QTL mapping of downy mildew resistance in foxtail millet by SLAF­seq and BSR-seq analysis.

KEY MESSAGE: Key message Three major QTLs for resistance to downy mildew were located within an 0.78 Mb interval on chromosome 8 in foxtail millet. Downy mildew, a disease caused by Sclerospora graminicola, is a serious problem that jeopardizes the yield and quality of foxtail millet. Breeding resistant varieties represents one of the most economical and effective solutions, yet there is a lack of molecular markers related to the resistance. Here, a mapping population comprising of 158 F6:7 recombinant inbred lines (RILs) was constructed from the crossing of G1 and JG21. Based on the specific locus amplified fragment sequencing results, a high-density linkage map of foxtail millet with 1031 bin markers, spanning 1041.66 cM was constructed. Based on the high-density linkage map and the phenotype data in four environments, a total of nine quantitative trait loci (QTL) associated with resistance to downy mildew were identified. Further BSR-seq confirmed the genomic regions containing the potential candidate genes related to downy mildew resistance. Interestingly, a 0.78-Mb interval between C8M257 and C8M268 on chromosome 8 was highlighted because of its presence in three major QTL, qDM8_1, qDM8_2, and qDM8_4, which contains 10 NBS-LRR genes. Haplotype analysis in RILs and natural population suggest that 9 SNP loci on Seita8G.199800, Seita8G.195900, Seita8G.198300, and Seita.8G199300 genes were significantly correlated with disease resistance. Furthermore, we found that those genes were taxon-specific by collinearity analysis of pearl millet and foxtail millet genomes. The identification of these new resistance QTL and the prediction of resistance genes against downy mildew will be useful in breeding for resistant varieties and the study of genetic mechanisms of downy mildew disease resistance in foxtail millet.

Quadrisphaera setariae sp. nov., polyphosphate-accumulating bacterium occurring as tetrad or aggregate cocci and isolated from Setaria viridis.

A Gram-stain-positive, orange-pigmented, aerobic, cocci (occurring in tetrads), non-spore-forming, non-motile bacterium, designated as DD2AT, was isolated from Setaria viridis collected at Dongguk University, Republic of Korea. Phylogenetic analysis based on the 16S rRNA gene revealed that strain DD2AT was most closely related to type strains of the genus Quadrisphaera. Strain DD2AT showed the highest 16S rRNA gene sequence similarities to Quadrisphaera oryzae TBRC 8486T (99.4 %) and Quadrisphaera granulorum JCM 16010T (98.8 %). Strain DD2AT also showed auto-aggregation ability. The digital DNA-DNA hybridization values between strain DD2AT and the reference strains, Q. oryzae TBRC 8486T and Q. granulorum JCM 16010T were 31.1 and 27.4 %, respectively. The average nucleotide identity values between strain DD2AT and Q. oryzae TBRC 8486T and Q. granulorum JCM 16010T were 86.3 and 84.1 %, respectively. The major polar lipids of strain DD2AT were diphosphatidylglycerol and phosphatidylglycerol. The major cellular fatty acid of strain DD2AT was anteiso-C15 : 0. The cell-wall peptidoglycan contained meso-diaminopimelic acid (which is a diagnostic cell-wall diamino acid), alanine and glutamic acid. The respiratory quinones was found to be menaquinone-8. The DNA G+C content of strain DD2AT was 74.8 mol%. On the basis of the findings of genotypic, phenotypic, chemotaxonomic and phylogenetic analyses, strain DD2AT was considered to represent a novel member in the genus Quadrisphaera, for which the name Quadrisphaera setariae sp. nov. is proposed. The type strain of Quadrisphaera setariae is DD2AT (=KACC 21165T=NBRC 113770T).

Deciphering the microbial and molecular responses of geographically diverse Setaria accessions grown in a nutrient-poor soil.

The microbial and molecular characterization of the ectorhizosphere is an important step towards developing a more complete understanding of how the cultivation of biofuel crops can be undertaken in nutrient poor environments. The ectorhizosphere of Setaria is of particular interest because the plant component of this plant-microbe system is an important agricultural grain crop and a model for biofuel grasses. Importantly, Setaria lends itself to high throughput molecular studies. As such, we have identified important intra- and interspecific microbial and molecular differences in the ectorhizospheres of three geographically distant Setaria italica accessions and their wild ancestor S. viridis. All were grown in a nutrient-poor soil with and without nutrient addition. To assess the contrasting impact of nutrient deficiency observed for two S. italica accessions, we quantitatively evaluated differences in soil organic matter, microbial community, and metabolite profiles. Together, these measurements suggest that rhizosphere priming differs with Setaria accession, which comes from alterations in microbial community abundances, specifically Actinobacteria and Proteobacteria populations. When globally comparing the metabolomic response of Setaria to nutrient addition, plants produced distinctly different metabolic profiles in the leaves and roots. With nutrient addition, increases of nitrogen containing metabolites were significantly higher in plant leaves and roots along with significant increases in tyrosine derived alkaloids, serotonin, and synephrine. Glycerol was also found to be significantly increased in the leaves as well as the ectorhizosphere. These differences provide insight into how C4 grasses adapt to changing nutrient availability in soils or with contrasting fertilization schemas. Gained knowledge could then be utilized in plant enhancement and bioengineering efforts to produce plants with superior traits when grown in nutrient poor soils.

Engineering Posttranslational Regulation of Glutamine Synthetase for Controllable Ammonia Production in the Plant Symbiont Azospirillum brasilense.

Nitrogen requirements for modern agriculture far exceed the levels of bioavailable nitrogen in most arable soils. As a result, the addition of nitrogen fertilizer is necessary to sustain productivity and yields, especially for cereal crops, the planet's major calorie suppliers. Given the unsustainability of industrial fertilizer production and application, engineering biological nitrogen fixation directly at the roots of plants has been a grand challenge for biotechnology. Here, we designed and tested a potentially broadly applicable metabolic engineering strategy for the overproduction of ammonia in the diazotrophic symbiont Azospirillum brasilense. Our approach is based on an engineered unidirectional adenylyltransferase (uAT) that posttranslationally modifies and deactivates glutamine synthetase (GS), a key regulator of nitrogen metabolism in the cell. We show that this circuit can be controlled inducibly, and we leveraged the inherent self-contained nature of our posttranslational approach to demonstrate that multicopy redundancy can improve strain evolutionary stability. uAT-engineered Azospirillum is capable of producing ammonia at rates of up to 500 µM h-1 unit of OD600 (optical density at 600 nm)-1. We demonstrated that when grown in coculture with the model monocot Setaria viridis, these strains increase the biomass and chlorophyll content of plants up to 54% and 71%, respectively, relative to the wild type (WT). Furthermore, we rigorously demonstrated direct transfer of atmospheric nitrogen to extracellular ammonia and then plant biomass using isotopic labeling: after 14 days of cocultivation with engineered uAT strains, 9% of chlorophyll nitrogen in Setaria seedlings was derived from diazotrophically fixed dinitrogen, whereas no nitrogen was incorporated in plants cocultivated with WT controls. This rational design for tunable ammonia overproduction is modular and flexible, and we envision that it could be deployable in a consortium of nitrogen-fixing symbiotic diazotrophs for plant fertilization. IMPORTANCE Nitrogen is the most limiting nutrient in modern agriculture. Free-living diazotrophs, such as Azospirillum, are common colonizers of cereal grasses and have the ability to fix nitrogen but natively do not release excess ammonia. Here, we used a rational engineering approach to generate ammonia-excreting strains of Azospirillum. Our design features posttranslational control of highly conserved central metabolism, enabling tunability and flexibility of circuit placement. We found that our strains promote the growth and health of the model grass S. viridis and rigorously demonstrated that in comparison to WT controls, our engineered strains can transfer nitrogen from 15N2 gas to plant biomass. Unlike previously reported ammonia-producing mutants, our rationally designed approach easily lends itself to further engineering opportunities and has the potential to be broadly deployable.

Identification of QTL for resistance to leaf blast in foxtail millet by genome re-sequencing analysis.

KEY MESSAGE: Three QTL for resistance to leaf blast were identified on chromosomes 1, 2, and 8 of the foxtail millet cultivar Yugu 5. Leaf blast disease of foxtail millet (Setaria italica) is caused by Pyricularia spp., can infect all the aboveground parts of plants, and is the most frequently observed blast disease in China. Lack of information on genetic control of disease resistance impedes developing leaf blast-resistant cultivars. An F6 recombinant inbred line (RIL) population from the cross Yugu 5 × Jigu 31 was phenotyped for its reactions to leaf blast in six field trials in the naturally diseased nurseries. An ultra-density genetic linkage map was constructed using 35,065 single nucleotide polymorphism (SNP) markers generated by sequencing of the RIL population. Three QTL, QLB-czas1, QLB-czas2, and QLB-cazas8, were detected in the genomic intervals of 276.6 kb, 1.62 Mb, and 1.75 Mb on chromosomes 1, 2, and 8 of Yugu 5, which explained 14-17% (2 environments), 9% (5 environments), and 12-20% (6 environments) of the phenotypic variations. Bulked segregant analysis (BSA) and RNA sequencing (BSR-Seq) method identified common SNPs that fell in the genomic region of QLB-czas8, providing additional evidence of localization of this QTL. Three and 19 predicted genes were annotated to be associated with disease resistance in the genomic intervals for QLB-czas2 and QLB-czas8. Due to their unique positions, these QTL appear to be new loci conferring resistance to leaf blast. The identification of these new resistance QTL will be useful in cultivar development and the study of the genetic control of blast resistance in foxtail millet.

Diverse Bacterial Genes Modulate Plant Root Association by Beneficial Bacteria.

The plant rhizosphere harbors a diverse population of microorganisms, including beneficial plant growth-promoting bacteria (PGPB), that colonize plant roots and enhance growth and productivity. In order to specifically define bacterial traits that contribute to this beneficial interaction, we used high-throughput transposon mutagenesis sequencing (TnSeq) in two model root-bacterium systems associated with Setaria viridis: Azoarcus olearius DQS4T and Herbaspirillum seropedicae SmR1. This approach identified ∼100 significant genes for each bacterium that appeared to confer a competitive advantage for root colonization. Most of the genes identified specifically in A. olearius encoded metabolism functions, whereas genes identified in H. seropedicae were motility related, suggesting that each strain requires unique functions for competitive root colonization. Genes were experimentally validated by site-directed mutagenesis, followed by inoculation of the mutated bacteria onto S. viridis roots individually, as well as in competition with the wild-type strain. The results identify key bacterial functions involved in iron uptake, polyhydroxybutyrate metabolism, and regulation of aromatic metabolism as important for root colonization. The hope is that by improving our understanding of the molecular mechanisms used by PGPB to colonize plants, we can increase the adoption of these bacteria in agriculture to improve the sustainability of modern cropping systems.IMPORTANCE There is growing interest in the use of associative, plant growth-promoting bacteria (PGPB) as biofertilizers to serve as a sustainable alternative for agriculture application. While a variety of mechanisms have been proposed to explain bacterial plant growth promotion, the molecular details of this process remain unclear. The current research supports the idea that PGPB use in agriculture will be promoted by gaining more knowledge as to how these bacteria colonize plants, promote growth, and do so consistently. Specifically, the research seeks to identify those bacterial genes involved in the ability of two, PGPB strains, Azoarcus olearius and Herbaspirillum seropedicae, to colonize the roots of the C4 model grass Setaria viridis. Applying a transposon mutagenesis (TnSeq) approach, we assigned phenotypes and function to genes that affect bacterial competitiveness during root colonization. The results suggest that each bacterial strain requires unique functions for root colonization but also suggests that a few, critical functions are needed by both bacteria, pointing to some common mechanisms. The hope is that such information can be exploited to improve the use and performance of PGPB in agriculture.

Hymenobacter setariae sp. nov., isolated from the ubiquitous weedy grass Setaria viridis.

A Gram-stain-negative, short-rod, aerobic, non-motile, red to pink-pigmented bacterium, designated Fur1T, was isolated from the dry spikelet clusters of a plant called Setaria viridis near Dongguk University. Phylogenetic analysis conducted based on 16S rRNA gene sequences indicated that strain Fur1T belonged to the genus Hymenobacter of the family Hymenobacteraceae. The 16S rRNA gene of Fur1T showed highest sequence similarity to those of Hymenobacter metalli KACC 17381T (97.5 %) and Hymenobacter marinus KACC 19042T (97.1 %). Growth occurred at 4-37 °C (optimum, 25-28 °C), up to 1.0 % NaCl (optimum, 0 %) and pH 5.5-9.0 (optimum, pH 6.0-7.5). The major fatty acids of strain Fur1T were identified as iso-C15 : 0, C16 : 1 ω5c, anteiso-C15 : 0, summed feature 3 (comprising C16 : 1 ω7c and/or C16 : 1 ω6c) and summed feature 4 (comprising anteiso-C17 : 1B and/or iso-C17 : 1I) as the major cellular fatty acids. The predominant respiratory quinone was identified as MK-7. The polar lipids were phosphatidylethanolamine, five unidentified aminophospholipids, two unidentified phospholipids, one unidentified glycolipid and one unidentified polar lipid. The genomic DNA G+C content based on the draft genome sequence was 58.7 mol%. DNA-DNA relatedness between strain Fur1T and its closest relative was below 70 %. Characterization based on phylogenetic, chemotaxonomic and phenotypic analyses clearly indicated that strain Fur1T represents a novel species of the genus Hymenobacter, for which the name Hymenobacter setariae sp. nov. is proposed. The type strain is Fur1T (=KACC 19903T=NBRC=113691T).

An improved protocol for efficient transformation and regeneration of Setaria italica.

KEY MESSAGE: An efficient and improved transformation method for functional genetics studies in S. italica, being a boon for the Setaria scientific community and for crop improvement. Foxtail millet (Setaria italica) is a short life cycle C4 plant, with sequenced genome, and a potential model plant for C4 species. S. italica is also important on a global food security and healthiness context due to its importance in arid and semi-arid areas. However, despite its importance, there are just few transformation protocols directed to this species. The current protocols reached about 5.5-9% of efficiency, which do not make it a valuable model organism. Different types of explants were used in the above mentioned methods, such as immature and mature inflorescence and shoot apex. However, these techniques have many limitations, such as unavailability of explants throughout the year and a crucial, laborious and considerable time-consuming selection. Aiming a simplified and efficient methodology, we adopted dry mature seeds as explants, which are available in abundance, are constant along the year and well responsive to tissue culture, in addition to a differentiated approach that reaches on an average 19.2% transformation efficiency of S. italica. Thus, we propose a protocol that optimizes the transformation efficiency of this cereal crop allowing a high increase on transformation and regeneration rates. Our transformation protocol provides an interesting tool for Setaria community research as well as enables new strategies for breeding enhanced productivity in the species.

An efficient Agrobacterium-mediated genetic transformation method for foxtail millet (Setaria italica L.).

KEY MESSAGE: A simple and robust Agrobacterium-mediated gene expression system in the C4 panicoid model crop, foxtail millet has been developed with up to 27 % transformation efficiency. Foxtail millet (Setaria italica L.) is a model crop to study C4 photosynthesis, abiotic stress tolerance, and bioenergy traits. Advances in molecular genetics and genomics had identified several potential genes in this crop that would serve as candidates for imparting climate-resilient traits in related millets, cereals, and biofuel crops. However, the lack of an efficient genetic transformation system has been impeding the functional characterization of these genes in foxtail millet per se. Given this, an easy and efficient regeneration and transformation protocol was optimized using mature seeds as a choicest explant. The suitability of secondary embryogenic calli over primary calli is underlined due to their high competence. The use of perfect combinations of plant growth regulators together with the ionic strength of organic and inorganics salts was found to influence regeneration and genetic transformation. We studied and optimized various crucial factors that affect the genetic transformation of foxtail millet calli using Agrobacterium tumefaciens-mediated approach. Secondary embryogenic calli and LBA44404 strain were found to be the best targets for transformation. The use of high sucrose and glucose, together with freshly prepared tobacco leaves extract, Silwet L-77 and acetosyringone, improved the efficiency of the genetic transformation of foxtail millet. Moreover, the use of an in vitro regeneration system with 84% callusing efficiency and 70-74% regeneration frequency led to a high recovery of transformants. Altogether, the present study reports a highly efficient (~ 27%) transformation system in foxtail millet that will expedite forward and reverse genetic studies in this important crop.

In-Situ Metabolomic Analysis of Setaria viridis Roots Colonized by Beneficial Endophytic Bacteria.

Over the past decades, crop yields have risen in parallel with increasing use of fossil fuel-derived nitrogen (N) fertilizers but with concomitant negative impacts on climate and water resources. There is a need for more sustainable agricultural practices, and biological nitrogen fixation (BNF) could be part of the solution. A variety of nitrogen-fixing, epiphytic, and endophytic plant growth-promoting bacteria (PGPB) are known to stimulate plant growth. However, compared with the rhizobium-legume symbiosis, little mechanistic information is available as to how PGPB affect plant metabolism. Therefore, we investigated the metabolic changes in roots of the model grass species Setaria viridis upon endophytic colonization by Herbaspirillum seropedicae SmR1 (fix+) or a fix- mutant strain (SmR54) compared with uninoculated roots. Endophytic colonization of the root is highly localized and, hence, analysis of whole-root segments dilutes the metabolic signature of those few cells impacted by the bacteria. Therefore, we utilized in-situ laser ablation electrospray ionization mass spectrometry to sample only those root segments at or adjacent to the sites of bacterial colonization. Metabolites involved in purine, zeatin, and riboflavin pathways were significantly more abundant in inoculated plants, while metabolites indicative of nitrogen, starch, and sucrose metabolism were reduced in roots inoculated with the fix- strain or uninoculated, presumably due to N limitation. Interestingly, compounds, involved in indole-alkaloid biosynthesis were more abundant in the roots colonized by the fix- strain, perhaps reflecting a plant defense response.

Amnibacterium setariae sp. nov., an endophytic actinobacterium isolated from dried foxtail.

A Gram-stain positive, short rod-shaped, aerobic, motile by means of gliding, yellow-pigmented actinobacterium, designated strain DD4aT, was isolated from dry yellow foxtail. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain DD4aT is closely related to Amnibacterium soli MB78T (98.4% similarity), Amnibacterium kyonggiense KSL51201-037T (98.2%) and Amnibacterium endophyticum 1T4Z-3T (97.43%). Strain DD4aT forms yellow colonies on R2A agar medium. The peptidoglycan was found to contains diaminopimelic acid (which is a diagnostic cell wall diamino acid), alanine, glutamic acid and lysine. The polar lipids diphosphatidylglycerol, phosphatidylglycerol, six unidentified glycolipids and an unidentified polar lipid were found to be present in strain DD4aT. The major cellular fatty acids anteiso-C15:0 (42.9%) and iso-C16:0 (34.6%) were found in strain DD4aT. The predominant respiratory quinones were found to be MK-11 and MK-12. The DNA G+C content of strain DD4aT is 73.9 mol%. DNA-DNA relatedness of strain DD4aT with A. soli MB78T, A. kyonggiense KSL51201-037T, and A. endophyticum 1T4Z-3T were 53.3% (± 1.1%), 47.0% (± 0.5%), and 47.9% (± 0.9%), respectively. The digital DNA-DNA hybridisation and average nucleotide identity values between strain DD4aT and A. kyonggiense KSL51201-037T were determined to be 26.1% and 82.7%. On the basis of phenotypic, genotypic, chemotaxonomic and phylogenetic analysis, DD4aT represents a novel member of the genus Amnibacterium, for which the name Amnibacterium setariae sp. nov., is proposed. The type strain of Amnibacterium setariae is DD4aT (= KACC 19817T = JCM 32878T).

Importance of Poly-3-Hydroxybutyrate Metabolism to the Ability of Herbaspirillum seropedicae To Promote Plant Growth.

Herbaspirillum seropedicae is an endophytic bacterium that establishes an association with a variety of plants, such as rice, corn, and sugarcane, and can significantly increase plant growth. H. seropedicae produces polyhydroxybutyrate (PHB), stored in the form of insoluble granules. Little information is available on the possible role of PHB in bacterial root colonization or in plant growth promotion. To investigate whether PHB is important for the association of H. seropedicae with plants, we inoculated roots of Setaria viridis with H. seropedicae strain SmR1 and mutants defective in PHB production (ΔphaP1, ΔphaP1 ΔphaP2, ΔphaC1, and ΔphaR) or mobilization (ΔphaZ1 ΔphaZ2). The strains producing large amounts of PHB colonized roots, significantly increasing root area and the number of lateral roots compared to those of PHB-negative strains. H. seropedicae grows under microaerobic conditions, which can be found in the rhizosphere. When grown under low-oxygen conditions, only the parental strain and ΔphaP2 mutant exhibited normal growth. The lack of normal growth under low oxygen correlated with the inability to stimulate plant growth, although there was no effect on the level of root colonization. The data suggest that PHB is produced in the root rhizosphere and plays a role in maintaining normal metabolism under microaerobic conditions. To confirm this, we screened for green fluorescent protein (GFP) expression under the control of the H. seropedicae promoters of the PHA synthase and PHA depolymerase genes in the rhizosphere. PHB synthesis is active on the root surface and later PHB depolymerase expression is activated.IMPORTANCE The application of bacteria as plant growth promoters is a sustainable alternative to mitigate the use of chemical fertilization in agriculture, reducing negative economic and environmental impacts. Several plant growth-promoting bacteria synthesize and accumulate the intracellular polymer polyhydroxybutyrate (PHB). However, the role of PHB in plant-bacterium interactions is poorly understood. In this study, applying the C4 model grass Setaria viridis and several mutants in the PHB metabolism of the endophyte Herbaspirillum seropedicae yielded new findings on the importance of PHB for bacterial colonization of S. viridis roots. Taken together, the results show that deletion of genes involved in the synthesis and degradation of PHB reduced the ability of the bacteria to enhance plant growth but with little effect on overall root colonization. The data suggest that PHB metabolism likely plays an important role in supporting specific metabolic routes utilized by the bacteria to stimulate plant growth.

Effect of Different Storage Conditions on Analytical and Sensory Quality of Thermally Processed, Milk-Based Germinated Foxtail Millet Porridge.

Foxtail millet porridge was prepared using germinated grains and milk and was evaluated for its storage stability after thermal processing at ultra-high temperatures (UHT) of 142 °C for 5 s and retort processing temperatures of 121.5 °C for 15 min. Various physical, chemical, and microbial changes of the porridge were studied for a storage period of 180 days at 25 ± 1 °C. Using consumer perception and survival analysis, the predicted shelf life of the UHT treated and retort processed foxtail millet porridge samples stored at 25 ± 1 °C was found to be 186 ± 9 days and 245 ± 15 days, respectively. Also, data from consumer liking, profiling, physical, chemical, and microbial parameters showed significant changes (P < 0.05) in the thermally treated packaged porridge samples over time. As the consumer overall acceptability decreased, the detection of positive attributes (thick and uniformly colored texture and appearance; grainy mouth texture; caramel taste and aroma) in the porridge decreased, while the detection of negative attributes (uneven, decolored, and curdled texture and appearance; sticky mouth texture; cooked, sour and off smell; cooked, sour and off taste) increased. The present study could establish a significant difference (P < 0.05) in the storage induced properties of UHT and retort processed porridge samples. The analytical evaluation of foxtail millet porridge found that UHT treated porridge was better in quality, but consumers preferred retort processed porridge. PRACTICAL APPLICATION: The quality and sensory attributes, evaluated for UHT treated and retort processed porridge samples during the storage period of 180 days, were found to be contradictory. Based on the results of CATA sensory analysis, the shelf life of UHT treated and retort processed porridge samples was predicted to be more than 6 months. Therefore, both UHT treatment and retort processing can be effectively applied to prepare a ready to eat milk based porridge using germinated foxtail millet grains.

Induced accumulation of tyramine, serotonin, and related amines in response to Bipolaris sorokiniana infection in barley.

The inducible metabolites were analyzed in barley leaves inoculated with Bipolaris sorokiniana, the causal agent of spot blotch of barley. HPLC analysis revealed that B. sorokiniana-infected leaves accumulated 4 hydrophilic compounds. They were purified by ODS column chromatography and preparative HPLC. Spectroscopic analyses revealed that they were tyramine (1), 3-(2-aminoethyl)-3-hydroxyindolin-2-one (2), serotonin (3), and 5,5'-dihydroxy-2,4'-bitryptamine (4). Among these, 2 and 4 have not been reported as natural products. They showed antifungal activity in an assay of inhibition of B. sorokiniana conidia germination, suggesting that they play a role in the chemical defense of barley as phytoalexins. The accumulation of 1-4 was examined also in the leaves of rice and foxtail millet. Rice leaves accumulated 2, 3, and 4, whereas foxtail millet leaves accumulated 3 and 4 in response to pathogen attack, suggesting the generality of accumulation of 3 and 4 in the Poaceae species.

The oil-contaminated soil diazotroph Azoarcus olearius DQS-4T is genetically and phenotypically similar to the model grass endophyte Azoarcus sp. BH72.

The genome of Azoarcus olearius DQS-4T , a N2 -fixing Betaproteobacterium isolated from oil-contaminated soil in Taiwan, was sequenced and compared with other Azoarcus strains. The genome sequence showed high synteny with Azoarcus sp. BH72, a model endophytic diazotroph, but low synteny with five non-plant-associated strains (Azoarcus CIB, Azoarcus EBN1, Azoarcus KH32C, A. toluclasticus MF63T and Azoarcus PA01). Average Nucleotide Identity (ANI) revealed that DQS-4T shares 98.98% identity with Azoarcus BH72, which should now be included in the species A. olearius. The genome of DQS-4T contained several genes related to plant colonization and plant growth promotion, such as nitrogen fixation, plant adhesion and root surface colonization. In accordance with the presence of these genes, DQS-4T colonized rice (Oryza sativa) and Setaria viridis, where it was observed within the intercellular spaces and aerenchyma mainly of the roots. Although they promote the growth of grasses, the mechanism(s) of plant growth promotion by A. olearius strains is unknown, as the genomes of DQS-4T and BH72 do not contain genes for indole acetic acid (IAA) synthesis nor phosphate solubilization. In spite of its original source, both the genome and behaviour of DQS-4T suggest that it has the capacity to be an endophytic, nitrogen-fixing plant growth-promoting bacterium.

Robust biological nitrogen fixation in a model grass-bacterial association.

Nitrogen-fixing rhizobacteria can promote plant growth; however, it is controversial whether biological nitrogen fixation (BNF) from associative interaction contributes to growth promotion. The roots of Setaria viridis, a model C4 grass, were effectively colonized by bacterial inoculants resulting in a significant enhancement of growth. Nitrogen-13 tracer studies provided direct evidence for tracer uptake by the host plant and incorporation into protein. Indeed, plants showed robust growth under nitrogen-limiting conditions when inoculated with an ammonium-excreting strain of Azospirillum brasilense. (11)C-labeling experiments showed that patterns in central carbon metabolism and resource allocation exhibited by nitrogen-starved plants were largely reversed by bacterial inoculation, such that they resembled plants grown under nitrogen-sufficient conditions. Adoption of S. viridis as a model should promote research into the mechanisms of associative nitrogen fixation with the ultimate goal of greater adoption of BNF for sustainable crop production.

Colonization and bioherbicidal activity on green foxtail by Pseudomonas fluorescens BRG100 in a pesta formulation.

Pseudomonas fluorescens BRG100 produces secondary metabolites with herbicidal activity on green foxtail ( Setaria viridis ), an important weed pest in Canadian agriculture. Five gfp transformants of P. fluorescens BRG100 were compared with the wild-type isolate for green foxtail root herbicide activity, i.e., root growth suppression, doubling time, carbon utilization, and colonization of green foxtail root (proximal and distal regions). The most revealing comparison between the wild type and its gfp transformants was herbicidal activity on green foxtail. Herbicidal activity of transformant gfp-7 was not significantly different from the uninoculated control, suggesting that insertion of the gfp gene may have interfered with a gene, or genes, vital to the bioherbicide process. Doubling time, carbon utilization, and colonization of green foxtail did not differ to a great extent between the wild type and the gfp transformants, indicating their suitability as conservatively tagged organisms for subsequent colonization-herbicidal activity studies. Accordingly, a pesta granule formulation delivered transformant gfp-2 to the seed coat and roots of green foxtail. Epifluorescent and confocal laser scanning microscopy revealed the transformant gfp-2 colonized the ventral portion of the seed coat, root hairs, and all areas of the root except the root cap region, where gfp-2 presumably exerted herbicidal effects. These results suggest that P. fluorescens BRG100 has considerable potential as a bioherbicide because of its successful colonization and suppressive activity on green foxtail root growth.

Differentiation of Magnaporthe species complex by rep-PCR genomic fingerprinting.

Rice blast disease, caused by the fungus Magnoporthe grisea is responsible for considerable damages on rice and leaf spot on some weeds in Iran and in other parts of the world. Infected samples were collected from rice and weeds including Digitaria sanguinalis (crabgrass), Setaria italica (foxtail millet), Echinochloa crus-galli (barnyard millet), and some unknown weeds during 1997-2005 and were preserved in collection of Mycology at the University of Tehran, Iran. In this study, genetic diversity of Magnaporthe grisea species complex isolates was studied based on DNA fingerprinting by rep-PCR, using of two primers including ERIC and BOX. The total DNA of 75 isolates was extracted and DNA fragments were amplified in a thermal cycler program using mentioned primers. Therefore, DNA fragments from 400 bp to 3000 bp were amplified. Based on cluster analysis for two primers (ERIC and BOX), eight fingerprinting groups (ctonal lineages) and sixty haplotypes were identified. "A" clonal lineage was containing the highest number of isolates and became dominant clonal lineages with 35 isolates from rice and 3 isolates from S. italica, whereas the highest number of isolates obtained from D. sanguinalis belonged to "E" clonal lineage and was the second largest clonal lineage. Approximately all of the M. grisea species complex isolates from crabgrass and some of unknown weeds were separated from other isolates in 42% similarity. As a result, asexual fertility causes low diversity in populations of M. grisea species complex and speciation could be one of the reasons of differentiation between isolates from D. sanguinalis with other isolates. Overall, these data indicated a low level of genetic diversity in the Iranian M. grisea species complex population similar to that reported in other countries.

Determining the fertility status of Setaria infecting Magnaporthe grisea isolates with standard testers and identification of tolerant cultivar of Setaria italica.

A total of 128 isolates of Setaria-infecting Magnaporthe grisea strains were obtained from different states of South India which includes sampling sites from Tamil Nadu, two from Karnataka, one from Andhra Pradesh and Kerala. Out of the selected 128 isolates 30 strains were tested with MAT1-1 and MAT1-2 fertile standard testers to determine their mating type. None of the 30 Setaria isolates produced perithecia with fertile testers. However, when monoconidial isolates were mated among themselves, isolates from the same field produced only barren perithecia and the tester isolates were able to mate readily with finger millet isolates. This is the first report of the mating-type studies on Setaria infecting Magnaporthe grisea with standard testers. This result indicates that the Setaria infecting population is infertile. In pathogenicity assay, it was found that 9 out of the 22 Setaria accessions were highly susceptible to Setaria strains of the blast fungus and seven cultivars/accessions were resistant to blast pathogen. Various virulence reactions were scored according to Standard Evaluation System.