| Soil is the place of material circulation and energy flow,with being the basis of ecosystem food chains.The various soil biogeochemical processes are traditionally thought of as a “black-box”,which involve hotspot areas terming microbial hotspots.These microbial hotspots with highly spatial and temporal heterogeneity mainly occur in interfaces of the root-soil and biopores,and have an extremely small proportion of total volume in soil.Therefore,exploring the formation mechanism of hotspot areas of biogeochemical reactions is currently the focus and difficulty in soil science.The addition of biochar,plant growth-promoting bacteria,and microplastics to the soil will not only form new microbial habitats,but also affect microbial colonization by influencing soil physicochemical properties.However,the mechanism by which these exogenous substances drive microbial hotspot formation is not yet clear.Using in situ soil zymogram and high-throughput sequencing technology,this study focusing heavy metal contaminated soils investigated the response and key drivers of microbial hotspots in the interfaces of the root-soil to addition of three exogenous substances(biochar,plant growth-promoting bacteria or microplastics),and further elucidated interaction mechanisms of biotic and abiotic factors.In addition,earthworms and ants are “ecosystem engineers”,and their pores are typical microbial hotspot.In situ experiment in the field was also conducted to reveal the influence mechanism of soil fauna activity on hotspot areas of biopores.The response of enzyme activity and microbial community structure to microenvironmental changes in the hotspots was elucidated.The main results were following:(1)The mechanism of soil microbial hotspot distribution characteristics in response to biochar addition under heavy metal was revealed.The results showed that the addition of biochar reduced the bioavailability of cadmium(Cd),lead(Pb),copper(Cu),and zinc(Zn)in the soil and improved soil nutrient.As a result,the heavy metal toxicity of alfalfa was reduced and the growth of alfalfa was promoted.The addition of biochar altered bacterial and fungal community compositions,and shaped the soil microbial communities.Co-occurrence network analysis showed that interactions among taxa of microbial(bacterial and fungal)groups were more complex,stable and closer in the rhizosphere than those in the bulk soil.Proteobacteria and Acidobacteria of bacteria and Basidiomycota of fungi were the key microbial taxa driving the formation of hotspots in rhizosphere,and these taxa were strongly related to the hotspots of enzyme activities.The addition of biochar significantly expanded the area of microbial hotspots in the root-soil interface,such as the increasing in hotspot area of alkaline phosphatase(ALP),acid phosphatase(ACP),and β-glucosidase(BG)activities.The analyses of random forest and Pearson’s correlation further suggested that areas of microbial hotspots were co-regulated by biotic(bacterial community structure and composition)and abiotic(soil soluble organic carbon and available heavy metals concentrations)factors.(2)The mechanism of the effect of biochar and rhizobia co-addition on microbial hotspot under heavy metal stress was illustrated.Compared with only additions of biochar or rhizobia,the combined addition of biochar and rhizobia significantly reduced the bioavailability of heavy metals(Cu,Zn,Pb,and Cd)in the soil,and decreased the concentration of heavy metals in plant tissues,ultimately promoting plant growth in soils contaminated with heavy metals.Furthermore,the rhizosphere hotspot areas of ACP,ALP,and BG significantly increased due to the reduction of heavy metals toxicity in the root-soil interface.The hotspot area was 1 to 3 times higher than the control after combined application of biochar and rhizobia.Given that biochar provided a favorable habitat and nutrients for rhizobia,the combined addition can promote microbial growth and reproduction,and increase the richness and diversity of bacterial communities in both rhizosphere and bulk soils.Co-occurrence network analysis showed that Proteobacter,Acidobacteria,Actinobacteria,Ascomycota and Basidiomycota were the key taxa in regulating the microbial community structure and composition,irrespective of hotspots or non-hotspots.(3)The mechanism of how three types of microplastics affected the spatial distribution of rhizosphere enzyme activity and bacterial community in heavy metal contaminated soils was explored.These three microplastics with different structural composition and physical characteristics had distinct effects on plants,soil and microorganisms.Non-degradable microplastics(polyethylene(PE)and polyvinyl chloride(PVC))directly or indirectly reduced the bioavailability of heavy metals in soil and the uptake of metals by plants,thus promoting maize growth,especially for root lengthening.Moreover,PE and PVC improved the complexity and stability of bacterial co-occurrence networks.In contrast,biodegradable microplastics(polylactic acid(PLA))inhibited plant growth.Due to the rapid degradation of PLA,their intermediates and final products largely altered the soil physicochemical properties,the availability of heavy metals and the unique microbial taxa,and decreased the abundance and diversity of bacteria in the rhizosphere soil.Moreover,microplastics particles may act individual habitats of microorganisms(Acidobacteria and Actinobacteria)forming unique microbial hotspots,which increased the hotspot areas of carbon(C),nitrogen(N),and phosphorus(P)related enzymes,and were 1.77～13.8,1.66～10.4,and1.31～14.5 times than control treatment,respectively.(4)Based on in situ field experiments,the hotspots of C-,N-,and P-related enzymes in earthworm burrows were visualized using in situ zymography,and their main drivers were revealed by high-throughput sequencing technology.Earthworm activity significantly improved soil quality by increasing nutrient content and decreasing p H in the biopores,which provided a favorable microenvironment for microbial growth and further improved soil ecological functions.Soil microorganisms preferentially inhabited the inner wall and the edge area of biopores forming microbial hotspots,thus improving the soil microbial biomass and the activities of C,N,and P cycle enzymes.Thaumarchaeota and Latescibacteria were the core groups of biopores,which played an important role in stimulating the formation of enzymatic hotspots.(5)The hotspot distribution of ALP activity was visualized in the inner wall of ant biopores in the field,and the interaction relationships of the abundance of pho D,the pho D-harboring microbial community composition and ALP enzyme activity were disentangled in hotspot soils in the ant biopores.The feeding activities of ants not only improved the soil nutrient and increased soil microbial biomass,but also formed microbial hotspots due to the presence of viscous secretions in the inner wall of ant biopores,further improving the hotspot areas of ALP activity.Moreover,the hotspot formation of ALP activity was dominantly regulated by rare taxa,genus Devosia of class Proteobacteria with a great of demand for nutrients,rather than the abundance of pho D.In summary,this study revealed the mechanism of exogenous additive influence on the microbial hotspots at the root-soil interface by in situ zymography and highthroughput sequencing,and clarified that biotic factor(bacterial key taxa and diversity)and abiotic factors(bioavailability of heavy metals)jointly derived the formation of the microbial hotspots in the rhizosphere.In addition,it was clarified that Thaumarchaeota and Latescibacteria were the core flora of earthworm biopores,which played an important role in stimulating the formation of microbial hotspots in earthworm biopores.The hotspots of alkaline phosphatase activity in ant biopores were regulated by pho D gene abundance and rare species.This study comprehensively analyzed the formation mechanism of microbial hotspots in rhizosphere and biopores,and provided key information and scientific basis for understanding the ecological and environmental effects such as carbon turnover and element cycling in hotspots. |
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