The Responses of Soil Microbial to Changed Rainfall and Increased Temperature in Desert Grassland in Northern China

Background: The study evaluates how rainfall change and temperature increase affect microbial communities in the desert grassland of Ningxia Autonomous Region, China to explore the soil microbial community and the relationships among the soil microbial community, chemical properties, soil respiration (SR) and plant biomass under the climate change. We established the eld experiment with ve levels of rainfall by rainout shelters and two levels of temperature by Open-Top Chamber (OTC). Results: The effect of temperature to soil microbial communities is not signicant, but with the continuous increase of rainfall, the microbial community gradually increases. Soil microbial diversity negatively correlated with soil CO 2 ux. The α-diversity of microbial communities positively correlated with above-living biomass (ALB) and soil temperature (ST), but negatively correlated with root biomass (RB). Conclusions: Both rainfall and temperature’s rising do not promote the soil community α-diversity, but it can promote soil microbial community β-diversity. Soil microbial communities show resistance to rainfall changing. Soil respiration (SR) will limit soil microbial diversity. Soil organic carbon (SOC), soil total nitrogen (STN), and soil total phosphorus (STP) will promote soil microbial abundance and diversity. ALB and ST will promote the soil α-diversity, but the effect of RB to soil microbial is opposite. These ndings maybe provide a reliable theoretical basis for formulating a reasonable response strategy in desert steppe ecosystems. (mean±SE). lowercase signicant between the variance of The are precipitation ve levels of R are 33%, 66%, 100%, 133%, and 166% of normal precipitation (recorded R33, R66, CK, R133, R166), two manipulated precipitation (i) annual (ii) (66% (iii) (iv) (133% (v) mm

signi cant to research microbial feedbacks, especially in ecosystem types under concurrent changes of temperature as well as rainfall changes in moisture and temperature conditions. It also affects aboveground plant communities [7] which manage the type and abundance of many organic substrates provided to soil microbial heterotrophs. So, climate change should also affect microbial communities indirectly by shifts in plant composition and productivity. The semi-arid steppe ecosystem in northern China is a crucial component of the Eurasian grassland biome [8]. The water is an important reason, and soil temperature is low from last autumn to early spring, limiting microbial activity [9]. The changing of moisture and temperature conditions also affect aboveground plant communities [10], which manage the type and abundance of many organic substrates provided to soil microbial heterotrophs. Hence, climatechanging should also impact on microbial communities indirectly through shifts in plant composition and productivity.
The previous studies have researched the soil microbial responding to climate change from different perspectives, and have achieved signi cant results. However, there is still some blind spot that needs further inquiry as follow: i. How to evaluate the soil microbial community in desert grassland ecosystems under the interaction of temperature and rainfall. As temperature and rainfall change continues, has sensitive indicators in components of the ecosystem changed?
ii. In the context of climate change, what are the correlations among soil respiration, soil microbial abundance, soil microbial diversity and soil microbial coverage?
iii. How do soil properties response to soil microbial abundance, soil microbial diversity and soil microbial coverage? iv. How plant biomass affects the soil microbial community?
To solve the above issues, this study takes the desert steppe (south edge of the Mu Us Sandy Land) of Yanchi County, Ningxia as the research object. We used OTC (Open-Top Chamber) to simulate temperature increase, shelters, and arti cial watering to simulate rainfall changes under the interaction of human factors: a) Dynamic changes of soil microbial in desert steppe ecosystems, and b) Ecological responses to desert steppe ecosystem. c) From the response of soil microbes to temperatures rising and rainfall change, nding the ecological sensitivity index among them. This research provides a reliable theoretical basis for formulating a reasonable response strategy in a desert steppe.

Study site
The study site is located in the desert steppe of Ningxia Autonomous Region, China (37°47′N, 107°25′E). The climate belongs to a typical continental climate with an average annual temperature of 8.1°C higher than 0°C. The annual accumulated temperature is 3430.3℃, and the annual rainfall is 295mm (average in 1981-2017). The rainfall of July to September accounts for approximately 61% of the whole year. The annual evaporation is 2131.8mm, and the frost-free period is about 162d. The zonal soil is light grey calcareous soil, sandy soil, and silt soil. Zonal vegetation is a desert steppe. It is mainly dominated by xerophyte and mesophyte. The Main distribution is perennial plants such as Stipa brevi ora, Cleistogenes squarrosa, Leymus scallions, Lespedeza davurica and annual plants such as Setaria viridis and Salsola Collina.

Experimental design
According to the meteorological monitoring from 1981 to 2017 in the study site, the annual average rainfall, ground temperature, and air temperature all showed a rising trend. Based on the 37-year average rainfall and uctuation extremes, 66% and 133% rainfall gradients were achieved using arti cial raincollecting greenhouses and sprinkler irrigation techniques to ensure that the rainfall treatment within the range of natural rainfall extremes. Due to the steady increase of ground temperature and atmospheric temperature, two temperature increase gradients are set, and the Open-Top Chamber (OTC) device is used to achieve a temperature increase about 2°C (data from preliminary experiment).
The designed rainout shelter was well-ventilated in November 2018 (Fig.1). Rainfall gradient was constructed by arti cial shelters and arti cial sprinklers. A two-factor completely randomised experimental design is used based on rainfall and temperature factor. Five levels of rainfall are 33% (R33), 66% (R66), 100% (CK), 133% (R133), and 166% (R166) of normal rainfall , two rainout shelters with manipulated rainfall doses (i) of 97 mm (33% of annual average) and (ii) 194 mm (66% of annual average), along with three unsheltered plots with manipulated rainfall doses (iii) of 295 mm (normal annual rainfall), (iv) 392 mm (133% of annual average) and (v) 490 mm (166% of annual average), increased rainfall due to watering pot. Temperature is two levels that the actual temperature (CK) and the interaction between the rainfall and temperature increase about 2°C (T) to achieve a temperature change by the OTC (Open-Top Chamber) in each plot. Each plot area is (6 x 6) m, and each treatment repeated three times, 15 plots in total (Temperature treatment included in rainfall treatment). On the 15 th and 30 th of each month, R33 and R66 of the actual rainfall during 1 st -15 th and 16 st -30 st of the month are collected from the actual rainfall respectively, and then evenly replenished to the plots containing R133 and R166 by a watering pot.

Collection of soil microbial samples
In each plot, including inner OTC, we take 30cm of soil from each sample plot and then divided it into three layers, which are 0-10cm, 10-20cm, and 20-30cm separately. We remove covering from the soil (plants, moss, visible roots, litter, and visible soil animals) and wipe the sample with alcohol cotton. After the alcohol has completely evaporated, a 6 cm diameter drill was used to soak the sample (S1 Canada). The steps are repeated every time the sample is changed. Three samples were taken from the same quadrant and mixed as one soil sample. Then, we mixed the soil into a 10ml centrifuge tube and transfer it to a -80°C refrigerator for determination of soil microbes. Table 1 and 2 show the sample's data analysis of soil.

Soil property analysis
Soil respiration (SR) measurement using open soil CO 2 ux system (LI-8100 Automated Soil CO 2 Flux System, Li-COR, Lincoln, NB, USA). A sample of each plot in the square, a circular PVC pipe with a diameter of 20 cm and a height of 3 cm was embedded in the soil to a depth of 12 cm , took off inside plants of circular pipe, and then SR was measured. The measurement frequency is one time /15 days, and the time point is 10am-2pm. A 30 cm soil (divide evenly into three layers, each layer is 10 cm) was drilled in each plot and then put into plastic bags separately to the laboratory to test the soil physical and chemical properties.
Soil organic carbon (SOC) was measured by the external heating method: potassium dichromate-sulfuric acid digestion, ammonium ferrous sulphate titration (TItrette 50 ml Automatic titrate) and soil total nitrogen (STN) were tested using an elemental analyser (Vario EL/micro cube, Germany). Soil total phosphorus (STP) was studied by Sulphuric acid-perchloric acid digestion, antimony molybdenum calorimetry, UV spectrophotometer determination (Hyener I5 Photometer). The soil pH value was measured by an aciditying agent (PHS-3C pH audiometer, China).

Plant biomass measurement
Plant biomass was measured in a 1m 2 quadrant which randomly selected in each plot at the end of July 2019. All plants in each plot were dug from the soil and then cut the aboveground living plant. Moreover, the plant roots were cut and sorted according to species and place them in their respective envelope. Finally, these species were taken into the laboratory and drying at 65℃ in the oven for 48h. Then the aboveground living biomass (ALB) and root biomass (RB) were calculated. Note: All plant samples collected from the desert grassland and the property right of grassland belongs to Ningxia University, so we can carry out experiments on grassland without license certi cate.

DNA extraction and PCR ampli cation
Microbial community genomic DNA was extracted from soil samples by using the MP Fast DNA Spin Kit for soil according to manufacturer's instructions. The DNA extract was checked on 1% agarose gel, and DNA concentration and purity were determined by NanoDrop 2000 UV-vis spectrophotometer (Thermo Scienti c, Wilmington, USA). The hypervariable region V3-V4 of the bacterial 16S rRNA gene were ampli ed with primer pairs 338F(5'-ACTCCTACGGGAGGCAGCAG-3')and806R(5'-GGACTACHVGGGTWTCTAAT-3') by an ABI GeneAmp® 9700 PCR thermocycler (ABI, CA, USA). The PCR ampli cation of 16S rRNA gene was performed as follows: initial denaturation at 95℃ for 3 min, followed by 27 cycles of denaturing at 95 ℃ for 30s, annealing at 55 ℃ for 30 s and extension at 72 ℃ for 45 s, and single extension at 72 ℃ for 10 min, and 10℃ until halted by the user. The PCR mixtures contain 5 × Transport FastPfu buffer 4 μL, 2.5 mM dNTPs, 2 μL , forward primer (5μM) 0.8 μL, reverse primer (5μM) 0.8 μL, TransStart FastPfu DNA Polymerase 0.4μL, template DNA 10ng, and nally did H 2 O up to 20 μL. PCR reactions were performed in triplicate. The PCR product was extracted from 2% agarose gel and puri ed using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to the manufacturer's instructions and quanti ed using Quantus™ Fluorometer (Promega, USA).

Statistical analysis
Statistical analyses were variously performed using the software packages R, Origin 2018 and Microsoft Excel. All data were analysed rst using a 'repeated measures' statement as well as obtain the standard errors of individual means and used to perform correlation analyses in SPSS 20.0. PCA eliminates redundant variables depending on other measured variables are based on canoco 5.

Soil microbial community
The α-diversity of soil microbial in our study, including three parts, which are community richness, community diversity and community coverage. The number of bacterial community's richness is obviously more than fungus communities, meanwhile, with the temperature and rainfall increasing, the highest community richness of fungus and bacteria are both in R33, and the lowest is in CK. The highest community diversity of fungus is both in CK, and the lowest are in TCK. The highest community coverage of fungus and bacteria are the same separately Fig.2 Under different interaction of temperature and rainfall treatments, the number of bacterials in the soil was signi cantly higher than the number of fungus. In the fungus communities, the total kinds of microbes are the highest under the R33, the number of common species of microbes under different treatments was 75, and the number of unique species of microbes under R166 was the highest. In the bacterial communities, the total kinds of microbes are also under the R33. The number of common species of microbials under different treatments is 2576, and the number of unique species is the highest under the interaction of normal rainfall and temperature (Fig.3).
In the fungal and bacterial communities, the distance between each sample point is the farthest under the interaction of R166 and temperature rising. Thus, the corresponding β-diversity is the highest. However, sample point has the shortest distance under R33, therefore, the corresponding β-diversity is the lowest (Fig.4).
In the fungus and bacteria communities, the effect of temperature of them is not signi cant, their abundance is the lowest under natural rainfall, and with the continuous increase and decrease of rainfall, the microbial abundance gradually increases (Fig.5).

Rank of different environmental factors on soil microbial community
In the fungal communities, soil microbial abundance (ace) positively correlated with ALB and SM, and the correlation was ALB > SM. Soil microbial abundance negatively correlated with other indicators, and the negative correlation is RB > SR> STP> SOC> STN> SM> pH> ST.
Soil microbial diversity positively correlated with ST and pH, and the correlation was ST> pH. Soil microbial diversity negatively correlated with other indicators, and the correlation is RB> SR > STP> SOC> STN> ST. Soil microbial coverage has no signi cant correlation with various indicators.
In the bacterial communities, soil microbial abundance positively correlated with ALB and soil microbial abundance negatively correlated with other indicators. The correlation is STP> ST> SOC> STN> SM> SR. Soil microbial diversity positively correlated with pH and ST, and the correlation was ST > pH. Soil microbial diversity negatively correlated with other indicators, and the correlation is ALB >STP > SOC> STN> SM> SR > pH. Soil microbial coverage positively correlated with SM, SOC, STN, STP, SR, ST, pH, and the correlation is STP>ST>SOC>STN>SM> SR. Soil microbial coverage negatively correlated with ALB Fig.5).

Discussion
The effect of temperature and rainfall on soil microbial community Global climate change characterized by climate warming has a signi cant impact on the global natural environment and social-economic activities, which becomes the research challenge of global sustainable development [11]. The search result of the net change primary productivity (NPP) shows that temperature rise is bene cial to plant growth and rainfall changes. Moreover, the extreme increase in climate events may cause grasslands to face higher levels of risk in future climate change scenarios [12]. The increase of atmospheric CO 2 concentration and temperature will affect the physiological growth process of plants and the structure as well as the function of terrestrial ecosystems through photosynthesis. However, as a feedback to terrestrial ecosystems, soil microorganisms will also change.
Soil microorganisms participate in biochemical cycles, the decomposition of soil organic matter and the formation of soil structure in the ecosystem are sensitive to environmental factors. Domestically and abroad comprehensive researches have found that rainfall changing and temperature increasing in the future are bound to have an impact on plant growth and soil metabolism, which will have a signi cant impact on soil microbial communities.
The number of bacterial communities abundance (ace) is signi cantly more than fungus communities. The reason maybe is that bacterial spores have strong ability of drought resistance. The α-diversity of fungus and bacterias' communities do not gradually increase with the rise of temperature and rainfall.
So, temperature and rainfall do not give any signi cant impact on microbial activities due to these indexes adapt to environmental changes caused by experimental treatment through self-regulation. Yet βdiversity of fungus and bacterial communities are highest in R166. Therefore, rainfall rising promote signi cant impact on microbial activities, which agreed with the previous studies.
In the fungus and bacteria communities, the distance between each sample point is the farthest under the interaction of R166 and temperature rising. Thus, the corresponding β-diversity is the highest, however, sample point has the shortest distance in R33. Therefore, the corresponding β-diversity is the lowest.
The total kinds of fungus and bacterials are both high under the R33, so rainfall reducing could promote the microbial species. The reason maybe is in environmental adaptability, microbes have better tolerance to environmental moisture changes, thus show resistance to rainfall changing.
The effect of temperature on the fungus and bacterials are not signi cant, and the abundance of fungus and bacterial communities are both the lowest under natural rainfall. With the continuous increasing and decreasing of rainfall, the microbial abundance gradually increases, hence the difference between nature rainfall and rainfall changes are signi cant. It is because of the change of rainfall will promote the increase of microbial abundance.
The relationship between soil microbial community and soil respiration Globally, in the different climate change, a better understanding of the mechanisms that regulate Rs dynamics is essential in predictions of future atmospheric C concentrations and accurate estimations of the C balance.
A primary function of soil microorganisms is the processing and restoration of key nutrients in the input of cuttings and the accumulation of soil organic matter. It is a storage reservoir of soil nutrients and an important source of nutrients available for plant growth. It can re ect the state of soil fertility and nutrient cycling meanwhile play the most prominent role in the cycle. Photoautotrophic microorganisms x CO 2 as organic matter, which can be consumed or respired by heterotrophic microorganisms. The nal product of respiration is CO 2 and new cytoplasm. More than 70% of CO 2 loss caused by soil respiration comes from soil microbial respiration.
In the present study, the fungal and bacterial communities, soil microbial abundance (ace) is positively related to ST but negatively related to soil moisture (SM) and soil CO 2 ux. Soil microbial diversity (shannon) is positively correlated with ST and negatively correlated with soil CO 2 ux and SM. Soil microbial coverage has no signi cant correlation with other factors in fungal communities, but was positively correlated with soil CO 2 ux and ST meanwhile negatively correlated with SM in bacterial communities.
The response of soil chemical properties to soil microbial community The soil microbial community's activity is a key factor affecting the soil environment. It is because soil microbes are a vital factor in participating in the process of soil nutrient cycling, and they react rapidly to changes in environmental factors.
The nding of this study (in the fungus' communities) shows that with the temperature and rainfall variation, SOC, STN, and STP will promote soil microbial abundance, diversity and coverage separately. In the bacterial communities, SOC, STN, and STP will promote soil microbial abundance and diversity, but limit the coverage. It was same as previous studies showing that soil microbial community, as an important soil biogeochemical cycles, soil formation as well as ecosystem resilience to the external environment and a driver of soil properties and processes [13]. Soil microbial community is an intrinsic sensitive factor of soil. Although it only accounts for a small part of it, as an essential source and reservoir of nutrients, it acts a vital role in the improvement of nutrient cycling and soil physical and chemical properties. It can directly re ect soil fertility [14]. Soil microbial community regarded as a part of available or labile soil organic matter. The small fraction of the total soil organic matter is a readily decomposed and concluded in nutrient cycling [15].
Meanwhile, nitrogen and phosphorus are also an essential nutrient which limits primary productivity of terrestrial ecosystems [16]. Soil microbes directly drive the soil carbon, nitrogen and phosphorus cycle process. Therefore, the study of its distribution characteristics under different rainfall and temperature is of great signi cance for understanding the soil carbon, nitrogen and phosphorus turnover [17]. Soil nitrogen and phosphorus turnover are serious microbial mediated processes, which controlled by a range of environmental factors, particularly in rainfall and temperature [18].
The effect of plant biomass to soil microbial community Composition changing of soil microbial community is not only in uenced by soil physicochemical factor (etc. pH, SOC, nutrient availability, SM and ST) but also by plant properties (etc. plant community type, plant microbial interactions, and plant functional traits). These demonstrated that plant identity was more important factors in deciding the soil microbial community structure than plant species diversity [19]. It has been proposed that plant above-living biomass and plant root biomass economics spectrum could provide a framework for better understand how vegetation composition in uences variation in soil microbial communities [20].
In the present study, the abundance, diversity, and coverage of fungal and bacterial communities were positively correlated with ALB but a negative correlation with RB. It was in agreement with previous studies showing that ALB will promote the characters of microbial communities [21]. The main driving factors on soil microbial community Soil temperature and moisture, changing caused by environmental factors may have an unpredictable effect on the soil microbial community and further affect the decomposition process of organic matter in the soil [22]. Although soil microorganisms are involved in most of the physiological, metabolic processes in the soil, there are still few studies on the response laws of soil microbial community structure and function under the background of global climate change, and how it affects the aboveground ecosystem processes. Related eld experiments to simulate global climate change show that increasing temperature can signi cantly affect the content of soil microbes in grassland and forest ecosystems [23]. For example, some researchers have found that the increase in air temperature has no noticeable effect on the soil microbial community in the heath forest. However, in the soil heating experiment, the biomass of the fungus group decreased, and the bacterial biomass increased [24]. Besides, some scholars believe that increasing temperature does not have a signi cant effect on the composition of the grassland soil microbial community and its biomass [25]. Increasing the temperature can increase the activity of the microbial community and increase the rate of metabolism. The soil microbial community will tend to adapt to a broader temperature range and a higher metabolic rate.
Meanwhile, in addition to the increase in temperature, changes in rainfall value and rainfall frequency will also affect the soil microbial community [26]. Field rainfall control experiments on grassland ecosystems show that seasonal changes in rainfall have a signi cant effect on changes in soil microbial community content. The experiments conducted by related researchers affect the water on soil microorganisms concluded that the water environment can signi cantly affect forest soil bacteria, but has no signi cant effect on grassland soil microorganisms [27]. However, through eld rainfall control experiments, Taylor concluded that neither rainfall decline nor rainfall increase has any signi cant effect on the biomass of soil microbial communities. Related research reports pointed out that changes in rainfall pattern will affect the ratio of fungus and bacteria, and also have a signi cant effect on soil microbial communities [28]. Most of the above reports are about the research and discussion of the soil microbial community under the effect of single climatic factors. The actual effect of the interaction of multiple climatic factors on the soil microbial community may be different from the effect of a single climatic factor. For example, the increasing temperature may increase soil microbial activity, but the impact of falling rainfall may mask this increase. The decrease in rainfall causes a decrease in soil moisture, which reduces the biomass and metabolic rate of litter, and then affects the soil microbial action. However, the interactive effects of such climatic factors have not been reported in terrestrial ecosystems.
In the present study, the lowest ace indices of fungus and bacteria are both in CK, which indicate an increase or decrease both rainfall and temperature could increase community abundance. In the bacterial and fungal community, the distance between samples gradually increases with the temperature and rainfall, so temperature and rainfall rising will promote community β-diversity. In the fungus and bacterial communities, abundances are negatively correlated with ST, so temperature rising will limit the abundance of fungus and bacteria, soil microbial diversity is negatively correlated with soil CO 2 ux and ST, so soil CO 2 ux and ST will limit soil microbial diversity. Soil microbial coverage was positively correlated with soil CO 2 ux, so the SR will promote the soil microbial coverage, which is correlated with the previous study.

Conclusions
This nding suggests that in general bacterial spores have strong ability of drought resistance, both rainfall and temperature's rising could not promote the soil community α-diversity. However, it can promote soil microbial community β-diversity. Fungus and bacteria have better tolerance to environmental moisture changing, thus show resistance to change of rainfall. The effect of temperature to fungus and bacteria are not signi cant, but the change of rainfall will promote the increase of microbial's abundance.
In the soil microbial communities, SR and ST will limit soil microbial diversity; meanwhile, the SR will promote the soil microbial coverage. ALB and ST promote the soil microbial α-diversity. These ndings maybe provide a reliable theoretical basis for formulating a reasonable response strategy in desert steppe ecosystems. Declarations LJP conceived and designed the experiments, prepared gures and/or tables, authored or reviewed drafts of the paper, and approved the nal draft.

Funding
The study was funded by the Science Foundation of Ningxia (2019AAC03042) for testing soil microbe by LJP who bought the testing materials. The National Natural Science Foundation of China (31660143) for testing soil properties, and the Top Discipline Construction Project of Pratacultural Science (NXYLXK2017A01) for testing plant properties by XYZ who pay for the testing materials. .

Availability of data
All data generated or analysed during this study are included in this published article and its supplementary information les.