Seasonal Change of Microbial Diversity and Its Relation with Soil Chemical Properties in Orchard

This study aimed to determine the microbial diversity of different soil depths (0-5 and 5-20 cm) in a subtropical orchard during different seasons (i.e., Spring, Summer and Autumn) for enrich the knowledgements on micorbes roles in orchard ecosystem balance. In tracking experiments conducted in an orchard (established in 1996), the phospholipid fatty acid (PLFA) biomarker method was employed to know soil microbial system. Total PLFAs concentration did not vary significantly between soil depths but changed between seasons. It peaked in the summer at 258.97 ± 23.48 μg g-1 soil from 0-5 cm and at 270.99 ± 58.94 μg g-1 soil from 5-20 cm. A total of 33 microbial fatty acid biomarkers were observed and identified in the sampled soil. Quantities of PLFAs for 29 microbe groups varied significantly between seasons, except for 15:0 iso 3OH, 15:1 iso G, 16:0 2OH, and 17:0 iso 3OH. The bacterial PLFAs and fungal and actinomycotic PLFAs in the orchard soil collected in Summer were significantly higher than in the Spring or Autumn (P < 0.01). The number of soil microorganism species (Richness) and the Simpson and Shannon-Wiener indexes were all the highest in summer. The total PLFAs, bacterial PLFAs, fungal PLFAs, actinomycotic PLFAs, Richness, or the Simpson and Shannon-Wiener indexes were all significantly negatively correlated with soil pH, total carbon (TOC), total nitrogen (TN) and cation-exchange capacity (CEC) (P < 0.05).


Introduction
5 Worldwide (Shanghai) Co., Ltd., Shanghai, China) solution was added before tightening the cap. The centrifuge 81 tube was shaken for 5 min at 100 rpm. After shaking, the tube was incubated in a CU600 thermostat water bath added to decrease the pH of the reaction. 3). After adding 10 mL of n-hexane (Merck Co., Darmstadt, German) 85 and mixing well, the tubes were centrifuged in an N00077 centrifuge for 15 min using the following settings: rotor: 86 #12,150; speed: 2000 rpm; time: 15 min; and temperature: 4°C. After centrifugation, the supernatant n-hexane 87 was transferred to a clean flask and air-dried under a fan. 4). The air-dried sample was re-suspended in 0.5 mL of 88 a mixture of n-hexane:methyl-tert-butyl ether (Tedia Co., Inc., Fairfield, OH) (1:1, v/v) for 3-5 min and transferred 89 to gas chromatography (GC) vials for PLFA determination.

109
The number of species (Richness), Simpson diversity, Shannon-Wiener diversity and Alatalo evenness were 110 used to calculate the ecological parameters of the microbial fatty acid biomarkers. The calculation equations are 111 expressed as follows:

112
The Simpson diversity index was calculated according to Pi D (1) ;

113
The Shannon-Wiener diversity index was calculated according to The Alatalo evenness index was calculated according to Ni is the content of the i th kind of phospholipid fatty acid (PLFA); and N is the total PLFA 116 content. 117 2.6. Statistical Analysis 118 An one-way ANOVA followed by least significant difference (LSD) multiple comparison test was used to establish 119 significant differences among the means of soil properties, microbial community indicators (Bacterial PLFAs, 120 Fungal PLFAs, Actinomycotic PLFAs, and B/F ratio of PLFAs), and microbial diversity indicators (Richness,

121
Simpson index, Shannon-Wiener index and Alatalo index) in different seasons. A two-way analysis was used to 122 establish significant differences in the total PLFA and individual PLFA variance between seasons and soil depths.

139
No significant difference of pH, TOC, C/N ratio, CEC, exchangeable Mg 2+ or Ca 2+ was found between soil 140 depths.

148
For the 5-20 cm soil depth, bacterial PLFAs, fungal PLFAs and actinomycotic PLFAs summit to 230.00, 31.15 and 149 9.83 g g -1 soil, respectively (Table 3). Microbial PLFAs contents (g g -1 soil) of different seasons sampled at 0-5 150 and 5-20 cm soil depths in orchard is shown in Table 4. Two-way analysis of variance for the effects of seasons

182
Usually, microbial population varies by moisture and temperature change among seasons. However, whether this 183 change could be found easily depend on how long the suitable season last and how hard the extreme water and 184 temperature condition affect on soil microorganisms. In subtropical mountain area, the soil was utilized as planting peach, obvious microbial change was confirmed in this article. And, principal component 1 explained remarkable change in microbial community was related to the peak soil microorganism growth in the Summer 189 because of comfortable temperature and rainfall.
index and Shannon-Wiener index were its major drivers (Table S1). This could be explained by the 192 microorganism propagating well and maintaining a good balance in the Summer in the tested orchard soil system.

193
The results of our study are consistent with the results of Zhu et al. [14] in an evergreen broadleaf forest and   (Table 1). Liu et al. [18] also found the microbial dominance index and Shannon-Wiener index to be negatively 208 related to soil NH4 + -N and NO3 --N in an apple system by observation on different growing periods. However, in the 209 view of change by utilization, Yao et al. [6] illustrated microbial biomass C, basal respiration and total PLFA to be 210 highly correlated with organic C and TN on red soil orchard ecosystem. In some ecosystem soil microbes grew 211 rapidly with energy and nutrition consumption [25,26]. It is clearly suggested that the propagation and growth of 212 soil microorganisms from Spring to Summer require energy from TOC and nutrition from N in an subtropical 213 orchard system. Microbes mainly act as "consumers" could be well documented.

215
In subtropical orchards, the temperature and humidity in the Summer are conducive to the growth of soil        .31 a TOC = total organic carbon, TN = total nitrogen, CEC = cation exchange capacity, C/N ratio = ratio of total organic 311 carbon to total nitrogen. Values followed by the same letter (s) in a low are not significantly different at P ＜ 0.05 312 using LSD post hoc tests. Case number is shown in parentheses

335
total nitrogen, CEC = cation exchange capacity, C/N ratio = ratio of total organic carbon to total nitrogen.