摘要
[目的]明确平板计数法中不同稀释梯度对土壤细菌数量和组成的影响规律,比较稀释平板计数法和高通量测序研究土地利用方式变化下土壤细菌群落的差异。[方法]针对不同土地利用方式下的4种土壤(次生林、健康蕉园、发病蕉园和水稻土),设置5个土壤悬液10^(-1)‒10^(-5)稀释梯度开展平板计数,获得平板上可培养细菌富集物并提取总DNA;同时直接提取原位土壤微生物总DNA,高通量测序细菌富集物DNA和土壤总DNA中的16S rRNA基因,研究不同土壤悬液稀释梯度下的可培养细菌群落和背景土壤细菌多样性,明确可培养细菌占土壤总细菌的比例以及多样性差异。[结果]次生林垦殖为蕉园土壤后土壤呼吸增幅最高,细菌数量降幅最高,稀释平板计数与实时荧光定量PCR(real-time fluorescent quantitative PCR)结果一致,但其他土地利用方式变化的稀释平板计数与实时荧光定量细菌数量的结果并不完全一致。连续稀释显著降低了可培养细菌多样性。与原位土壤总细菌相比,土壤可培养细菌群落Chao 1指数降幅高达86%‒98%。与土壤悬液稀释10倍(10^(-1))相比,稀释100‒10万倍(10^(‒2)‒10^(‒5))后可培养细菌群落Chao 1指数降幅高达35%‒60%。连续梯度稀释也降低了物种多样性,所有稀释梯度共检测到315‒401个微生物属,共有属仅为21‒38个,而10^(-1)梯度独有属有92‒210个,10^(‒2)‒10^(‒5)梯度独有属为2‒59个。4种土壤中可培养细菌占背景土壤总细菌比例范围为:16.1%‒47.7%(门水平),7.4%‒30.9%(属水平)。两两比较不同土地利用方式间的显著差异物种,可培养方法获得的显著增加和减少的物种数量仅为高通量直接测序原位土壤结果的9.7%和22.9%;免培养和可培养均发现了一些共同的差异物种,包括Bacillaceae、Micrococcaceae、Microbacterium、Comamonadaceae和Burkholderiales。[结论]传统稀释平板计数可培养细菌数量过程中,10
[Objective]To assess the effect of different dilution gradients of dilution-plate method on the number and composition of soil bacteria,and to study the differences in soil bacterial communities under distinct land use scenarios by comparing plate-dilution method and high-throughput sequencing.[Methods]With four types of soil(secondary forest soil,healthy banana soil,diseased banana soil and paddy soil)collected,soil suspensions of five dilution gradients(10^(-1)–10^(-5))were prepared.Classic plate-dilution method was employed to obtain culturable bacteria,followed by colony-counting and DNA extraction.In addition,the total bacterial DNA in background soils was extracted.Then high-throughput sequencing of 16S rRNA gene was performed to study the diversity of bacterial communities at different dilution gradients and in background soils,proportion of culturable bacteria in total soil bacteria,and species difference.[Results]The highest increase in soil respiration was found after the conversion of secondary forest to banana plantations,and both dilution-plate method and real-time fluorescence quantitative PCR found the highest decrease in bacterial counts.However,the results of these two methods were not entirely consistent for other land use changes affecting soil bacterial populations.The Chao 1 index of the soil culturable bacterial community was reduced by 86%–98%compared with that of the total background soil bacteria.Dilutions of 100–100000 times(10^(-2)‒10^(-5))lowered the Chao 1 index of the culturable bacterial community by 35%–60%compared with dilutions of 10 times of soil suspensions.Gradient dilution also reduced the species diversity.Dilution-plate method detected a total of 315–401 microbial genera,with only 21–38 genera common to all dilution gradients,compared with 92–210 genera unique to the 10^(-1) gradient and 2–59 genera unique to the 10^(-2)‒10^(-5) gradients.The proportions of culturable bacteria in the four types of background soils were in the range of 16.1%–47.7%(phylu
作者
李凯凯
曹伟伟
文昌丽
周晓丽
徐修远
邱旭
赵悦蓓
贾仲君
孟磊
LI Kaikai;CAO Weiwei;WEN Changli;ZHOU Xiaoli;XU Xiuyuan;QIU Xu;ZHAO Yuebei;JIA Zhongjun;MENG Lei(College of Tropical Crops,Hainan University,Haikou 570208,Hainan,China;State Key Laboratory of Soil and Sustainable Agriculture,Institute of Soil Science,Chinese Academy of Sciences,Nanjing 210008,Jiangsu,China;University of Chinese Academy of Sciences,Beijing 100049,China;School of Marine and Biological Engineering,Yancheng Teachers University,Yancheng 224007,Jiangsu,China)
出处
《微生物学报》
CAS
CSCD
北大核心
2022年第11期4447-4464,共18页
Acta Microbiologica Sinica
基金
国家自然科学基金(32072688,318723623)
江苏省农业科技自主创新资金[CX(19)1006]。
