Regulation of NF-κB signaling by NLRC (NLRC3-like) gene in the common carp (Cyprinus carpio).

Among teleost NLRs, NLR-C subfamily is a large group of proteins that were teleost-specific and evolution analysis showed that NLR-Cs are most likely to evolve from NLRC3 gene (thus also called as NLRC3Ls). Presently, although there have been rich studies investigating teleost NLRC3 and NLRC3L, the data on the regulatory mechanism was limited. In this study, immune regulation of inflammatory signaling pathway mediated by common carp NLRC3L gene (CcNLRC) has been investigated. Confocal microscopy analysis showed that CcNLRC was located in cytoplasm, and in HEK293T cells, dual-luciferase reporter assay showed the regulation of NF-κB signaling by CcNLRC, in which CcNLRC could alter/decrease RIPK2-induced activation of NF-κB. These results indicated that CcNLRC may function as a negative NLR in the regulation of inflammatory response in common carp. Our data will allow to gain more insights into the molecular mechanism of teleost specific NLR (NLRC3L).

Transcriptome analysis of the hepatopancreas in Penaeus vannamei under experimental infection with Enterocytozoon hepatopenaei (EHP).

Enterocytozoon hepatopenaei (EHP) is a specialized intracellular parasite that mainly resides in the hepatopancreas of shrimp, causing significant growth retardation in shrimp. In this study, Penaeus vannamei was infected with EHP through an artificial challenge experiment, and the different genes and pathways in the hepatopancreas between EHP-infected and healthy shrimp were analyzed by transcriptome sequencing. The results showed that a total of 240 significantly differentially expressed genes were obtained, including 99 up-regulated genes and 141 down-regulated genes. Immune-related genes such as Astakine, lysozyme, NACHT, LRR, and PYD domains-containing protein 3 (NLRP3), and macrophage mannose receptor 1-like (MMR) were up-regulated, and the expression levels of lipid metabolism-related genes pancreatic lipase-related protein 2 (PLRP2), lysosomal acid lipase (LIPA), and adiponectin receptor protein (AdipoR) were also increased. However, several genes were down-regulated in carbohydrate and protein metabolism, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), trypsin-1, and delta-1-pyrroline-5-carboxylate synthase (ALDH18A1). The results suggested that EHP infection of shrimps could significantly activate the immune system, but the energy and material metabolism processes were disturbed. This study identified a substantial number of genes and pathways associated with EHP infection, providing a valuable resource for revealing the molecular mechanism of growth retardation in shrimp.

Identification of CCCH-type zinc finger antiviral protein 1 (ZAP) gene from Pacific white shrimp (Penaeus vannamei): Characterization and expression analysis in response to viral infection.

Zinc-finger proteins (ZFPs) are a huge family that exert multiple roles in the cells. ZFPs could be divided into nine types based on the numbers and positions of conserved Cys and His residues, in which CCCH-type ZFP was one of the most widely studied types. CCCH-type zinc finger antiviral protein 1 (ZAP), a CCCH-type ZFP that can inhibit the replication of certain RNA viruses and DNA viruses by mediating degradation of viral RNA and repressing mRNA translation, plays significant roles in the host innate immune defenses against viral infections. Presently, there have been numerous reports investigating the antiviral ability of ZAP, while no data is available about ZAP gene in the species of shrimps or even crustaceans. In this study, a novel protein containing CCCH-type zinc finger motifs (ZnF-CCCH), CCCH-type zinc finger antiviral protein 1 (ZAP) gene, was identified from Pacific white shrimp (Penaeus vannamei) and its role in antiviral immunity was further investigated. Similar to mammalian ZAPs, in addition to ZnF-CCCH, PvZAP also possesses central WWE domains and C-terminal PARP domain. Phylogenetic analysis showed that PvZAP was close to that of the crustacean Pacific oyster, separating from the cluster of vertebrate ZAP proteins. Upon in vivo infection by IHHNV, gene expression of PvZAP was strongly up-regulated in the hepatopancreas and gills of both adult and juvenile shrimps, where adult individuals showed higher fold changes of up-regulation than in juvenile individuals. These results suggested that PvZAP might play an important role in the innate immune defense of Pacific white shrimp against IHHNV infection. This allows us to gain new insights into the immunological function of ZAP in the innate immunity of shrimp species and even crustaceans.

Development of a duplex PCR for the simultaneous detection of EHP and IHHNV and analysis of the correlation between these two pathogens.

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the linearly single-stranded DNA viruses. Ecytonucleospora hepatopenaei (EHP) is an intracellular parasitic microsporidian. IHHNV and EHP are pathogens that have been widely prevalent in shrimp farming. Both of them are associated with growth retardation of the penaeid shrimp, which causes serious economic losses to shrimp farming. Shrimp can be co-infected with IHHNV and EHP. In this study, a rapid duplex polymerase chain reaction (PCR) was developed and optimized for the simultaneous detection of EHP and IHHNV. The detection limit of the duplex PCR could reach 1.5 × 102 copies for EHP and IHHNV. A total of 578 Litopenaeus vannamei samples were detected by the established duplex PCR detection method. The results suggested that 398 samples were infected with EHP, 362 samples were infected with IHHNV, and 265 samples were co-infected with EHP and IHHNV. The case-control analysis of the detected shrimp samples showed a certain synergistic effect between EHP and IHHNV.

Study of infectious hypodermal and hematopoietic necrosis virus (IHHNV) infection in different organs of Penaeus vannamei.

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) is a major viral pathogen in cultured shrimp. It is generally believed that the target organs of IHHNV in shrimp include tissues of ectodermal and mesodermal origin, but do not normally include organ systems of endodermal origin, such as hepatopancreas. In this study, the feeding challenge of IHHNV in different organs (pleopods, muscles, gills, and hepatopancreas) of Penaeus vannamei was studied. The PCR results showed that hepatopancreas of P. vannamei had the strongest IHHNV positivity (100% positive, 19.4 copies/mg) in the feeding challenge experiment. Gills and pleopods had similar infectivity to IHHNV (86.7% positive, 10.6 and 10.5 copies/mg). Among the four organs tested in this study, the IHHNV positivity of muscles was the weakest (33.3% positive, 4.7 copies/mg). The IHHNV infection to hepatopancreas of P. vannamei was also histological confirmed. Our current data indicated that the shrimp tissues derived from the endoderm such as hepatopancreas could also be infected by IHHNV.

Transcriptome analysis of IHHNV infection in Penaeus vannamei at different developmental stages.

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) is the smallest known virus in shrimp, which causes runt-deformity syndrome (RDS) and leads to huge economic loss every year in penaeid shrimp farming. Previous studies have shown that the juvenile Penaeus vannamei is more susceptible to IHHNV infection than the adults, but the mechanism is still unclear. In order to investigate the mechanism of pathogenic differences in IHHNV infection of P. vannamei at different developmental stages, the juvenile and adult P. vannamei were studied by transcriptome high-throughput sequencing to analyze their response to IHHNV infection. GO and KEGG enrichment were analyzed to search for differentially expressed genes (DEGs) related to immunity, growth and metabolism. The results showed that many immune-related genes of the juvenile and adult P. vannamei responded differently to IHHNV infection. For the adult P. vannamei, the expression of most immune-related genes was significantly up-regulated, which means that a cellular defense response was triggered after IHHNV infection. However, most immune-related genes in juvenile P. vannamei were inhibited, indicating that the immune system of juvenile the P. vannamei is imperfect and makes it to be more susceptible to IHHNV. Similarly, the growth-related genes of P. vannamei were changed during IHHNV infection. For the juvenile P. vannamei, the growth-related genes were significantly down-regulated, which resulted in a growth hormone disorder and prevented the juvenile P. vannamei from growth. In the adult P. vannamei, most molting-related genes were significantly up-regulated, indicating that IHHNV infection leads the adult P. vannamei to early molting to eliminate pathogen in the body. Metabolic process data showed that energy metabolism pathway was affected when P. vannamei infected with IHHNV. The adult P. vannamei infected with IHHNV can cause energetically costly and lead to the disturbance of the metabolism, activate complex immune systems to resist the invasion of pathogens. The results of this study clarified the response mechanism of P. vannamei at different developmental stages to IHHNV infection, which can provide new insights to IHHNV effective control and a reference for the study of sensitive period of different shrimp virus to host infection.

Summary and comparison of the perforin in teleosts and mammals: A review.

Perforin, a pore-forming glycoprotein, has been demonstrated to play key roles in clearing virus-infected cells and tumour cells due to its ability of forming 'pores' on the cell membranes. Additionally, perforin is also found to be associated with human diseases such as tumours, virus infections, immune rejection and some autoimmune diseases. Until now, plenty of perforin genes have been identified in vertebrates, especially the mammals and teleost fish. Conversely, vertebrate homologue of perforin gene was not identified in the invertebrates. Although recently there have been several reviews focusing on perforin and granzymes in mammals, no one highlighted the current advances of perforin in the other vertebrates. Here, in addition to mammalian perforin, the structure, evolution, tissue distribution and function of perforin in bony fish are summarized, respectively, which will allow us to gain more insights into the perforin in lower animals and the evolution of this important pore-forming protein across vertebrates.

Development of a PCR assay for the effective detection of Enterocytozoon hepatopenaei (EHP) and investigation of EHP prevalence in Shandong Province, China.

Enterocytozoon hepatopenaei (EHP), a recently reported pathogen in the penaeid shrimp, is spreading widely and seriously threatening Penaeus (Litopenaeus) vannamei aquaculture. This study aimed to develop a new and more sensitive polymerase chain reaction (PCR) method for the effective detection of EHP. An EHP PCR assay with a pair of primers specifically amplifying a 358 bp EHP DNA fragment was developed, which was demonstrated to be capable of detecting as low as 2 × 101 copies of EHP and is specific for EHP without cross reaction with DNA samples prepared from five common shrimp pathogens, including white spot syndrome virus (WSSV), infectious hypodermal and haematopoietic virus (IHHNV), hepatopancreatic parvovirus (HPV), infectious myonecrosis virus (IMNV), and yellow head virus (YHV). This new assay is more specific and more sensitive than the previously published EHP PCR methods. With the PCR assay developed in this study, we investigated the prevalence of EHP in four areas of Shandong, China by testing a total of 639 shrimp samples collected from Yantai, Binzhou, Dongying, and Weifang. The results showed that the EHP positive rate reached 51.2%, indicating that EHP is prevalent in shrimp culture in China.

Research progress on hosts and carriers, prevalence, virulence of infectious hypodermal and hematopoietic necrosis virus (IHHNV).

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the major viral pathogens of penaeid shrimp and it has spread worldwide. IHHNV causes substantial economic loss to the shrimp farming industry and has been listed as a notifiable crustacean disease pathogen by the World Organization for Animal Health (OIE). In this paper, we reviewed studies on the hosts and carriers, prevalence, genotypes and virulence of IHHNV. The pathogenesis mechanisms of IHHNV and the viral interference between IHHNV and white spot syndrome virus (WSSV) were also discussed. The mechanism of IHHNV infection and its virulence difference in different hosts and different developmental stages have not been fully studied yet. The mechanisms underlying viral interference between IHHNV and WSSV are not yet fully understood. Further studies are needed to elucidate the precise molecular mechanisms underlying IHHNV infection and to apply the insights gained from such studies for the effective control and prevention of IHHNV disease.

Transcriptome analysis of Procambarus clarkii infected with infectious hypodermal and haematopoietic necrosis virus.

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) is a major viral pathogen in cultured penaeid shrimp. IHHNV has many hosts, mainly including crustaceans. It has recently been reported that Procambarus clarkii can be infected by IHHNV. In the present study, we studied the hepatopancreas of P. clarkii by transcriptome high-throughput sequencing to analyze the response of P. clarkii to IHHNV infection. After de novo assembly, there were 400,340,760 clean reads. A total of 237 differentially expressed genes (DEGs) were obtained, including 77 significantly up-regulated unigenes and 160 significantly down-regulated ones. The expression levels of 12 immune-related DEGs were validated by qRT-PCR, substantiating the reliability of RNA-Seq results. The enrichment analysis of DEGs showed that the immune-related pathways were closely related to apoptosis and phagocytosis. Moreover, a large number of pathways related to metabolic function were down-regulated, suggesting that IHHNV infection might affect the growth of P. clarkii.

Expression and active testing of VP7 from GCRV (Grass carp reovirus) fused with cholera toxin B subunit in rice calli.

Grass carp reovirus (GCRV) is one of the most serious pathogens threatening grass carp (Ctenopharyngodon idellus) production and results in high mortality in China. VP7 from GCRV is involved in viral infection and could be suitable for developing vaccines for the control of GCRV infection. To obtain a genetically engineered vaccine and a plant-based oral vaccine and to evaluate their immune efficacy as an oral vaccine against GCRV, cholera toxin B subunit (CTB) of Vibrio cholerae fused to VP7 (CTB-VP7) was transformed into BL21(DE3) for expression. SDS-PAGE and Western blotting showed that the purified CTB-VP7 fusion protein (rCTB-VP7) was approximately 49.0 kDa. Meanwhile, CTB-VP7 was transformed into rice callus cells by Agrobacterium tumefaciens-mediated gene transformation. CTB-VP7 was integrated into the nuclear genome by PCR, and mRNA transcripts of CTB-VP7 were detected. ELISA and Western blot analyses revealed that the CTB-VP7 fusion protein (CTB-VP7) could be expressed in rice callus lines. The level of expression was determined to be 1.54% ±â€¯0.43 of the total soluble protein. CTB-VP7 showed a binding affinity for monosialoganglioside(GM1), a receptor for CTB. CTB-VP7 showed a higher affinity towards GM1 compared to rCTB-VP7. CTB-VP7 bonded to GM1 with different affinities under different temperatures. Maximum binding of CTB-VP7 to GM1 was reported to occur within 2 h at 37 °C, and approximately half of the binding affinity remained at 25 °C. Our results suggest that CTB-VP7 could be produced in rice calli, increasing the possibility that edible plants can be employed in mucosal vaccines for protection against GCRV in aquaculture.

A novel real-time PCR approach for detection of infectious hypodermal and haematopoietic necrosis virus (IHHNV) in the freshwater crayfish Procambarus clarkii.

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) infects many crustacean hosts, including cultured penaeid shrimp. In the present study, we aimed to develop a novel sensitive SYBR Green-based real-time PCR method to specifically amplify DNA fragments of IHHNV. Our newly developed real-time PCR method with a 195-bp amplicon specifically detected IHHNV and showed no cross reaction with white spot syndrome virus (WSSV), hepatopancreatic parvovirus (HPV), Enterocytozoon hepatopenaei (EHP), infectious myonecrosis virus (IMNV) and yellow-head virus (YHV). This method could detect as low as one single copy of IHHNV plasmid DNA, more sensitive than other SYBR Green-based real-time PCR methods and less expensive and more convenient than the TaqMan probe-based real-time PCR. Moreover, our data using the newly designed method showed that 80% of IHHNV-fed Procambarus clarkii samples were IHHNV positive. Our findings further confirmed that P. clarkii can be infected by IHHNV.

A real-time PCR for the detection of infectious myonecrosis virus in penaeid shrimp.

Infectious myonecrosis virus (IMNV) is a recently observed shrimp virus, which threats the cultured Litopenaeus vannamei and can cause huge economic loss in shrimp farming industry. The specific aim of this study was to develop a new sensitive real-time PCR method for the specific detection of shrimp IMNV. A real-time PCR assay with a pair of primers to specifically amplify a 101bp IMNV cDNA fragment and a corresponding TaqMan probe was developed, which shown to be specific for IMNV without cross reaction with DNA samples prepared from four other shrimp viruses including white spot syndrome virus (WSSV), hepatopancreatic parvovirus (HPV), monodon baculovirus (MBV), and infectious hypodermal and haematopoietic virus (IHHNV). The method could detect as low as one single copy of IMNV plasmid cDNA.

Enterocytozoon hepatopenaei (EHP) Infection Alters the Metabolic Processes and Induces Oxidative Stress in Penaeus vannamei.

Enterocytozoon hepatopenaei (EHP) is highly contagious and can cause hepatopancreatic microsporidiosis (HPM), which is typically characterized by the slow growth of shrimp. In this study, the differences in histology, metabolism, oxidative stress and growth between healthy and EHP-infected Penaeus vannamei were analyzed using an EHP challenge experiment. Histology showed that EHP caused lesions in the hepatic tubules of P. vannamei, such as hepatic tubular atrophy and epithelial cell shedding, with mature spores. Meanwhile, white feces may appear when the infection is severe. Furthermore, the content of total protein, glycogen, ATP and glucose in the EHP challenge group was significantly reduced. The qPCR results showed that EHP infection changed the expression of key genes in glucose metabolism, among which hexokinase (HK), phosphofructokinase (PFK), pyruvatekinase (PK), citrate synthase (CS) and isocitric dehydrogenase (IDH) were significantly down-regulated, while phosphoenolpyruvate carboxykinase (PEPCK), fructose bisphosphatase (FBP) and glucose-6-phosphatase (G6P) were significantly up-regulated. Obviously, the expression of growth-related genes was disordered. Simultaneously, the antioxidant genes manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPX), glutathione-S-transferases (GST) and nuclear factor E2-related factor2 (Nrf2) were up-regulated to varying degrees in the EHP challenge group, and EHP infection induced significant increases in the oxidative damage products lipid peroxide (LPO) and malondialdehyde (MDA). Ultimately, the shrimp weight of the challenge group was 6.85 ± 0.86 g, which was significantly lower than that of the control group (8.95 ± 0.75 g). Taken together, we speculate that EHP changes the substance metabolism and growth process by causing oxidative damage to the hepatopancreas, which may lead to the growth retardation of P. vannamei.

[Effects of Different Land Use Practices on Nitrogen Loss from Runoff During Rainfall Events].

To understand the effect of nitrogen from runoff during rainfall events for different land uses, sub-catchments A and B in the small Shipanqiu watershed in Zhong County, Chongqing-which were managed using different land use practices-were taken as research objects. Runoff flow and nitrogen levels at the outlet of the catchment were monitored. Sub-catchment A is an agroforestry-water complex and sub-catchment B is the site of traditional agriculture. EMC was used to evaluate the average concentration of runoff nitrogen during rainfall events, and the effect of this runoff nitrogen on the small watershed with different land use systems was analyzed. The results showed that the TN concentration in catchment B (1.37-15.17 mg·L-1) > catchment A (0.84-9.28 mg·L-1); the ratio of the first peak to the second peak in catchment A was 62%, which was far less than the 97% in catchment B; the average DN/TN values were 69% and 75% in catchments A and B, respectively; and the average NN/DN values were 67% and 80% in catchments A and B, respectively. The different land use practices have significant impacts on nitrogen loss. Compared with the catchment where traditional agricultural practices were followed, the agroforestry-water complex catchment effectively reduced the loss of nitrogen and decreased the first TN peak value and DN/TN and NN/DN values. This study provides a scientific basis for the prevention and control of non-point source pollution in small watersheds in the area of the Three Gorges Reservoir.

B subunit of cholera toxin fused with VP7 from GCRV (grass carp reovirus) was expressed in E. coli and folds into an active protein.

Grass carp reovirus (GCRV) is one of the most serious pathogens threatening grass carp (Ctenopharyngodon idella) production and results in high mortality in China. To obtain a genetically engineered oral vaccine against GCRV, the cholera toxin B subunit (CTB) of Vibrio cholerae was fused to VP7 (CTB-VP7) and transformed into BL21(DE3) for expression. SDS-PAGE and Western blotting showed that the purified rCTB-VP7 fusion protein (rCTB-VP7) was approximately 49.0 kDa. The monomeric nature of rCTB-VP7 through multistage purification showed a binding affinity for GM1, a receptor for biologically active CTB. rCTB-VP7 is not vulnerable to disassembly by SDS but is vulnerable to disassembly by 2-mercaptoethanol. rCTB-VP7 is stable and highly active at room temperature. The binding affinity experiment between rCTB-VP7 and GM1 also confirms the effects of acid and alkalinity in solution on the structure of rCTB-VP7. rCTB-VP7 bound to GM1 with different affinities under different temperatures and pH values. Prokaryotic expression of rCTB-VP7 was characterized by high expression and easy purification and had a strong binding force with GM1 at 37 °C and pH 7.4. Our results suggest that rCTB-VP7 has the potential as an oral vaccine for protection against GCRV in aquaculture.

[Characteristics of Nitrogen and Phosphorus Output and Loss Flux in the Shipanqiu Watershed, Three Gorges Reservoir Area].

As the source of non-point pollution in the Three Gorges Reservoir Area, small watershed is a key control object in alleviating deterioration of water quality. In the Three Gorges Reservoir Area, the Shipanqiu small watershed with various land-use types was selected as the research object, and the water quantity and quality of the outlet section of the watershed were continuously monitored. We carried out analysis of the small watershed runoff loss and nitrogen and phosphorus pollutants with concentration, analyzed the morphology change characteristics of runoff erosion, calculated the small watershed of pollutant emission flux, and analyzed the nitrogen and phosphorus nutrient loss and main human and natural factors, especially in the Three Gorges Reservoir Area of agriculture where nonpoint source pollution research has important practical significance. The results showed that the rainfall in the watershed varied significantly with the seasons, and the rainfall was mainly distributed from April to June, which was the main output period of nitrogen and phosphorus loss in the small watershed, accounting for 58.94% and 67.60% of the total nitrogen and phosphorus load, respectively, in the whole year. The total annual runoff in the Shipanqiu small watershed was 8.02×104 m3, and the annual total nitrogen loss flux was 5.04 kg·hm-2, of which nitrate nitrogen (2.54 kg·hm-2) was the main part. The total phosphorus output was 0.534 kg·hm-2, and the soluble total phosphorus (0.422kg·hm-2) accounted for 79.00% of the total phosphorus flux. The loss flux of total nitrogen was 9.51 times that of total phosphorus, and the non-point source pollution risk of nitrogen was much greater than that of phosphorus. Therefore, for the Shipanqiu small watershed, it is especially important to prevent nitrogen loss in paddy fields when fertilization and rainfall coincide.

[Effect of Optimized Fertilization and Biochar Application on Phosphorus Loss in Purple Soil Sloping Farmland].

Phosphorus is an essential nutrient for crop growth, but the input of excess phosphorus is a significant cause of eutrophication. This study explored the relationship between fertilization methods and phosphorus loss in actual production, providing a theoretical basis for scientific fertilization and rational reduction of fertilizer application. In the experiment, a wild-type OD flow plot was used to monitor the occurrence of multiple rainfall runoff and sediment yield in purple soil sloping farmland in 2017-2018. Four different schemes of non-fertilizer treatment, conventional fertilization treatment, optimized fertilization treatment, and reduced fertilization combined with biochar were studied. The effects of soil flow, surface runoff, and sediment phosphorus loss on purple soil sloping farmland were analyzed. The results showed that:①The total yield of each treatment was optimized (20737.23 L) > conventional (18513.17 L) > CK (18134.58 L) > biochar (13594.85 L), and the total sediment yield of each treatment was CK (1998 kg·hm-2) > biochar (1884 kg·hm-2) > optimized (1681 kg·hm-2) > conventional (910 kg·hm-2). The middle stream of soil is the main type of runoff in the rainy season, accounting for 60.14%-87.34% of the total output flow. The total amount of sediment produced by each treatment was not significantly different from that of the conventional treatment (P>0.05). ②The flux of total phosphorus loss in each treatment was characterized by sediment > surface runoff > soil middle flow. Phosphorus lost through the middle stream of soil is the least, accounting for only 2.63%-12.91% of the flux of total phosphorus loss, while the flux of sediment loss of phosphorus can reach 63.74%-78.74%, and thus is the main output route of soil phosphorus loss. ③The application of biochar can effectively reduce the abortion flow in the soil of purple soil sloping land, and the loss flux of orthophosphate in the middle stream, which are 49.94% and 56.45% lower than the conventional treatment, respectively. However, the interception effect on surface runoff is not good, and there is no significant influence on the flux loss of particulate phosphorus. At the same time, the flux of total phosphorus in surface runoff and sediment is significantly increased by 73.28% and 123.53%, respectively, compared with conventional treatment (P<0.05). Therefore, to control the loss of phosphorus in purple soil sloping farmland in southwest China, we should focus on reducing the occurrence of soil sediment loss. Bio-carbon should be further optimized in the practical application of agricultural production with the phosphorus fertilizer input ratio.

[Response of Nitrogen Loss Flux in Purple Soil Sloping Field to Reduced Fertilizer and Combining Straw].

The purple soil sloping field is considered as the main source of sediment and non-point source pollution in the Three Gorges Reservoir area. To prevent and control the non-point source pollution, it is indispensable to explore the characteristics of nitrogen loss in the overland flow and interflow of purple soil sloping field in Three Gorges Reservoir area. The purple soil sloping runoff plots, located in the Shibaozhai Experimental Station of Chengdu Institute of Chinese Academy of Sciences in Zhongxian County, Chongqing, were studied. The experiment included no fertilization treatment (CK), traditional fertilization treatment (T1), amended fertilization treatment (T2), and reduced fertilizer with straw treatment (T3). According to the data of volume of the interflow and runoff and the leach concentration and flux of nitrogen forms under rapeseed-maize rotation system, the response of nitrogen leaching flux to reduce fertilizer with straw application can be definite in purple soil sloping plots. The results show that the ratio of interflow to total runoff is 60.14%-88.56%, and the flux of nitrogen leaching in the interflow accounts for 72.88%-92.35% of total nitrogen loss flux. Ammonium was mainly leached by the overland flow. In addition, nitrate was mainly leached by the interflow and was the main form of nitrogen leaching. The fluxes of ammonium and nitrate under different treatments followed the order T1 > T2 > T3 > CK. The total nitrogen flux of T3 was 20.07 kg·(hm2·a)-1, which was 43.59% and 39.55% lower than that of T1 and T2, respectively. The reduced fertilizer with straw application significantly decreased the leaching flux of ammonium, nitrate, and total nitrogen, and weakened the effect on runoff nitrogen leaching in the purple soil sloping plots.

[Effect of Fertilizer Reduction and Biochar Application on Soil Nitrogen Loss in Purple Upland].

This study seeks to clarify the effect of biochar application on nitrogen loss patterns and flux in purple arid sloping land, so as to provide a scientific basis for improving the quality of farmland and reducing the risk of agricultural non-point source pollution in purple arid sloping land. The effects of four treatments on surface runoff and soil nitrogen loss patterns and fluxes in purple arid sloping land were studied by field experiments, including no fertilization (control), conventional fertilization, optimized fertilization, and biochar (fertilizer reduction and biochar application). The results showed that:① Of the fertilization treatments, the total runoff of conventional treatment was the highest at 16133 L·a-1, and the total runoff of biochar treatment was the lowest at 11893 L·a-1. In each fertilization treatment, soil midstream was the main mode of runoff, accounting for 61.80%-68.60% of the total loss. Compared with the control (no fertilization treatment), the sediment loss in other fertilization treatments was decreased, with conventional treatment showing the most significant effect. ② Ammonium nitrogen is mainly lost by surface runoff, accounting for 86.51%-96.58% of the total loss flux. Biochar treatment had the highest loss flux at 0.69 kg·(hm2·a)-1, and the control treatment had the lowest at 0.17 kg·(hm2·a)-1. ③ The concentration of granular nitrogen in the production flow of each fertilization treatment was higher than that of the control treatment, and the loss flux of granular nitrogen in the conventional fertilization treatment was the highest at 2.87 kg·(hm2·a)-1. ④ There was a significant positive correlation between total nitrogen concentration and nitrate nitrogen concentration in the soil midstream and surface runoff of each fertilization treatment (P<0.01). Nitrate nitrogen is the main form of total nitrogen loss, and both take soil midstream as the main way of loss. The total nitrogen loss through soil flow accounted for 72.86%-89.13%, and that of conventional fertilization was the highest at 35.58 kg·(hm2·a)-1, whereas that of biochar treatment was the lowest at 21.49 kg·(hm2·a)-1. Reducing the amount of fertilizer and applying biochar can significantly reduce the runoff and nitrogen flux, and effectively prevent and control the risk of agricultural non-point source pollution.