Project description:Sugarcane plantlets from a variety with high inputs of N obtained from BNF (genotype SP70-1143, CTC, Brazil) free of microorganisms were obtained by sterile meristem culture and micropropagation according to the method of Hendre et al. (1983). In vitro-grown SP70-1143 rooted sugarcane plantlets were inoculated as described by James et al. (1994) with 0.1 ml of 106–107 bacterial suspension. Controls were inoculated with medium only. Endophytic diazotrophic bacteria used were Gluconacetobacter diazotrophicus (PAL5 strain) or a mixture of Herbaspirillum seropedicae (HRC54 strain) and H. rubrisubalbicans (HCC103 strain). All plants were maintained at 30°C with an irradiance of 60 µmol photons m–2 s–1 for 12 h d–1. One day after the inoculation, plant tissues were examined for bacterial colonization by the Most Probable Number (MPN) estimation, according to the methods of Reis et al. (1994) and plantlets were collected and immediately frozen in liquid nitrogen. Five plantlets were polled for each treatment. Extraction of total RNA was performed separately on each sample pool. Keywords: comparison of associations with different endophytic bacterias
Project description:Citrus variegated chlorosis (CVC), caused by Xylella fastidiosa, is an important citrus disease that produces chlorotic injuries on leaves and reduced fruit size. This bacterium colonizes plant xylem, thereby interrupting sap flow. Other disease symptoms depend on environmental factors, since asymptomatic and symptomatic CVC plants may be genetically similar. The endophytic microbiome comprises many microbial species that may interact with pathogens, reducing disease symptoms and improving plant growth. However, the genetic and physiological mechanisms that underlie this interaction are largely unknown. In this study, the citrus endophytic bacterium Methylobacterium mesophilicum SR1.6/6 was isolated from healthy plants. This bacterium was able to colonize citrus xylem and could be transferred from plant to plant by Bucephalogonia xanthopis (Insecta), suggesting that this endophytic bacterium may interact with X. fastidiosa in planta, as a result of co-transmission by the same insect vector. To better understand how X. fastidiosa genetic responds to the presence of M. mesophilicum in the same environment, we used microarrays to evaluate the transcriptional profile of X. fastidiosa, after in vitro co-cultivation with M. mesophilicum SR1.6/6. The results showed that during co-cultivation with M. mesophilicum, X. fastidiosa downregulated genes related to growth, while genes related to energy production (cellular respiration) and transport were upregulated. Moreover, X. fastidiosa modulates genes associated with molecular recognition, nutrient competition and the stress response, suggesting the existence of a specific adaptive response to the presence of M. mesophilicum in the culture medium
Project description:Citrus variegated chlorosis (CVC), caused by Xylella fastidiosa, is an important citrus disease that produces chlorotic injuries on leaves and reduced fruit size. This bacterium colonizes plant xylem, thereby interrupting sap flow. Other disease symptoms depend on environmental factors, since asymptomatic and symptomatic CVC plants may be genetically similar. The endophytic microbiome comprises many microbial species that may interact with pathogens, reducing disease symptoms and improving plant growth. However, the genetic and physiological mechanisms that underlie this interaction are largely unknown. In this study, the citrus endophytic bacterium Methylobacterium mesophilicum SR1.6/6 was isolated from healthy plants. This bacterium was able to colonize citrus xylem and could be transferred from plant to plant by Bucephalogonia xanthopis (Insecta), suggesting that this endophytic bacterium may interact with X. fastidiosa in planta, as a result of co-transmission by the same insect vector. To better understand how X. fastidiosa genetic responds to the presence of M. mesophilicum in the same environment, we used microarrays to evaluate the transcriptional profile of X. fastidiosa, after in vitro co-cultivation with M. mesophilicum SR1.6/6. The results showed that during co-cultivation with M. mesophilicum, X. fastidiosa downregulated genes related to growth, while genes related to energy production (cellular respiration) and transport were upregulated. Moreover, X. fastidiosa modulates genes associated with molecular recognition, nutrient competition and the stress response, suggesting the existence of a specific adaptive response to the presence of M. mesophilicum in the culture medium To evaluate and compare the Xylella fastidiosa 9a5c transcriptome profiles from bacteria cultured in PW and in co-cultive with Methylobacterium mesophilicum SR1.6/6, bacterial cultures were harvested for total RNA extraction after 24h of inoculation. Samples from the resulting RNAs were then used in competitive hybridizations against Xf microarrays. Replicated experiments were performed with RNA preparations from cells in each treatment and since each microarray carried two replicas of each spotted gene, we ended up with a series of 4 independent readings for each gene present in the microarrays. Images were analyzed with the TIGR Spotfinder program (v.2.2.4). All spots with median values lower than the median local background plus two Standard Deviations have been flagged and excluded from further analyses. Replicated experiments were performed with two independent RNA preparations from cells cultivated in each medium. For each pair of RNA preparations, two independent hybridizations were performed, with dye swaps within each pair. The results from each hybridization were submitted to a series of mathematical transformations with the aid of the software TIGR MIDAS v.2.19. These included filtering out all spots whose integrated intensities were below 10,000 a/d units, normalization between the two channels with the aid of the Lowess algorithm and SD regularization of the Cy5/Cy3 ratios across all sectors (blocks) of the array. Finally, the results from each individual experiment were loaded into the software TIGR Multi-Experiment Viewer (TMEV), v.3.01. Experiments were then normalized and genes that displayed statistically significant modulation were identified with the aid of the one-class option of the Significance Analysis of Microarrays (SAM) test, described by Tusher et al. (2001). The δ factor of the SAM test was adjusted to guarantee a False Discovery Rate (FDR) < 1.
Project description:Endophytic bacteria influence plant growth and development and therefore are an attractive resource for applications in agriculture. However, little is known about the impact of these microorganisms on secondary metabolite (SM) production by medicinal plants. Here we assessed, for the first time, the effects of root endophytic bacteria on the modulation of SMs in the medicinal plant Lithospermum officinale (Boraginaceae family), with a focus on the naphthoquinones alkannin/shikonin (A/S). The study was conducted using a newly developed in vitro system as well as in the greenhouse. Targeted and non-targeted metabolomics approaches were used and supported by expression analysis of the gene PGT, encoding a key enzyme in the A/S biosynthesis pathway. Three bacterial strains, Chitinophaga sp. R-73072, Xanthomonas sp. R-73098 and Pseudomonas sp. R-71838 induced a significant increase of diverse SMs, including A/S, in L. officinale in both systems, demonstrating the strength of our approach for screening A/S derivative-inducing bacteria. Our results highlight the impact of root-endophytic bacteria on secondary metabolism in plants and indicate that production of A/S derivatives in planta likely involves cross-modulation of different metabolic pathways that can be manipulated by bacterial endophytes.
Project description:The experiments were performed to understand the molecular basis of plant growth promotion in rice by Rhodotorula mucilaginosa JGTA-S1, an endophytic yeast from Typha angustifolia.