbGID1s genes were obtained by homologous cloning method.Bioinformatics analysis software was used to construct the gene structure and design the target sites;construction of sgRNA expression cassettes with targets into CRISPR/Cas9 expression vectors,through the mediation of Agrobacterium,the CRISPR/Cas9 expression vector was transferred into the cotyledons of P.betulifolia.Results showed that four PbGID1s were successfully cloned from P.betulifolia plants and named as PbGID1b-1,PbGID1b-2,PbGID1c-1 and PbGID1c-2. They all consisted of two exons and one intron found by gene structure analysis.Amino acid sequence comparison showed that all PbGID1s had the HGG and GXSXG conserved domains.Five gRNAs that could potentially edit all 4 PbGID1s simultaneously were successfully constructed into a single CRISPR/Cas9 vector,pYLCRISPR/Cas9P_35S-N.The results of the genetic transformation test of P.betulifolia showed that a total of 595 cotyledons of P.betulifolia were infiltrated,176 resistant buds and 33 positive plantlets were obtained,and the transformation efficiency reached 5.55%.A CRISPR/Cas9 vector was successfully constructed that could simultaneously target the PbGID1s family genes of P.betulifolia.Through the mediation of Agrobacterium,the vector was successfully transformed into P.betulifolia cotyledons,and positive plants were obtained.

In order to reveal the effect and molecular mechanism of salicylic acid-induced resistance to black spot disease of Yali,the leaves and fruits of Yali were used as experimental material which were collected from the experimental orchard of Hebei Agricultural University.Tissue isolation method was used to isolate and purify the pathogen of black spot disease and detected its pathogenicity.Morphological observation and multi-genes of ITS,HIS,RPB2 and ACT were used to identify the pathogen of black spot disease.The detached leaves of Yali were inoculated with conidia suspension of Alternaria alternata by acupuncture for 0,6,12,24,48,72,96 and 120 h,respectively.Real-time RT-PCR was used to analyse the expression of related genes which involved in salicylic acid signaling,and high-performance liquid chromatography (HPLC) was used to detect the content of endogenous SA at 0 and 72 h,respectively.Exogenous SA treatment with different concentrations (0,0.002,0.02,0.2,2.0,10.0,20.0 mmol/L) were used to identify the effect to the mycelial growth of A.alternata;Exogenous SA treated Yali fruit with different concentrations (0,0.02,0.2,2.0 mmol/L),respectively,and inoculated with A.alternata to detect the effect of disease resistance.The results showed that the pathogen of Yali black spot disease was A.alternata. Detached leaves of Yali were inoculated with A.alternata for 72 h,the free state SA content increased from 0 mg/g to 0.02 mg/g and the binding state SA content increased from 0.47 mg/g to 1.55 mg/g,and for 96 h compared with 0 h,the expression of Pbrgene12425,Pbrgene6286, increased 5.48,4.66 times,respectively,the expression of Pbrgene8895 and Pbrgene43605 increased 7.90 and 10.0 times for 120 h.Exogenous 0.2 mmol/L SA significantly increased the resistance of Yali fruit to black spot disease.In the process of Yali resistance to black spot disease,the free state SA content of Yali leaves significantly increased,and the related genes in SA signaling were induced to up-expressed,exogenous 0.2 mmol/L SA significantly increased the resistance of Yali to black spot disease.

The effects of 1-MCP and ethephon on the main physiological quality indexes and softening related indexes of Postharvest Pyrus ussuriensis during normal temperature storage were studied, and the mechanism of 1-MCP and ethephon on softening regulation of Pyrus ussuriensis was discussed, so as to provide theoretical basis for regulating the ripening and softening process of Pyrus ussuriensis. Five kinds of Pyrus ussuriensis, Nanguo pear, Jingbai pear, Huagai pear, Jianba pear and An pear, were fumigated with 0.5 μL/L 1-MCP for 24 h and 1.0 g/kg ethephon solution respectively. The changes of fruit firmness, respiration rate, ethylene production, soluble solids, titratable acid, vitamin C content, water-soluble pectin, cellulose, starch content and softening related enzyme activities of polygalacturonase(PG), β-galactosidase(β-Gal), cellulase(CL)and amylase(AM)were studied during normal temperature storage((20±1)℃). 1-MCP treatment significantly delayed the decline of firmness, ethylene production, respiration rate, water soluble pectin(WSP), cellulose and starch content of Pyrus ussuriensis, and decreased the activities of PG, β-Gal, CL and AM enzyme, but there was little difference between ethephon treatment group and control group. 1-MCP treatment could alleviate the degradation of cell wall substances by inhibiting the activities of cell wall related degrading enzymes and reducing ethylene production, thus effectively delaying the fruit softening process of Pyrus ussuriensis. However, ethephon had no significant effect on the physiological changes of fruit softening and cell wall degradation during storage.

In order to investigate the effects of different materials of modified atmosphere packaging on the postharvest physiology and storage quality of Hongxiangsu pear, six kinds of packaging treatments, such as CK, high exudation CO₂ bag, PE bag, PE bag + 1-MCP preservative, PE bag + ethylene absorbent, PVC bag, were studied. The fruits of Hongxiangsu pear were refrigerated at (0±0.5)℃ for 120, 180, 240 d. After being taken out, the fruits were balanced at 20℃ for 24 h and stored for 7 d. The changes of respiration rate, ethylene release, ethanol and acetaldehyde contents, fruit appearance and internal quality were measured and the contents of CO₂, O₂ and ethylene in different bags were monitored regularly. The results showed that compared with CK and high exudation CO₂ bag, PE bag and PVC bag had good air conditioning ability, which could significantly reduce the volume fraction of O₂ and increase the volume fraction of CO₂, but the ethylene concentration in the bag was also relatively high, while the ethylene concentration in PE bag + ethylene absorbent bag was relatively low. Compared with CK fruit, the other four kinds of packaging treatments except high exudation CO₂ bag could delay the decline of fruit firmness and the yellowing of peel in the middle and later storage periods, maintain relatively high soluble solid content. Among them, the PE bag+1-MCP preservative not only maintained better appearance and normal taste flavor of Hongxiangsu pear fruit during cold storage and shelf life, but also significantly reduced the fruit respiration rate, ethylene release, mass fraction of ethanol and acetaldehyde. However, when the fruits were stored in PVC bags for 240 d and stored at 20℃ for 7 d shelves, some fruits showed browning and abnormal flavor. For Hongxiangsu pear stored in medium and long term, the PE bag+1-MCP preservative treatment had the best storage effect, followed by single PE bag and PE bag + ethylene absorbent. PVC bag was not recommended for Hongxiangsu pear.

In order to explore the relationship between the superficial scald and related physiological indexes of Dangshansu pear during storage, the mineral elements(N, P, K, Mg, Ca) content, respiration intensity, ethanol content, relative conductivity(cell membrane permeability), total phenol content, polyphenol oxidase activity(PPO activity), chlorophyll fluorescence parameters and related physiological indexes of Dangshansu pear fruits with different levels of superficial scald were measured, and the correlation between the various physiological indicators and the superficial scald progression was analyzed. The results showed that the content of Ca in fruits of different grades of superficial scald was significantly lower than that of normal fruits(no superficial scald), while the content of N, K, Mg and P were higher than that of normal fruits. There was an extremely significant positive correlation between the incidence degree of superficial scald and N content, and an extremely significant negative correlation between the incidence degree of superficial scald and Ca content. Results showed that the fruit respiration intensity, ethanol content(peel), relative electric conductivity(flesh), total phenol content(peel and core), PPO activity(peel, flesh, core) and incidence degree of superficial scald were very significant positive correlation, both the chlorophyll fluorescence parameters(Fo, Fm, Fv, Fv/Fm, Fv/Fo) of normal fruit peel were significantly higher than superficial scald fruit. It was preliminarily determined that the higher N content and the lower Ca content in the fruit were the main causes of the superficial scald in the late stage of storage. The more serious the superficial scald was, the higher the respiration intensity, ethanol content, tissue membrane permeability and PPO activity were, and the lower the chlorophyll fluorescence parameters of peel(Fm, Fo) were. Therefore, the occurrence of superficial scald during storage can be reduced by regulating mineral nutrients or post harvest calcium supplementation before harvest, so as to improve fruit storage quality.

In order to investigate the incidence of known viral diseases of pear in Tianjin Region,the total RNA of snow pear leaves was extracted by improved CTAB method. And the latent virus species of the 70 pear trees from three regions of Tianjin were detected through RT-PCR identification,including Apple cholortotic leaf spot virus (ACLSV), Apple scar skin viroid (ASSVd), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV) and Apple dimple fruit viroid (ADFVd). The results showed that the improved CTAB-mercaptoethanol method was a kind of comparatively ideal method for total RNA extraction from snow pear,the RNA had less impurity, the rate of total RNA extrate was higher, and the mean concentration was 73.880 μg/g. The obtained RNA meted the requirements for the next analysis. By RT-PCR technique the infection rates of the 70 pear trees with the above five viruses were 47.1%,11.4%,10.0%,8.6%,and 1.4%,respectively,and the mixed infection rate was 28.6% on average. The higher the age of pear trees, the higher the virus carrying rate. And the sensitivity of different varieties to viruses was different. The results showed that the pear virus disease in Tianjin was common, in which apple chlorotic leaf spot virus was the main type of virus, and had a certain proportion of multiple virus complex infection. The low level of pear planting management and the lack of standardized detection and monitoring of pear introduction among regions could lead to the occurrence and spread of pear virus disease. It was very unfavorable for the healthy development of Tianjin pear industry.

To clarify the role of chitinase in the process of pear resistance disease, the total RNA of Yali was extracted and specifi c primers were designed to amplify the coding region of PbChi Ⅱ protein by RT-PCR,analysed the expression pattern of PbChi Ⅱ after treatment with pathogens. The prokaryotic expression vector pET30a-PbChi Ⅱ was constructed and the recombinant protein was expressed in E.coli BL21 (DE3). The recombinant protein growth ability was analyzed under abiotic stress. The results showed that the length of PbChi Ⅱ (GenBank accession Number:KP876485) gene was 969 bp,Quantitative Real-time PCR (qRT-PCR) analysis revealed that the expression of PbChi Ⅱ was regulated by pathogens,and enhanced up to the peak at 48 h after treatment with Venturia nashicola during 96 h. The PbChi Ⅱ gene was successfully subcloned into the expression vector pET30a. SDS-PAGE analysis showed that a specific recombinant protein of approximately 35.53 kDa was produced in the BL21 (DE3) with the prokaryotic expression vector pET30a-PbChi Ⅱ in 37℃ with 1.0 mmol/L IPTG for 2 hours. This protein enhanced the stress of isolate with NaCl,CuCl₂,CdCl₂ and ZnSO₄. This research provided basic references for further study the function of PbChi Ⅱ.

In order to understudy the relationship between calcium and browning spot,Ca(NO₃)₂ had been used to treat the Huangguan fruit before bagging and the rate of browning spot was counted and Ca compounds were determined.The results indicated that exogenous calcium supply in different treatment decreased the incidence of browning spot,dip fruit and spraying in the leaves of exogenous calcium were significant differences compared withthree layers bag fruits,but all of treatment were significantly higher than without bag fruit.After flower 40-80 d,the total calcium and water-soluble calcium content of calcium treatment fruit were higher than those in three layers bag fruits.At the same time,dip fruit and three layers bag fruits were significant differences.After flower 90 d to fruit maturity,the total calcium and water-soluble calcium content in Ca(NO₃)₂ treatment were lower than without bagfruit.All types of calcium treatment were significantly increase in NaCl-soluble calcium and hydrochloric acid-soluble calcium content,but did not affect the content of acetic acid-soluble calcium.In summary,calcium treatment increased total calcium and water-,NaCl-and hydrochloric acid-soluble calcium,and the changes of these calcium concentrations have closely related with reduction of browning spot.

In order to reveal the expression characteristic of genes (PAL1,PAL2,CAD,4CL,POD) related to lignin biosynthesis in brown pericarp mutant of Suisho pear,and provide the basis for further studying pear brown pericarp formation mechanism.RNA was extracted from different tissues according to RNA extraction kit (EASY spin,Biomed),primers were designed with Primer 5.0 and DNAMAN,expression pattern of genes were analyzed by QRT-PCR.Among the different tissues and organs,the highest relative expression of PAL1,PAL2,CAD,4CL,POD gene appeared in peels.In peels and pulps,the relative expression of POD gene was extremely significantly higher than the other genes.In flowers,leaves and branches phloem,the relative expression of CAD was extremely significantly higher than the other genes.The study suggested that the expression pattern of PAL,CAD,4CL and POD had significant tissue-specific in the five selected tissues and organs,and these genes may be associated with the formation of Suisho pear brown peel closely.

Yali pear belongs to the kind of gametophytic self-incompatibility,the percentage of self-pollinated fruit set is extremely low.Our previous research showed that sprayed polyamine in a concentration range at florescence could break self-incompatibility of Yali pear.In order to explore the physiological and biochemical mechanism,the effects of exogenous Spd on the pollen tube growth and stylar protein expression of the Yali pear were studied by two-dimensional gel electrophoresis and mass spectrum analysis technology.The main results were as follows:The pollen tube length were extremely significantly increased after suitable concentration of Spd treatment,the best effects of exogenous Spd were 0.25 mmol/L Spd treatment,the number of pollen tube reached to the bottom of style were the most after 72 hours of treatment.Three specific proteins were found in the electrophoretogram.Two protein spots were obtained by the mass spectrometry results,which were predicted protein and putative protein respectively.