Reference genes with stable expression are required to normalize expression differences of target genes in qPCR experiments. Several procedures and companion software have been proposed to find the ...most stable genes. Model based procedures are attractive because they provide a solid statistical framework. NormFinder, a widely used software, uses a model based method. The pairwise comparison procedure implemented in GeNorm is a simpler procedure but one of the most extensively used. In the present work a statistical approach based in Maximum Likelihood estimation under mixed models was tested and compared with NormFinder and geNorm softwares. Sixteen candidate genes were tested in whole blood samples from control and heat stressed sheep.
A model including gene and treatment as fixed effects, sample (animal), gene by treatment, gene by sample and treatment by sample interactions as random effects with heteroskedastic residual variance in gene by treatment levels was selected using goodness of fit and predictive ability criteria among a variety of models. Mean Square Error obtained under the selected model was used as indicator of gene expression stability. Genes top and bottom ranked by the three approaches were similar; however, notable differences for the best pair of genes selected for each method and the remaining genes of the rankings were shown. Differences among the expression values of normalized targets for each statistical approach were also found.
Optimal statistical properties of Maximum Likelihood estimation joined to mixed model flexibility allow for more accurate estimation of expression stability of genes under many different situations. Accurate selection of reference genes has a direct impact over the normalized expression values of a given target gene. This may be critical when the aim of the study is to compare expression rate differences among samples under different environmental conditions, tissues, cell types or genotypes. To select reference genes not only statistical but also functional and biological criteria should be considered. Under the method here proposed SDHA/MDH1 have arisen as the best set of reference genes to be used in qPCR assays to study heat shock in ovine blood samples.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Summary
Biological control of plant diseases has gained acceptance in recent years.
B
acillus subtilis
UMAF
6639 is an antagonistic strain specifically selected for the efficient control of the ...cucurbit powdery mildew fungus
P
odosphaera fusca
, which is a major threat to cucurbits worldwide. The antagonistic activity relies on the production of the antifungal compounds iturin and fengycin. In a previous study, we found that
UMAF
6639 was able to induce systemic resistance (
ISR
) in melon and provide additional protection against powdery mildew. In the present work, we further investigated in detail this second mechanism of biocontrol by
UMAF
6639. First, we examined the signalling pathways elicited by
UMAF
6639 in melon plants, as well as the defence mechanisms activated in response to
P
. fusca
. Second, we analysed the role of the lipopeptides produced by
UMAF
6639 as potential determinants for
ISR
activation. Our results demonstrated that
UMAF
6639 confers protection against cucurbit powdery mildew by activation of jasmonate‐ and salicylic acid‐dependent defence responses, which include the production of reactive oxygen species and cell wall reinforcement. We also showed that surfactin lipopeptide is a major determinant for stimulation of the immune response. These results reinforce the biotechnological potential of
UMAF
6639 as a biological control agent.
Biological control of plant diseases has gained acceptance in recent years. Bacillus subtilis UMAF 6639 is an antagonistic strain specifically selected for the efficient control of the cucurbit ...powdery mildew fungus Podosphaera fusca, which is a major threat to cucurbits worldwide. The antagonistic activity relies on the production of the antifungal compounds iturin and fengycin. In a previous study, we found that UMAF 6639 was able to induce systemic resistance (ISR) in melon and provide additional protection against powdery mildew. In the present work, we further investigated in detail this second mechanism of biocontrol by UMAF 6639. First, we examined the signalling pathways elicited by UMAF 6639 in melon plants, as well as the defence mechanisms activated in response to P. fusca. Second, we analysed the role of the lipopeptides produced by UMAF 6639 as potential determinants for ISR activation. Our results demonstrated that UMAF 6639 confers protection against cucurbit powdery mildew by activation of jasmonate- and salicylic acid-dependent defence responses, which include the production of reactive oxygen species and cell wall reinforcement. We also showed that surfactin lipopeptide is a major determinant for stimulation of the immune response. These results reinforce the biotechnological potential of UMAF 6639 as a biological control agent. In the present work we investigated in detail the mechanisms by which the antagonistic strain Bacillus subtilis UMAF6639 is also able to induce systemic resistance in melon and provide protection against powdery mildew. Our results demonstrated that UMAF6639 combats disease by activation of jasmonate- and salicylic acid-dependent defence responses, which include the production of reactive oxygen species and cell wall reinforcement. We also showed that surfactin lipopeptide is a major determinant for stimulation of the plant immune response. These results reinforce the biotechnological potential of UMAF6639 as a biological control agent.
Biological control of plant diseases has gained acceptance in recent years. Bacillus subtilis UMAF6639 is an antagonistic strain specifically selected for the efficient control of the cucurbit ...powdery mildew fungus Podosphaera fusca, which is a major threat to cucurbits worldwide. The antagonistic activity relies on the production of the antifungal compounds iturin and fengycin. In a previous study, we found that UMAF6639 was able to induce systemic resistance (ISR) in melon and provide additional protection against powdery mildew. In the present work, we further investigated in detail this second mechanism of biocontrol by UMAF6639. First, we examined the signalling pathways elicited by UMAF6639 in melon plants, as well as the defence mechanisms activated in response to P. fusca. Second, we analysed the role of the lipopeptides produced by UMAF6639 as potential determinants for ISR activation. Our results demonstrated that UMAF6639 confers protection against cucurbit powdery mildew by activation of jasmonate‐ and salicylic acid‐dependent defence responses, which include the production of reactive oxygen species and cell wall reinforcement. We also showed that surfactin lipopeptide is a major determinant for stimulation of the immune response. These results reinforce the biotechnological potential of UMAF6639 as a biological control agent.
Agrobacterium spp. are routinely used in plant transformation to introduce genes of interest in valuable economic species. However, several agrobacteria species are also plant pathogens with ability ...to survive in different environments including the inner part of the plants. To avoid the release of genetic modified bacteria a successful plant transformation protocol must include the total elimination of agrobacteria by the use of antibiotics. Because sometimes these antibiotics failed in removing the bacteria entirely, confirmation of agrobacteria absence after plant transformation and regeneration is required. Different methodologies can be used for this purpose: isolation techniques followed by identification are used if detection of viable and culturable bacteria is necessary and techniques based on the polymerase chain reaction can be used to detect agrobacteria independently of their physiological state. Here we present several protocols to detect Agrobacterium in tissues of transformed plants as well as methods to identify the strains isolated. These identification methods can help to elucidate if they are the engineered bacteria used in the transformation process or just part of the natural endophytic microbiota.
Abstract
Laboratory experiments were conducted to study the effects of various
trapping and endoparasitic nematophagous fungi (NF) isolated from Florida
citrus orchards on five entomopathogenic ...nematode (EPN) species that show
various distributions across Florida's citrus industry. Four trapping NF
(Arthrobotrys oligospora, A. dactyloides, A. musiformis and Gamsylella
gephyropaga) and two endoparasitic NF (Catenaria sp. and Myzocytium sp.)
were tested against Steinernema diaprepesi (Sd), S. glaseri (Sg), S.
riobrave (Sr), Heterorhabditis zealandica (Hz) and H. indica (Hi). Fungi
were added to soil microcosms either as a pure culture on agar plugs
(trapping NF) or as fungal-colonised nematodes (endoparasitic NF) on agar
plugs, concurrently with 2000 EPN of a given species. After 7 or 14 days
exposure, nematodes were recovered from the soil using Baermann funnels. The
recovery of all EPN species was reduced between 56-92% by G. gephyropaga.
Neither Sd or Sg were affected by any species of Arthrobotrys, whereas A.
musiformis reduced recovery of all other EPN and A. oligospora reduced
numbers of all other species except Hi. Both endoparasitic NF reduced the
recovery of all EPN except Hi by at least 82%. The data are consistent with
the hypothesis that NF may play a role in regulating regional patterns of
abundance and diversity of EPN species in Florida, which in turn regulate
the abundance of a major citrus pest, the Diaprepes root weevil, Diaprepes
abbreviatus.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Xanthomonas arboricola is a species in genus Xanthomonas which is mainly comprised of plant pathogens. Among the members of this taxon, X. arboricola pv. pruni, the causal agent of bacterial spot ...disease of stone fruits and almond, is distributed worldwide although it is considered a quarantine pathogen in the European Union. Herein, we report the draft genome sequence, the classification, the annotation and the sequence analyses of a virulent strain, IVIA 2626.1, and an avirulent strain, CITA 44, of X. arboricola associated with Prunus spp. The draft genome sequence of IVIA 2626.1 consists of 5,027,671 bp, 4,720 protein coding genes and 50 RNA encoding genes. The draft genome sequence of strain CITA 44 consists of 4,760,482 bp, 4,250 protein coding genes and 56 RNA coding genes. Initial comparative analyses reveals differences in the presence of structural and regulatory components of the type IV pilus, the type III secretion system, the type III effectors as well as variations in the number of the type IV secretion systems. The genome sequence data for these strains will facilitate the development of molecular diagnostics protocols that differentiate virulent and avirulent strains. In addition, comparative genome analysis will provide insights into the plant-pathogen interaction during the bacterial spot disease process.
A microtiter system for biovar characterization of Agrobacterium strains which simplifies the analysis of a large number of isolates is described. This method is based on incubation of bacterial ...strains in microplate wells previously amended with media specifically used by the different Agrobacterium biovars. More than 150 purified Agrobacterium strains isolated from the most common host plants were analysed by the microtiter system. It proved to be an excellent tool using less reagents, time and space for incubation in comparison with the traditional method.PUBLICATION ABSTRACT
Agrobacterium tumefaciens was isolated from stem tumors of several rose cultivars showing that the bacterium is the causal agent of aerial galls in rose plants. No differences were observed in the ...characteristics of the Agrobacterium isolates from crown or aerial galls. Stem inoculation of ten rose cultivars showed that all of them were susceptible to A. tumefaciens but differences in the size of the resulting tumors were observed. The movement of A. tumefaciens in rose plants was demonstrated using two wild type strains and two antibiotic resistant mutants. Three months after inoculation, the inoculated strains were recovered in the roots, crown and below and above the inoculation site but low numbers of pathogenic Agrobacterium cells were isolated. New tumors appeared in 5% of the noninoculated wounds. A. tumefaciens was isolated from the stem at different distances from the tumor in naturally infected plants. In symptomless commercial plants, the isolation from the roots, crown and at different stem levels demonstrated the existence of systemic and latent infections in rose. Direct isolation using a nonselective and selective media with or without a previous enrichment step were efficient methods for isolating tumorigenic Agrobacterium from the different parts of rose plants.PUBLICATION ABSTRACT
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