| Chengdu plain is one of the most important grain-producing areas in China,and the rice-wheat rotation system plays key role of the crop planting patterns.Since long-term application of chemical fertilizer has resulted in soil acidification and soil fertility reduction,which became the key limiting factor affecting the agricultural sustainable development.Application of organic fertilizer properly or combination of organic fertilizer and chemical fertilizer is a good way to reduce chemical fertilizer usage.Biogas manure,a high quality fertilizer resource,can improve soil fertility,and increase crop production.In this paper,the field experiment included six fertilizer treatments,i.e.,low amount of biogas manure(BL),medium quantity of biogas manure(BM),high quantity of biogas manure(BH),biogas manure combined with chemical nitrogen fertilizer(BN),chemical fertilizer(NPK),and no fertilizer treatment(CK)were carried out in Chengdu plain from 2014 to 2016,the effects of biogas manure application in rice-wheat rotation system on crop yields,soil and crop quality,soil microbial property has been revealed,and soil ammonia-oxidizing microorganisms were detected by PCR-DGGE and clone sequencing technology at wheat tillering stage in 2014,soil bacterial and fungal population communities at harvest periods was discussed by using Miseq sequencing technique.The main results were shown as the below:Compared with no fertilizer treatment(CK),the application of biogas manure significantly increased the crop yields.The highest rice and wheat yields appeared in NPK treatment in 2014a,while in 2015a and 2016a,BN treatment got the highest rice and wheat yield,and the rice yield was 7554.52 kg-hm"2 and 7985.85 kg·hm-2 in 2015a and 2016a,and increased by 73.05%and 73.05%over CK,respectively;wheat yield was 4176.80 kg-hm"2 and 4708.65 kg·hm-2 in 2015a and 2016a,increased by 73.05%and 73.05%compared with CK,respectively.The results also showed that application of biogas manure could improve the yield components,which was good consistency with yield result.Biogas manure also increased the crop nutrient content,the results revealed that the crude protein content of rice grains was the highest in NPK treatment in 2014a,followed by BH treatment,but there was not significant difference between treatments BH and NPK.however,treatment BH had the highest crude protein content of rice grains both in 2015 and 2016,and was 7.71%and 7.07%,increased by 26.88%and 36.49%over CK,respectively.The crude protein content in wheat flour was the highest in NPK treatment in 2014a,but there was not significant difference with BH and NPK.BH and BN treatments had the highest crude protein content in wheat flour both in 2015 and 2016,and was 11.36%and 11.20%,and increased by 31.46%and 24.22%with CK,respectively.The results showed that Cd has not been detected in all the rice grains of different treatments from 2014a to 2016a.The content of Cr,Cu,Pb,and Zn in rice grains ranged from 0.138-0.259 mg·kg-1,2.875-4.075 mg·kg-1,0.078-0.139 mg·kg-1,17.806-41.825 mg·kg-1,respectively.Similarly,Cd has not been detected in wheat flour;the content of Cr,Cu,Pb and Zn in wheat flour ranged from 0.146-0.330 mg·kg-1,0.212-0.392 mg·kg-1,3.539-5.181 mg·kg-1,0.064-0.169 mg·kg-1,and 17.060-38.583 mg·kg-1.The content of all heavy metal in both rice grains and wheat flour was lower than the state food and heavy metal content restrict standard for grain(NY861-2004).Compared with CK,application of biogas manure significantly increased the soil nutrient contents,treatments BH and BN had good treatment effects.The soil TN,OM,A-P,A-K,NO3-N,NH4-N contents were increased by BN treatment from 1.65%-78.38%,30.02%-76.87%,19.53%-295.74%,19.16%-31.92%,10.38%-28.03%,19.16%-21.94%at rice season from 2014 to 2016,respectively;and which were increased by BN treatment from 7.52%-41.33%,13.44%-79.03%,6.13%-78.58%,20.99%-34.38%,20.49%-36.02%,and 6.13%-31.52%at wheat season from 2014 to 2016,respectively。In wheat season,the highest soil total nitrogen content and soil organic matter content was presented to the BH treatment from 2014a to 2016a;the highest available phosphorus content was presented to BH,BH in 2014a,2015a,and to NPK treatment in 2016a;the highest soil available potassium content was showed in BN,NPK and BH treatment;the highest soil nitrate content was showed in NPK treatment in the first two years,and in BH treatment in 2016;the highest soil ammonium nitrogen content was presented to treatment BH,BN and BH.Overall,fertilization significantly increased the soil nutrient contents,especially the BH and BN treatments.The quantity of soil Cd,Cr,Cu,Pb and Zn content in rice season ranged from 0.136-0.338 mg·kg-1,50.475-142.547 mg·kg-1,9.755-18.038 mg·kg-1,19.453-43.858 mg·kg-1,83.793-171.925 mg·kg-1;the quantity of soil Cd,Cr,Cu,Pb and Zn content in wheat season ranged from 0.146-0.227 mg·kg-1,34.128-139.367 mg·kg-1,9.728-17.029 mg·kg-1,19.560-52.708 mg·kg-1,90.053-158.567 mg·kg-1,which suggested that application of biogas manure did not cause soil heavy metal pollution.From 2014 to 2016,the soil Cd,Cr,Cu and Pb content tended to decrease,except the soil Zn content,all the heavy metal content of tested soil was far below the second level of soil environment quality standard(GB15618-1995).To understand the effect of different fertilizing treatments on soil nitrification intensity,soil nitrification intensity,16S rDNA PCR-DGGE for AOB communities,amoA PCR-DGGE for AOA communities was analyzed at wheat tillering stage in 2014a;then the representative DGGE bands were cloned and sequenced,the phylogenic tree was constructed.The results showed that compare to CK,fertilization soil significantly improved soil nitrification intensity(P<0.05).The soil nitrification intensity varied as the following:BH(54.83%)>NPK(52.12%)>BM(48.56%)>BN(47.78%)>BL(44.75%)>CK(38.93%).Based on the PCR-DGGE patterns,the AOB and AOA diversity index(H)was determined,the results showed that the H value of AOB changed obviously(p<0.05);however,the H value of AOA among different treatments changed slightly.Sequencing and phylogeny analysis showed that AOB was very diverse at wheat tillering stage,and two novel AOB clusters were formed;AOA among the different treatment soils belonged to Soil/Sediment-associated cluster,and were very similar.The application of biogas manure significantly increased soil microbial biomass carbon(SMBC),soil microbial biomass nitrogen(SMBN),and soil microbial phosphorus(SMBP)content at harvest periods.From 2014 to 2016,treatment BH had the highest SMBC content in rice season,increased by 108.38%,94.14%and 108.38%than CK,respectively;treatment BH got the highest SMBN content,increased by 92.03%,120.49%and 92.03%over CK,respectively;the highest SMBP was appeared in NPK treatment in 2014,and appeared in BH treatment both in 2015a and 2016a,and was increased by 109.10%,67.90%and 109.10%than CK.In wheat season from 2014a to 2016a,treatment BH had the highest SMBC content,increased by 91.67%,136.40%and 91.67%than that of CK,respectively;the highest SMBN content presented to treatment BH,and increased by 109.10%,30.58%and 109.10%than CK,respectively;the highest SMBP,presented to treatment BH,and increased by 133.00%,67.90%and 56.26%than CK.Application of biogas manure can increase soil hydrolase and redox enzyme activities significantly.Treatment BH and BN could significantly increase soil invertase,amylase,enzyme lactase,cellulose,catalase,neutral phosphatase,protease and catalase activities,and treatment NPK significantly improved the soil urease activity in 2014a,but decreased the soil invertase and protease activity significantly both in 2015a and 2016a.Miseq sequencing analysis showed that,at the 97%level,7114 OTUs were affiliated to the bacteria,divided into 43 phylum,included ’101 classes,229 orders,434 families,734 genera,and 1576 species;3727 OTUs were affiliated to the fungi,divided into 8 phylum,contained 32 classes,105 orders,249 families,522 genera,and 835 species.At the phylum level,Proteobacteria was the most dominante one,and followed by Acidobacteria,actinomyces,Chloroflexi and bacteroidetes.At the genus level,subgroup-6 affiliated with Acidobacteria was the predominant bacterial group.Among the soil fungal groups,Ascomycetes was the most predominant one,and then Zygomycota and Basidiomycota.At the genus level,Mortierella affiliated with zygomycetes was the predominant fungal group.PCA and Heatmap analysis showed that the bacterial communities among the treatments applying biogas manure singly were very similar,while the bacterial groups in BN treatment was more similar to NPK treatment;the fungal communities of the biogas manue treatments were more different from that in BN and NPK treatment.RDA results showed the organic matter content and available P were the key factors affecting the bacterial communities in rice season,and the organic matter content was more positive to the bacterial communities among of the biogas manure applying treatments;however,available P was more positive to the bacterial communities in NPK treatments.The total N was the key factor affecting bacterial communities in wheat season,while organic matter,total N and available P were the most important factors affected fungal community structures.Overall,the biogas manure combined with chemical nitrogen fertilizer(BN)treatment was the proper fertilization schedule in Chengdu plain,there was about 178 kg·hm-2·a-1 and 178 kg·hm-2·a-1 of urea could be substituted by biogas manure,respectively;and phosphate and potash could be saved in a period. |
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