Village Settlements in Mountainous Tropical Areas, Hotspots of Fecal Contamination as Evidenced by Escherichia coli and Stanol Concentrations in Stormwater Pulses.

Fecal bacteria in surface water may indicate threats to human health. Our hypothesis is that village settlements in tropical rural areas are major hotspots of fecal contamination because of the number of domestic animals usually roaming in the alleys and the lack of fecal matter treatment before entering the river network. By jointly monitoring the dynamics of Escherichia coli and of seven stanol compounds during four flood events (July-August 2016) at the outlet of a ditch draining sewage and surface runoff out of a village of Northern Lao PDR, our objectives were (1) to assess the range of E. coli concentration in the surface runoff washing off from a village settlement and (2) to identify the major contributory sources of fecal contamination using stanol compounds during flood events. E. coli pulses ranged from 4.7 × 104 to 3.2 × 106 most probable number (MPN) 100 mL-1, with particle-attached E. coli ranging from 83 to 100%. Major contributory feces sources were chickens and humans (about 66 and 29%, respectively), with the highest percentage switching from the human pole to the chicken pole during flood events. Concentrations indicate a severe fecal contamination of surface water during flood events and suggest that villages may be considered as major hotspots of fecal contamination pulses into the river network and thus as point sources in hydrological models.

Distribution of Burkholderia pseudomallei within a 300-cm deep soil profile: implications for environmental sampling.

The environmental distribution of Burkholderia pseudomallei, the causative agent of melioidosis, remains poorly understood. B. pseudomallei is known to have the ability to occupy a variety of environmental niches, particularly in soil. This paper provides novel information about a putative association of soil biogeochemical heterogeneity and the vertical distribution of B. pseudomallei. We investigated (1) the distribution of B. pseudomallei along a 300-cm deep soil profile together with the variation of a range of soil physico-chemical properties; (2) whether correlations between the distribution of B. pseudomallei and soil physico-chemical properties exist and (3) when they exist, what such correlations indicate with regards to the environmental conditions conducive to the occurrence of B. pseudomallei in soils. Unexpectedly, the highest concentrations of B. pseudomallei were observed between 100 and 200 cm below the soil surface. Our results indicate that unravelling the environmental conditions favorable to B. pseudomallei entails considering many aspects of the actual complexity of soil. Important recommendations regarding environmental sampling for B. pseudomallei can be drawn from this work, in particular that collecting samples down to the water table is of foremost importance, as groundwater persistence appears to be a controlling factor of the occurrence of B. pseudomallei in soil.

Effects of hydrological regime and land use on in-stream Escherichia coli concentration in the Mekong basin, Lao PDR.

In the basin of Mekong, over 70 million people rely on unimproved surface water for their domestic requirements. Surface water is often contaminated with fecal matter and yet little information exists on the underlying mechanisms of fecal contamination in tropical conditions at large watershed scales. Our objectives were to (1) investigate the seasonality of fecal contamination using Escherichia coli as fecal indicator bacteria (FIB), and (2) establish links between the fecal contamination in stream water and its controlling factors (hydrology and land use). We present the results of (1) a sampling campaign at the outlet of 19 catchments across Lao PDR, in both the dry and the rainy seasons of 2016, and (2) a 10-day interval monitoring conducted in 2017 and 2018 at three point locations of three rivers (Nam Ou, Nam Suang, and Mekong) in northern Lao PDR. Our results show the presence of fecal contamination at most of the sampled sites, with a seasonality characterized by higher and extreme E. coli concentrations occurring during the rainy season. The highest E. coli concentrations, strongly correlated with total suspended sediment concentrations, were measured in catchments dominated by unstocked forest areas, especially in mountainous northern Lao PDR and in Vientiane province.

Whole-Genome Assemblies of 16 Burkholderia pseudomallei Isolates from Rivers in Laos.

We report 16 Burkholderia pseudomallei genomes, including 5 new multilocus sequence types, isolated from rivers in Laos. The environmental bacterium B. pseudomallei causes melioidosis, a serious infectious disease in tropical and subtropical regions. The isolates are geographically clustered in one clade from around Vientiane, Laos, and one clade from further south.

Digging Deeper for Agricultural Resources, the Value of Deep Rooting.

In the quest for sustainable intensification of crop production, we discuss the option of extending the root depth of crops to increase the volume of soil exploited by their root systems. We discuss the evidence that deeper rooting can be obtained by appropriate choice of crop species, by plant breeding, or crop management and its potential contributions to production and sustainable development goals. Many studies highlight the potentials of deeper rooting, but we evaluate its contributions to sustainable intensification of crop production, the causes of the limited research into deep rooting of crops, and the research priorities to fill the knowledge gaps.

Evaluation of consensus method for the culture of Burkholderia pseudomallei in soil samples from Laos.

Background: We have previously shown that PCR following enrichment culture is the most sensitive method to detect Burkholderia pseudomallei in environmental samples. Here we report an evaluation of the published consensus method for the culture of B. pseudomallei from Lao soil in comparison with our conventional culture method and with PCR with or without prior broth enrichment. Methods: One hundred soil samples were collected from a field known to contain B. pseudomallei and processed by: (i) the conventional method, (ii-iii) the consensus method using media prepared in either Laos or Thailand, and (iv) the consensus method performed in Thailand, as well as by (v) PCR following direct extraction of DNA from soil and (vi) PCR following broth pre-enrichment. Results: The numbers of samples in which B. pseudomallei was detected were 42, 10, 7, 6, 6 and 84, respectively. However, two samples were positive by the consensus method but negative by conventional culture, and one sample was negative by PCR following enrichment although B. pseudomallei was isolated by the conventional culture method. Conclusions/Discussion: The results show that no single method will detect all environmental samples that contain B. pseudomallei. People conducting environmental surveys for this organism should be aware of the possibility of false-negative results using the consensus culture method. An approach that entails screening using PCR after enrichment, followed by the evaluation of a range of different culture methods on PCR-positive samples to determine which works best in each setting, is recommended.

Rivers as carriers and potential sentinels for Burkholderia pseudomallei in Laos.

Burkholderia pseudomallei, causative agent of the often fatal disease melioidosis, dwells in tropical soils and has been found in freshwater bodies. To investigate whether rivers are potential habitats or carriers for B. pseudomallei and to assess its geographical distribution in Laos, we studied 23 rivers including the Mekong, applying culture-based detection methods and PCR to water filters and streambed sediments. B. pseudomallei was present in 9% of the rivers in the dry season and in 57% in the rainy season. We found the pathogen exclusively in Southern and Central Laos, and mainly in turbid river water, while sediments were positive in 35% of the B. pseudomallei-positive sites. Our results provide evidence for a heterogeneous temporal and spatial distribution of B. pseudomallei in rivers in Laos with a clear north-south contrast. The seasonal dynamics and predominant occurrence of B. pseudomallei in particle-rich water suggest that this pathogen is washed out with eroded soil during periods of heavy rainfall and transported by rivers, while river sediments do not seem to be permanent habitats for B. pseudomallei. Rivers may thus be useful to assess the distribution and aquatic dispersal of B. pseudomallei and other environmental pathogens in their catchment area and beyond.

Linking crop structure, throughfall, soil surface conditions, runoff and soil detachment: 10 land uses analyzed in Northern Laos.

In Montane Southeast Asia, deforestation and unsuitable combinations of crops and agricultural practices degrade soils at an unprecedented rate. Typically, smallholder farmers gain income from "available" land by replacing fallow or secondary forest by perennial crops. We aimed to understand how these practices increase or reduce soil erosion. Ten land uses were monitored in Northern Laos during the 2015 monsoon, using local farmers' fields. Experiments included plots of the conventional system (food crops and fallow), and land uses corresponding to new market opportunities (e.g. commercial tree plantations). Land uses were characterized by measuring plant cover and plant mean height per vegetation layer. Recorded meteorological variables included rainfall intensity, throughfall amount, throughfall kinetic energy (TKE), and raindrop size. Runoff coefficient, soil loss, and the percentage areas of soil surface types (free aggregates and gravel; crusts; macro-faunal, vegetal and pedestal features; plant litter) were derived from observations and measurements in 1-m2 micro-plots. Relationships between these variables were explored with multiple regression analyses. Our results indicate that TKE induces soil crusting and soil loss. By reducing rainfall infiltration, crusted area enhances runoff, which removes and transports soil particles detached by splash over non-crusted areas. TKE is lower under land uses reducing the velocity of raindrops and/or preventing an increase in their size. Optimal vegetation structures combine minimum height of the lowest layer (to reduce drop velocity at ground level) and maximum coverage (to intercept the largest amount of rainfall), as exemplified by broom grass (Thysanolaena latifolia). In contrast, high canopies with large leaves will increase TKE by enlarging raindrops, as exemplified by teak trees (Tectona grandis), unless a protective understorey exists under the trees. Policies that ban the burning of multi-layered vegetation structure under tree plantations should be enforced. Shade-tolerant shrubs and grasses with potential economic return could be promoted as understorey.

From shifting cultivation to teak plantation: effect on overland flow and sediment yield in a montane tropical catchment.

Soil erosion supplies large quantities of sediments to rivers of Southeastern Asia. It reduces soil fertility of agro-ecosystems located on hillslopes, and it degrades, downstream, water resource quality and leads to the siltation of reservoirs. An increase in the surface area covered with commercial perennial monocultures such as teak plantations is currently observed at the expanse of traditional slash-and-burn cultivation systems in steep montane environments of these regions. The impacts of land-use change on the hydrological response and sediment yields have been investigated in a representative catchment of Laos monitored for 13 years. After the gradual conversion of rice-based shifting cultivation to teak plantation-based systems, overland flow contribution to stream flow increased from 16 to 31% and sediment yield raised from 98 to 609 Mg km-2. This result is explained by the higher kinetic energy of raindrops falling from the canopy, the virtual absence of understorey vegetation cover to dissipate drop energy and the formation of an impermeable surface crust accelerating the formation and concentration of overland flow. The 25-to-50% lower 137Cs activities measured in soils collected under mature teak plantations compared to soils under other land uses illustrate the severity of soil erosion processes occurring in teak plantations.

Hydrological modeling of Fecal Indicator Bacteria in a tropical mountain catchment.

The occurrence of pathogen bacteria in surface waters is a threat to public health worldwide. In particular, inadequate sanitation resulting in high contamination of surface water with pathogens of fecal origin is a serious issue in developing countries such as Lao P.D.R. Despite the health implications of the consumption of contaminated surface water, the environmental fate and transport of pathogens of fecal origin and their indicators (Fecal Indicator Bacteria or FIB) are still poorly known in tropical areas. In this study, we used measurements of flow rates, suspended sediments and of the FIB Escherichia coli (E. coli) in a 60-ha catchment in Northern Laos to explore the ability of the Soil and Water Assessment Tool (SWAT) to simulate watershed-scale FIB fate and transport. We assessed the influences of 3 in-stream processes, namely bacteria deposition and resuspension, bacterial regrowth, and hyporheic exchange (i.e. transient storage) on predicted FIB numbers. We showed that the SWAT model in its original version does not correctly simulate small E. coli numbers during the dry season. We showed that model's performance could be improved when considering the release of E. coli together with sediment resuspension. We demonstrated that the hyporheic exchange of bacteria across the Sediment-Water Interface (SWI) should be considered when simulating FIB concentration not only during wet weather, but also during the dry season, or baseflow period. In contrast, the implementation of the regrowth process did not improve the model during the dry season without inducing an overestimation during the wet season. This work thus underlines the importance of taking into account in-stream processes, such as deposition and resuspension, regrowth and hyporheic exchange, when using SWAT to simulate FIB dynamics in surface waters.

Hydrological Regime and Water Shortage as Drivers of the Seasonal Incidence of Diarrheal Diseases in a Tropical Montane Environment.

BACKGROUND: The global burden of diarrhea is a leading cause of morbidity and mortality worldwide. In montane areas of South-East Asia such as northern Laos, recent changes in land use have induced increased runoff, soil erosion and in-stream suspended sediment loads, and potential pathogen dissemination. To our knowledge, few studies have related diarrhea incidences to catchment scale hydrological factors such as river discharge, and loads of suspended sediment and of Fecal Indicator Bacteria (FIB) such as Escherichia coli, together with sociological factors such as hygiene practices. We hypothesized that climate factors combined with human behavior control diarrhea incidence, either because higher rainfall, leading to higher stream discharges, suspended sediment loads and FIB counts, are associated with higher numbers of reported diarrhea cases during the rainy season, or because water shortage leads to the use of less safe water sources during the dry season. Using E. coli as a FIB, the objectives of this study were thus (1) to characterize the epidemiological dynamics of diarrhea in Northern Laos, and (2) to identify which hydro-meteorological and sociological risk factors were associated with diarrhea epidemics. METHODS: Considering two unconnected river catchments of 22 and 7,448 km2, respectively, we conducted a retrospective time series analysis of meteorological variables (rainfall, air temperature), hydrological variables (discharge, suspended sediments, FIB counts, water temperature), and the number of diarrheal disease cases reported at 6 health centers located in the 5 southern districts of the Luang Prabang Province, Lao PDR. We also examined the socio-demographic factors potentially affecting vulnerability to the effect of the climate factors, such as drinking water sources, hygiene habits, and recreational water exposure. RESULTS: Using thus a mixed methods approach, we found E. coli to be present all year long (100-1,000 Most Probable Number or MPN 100 mL-1) indicating that fecal contamination is ubiquitous and constant. We found that populations switch their water supply from wells to surface water during drought periods, the latter of which appear to be at higher risk of bacterial contamination than municipal water fountains. We thus found that water shortage in the Luang Prabang area triggers diarrhea peaks during the dry and hot season and that rainfall and aquifer refill ends the epidemic during the wet season. The temporal trends of reported daily diarrhea cases were generally bimodal with hospital admissions peaking in February-March and later in May-July. Annual incidence rates were higher in more densely populated areas and mostly concerned the 0-4 age group and male patients. CONCLUSIONS: We found that anthropogenic drivers, such as hygiene practices, were at least as important as environmental drivers in determining the seasonal pattern of a diarrhea epidemic. For diarrheal disease risk monitoring, discharge or groundwater level can be considered as relevant proxies. These variables should be monitored in the framework of an early warning system provided that a tradeoff is found between the size of the monitored catchment and the frequency of the measurement.

Effect of land use and hydrological processes on Escherichia coli concentrations in streams of tropical, humid headwater catchments.

Lack of access to clean water and adequate sanitation continues to be a major brake on development. Here we present the results of a 12-month investigation into the dynamics of Escherichia coli, a commonly used indicator of faecal contamination in water supplies, in three small, rural catchments in Laos, Thailand and Vietnam. We show that land use and hydrology are major controlling factors of E. coli concentrations in streamwater and that the relative importance of these two factors varies between the dry and wet seasons. In all three catchments, the highest concentrations were observed during the wet season when storm events and overland flow were highest. However, smaller peaks of E. coli concentration were also observed during the dry season. These latter correspond to periods of intense farming activities and small, episodic rain events. Furthermore, vegetation type, through land use and soil surface crusting, combined with mammalian presence play an important role in determining E. coli loads in the streams. Finally, sampling during stormflow revealed the importance of having appropriate sampling protocols if information on maximum contamination levels is required as grab sampling at a fixed time step may miss important peaks in E. coli numbers.

Understanding deep roots and their functions in ecosystems: an advocacy for more unconventional research.

Background Deep roots are a common trait among a wide range of plant species and biomes, and are pivotal to the very existence of ecosystem services such as pedogenesis, groundwater and streamflow regulation, soil carbon sequestration and moisture content in the lower troposphere. Notwithstanding the growing realization of the functional significance of deep roots across disciplines such as soil science, agronomy, hydrology, ecophysiology or climatology, research efforts allocated to the study of deep roots remain incommensurate with those devoted to shallow roots. This is due in part to the fact that, despite technological advances, observing and measuring deep roots remains challenging. Scope Here, other reasons that explain why there are still so many fundamental unresolved questions related to deep roots are discussed. These include the fact that a number of hypotheses and models that are widely considered as verified and sufficiently robust are only partly supported by data. Evidence has accumulated that deep rooting could be a more widespread and important trait among plants than usually considered based on the share of biomass that it represents. Examples that indicate that plant roots have different structures and play different roles with respect to major biochemical cycles depending on their position within the soil profile are also examined and discussed. Conclusions Current knowledge gaps are identified and new lines of research for improving our understanding of the processes that drive deep root growth and functioning are proposed. This ultimately leads to a reflection on an alternative paradigm that could be used in the future as a unifying framework to describe and analyse deep rooting. Despite the many hurdles that pave the way to a practical understanding of deep rooting functions, it is anticipated that, in the relatively near future, increased knowledge about the deep rooting traits of a variety of plants and crops will have direct and tangible influence on how we manage natural and cultivated ecosystems.

Land use and soil type determine the presence of the pathogen Burkholderia pseudomallei in tropical rivers.

Burkholderia pseudomallei is the bacterium that causes melioidosis in humans. While B. pseudomallei is known to be endemic in South East Asia (SEA), the occurrence of the disease in other parts of the tropics points towards a potentially large global distribution. We investigated the environmental factors that influence the presence (and absence) of B. pseudomallei in a tropical watershed in SEA. Our main objective was to determine whether there is a link between the presence of the organism in the hydrographic network and the upstream soil and land-use type. The presence of B. pseudomallei was determined using a specific quantitative real-time PCR assay following enrichment culture. Land use, soil, geomorphology, and environmental data were then analyzed using partial least squares discriminant analysis (PLSDA) to compare the B. pseudomallei positive and negative sites. Soil type in the surrounding catchment and turbidity had a strong positive influence on the presence (acrisols and luvisols) or absence (ferralsols) of B. pseudomallei. Given the strong apparent links between soil characteristics, water turbidity, and the presence/absence of B. pseudomallei, actions to raise public awareness about factors increasing the risk of exposure should be undertaken in order to reduce the incidence of melioidosis in regions of endemicity.

Seasonal Patterns of Fine Root Production and Turnover in a Mature Rubber Tree (Hevea brasiliensis Müll. Arg.) Stand- Differentiation with Soil Depth and Implications for Soil Carbon Stocks.

Fine root dynamics is a main driver of soil carbon stocks, particularly in tropical forests, yet major uncertainties still surround estimates of fine root production and turnover. This lack of knowledge is largely due to the fact that studying root dynamics in situ, particularly deep in the soil, remains highly challenging. We explored the interactions between fine root dynamics, soil depth, and rainfall in mature rubber trees (Hevea brasiliensis Müll. Arg.) exposed to sub-optimal edaphic and climatic conditions. A root observation access well was installed in northern Thailand to monitor root dynamics along a 4.5 m deep soil profile. Image-based measurements of root elongation and lifespan of individual roots were carried out at monthly intervals over 3 years. Soil depth was found to have a significant effect on root turnover. Surprisingly, root turnover increased with soil depth and root half-life was 16, 6-8, and only 4 months at 0.5, 1.0, 2.5, and 3.0 m deep, respectively (with the exception of roots at 4.5 m which had a half-life similar to that found between depths of 1.0 and 2.5 m). Within the first two meters of the soil profile, the highest rates of root emergence occurred about 3 months after the onset of the rainy season, while deeper in the soil, root emergence was not linked to the rainfall pattern. Root emergence was limited during leaf flushing (between March and May), particularly within the first two meters of the profile. Between soil depths of 0.5 and 2.0 m, root mortality appeared independent of variations in root emergence, but below 2.0 m, peaks in root emergence and death were synchronized. Shallow parts of the root system were more responsive to rainfall than their deeper counterparts. Increased root emergence in deep soil toward the onset of the dry season could correspond to a drought acclimation mechanism, with the relative importance of deep water capture increasing once rainfall ceased. The considerable soil depth regularly explored by fine roots, even though significantly less than in surface layers in terms of root length density and biomass, will impact strongly the evaluation of soil carbon stocks.

Evaluation of Molecular Methods To Improve the Detection of Burkholderia pseudomallei in Soil and Water Samples from Laos.

Burkholderia pseudomallei is the cause of melioidosis, a severe and potentially fatal disease of humans and animals. It is endemic in northern Australia and Southeast Asia and is found in soil and surface water. The environmental distribution of B. pseudomallei worldwide and within countries where it is endemic, such as the Lao People's Democratic Republic (Laos), remains unclear. However, this knowledge is important to our understanding of the ecology and epidemiology of B. pseudomallei and to facilitate public health interventions. Sensitive and specific methods to detect B. pseudomallei in environmental samples are therefore needed. The aim of this study was to compare molecular and culture-based methods for the detection of B. pseudomallei in soil and surface water in order to identify the optimal approach for future environmental studies in Laos. Molecular detection by quantitative real-time PCR (qPCR) was attempted after DNA extraction directly from soil or water samples or after an overnight enrichment step. The positivity rates obtained by qPCR were compared to those obtained by different culture techniques. The rate of detection from soil samples by qPCR following culture enrichment was significantly higher (84/100) than that by individual culture methods and all culture methods combined (44/100; P < 0.001). Similarly, qPCR following enrichment was the most sensitive method for filtered river water compared with the sensitivity of the individual methods and all individual methods combined. In conclusion, molecular detection following an enrichment step has proven to be a sensitive and reliable approach for B. pseudomallei detection in Lao environmental samples and is recommended as the preferred method for future surveys.

How to study deep roots-and why it matters.

The drivers underlying the development of deep root systems, whether genetic or environmental, are poorly understood but evidence has accumulated that deep rooting could be a more widespread and important trait among plants than commonly anticipated from their share of root biomass. Even though a distinct classification of "deep roots" is missing to date, deep roots provide important functions for individual plants such as nutrient and water uptake but can also shape plant communities by hydraulic lift (HL). Subterranean fauna and microbial communities are highly influenced by resources provided in the deep rhizosphere and deep roots can influence soil pedogenesis and carbon storage.Despite recent technological advances, the study of deep roots and their rhizosphere remains inherently time-consuming, technically demanding and costly, which explains why deep roots have yet to be given the attention they deserve. While state-of-the-art technologies are promising for laboratory studies involving relatively small soil volumes, they remain of limited use for the in situ observation of deep roots. Thus, basic techniques such as destructive sampling or observations at transparent interfaces with the soil (e.g., root windows) which have been known and used for decades to observe roots near the soil surface, must be adapted to the specific requirements of deep root observation. In this review, we successively address major physical, biogeochemical and ecological functions of deep roots to emphasize the significance of deep roots and to illustrate the yet limited knowledge. In the second part we describe the main methodological options to observe and measure deep roots, providing researchers interested in the field of deep root/rhizosphere studies with a comprehensive overview. Addressed methodologies are: excavations, trenches and soil coring approaches, minirhizotrons (MR), access shafts, caves and mines, and indirect approaches such as tracer-based techniques.