Application Note

pdfRural landscapes degenerated by human activities pose restoration challenges as well as opportunities to study the stability and resilience of ecosystems. Soil nutrients and microbial communities are the two key factors in revegetation of barren environments. Ecological stoichiometry plays an important role in ecosystem function and limitation, but the relationships between above- and belowground stoichiometry and the bacterial communities in a specific region may be poorly understood.

Researchers at Nanjing Agricultural University explored the use of pepino (Solanum muricatum) planting for restoration of the karst landscape of southern China. They planted pepino in 2 of 3 selected quadrats in their study area and after 4 months, collected bulk soil samples from depth of 0~10 cm from each selected quadrats as well as samples directly adhered to the roots of the pepino (rhizosphere soil).

The researchers examined the stoichiometric traits between soil and foliage and found the soil had a relatively high pH, low fertility, and coarse texture. Foliar N:P ratio and the correlations with soil nitrogen and phosphorus suggested nitrogen limitation and the planting of pepino increased soil urease activity and decreased catalase activity.

Next they extracted DNA directly from the soil samples and used 16S microbial sequencing to determine diversity and abundance of bacteria in the karst soil. Primers targeting the regions (V3-V4) of the 16S rRNA gene were used for PCR because sequences in that regions provided the greatest diversity at the domain and bacteria phylum levels.


Compared with S1 (not planted), more than 1,000 additional OTUs
were observed in bulk soils S2 and S3 (pepino planted quadrats)

LC Sciences


(a) Rarefaction curves showing the observed OTU richness (97% identity) of the 16S rRNA gene with increasing sequencing depth. (bf) Percentage of different bacteria phyla in each soil sample. Data are means ± standard error (n = 3). One-way ANOVA followed by Duncan test (p = 0.05) was done for each bacterial phylum separately. Sequences that could not be classified into any known groups were labeled “Others”.

Higher diversity of bacteria was determined in the pepino rhizosphere than bulk soil. Proteobacteria, Acidobacteria, Actinobacteria and Bacteroidetes were the dominant phyla in all samples, accounting for more than 80% of the reads. On a genus level, all 625 detected genera were found in all rhizosphere and bulk soils, and 63 genera showed significant differences among samples.

The rhizosphere soil in quadrat T2 (RS2) had the higher Shannon and Chao 1 indices than the corresponding bulk soil (S2), indicating that the growth of pepino increased diversity of soil bacterial communities (RS2 had the highest Shannon and Chao 1 indices of all soils)

Comparison of the estimated operational taxonomic unit (OTU) richness and the diversity indices of the 16S rRNA gene libraries for clustering at 97% identity as obtained from the pyrosequecing analysis.


Observed OTUsShannon indexChao 1 indexSimpson’s diversity index (10−2)

Means (n = 3). Different letters in a single column indicate significant (p ≤ 0.05) differences among the soils.


Similar results were obtained in comparison between the bulk and rhizosphere soils in T3 (agricultural quadrat), albeit to a slightly lesser degree than in natural T2 soil. The Simpson index was reverse of diversity (the lower the index, the greater biodiversity), and the lowest Simpson index in RS2 confirmed the results derived from the Shannon and Chao 1 indices.

This study increased our understanding of the diversity and structure of bacterial communities in karst soil and the important changes caused by pepino planting.

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