Colonization Of Wheat Roots Research Articles

Flg22-facilitated PGPR colonization in root tips and control of root rot.

Plant root border cells (RBCs) prevent the colonization of plant growth-promoting rhizobacteria (PGPR) at the root tip, rendering the PGPR unable to effectively control pathogens infecting the root tip. In this study, we engineered four strains of Pseudomonas sp. UW4, a typical PGPR strain, each carrying an enhanced green fluorescent protein (EGFP)-expressing plasmid. The UW4E strain harboured only the plasmid, whereas the UW4E-flg22 strain expressed a secreted EGFP-Flg22 fusion protein, the UW4E-Flg(flg22) strain expressed a non-secreted Flg22, and the UW4E-flg22-D strain expressed a secreted Flg22-DNase fusion protein. UW4E-flg22 and UW4E-flg22-D, which secreted Flg22, induced an immune response in wheat RBCs and colonized wheat root tips, whereas the other strains, which did not secrete Flg22, failed to elicit this response and did not colonize wheat root tips. The immune response revealed that wheat RBCs synthesized mucilage, extracellular DNA, and reactive oxygen species. Furthermore, the Flg22-secreting strains showed a 33.8%-93.8% higher colonization of wheat root tips and reduced the root rot incidence caused by Rhizoctonia solani and Fusarium pseudograminearum by 24.6%-35.7% compared to the non-Flg22-secreting strains in pot trials. There was a negative correlation between the incidence of wheat root rot and colonization of wheat root tips by these strains. In contrast, wheat root length and dry weight were positively correlated with the colonization of wheat root tips by these strains. These results demonstrate that engineered secretion of Flg22 by PGPR is an effective strategy for controlling root rot and improving plant growth.

The potential of Pseudomonas fluorescens SBW25 to produce viscosin enhances wheat root colonization and shapes root-associated microbial communities in a plant genotype-dependent manner in soil systems.

Understanding parameters governing microbiome assembly on plant roots is critical for successfully exploiting beneficial plant-microbe interactions for improved plant growth under low-input conditions. While it is well-known from in vitro studies that specialized metabolites are important for plant-microbe interactions, e.g., root colonization, studies on the ecological role under natural soil conditions are limited. This might explain the often-low translational power from laboratory testing to field performance of microbial inoculants. Here, we showed that viscosin synthesis potential results in a differential impact on the microbiome assembly dependent on wheat cultivar, unlinked to colonization potential. Overall, our study provides novel insights into factors governing microbial assembly on plant roots, and how this has a derived but differential effect on the bacterial and protist communities.

A potent endophytic fungus Purpureocillium lilacinum YZ1 protects against Fusarium infection in field-grown wheat.

Fusarium diseases pose a severe global threat to major cereal crops, particularly wheat. Existing biocontrol strains against Fusarium diseases are believed to primarily rely on antagonistic mechanisms, but not widely used under field conditions. Here, we report an endophytic fungus, Purpureocillium lilacinum YZ1, that shows promise in combating wheat Fusarium diseases. Under glasshouse conditions, YZ1 inoculation increased the survival rate of Fusarium graminearum (Fg)-infected wheat seedlings from 0% to > 60% at the seedling stage, and reduced spikelet infections by 70.8% during anthesis. In field trials, the application of YZ1 resulted in an impressive 89.0% reduction in Fg-susceptible spikelets. While a slight antagonistic effect of YZ1 against Fg was observed on plates, the induction of wheat systemic resistance by YZ1, which is distantly effective, non-specific, and long-lasting, appeared to be a key contributor to YZ1's biocontrol capabilities. Utilizing three imaging methods, we confirmed YZ1 as a potent endophyte capable of rapid colonization of wheat roots, and systematically spreading to the stem and leaves. Integrating dual RNA-Seq, photosynthesis measurements and cell wall visualization supported the link between YZ1's growth-promoting abilities and the activation of wheat systemic resistance. In conclusion, endophytes such as YZ1, which exhibits non-antagonistic mechanisms, hold significant potential for industrial-scale biocontrol applications.

Orfamide production in Pseudomonas protegens CHA0T promotes rhizospheric colonization and influences assemblage of the bacterial community of wheat roots in soil.

Orfamides (Ofa) are a family of closely related cyclic lipopeptides (cLPs) produced by different soil-inhabiting and plant beneficial strains of the Pseudomonas protegens subgroup. Orfamides are required for swarming motility, they mediate toxicity on oomycetes, algae, and insects, and they can act as ISR elicitors. Ofa biosynthesis depends on a non-ribosomal peptide synthetase gene cluster whose expression is tightly controlled by the post-transcriptional regulatory cascade Gac-Rsm. Although the biological properties and physiological role of Ofa have been well characterized in vitro, the role of Ofa in the fitness of the producing bacterium in its niche has not been studied so far. Here, we used an Ofa biosynthesis-deficient mutant (ΔofaABC) derived from the biocontrol model strain P. protegens CHA0T to explore the relevance of this cLP in soil for the survival of strain CHA0T, its contribution to the bacterial competitiveness for colonization of wheat roots, and its impact on the corresponding rhizobacterial community structure. To facilitate CFU counts in non-sterile soil and root samples we used chromosomally tagged versions of CHA0T and its ΔofaABC mutant with mini-Tn7 constructs carrying antibiotic resistance markers. We confirmed that the tagged Ofa− mutant does not show drop-collapse activity neither it can swarm in semi-solid medium. We also confirmed that Ofa can be exploited in vitro as a social good by the Ofa− mutant to resume swarming in the presence of CHA0T cells. We found that Ofa production did not confer any advantage for bacterial survival along a period of 5 weeks after soil inoculation. However, we observed that Ofa production was associated with a higher competitiveness for colonization of the wheat rhizosphere. Expression of the ofa biosynthetic operon, by means of a transcriptional Pofa-gfp reporter fusion, was detected in the rhizoplane of colonized wheat roots, suggesting that Ofa is produced in situ on the surface of colonized wheat roots. Finally, we observed that seed inoculation with the wild type Ofa producer strain CHA0T resulted in a differential assemblage of the bacterial community of the wheat rhizosphere with respect to that of seeds bacterized with the Ofa deficient mutant. Overall, we provide evidence that Ofa is important for the competitiveness of P. protegens CHA0T cells to colonize the surface of wheat roots and that Ofa production in the rhizosphere can impact the structure of the assembled bacterial microbiome.

Mycorrhizal effects on crop yield and soil ecosystem functions in a long-term tillage and fertilization experiment.

It is well understood that agricultural management influences arbuscular mycorrhizal (AM) fungi, but there is controversy about whether farmers should manage for AM symbiosis. We assessed AM fungal communities colonizing wheat roots for three consecutive years in a long-term (> 14 yr) tillage and fertilization experiment. Relationships among mycorrhizas, crop performance, and soil ecosystem functions were quantified. Tillage, fertilizers and continuous monoculture all reduced AM fungal richness and shifted community composition toward dominance of a few ruderal taxa. Rhizophagus and Dominikia were depressed by tillage and/or fertilization, and their abundances as well as AM fungal richness correlated positively with soil aggregate stability and nutrient cycling functions across all or no-tilled samples. In the field, wheat yield was unrelated to AM fungal abundance and correlated negatively with AM fungal richness. In a complementary glasshouse study, wheat biomass was enhanced by soil inoculum from unfertilized, no-till plots while neutral to depressed growth was observed in wheat inoculated with soils from fertilized and conventionally tilled plots. This study demonstrates contrasting impacts of low-input and conventional agricultural practices on AM symbiosis and highlights the importance of considering both crop yield and soil ecosystem functions when managing mycorrhizas for more sustainable agroecosystems.

Increasing diversity gradient in lentil mixtures for intercropping with wheat reveals an increase in lentil biomass with minimal impact on mycorrhizal activity

The management of arbuscular mycorrhizal fungi (AMF) is crucial for sustainable agricultural systems, given its beneficial impacts on soil health and crop productivity. This study aimed to assess the effect of lentil (Lens culinaris Medik.) mixtures for intercropping with durum wheat (Triticum durum Desf.) on mycorrhiza and investigate the role of legume intraspecific diversity in mycorrhizal development and crop biomass production. Since both intraspecific and interspecific diversity (i.e., cultivar mixtures and intercropping) have an effect in shaping soil microbiota in the rhizosphere, we hypothesised that increasing the level of diversification in the system enhances the provision of services (e.g., aboveground biomass accumulation), due to enhanced microbial activity and diversity. Specifically, increasing diversity within lentil cultivar mixtures strengthens the affinity of wheat for mycorrhiza, thereby influencing wheat mycorrhization in wheat-lentil intercrops. To test our hypothesis, we designed three lentil cultivar mixtures with increasing diversity levels using a trait-based approach, drawing from a pool of Italian lentil landraces characterised in a previous study. As an additional factor, we intercropped lentil mixtures with wheat and compared it with sole wheat and sole lentil commercial cultivar. In both experimental years, intercropping lentil mixtures with wheat consistently yielded higher soil mycorrhization levels (46% in 2021 and 52% in 2022) compared to sole wheat cultivation (23% in 2021 and 34 % in 2022). Nevertheless, AMF colonisation in lentil roots was significantly higher in sole crops (55% in 2021 and 70% in 2022, except for the commercial cultivar) compared to intercrops (35% in 2021 and 60% in 2022). This difference could be attributed to the presence of wheat, which might have suppressed the capacity of lentils to establish successful symbiosis with mycorrhiza. On the other hand, AMF colonisation of wheat roots in 2022 was significantly higher (52%) when intercropped with lentils than when cultivated as sole wheat (40%). Concerning biomass production in lentil mixtures in 2022, we observed a positive correlation between lentil biomass and the number of functional groups in the mixtures, both as pure stands (R2 = 0.19, p = 0.06) and intercrops (R2 = 0.29, p = 0.01). Additionally, in 2022, the total biomass of the intercropped system increased with the increase in diversity. Further experiments, incorporating microbial genetic analysis, are necessary to uncover potential associations between functional mixtures and AMF composition.

Function of the endophytic fungus Acrophialophora jodhpurensis, methionine, and nitric oxide in wheat resistance induction against Fusarium graminearum via interplay of reactive oxygen species and iron

In this study, the effect of Acrophialophora jodhpurensis, methionine, and sodium nitroprusside (as a NO donor) in induction of resistance against Fusarium graminearum, as a hemibiotrophic phytopathogenic fungus causing wheat root and crown rot, was investigated in vivo. Also, the effect of A. jodhpurensis was investigated on the mycelial growth, spore germination and hyphal structures of F. graminearum in vitro. The obtained results showed that A. jodhpurensis reduced mycelial growth and spore germination of the pathogen. Also, the spores and hyphae of the pathogen were swollen and deformed in the presence of A. jodhpurensis. Wheat root colonization by A. jodhpurensis and foliar application of methionine and sodium nitroprusside reduced the disease severity of F. graminearum and increased the plant growth parameters, such as shoot and root weight compared to the controls. Application of A. jodhpurensis, methionine and sodium nitroprusside had a significant effect in induction of resistance against F. graminearum via increasing superoxide (O2⋅-), hydrogen peroxide (H2O2), iron ions, phenolic contents, induction of antioxidant enzymes such as catalase (CAT), guaiacol peroxidase (GPX), ascorbate peroxidase (APX) and superoxide dismutase (SOD) activities, membrane stability index (MSI) and relative water content (RWC) compared to the plants only inoculated with F. graminearum and uninoculated controls. Simultaneous application of A. jodhpurensis and sodium nitroprusside was more effective than simultaneous application of A. jodhpurensis and methionine. Also, simultaneous application of A. jodhpurensis, methionine and sodium nitroprusside treatments showed more effect on the plant growth parameters and resistance induction against the pathogen compared to the other treatments. Therefore, the combined A. jodhpurensis, methionine and sodium nitroprusside treatment can be used for increasing the plant growth parameters and protecting wheat plants against F. graminearum.

Azospirillum brasilense and Zinc Rates Effect on Fungal Root Colonization and Yield of Wheat-Maize in Tropical Savannah Conditions.

A successful microbial inoculant can increase root colonization and establish a positive interaction with native microorganisms to promote growth and productivity of cereal crops. Zinc (Zn) is an intensively reported deficient nutrient for maize and wheat production in Brazilian Cerrado. It can be sustainably managed by inoculation with plant growth-promoting bacteria and their symbiotic association with other microorganisms such as arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSE). The objective of this study was to evaluate the effect of Azospirillum brasilense inoculation and residual Zn rates on root colonization and grain yield of maize and wheat in succession under the tropical conditions of Brazil. These experiments were conducted in a randomized block design with four replications and arranged in a 5 × 2 factorial scheme. The treatments consisted of five Zn rates (0, 2, 4, 6 and 8 kg ha-1) applied from zinc sulfate in maize and residual on wheat and without and with seed inoculation of A. brasilense. The results indicated that root colonization by AMF and DSE in maize-wheat cropping system were significantly increased with interaction of Zn rates and inoculation treatments. Inoculation with A. brasilense at residual Zn rates of 4 kg ha-1 increased root colonization by AMF under maize cultivation. Similarly, inoculation with A. brasilense at residual Zn rates of 2 and 4 kg ha-1 reduced root colonization by DSE under wheat in succession. The leaf chlorophyll index and leaf Zn concentration were increased with inoculation of the A. brasilense and residual Zn rates. The inoculation did not influence AMF spore production and CO2-C in both crops. The grain yield and yield components of maize-wheat were increased with the inoculation of A. brasilense under residual Zn rates of 3 to 4 kg ha-1 in tropical savannah conditions. Inoculation with A. brasilense under residual Zn rates up to 4 kg ha-1 promoted root colonization by AMF and DSE in the maize cropping season. While the inoculation with A. brasilense under 2 and 4 kg ha-1 residual Zn rates reduced root colonization by AMF and DSE in the wheat cropping season. Therefore, inoculation with A. brasilense in combination with Zn fertilization could consider a sustainable approach to increase the yield and performance of the maize-wheat cropping system in the tropical savannah conditions of Brazil.

Bacterial secondary metabolites: Possible mechanism for weed suppression in wheat.

Chemical weed control is an effective method, but has proved hazardous for humans, environment, and soil biodiversity. Use of allelopathic bacteria (AB), may be more efficient and sustainable weed control measure. The bacterial inoculants never studied in context of their interaction with weed root exudates and precursor-dependent production of the natural phytotoxins (cyanide, cytolytic enzymes and auxin) by these stains to understand their weed suppression and wheat growth promotion abilities. Therefore, root exudates of Avena fatua, Phalaris minor, Rumex dentatus and wheat were quantified and their role in microbial root colonization and secondary metabolites production i.e. cyanide, cytolitic enzymes, phenolics and elevated auxin concentration was studied. The results depicted L-tryptophan and glycine as major contributor of elevated cyanide and elevated levels in weed rhizosphere by the studied Pseudomonas strains, through their higher root colonization ability in weeds as compared to wheat. Furthermore, the higher root colonization also enhanced p-coumaric acid (photosynthesis inhibitor by impairing cytochrome c oxidase activity in plants), and cytolytic enzymes (root cell wall degradation) concentration in weed rhizosphere. In conclusion, the differential root colonization of wheat and weeds by these strains is responsible for enhancing weed suppression (enhancing phytotoxic effect) and wheat growth promotion (lowering phytotoxic effect).

Rhizobium leguminosarum symbiovar viciae strains are natural wheat endophytes that can stimulate root development.

Although rhizobia that establish a nitrogen-fixing symbiosis with legumes are also known to promote growth in non-legumes, studies on rhizobial associations with wheat roots are scarce. We searched for Rhizobium leguminosarum symbiovar viciae (Rlv) strains naturally competent to endophytically colonize wheat roots. We isolated 20 strains from surface-sterilized wheat roots and found a low diversity of Rlv compared to that observed in the Rlv species complex. We tested the ability of a subset of these Rlv for wheat root colonization when co-inoculated with other Rlv. Only a few strains, including those isolated from wheat roots, and one strain isolated from pea nodules, were efficient in colonizing roots in co-inoculation conditions, while all the strains tested in single strain inoculation conditions were found to colonize the surface and interior of roots. Furthermore, Rlv strains isolated from wheat roots were able to stimulate root development and early arbuscular mycorrhizal fungi colonization. These responses were strain and host genotype dependent. Our results suggest that wheat can be an alternative host for Rlv; nevertheless, there is a strong competition between Rlv strains for wheat root colonization. In addition, we showed that Rlv are endophytic wheat root bacteria with potential ability to modify wheat development.

Bacillus subtilis HG-15, a Halotolerant Rhizoplane Bacterium, Promotes Growth and Salinity Tolerance in Wheat (Triticum aestivum).

Certain plant growth-promoting bacteria (PGPB) reduce salt stress damage in plants. Bacillus subtilis HG-15 is a halotolerant bacterium (able to withstand NaCl concentrations as high as 30%) isolated from the wheat rhizoplane in the Yellow River delta. A qualitative and quantitative investigation of the plant growth-promoting characteristics of this strain confirmed nitrogen fixation, potassium dissolution, ammonia, plant hormone, ACC deaminase, and proline production abilities. B. subtilis HG-15 colonization of wheat roots, stems, and leaves was examined via scanning electron microscopy, rep-PCR, and double antibiotic screening. After inoculation with the B. subtilis HG-15 strain, the pH (1.08–2.69%), electrical conductivity (3.17–11.48%), and Na+ (12.98–15.55%) concentrations of rhizosphere soil significantly decreased (p < 0.05). Under no-salt stress (0.15% NaCl), low-salt stress (0.25% NaCl), and high-salt stress (0.35% NaCl) conditions, this strain also significantly increased (p < 0.05) the dry weight (17.76%, 24.46%, and 9.31%), fresh weight (12.80%, 20.48%, and 7.43%), plant height (7.79%, 5.86%, and 13.13%), and root length (10.28%, 17.87%, and 48.95%). Our results indicated that B. subtilis HG-15 can effectively improve the growth of wheat and elicit induced systemic tolerance in these plants, thus showing its potential as a microbial inoculant that can protect wheat under salt stress conditions.

LC_Glucose-Inhibited Division Protein Is Required for Motility, Biofilm Formation, and Stress Response in Lysobacter capsici X2-3.

Glucose-inhibited division protein (GidA) plays a critical role in the growth, stress response, and virulence of bacteria. However, how gidA may affect plant growth-promoting bacteria (PGPB) is still not clear. Our study aimed to describe the regulatory function of the gidA gene in Lysobacter capsici, which produces a variety of lytic enzymes and novel antibiotics. Here, we generated an LC_GidA mutant, MT16, and an LC_GidA complemented strain, Com-16, by plasmid integration. The deletion of LC_GidA resulted in an attenuation of the bacterial growth rate, motility, and biofilm formation of L. capsici. Root colonization assays demonstrated that the LC_GidA mutant showed reduced colonization of wheat roots. In addition, disruption of LC_GidA showed a clear diminution of survival in the presence of high temperature, high salt, and different pH conditions. The downregulated expression of genes related to DNA replication, cell division, motility, and biofilm formation was further validated by real-time quantitative PCR (RT–qPCR). Together, understanding the regulatory function of GidA is helpful for improving the biocontrol of crop diseases and has strong potential for biological applications.

CdgC, a Cyclic-di-GMP Diguanylate Cyclase of Azospirillum baldaniorum Is Involved in Internalization to Wheat Roots.

Azospirillum baldaniorum is a plant growth-promoting rhizobacterium (PGPR) capable of fixing nitrogen, the synthesis of several phytohormones including indole-acetic acid, and induction of plant defenses against phytopathogens. To establish a successful and prolonged bacteria-plant interaction, A. baldaniorum can form biofilms, bacterial communities embedded in a self-made matrix formed by extracellular polymeric substances which provide favorable conditions for survival. A key modulator of biofilm formation is the second messenger bis-(3′–5′)-cyclic-dimeric-GMP (c-di-GMP), which is synthesized by diguanylate cyclases (DGC) and degraded by specific phosphodiesterases. In this study, we analyzed the contribution of a previously uncharacterized diguanylate cyclase designated CdgC, to biofilm formation and bacterial-plant interaction dynamics. We showed that CdgC is capable of altering c-di-GMP levels in a heterologous host, strongly supporting its function as a DGC. The deletion of cdgC resulted in alterations in the three-dimensional structure of biofilms in a nitrogen-source dependent manner. CdgC was required for optimal colonization of wheat roots. Since we also observed that CdgC played an important role in exopolysaccharide production, we propose that this signaling protein activates a physiological response that results in the strong attachment of bacteria to the roots, ultimately contributing to an optimal bacterium-plant interaction. Our results demonstrate that the ubiquitous second messenger c-di-GMP is a key factor in promoting plant colonization by the PGPR A. baldaniorum by allowing proficient internalization in wheat roots. Understanding the molecular basis of PGPR-plant interactions will enable the design of better biotechnological strategies of agro-industrial interest.

Phytohormone Production Profiles in Trichoderma Species and Their Relationship to Wheat Plant Responses to Water Stress.

The production of eight phytohormones by Trichoderma species is described, as well as the 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase (ACCD) activity, which diverts the ethylene biosynthetic pathway in plants. The use of the Trichoderma strains T. virens T49, T. longibrachiatum T68, T. spirale T75 and T. harzianum T115 served to demonstrate the diverse production of the phytohormones gibberellins (GA) GA1 and GA4, abscisic acid (ABA), salicylic acid (SA), auxin (indole-3-acetic acid: IAA) and the cytokinins (CK) dihydrozeatin (DHZ), isopenteniladenine (iP) and trans-zeatin (tZ) in this genus. Such production is dependent on strain and/or culture medium. These four strains showed different degrees of wheat root colonization. Fresh and dry weights, conductance, H2O2 content and antioxidant activities such as superoxide dismutase, peroxidase and catalase were analyzed, under optimal irrigation and water stress conditions, on 30-days-old wheat plants treated with four-day-old Trichoderma cultures, obtained from potato dextrose broth (PDB) and PDB-tryptophan (Trp). The application of Trichoderma PDB cultures to wheat plants could be linked to the plants’ ability to adapt the antioxidant machinery and to tolerate water stress. Plants treated with PDB cultures of T49 and T115 had the significantly highest weights under water stress. Compared to controls, treatments with strains T68 and T75, with constrained GA1 and GA4 production, resulted in smaller plants regardless of fungal growth medium and irrigation regime.

Straw Competition and Wheat Root Endophytism of Trichoderma gamsii T6085 as Useful Traits in the Biological Control of Fusarium Head Blight.

Trichoderma gamsii T6085 has been investigated for many years as a beneficial isolate for use in the biocontrol of Fusarium head blight (FHB) of wheat caused primarily by Fusarium graminearum. Previous work focused on application of T6085 to wheat spikes at anthesis, whereas application to soil before or at sowing has received limited attention. In the present study, the competitive ability of T6085 on plant residues against F. graminearum was investigated. Results showed a significant reduction of wheat straw colonization by the pathogen and of the development of perithecia, not only when T6085 was applied alone but also in the presence of a F. oxysporum isolate (7121), well known as a natural competitor on wheat plant residues. T6085 was able to endophytically colonize wheat roots, resulting in internal colonization of the radical cortex area, without reaching the vascular system, as confirmed by confocal microscopy. This intimate interaction with the plant resulted in a significant increase of the expression of the plant defense-related genes PAL1 and PR1. Taken together, competitive ability, endophytic behavior, and host resistance induction represent three important traits that can be of great use in the application of T6085 against FHB not only on spikes at anthesis but potentially also in soil before or at sowing.

Identification of genes involved in regulating MnSOD2 production and root colonization in Bacillus cereus 905

sodA2-encoding manganese-containing superoxide dismutase (MnSOD2) in Bacillus cereus 905 plays an essential role in antioxidative stress, nutrition utilization, rhizosphere and phyllosphere colonization. However, the genes involved in regulating the sodA2 expression have not been clearly elucidated in B. cereus. In this study, a genome-wide random insertion mutagenesis was constructed by using transposon TnYLB-1 to identify novel genes regulating the sodA2 expression. Seven mutants that changed the sodA2 expression at both mRNA and protein levels were finally obtained. Sequence analysis and BLAST data showed that the genes disrupted by TnYLB-1 in B. cereus 905 shared high conservations with those in the B. cereus type strain, ATCC 14579. These genes encode heat-inducible transcription repressor, chloride channel protein, recombinase A, ferrous iron transport protein, nucleoside diphosphate kinase, and histidine ammonia-lyase. Besides, we also provided the evidence that the genes regulating the sodA2 expression could influence colonization ability of B. cereus 905 on wheat roots. Specifically, those genes downregulating the sodA2 expression significantly reduced bacterial colonization on wheat roots, and mutants with increased MnSOD2 activities could enhance bacterial population densities on wheat roots to a certain degree. Our work provided information that multiple genes are involved in MnSOD2 production and wheat root colonization. The molecular regulatory pathways through which these genes modulate the sodA2 expression and root colonization need to be investigated extensively in the future.

Expression and function of the cdgD gene, encoding a CHASE\u2013PAS-DGC-EAL domain protein, in Azospirillum brasilense

The plant growth-promoting bacterium Azospirillum brasilense contains several genes encoding proteins involved in the biosynthesis and degradation of the second messenger cyclic-di-GMP, which may control key bacterial functions, such as biofilm formation and motility. Here, we analysed the function and expression of the cdgD gene, encoding a multidomain protein that includes GGDEF-EAL domains and CHASE and PAS domains. An insertional cdgD gene mutant was constructed, and analysis of biofilm and extracellular polymeric substance production, as well as the motility phenotype indicated that cdgD encoded a functional diguanylate protein. These results were correlated with a reduced overall cellular concentration of cyclic-di-GMP in the mutant over 48 h compared with that observed in the wild-type strain, which was recovered in the complemented strain. In addition, cdgD gene expression was measured in cells growing under planktonic or biofilm conditions, and differential expression was observed when KNO3 or NH4Cl was added to the minimal medium as a nitrogen source. The transcriptional fusion of the cdgD promoter with the gene encoding the autofluorescent mCherry protein indicated that the cdgD gene was expressed both under abiotic conditions and in association with wheat roots. Reduced colonization of wheat roots was observed for the mutant compared with the wild-type strain grown in the same soil conditions. The Azospirillum-plant association begins with the motility of the bacterium towards the plant rhizosphere followed by the adsorption and adherence of these bacteria to plant roots. Therefore, it is important to study the genes that contribute to this initial interaction of the bacterium with its host plant.

Dynamics of wheat root colonization by arbuscular mycorrhizal fungi under contrasting soil tillage systems and its impact on grain yield

In this work, we investigated the effect of the soil tillage farming system on the variability of arbuscular mycorrhizal fungi (AMF) root colonization of a local durum wheat variety, conducted during five years of experimental trial in a semi-arid region of eastern Algeria. No tillage farming system improved the AMF symbiotic activity from the fourth year of testing with the appearance of high root percentage of arbuscules at tillering and grain filling stage. Compared to that, the conventional tillage was marked by the observation of a high root percentage of vesicles especially at grain filling. Despite inter-annual fluctuations, the no tillage farming system improved the average yield of the five trial years by 20%. Multiple regressions analyses using the stepwise selection method were made to formulate an equation linking grain yield and the AMF symbiotic components specific to each tillage farming system.

Quantification of Pseudomonas protegens FD6 and Bacillus subtilis NCD-2 in soil and the wheat rhizosphere and suppression of root pathogenic Rhizoctonia solani AG-8

Bacillus subtilis strain NCD-2 and Pseudomonas protegens strain FD6 produce a range of antimicrobial metabolites known to be effective against soil-borne plant pathogens. Quantitative qPCR assays were designed from sequence characterized amplified fragment length polymorphisms (AFLP) diagnostic for each inoculant genotype. The qPCR minimum detection thresholds (MDT) for NCD-2 and FD6 ranged between 5.2 × 103 (log 3.71) and 1.5 × 106 (log 6.18) genome (cell) equivalents g−1 of soil or wheat root tissue and were independent of soil type. Wheat rhizosphere populations of strains FD6 and NCD-2 significantly declined during the first 14 days postinoculation and then remained stable for the next 14 days. Colonization of wheat roots, rhizosphere soil and bulk (non-rhizosphere) soil by FD6 was significantly greater than that of NCD-2. Strains FD6 and NCD-2 significantly decreased in vitro growth of wheat root pathogenic Rhizoctonia solani AG-8, Pythium irregulare and Gaeumannomyces graminis var. tritici, fungal antibiosis by FD6 greater than NCD-2. Wheat rhizosphere colonization by NCD-2 and FD6 significantly decreased abundance of R. solani AG-8 in the rhizosphere by 3- and 13-fold respectively, pathogen suppression increasing (21%–29%) foliar biomass of wheat seedlings to levels not significantly different to the non-diseased control. The qPCR assays developed in this study were used to monitor soil and rhizosphere populations of P. protegens FD6 and B. subtilis NCD-2 and their suppressive efficacies against R. solani AG-8 in wheat. These assays can be applied to monitor the dynamics of these inoculants in suppressing Rhizoctonia root rot and other crop diseases.

PcsR2 Is a LuxR-Type Regulator That Is Upregulated on Wheat Roots and Is Unique to Pseudomonas chlororaphis.

LuxR solos are common in plant-associated bacteria and increasingly recognized for playing important roles in plant-microbe interkingdom signaling. Unlike the LuxR-type transcriptional regulators of prototype LuxR/LuxI quorum sensing systems, luxR solos do not have a LuxI-type autoinducer synthase gene associated with them. LuxR solos in plant-pathogenic bacteria are important for virulence and in plant endosymbionts contribute to symbiosis. In the present study, we characterized an atypical LuxR solo, PcsR2, in the biological control species Pseudomonas chlororaphis 30–84 that is highly conserved among sequenced P. chlororaphis strains. Unlike most LuxR solos in the plant-associated bacteria characterized to date, pcsR2 is not associated with a proline iminopeptidase gene and the protein has an atypical N-terminal binding domain. We created a pcsR2 deletion mutant and used quantitative RT-PCR to show that the expression of pcsR2 and genes in the operon immediately downstream was upregulated ∼10-fold when the wild type strain was grown on wheat roots compared to planktonic culture. PcsR2 was involved in upregulation. Using a GFP transcriptional reporter, we found that expression of pcsR2 responded specifically to root-derived substrates as compared to leaf-derived substrates but not to endogenous AHLs. Compared to the wild type, the mutant was impaired in the ability to utilize root carbon and nitrogen sources in wheat root macerate and to colonize wheat roots. Phenazine production and most biofilm traits previously shown to be correlated with phenazine production also were diminished in the mutant. Gene expression of several of the proteins in the phenazine regulatory network including PhzR, Pip (phenazine inducing protein) and RpeA/RpeB were reduced in the mutant, and overexpression of these genes in trans restored phenazine production in the mutant to wild-type levels, indicating PcsR2 affects the activity of the these regulatory genes upstream of RpeA/RpeB via an undetermined mechanism. Our results indicate PcsR2 upregulates the expression of the adjacent operon in response to unknown wheat root-derived signals and belongs to a novel subfamily of LuxR-type transcriptional regulators found in sequenced P. chlororaphis strains.