In this study,based on the results of conventional pathogen isolation,identification and pathogenicity test,the methods of denaturing gradient gel electrophoresis(DGGE) and principal component analysis(PCA)were applied to determine the population structure of pathogenic Fusarium species associated with soybean root rot in a continuous cropping soybean field located at the Hailun Experimental Station of Agricultural Ecology in Northeast China.After fungal isolation and morphological identification,six Fusarium species were determined,including F.avenaceum,F.equiseti,F.graminearum,F.solani,F.oxysporum and F.verticillioides.The maximum isolation ratio was 56.7% of F.oxysporum.The DGGE fingerprint offered 13 bands about tef-1 sequence of Fusarium,and eight bands had been cloned,sequenced and aligned successfully.Besides the above six Fusarium species determined by conventional isolation and identification,additionally the other two Fusarium species were identified,namely F.culmorum and F.oxysporum-like species.Meanwhile,the percentage of F.oxysporum decreased to 37.4%.According to principal component analysis,the first principal component(PC1) accounted for 91.2% of variation in all data,which separated the Fusarium populations into pathogenic group and nonpathogenic group.The second principal component(PC2) weighed 5.8% of all variation,which could adjust the error evaluation of pathogenicity.In conclusion,F.oxysporum,F.graminearum and F.avenaceum were confirmed as the pathogenic fungi of soybean root rot in this experimental station with F.oxysporum as the dominant pathogen.

The effect of conservation tillage on crop growth and soil microbial community play an important role in soil management.A long-term tillage experiment was conducted at Hailun Agro-Ecological Experiment Station of the Chinese Academy of Sciences.Tillage systems included no-tillage(NT),reduced tillage(RT)and rotary tillage(ROT).The study was to determine soybean growth and yield under conservation tillage,and analyze soil microbial diversity with Real-time QPCR and denaturing gradient gel electrophoresis(DGGE).NT and RT inhibited soybean growth and increased the disease severity of root rot at seedling stage.Soybean growth and yield was significantly increased under NT and RT compared with ROT at maturing stage.The highest yield of 2 507.3 kg·ha-1 was obtained under RT,which was increased by 3.9% compared with ROT.The three tillage systems had no significant effect on the amount of bacteria,and the amount of fungi was NTRTROT(P0.05).The colony forming units(CFU)and the genomic DNA quality of soil fungi were increased by 31.9% and 13.3% under NT compared with ROT,respectively.The ratio of bacteria and fungi was increased significantly under RT and ROT.DGGE fingerprint showed that the soil bacterial diversity had no significant difference under three tillage systems,however,NT and RT increased soil fungal diversity.Principal component analysis(PCA)of DGGE banding patterns indicated that the bacterial community structure had a higher similarity under RT and ROT,and significant differences were observed on the fungal community structure under three tillage systems.

Base on the internal transcribed spacers (ITS) sequences of Trichoderma spp.,a pairs of specificity primers(DG and DT)were designed for the real-time quantitative PCR assay of soil Trichoderma. The optimal reaction system of real-time quantitative PCR was established. The masses of genomic DNA of Trichoderma spp. in soybean filed were quantified by using this reaction system,and the genomic DNA was extracted from three long-term fertilization management; no fertilization(NF),chemical fertilization applied with nitrogen and phosphorus(NP)and chemical N,P combined with pig manure(NPM). Primers DG and DT possessed a good specificity to the amplification of Trichoderma spp..The correlation coefficient(R2) and slope of the equation from the standard curve were 0.993 and -0.282 9 respectively,and the melting curve only showed a single peak. At seedling stage of soybean growth stage,the mass of genomic DNA of Trichoderma spp. in NF,NP and NPM treatments were 0.4 ng,0.8 ng and 1.2 ng per gram of soil,respectively. The mass in NPM measure was higher than that in NF and NP measure significantly (P<0.05).

At the five growing stages,the abundance of Fusarium population in black soil under three fertilization managements was quantified using the Plating Counting method and Real-time QPCR method.Fertilization management included no fertilizer(NF),nitrogen and phosphorous chemical fertilizer(NP) and chemical fertilizer with manure(NPM).The result of Plating Counting method showed that the maximum of Fusarium CFU in NF and NP treatment was 1.5×103 and 3.1×103 g-1 dry soil at R1 stage.The peak value in NPM treatment was 1.9×103 at V3 stage.However,the dynamic trend of Fusarium CFU in each treatment during the growth stages or the difference of CFU values at the same stage was not significant,indicating the quantified detection of Fusarium abundance was infeasible using the Plating Counting method.The result of Real-time QPCR method indicated that the peak values of Fusarium DNA amount in the three treatments occurred at the same stage.The values of DNA quantities in the three treatments were 99.6,77.1 and 140.8 pg per gram dry soil,respectively.Meanwhile,the dynamic trend of Fusarium abundance in three fertilization treatments was similar.The amount was increased from V3 stage and then decreased after their peak values.It indicated that this method was sensitive to investigate the abundance of soil Fusarium population;and the fertilizer not only could affect the amount of Fusarium DNA,but also Time new romeen.

For establishing the system of quantitative analysis for soil bacteria population,3 soil treatments including the sample P1 and P2 which was polluted by polycyclic aromatic hydrocarbon( PAH) ,and sample C from bare land were sampled and analyzed. Using the molecular cloning method,the standard substance of real-time PCR was structured,and the relevant parameter was optimized including the linearity( R 2 ) ,amplification efficiency( E) and slope( S) of the standard curve. The results indicated that the relevant parameter is regular for real-time PCR,which R 2 was 0. 985,E was 101. 7 % and S was 3. 282. The quantity of bacteria cell in soil sample P1,P2 and C was quantitated as 1. 4 × 10 9 ,4. 0 ×10 9 and 5. 0 × 10 9 respectively. It indicated that the abundance of soil bacteria could be quantitated by the established system of real-time PCR using unknown sensitive template.

【Objective】The study was done in order to explore the effect of natural sunlight hours on soybean yield and chemical quality in the pod stage,and to provide reference for cultural practices of high yield and superior quality soybean with high oil or protein content.【Method】The effect of different sunlight hours controlled by human on soybean yield,oil and protein content was conducted in the field with random blocks design.【Result】The results showed that the effects of different sunlight hours on soybean yield and chemical quality with high oil or protein content(Dongnong 434 and Dongnong 42)were different.Soybean yield of the two cultivars declined evidently with shortened sunlight hours in the pod stage,and declined level of Dongnong 42 was higher than that of Dongnong 434.Oil and protein content of the two cultivars were not changed distinctly,and gross contents of oil and protein of Dongnong 434 and Dongnong 42 were improved by proper shortened sunlight hours in the pod stage.【Conclusion】These indicated that the effect of natural sunlight hours in the pod stage on soybean yield was visible,but that of chemical quality wasn't obvious.

On the long-term experiment in situ,soybean rhizosphere soils in different growth stages of soybean continual cropping for 16 years(S-S...S),wheat-corn-soybean(W-C-S...W-C-S),corn-soybean(C-S...C-S)and corn-soybean-soybean (C-S-S...C-S-S) were sampled for the investigation of soil fungi and the main groups,to study the effect of soybean long term continual cropping on the fungi main groups change of soybean rhizosphere.The results show that:the total CFU/g dry soil of fungi descending order in different soybean rotation systems was CS-S...C-S-S C-S...C-S W-C-S...W-C-S S-S...S;the quantity of Penicillium spp.in different soybean rotations from high to low was C-S-S...C-S-S W-C-S...WC-S C-S...C-S S-S...S;in all the soybean growth stages,the quantity dynamic of Trichoderma spp.in different soybean rotation systems varied,and the descending order of Trichoderma spp.quantity was C-S...C-S S-S...S C-S-S...C-S-S W-C-S...W-C-S;the quantity of Fusarium spp.was highest in S-S...S,and all lower than W-C-S...W-C-S after V5,the quantity of Fusarium spp.was higher at R5 and R8 in W-C-S...W-C-S than others.

Fusarium root rot of soybean is one of the soil-borne diseases which is difficult to control. In soft, distribution of Fusarium is surprisingly diverse,which can efficiently influence the pathogenic Fusarium populations and host resistance. Isolation of Fusarium is very useful in the study of soil Fusarium diversity. Many methods for the isolation of Fusarium have been reported,but the efficiency was certainly different. In this paper,some related contents which affect the effeciency of Fusarium separation were researched, and dilution plate method and direct soil plating method were compared. The results showed that bacteria could be inhibited excellently when the concentration of antibiotic reached fourfold, but there was no significant impact on the number of fungi. Interestingly ,it seems that different combination of antibiotics had effect on Fusarium isolation. Between colony forming units (CFU) of fungi and different sample volume,well linear relationship were de tected in both sterile water and Water Agar treatment(R2 ＞ 0.9). The isolation rate of Fusarium in sterile water treatment was 21.0%, a bit higher than that in Water Agar treatment, which was 10.0%. No significant effects on Fusarium isolation between two culture media were observed. Compared with Malachite Green Agar (MGA),Peptone PCNB Agar (PPA) behaved higher isolation rate of Fusarium which was 20.93%. The difference between Fusarium isolation rates derived from two culture methods were significant. The isolation rate of Fusariurn derived from direct soil plating method were all above 60.0% ,which is significantly higher than that derived from dilution plate method which were all below 6%.

Proper agronomical management practices could reduce yield loss of continuous cropping soybean.In order to make clear out the effect of appending fertilizer on soybean at the black soil plain area,foliar fertilizer sprayed and nitrogen fertilizer applied at later growth stage,fresh weight of soybean root nodules,dry weight above and under ground,leaf area index and yield of soybean were investigated.The result showed that fresh weight of soybean root nodules by appending fertilizer later were increased which was as 1.3-1.6 times of control,dry weight above and under ground were significantly higher than control,and leaf area index was increased.Soybean yield of appending fertilizer later was increased at above 11% of control at the black soil plain area,and the soybean disease and pest seeds rate of that were reduced.Therefore,soybean commercial quality was increased.Soybean growth and development indexes and yield wasn′t significantly affected by foliar fertilizer sprayed and nitrogen fertilizer applied in soil at later growth stage.