Interacting effect of diclofop-methyl on the rice rhizosphere microbiome and denitrification

Highlights

• The rhizosphere microbiome acts as a highly evolved external functional milieu for plants.

• The interactions between rhizosphere microbiota, exudates and plant stress remain poorly understood.

• Herbicide (diclofop-methyl) affected rice root exudates.

• The richness and diversity of rhizosphere microorganisms were significantly affected by root exudates.

• The residual diclofop-methyl affected the rice root microbiome community and promoted the potential soil denitrification.

Abstract

A better knowledge of the intertwined effects of herbicides on plant physiology and microbiome as well as nutrient biogeochemical cycles are needed for environmental management. Here we studied the influence of herbicide diclofop-methyl (DM) on the rice root microbiome and its relationship with N cycle. To do so, we exposed rice seedlings to 100 μg/L DM and studied rhizosphere microbiota using MiSeq-pyrosequencing, root exudation by GC–MS, and denitrification activity by ¹⁵N isotope-tracing and qRT-PCR. The richness and diversity of rhizosphere microorganisms, significantly increased after DM exposure combined with an increase in root exudation of amino acids, sugars, and fatty acids. Transcription of denitrification-related gene and denitrification rate increased significantly in the rice rhizosphere. Our results suggest that DM strongly influenced the root exudation of bacteria nutrients, which affected root microbiome community and potentially influenced N cycle in rice rhizosphere.

Introduction

The rhizosphere microbiome acts as a highly evolved external functional milieu for plants. Root microbiota plays critical role in plant growth [1,2], development (e.g. flowering time) [3], and pathogen resistance [4]. Plant-microbiota interactions in the rhizosphere also drive global biogeochemical cycles of N as they affect biological N utilization, as well as N loss [5,6] and anaerobic ammonium oxidation [6]. It has been shown that the community structure, abundance and function of root microbiota are affected by several biotic factors (plant phenotype and development stages) and abiotic factors (N limitation, anoxia, nanoparticles, pesticides) [[7], [8], [9], [10]]. Previous studies have shown that pesticides could also influence soil N cycling. For instance, Tenuta et al. found that the application of glyphosate in a grass sward increase the denitrification rate by 20- to 30-fold after a 14- and 49-d application compared to herbicide-untreated grass sward, which might result in greater nitrous oxide emission and N loss from soil [11]. It was also reported that diclofop-methyl (DM) decreased nitrification of urea nitrogen in soils [12]. Therefore, more studies in this area are critically needed in view of the importance of microbiota for plant growth and the potential negative side-effect of pesticides on the plant microbiome of target and non-target plants [13].

In Asian subtropical regions, Oryza sativa (rice) is one of the most important staple food and crop rotation is frequently used to increase the land utilization rate and improve soil fertility. Therefore, paddy soils used for farming other crops, e.g., soybean, oilseed rape and wheat and barley, which are heavily sprayed by DM, can in turn contaminate rice cultures [14]. DM, which is widely used to control graminaceous weeds, can inhibit acetyl-CoA carboxylase activity and decrease fatty acid synthesis in rice [14]. Moreover, residual DM in soil can affect a wide range of metabolic pathways and increase organic acid exudation in rice [[15], [16], [17]]. Since plant exudates (e.g., sugars, organic acids and amino acids) are the key drivers of the rhizosphere microbiome composition, the reported DM effect on root exudation raises the possibility that complex plant-microbiota interactions could modulate DM toxicity in rice as well as the abundance of specialized microorganisms affecting the biogeochemical cycles of nutrients and plant growth [18]. Although previous studies have shown that N cycling in soils could be affected by pesticide like glyphosate or DM [11,12], the interactions between rhizosphere microbiota, plant exudates and the presence of these pesticides remain largely enigmatic.

To better understand the interactions between soil N cycle and residual DM in rice cultures, we studied the effect of DM exposure on root exudation, rhizosphere microbiome and potential N loss in laboratory cultures of rice. The effect of DM on the rice rhizosphere and soil microbial community was examined using Illumina sequencing. The composition and concentrations of root exudates were also studied by GC–MS. Moreover, we evaluated the effects of DM exposure and soil microbial community on potential soil denitrification (and fertilizer loss) using qRT-PCR (transcription of a key denitrification-related gene) and ¹⁵N isotope-tracing by mass spectrometry.