Production of Agrocinopine A by Ipomoea batatas Agrocinopine Synthase in Transgenic Tobacco and Its Effect on the Rhizosphere Microbial Community.

Agrobacterium tumefaciens is a bacterial pathogen that causes crown gall disease on a wide range of eudicot plants by genetic transformation. Besides T-DNA integrated by natural transformation of plant vegetative tissues by pathogenic Agrobacterium spp., previous reports have indicated that T-DNA sequences originating from an ancestral Agrobacterium sp. are present in the genomes of all cultivated sweet potato (Ipomoea batatas) varieties analyzed. Expression of an Agrobacterium-derived agrocinopine synthase (ACS) gene was detected in leaf and root tissues of sweet potato, suggesting that the plant can produce agrocinopine, a sugar-phosphodiester opine considered to be utilized by some strains of Agrobacterium spp. in crown gall. To validate the product synthesized by Ipomoea batatas ACS (IbACS), we introduced IbACS into tobacco under a constitutive promoter. High-voltage paper electrophoresis followed by alkaline silver nitrate staining detected the production of an agrocinopine-like substance in IbACS1-expressing tobacco, and further mass spectrometry and nuclear magnetic resonance analyses of the product confirmed that IbACS can produce agrocinopine A from natural plant substrates. The partially purified compound was biologically active in an agrocinopine A bioassay. A 16S ribosomal RNA amplicon sequencing and meta-transcriptome analysis revealed that the rhizosphere microbial community of tobacco was affected by the expression of IbACS. A new species of Leifsonia (actinobacteria) was isolated as an enriched bacterium in the rhizosphere of IbACS1-expressing tobacco. This Leifsonia sp. can catabolize agrocinopine A produced in tobacco, indicating that the production of agrocinopine A attracts rhizosphere bacteria that can utilize this sugar-phosphodiester. These results suggest a potential role of IbACS conserved among sweet potato cultivars in manipulating their microbial community.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Ability to produce indole acetic acid is associated with improved phosphate solubilising activity of rhizobacteria.

Indole acetic acid (IAA) can upregulate genes encoding enzymes responsible for the synthesis of carboxylates involved in phosphorus (P) solubilisation. Here, we investigated whether IAA and its precursor affect the P-solubilising activity of rhizobacteria. A total of 841 rhizobacteria were obtained using taxonomically selective and enrichment isolation methods. Phylogenetic analysis revealed 15 genera of phosphate solubilising bacteria (PSB) capable of producing a wide range of IAA concentrations between 4.1 and 67.2 µg mL-1 in vitro. Addition of L-tryptophan to growth media improved the P-solubilising activity of PSB that were able to produce IAA greater than 20 µg mL-1. This effect was connected to the drop of pH and release of a high concentration of carboxylates, comprising α-ketoglutarate, cis-aconitate, citrate, malate and succinate. An increase in production of organic acids rather than IAA production per se appears to result in the improved P solubilisation in PSB.

Near-Complete Genomes of Two Trichoderma Species: A Resource for Biological Control of Plant Pathogens.

Trichoderma species are widely used to control fungal and nematode diseases of crops. To date, only one complete Trichoderma genome has been sequenced, T. reesei QM6a, a model fungus for industrial enzyme production, while the species or strains used for biological control of plant diseases are only available as draft genomes. Previously, we demonstrated that two Trichoderma strains (T. afroharzianum and T. cyanodichotomus) provide effective control of nematode and fungal plant pathogens. Based on deep sequencing using Illumina and Pacbio platforms, we have assembled high-quality genomes of the above two strains, with contig N50 reaching 4.2 and 1.7 Mbp, respectively, which is greater than those of published draft genomes. The genome data will provide a resource to assist research on the biological control mechanisms of Trichoderma spp.

Agrocinopine C, a Ti-plasmid-coded enzyme-product, is a 2-O, 6-O linked phosphodiester of D-Glucose and sucrose.

Agrocinopine C is a small molecule found in crown gall tumours induced by pathogenic Agrobacterium radiobacter carrying the tumour-inducing plasmid pTi Bo542. This phosphodiester opine was isolated (at 0.02 g/100 g fresh wt.) from sunflower (Helianthus annuus L.) galls. It is structurally related to agrocinopine A and is a glucose-2-phosphodiester linked to the C6-hydroxy-methyl group of the glucose moiety of sucrose. Sugar-2-phosphates are uncommon in plant tissues, whether transformed by Agrobacterium or not. 1H and 31P NMR signal multiplicity indicates five-fold anomeric complexity of agrocinopine C in solution, implying that the permeases taking up these sucrose-phosphodiesters could recognise any one of the five anomers. Data suggests that the open chain aldehyde forms of the 2-phosphorylated opines agrocinopine C and agrocinopine A and the corresponding phosphorylated glucose-2-phosphoramidate component of the antibiotic agrocin 84 play a central role in agrocin's selective toxicity to certain strains of Agrobacterium after uptake via Ti plasmid-encoded permeases.

Rhizobial diversity is associated with inoculation history at a two-continent scale.

A total of 120 Mesorhizobium strains collected from the central dry zone of Myanmar were analyzed in a pot experiment to evaluate nodulation and symbiotic effectiveness (SE%) in chickpea plants. Phylogenetic analyses revealed all strains belonged to the genus Mesorhizobium according to 16-23S rDNA IGS and the majority of chickpea nodulating rhizobia in Myanmar soils were most closely related to M. gobiense, M. muleiense, M. silamurunense, M. tamadayense and M. temperatum. Around two-thirds of the Myanmar strains (68%) were most closely related to Indian strain IC-2058 (CA-181), which is also most closely related to M. gobiense. There were no strains that were closely related to the cognate rhizobial species to nodulate chickpea: M. ciceri and M. mediterraneum. Strains with diverse 16S-23S rDNA IGS shared similar nodC and nifH gene sequences with chickpea symbionts. Detailed sequence analysis of nodC and nifH found that the strains in Myanmar were somewhat divergent from the group including M. ciceri and were more closely related to M. muleiense and IC-2058. A cross-continent analysis between strains isolated in Australia compared with Myanmar found that there was little overlap in species, where Australian soils were dominated with M. ciceri, M. temperatum and M. huakuii. The only co-occurring species found in both Myanmar and Australia were M. tamadayense and M. silumurunense. Continued inoculation with CC1192 may have reduced diversity of chickpea strains in Australian soils. Isolated strains in Australian and Myanmar had similar adaptive traits, which in some cases were also phylogenetically related. The genetic discrepancy between chickpea nodulating strains in Australia and Myanmar is not only due to inoculation history but to adaptation to soil conditions and crop management over a long period, and there has been virtually no loss of symbiotic efficiency over this time in strains isolated from soils in Myanmar.

Trichoderma atroviride seed dressing influenced the fungal community and pathogenic fungi in the wheat rhizosphere.

Fusarium crown rot and wheat sharp eyespot are major soil-borne diseases of wheat, causing serious losses to wheat yield in China. We applied high-throughput sequencing combined with qPCR to determine the effect of winter wheat seed dressing, with either Trichoderma atroviride HB20111 spore suspension or a chemical fungicide consisting of 6% tebuconazole, on the fungal community composition and absolute content of pathogens Fusarium pseudograminearum and Rhizoctonia cerealis in the rhizosphere at 180 days after planting. The results showed that the Trichoderma and chemical fungicide significantly reduced the amount of F. pseudograminearum in the rhizosphere soil (p < 0.05), and also changed the composition and structure of the fungal community. In addition, field disease investigation and yield measurement showed that T. atroviride HB20111 treatment reduced the whiteheads with an average control effect of 60.1%, 14.9% higher than the chemical treatment; T. atroviride HB20111 increased yield by 7.7%, which was slightly more than the chemical treatment. Therefore, T. atroviride HB20111 was found to have the potential to replace chemical fungicides to control an extended range of soil-borne diseases of wheat and to improve wheat yield.

Plant growth-promoting rhizobacteria Burkholderia vietnamiensis B418 inhibits root-knot nematode on watermelon by modifying the rhizosphere microbial community.

Burkholderia vietnamiensis B418 is a multifunctional plant growth-promoting rhizobacteria (PGPR) strain with nitrogen-fixing and phosphate-solubilizing capability which can be employed for root-knot nematode (RKN) management on various crops and vegetables. Here we investigated the control efficacy of B. vietnamiensis B418 inoculation against RKN on watermelon, applied either alone or combined with nematicides fosthiazate or avermectin, and their effects on bacterial and fungal microbiomes in rhizosphere soil. The results of field experiments showed individual application of B418 displayed the highest control efficacy against RKN by 71.15%. The combinations with fosthiazate and avermectin exhibited slight incompatibility with lower inhibitory effects of 62.71% and 67.87%, respectively, which were still notably higher than these nematicides applied separately. Analysis of microbiome assemblages revealed B418 inoculation resulted in a slight reduction for bacterial community and a significant increment for fungal community, suggesting that B418 could compete with other bacteria and stimulate fungal diversity in rhizosphere. The relative abundance of Xanthomonadales, Gemmatimonadales and Sphingomonadales increased while that of Actinomycetales reduced with B418 inoculation. The predominate Sordariomycetes of fungal community decreased dramatically in control treatment with B418 inoculation whereas there were increments in fosthiazate and avermectin treatments. Additionally, nitrogen (N) cycling by soil microbes was estimated by quantifying the abundance of microbial functional genes involved in N-transformation processes as B418 has the capability of N-fixation. The copy number of N-fixing gene nifH increased with B418 inoculation, and the highest increment reached 35.66% in control treatment. Our results demonstrate that B. vietnamiensis B418 is an effective biological nematicide for nematode management, which acts through the modulation of rhizosphere microbial community.

Near-Complete Genome Assembly of the Grapevine Crown Gall Pathogen Allorhizobium vitis Strain K377.

Here, we report the annotated, near-complete genome sequence of Allorhizobium vitis K377, a phytopathogenic Rhizobiales strain isolated from a grapevine in South Australia. The assembled genome sequence is 6.40 Mb long, with 5,855 predicted protein-coding sequences, 56 tRNAs, and 12 rRNAs, and contains ttuC (tartrate metabolism; chromosomal) and nopaline synthesis, uptake, and catabolic genes (tumor-inducing plasmid-encoded).

Complete Genome Sequence of Allorhizobium vitis Strain K306, the Causal Agent of Grapevine Crown Gall.

Here, we present the annotated complete genome sequence of Allorhizobium vitis K306, a phytopathogenic strain causing crown gall of grapevine. The A. vitis K306 genome is 5.79 Mb long with 5,199 predicted protein-coding genes and contains 2 circular chromosomes of 3.8 Mb and 1.1 Mb and 2 plasmids, namely, pTiK306 and pTrK306, that are 262 kb and 581 kb, respectively.

Trichoderma harzianum Inoculation Reduces the Incidence of Clubroot Disease in Chinese Cabbage by Regulating the Rhizosphere Microbial Community.

Clubroot is a disease of cruciferous crops that causes significant economic losses to vegetable production worldwide. We applied high-throughput amplicon sequencing technology to quantify the effect of Trichodermaharzianum LTR-2 inoculation on the rhizosphere community of Chinese cabbage (Brassica rapa subsp. pekinensis cv. Jiaozhou) in a commercial production area. T. harzianum inoculation of cabbage reduced the incidence of clubroot disease by 45.4% (p < 0.05). The disease control efficacy (PDIDS) was 63%. This reduction in disease incidence and severity coincided with a drastic reduction in both the relative abundance of Plasmodiaphora brassicae, the causative pathogen of cabbage clubroot disease, and its copy number in rhizosphere soil. Pathogenic fungi Alternaria and Fusarium were also negatively associated with Trichoderma inoculation according to co-occurrence network analysis. Inoculation drastically reduced the relative abundance of the dominant bacterial genera Delftia and Pseudomonas, whilst increasing others including Bacillus. Our results demonstrate that T. harzianum LTR-2 is an effective biological control agent for cabbage clubroot, which acts through modulation of the soil and rhizosphere microbial community.

Nitrifying Microbes in the Rhizosphere of Perennial Grasses are Modified by Biological Nitrification Inhibition.

Soil nitrification (microbial oxidation of ammonium to nitrate) can lead to nitrogen leaching and environmental pollution. A number of plant species are able to suppress soil nitrifiers by exuding inhibitors from roots, a process called biological nitrification inhibition (BNI). However, the BNI activity of perennial grasses in the nutrient-poor soils of Australia and the effects of BNI activity on nitrifying microbes in the rhizosphere microbiome have not been well studied. Here we evaluated the BNI capacity of bermudagrass (Cynodon dactylon L.), St. Augustinegrass (Stenotaphrum secundatum (Walt.) Kuntze), saltwater couch (Sporobolus virginicus), seashore paspalum (Paspalum vaginatum Swartz.), and kikuyu grass (Pennisetum clandestinum) compared with the known positive control, koronivia grass (Brachiaria humidicola). The microbial communities were analysed by sequencing 16S rRNA genes. St. Augustinegrass and bermudagrass showed high BNI activity, about 80 to 90% of koronivia grass. All the three grasses with stronger BNI capacities suppressed the populations of Nitrospira in the rhizosphere, a bacteria genus with a nitrite-oxidizing function, but not all of the potential ammonia-oxidizing archaea. The rhizosphere of saltwater couch and seashore paspalum exerted a weak recruitment effect on the soil microbiome. Our results demonstrate that BNI activity of perennial grasses played a vital role in modulating nitrification-associated microbial populations.

Agrocin 108 is a 5'-cytidine nucleotide bacteriocin containing a carbocyclic phosphoryl-ascorbate group.

Agrocin 108 is a 3'-O-ß-D-xylopyranosyl-cytidine-5'-O-phosphodiester of an ascorbate-carbocyclic cyclopentenone analogue, with bacteriocin-like properties. This bacteriocin exhibits orders of magnitude greater than the inhibition zone diameter towards the indicator strain than either ampicillin or streptomycin. It has been isolated from cultures of Rhizobium rhizogenes strain K108. The structure of the agrocin 108 without detail, has been previously published. We now report a detailed structure elucidation, including the hitherto undetermined residual 5'-phospho-diester fragment by a combination of 1D and 2D NMR studies at various pH values in H2O/D2O, high resolution MS, pKa determination, and chemical degradation.