No document with DOI "10.1.1.864.437"Trichoderma parareesei and Transformation of steroids by trichoderma hamatum reesei teleomorph Hypocrea jecorina produce cellulases and xylanases of industrial interest. Here, the anamorphic strain T6 formerly T. A Trichoderma microarray was used to examine the transcriptomic changes in T6 at 20 h of interaction with tomato plants. Out of a total 34, Trichoderma probe sets transformation of steroids by trichoderma hamatum transcormation the microarray, showed a significant change of at least 2-fold danabol ds 10mg reviews expression in the presence of tomato plants, with most of them being downregulated. Time course expression patterns 0 to 6 days observed for defense-related genes suggest that T6 was able to prime defense responses in the tomato plants transfor,ation biotic and abiotic stresses. Our study demonstrates that the T.
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Trichoderma parareesei and Trichoderma reesei teleomorph Hypocrea jecorina produce cellulases and xylanases of industrial interest. Here, the anamorphic strain T6 formerly T. A Trichoderma microarray was used to examine the transcriptomic changes in T6 at 20 h of interaction with tomato plants.
Out of a total 34, Trichoderma probe sets deposited on the microarray, showed a significant change of at least 2-fold in expression in the presence of tomato plants, with most of them being downregulated. Time course expression patterns 0 to 6 days observed for defense-related genes suggest that T6 was able to prime defense responses in the tomato plants against biotic and abiotic stresses.
Our study demonstrates that the T. Trichoderma is a genus of filamentous fungi that includes strains used as biocontrol agents in agriculture due to their antagonistic abilities against phytopathogenic fungi and oomycetes 1. In addition, Trichoderma strains are also able to stimulate defense responses in plants, inducing resistance to biotic and abiotic stresses, and to promote plant growth 5 — 7. Trichoderma reesei teleomorph Hypocrea jecorina is widely used in the enzyme industry for the production of cellulases 17 — 19 , and it has also been used as a cell factory to express numerous recombinant proteins Trichoderma pseudokoningii is another interesting species within the clade Longibrachiatum with the ability to inhibit mycorrhizal spore germination 24 , produce peptaibols able to induce defense responses in plants 25 , and trigger apoptosis in phytopathogenic fungi To date, no studies on the interaction of these two species with living plants have been reported.
The aim of the present study was to analyze the biocontrol potential of T. A Trichoderma high-density oligonucleotide HDO microarray was used to examine the transcriptomic profiles in T. Physiological parameters and expression changes of plant defense marker genes linked to biotic and environmental stresses were used to analyze the response of the tomato plants to T.
This paper reports that the T. Microorganisms and tomato seeds. Trichoderma parareesei formerly T. Spores from 7-day-old PDA plates were harvested by adding 5 ml of water to the plates and scraping the culture with a rubber spatula. These suspensions were filtered through a double layer of cheesecloth to separate large mycelial fragments from conidia.
Spore concentrations were calculated using a counting chamber, and suspensions were used to inoculate the medium. Rhizoctonia solani 19, Botrytis cinerea B The mycelium was collected by filtration, washed with distilled water, frozen, and lyophilized.
Fungal genomic DNA was isolated according to previously described protocols In vitro confrontation assays between T6 and the pathogens P. Growth assays on cellophane sheets and kDa-cutoff dialysis cellulose membranes were carried out on PDA for P. Each pathogen was tested in triplicate. Growth diameters were measured after 72 h for P. The results are expressed as the percentage of growth inhibition of each pathogen by T6, with respect to the mean colony diameters of each pathogen grown alone.
Trichoderma -plant interaction in hydroponic culture for microarray experiments. Control mycelia were grown under the same conditions but in the absence of tomato plants.
After this period, the T6 mycelium was collected by filtration the mycelium on the plant roots was recovered with a direct jet of water , washed, frozen, and lyophilized.
Digitization of the fluorescent signals emitted after the hybridization was performed using an Axon GenePix B scanner with NimbleScan 2. The normalized values for each probe obtained from the 6 microarrays were scaled in the range of 0 to 1 to compensate for sequence-specific sensitivity.
The processed data for the different probes within a probe set were summed to produce an expression measure. Finally, a multiclass significance analysis of microarray SAM test was carried out on the expression values using a false-discovery rate FDR of 0. Real-time PCR quantification of transcripts. Real-time PCRs were performed with the cDNAs of four pooled biological replicates for each condition, with the exception of two pooled biological replicates used to analyze plant defense gene expression from in vitro cultures.
All PCRs were performed in triplicate, and the primers used are given in Table S1 in the supplemental material. Mycelial plugs with a diameter of 0. MM plates supplemented only with agar were used as controls. The growth of T. Experiments were performed in triplicate.
The culture media were filtered through sterile filter paper. An in vitro assay was carried out to analyze the effect of T6 on tomato seedlings. Experiments were performed in triplicate, and the plates were photographed at 4 days after T6 inoculation. Plants were grown for 7 days before T6 inoculation and photographed 2 days later. The ability of T6 to promote the growth of tomato plants was also evaluated in in vivo assays, as previously described To analyze the salt stress tolerance of tomato plants grown from T6-treated seeds or untreated seeds control , 4-week-old plants were treated daily with 5 ml of either water or mM NaCl for 10 days, and measurements of stem height and main root length were taken at this time.
Quantification of marker genes in tomato. Defense marker gene expression was checked by real-time PCR in tomato seedlings grown under in vitro and in vivo conditions. The aerial part of each tomato seedling was collected at 6 days after inoculation with T6 or water control , and it was used for RNA extraction with TRIzol reagent.
The aerial part of each tomato plant was sampled at 0, 8, 24, and 48 h and 6 days and used for RNA extraction. The primer pairs used for real-time PCR of these genes, under the conditions indicated above, are shown in Table S1 in the supplemental material.
After 3 days, plants were leaf inoculated with B. The appearance of necrotic spots was assessed at 4 days after inoculation. Values of 0 absence of reaction to 7 high necrotic reaction more than 1. The sequences of the tef1 , cal1 , and las1 fragments were deposited in the GenBank database under accession numbers KF , KF , and KF , respectively.
Molecular characterization of the T6 strain. Strain T6, formerly identified as T. The sequences of the tef1 , cal1 , and las1 gene fragments were obtained, and the fourth intron of tef1 and the second and third introns of cal1 served to identify strain T6 as T. Antagonistic activity of the T6 strain.
Plate confrontation experiments between T6 and the pathogens P. The T6 strain antagonized the three pathogens in all the dual cultures, although different behaviors were observed: T6 completely overgrew the colonies of P. Antagonism assays were also performed by growing T6 on cellophane and cellulose kDa-cutoff membranes to allow the diffusion of Trichoderma extracellular compounds into the medium. After removal of the membranes containing the mycelium, the effect of hydrolytic enzymes plus metabolites cellophane or only metabolites cellulose on the growth of the three pathogens was determined.
Table 1 summarizes the percentages of growth inhibition of P. It may be observed that the inhibition values for the three pathogens on cellophane were always higher than the inhibition values on cellulose. The highest inhibition values corresponded to P. Transcriptional response of T.
A transcriptomic analysis using a Trichoderma HDO microarray containing 34, probe sets was performed to ascertain the physiological and biochemical changes produced in T. Transcriptomic changes detected in T. A total of probe sets 0. Thirty-three of these were upregulated, whereas were downregulated.
A majority of the differentially expressed probe sets GO terms were assigned to of the probe sets 18 up- and downregulated probe sets. We then examined whether these differentially expressed probe sets were associated with similar GO categories.
This analysis revealed that no categories were significantly overrepresented in T. Accordingly, all the differentially expressed probe sets were analyzed independently and then grouped into several physiological processes, with T6 carbohydrate metabolism and transport being the most affected Table 2. More detailed information about up- and downregulated probe sets, with indication of their corresponding orthologous numbering in the genomes of T.
Regarding transport processes, although the upregulation of two probe sets related to ABC transporters and two probe sets related to the major facilitator superfamily MFS transporters was detected, we also observed the downregulation of 16 probe sets related to other MFS transporters. In addition, we detected the downregulation of the genes involved in the metabolism of fatty acids and lipids, amino acids and proteins, nucleotides and nucleic acids, vitamins, and inorganic and nitrogen compounds, genes involved in energetic and secondary metabolism, and genes involved in several cellular processes, signaling e.
Summary of the physiological processes expressed differentially for Trichoderma parareesei T6 in response to the presence of tomato plants in the culture medium in comparison to the basal medium. To confirm the microarray results, quantitative real-time PCR was performed to analyze the expression of 9 genes: Expression of these genes correlated well with the data from the microarray experiments see Fig.
S1 in the supplemental material. Effect of tomato plants on the growth of T. An in vitro assay was performed to evaluate the influence of different plant polymers on the growth of T. Fungal growth was significantly enhanced, in comparison with that on MM plates, when 0.
T6 growth in the presence of tomato plant exudates was also evaluated on well E plates, as described in Materials and Methods. S2 in the supplemental material. We studied the effect of T6 on the development of tomato seedling roots using an in vitro assay.
This strain favored seedling root development, increasing the number and length of the lateral roots compared to those under the control condition Fig. Effect of Trichoderma parareesei T6 on tomato seedlings. In vivo assay results revealed that there were no significant differences in stem and root lengths of 4-week-old tomato plants between the two conditions Table 4.
Plants from T6 conidium-treated seeds had stem and root lengths of Effect of Trichoderma parareesei T6 and salt stress on the growth of tomato plants. To test whether the tomato response to T. Marker gene expression in tomato seedlings from in vitro assays is shown in Fig.