The functional identification and evaluation of endophytic bacteria sourced from the roots of tolerant Achyranthes bidentata to overcome monoculture problems of Rehmannia glutinosa.

The isolation and identification of plant growth-promoting endophytic bacteria (PGPEB) from Achyranthes bidentata roots have profound theoretical and practical implications in ecological agriculture, particularly as bio-inoculants to address challenges associated with continuous monoculture. Our research revealed a significant increase in the abundance of these beneficial bacteria in A. bidentata rhizosphere soil under prolonged monoculture conditions, as shown by bioinformatics analysis. Subsequently, we isolated 563 strains of endophytic bacteria from A. bidentata roots. Functional characterization highlighted diverse plant growth-promoting traits among these bacteria, including the secretion of indole-3-acetic acid (IAA) ranging from 68.01 to 73.25 mg/L, phosphorus and potassium solubilization capacities, and antagonistic activity against pathogenic fungi (21.54%-50.81%). Through 16S rDNA sequencing, we identified nine strains exhibiting biocontrol and growth-promoting potential. Introduction of a synthetic microbial consortium (SMC) in pot experiments significantly increased root biomass by 48.19% in A. bidentata and 27.01% in replanted Rehmannia glutinosa. These findings provide innovative insights and strategies for addressing continuous cropping challenges, highlighting the practical promise of PGPEB from A. bidentata in ecological agriculture to overcome replanting obstacles for non-host plants like R. glutinosa, thereby promoting robust growth in medicinal plants.

Glutathione S-transferase activity facilitates rice tolerance to the barnyard grass root exudate DIMBOA.

BACKGROUND: In paddy fields, the noxious weed barnyard grass secretes 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) to interfere with rice growth. Rice is unable to synthesize DIMBOA. Rice cultivars with high or low levels of allelopathy may respond differently to DIMBOA. RESULTS: In this study, we found that low concentrations of DIMBOA (≤ 0.06 mM) promoted seedling growth in allelopathic rice PI312777, while DIMBOA (≤ 0.08 mM) had no significant influence on the nonallelopathic rice Lemont. DIMBOA treatment caused changes in the expression of a large number of glutathione S-transferase (GST) proteins, which resulting in enrichment of the glutathione metabolic pathway. This pathway facilitates plant detoxification of heterologous substances. The basal levels of GST activity in Lemont were significantly higher than those in PI312777, while GST activity in PI312777 was slightly induced by increasing DIMBOA concentrations. Overexpression of GST genes (Os09g0367700 and Os01g0949800) in these two cultivars enhanced rice resistance to DIMBOA. CONCLUSIONS: Taken together, our results indicated that different rice accessions with different levels of allelopathy have variable tolerance to DIMBOA. Lemont had higher GST activity, which helped it tolerate DIMBOA, while PI312777 had lower GST activity that was more inducible. The enhancement of GST expression facilitates rice tolerance to DIMBOA toxins from barnyard grass root exudates.

Intercropping with Achyranthes bidentata alleviates Rehmannia glutinosa consecutive monoculture problem by reestablishing rhizosphere microenvironment.

Background: The consecutive monoculture of Rehmannia glutinosa leads to a serious decrease in its production and quality. Previous studies have demonstrated that intercropping altered species diversity and rhizosphere microbial diversity. However, it remained unknown whether the impaired growth of monocultured plants could be restored by enhanced belowground interspecific interactions. Method: In the present research, a continuous cropping facilitator Achyranthes bidentata was intercropped with R. glutinosa under pot conditions, and three different types of root barrier treatments were set, including that complete belowground interaction (N), partial belowground interaction (S), and no belowground interspecies interaction (M), with the aims to investigate belowground interaction and the underlying mechanism of alleviated replanting disease of R. glutinosa by intercropping with A. bidentata. Results: The results showed that the land equivalent ratio (LER) of the two years was 1.17, and the system productivity index (SPI) increased by 16.92 % under S treatment, whereas no significant difference was found in N and M regimes. In the rhizosphere soil, intercropping systems had significantly increased the contents of sugars and malic acid in the soil of R. glutinosa, together with the content of organic matter and the invertase and urease activities. Meanwhile, intercropping increased the community diversity of fungi and bacteria, and the relative abundance of potential beneficial bacteria, such as Bacillus, Nitrospira, and Sphingomonas, despite the pathogenic Fusarium oxysporum was still the dominant genus in the rhizospheric soil of R. glutinosa under various treatments. The results of antagonism experiments and exogenous addition of specific bacteria showed that Bacillus spp. isolated from rhizosphere soil had a significant antagonistic effect on the pathogen of R. glutinosa. Conlusion: Taken together, our study indicated that the R. glutinosa//A. bidentata intercropping systems alleviate the consecutive monoculture problem of R. glutinosa by recruiting beneficial bacteria. The studies we have conducted have a positive effect on sustainable agricultural development.

Spatial-Temporal Distribution of Allelopathic Rice Roots in Paddy Soil and Its Impact on Weed-Suppressive Activity at the Seedling Stages.

Background: Allelochemicals secreted by allelopathic rice roots are transmitted to the receptor rhizosphere through the soil medium to inhibit the growth of the surrounding weeds. This research aimed to explore the relationships between the spatial-temporal distribution of rice roots in soil and weed-suppression ability at its seedling stage. Results: This study first examined the root distribution of three rice cultivars in paddy soil in both vertical and horizontal directions at 3-6 leaf stage. Then, an experiment using rice-barnyardgrass mixed culture was conducted to analyze the allelopathic potential and allelochemical content secreted by rice roots in different lateral soil layers. The results showed that allelopathic rice had a smaller root diameter and larger root length density, root surface area density, and root dry weight density than those of non-allelopathic rice, in the top 5 cm at 5- and 6-leaf stages. In particular, there were significant differences in root distribution at the horizontal distance of 6-12 cm. Besides, allelopathic rice significantly inhibited the above-ground growth of barnyardgrass co-cultured at 12 cm lateral distance in situ, and the content of benzoic acid derivatives in allelopathic rice in a 6-12 cm soil circle was higher than that observed at 0-6 cm distance. Moreover, correlation analysis confirmed that the distribution of roots in the horizontal distance was significantly correlated with weed inhibition effect and allelochemical content. Conclusion: These results implied that spatial distribution of allelopathic rice roots in paddy soil, particularly at the lateral distance, appears to have important impact on its weed-suppressive activity at the seedling stage, suggesting that modifying root distribution in soil may be a novel method to strengthen the ability of rice seedlings to resist paddy weeds.

Deciphering the Molecular Mechanisms of Chilling Tolerance in Lsi1-Overexpressing Rice.

Improving tolerance to low-temperature stress during the rice seedling stage is of great significance in agricultural science. In this study, using the low silicon gene 1 (Lsi1)-overexpressing (Dular-OE) and wild-type rice (Dular-WT), we showed that Lsi1 overexpression enhances chilling tolerance in Dular-OE. The overexpression of the Lsi1 increases silicon absorption, but it was not the main reason for chilling tolerance in Dular-OE. Instead, our data suggest that the overexpression of a Lsi1-encoding NIP and its interaction with key proteins lead to chilling tolerance in Dular-OE. Additionally, we show that the high-mobility group protein (HMG1) binds to the promoter of Lsi1, positively regulating its expression. Moreover, Nod26-like major intrinsic protein (NIP)'s interaction with α and ß subunits of ATP synthase and the 14-3-3f protein was validated by co-immunoprecipitation (Co-IP), bimolecular fluorescent complementary (BiFC), and GST-pulldown assays. Western blotting revealed that the overexpression of NIP positively regulates the ATP-synthase subunits that subsequently upregulate calcineurin B-like interacting protein kinases (CIPK) negatively regulating 14-3-3f. Overall, these NIP-mediated changes trigger corresponding pathways in an orderly manner, enhancing chilling tolerance in Dular-OE.

Comparison of Silicon-Evoked Responses on Arsenic Stress between Different Dular Rice Genotypes.

Arsenic is one of the most hazardous metalloids in nature, and due to its high water solubility, it is one of the most important causes of pollution. However, silicon reduces the uptake and transport of arsenic in rice. This study investigates the interaction of different arsenic and silicon levels on dry weight, protein content, and concentrations of arsenic and silicon in two different rice shoots and roots of Dular wild-type (DU-WT) and Dular Lsi1-overexpressed (DU-OE) rice. It should be noted that all seedlings were subjected to four different treatments. For RNA-seq and qPCR, the DU-WT genotype was selected as the control and DU-OE as the treatment. With the addition of silicone treatment, dry weight and protein content in the shoots and roots of both rice lines were increased, while the concentration of arsenic in these two organs was decreased. When seedlings were exposed to arsenic treatments, protein content, silicon concentration, and dry weight were decreased in both roots and shoots, while arsenic concentration was increased in both rice genotypes. The RNA-seq in DU-OE showed 5823 differentially expressed genes (DEGs), of which 2604 were up-regulated and 3219 down-regulated. Treatment of rice by arsenic and silicon has changed the expression of genes encoding cytokinin-responsive GATA transcription factor 1, protein IN2-1 homolog B, calcium-binding EGF domain-containing protein, Os01g0369700 protein, probable glutathione S-transferase GSTU1, glutathione S-transferase protein, Os09g0367700 protein, isocitrate dehydrogenase (NADP), and Os08g0522400 protein in the root of DU-OE. The present study's findings showed that in the presence of silicon, the transgenic genotype is much more resistant to arsenic than the wild genotype of Dular rice.

Serine hydroxymethyltransferase localised in the endoplasmic reticulum plays a role in scavenging H2O2 to enhance rice chilling tolerance.

BACKGROUND: Rice is a chilling-sensitive crop that would suffer serious damage from low temperatures. Overexpression of the Lsi1 gene (Lsi1-OX) in rice enhances its chilling tolerance. This study revealed that a serine hydroxymethyltransferase (OsSHMT) mainly localised in the endoplasmic reticulum (ER) is involved in increasing tolerance to chilling. RESULTS: A higher transcription level of OsSHMT was detected in Lsi1-OX rice than in the wild type. Histone H1 and nucleic acid binding protein were found to bind to the promoter region of OsSHMT and regulate its expression, and the transcription levels of these proteins were also up-regulated in the Lsi1-OX rice. Moreover, OsSHMT interacts with ATP synthase subunit α, heat shock protein Hsp70, mitochondrial substrate carrier family protein, ascorbate peroxidase 1 and ATP synthase subunit ß. Lsi1-encoded protein OsNIP2;1 also interacts with ATP synthase subunit ß, and the coordination of these proteins appears to function in reducing reactive oxygen species, as the H2O2 content of transgenic OsSHMT Arabidopsis thaliana was lower than that of the non-transgenic line under chilling treatment. CONCLUSIONS: Our results indicate that ER-localised OsSHMT plays a role in scavenging H2O2 to enhance the chilling tolerance of Lsi1-OX rice and that ATP synthase subunit ß is an intermediate junction between OsNIP2;1 and OsSHMT.

MYB57 transcriptionally regulates MAPK11 to interact with PAL2;3 and modulate rice allelopathy.

Rice allelopathy is a natural method of weed control that is regarded as an eco-friendly practice in agroecology. The allelopathic potential of rice is regulated by various genes, including those that encode transcription factors. Our study characterized a MYB transcription factor, OsMYB57, to explore its role in the regulation of rice allelopathy. Increasing the expression of OsMYB57 in rice using the transcription activator VP64 resulted in increased inhibitory ratios against barnyardgrass. The gene expression levels of OsPAL, OsC4H, OsOMT, and OsCAD from the phenylpropanoid pathway were also up-regulated, and the content of l-phenylalanine increased. Chromatin immunoprecipitation incorporated with HiSeq demonstrated that OsMYB57 transcriptionally regulated a mitogen-activated protein kinase (OsMAPK11); in addition, OsMAPK11 interacted with OsPAL2;3. The expression of OsPAL2;3was higher in the allelopathic rice PI312777 than in the non-allelopathic rice Lemont, and OsPAL2;3 was negatively regulated by Whirly transcription factors. Moreover, microbes with weed-suppression potential, including Penicillium spp. and Bacillus spp., were assembled in the rhizosphere of the rice accession Kitaake with increased expression of OsMYB57, and were responsible for phenolic acid induction. Our findings suggest that OsMYB57 positively regulates rice allelopathy, providing an option for the improvement of rice allelopathic traits through genetic modification.

Transcriptome analysis reveals that barnyard grass exudates increase the allelopathic potential of allelopathic and non-allelopathic rice (Oryza sativa) accessions.

BACKGROUND: Allelopathy in rice (Oryza sativa) is a chemically induced response that is elevated by the exogenous application of chemical compounds and barnyard grass root exudates. An in-depth understanding of the response mechanisms of rice to chemical induction is necessary for the identification of target genes for increasing the allelopathic potential of rice. However, no previous studies have evaluated the transcriptomic changes associated with allelopathy in rice in response to barnyard grass exudates treatment. Thus, the aim of the present study was to reveal differentially expressed genes (DEGs) in allelopathic and non-allelopathic rice seedlings treated with barnyard grass exudates to identify target allelopathy genes. RESULTS: The inhibitory effect of the culture solutions on the allelopathic rice accession PI312777 (PI) and the non-allelopathic rice accession Lemont (LE) significantly increased (P < 0.05) after treatment with barnyard grass root exudates. The RNA sequencing results revealed that 14,891 genes in PI(+B) vs. LE(+B), 12,505 genes in PI(+B) vs. PI(-B), and 5857 genes in LE(+B) vs. LE(-B) were differentially expressed following root exudates treatment. These DEGs were classified into three categories and 32 functional groups, i.e., 12 groups in the biological process category, 12 groups in the cellular component category, and eight groups in the molecular function category. There were 5857 and 2846 upregulated genes and 135 and 50 upregulated Gene Ontology terms (P < 0.05) in the biological process category in PI(+B) vs. PI(-B) and LE(+B) vs. LE(-B), respectively. These results indicated that the allelopathic accession PI is more sensitive than the non-allelopathic accession LE to exogenous root exudates treatment. Genes related to rice allelochemical-related biosynthesis pathways, particularly the shikimic acid and acetic acid pathways, were significantly differentially expressed in both rice accessions. These findings suggested that phenolic acids, fatty acids, and flavonoids, which constitute the downstream metabolites of the shikimic acid and acetic acid pathways, are significantly expressed in response to root exudates of barnyard grass. CONCLUSIONS: The allelopathic potential of both rice accessions could be significantly enhanced by barnyard grass root exudates application. Furthermore, genes related to the biosynthesis pathways of reported rice allelochemicals were significantly differentially expressed in both accessions. Phenylalanine ammonia lyase was determined to be a potential target for the regulation of chemical induction.