scholarly journals Barley Nepenthesin-Like Aspartic Protease HvNEP-1 Degrades Fusarium Phytase, Impairs Toxin Production, and Suppresses the Fungal Growth

2021 ◽  
Vol 12 ◽  
Author(s):  
Zelalem Eshetu Bekalu ◽  
Giuseppe Dionisio ◽  
Claus Krogh Madsen ◽  
Thomas Etzerodt ◽  
Inge S. Fomsgaard ◽  
...  
Keyword(s):  
Acid Phosphatase ◽  
Pichia Pastoris ◽  
Fusarium Culmorum ◽  
Fungal Growth ◽  
Distinct Group ◽  
Toxin Production ◽  
Pcr Analysis ◽  
Aspartic Proteases ◽  
Inorganic Phosphorous

Nepenthesins are categorized under the subfamily of the nepenthesin-like plant aspartic proteases (PAPs) that form a distinct group of atypical PAPs. This study describes the effect of nepenthesin 1 (HvNEP-1) protease from barley (Hordeum vulgare L.) on fungal histidine acid phosphatase (HAP) phytase activity. Signal peptide lacking HvNEP-1 was expressed in Pichia pastoris and biochemically characterized. Recombinant HvNEP-1 (rHvNEP-1) strongly inhibited the activity of Aspergillus and Fusarium phytases, which are enzymes that release inorganic phosphorous from phytic acid. Moreover, rHvNEP-1 suppressed in vitro fungal growth and strongly reduced the production of mycotoxin, 15-acetyldeoxynivalenol (15-ADON), from Fusarium graminearum. The quantitative PCR analysis of trichothecene biosynthesis genes (TRI) confirmed that rHvNEP-1 strongly repressed the expression of TRI4, TRI5, TRI6, and TRI12 in F. graminearum. The co-incubation of rHvNEP-1 with recombinant F. graminearum (rFgPHY1) and Fusarium culmorum (FcPHY1) phytases induced substantial degradation of both Fusarium phytases, indicating that HvNEP-1-mediated proteolysis of the fungal phytases contributes to the HvNEP-1-based suppression of Fusarium.

scholarly journals Investigating Useful Properties of Four Streptomyces Strains Active against Fusarium graminearum Growth and Deoxynivalenol Production on Wheat Grains by qPCR

Toxins ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 560
Author(s):  
Elena Maria Colombo ◽  
Andrea Kunova ◽  
Claudio Gardana ◽  
Cristina Pizzatti ◽  
Paolo Simonetti ◽  
...  
Keyword(s):  
Fusarium Graminearum ◽  
Plant Pathogens ◽  
Fungal Growth ◽  
Toxin Production ◽  
Fungal Mycelium ◽  
Head Blight ◽  
Wheat Grains ◽  
Streptomyces Spp ◽  
Dual Plate

Streptomyces spp. can be exploited as biocontrol agents (BCAs) against plant pathogens such as Fusarium graminearum, the main causal agent of Fusarium head blight (FHB) and against the contamination of grains with deoxynivalenol (DON). In the present research, four Streptomyces strains active against F. graminearum in dual plate assays were characterized for their ability to colonize detached wheat grains in the presence of F. graminearum and to limit DON production. The pathogen and BCA abundance were assessed by a quantitative real-time PCR, while DON production was assessed by HPLC quantification and compared to ergosterol to correlate the toxin production to the amount of fungal mycelium. Fungal growth and mycotoxin production were assessed with both co-inoculation and late inoculation of the BCAs in vitro (three days post-Fusarium inoculation) to test the interaction between the fungus and the bacteria. The level of inhibition of the pathogen and the toxin production were strain-specific. Overall, a higher level of DON inhibition (up to 99%) and a strong reduction in fungal biomass (up to 71%) were achieved when streptomycetes were co-inoculated with the fungus. This research enabled studying the antifungal efficacy of the four Streptomyces strains and monitoring their development in DON-inducing conditions.

scholarly journals Development and Use of a Reverse Transcription-PCR Assay To Study Expression of Tri5 byFusarium Species In Vitro and In Planta

1999 ◽  
Vol 65 (9) ◽  
pp. 3850-3854 ◽  
Author(s):  
F. M. Doohan ◽  
G. Weston ◽  
H. N. Rezanoor ◽  
D. W. Parry ◽  
P. Nicholson
Keyword(s):  
Reverse Transcription ◽  
Infected Plant ◽  
Fusarium Culmorum ◽  
Pcr Analysis ◽  
Growth Stages ◽  
In Planta ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Glucuronidase Activity

ABSTRACT The Tri5 gene encodes trichodiene synthase, which catalyzes the first reaction in the trichothecene biosynthetic pathway. In vitro, a direct relationship was observed between Tri5expression and the increase in deoxynivalenol production over time. We developed a reverse transcription (RT)-PCR assay to quantifyTri5 gene expression in trichothecene-producing strains ofFusarium species. We observed an increase inTri5 expression following treatment of Fusarium culmorum with fungicides, and we also observed an inverse relationship between Tri5 expression and biomass, as measured by β-d-glucuronidase activity, during colonization of wheat (cv. Avalon) seedlings by F. culmorum. RT-PCR analysis also showed that for ears of wheat cv. Avalon inoculated with F. culmorum, there were different levels of Tri5 expression in grain and chaff at later growth stages. We used the Tri5-specific primers to develop a PCR assay to detect trichothecene-producing Fusariumspecies in infected plant material.

scholarly journals Antifungal Activity of Chitosan Oligomers–Amino Acid Conjugate Complexes against Fusarium culmorum in Spelt (Triticum spelta L.)

Agronomy ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1427
Author(s):  
Laura Buzón-Durán ◽  
Jesús Martín-Gil ◽  
José Luis Marcos-Robles ◽  
Ángel Fombellida-Villafruela ◽  
Eduardo Pérez-Lebeña ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Antifungal Activity ◽  
Fusarium Culmorum ◽  
Fungal Growth ◽  
Control Efficacy ◽  
Triticum Spelta ◽  
Amino Acid Conjugate ◽  
Acid Conjugate

Fusarium head blight (FHB) is a complex disease of cereals caused by Fusarium species, which causes severe damages in terms of yield quality and quantity worldwide, and which produces mycotoxin contamination, posing a serious threat to public health. In the study presented herein, the antifungal activity against Fusarium culmorum of chitosan oligomers (COS)–amino acid conjugate complexes was investigated both in vitro and in vivo. The amino acids assayed were cysteine, glycine, proline and tyrosine. In vitro tests showed an enhancement of mycelial growth inhibition, with EC50 and EC90 effective concentration values ranging from 320 to 948 µg·mL−1 and from 1107 to 1407 µg·mL−1 respectively, for the conjugate complexes, as a result of the synergistic behavior between COS and the amino acids, tentatively ascribed to enhanced cell membrane damage originating from lipid peroxidation. Tests on colonies showed a maximum percentage reduction in the number of colonies at 1500 µg·mL−1 concentration, while grain tests were found to inhibit fungal growth, reducing deoxynivalenol content by 89%. The formulation that showed the best performance, i.e., the conjugate complex based on COS and tyrosine, was further investigated in a small-scale field trial with artificially inoculated spelt (Triticum spelta L.), and as a seed treatment to inhibit fungal growth in spelt seedlings. The field experiment showed that the chosen formulation induced a decrease in disease severity, with a control efficacy of 83.5%, while the seed tests showed that the treatment did not affect the percentage of germination and resulted in a lower incidence of root rot caused by the pathogen, albeit with a lower control efficacy (50%). Consequently, the reported conjugate complexes hold enough promise for crop protection applications to deserve further examination in larger field trials, with other Fusarium spp. pathogens and/or Triticum species.

scholarly journals Deoxynivalenol and Nivalenol Production by Fusarium culmorum Isolates Differing in Aggressiveness Toward Winter Rye

1998 ◽  
Vol 88 (9) ◽  
pp. 879-884 ◽  
Author(s):  
G. Gang ◽  
T. Miedaner ◽  
U. Schuhmacher ◽  
M. Schollenberger ◽  
H. H. Geiger
Keyword(s):  
Genetic Variation ◽  
The Netherlands ◽  
Fungal Biomass ◽  
Fusarium Culmorum ◽  
Toxin Production ◽  
Field Trials ◽  
In Vitro Assays ◽  
Winter Rye ◽  
Field Samples

A susceptible synthetic winter rye population was inoculated with 42 isolates of Fusarium culmorum, originating from nine European countries and Australia, at two field locations in Germany. Significant (P = 0.01) genetic variation in aggressiveness of isolates of F. culmorum was observed across both field locations. Field samples were used to determine deoxynivalenol (DON), nivalenol (NIV), and ergosterol (ERG) contents. The 42 isolates also were incubated on rye grain in vitro, and DON and NIV contents were analyzed. Thirty-four isolates produced DON, and seven isolates produced NIV at both field locations and in vitro. Mean DON contents ranged from 0.5 to 64.6 mg/kg in grain from field trials and from 0.3 to 376.3 mg/kg in grain incubated in vitro; mean NIV contents ranged from 17.6 to 30.4 mg/kg in grain from field trials and from 0.8 to 381.0 mg/kg in grain incubated in vitro. No correlation was found between the DON content of field-grown grain and grain incubated in vitro. NIV-producing isolates originated from the Netherlands, Germany, Italy, and Australia. More aggressive isolates produced higher mean DON contents in grain in field trials (r = 0.69; P = 0.01). However, DON production rate per unit of fungal biomass, estimated as the DON/ERG ratio at harvest, was not correlated with aggressiveness. Toxin production seemed to be a common feature in F. culmorum. In vitro assays reliably distinguished DON- and NIV-producing types of F. culmorum; however, these assays could not predict production of DON by these isolates in the field.

Effects of Hebeloma arenosa and phosphorus fertility on root acid phosphatase activity of red pine (Pinus resinosa) seedlings

1991 ◽  
Vol 69 (2) ◽  
pp. 380-383 ◽  
Author(s):  
Janet MacFall ◽  
Steven A. Slack ◽  
Jaya Iyer
Keyword(s):  
Enzyme Activity ◽  
Acid Phosphatase ◽  
Phosphatase Activity ◽  
Acid Phosphatase Activity ◽  
Fungal Growth ◽  
Pinus Resinosa ◽  
Ectomycorrhizal Fungus ◽  
Red Pine ◽  
Nitrophenyl Phosphate

The ectomycorrhizal fungus Hebeloma arenosa Burdsall, MacFall & Albers was assayed for surface-accessible acid phosphatase activity in vitro on roots of red pine (Pinus resinosa Ait.) seedlings. Hebeloma arenosa was grown in defined liquid media containing 0, 17, 34, 68, or 136 mg/L phosphorus for 4 weeks. When assayed for acid phosphatase activity with p-nitrophenyl phosphate, 7.3 μmol of orthophosphate were released per gram dry weight of fungal tissue. There was no effect of added P on enzyme activity, excluding the treatment with no added P in which there was negligible fungal growth. Red pine seedlings were grown in Sparta loamy fine sand amended with 0, 17, 34, 68, or 136 mg/kg P as superphosphate, with and without H. arenosa inoculum. Mycorrhizal roots had greater enzyme activity than nonmycorrhizal roots of seedlings grown in similarly P-amended soil. This was determined by the following three assays: orthophosphate release from two salts of myoinosital hexaphosphate (Na and KMg) and from p-nitrophenyl phosphate. It is suggested that greater acid phosphatase activity by roots mycorrhizal with H. arenosa is one mechanism for improved P nutrition through the formation of a pool of P released from sources unavailable for direct intake.

scholarly journals Effect of Naturally Occurring Compounds on Fumonisin Production and fum Gene Expression in Fusarium verticillioides

Agronomy ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1060
Author(s):  
Davide Ferrigo ◽  
Sharda Bharti ◽  
Massimiliano Mondin ◽  
Alessandro Raiola
Keyword(s):  
Gene Expression ◽  
Phenolic Compounds ◽  
Ellagic Acid ◽  
Fusarium Verticillioides ◽  
Fungal Growth ◽  
Toxin Production ◽  
Promising Alternative ◽  
Mycotoxin Biosynthesis

Fusarium verticillioides, one of the most common pathogens in maize, is responsible for yield losses and reduced kernel quality due to contamination by fumonisins (FBs). Two F. verticillioides isolates that differed in their ability to produce FBs were treated with a selection of eight natural phenolic compounds with the aim of identifying those that were able to decrease toxin production at concentrations that had a limited effect on fungal growth. Among the tested compounds, ellagic acid and isoeugenol, which turned out to be the most effective molecules against fungal growth, were assayed at lower concentrations, while the first retained its ability to inhibit toxin production in vitro, the latter improved both the fungal growth and FB accumulation. The effect of the most effective phenolic compounds on FB accumulation was also tested on maize kernels to highlight the importance of appropriate dosages in order to avoid conditions that are able to promote mycotoxin biosynthesis. An expression analysis of genes involved in FB production allowed more detailed insights into the mechanisms underlying the inhibition of FBs by phenolic compounds. The expression of the fum gene was generally down-regulated by the treatments; however, some treatments in the low-producing F. verticillioides strain up-regulated fum gene expression without improving FB production. This study showed that although different phenolic compounds are effective for FB reduction, they can modulate biosynthesis at the transcription level in opposite manners depending on strain. In conclusion, on the basis of in vitro and in vivo screening, two out of the eight tested phenols (ellagic acid and carvacrol) appear to be promising alternative molecules for the control of FB occurrence in maize.

scholarly journals Antifungal Activity against Fusarium culmorum of Stevioside, Silybum marianum Seed Extracts, and Their Conjugate Complexes

Antibiotics ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 440 ◽  
Author(s):  
Laura Buzón-Durán ◽  
Jesús Martín-Gil ◽  
María del Carmen Ramos-Sánchez ◽  
Eduardo Pérez-Lebeña ◽  
José Luis Marcos-Robles ◽  
...  
Keyword(s):  
Fusarium Culmorum ◽  
Fungal Growth ◽  
Stevia Rebaudiana ◽  
Molar Ratio ◽  
Silybum Marianum ◽  
Stored Grain ◽  
Head Blight ◽  
Wheat Seedlings ◽  
Stevia Rebaudiana Bertoni

Fusarium head blight (FHB) is a disease that poses a major challenge in cereal production that has important food and feed safety implications due to trichothecene contamination. In this study, the effect of stevioside—a glycoside found in the leaves of candyleaf (Stevia rebaudiana Bertoni)—was evaluated in vitro against Fusarium culmorum (W.G. Smith) Sacc., alone and in combination (in a 1:1 molar ratio) with polyphenols obtained from milk thistle seeds (Silybum marianum (L.) Gaertn). Different concentrations, ranging from 32 to 512 µg·mL−1, were assayed, finding EC50 and EC90 inhibitory concentrations of 156 and 221 µg·mL−1, respectively, for the treatment based only on stevioside, and EC50 and EC90 values of 123 and 160 µg·mL−1, respectively, for the treatment based on the stevioside–polyphenol conjugate complexes. Colony formation inhibition results were consistent, reaching full inhibition at 256 µg·mL−1. Given that synergistic behavior was observed for this latter formulation (SF = 1.43, according to Wadley’s method), it was further assessed for grain protection at storage, mostly directed against mycotoxin contamination caused by the aforementioned phytopathogen, confirming that it could inhibit fungal growth and avoid trichothecene contamination. Moreover, seed tests showed that the treatment did not affect the percentage of germination, and it resulted in a lower incidence of root rot caused by the pathogen in Kamut and winter wheat seedlings. Hence, the application of these stevioside–S. marianum seed extract conjugate complexes may be put forward as a promising and environmentally friendly treatment for the protection of cereal crops and stored grain against FHB.

Cloning and Expression of Recombinant Human Insulin Gene in Pichia pastoris

2019 ◽  
Vol 10 (01) ◽  
Author(s):  
Rafid A. Abdulkareem
Keyword(s):  
Pichia Pastoris ◽  
Human Insulin ◽  
Expression Vector ◽  
Expression System ◽  
Insulin Gene ◽  
Cloning And Expression ◽  
Pcr Analysis ◽  
Major Band ◽  
Human Insulin Gene ◽  
Pichia Pastoris Expression System

The main goal of the current study was cloning and expression of the human insulin gene in Pichia pastoris expression system, using genetic engineering techniques and its treatment application. Total RNA was purified from fresh normal human pancreatic tissue. RNA of good quality was chosen to obtain a first single strand cDNA. Human preproinsulin gene was amplified from cDNA strand, by using two sets of specific primers contain EcoR1 and Notl restriction sites. The amplified preproinsulin gene fragment was double digested with EcoRI and Not 1 restriction enzymes, then inserted into pPIC9K expression vector. The new pPIC9K-hpi constructive expression vector was transformed by the heat-shock method into the E.coli DH5α competent cells. pPic9k –hpi, which was propagated in the positive transformant E. coli cells, was isolated from cells and then linearised by restriction enzyme SalI, then transformed into Pichia pastoris GS115 using electroporation method. Genomic DNA of His+ transformants cell was extracted and used as a template for PCR analysis. The results showed, that the pPic9k – hpi was successfully integrated into the P. pastoris genome, for selected His+ transformants clones on the anticipated band at 330 bp, which is corresponded to the theoretical molecular size of the human insulin gene. To follow the insulin expression in transformans, Tricine–SDS gel electrophoresis and Western blot analysis were conducted. The results showed a successful expression of recombinant protein was detected by the presence of a single major band with about (5.8 KDa) on the gel. These bands correspond well with the size of human insulin with the theoretical molecular weight (5.8 KDa).

Healing effects of curcumin nanoparticles in deep tissue injury mouse model.

2020 ◽  
Vol 18 ◽  
Author(s):  
Zirui Zhang ◽  
Shangcong Han ◽  
Panpan Liu ◽  
Xu Yang ◽  
Jing Han ◽  
...  
Keyword(s):  
Wound Healing ◽  
Mrna Expression ◽  
Tissue Injury ◽  
Inflammatory Cells ◽  
Pcr Analysis ◽  
Protective Effects ◽  
Deep Tissue ◽  
Deep Tissue Injury

Background: Chronic inflammation and lack of angiogenesis are the important pathological mechanisms in deep tissue injury (DTI). Curcumin is a well-known anti-inflammatory and antioxidant agent. However, curcumin is unstable under acidic and alkaline conditions, and can be rapidly metabolized and excreted in the bile, which shortens its bioactivity and efficacy. Objective: This study aimed to prepare curcumin-loaded poly (lactic-co-glycolic acid) nanoparticles (CPNPs) and to elucidate the protective effects and underlying mechanisms of wound healing in DTI models. Methods: CPNPs were evaluated for particle size, biocompatibility, in vitro drug release and their effect on in vivo wound healing. Results : The results of in vivo wound closure analysis revealed that CPNP treatments significantly improved wound contraction rates (p<0.01) at a faster rate than other three treatment groups. H&E staining revealed that CPNP treatments resulted in complete epithelialization and thick granulation tissue formation, whereas control groups resulted in a lack of compact epithelialization and persistence of inflammatory cells within the wound sites. Quantitative real-time PCR analysis showed that treatment with CPNPs suppressed IL-6 and TNF-α mRNA expression, and up-regulated TGF-β, VEGF-A and IL-10 mRNA expression. Western blot analysis showed up-regulated protein expression of TGF-β, VEGF-A and phosphorylatedSTAT3. Conclusion: Our results showed that CPNPs enhanced wound healing in DTI models, through modulation of the JAK2/STAT3 signalling pathway and subsequent upregulation of pro-healing factors.

scholarly journals The Potential of Rhizoctonia-Like Fungi for the Biological Protection of Cereals against Fungal Pathogens

Plants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 349
Author(s):  
Dominik Bleša ◽  
Pavel Matušinský ◽  
Romana Sedmíková ◽  
Milan Baláž
Keyword(s):  
Biological Control ◽  
Fungal Pathogens ◽  
Plant Pathogens ◽  
Higher Plants ◽  
Fusarium Culmorum ◽  
Suppressive Effect ◽  
Plant Production ◽  
Shoot Biomass ◽  
Organic Substrates

The use of biological control is becoming a common practice in plant production. One overlooked group of organisms potentially suitable for biological control are Rhizoctonia-like (Rh-like) fungi. Some of them are capable of forming endophytic associations with a large group of higher plants as well as mycorrhizal symbioses. Various benefits of endophytic associations were proved, including amelioration of devastating effects of pathogens such as Fusarium culmorum. The advantage of Rh-like endophytes over strictly biotrophic mycorrhizal organisms is the possibility of their cultivation on organic substrates, which makes their use more suitable for production. We focused on abilities of five Rh-like fungi isolated from orchid mycorrhizas, endophytic fungi Serendipita indica, Microdochium bolleyi and pathogenic Ceratobasidium cereale to inhibit the growth of pathogenic F. culmorum or Pyrenophora teres in vitro. We also analysed their suppressive effect on wheat infection by F. culmorum in a growth chamber, as well as an effect on barley under field conditions. Some of the Rh-like fungi affected the growth of plant pathogens in vitro, then the interaction with plants was tested. Beneficial effect was especially noted in the pot experiments, where wheat plants were negatively influenced by F. culmorum. Inoculation with S. indica caused higher dry shoot biomass in comparison to plants treated with fungicide. Prospective for future work are the effects of these endophytes on plant signalling pathways, factors affecting the level of colonization and surviving of infectious particles.

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