Transgenic expression of lactoferrin imparts enhanced resistance to head blight of wheat caused by Fusarium graminearum

<p>Abstract</p> <p>Background</p> <p>The development of plant gene transfer systems has allowed for the introgression of alien genes into plant genomes for novel disease control strategies, thus providing a mechanism for broadening the genetic resources available to plant breeders. Using the tools of plant genetic engineering, a broad-spectrum antimicrobial gene was tested for resistance against head blight caused by <it>Fusarium graminearum </it>Schwabe, a devastating disease of wheat (<it>Triticum </it><it>aestivum </it>L.) and barley (<it>Hordeum vulgare </it>L.) that reduces both grain yield and quality.</p> <p>Results</p> <p>A construct containing a bovine lactoferrin cDNA was used to transform wheat using an <it>Agrobacterium</it>-mediated DNA transfer system to express this antimicrobial protein in transgenic wheat. Transformants were analyzed by Northern and Western blots to determine lactoferrin gene expression levels and were inoculated with the head blight disease fungus <it>F</it>. <it>graminearum</it>. Transgenic wheat showed a significant reduction of disease incidence caused by <it>F. graminearum </it>compared to control wheat plants. The level of resistance in the highly susceptible wheat cultivar Bobwhite was significantly higher in transgenic plants compared to control Bobwhite and two untransformed commercial wheat cultivars, susceptible Wheaton and tolerant ND 2710. Quantification of the expressed lactoferrin protein by ELISA in transgenic wheat indicated a positive correlation between the lactoferrin gene expression levels and the levels of disease resistance.</p> <p>Conclusions</p> <p>Introgression of the lactoferrin gene into elite commercial wheat, barley and other susceptible cereals may enhance resistance to <it>F. graminearum</it>.</p

Genetic transformation of \u3ci\u3eFusarium oxysporum\u3c/i\u3e f.sp. \u3ci\u3egladioli\u3c/i\u3e with \u3ci\u3eAgrobacterium\u3c/i\u3e to study pathogenesis in \u3ci\u3eGladiolus\u3c/i\u3e

Fusarium rot caused by Fusarium oxysporum f.sp. gladioli (Fog) is one of the most serious diseases of Gladiolus, both in the field and in bulbs in storage. In order to study the mechanisms of pathogenesis of this fungus, we have transformed Fog with Agrobacterium tumefaciens binary vectors containing the hygromycin B phosphotransferase (hph) gene and fluorescence reporter genes EGFP (green), EYFP (yellow) or ECFP (cyan) using the AGL-1 strain of A. tumefaciens. Hygromycin B (100 μg/ml) resistant colonies were observed only when acetosyringone was added to the co-cultivation medium. Transformed colonies are more clearly visible when co-cultivated on cellophane membrane than on Hybond -N+ membrane. Transformed lines were stably maintained through four serial passages on medium containing hygromycin B, and they expressed green, yellow or cyano fluorescence. PCR with hph-specific primers and Southern blotting with an hph-specific probe were positive for HygR lines but not for the untransformed isolate. The cyano fluorescence of the ECFP-transformed isolate was clearly distinguishable from the green autofluorescence of Gladiolus roots, signifying the potential of these lines for further histopathological investigations. Transformed lines will be useful for identifying pathogenicity related genes, screening transgenic resistance, and in studies of host-pathogen interactions

Gene expression profiling of the plant pathogenic basidiomycetous fungus \u3ci\u3eRhizoctonia solani\u3c/i\u3e AG 4 reveals putative virulence factors

Rhizoctonia solani is a ubiquitous basidiomycetous soilborne fungal pathogen causing damping- off of seedlings, aerial blights and postharvest diseases. To gain insight into the molecular mechanisms of pathogenesis a global approach based on analysis of expressed sequence tags (ESTs) was undertaken. To get broad gene-expression coverage, two normalized EST libraries were developed from mycelia grown under high nitrogen-induced virulent and low nitrogen/methylglucose-induced hypovirulent conditions. A pilot-scale assessment of gene diversity was made from the sequence analyses of the two libraries. A total of 2280 cDNA clones was sequenced that corresponded to 220 unique sequence sets or clusters (contigs) and 805 singlets, making up a total of 1025 unique genes identified from the two virulence-differentiated cDNA libraries. From the total sequences, 295 genes (38.7%) exhibited strong similarities with genes in public databases and were categorized into 11 functional groups. Approximately 61.3% of the R. solani ESTs have no apparent homologs in publicly available fungal genome databases and are considered unique genes. We have identified several cDNAs with potential roles in fungal pathogenicity, virulence, signal transduction, vegetative incompatibility and mating, drug resistance, lignin degradation, bioremediation and morphological differentiation. A codon-usage table has been formulated based on 14 694 R. solani EST codons. Further analysis of ESTs might provide insights into virulence mechanisms of R. solani AG 4 as well as roles of these genes in development, saprophytic colonization and ecological adaptation of this important fungal plant pathogen

Long-term cryopreservation of non-spore-forming fungi in Microbank™ beads for plant pathological investigations

Esta publicación surgió como una colaboración indirecta durante mi tiempo como estudiante de doctorado, en el cual me encontraba en el USDA ARS. El investigador principal incluyó mi afiliación como estudiante de NC State, y se omitió por error la afiliación secundaria en la Universidad de Costa Rica.Long-term preservation of experimental fungi without genetic, morphological, and pathogenic changes is of paramount importance in mycological and plant pathological investigations. Several cryogenic and non-cryo- genic methods are available for the preservation of fungi, but the methods can be cumbersome, hazardous, expensive, and often not suitable for long-term storage of non-spore-forming (sterile) fungi. A method of pre- servation of spore-forming fungi in commercially available porous beads (Micrbank™) under cryogenic condition was successfully tested for three non-spore-forming basidiomycetes genera: Rhizoctonia solani (teleomorph: Thanatephorus cucumeris) (n = 19), Ceratobasidium species (n = 1), and Waitea circinata (n = 3), and a non-spore forming ascomycetes, Sclerotinia sclerotiorum (n = 1). For comparison, spore-forming ascomycetous fungi, Alternaria alternata (n = 1), Bauveria basiana (n = 2), Botrytis cinerea (n = 1), Fusarium oxysporum f.sp. gladiolii (n = 1), Trichoderma spp. (n = 3), and Thielaviopsis basicola (n = 2) were also cryopreserved in Microbank beads. Viable fungal isolates of all test species were retrieved after five years of storage at −80 °C, which was longer than the viabilities of the corresponding isolates cryopreserved in agar plugs or colonized wheat seeds. Fungi revived from the Microbank beads maintained identical morphology and cultural characteristics of the parent isolates. Randomly selected Rhizoctonia isolates revived from the Microbank beads maintained respective pathological properties of the parent isolates; also, no mutation was detected in the internal transcribed spacer (ITS) ribosomal DNA when compared with respective cultures maintained at ambient temperature. This finding demonstrated the utility of cryopreservation in Microbank beads as a convenient alternative to conventional long-term preservation of a wide group of fungal cultures for plant pathological investigations and serves as the first report of using porous beads under cryogenic conditions for long-term storage of sterile fungi.UCR::Vicerrectoría de Docencia::Ciencias Agroalimentarias::Facultad de Ciencias Agroalimentarias::Escuela de Agronomí

Soybean Nodule-Associated Non-Rhizobial Bacteria Inhibit Plant Pathogens and Induce Growth Promotion in Tomato

The root nodules are a unique environment formed on legume roots through a highly specific symbiotic relationship between leguminous plants and nodule-inducing bacteria. Previously, Rhizobia were presumed to be the only group of bacteria residing within nodules. However, recent studies discovered diverse groups of bacteria within the legume nodules. In this report soybean nodule-associated bacteria were studied in an effort to identify beneficial bacteria for plant disease control and growth promotion. Analysis of surface-sterilized single nodules showed bacterial diversity of the nodule microbiome. Five hundred non-rhizobial colonies from 10 nodules, 50 colonies per nodule, were tested individually against the tomato wilt causing bacterial pathogen Clavibacter michiganensis subsp. michiganensis (Cmm) for inhibition of pathogen growth. From the initial screening, 54 isolates were selected based on significant growth inhibition of Cmm. These isolates were further tested in vitro on another bacterial pathogen Pseudomonas syringae pv. tomato (Pst) and two fungal pathogens Rhizoctonia solani and Sclerotinia sclerotiorum. Bacterial metabolites were extracted from 15 selected isolates with ethanol and tested against pathogen Cmm and Pst. These isolates were identified by using MALDI-TOF mass spectrometry and 16S rRNA gene sequencing. Pseudomonas spp. were the dominant soybean nodule-associated non-rhizobial bacterial group. Several isolates imparted significant protection against pathogens and/or plant growth promotion on tomato seedlings. The most promising nodule-associated bacterial isolate that suppressed both Cmm and Pst in vitro and Pst in tomato seedlings was identified as a Proteus species. Isolation and identification of beneficial nodule-associated bacteria established the foundation for further exploration of potential nodule-associated bacteria for plant protection and growth promotion

Transgenic expression of lactoferrin imparts enhanced resistance to head blight of wheat caused by \u3ci\u3eFusarium graminearum\u3c/i\u3e

Background: The development of plant gene transfer systems has allowed for the introgression of alien genes into plant genomes for novel disease control strategies, thus providing a mechanism for broadening the genetic resources available to plant breeders. Using the tools of plant genetic engineering, a broad-spectrum antimicrobial gene was tested for resistance against head blight caused by Fusarium graminearum Schwabe, a devastating disease of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) that reduces both grain yield and quality. Results: A construct containing a bovine lactoferrin cDNA was used to transform wheat using an Agrobacterium-mediated DNA transfer system to express this antimicrobial protein in transgenic wheat. Transformants were analyzed by Northern and Western blots to determine lactoferrin gene expression levels and were inoculated with the head blight disease fungus F. graminearum. Transgenic wheat showed a significant reduction of disease incidence caused by F. graminearum compared to control wheat plants. The level of resistance in the highly susceptible wheat cultivar Bobwhite was significantly higher in transgenic plants compared to control Bobwhite and two untransformed commercial wheat cultivars, susceptible Wheaton and tolerant ND 2710. Quantification of the expressed lactoferrin protein by ELISA in transgenic wheat indicated a positive correlation between the lactoferrin gene expression levels and the levels of disease resistance. Conclusions: Introgression of the lactoferrin gene into elite commercial wheat, barley and other susceptible cereals may enhance resistance to F. graminearum

Soybean Nodule-Associated Non-Rhizobial Bacteria Inhibit Plant Pathogens and Induce Growth Promotion in Tomato

The root nodules are a unique environment formed on legume roots through a highly specific symbiotic relationship between leguminous plants and nodule-inducing bacteria. Previously, Rhizobia were presumed to be the only group of bacteria residing within nodules. However, recent studies discovered diverse groups of bacteria within the legume nodules. In this report soybean nodule-associated bacteria were studied in an effort to identify beneficial bacteria for plant disease control and growth promotion. Analysis of surface-sterilized single nodules showed bacterial diversity of the nodule microbiome. Five hundred non-rhizobial colonies from 10 nodules, 50 colonies per nodule, were tested individually against the tomato wilt causing bacterial pathogen Clavibacter michiganensis subsp. michiganensis (Cmm) for inhibition of pathogen growth. From the initial screening, 54 isolates were selected based on significant growth inhibition of Cmm. These isolates were further tested in vitro on another bacterial pathogen Pseudomonas syringae pv. tomato (Pst) and two fungal pathogens Rhizoctonia solani and Sclerotinia sclerotiorum. Bacterial metabolites were extracted from 15 selected isolates with ethanol and tested against pathogen Cmm and Pst. These isolates were identified by using MALDI-TOF mass spectrometry and 16S rRNA gene sequencing. Pseudomonas spp. were the dominant soybean nodule-associated non-rhizobial bacterial group. Several isolates imparted significant protection against pathogens and/or plant growth promotion on tomato seedlings. The most promising nodule-associated bacterial isolate that suppressed both Cmm and Pst in vitro and Pst in tomato seedlings was identified as a Proteus species. Isolation and identification of beneficial nodule-associated bacteria established the foundation for further exploration of potential nodule-associated bacteria for plant protection and growth promotion

Penicillium pinophilum has the potential to reduce damping-off caused by Rhizoctonia solani in sugar beet

© 2021 Springer. This is the accepted manuscript version of an article which has been published in final form at https://doi.org/10.1007/s12355-021-00958-8Rhizoctonia solani is an economically important pathogen of sugar beet (Beta vulgaris L.) causing seedling damping-off, and root and crown rot. Cultural practices, partially resistant cultivars, and fungicides are among the methods most used to manage R. solani. Penicillium pinophilum, a potential biocontrol agent for Rhizoctonia damping-off, was isolated from sugar beet. Our objective was to evaluate the biocontrol potential of P. pinophilum against R. solani AG 2–2 under laboratory and greenhouse conditions. In vitro co-culture of both fungi showed that R. solani growth was inhibited by P. pinophilum. A greenhouse inoculation study was done using sclerotia of R. solani and a conidia suspension of P. pinophilum to evaluate the response of a Rhizoctonia susceptible cultivar. Treatments included R. solani sclerotia, P. pinophilum conidia suspension, a combination of R. solani sclerotia with P. pinophilum conidia suspension, and a mock inoculation with water (control). One 2-cm deep furrow was made in the middle of peat filled trays into which 10 seeds were planted. Each treatment was applied adjacent to each seed and covered with peat. There were four replicates per treatment arranged in a completely randomized design. The sole sclerotia treatment caused 75% damping-off and severe root rot on surviving plants whereas the combination of sclerotia with P. pinophilum conidia suspension reduced damping-off by 50%. No damping-off incidences were observed with the P. pinophilum conidia suspension or the mock-inoculated control. It was concluded that P. pinophilum has the potential to reduce damping-off caused by R. solani but use of the most appropriate P. pinophilum concentration and its mitigation mechanisms need further studies.Peer reviewedFinal Accepted Versio