Validation of Bacteroidales-based microbial source tracking markers for pig fecal pollution and their application in two rivers of North China

In China,pig feces is the predominant source of excrement produced b y animal husbandry.Improper use or direct discharge of pig feces can result in contamination of natural water systems.Microbial source tracking(MST)technology can identify the sources of fecal pollution in environmental water,and contribute to the management of pig fecal pollution by local environmental protection agencies.However,the accuracy of such assays can be context-dependent,and they have not been comprehensively evaluated under Chinese conditions.We aimed to compare the performance of five previously reported nig-specific MST assays(PF,Pig-Bac 1 STBR,Pig-Bac2 SYBR,Pig-1-Bac TaqMan,and Pig-2-Bac TaqMan,which are based on Bacteroidales 16S rRNA gene markers)and apply them in two rivers of North China.We collected a total of 173 fecal samples from pigs,cows,goats,chickens,humans,and horses across China.The PF assay optimized in this study showed outstanding qualitative performance and achieved 100%specificity and sensitivity.However,the two SYBR green qPCR assays(Pig-Bac1 SYBR and Pig-Bac2 SYBR)cross-reacted with most non-pig fecal samples.In contrast,both the Pig-l-Bac TaqMan and Pig-2-Bacr TaqMan assays gave 100%specificity and sensitivity.Of these,the Pig-2-Bac TaqMan assay showed higher reproducibility.Our results regarding the specificity of these pig-specific MST assays differ from those reported in Thailand,Japan,and America.Using the PF and Pig-2-Bac TaqMan assays,a field test comparing the levels of pig fecal pollution in rivers near a pig farm before and after comprehensive environmental pollution governance indicated that pig fecal pollution was effectively controlled at this location.

Assembly process and source tracking of microbial communities in sediments of Dongting Lake

●Soil erosion resulted in homogenization of bacterial communities in the watershed.●Microbial community heterogeneity among erosion sites made soil tracing possible.●Assembly process results showed that the tracking results can achieve high precision.●Dryland was the main source of sediment deposition based on the result of FEAST.Sediment source tracing can accurately provide a theoretical basis for controlling soil erosion effectively,by identifying the most serious types of land use.Traditional sediment tracing methods are based on physical,chemical,biological,and composite fingerprinting,which have not included microbes.As high-throughput sequencing becomes more prevalent,microorganisms can provide more information than what we think.Thus,whether the microorganism can also be used as a special fingerprint factor for sediment source identification during soil erosion,we have tested it by using microbial source tracking tool FEAST to quantify the microbe contribution from five types of eroded land(including dryland,urban,paddy field,forest and grassland)to the depositional areas(Niubitan)in the Yuanjiang basin.The source microbial community in the erosive area was heterogeneous,and assembly process analysis further demonstrated that the source tracking results could reach higher accuracy.The results of FEAST showed that dryland(35.50%),urban(17.21%),paddy field(8.14%),and forest(1.07%)were the major contributors to Niubitan.Our results follow the general soil erosion rules and prove its validity.Taken together,a new perspective is provided by these results for tracing sediment sources in erosion-sedimentary systems.

Microbial diversity accumulates in a downstream direction in the Three Gorges Reservoir

Organic and inorganic materials migrate downstream and have important roles in regulating environmental health in the river networks.However,it remains unclear whether and how a mixture of materials(i.e.,microbial species)from various upstream habitats contribute to microbial community coalescence upstream of a dam.Here we track the spatial variation in microbial abundance and diversity in the Three Gorges Reservoir based on quantitative PCR and 16 S rRNA gene high-throughput sequencing data.We further quantitatively assess the relative contributions of microbial species from mainstem,its tributaries,and the surrounding riverbank soils to the area immediately upstream of the Three Gorges Dam(TGD).We found an increase of microbial diversity and the convergent microbial distribution pattern in areas immediately upstream of TGD,suggesting this area become a new confluence for microbial diversity immigrating from upstream.Indeed,the number of shared species increased from upstream to TGD but unique species decreased,indicating immigration of various sources of microbial species overwhelms local environmental conditions in structuring microbial community close to TGD.By quantifying the sources of microbial species close to TGD,we found little contribution from soils as compared to tributaries,especially for sites closer to TGD,suggesting tributary microbes have greater influence on microbial diversity and environmental health in the Three Gorges Reservoir.Collectively,our results suggest that tracking microbial geographic origin and evaluating accumulating effects of microbial diversity shed light on the ecological processes in microbial communities and provide information for regulating aquatic ecological health.

The spatial variation of soil bacterial community assembly processes affects the accuracy of source tracking in ten major Chinese cities

Urban soils harbor billions of bacterial cells and millions of species.However,the distribution patterns and assembly processes of bacterial communities remain largely uncharacterized in urban soils.It is also unknown if we can use the bacteria to track soil sources to certain cities and districts.Here,Illumina MiSeq sequencing was used to survey soil bacterial communities from 529 random plots spanning 61 districts and 10 major cities in China.Over a 3,000 km range,community similarity declined with increasing geographic distance(Mantel r=0.62),and community composition was clustered by city(R^(2)=0.50).Within cities(<100 km),the aforementioned biogeographic patterns were weakened.Process analysis showed that homogenizing dispersal and dispersal limitation dominated soil bacterial assembly at small and large spatial scales,respectively.Accordingly,the probabilities of accurately tracking random soil sources to certain cities and districts were 90.0% and 66.7%,respectively.When the tested samples originated from cities that were more than 1,265 km apart,the soil sources could be identified with nearly 100% accuracy.Overall,this study demonstrates the strong distance-decay relationship and the clear geographic zoning of urban soil bacterial communities among cities.The varied importance of different community assembly processes at multiple spatial scales strongly affects the accuracy of microbial source tracking.