Transcriptional profiling of Bacillus amyloliquefaciens FZB42 in response to seed and root exudates collected under different nutrient regimes

Plant growth-promoting rhizobacteria (PGPR) live in close association with plants and improve their growth. Bacillus amyloliquefaciens strain FZB42 is a prominent plant root-colonizing bacterium that is able to stimulate the growth of maize. To decipher the molecular cross-talk between B. amyloliquefaciens and crop plants, an exploratory analysis of the effect of seed and root exudates on the transcriptome of Bacillus amyloliquefaciens FZB42 was performed. Root exudates were collected from maize plants grown in an axenic hydroponic system under nutrient sufficiency or under deficient supply of nitrogen (N), phosphorus (P), iron (Fe) or potassium (K). An analysis of primary metabolites in the exudates was carried out, compared between treatments, and correlated with the transcriptional profiles of Bacillus amyloliquefaciens FZB42 that were gained after incubation of the bacterial culture with the root exudates. Higher exudation rates of citrate were found under Fe deficiency and greater release of ã-amino butyric acid under P deficiency. Based on a negative correlation observed between the average diffusion coefficient of N, P, K, and Fe in soils and the exudation rates of primary metabolites under conditions of N, P, K, or Fe deficiency, it was hypothesized that the exudation of sugars, amino acids and organic acids may reflect the availability and mobility of plant nutrients in soils. In the presence of seed and root exudates collected from nutrient-sufficient plants, genes involved in spore germination, transport and utilization of nutrients, biosynthesis pathways, multidrug transporters, motility and competence development were differentially expressed. In comparison to P, Fe and K, N-deficient maize root exudates caused a more distinguished change in the transcriptome of bacteria when they were in the logarithmic growth phase. During this growth phase, a number of genes coding for ribosomal proteins were down-regulated by N-deficient maize root exudates, indicating that bacterial activity was repressed. Exclusively in the presence of P-deficient maize root exudates, several genes associated to bacterial motility were induced. Moreover, a gene involved in the biosynthesis of the auxin precursor tryptophan was up-regulated by all deficiency treatments. In the transitional growth phase of Bacillus amyloliquefaciens FZB42, several genes were commonly down-regulated in different deficiency treatments. This finding is in agreement with previous studies showing that quorum-sensing and starvation-sensing are integrated to regulate cell entry into the transient phase. Taken together, this is the first study comparing the effect of different nutrient deficiencies on the composition of primary metabolites in root exudates of one plant species and evaluating systematically the transcriptional response of a Gram-positive PGPR to seed and root exudates collected from plants grown under different nutrient regimes. This analysis provides new information about the early communication between plant roots and PGPR and points to involved genes and processes that merit further investigation.Pflanzenwachstumsfördernde Rhizosphärenbakterien (plant growth-promoting rhizobacteria, PGPR) leben in enger Assoziation mit Pflanzen und verbessern deren Wachstum. Bacillus amyloliquefaciens FZB42 ist ein prominenter Vertreter wurzelkolonisierender Bakterien, die das Wachstum von Maispflanzen stimulieren. Um einen Beitrag zur Aufklärung des molekularen Signalaustausches zwischen Bacillus amyloliquefaciens und Kulturpflanzen zu leisten, wurde der Einfluss von Samen- und Wurzelexsudaten auf die Gesamtheit der exprimierten Gene (Transkriptom) von Bacillus amyloliquefaciens FZB42 untersucht. In einer axenischen Nährlösungskultur wurden Wurzelexsudate von Maispflanzen gesammelt, die unter ausreichender Nährstoffversorgung oder unter Mangel an Stickstoff (N), Phosphor (P), Eisen (Fe) oder Kalium (K) angezogen wurden. Die gesammelten Exsudate wurden hinsichtlich ihrer Zusammensetzung an primären Metaboliten charakterisiert und dann in Bezug zu den Transkriptionsprofilen der Bakterien gesetzt. Unter Fe-Mangel kam es zu höheren Exsudationsraten an Citrat und unter P-Mangel zu verstärkter Abgabe an γ-Aminobuttersäure. Auf Grundlage einer negativen Korrelation zwischen dem durchschnittlichen Diffusionskoeffizienten von N, P, K und Fe in Böden und den Exsudationsraten von Primärmetaboliten unter N-, P-, K- oder Fe-Mangel wurde die Hypothese aufgestellt, dass die Abgabe von Zuckern, Aminosäuren und organischen Säuren die Verfügbarkeit und Mobilität dieser Pflanzenährstoffe in Böden widerspiegelt. Einige Gene der Sporenbildung und -keimung, des Nährstofftransportes und Stoffwechsels, verschiedener Biosynthesewege, sowie der Mobilität und Kompetenzentwicklung waren nach Inkubation der Bakterien mit Samen- oder Wurzelexsudaten differentiell exprimiert, wenn die Pflanzen keinen Nährstoffmangel hatten. Im Vergleich zu Exsudaten aus P-, Fe- oder K-Mangelpflanzen, führten Exudate von N-Mangelpflanzen zu einer stärkeren Veränderung des bakteriellen Transkriptoms, wenn sich die Bakterien in der logarithmischen Wachstumsphase befanden. Während dieser Wachstumsphase wurde durch N-Mangelexsudate eine Vielzahl von Genen reprimiert, die ribosomale Proteine kodieren, wodurch sich andeutet, dass die bakterielle Aktivität gehemmt wurde. Nur in Gegenwart von P-Mangelexsudaten wurden bakterielle Gene induziert, die mit der Motilität der Bakterien zusammenhängen. Darüber hinaus wurde durch alle Exsudate von Mangelpflanzen ein Gen induziert, das an der Biosyntheses der Auxinvorstufe Tryptophan beteiligt ist. In der stationären Wachstumsphase von Bacillus amyloliquefaciens FZB42 gab es nur geringe Unterschiede in der Expression von Genen durch die unterschiedlichen Exsudate. Dies ist in Übereinstimmung mit früheren Arbeiten, die zeigen, dass ?quorum-sensing? und ?Mangel-sensing? integriert werden, um den Eintritt der Bakterien in die stationäre Wachstumsphase einzuleiten. Diese Arbeit vergleicht zum ersten Mal den Einfluss unterschiedlicher Nährstoffmangelsituationen auf das Profil primärer Metabolite in Wurzelexsudaten in einer Pflanzenart und untersucht systematisch die transkriptionelle Reaktion eines Grampositiven, wachstumsfördernden Bakteriums auf Samen- und Wurzelexsudate von Pflanzen in unterschiedlicher Nährstoffmangelsituationen. Mit dem Hinweis auf Gene und Prozesse, die am molekularen Signalaustausch zwischen Pflanzenwurzeln und PGPR beteiligt sein könnten, leistet diese Arbeit einen Beitrag zum besseren Verständnis der möglichen Kommunikation der beiden Partner innerhalb ihrer Assoziation

Molecular diagnostics of banana Fusarium wilt targeting Secreted-in-Xylem genes

Fusarium wilt is currently spreading in banana growing regions around the world leading to substantial losses. The disease is caused by the fungus Fusarium oxysporum f. sp. cubense (Foc), which is further classified into distinct races according to the banana varieties that they infect. Cavendish banana is resistant to Foc race 1, to which the popular Gros Michel variety succumbed last century. Cavendish effectively saved the banana industry, and became the most cultivated commercial variety worldwide. However, Foc tropical race 4 (TR4) subsequently emerged in Southeast Asia, causing significant yield losses due to its high level of aggressiveness to Cavendish and other commonly grown varieties. Preventing further spread is crucially important in the absence of effective control methods or resistant market-acceptable banana varieties. Implementation of quarantine and containment measures depends on early detection of the pathogen through reliable diagnostics. In this study, we tested the hypothesis that secreted in xylem (SIX) genes, which currently comprise the only known family of effectors in F. oxysporum, contain polymorphisms to allow the design of molecular diagnostic assays that distinguish races and relevant VCGs of Foc. We present specific and reproducible diagnostic assays based on conventional PCR targeting SIX genes, using as templates DNA extracted from pure Foc cultures. Sets of primers specifically amplify regions of: SIX6 in Foc race 1, SIX1 gene in TR4, SIX8 in subtropical race 4, SIX9/SIX10 in Foc VCG 0121, and SIX13 in Foc VCG 0122. These assays include simplex and duplex PCRs, with additional restriction digestion steps applied to amplification products of genes SIX1 and SIX13. Assay validations were conducted to a high international standard including the use of 250 Fusarium spp. isolates representing 16 distinct Fusarium species, 59 isolates of F. oxysporum, and 21 different vegetative compatibility groups (VCGs). Tested parameters included inter and intraspecific analytical specificity, sensitivity, robustness, repeatability and reproducibility. The resulting suite of assays is able to reliably and accurately detect R1, STR4, TR4 as well as two VCGs (0121 and 0122) causing Fusarium wilt in bananas

Rhizobium strains competitiveness on bean nodulation in Cerrado soils

The objective of this work was to identify the most competitive and effective Rhizobium strains in order to increase common bean yield by nitrogen fixation as alternative or complementation to the nitrogen fertilization. Competitiveness tests were lead in axenic conditions, in Cerrado soil pots and in three field experiments, with native Rhizobium strains that were previously identified, according to their effectiveness and genetic variability. The identification of strains in nodules was performed using serological tests (axenic conditions) - agglutination and enzyme linked immunosorbent (Elisa) assays - and random amplified polymorfic DNA (RAPD) (Cerrado soil). Plant yield was determined using the dry weight (greenhouse conditions), total N and grain yield (field experiments). Among the analyzed Rhizobium strains, native strain SLA 2.2 and commercial strain CIAT 899 were the dominant nodules in plants of the most productive plots, presenting yield productivity similar or higher to those obtained in treatments where 20 kg ha-1 of N were applied.O objetivo deste trabalho foi identificar as estirpes de Rhizobium mais efetivas e competitivas, a fim de maximizar a produtividade do feijoeiro por meio da fixação de nitrogênio, como alternativa à adubação nitrogenada. Foram conduzidos testes de competitividade em condições axênicas, em vasos com solo do Cerrado e em três experimentos de campo, com estirpes de Rhizobium nativas, previamente selecionadas quanto à efetividade e à variabilidade genética. A identificação das estirpes nos nódulos foi efetuada por meio das técnicas de aglutinação e ensaio imunoabsorvente de ligação de enzimas (Elisa), em condições de casa de vegetação, e pela técnica de DNA polimórfico amplificado ao acaso (RAPD), em solo de Cerrado. A produtividade das plantas foi determinada pela produção de matéria seca, teor de N e produção de grãos (condições de campo). A estirpe nativa SLA 2.2 e a estirpe comercial CIAT 899 foram dominantes nos nódulos das plantas das parcelas mais produtivas, com índices de produtividade iguais ou superiores aos obtidos nos tratamentos em que foram aplicados 20 kg ha-1 de N

Development of marker genes for jasmonic acid signaling in shoots and roots of wheat

The jasmonic acid (JA) signaling pathway plays key roles in a diverse array of plant development, reproduction, and responses to biotic and abiotic stresses. Most of our understanding of the JA signaling pathway derives from the dicot model plant Arabidopsis thaliana, while corresponding knowledge in wheat is somewhat limited. In this study, the expression of 41 genes implicated in the JA signaling pathway has been assessed on 10day-old bread wheat seedlings, 24h, 48h, and 72h after methyl-jasmonate (MeJA) treatment using quantitative real-time PCR. The examined genes have been previously reported to be involved in JA biosynthesis and catabolism, JA perception and signaling, and pathogen defense in wheat shoots and roots. This study provides evidence to suggest that the effect of MeJA treatment is more prominent in shoots than roots of wheat seedlings, and substantial regulation of the JA pathway-dependent defense genes occurs at 72h after MeJA treatment. Results show that the expression of 22 genes was significantly affected by MeJA treatment in wheat shoots. However, only PR1.1 and PR3 were significantly differentially expressed in wheat roots, both at 24h post-MeJA treatment, with other genes showing large variation in their gene expression in roots. While providing marker genes on JA signaling in wheat, future work may focus on elucidating the regulatory function of JA-modulated transcription factors, some of which have well-studied potential orthologs in Arabidopsis

Capacidade competitiva de estirpes de Rhizobium na nodulação do feijoeiro em solos de Cerrado

The objective of this work was to identify the most competitive and effective Rhizobium strains in order to increase common bean yield by nitrogen fixation as alternative or complementation to the nitrogen fertilization. Competitiveness tests were lead in axenic conditions, in Cerrado soil pots and in three field experiments, with native Rhizobium strains that were previously identified, according to their effectiveness and genetic variability. The identification of strains in nodules was performed using serological tests (axenic conditions) – agglutination and enzyme linked immunosorbent (Elisa) assays – and random amplified polymorfic DNA (RAPD) (Cerrado soil). Plant yield was determined using the dry weight (greenhouse conditions), total N and grain yield (field experiments). Among the analyzed Rhizobium strains, native strain SLA 2.2 and commercial strain CIAT 899 were the dominant nodules in plants of the most productive plots, presenting yield productivity similar or higher to those obtained in treatments where 20 kg ha-1 of N were applied.O objetivo deste trabalho foi identificar as estirpes de Rhizobium mais efetivas e competitivas, a fim de maximizar a produtividade do feijoeiro por meio da fixação de nitrogênio, como alternativa à adubação nitrogenada. Foram conduzidos testes de competitividade em condições axênicas, em vasos com solo do Cerrado e em três experimentos de campo, com estirpes de Rhizobium nativas, previamente selecionadas quanto à efetividade e à variabilidade genética. A identificação das estirpes nos nódulos foi efetuada por meio das técnicas de aglutinação e ensaio imunoabsorvente de ligação de enzimas (Elisa), em condições de casa de vegetação, e pela técnica de DNA polimórfico amplificado ao acaso (RAPD), em solo de Cerrado. A produtividade das plantas foi determinada pela produção de matéria seca, teor de N e produção de grãos (condições de campo). A estirpe nativa SLA 2.2 e a estirpe comercial CIAT 899 foram dominantes nos nódulos das plantas das parcelas mais produtivas, com índices de produtividade iguais ou superiores aos obtidos nos tratamentos em que foram aplicados 20 kg ha-1 de N

Strategic tillage in conservation agricultural systems of north-eastern Australia: why, where, when and how?

Farmers often resort to an occasional tillage (strategic tillage (ST)) operation to combat constraints of no-tillage (NT) farming systems. There are conflicting reports regarding impacts of ST and a lack of knowledge around when, where and how ST is implemented to maximise its benefits without impacting negatively on soil and environment. We established 14 experiments during 2012–2015 on farms with long-term history of continuous NT to (i) quantify the associated risks and benefits to crop productivity, soil and environmental health and (ii) explore key factors that need to be considered in decisions to implement ST in an otherwise NT system. Results showed that introduction of ST reduced weed populations and improved crop productivity and profitability in the first year after tillage, with no impact in subsequent 4 years. Soil properties were not impacted in Vertosols; however, Sodosols and Dermosols suffered short-term negative soil health impacts (e.g. increased bulk density). A Sodosol and a Dermosol also posed higher risks of runoff and associated loss of nutrients and sediment during intense rainfall after ST. The ST reduced plant available water in the short term, which could result in unreliable sowing opportunities for the following crop especially in semi-arid climate that prevails in north-eastern Australia. The results show that generally, there were no significant differences in crop productivity and soil health between tillage implements and tillage frequencies between ST and NT. The study suggests that ST can be a viable strategy to manage constraints of NT systems, with few short-term soil and environmental costs and some benefits such as short-term farm productivity and profitability and reduced reliance on herbicides