Combined assembly of long and short sequencing reads improve the efficiency of exploring the soil metagenome

Background Advances in DNA sequencing technologies have transformed our capacity to perform life science research, decipher the dynamics of complex soil microbial communities and exploit them for plant disease management. However, soil is a complex conglomerate, which makes functional metagenomics studies very challenging. Results Metagenomes were assembled by long-read (PacBio, PB), short-read (Illumina, IL), and mixture of PB and IL (PI) sequencing of soil DNA samples were compared. Ortholog analyses and functional annotation revealed that the PI approach significantly increased the contig length of the metagenomic sequences compared to IL and enlarged the gene pool compared to PB. The PI approach also offered comparable or higher species abundance than either PB or IL alone, and showed significant advantages for studying natural product biosynthetic genes in the soil microbiomes. Conclusion Our results provide an effective strategy for combining long and short-read DNA sequencing data to explore and distill the maximum information out of soil metagenomics.

Understanding the Responses of Soil Bacterial Communities to Long-Term Fertilization Regimes Using DNA and RNA Sequencing

Studies of soil DNA-based and RNA-based bacterial communities under contrasting long-term fertilization regimes can provide valuable insights into how agricultural management affects soil microbial structure and functional diversity. In this study, soil bacterial communities subjected to six fertility treatments in an alkaline soil over 27 years were investigated by 454 pyrosequencing based on 16S rDNA and 16S rRNA. Long-term fertilization showed significant influences on the diversity of the soil DNA-based bacteria, as well as on their RNA-based members. The top five phyla (Proteobacteria, Acidobacteria, Chloroflexi, Actinobacteria, and Planctomycetes) were found in both the DNA- and RNA-based samples. However, the relative abundances of these phyla at both DNA and RNA levels were showed significantly different. Analysis results showed that the diversity of the 16S rRNA samples was consistently lower than that of the rDNA samples, however, 16S rRNA samples had higher relative abundance. PICRUSt analysis indicated that glycan biosynthesis and metabolism were detected mainly in the DNA samples, while metabolism and degradation of xenobiotics and the metabolism of amino acids, terpenoids and polyketides were relatively higher in the RNA samples. Bacilli were significantly more abundant in all the OM-fertilized soils. Redundancy analysis indicated that the relative abundances of both DNA- and RNA-based bacterial groups were correlated with soil total organic carbon content, nitrogen content, Olsen-P, and soil pH. Moreover, the RNA-based Bacilli were positively correlated with available phosphorus (Olsen-P).

Soil Health Check-Up of Conservation Agriculture Farming Systems in Brazil

Conservation agriculture has been promoted as the main strategy to regenerate soil life but its effect on soil enzyme activity remains little documented. This study investigated the β-glucosidase and arylsulfatase enzymes as tools to evaluate soil health at the field level. Croplands in four main grain-producing states in Brazil were selected for this study. In each cropland, three environments (high yield (HYE), medium yield (MYE), and low yield (LYE)) were delineated for soil sampling to determine soil chemical attributes and enzyme activity. In one of these fields with a large temporal database, soil DNA characterization was also undertaken. The two soil enzymes investigated were affected by a range of soil attributes and the most important of these were identified. Around 40% of the data points sampled had low soil organic matter content; these were associated with low enzyme activity. Furthermore, in HYE there was more biodiversity and a higher presence of plant-growth promoters, while in LYE there were more plant pathogenic organisms.

16S Metagenomics of Nitrifying Bacteria and Archaea Inhabiting Maize Rhizosphere and the Influencing Environmental Factors

The maize rhizosphere soil is unique with diverse microorganisms. Nitrifying bacteria and archaea are ubiquitous and can transform ammonia locked up in soil or manure into nitrate; a more soluble form of nitrogen. However, nitrifying bacteria and archaea inhabiting maize rhizosphere are yet to be identified. We elucidate the diversity and abundance of nitrifying bacteria and archaea associated with maize rhizosphere across different growth stages using 16S metagenomics sequencing. Also, the influence of environmental factors on the nitrifying communities was evaluated. The maize rhizosphere soil was collected from North-West University, Molelwane, South Africa. DNA was extracted using Nucleospin Soil DNA extraction kit and the V3-V4 hypervariable region was sequenced on Illumina Miseq platform. MG-RAST was used to analyze the raw sequences. The environmental factors were measured using standard procedure. The result revealed 9 genera of nitrifying bacteria; Nitrospira, Nitrosospira, Nitrobacter, Nitrosovibrio, Nitrosomonas, Nitrosococcus, Nitrococcus, unclassified (derived from Nitrosomonadales), unclassified (derived from Nitrosomonadaceae) and 1 archaeon Candidatus Nitrososphaera. The Nitrospirae phyla group which had the most nitrifying bacteria was more abundant at the tasselling stage (67.94%). Alpha diversity showed no significant difference. However, the Beta diversity showed significant difference (P=0.01, R=0.58) across the growth stages. The growth stages had no significant effect on the diversity of nitrifying bacteria and archaea, but the tasselling stage had the most abundant. A correlation was observed among some of the environmental factors. The research outcome can be put into consideration while carrying out a biotechnological process that involves nitrifying bacteria and archaea.

Soil fungal community structure and seasonal diversity following application of organic amendments of different quality under maize cropping in Zimbabwe

Recent advocacy for Integrated Soil Fertility Management (ISFM) in smallholder farming systems in east and southern Africa show substantial evidence of increased and sustained crop yields associated with enhanced soil productivity. However, the impact ISFM on soil fungi has received limited attention, yet fungi play key roles in crop growth. Following total soil DNA extraction with ZR soil microbe miniprep kit, illumina sequencing was used to, examine the fungal communities (ITS1F) under a maize crop following co-application of organic nutrient resources including Crotalaria juncea, cattle manure and maize stover with inorganic fertilizers at three-time periods (T1-December, T2-January, and T3-February) in Zimbabwe. Ninety-five fungal species were identified that were assigned to Ascomycota (>90%), Basidiomycota (7%) and Zygomycota (1%). At T1, Ascomycota and Basidiomycota were identified across treatments, with Ascomycota attaining > 93% frequency. Fungal succession was noted and involved reduction of Ascomycota coupled by increase in Basidiomycota under the different treatments. For example at T3, Basidiomycota increased to 34% while Ascomycota declined to 66% under manure but remained unchanged in other two organics. Pre-season mineral nitrogen (N) associated with the ‘Birch effect’ apparently influenced the fungal community structure at T1 while readily available fertilizer N was critical at T2 and T3. The low-quality maize stover promoted the presence of Exophiala sp SST 2011 and this was linked to N immobilization. The impact of N addition was more pronounced under medium (manure) to low-quality (maize stover) resources. Fungi required phosphorus (P) and N for survival while their proliferation was dependent on substrate availability linked to resource quality. Interactive-forward test indicated that soil available P and N were most influential (P < 0.05) factors shaping fungal communities. Co-application of medium to high quality organic and inorganic resources show promise as a sustainable entry point towards enhancing belowground fungal diversity critical in driving nutrient supply.

Changes in Soil Prokaryotic Diversity in Response to Land-Use Changes in Sub-Saharan Africa

Sub-Saharan Africa is one of the most severely affected regions regarding soil degradation, a global issue with the loss of nutrients caused by inappropriate management, leading to low agricultural productivity. Here we asked the question of how soil prokaryotic communities are affected by shifts in land use management and subsequent losses in soil organic carbon. We sampled soils from three sites in Zambia which have neighboring natural and managed sites. After the measurement of soil properties, soil DNA was sequenced, targeting the 16S rRNA gene. As expected, total carbon in soil was decreased in the managed sites, with significant reductions of bacterial biomass. However, the diversity indices in the managed soils were higher than in natural soils. Particularly, the relative abundance of nitrifiers was increased in the managed soils, most likely as a result of fertilization. However also other bacteria, e.g., those which formed tight interactions with the cultivated crops including the genera Balneimonas, and Bacillus, were increased in the managed soils. In contrast bacteria belonging to the family Chloroflexi, which were high in abundance in the natural soil were outcompeted by other prokaryotes in the managed soils most likely as a result of changes in the amount of soil organic carbon. Overall, our results suggest that we need to discuss the trends of prokaryotic diversity separately from those for prokaryotic abundance. Even when bacterial abundances were decreased in the managed soils, nitrifiers’ relative abundance and diversity increased in our experiment, suggesting the possible alteration of the nitrogen cycle in managed soils in sub-Saharan Africa.

Highly comparable metabarcoding results from MGI-Tech and Illumina sequencing platforms

With the developments in DNA nanoball sequencing technologies and the emergence of new platforms, there is an increasing interest in their performance in comparison with the widely used sequencing-by-synthesis methods. Here, we test the consistency of metabarcoding results from DNBSEQ-G400RS (DNA nanoball sequencing platform by MGI-Tech) and NovaSeq 6000 (sequencing-by-synthesis platform by Illumina) platforms using technical replicates of DNA libraries that consist of COI gene amplicons from 120 soil DNA samples. By subjecting raw sequencing data from both platforms to a uniform bioinformatics processing, we found that the proportion of high-quality reads passing through the filtering steps was similar in both datasets. Per-sample operational taxonomic unit (OTU) and amplicon sequence variant (ASV) richness patterns were highly correlated, but sequencing data from DNBSEQ-G400RS harbored a higher number of OTUs. This may be related to the lower dominance of most common OTUs in DNBSEQ data set (thus revealing higher richness by detecting rare taxa) and/or to a lower effective read quality leading to generation of spurious OTUs. However, there was no statistical difference in the ASV and post-clustered ASV richness between platforms, suggesting that additional denoising step in the ASV workflow had effectively removed the ‘noisy’ reads. Both OTU-based and ASV-based composition were strongly correlated between the sequencing platforms, with essentially interchangeable results. Therefore, we conclude that DNBSEQ-G400RS and NovaSeq 6000 are both equally efficient high-throughput sequencing platforms to be utilized in studies aiming to apply the metabarcoding approach, but the main benefit of the former is related to lower sequencing cost.

Effects of Sugarcane and Soybean Intercropping on the Nitrogen-Fixing Bacterial Community in the Rhizosphere

Intercropping between sugarcane and soybean is widely used to increase crop yield and promote the sustainable development of the sugarcane industry. However, our understanding of the soil microenvironment in intercropping systems, especially the effect of crop varieties on rhizosphere soil bacterial communities, remains poor. We selected two excellent sugarcane cultivars, Zhongzhe1 (ZZ1) and Zhongzhe9 (ZZ9), from Guangxi and the local soybean variety GUIZAO2 from Guangxi for field interplanting experiments. These two cultivars of sugarcane have good drought resistance. Rhizosphere soil samples were collected from the two intercropping systems to measure physicochemical properties and soil enzyme activities and to extract total soil DNA for high-throughput sequencing. We found that the diversity of the rhizosphere bacterial community was significantly different between the two intercropping systems. Compared with ZZ1, the ZZ9 intercropping system enriched the nitrogen-fixing bacteria, increasing the available nitrogen content by 18% compared with that with ZZ1. In addition, ZZ9 intercropping with soybean formed a more compact rhizosphere environment than ZZ1, thus providing favorable conditions for sugarcane growth. These results provide guidance for the sugarcane industry, especially for the management of sugarcane and soybean intercropping in Guangxi, China.

Exploring plant diversity through soil DNA in Thai national parks for influencing land reform and agriculture planning

Background The severe deforestation, as indicated in national forest data, is a recurring problem in many areas of Northern Thailand, including Doi Suthep-Pui National Park. Agricultural expansion in these areas, is one of the major drivers of deforestation, having adverse consequences on local plant biodiversity. Conserving biodiversity is mainly dependent on the biological monitoring of species distribution and population sizes. However, the existing conventional approaches for monitoring biodiversity are rather limited. Methods Here, we explored soil DNA at four forest types in Doi Suthep-Pui National Park in Northern Thailand. Three soil samples, composed of different soil cores mixed together, per sampling location were collected. Soil biodiversity was investigated through eDNA metabarcoding analysis using primers targeting the P6 loop of the plastid DNA trnL (UAA) intron. Results The distribution of taxa for each sample was found to be similar between replicates. A strong congruence between the conventional morphology- and eDNA-based data of plant diversity in the studied areas was observed. All species recorded by conventional survey with DNA data deposited in the GenBank were detected through the eDNA analysis. Moreover, traces of crops, such as lettuce, maize, wheat and soybean, which were not expected and were not visually detected in the forest area, were identified. It is noteworthy that neighboring land and areas in the studied National Park were once used for crop cultivation, and even to date there is still agricultural land within a 5–10 km radius from the forest sites where the soil samples were collected. The presence of cultivated area near the forest may suggest that we are now facing agricultural intensification leading to deforestation. Land reform for agriculture usage necessitates coordinated planning in order to preserve the forest area. In that context, the eDNA-based data would be useful for influencing policies and management towards this goal.