Molecular Soil Biology 2026, Vol.17, No.1, 12-25 http://bioscipublisher.com/index.php/msb 15 3 Overview of the Study Area and Experimental Design 3.1 Natural conditions and soil types in the study area The main areas of subtropical rice cultivation in China are distributed in the middle and lower reaches of the Yangtze River to the south of the country. These regions generally have hot and humid climates with long frost-free periods, and double-cropping is quite common, such as double rice crops or rice-crop rotation with other crops (Chen et al., 2020). Taking the Changsha Agricultural Environmental Observation Research Station of the China Flux Observation Research Alliance as an example, this site is often used to represent the typical agricultural ecological environment of the mid-subtropical hilly areas. The local area has a humid monsoon climate, with an average annual temperature of approximately 17.5 ℃and an annual rainfall of about 1330 mm, and the frost-free period is close to 280 days. The soil in the paddy fields at this site is typical paddy soil, with a plough layer thickness of approximately 0.2 m. It also records basic physical and chemical indicators such as organic matter, total nitrogen, and particle composition, and is therefore often used as a reference background for the study of the microenvironment of subtropical paddy fields. From the perspective of soil origin, many paddy fields in the south originally evolved from red soil or red-yellow soil parent materials. After long-term cultivation and repeated flooding, they gradually form characteristic layers of paddy soil, where iron and manganese patches and pH gradients are quite common (Huang et al., 2018). These properties often affect the redox cycle and are closely related to the iron reduction process and methane production. 3.2 Irrigation treatment setup and field experiment layout In field studies, if one wants to compare the effects of different irrigation methods, a continuous flooding (CF or FI) treatment is usually set as a control first, and then several different intensity or frequency dry-wet alternating irrigation treatments are arranged. A "safe AWD" experiment was conducted in Guangzhou, South China, which can serve as an example (Lampayan et al., 2015). The experiment was carried out in two seasons in 2014: in the early season, a randomized block design was used to compare AWD15, AWD30 and continuous flooding; in the late season, a split-plot design was adopted, with AWD15, AWD30, CF and the commonly used irrigation method by farmers (FP) as the main plots, and different rice varieties were also included. Throughout the process, the field water level and soil water potential were continuously recorded, and methane emissions were monitored at fixed time intervals. At the same time, crop growth, yield and water productivity were observed. Regarding the irrigation threshold, "safe AWD" usually considers a depth of about 15 cm below the soil surface as a relatively safe re-irrigation level. However, during the sensitive period from heading to flowering, a shallow water layer is generally required to avoid obvious water stress, and this point is often mentioned in many reviews and management recommendations (Carrijo et al., 2017). 3.3 Sample collection and data acquisition methods The measurement of greenhouse gas fluxes in paddy fields typically involves two approaches: one is the static chamber or automatic chamber method, and the other is the eddy correlation method. The static chamber method is more common, where a chamber is placed in the field plot and the gas inside the chamber is sampled at regular intervals, and then the concentration changes are measured using gas chromatography, and the flux is calculated through regression (Figure 2) (Butterbach-Bahl et al., 2016). In contrast, the eddy correlation method can continuously monitor the gas exchange at the entire field scale, but it requires high equipment standards, and has stricter requirements for terrain conditions and data processing. Regarding the manual chamber method, some operational guidelines specifically emphasize the arrangement of sampling times, the airtightness of the chamber, and the standardization of flux calculation methods; there have also been studies specifically comparing the scale differences between the static chamber and eddy correlation methods in paddy field methane monitoring (Dengel et al., 2019). As for the acquisition of microbial data, the common practice is to collect surface or rhizosphere soil samples, first using 16S rRNA amplicon sequencing to analyze the community structure, and then detecting the abundance of mcrA and pmoA through qPCR. If a deeper understanding of functional changes is needed, combined macro- or transcriptomic analysis can be conducted, which allows for the simultaneous explanation of microbial communities, functional genes, and gas fluxes within the same framework.
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