| In southeast China, seasonal drought exerts a tremendous influence on physiological metabolism, growth, yield, and corn quality of crops, especially on lowland rice. Continuously double rice systems are prevailing in these areas. Unfortunately, the second crop season(late rice) is mainly grown in drought periods in these areas. So, the growth and production of rice cultivation in second cropping season is affected greatly by water limited. From 2003 to 2006, we carried out a long-term field experiment to compare the agronomic performance of rice in both early-rice and late-rice seasons. In this experiment, we set up a new farming and water management system, which is the rotation of rice cultivation under flooded(the first cropping season, early rice) and non-flooded(the second cropping season) conditions. In the second cropping season, we considered several options to reduce water consuming, which included rice cultivation under non-flooded condition, mulching with rice straw and no-tillage before transplanting of late-rice.Understanding the effects of alternating conditions in paddy soil on the microbial community would increase our knowledge about adaptation mechanisms of microorganisms developed in response to environmental stress and organic matter inputs. Rice cultivated in non-flooded(NF).and flooded conditions(CF) cause to a different living environment for microbes. Until now, except for a few preliminary studies, only few detailed research has been conducted to examine the microorganisms in non-flooded paddies, including their adaptive mechanisms and physiological responses to oxidative and water stress. Fluorescence in situ hybridization(FISH) has emerged as a popular technique for identifying microbial cells. In soil samples, however, cell losses, cell clumps, and low signal-to-background ratio have often been mentioned as major problems for application of standard FISH techniques. FISH with catalyzed reporter deposition(CARD-FISH) has the potential to overcome this limitation. We improved the analysis methods of FISH and CARD-FISH for the identification of paddy soil microbes. The results of this study are presented as follows:1. Water consumption and water use efficiency under different water management systemsWater consumption and water use efficiency under different water management systems were tested in the second cropping seasons both in 2003 and 2004. Irrigation water was 19950 m3 ha-1 in 2003 and 15850 m3 ha-1 in 2004 in the CF treatments and 7200 m3 ha-1 in 2003 and 5045 m3 ha-1 in 2004 in the non-flooded rice fields(NF-ZM and NF-SM, represented rice cultivation under non-flooded condition without mulching and with rice straw mulching repectively). The field measurements showed that average water seepage of 2003 and 2004 was 13442 m3 ha-1 in the CF treatment, 5510 m3 ha-1 in the NF-ZM treatment and 5424 m3 ha-1 in the NF-SM treatment. Rice straw mulching decreased evapotranspiration by 33% and 63%(in 2003), 36.5% and 57.1%(in 2004) to the NF-ZM treatment and CF treatment, respectively. Compared with the NF-ZM treatment, mulch application significantly increased the leaf area per plant, main root length, tap root length and root dry weight per plant of crop. The yield of the NF-SM treatment(2003: 6489 kg/hm2; 2004: 8574.8 kg/hm2) was similar with the value of the CF treatment(2003: 6811.5; 2004: 8630.5 kg/hm2), and much higher than the NF-ZM treatment(2003: 4716; 2004: 6394.8 kg/hm2). The order of irrigation water use efficiency(IWUE) and water use efficiency(WUE) were as follows: NF-SM>NF-ZM>CF.2. Crop productivity under different rice cultivation systems from 2003 to 2006.1) There was no clear tendency within each treatment both for late rice and early rice. The new system(flooded-non flooded cultivation system) in the seasonal drought area with rice-rice rotation might be very important for water-saving cultivation of rice and sustainable agriculture in Southeast China.2) For 4 late rice seasons, the yields of FC treatment were higher than others. There was no significant between the yields of FC and NF-SM treatments. As a whole, the order of yields among different treatments was FC>NF-SM>NF-ZM. For the NF-ZM treatment, the yield of no-till system was higher than conventional tillage system in 2003, 2004 and 2006. For early rice, the yield of no-till system was higher than conventional tillage system in all growing seasons. Furthermore, there was significant difference in 2005 and 2006 between conventional tillage system and no-till system. 3. Soil microbial characteristics under different water-saving rice cultivation systems1) The improvement of(CARD-) FISH procedure for the identification of paddy soil microbesConsidering the special and complex characteristics of soil compared with other environmental samples(water, marine sediments and so on), we performed some tests in our samples of paddy soil. The detection rate increased from 14.3% to 20.6% with FISH protocol and ultrasonic treatment dispersed, as well as 79.8%-91.2% with CARD-FISH. Samples that were not treated with lysozyme showed lower detection rates and lower hybridization signals after FISH and CARD-FISH. Detection rates of a EUB338-HRP with CARD increased from 33.6% of DAPI-stained cells in the samples to 91.2% after 60 min of permeabilization. The low-gelling-point agarose was also very important to prevent the cell loss during cell wall permeabilization, hybridization and washing procedures. Both with FISH and CARD-FISH protocols, a strong decline in cell numbers was observed in samples that were not embedded with agarose. The results showed that the ultrasonic-dispersing, lysozyme incubation and low-gelling-point agarose embedded were all the crucial steps for both FISH and CARD-FISH in our soil samples. Another improvement of the method was the higher temperature we used during signal amplification and washing. We increased the temperature to 46℃and 48℃respectively during hybridization and washing procedures, instead of the temperature(35℃and 37℃) used widely before. Compared with the single monolabeled oligonucleotide FISH probes, the CARD-FISH with HRP-labeled protocols could increase the detection rates greatly in the paddies we studied.2) Identification of bacteria and archaea in paddies with CARD-FISHIn this study, bacteria and archaea in paddy field soil was studied by CARD-FISH with horseradish peroxidase(HRP) oligonucleotide probes and tyramide signal amplification. The approach greatly enhanced the sensitivity and applicability of FISH with paddies samples, allowing confident identification and enumeration of bacteria and archaea cells in paddy soil under different treatments. Quantitative whole-cell hybridization experiments accounted for 90 to 100% of the total 4', 6-diamidino-2-phenylindole(DAPI)-stained cells in most samples. We tested two layers of soil samples both on filters and slides. Overall, the intensities of bacteria cells detected by CARD-FISH on membrane filters were higher than on slides. For the surface soil samples(0-7cm), total cell counts after DAPI-staining on filters were in the range of 6.199×108 to 8.25×109 cells g-1 soil, and with the slides were 5.71×108 to 7.34×109 cells g-1 soil. At the bottom layer of soil cores(7-14cm), the values were in the range of 3.21×108 to 4.17×109 cells g-1 soil and 2.44×108 to 3.17×109 cells g-1 soil correspondingly. The abundances of the surface samples with the CARD-FISH using HRP-EUB338 probes were much higher than the bottom samples, either with the membrane filters or slides. The highest cells abundance reached to 7.93×108 cells g-1 soil in the NF-SM treatment. Among the different treatments of surface layer, either with membrane filters or slides, the order of cells abundance with CARD-FISH was NF-SM>NF-ZM>CF. However, it changed to NF-SM>CF>NF-ZM at the bottom layer. In the surface samples, we could not detect any stained cells with ARC-915 at the NF treatments(NF-SM and NF-ZM). The highest abundance of archaea cells was 10.01×106 cells g-1 soil in the bottom samples of the CF treatment.3) Detection of methanogen with specific probesSpecific probes were used to study methanogen in FC and NF-ZM treatments. The results showed that the abundance of all methanogen species in the bottom soil layer(7-14cm) was much higher than the surface layer(0-7cm). Under 4℃conditions, the abundances of cells in the surface layer of CF treatment with ARC915, MX825, MS1414 and MSMX860 were 2.16×l07cell/g soil, 1.63×107 cell/g soil, 0.44×107 cell/g soil and 0.59×l07 cell/g soil respectively. The cell abundance was much higher in the bottom layer. For the soils of FC treatment, after 48h under room temperature within closed condition, cell abundance increased significantly. There was no significant difference after 48h and 168h incubation under room temperature.
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