【作者】 刘娜；

【导师】 王文兴； 戴九兰；

【作者基本信息】 山东大学 ， 环境科学， 2020， 博士

【摘要】 当前我国部分区域耕地重金属汞/镉污染问题突出,由土壤污染引发的农产品安全问题频发。继“镉米”之后,“汞麦”“镉麦”事件已成为环境、健康等领域关注的焦点。基于我国人多耕地少的现状,必须寻找“边生产边修复”模式实施土壤污染防治,其中种植重金属低积累小麦品种是最优选途径。目前对低积累小麦品种筛选尚未建立统一的标准体系,对低积累品种根际微环境特征尚不清楚。因此本研究以黄淮海冬麦区推广小麦品种为研究对象,通过盆栽和大田试验,对小麦品种汞/镉积累特性进行研究,筛选低汞/镉积累品种,进而比较高低汞/镉积累品种根际微环境差异,揭示低汞/镉积累品种根际微环境特征。本文收集黄淮海冬麦区推广小麦品种,通过室内盆栽试验,设置3个汞处理梯度(0、1 mgkg-1、5 mgkg-1),研究小麦形态性状、生物量性状和产量相关性状等1 1个性状对汞胁迫的响应,计算各个性状的汞耐性系数、汞耐性指数、遗传变异系数和广义遗传力,构建改进隶属函数,建立小麦品种汞耐性评价体系,对耐汞小麦品种进行初步筛选。研究结果表明,低汞胁迫显著增加了小麦株高和第一节结长,而显著降低了小穗数。高汞胁迫显著增加了小麦株高,降低了小麦产量相关性状(穗粒数、谷物鲜重和谷物干重)。对具有中高等广义遗传力(0.31-0.68)的1 1个性状构建改进隶属函数,通过判别分析建立小麦品种汞耐性评价体系-判别函数。低汞胁迫下,汞耐性判别函数自变量为有效分蘖数汞耐性系数、谷物鲜重汞耐性系数和生物量干重汞耐性系数;高汞胁迫下,判别函数变量包括谷物干重汞耐性系数和生物量干重汞耐性系数。判别函数值越大,小麦品种汞耐性越强。低汞和高汞胁迫下,判别函数对小麦品种汞耐性评价的正确率分别为90.0%和83.3%。该评价体系突破了以往以产量为评价指标的简单鉴定模式。结合小麦品种汞耐性、籽粒汞浓度与富集因子,小麦品种农大3163(ND3163)和藁城8901(GC8901)被筛选为耐汞且低汞积累小麦品种,济麦21(JM21)和泰山21(TS21)为汞敏感且高汞积累小麦品种,该研究填补了黄淮海冬麦区推广小麦品种汞耐性和汞积累特性的研究空缺,为轻中度汞污染农田的安全利用提供了种质资源。进一步以筛选的低汞(DA3163和GC8901)和高汞(JM21和TS21)积累小麦品种为研究对象,揭示小麦高低汞积累品种汞积累和转运能力差异,明确小麦品种籽粒低汞积累是由于品种的低吸收还是向地上部转移较少所致。此外,本文也研究了高低汞积累品种根际土壤有机酸和微生物差异,阐明低汞积累小麦品种根际微环境特征。结果表明,低汞胁迫下,低汞积累品种DA3163和GC8901根汞浓度显著低于高汞积累品种JM21和TS21;高汞胁迫下,四个品种根汞浓度无显著差异,而两个低汞积累品种汞由根向茎、叶、籽粒的转移因子显著低于高汞积累品种,说明低汞胁迫下,小麦籽粒低汞积累是由于根部低汞吸收所致,而高汞胁迫下,小麦品种籽粒低汞积累是由于向地上部转移较低所致。低汞胁迫下,低汞积累品种ND3163和GC8901根际土壤柠檬酸和草酸浓度,革兰氏阳性菌、革兰氏阴性菌和真菌丰度显著低于高汞积累品种JM21;低汞积累品种GC8901根际土壤原生动物丰度显著高于高汞积累品种。低汞积累小麦品种根部分泌较少的柠檬酸和草酸,并且根际土壤原生动物丰度较高,明确根际土壤有机酸和微生物对汞在小麦中积累和转移的影响可为降低小麦籽粒汞浓度提供理论依据。本文通过3个田间小区试验对黄淮海冬麦区72个主推小麦品种籽粒镉积累特性进行鉴定,研究小麦品种镉积累稳定性,探究小麦品种吸收镉与微量元素(锌、铜、铁、锰和硼)的关系。对小麦性状与籽粒镉浓度相关性进行分析,以期为低镉积累小麦品种筛选提供简单快捷的筛选方法。依据小麦籽粒镉浓度值聚类分析,将轻度镉污染农田-试验田1中种植的小麦品种分为超低镉、低镉、中等镉、高镉和超高镉积累品种,其中超低镉和低镉积累品种24个,其籽粒镉浓度低于安全食用限值(0.1 mg kg-1,GB2762-2017)。对小麦品种镉积累稳定性研究表明,试验田1中筛选的24个低镉积累品种中有9个品种低镉积累特性不稳定,在试验田2和试验田3表现出中等镉、高镉或超高镉积累特性。对相对稳定的低镉和高镉积累品种籽粒锌、铜、铁、锰和硼浓度进行比较,其中5个低镉积累品种在至少两块试验农田其籽粒锌浓度显著低于高镉积累品种,另外1个低镉积累品种籽粒锌、铜和锰浓度均显著低于高镉积累品种。小麦籽粒铁浓度在低镉和高镉积累品种中无显著差异。最终筛选出9个低镉积累特性相对稳定且中高等微量元素积累品种,提供小麦品种清单,推荐用于山东省轻中度镉污染农田的安全利用。这9个品种在三个试验农田均有较高的千粒重和产量。对小麦农艺性状与籽粒镉浓度相关性研究表明,小麦籽粒镉浓度与茎、根干重呈显著正相关关系,与穗长呈显著负相关关系,与小麦产量无显著相关性,较长穗长的小麦品种或许具有低镉积累特性。筛选低镉积累小麦品种要尤为关注小麦低镉积累稳定性和对微量元素锌的积累能力。上述研究发现,一些低镉积累小麦品种同时也低锌吸收,本文以不同镉/锌积累能力小麦品种为研究对象,通过高通量测序技术对其根际细菌多样性和群落结构差异进行研究,揭示根际细菌与小麦籽粒吸收积累镉/锌的潜在关系。结果表明,在轻度镉污染农田,不同镉/锌积累能力小麦品种根际细菌主要菌门均为变形菌门(Proteobacteria)、放线菌门(Actinobacteria)、酸杆菌门(Acidobacteria)、拟杆菌门(Bacteroidetes)、芽单胞菌门(Gemmatimonadetes)、绿弯菌门(Chloroflexi)、疣微菌门(Verrucomicrobia)、浮霉菌门(Planctomycetes)和厚壁菌门(Firmicutes),这9个菌门占根际细菌95.0%以上。低镉积累品种根际细菌多样性显著高于高镉积累品种,其根际土壤芽单胞菌门(Gemmatimonadetes)、绿弯菌门(Chloroflexi)、浮霉菌门(Planctomycetes)和厚壁菌门(Firmicutes)相对丰度显著高于高镉积累品种;高锌积累品种根际土壤变形菌门(Proteobacteria)相对丰度显著高于低锌积累品种。本文进一步对丰度较高的放线菌门(Actinobacteria)、酸杆菌门(Acidobacteria)和芽单胞菌门(Gemmatimonadetes)在纲、目、科水平的差异进行研究。在科水平,低镉积累品种根际土壤芽单胞菌科(Gemmatimonadaceae)、鞘脂单胞菌科(Sphingomonadaceae)、拜叶林克氏菌科(Beijerinckiaceae)、亚硝化单胞菌科(Nitrosomonadaceae)、Nocardioidaceae 科、Intrasporangiaceae 科、微单孢菌科(Micromonosporaceae)、Pseudonocardiaceae 科、地嗜皮菌科(Geodermatophil aceae)和Gaiellaceae科相对丰度显著高于高镉积累品种;高锌积累品种根际土壤Rhodanobacteraceae科相对丰度显著高于低锌积累品种。低镉高锌积累品种根际土壤α-变形菌纲和芽单胞菌科相对丰度高于低镉低锌和高镉高锌积累品种。Tax4Fun功能预测结果表明,低镉高锌积累品种根际土壤硝化功能类细菌相对丰度较高,其需氧氨氧化功能菌和硝化功能菌相对丰度高于高镉高锌积累品种,亚硝酸盐氧化菌相对丰度高于低镉低锌积累品种。本研究首次揭示了小麦低辐高锌积累的品种差异可能与根际土壤某些关键细菌类群的丰度相关,根际土壤较高丰度的α-变形菌纲和芽单胞菌科细菌或许有利于小麦品种籽粒低镉且高锌积累。本文搜集2010-2020年发表的所有关于中国小麦品种吸收和积累镉的田间试验论文,提取籽粒镉浓度、土壤镉浓度、籽粒镉富集因子、土壤理化性质(pH、有机质、阳离子交换量、速效钾、碱解氮)等指标,对数据进行Meta分析。对搜集的895对小麦和土壤样品分析籽粒镉富集因子与土壤理化性相关性,结果表明,小麦籽粒镉富集因子与土壤镉浓度呈显著正相关关系,与土壤pH、有机质和碱解氮呈负相关关系,与阳离子交换量和速效钾无显著相关性。土壤pH>6.5时,小麦籽粒镉富集因子<1的比例达99.0%,主要集中在0-0.1之间,比例为71.9%;5.5<pH<6.5时,小麦籽粒镉富集因子<1的比例为72.7%,主要集中在0-0.6之间,比例为72.0%;pH<5.5时,小麦籽粒镉富集因子<1的比例仅为5.90%。土壤pH>6.5时,将小麦籽粒镉富集因子<1作为筛选低镉积累小麦品种指标之一无意义。土壤有机质、碱解氮较丰富的土壤中,小麦籽粒镉富集因子<1的比例分别为95.0%和91.5%。在pH较高,有机质和碱解氮较丰富的农田更有利于确保小麦镉食用安全。更多还原

【Abstract】 Some agricultural soils in China have been seriously contaminated with heavy metals,particularly for cadmium and mercury.The safety problems of agricultural products caused by contaminated soil frequently occurred.The "Hg wheat" and "Cd wheat" have attracted public attention.Soil pollution prevention and remediation must be implemented.Screening and planting low heavy metal accumulating wheat cultivars may be an effective approach to solve the above problem.However,the unified selection criteria for low-accumulating-cultivar remain to be established and the mechanism of low accumulation mercury or cadmium in wheat cultivars is not clear.So we selected generalized winter wheat cultivars in Huang-Huai-Hai Plain to investigate the accumulation characteristics of mercury or cadmium among different wheat cultivars,and screened low mercury or cadmium accumulating cultivars.We further investigated the difference of rhizospheric environment between high-and low-accumulating-cultivar to reveal why some wheat cultivars accumulate lower mercury or cadmium than other cultivars.The generalized winter wheat cultivars in Huang-Huai-Hai areas of China were evaluated for mercury resistance and mercury accumulation in grain under greenhouse conditions.The mercury treatments were assigned to three levels:0.1 and 5 mg kg-1.The responses of 11 wheat traits to mercury stress were investigated,including morphological traits,biomass traits and yield-related traits.The mercury resistance coefficient,resistance index,genetic variation coefficient and broad sense heritability of 11 investigated traits were calculated to establish an evaluation method for mercury resistance of wheat cultivars.Results demonstrated that the plant height and uppermost internode length significantly increased,while spikelet number significantly decreased under low mercury stress.Under high mercury stress,the plant height significantly increased,while yield-related traits,including total grain number,fresh grain yield,and dry grain yield,significantly decreased.An improved membership function was constructed with 11 traits of medium to high heritability(0.31-0.68).and then a mercury tolerance evaluation system for wheat cultivars was established through discriminant analysis.Discriminant analysis integrated the mercury resistance coefficients of effective tiller number,fresh grain yield.and dry biomass into the discriminant function under low mercury stress and the mercury resistance coefficients of dry grain yield and dry biomass under high mercury stress.The correct rates of identification were respective 90.0%and 83.3%under low and high mercury stress.The evalution system broke through the simple identification model with yield as a mere evaluation index.Based on the mercury resistance,mercury concentration and bio-concentration factor in wheat grain,ultimately,wheat cultivars Nongda-3163(ND3163)and Gaocheng-8901(GC8901)were screened for high mercury resistance and low mercury accumulation,and the cultivars Jimai-21(JM21)and Taishan-21(TS21)were mercury sensitive and high mercury accumulation.This research fills the knowledge gap on mercury resistance and mercury accumulation characteristics of wheat cultivars generalized in Huang-Huai-Hai areas,and provides germplasm resources for the safe production of slightly or moderately mercury contaminated farmland.In this study,we further selected two low-mercury-accumulating wheat cultivars(ND3163,GC8901)and two high-mercury-accumulating wheat cultivars(JM21.TS21)to clarify the reason of low mercury accumulation in wheat cultivars:low uptake or less translocation from root to aboveground parts.The difference of rhizospheric organic acids and microorganisms between low-and high-mercury-accumulating cultivars were also investigated to explore the specific rhizosphere microenvironment of low-mercury-accumulating cultivars.Under low mercury stress,low-mercury-accumulating cultivars ND3163 and GC8901 accumulated significantly lower mercury concentrations in their roots than high-mercury-accumulating cultivars JM21 and TS21.Under high mercury stress,no significant difference was detected in mercury concentrations in the roots among four cultivars,but ND3163 and GC8901 had significantly lower translocation factors from roots to leaves,glumes,and grains than JM21 and TS21.This shows that under low mercury stress,the low mercury accumulation in wheat grains is due to low mercury uptake in the roots,while under high mercury stress,the low mercury accumulation in wheat grains is due to lower translocation factor from root to the aboveground parts.Under low mercury stress,ND3163 and GC8901 secreted lower amounts of oxalic acid and citric acid in rhizosphere soil than JM21 and TS21.The abundances of gram positive bacteria,gram negative bacteria and fungi in rhizosphere soil of ND3163 and GC8901 were significantly lower than those of JM21,while abundance of protozoa in rhizosphere soil of GC8901 was significantly higher than in high-mercury-accumulating cultivars.The specific rhizosphere microenvironment of low-mercury-accumulating cultivars was less citric acid and oxalic acid,and the higher abundance of protozoa.Improving the beneficial relationship among rhizospheric organic acids,microorganisms and wheat cultivars is a realizable strategy to ensure wheat safety for mercury.Cadmium concentrations in 72 wheat cultivars generalized in Huanghuaihai area were identified by field experiments(field 1),and the stability of cadmium accumulation in these cultivars was tested in fields 2 and 3.The effects of cadmium on microelements(zinc,copper,iron,manganese,and boron)uptake in grains were also investigated.The correlation between agronomic traits and grain cadmium concentration was discussed to provide a simple and quick method to screen low-cadmium-accumulating wheat cultivars.Cluster analysis based on grain cadmium concentrations divided all wheat cultivars in field 1 into 5 categories:extremely low-cadmium-accumulating cultivar,low-cadmium-accumulating cultivar,medium-cadmium-accumulating cultivar,high-cadmium-accumulating cultivar and extremely high-cadmium-accumulating cultivar.A total of 24 wheat cultivars were screened for extremely low-cadmium-or low-cadmium-accumulating cultivars,and the cadmium concentrations in grains of these cultivars were below 0.1 mg kg-1,which is the maximum permissible concentration(MPC)for cadmium in wheat grain in China(GB2762-201 7).Nine of the 24 low-cadmium-accumulating wheat cultivars identified in field 1 exhibited unstably low cadmium accumulation characteristics in fields 2 and 3.We further investigated the differences of microelements concentrations in grain between low-and high-cadmium-accumulating cultivars.The results showed that five low-cadmium-accumulating cultivars contained significantly lower zinc concentrations in grain at least two experimental fields than high-cadmium-accumulating cultivars.Another one low-cadmium-accumulating cultivar also had lower zinc,copper and manganese concentrations in grain than high-cadmium-accumulating cultivars.There is no significant difference in grain iron concentrations between low-and high-cadmium-accumulating cultivars.Ultimately,nine wheat cultivars showed relatively stable low-cadmium and moderately high microelements accumulation in grain,which were recommended for planting in slightly and moderately cadmium contaminated farmland for food safety and high quality.The thousand kernel weights and yields of these nine wheat cultivars were relatively high in three fields.The correlations between wheat agronomic traits and grain cadmium concentration showed that grain cadmium concentration correlated positively with the dry weight of stem and root.and negatively with spike length.There was no correlation between grain cadmium concentration and wheat yield.Therefore,screening wheat cultivars with low cadmium concentration and high yield was feasible.Wheat cultivars with longer spike tended to accumulate less cadmium in grains.This study revealed that screening low-cadmium-accumulating wheat cultivars should pay particular attention to the stability of low-Cd accumulation characteristics and grain zinc concentrations.We found that some low-cadmium-accumulating wheat cultivars also have low zinc uptake.In this paper,high-throughput sequencing technology was used to study the differences in rhizobacteria diversity and community structure among wheat cultivars with different cadmium and zinc accumulation abilities,to explore the correlations between rhizobacteria and grain cadmium and zinc concentrations.The dominant phylum of rhizobacteria in wheat were Proteobacteria,Actinobacteria,Acidobacteria,Bacteroidetes,Gemmatimonadetes,Chloroflexi,Verrucomicrobia,Planctomycete and Firmicutes,which accounted for more than 95.0%of the total rhizobacterial communities.Low-cadmium-accumulating cultivars had significantly higher rhizobacteria diversity than high-cadmium-accumulating cultivar.At the phylum level,the relative abundances of Gemmatimonadetes,Chloroflexi,Planctomycete and Firmicutes were higher in rhizosphere soil of low-cadmium-accumulating cultivars than high-cadmium-accumulating cultivar.The relative abundances of Proteobacteria was higher in rhizosphere soil of high-zinc-accumulating cultivar than low-zinc-accumulating cultivars.At the family level,the relative abundances of Gemmatimonadaceae,Sphingomonadaceae,Beijerinckiaceae,Nitrosomonadaceae,Nocardioidaceae,Intrasporangiaceae,Micromonosporaceae,Pseudonocardiaceae.Geodermatophilaceae,Gaiellaceae in rhizosphere soil of low-cadmium-accumulating cultivars were significantly higher than that in rhizosphere soil of high-cadmium-accumulating cultivar.The relative abundances of the family of Xanthomonadales was significantly higher in high-zinc-accumulating cultivars than in low-zinc-accumulating cultivars.The low-cadmium-and high-zinc-accumulating cultivars had significantly higher relative abundances of Alphaproteobacteria and Gemmatimonadaceae than low-cadmium-and low-zinc-accumulating cultivars and high-cadmium-and high-zinc-accumulating cultivars.Tax4Fun function prediction showed that low-cadmium-and high-zinc-accumulating cultivars had higher relative abundance of aerobic_ammonia_oxidation and nitrification functional bacteria in rhizosphere soil than high-cadmium-and high-zinc-accumulating cultivars,and higher relative abundance of aerobic_nitrite_oxidation functional bacteria than low-cadmium-and low-zinc-accumulating cultivars.The higher abundances of Alphaproteobacteria and Gemmatimonadaceae in rhizosphere soil are beneficial to low cadmium and high zinc accumulation in wheat grain.We collected all field papers on the accumulation of cadmium in Chinese wheat cultivars published from 2010 to 2020 for Meta analysis,recording grain cadmium concentration,soil cadmium concentration,grain cadmium bio-concentration factor,and soil physical and chemical properties(pH,organic matter,cation exchange capacity,available potassium,alkaline nitrogen).Correlations between the cadmium bio-concentration factor and the soil physical and chemical properties were analyzed.Results showed that cadmium bio-concentration factor in wheat grain correlated positively with soil cadmium concentration,and negatively with soil pH,organic matter,and alkaline nitrogen.No significant correlations were found between cadmium bio-concentration factor of wheat grain and cation exchange capacity and available potassium.When soil pH>6.5.the proportion of cadmium bio-concentration factor<1 was 99.0%,mainly ranged from 0 to 0.1 with the proportion of 71.9%.When 5.5<pH<6.5,the proportion of cadmium bio-concentration factor<1 was 72.7%,mainly ranged from 0 to 0.6 with the proportion of 72.0%.When soil pH<5.5,the proportion of cadmium bio-concentration factor<1 was only 5.90%.It is not effective to regard cadmium bio-concentration factor<1 as one of the indicators for screening low-cadmium-accumulating wheat cultivars when soil pH>6.5.When soil organic matter and alkaline nitrogen were rich,the proportion of cadmium bio-concentration factor<1 was 95.0%and 91.5%,respectively.Farmland with higher pH,organic matter and alkaline nitrogen is more conducive to ensuring the safety of wheat consumption for cadmium.更多还原