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Protoplast fusion stages: (A) and (B) showing the released protoplasts from parent strains T. harzianum and T. viride, (C) and (D) showing fusion of protoplasts of parental strains after treatment with PEG.
Source publication
Biological control plays a crucial role in grapevine pathogens disease management. The cell-wall degrading enzymes
chitinase, cellulase and b-glucanase have been suggested to be essential for the mycoparasitism activity of Trichoderma
species against grapevine fungal pathogens. In order to develop a useful strain as a single source of these vital e...
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Citrus fruit can be infected by many fungal pathogens, and these pathogens cause considerable losses during storage and transportation. Recently, biological control methods of postharvest diseases are getting more prevalent due to the effects of chemical residues on fruits. The present study was carried out to determine the antifungal activities of...
Twelve isolates of Trichoderma spp. isolated from tobacco rhizosphere were evaluated for their ability to produce chitinase and β-1,3-glucanase extracellular hydrolytic enzymes. Isolates ThJt1 and TvHt2, out of 12 isolates, produced maximum activities of chitinase and β-1,3-glucanase, respectively. In vitro production of chitinase and β-1,3-glucana...
Fusarium udum, Fusarium oxysporum f. sp. ciceri and F.oxysporum f.sp.lentis cause severe economic loss to pigeonpea, chickpea and lentil crops. Forty three isolates of Trichoderma were screened for their antagonistic ability against the test pathogens. Potent strains were identified on the basis of in vitro antagonistic ability and fungicide tolera...
Citations
... It is clear that MNF-MAS-Tricho1, MNF-MAS-Tricho 2 and MNF-MAS-Tricho 3isolates were the most productive. Similarly, it is scientifically proven that the Trichoderma fungus is one of the most cellulose-producing species due to its rapid growth and abundance of cells (Hassan et al. 2014). It has also become noteworthy to use the Trichoderma fungus in the biological control of many fungal diseases due to the ability of Trichoderma to degrade the cell wall of these plant-pathogenic fungi, such as Pythium and Phanerochaete (Mazrou et al. 2020). ...
... Our results exhibited high diversity among Trichoderma isolates that ranged from 57 to 88%. These results indicated that the rep-PCR is a highly reproducible method for the characterization of fungal species (Hassan 2014). ...
Background
The phylogenetic relationships and phylogeny of twenty-six Trichoderma species collected from various Egyptian locations were investigated. The genetic diversity among the examined isolates was tested using the rep-PCR marker. Trichoderma species were screened for their cellulase activities.
Results
Three isolates demonstrated highly significant FPase activities, namely MNF-MAS-Tricho 1, MNF-MAS-Tricho 2, and MNF-MAS-Tricho 3 (0.50, 0.39, and 0.49 IU ml ⁻¹ , respectively). MNF-MAS-Tricho1 showed the highest significant CMCase activity (0.80 IU ml ⁻¹ ). Concerning β-glucosidase, MNF-MAS-Tricho 1 was the highest (0.78 IU ml ⁻¹ ), while MNF-MSH-Trich 11 and MNF-MAS-Tricho 15 were the lowest (0.36 IU mL ⁻¹ ). The percentage of polymorphism ranged from 46.15 to 83.33%. (GTG)5 marker produced the greatest number of polymorphic loci (13 loci out of 18 loci) with about 83.33% polymorphism, followed by rep-10 with 69.2% polymorphism. Furthermore, the polymorphism information content (PIC) estimates ranged between 0.285 for Rep-10 and 0.340 for (GTG) 5 with an average of 0.306. The tested primers exhibited high discriminating and resolving powers.
Conclusion
The findings of this investigation were used to classify Trichoderma species, evaluate their genetic variability using ITS sequencing, rep-PCR, and measure their cellulase activities. These markers can facilitate more rapid and less complicated studies of Trichoderma population dynamics and evaluate their establishment after release into agricultural environments. The results will help to evaluate the genetic diversity of Trichoderma in future research.
... A variety of techniques are used to develop industrially important microorganisms, protoplast fusion is the most straightforward and widely applied method which permits the recombination of genomes from many parental strains [7,8]. It can be seen as one of the recombinant DNA technologies that offer the ability to enhance the dosage and expression of genes from potent promoters, eliminate undesirable genes from the fungal genome, modify metabolic pathways, and create fungal strains that can generate heterologous proteins [9]. ...
... Isolation, fusion, and regeneration of protoplasts have been achieved in the genus Aspergillus mainly in the strain improvement program to enhance enzyme production [31]. The contrasting character of the parental strains is required for selecting the fusants [9]. In that light and according to the morphological and growth rate differences between the Aspergillus spp., we conducted protoplast fusion and the resultant fusants tested for cellulase activity as well as the parental strains to observe strain improvement. ...
... On the other hand, the fusant PFOT showed an unremarkable increase and the fusant PFFT exhibited a decrease in activity as compared to the parents. These findings suggest that protoplast fusion may involve partial or total genetic recombination, which may have had unfavorable consequences for fusants which is consistent with [9]. ...
Background
Lignocellulosic biomass provides a great starting point for the production of energy, chemicals, and fuels. The major component of lignocellulosic biomass is cellulose, the employment of highly effective enzymatic cocktails, which can be produced by a variety of microorganisms including species of the genus Aspergillus, is necessary for its utilization in a more productive manner. In this regard, molecular biology techniques should be utilized to promote the economics of enzyme production, whereas strategies like protoplast fusion could be employed to improve the efficacy of the hydrolytic process.
Results
The current study focuses on cellulase production in Aspergillus species using intrageneric protoplast fusion, statistical optimization of growth parameters, and determination of antioxidant activity of fermentation hydrolysate. Protoplast fusion was conducted between A. flavus X A. terreus (PFFT), A. nidulans X A. tamarii (PFNT) and A. oryzae X A. tubingensis (PFOT), and the resultant fusant PFNT revealed higher activity level compared with the other fusants. Thus, this study aimed to optimize lignocellulosic wastes-based medium for cellulase production by Aspergillus spp. fusant (PFNT) and studying the antioxidant effect of fermentation hydrolysate. The experimental strategy Plackett-Burman (PBD) was used to assess how culture conditions affected cellulase output, the best level of the three major variables namely, SCB, pH, and incubation temperature were then determined using Box-Behnken design (BBD). Consequently, by utilizing an optimized medium instead of a basal medium, cellulase activity increased from 3.11 U/ml to 7.689 U/ml CMCase. The following medium composition was thought to be ideal based on this optimization: sugarcane bagasse (SCB), 6.82 gm; wheat bran (WB), 4; Moisture, 80%; pH, 4; inoculum size, (3 × 10⁶ spores/ml); and incubation Temp. 31.8 °C for 4 days and the fermentation hydrolysate has 28.13% scavenging activities.
Conclusion
The results obtained in this study demonstrated the significant activity of the selected fusant and the higher sugar yield from cellulose hydrolysis over its parental strains, suggesting the possibility of enhancing cellulase activity by protoplast fusion using an experimental strategy and the fermentation hydrolysate showed antioxidant activity.
... Various strategies for improving the crop growth promotion and biocontrol efficacies of Trichoderma have been reviewed (Fraceto et al., 2018;Ferreira and Musumeci, 2021;Guzmán-Guzmán et al., 2023;Xiao et al., 2023). They include Trichoderma strain improvement (Szekeres et al., 2004;Montero-Barrientos et al., 2011;Hassan, 2014), formulation of Trichoderma with bioactive compounds (Yang et al., 2011;Nandini et al., 2017;Mbarga et al., 2020), co-application of Trichoderma with other beneficial microbes (Rudresh et al., 2005;Shanmugaiah et al., 2009;Mweetwa et al., 2016;Ghorchiani et al., 2018;Karuppiah et al., 2019;Szczałba et al., 2019;Konappa et al., 2020), and combining Trichoderma application with organic amendments (Haggag and Saber, 2000;Miranda et al., 2006;Joshi et al., 2009;Yang et al., 2011;Bhadauria et al., 2012;Hannan et al., 2012;Domínguez et al., 2014;Blaya et al., 2016;de Araujo et al., 2019;Sani et al., 2020;Da Silva et al., 2022;Amanullah and Khan, 2023). However, these strategies have their own limitations, calling for continued research to develop highly efficacious biocontrol products and application strategies that are economically viable, technically simple, regulatorily neutral, and easily adoptable. ...
Trichoderma spp. are widely used to enhance crop growth and suppress diverse diseases. However, inconsistent field efficacy remains a major barrier to their use as a reliable alternative to synthetic pesticides. Various strategies have been investigated to enhance the robustness of their application. Here, we evaluated how T. virens application methods (pre-, at-, and post-transplant) affect the growth of two tomato varieties and their rhizosphere fungal and bacterial communities. Although the greatest rhizosphere abundance of T. virens was observed in the post-transplant application, the at-transplant application promoted tomato growth the most, indicating that greater rhizosphere abundance does not necessarily result in better tomato growth. None of the application methods significantly altered the global rhizosphere fungal and bacterial communities of the tested varieties. Changes in specific microbial genera and guilds may underpin the enhanced tomato growth. We also investigated whether the resulting microbiome changes affect the mycelial growth and conidial germination of Fusarium oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicis-lycopersici , soilborne fungal pathogens of tomato, upon exposure to volatile compounds emitted by culturable rhizosphere microbes and metabolites extracted from the rhizosphere soils after Trichoderma treatments. Volatile compounds produced by cultured rhizosphere microbes after the at-transplant application suppressed the mycelial growth of both pathogens better than those after the other treatments. Similarly, water-soluble metabolites extracted from the rhizosphere soil samples after the at-transplant application most effectively suppressed the germination rate of F. oxysporum spores. Overall, our results suggest that the at-transplant application is most advantageous for promoting the growth of the tested tomato varieties and building soil suppressiveness against the tested fusaria. However, further studies are needed before applying this method to support tomato production. We discuss critical future questions.
... Correspondingly, introduction of the chit42 gene to plants increased their resistances to fungal pathogens (Lorito et al., 1998). Interspecific and intraspecific protoplasmic fusions were reported to enhance chitinase activity and antagonistic activity in T. harzianum (Balasubramanian et al., 2012;Hassan, 2014). On the other hand, rational genetic engineering has also been used to improve the chitinase activity of T. harzianum strains. ...
In the control of plant diseases, biocontrol has the advantages of being efficient and safe for human health and the environment. The filamentous fungus Trichoderma harzianum and its closely related species can inhibit the growth of many phytopathogenic fungi, and have been developed as commercial biocontrol agents for decades. In this review, we summarize studies on T. harzianum species complex from the perspective of strain improvement. To elevate the biocontrol ability, the production of extracellular proteins and compounds with antimicrobial or plant immunity-eliciting activities need to be enhanced. In addition, resistance to various environmental stressors should be strengthened. Engineering the gene regulatory system has the potential to modulate a variety of biological processes related to biocontrol. With the rapidly developing technologies for fungal genetic engineering, T. harzianum strains with increased biocontrol activities are expected to be constructed to promote the sustainable development of agriculture.
... The process of protoplast fusion is well-known for the role it plays in the creation of cybrids in addition to hybrids because of the widespread dissemination of this information. When it comes to phytoremediation, protoplast fusion is among the most essential strategies for enhancing the desirable qualities of species (Hassan, 2014). Scientists used electro-fusion to fuse different protoplasts from hyper-accumulator species to create more effective remediating plants for zinc uptake, such as Thlaspi caerulencens and B. napus (Brewer et al., 1999). ...
Soil and water pollution, rapid industrialization, contaminated irrigation-water, increased waste-production and surge in agricultural land leads to the accumulation of Heavy Metals (HM) with time. HM contamination has raised concern over the past years and new remediation strategies are required to deal with it. HM-contaminated soil is often used for the production of food, which makes a gateway for toxic metals into the food-chain, thereby affecting food security and human health. To avoid HM-toxicity, decontamination of important resources is essential. Therefore, exploring phytoremediation for the removal, decomposition and detoxification of hazardous metals from HM-contaminated sites is of great significance. Hyper-accumulator plants can efficiently remove HMs. However, despite many hyper-accumulator plant species, there is a research gap in the studies of phytotechnology. Hence biotechnological efforts advocating omics studies i.e. genomics, transcriptomics, proteomics, metabolomics and phenomics are in order, the purpose being to select and enhance a plant's potential for the process of phytoremediation to be more effective. There is a need to study newly developed high-efficiency hyper-accumulator plants as HM-decontaminator candidates for phytoremediation and phytomining. Therefore, this review focuses on various strategies and bio-technological methods for the removal of HM contaminants from sites, with emphasis on the advancement of phytoremediation, along with applications in cleaning up various toxic pollutants.
... Fusion of protoplast from M. ruber and P. ostreatus may result in genetic inference at the metabolic level, which was the reason behind the elevation of pigment in fusant colonies. A recent study observed a variation or stimulation of pigment production during protoplast hybridization in other species of fungi [22]. The proximate composition of parental and fusant strains contains 7.28% of moisture, 4.87 % f ash content, 11.01% of cured protein, 7.16% of curde fat, 51.01% of carbohydrates, and 8.43% of crude ber, respectively. ...
Protoplast fusion is a crucial technique for strain enhancement that brings genetic recombination to filamentous fungus and creates hybrid strains. In this present study, the intergenic fusion between M. ruber and P. ostreatus has been carried out to enhance pigments, secondary metabolites, and proximate values. Protoplasts from M. ruber and P. ostreatus were isolated and an equal amount of protoplast was taken and performed protoplast fusion. The regenerated colonies were plated in potato dextrose agar with selected biological markers (nystatin and fluconazole) for the confirmation of the hybrid. In a standardized condition, the protoplast yield of M. ruber and P. ostreatus was 2.31×10 ⁷ cells/ml and 2.33×10 ⁷ cells/ml, respectively. Acidic pH yielded high amounts of protoplasts than neutral and alkaline pH. KCl showed a high yield of protoplasts 2.36×10 ⁶ cells /ml from M. ruber and 2.46×10 ⁷ cells/ml from P. ostreatus . The colony resistance to nystatin and fluconazole was confirmed that the obtained regenerated colonies were fusant. The fusant has no septation in its transverse walls with lipid droplets. Fusion of protoplast from M. ruber and P. ostreatus resulted in genetic inference at the metabolic levels, explaining the elevation of pigment in fusant colonies. The proximate composition of fusant strains was higher. In conclusion, we have developed a new strain with genetic variance capable of producing an increased level of pigment with nutritional value through protoplast fusion that allows the generation of fusant with the characteristics of parent strains and elucidates the effectiveness of fusant for commercial applications.
... against grapevine pathogens, Macrophomina phaseolina, Pythium ultimum, and Sclerotium rolfsii. They showed higher production of β-1,3-glucanase, chitinase, and protease (Hassan, 2014). ...
Biocontrol Mechanisms of Endophytic Microorganisms introduces endophytic microorganisms, colonization, diversity and distribution, describes the isolation and identification of endophytic microorganisms by traditional cultivation and by next generation sequencing technologies, and covers biocontrol mechanisms, bacterial priming, endophytic based methods, the significance on fungi, and metabolite based formulations. The book concludes with chapters on biofilms, microbiota and safety issues of microorganisms. The intensive use of chemicals to control these plant pathogens has resulted in negative consequences such as the release of toxic chemicals in the environment, reduced soil fertility and human health problems. Therefore, environmentally-friendly and sustainable replacement of chemical fertilizers or pesticides is highly challenging.
... Moreover, this technique has previously been used for genetic manipulation via the interspecific or selffusion of protoplasts to modify industrially important fungi (Dahlmann et al. 2015). Protoplast fusion is also an extremely valuable tool in the development of fungal pathogen strains for commercialisation as biocontrol agents (Hassan 2014). nitrate non-utilising (nit) mutants that are unable to grow on media containing chlorate can be used for visual selection of positive hybrids in several fungal species through complementation testing via protoplast fusion (Parthiban et al. 2018). ...
The entomopathogenic fungal genus Lecanicillium Gams and Zare (formerly classified as the species Verticillium lecanii) includes species that are highly pathogenic to many insect genera. In this study, we identified six Lecanicilliumspp. isolated strains (designated as V1-V6) belonging to L. lecanii (V1, V3 and V5) and L. attenuatum (V2, V4 and V6). In addition, these strains were used to obtain new strains via protoplast fusion, and nit mutants were used for protoplast selection. Genetic recombination of the hybrid strains was determined using the random amplified polymorphic DNA(RAPD) technique. We obtained nine stable fusant strains from 176 new hybrid strains, which were termed V12-10, V14-3, V16-4, V23-6, V25-8, V34-14, V36-5, V45-16 and V56-7. Morphological characteristics varied between the hybrid and parental strains. Genomic DNA analysis of the fusants also showed genetic recombination. The median lethal concentration (LC50) for the fusants were lower than that for parental strains, and the median survival time (LT50) for the fusants were reduced compared with that for parental strains. Thus, these results showed that we produced new, more virulent hybrid Lecanicillium spp. strainsas biological control agents via intraspecific protoplast fusion.
... Trichoderma geus was first explained in a monograph by Rifai (1969) as morphologically identifying and characterising. The fungi (Trichoderma spp.) are a different collection of freeliving fungi in the family Hypocreaceae, generally found in totally soil-borne filamentous saprophytes, simply known in the culture medium where they develop large amounts of features from the phialides of the rich or slightly branched condio conidia, the thin, green, yellowish or white conidia (Atanasova et al, 2013;Hassan, 2014;Holzlechner et al, 2016;Mohan, 2017). Trichoderma colonies many ecological vocations where they play a key role; they has been identified as effective biocontrol agents for plant pathogens, secondary metabolite production, and bioremediation (Schuster and Schmoll, 2010;Mukherjee et al, 2013;B³aszczyk et al, 2014). ...
The study aimed to investigate the isolates of Trichoderma that produce emodin by High-performance Liquid Chromatography (HPLC). Trichoderma specimen were collected from University Gardens of Wassit Soil and identified dependent on morphological features and tested to production emodin by HPLC, then two Trichoderma isolates which produced a high concentration of emodin diagnosed for the species using PCR-ITS region. Twenty isolates were identified as Trichoderma according to morphological and microscopic features. In results eight Trichoderma isolates are show differences between the concentrations of secondary metabolic compound (emodin) among the twenty isolates. However, the total concentration of this compound in the extracts of specimen 1, specimen 2, specimen 3, specimen 4, specimen 5, specimen 6, specimen 7 and specimen 8 were (0.0009µg/ml), (0.1345µg/ml), (0.0041µg/ml), (0.0143µg/ml), (8.86666µg/ml), (0.0005µg/ml), (5.0590µg/ml) and (8.96203µg/ml), respectively. At the final concentration of Trichoderma isolates, genomic DNA has been extracted (400 to 600 µg) /(2 to 3g) of wet mycelium and at a purity of 1.6 to1.8 and the results of amplifying Trichoderma DNA samples by using the two universal primers ITS-1 and ITS-4 showed a single unique band consistent with T. harzianum, which with other isolates of the Trichoderma were missing, were identified successfully.
... With the aim of increasing the use of this eco-friendly technology, strategies including the development of effective fermentation processes , microbial stabilization protocols (Bejarano et al., 2017) and specific formulations (Angeli et al., 2017) have been employed to enhance the power of BCAs against diverse pests and diseases in agriculture. Furthermore, classical genetic improvement approaches have been applied to increase the most desirable phenotypic characteristics of effective BCAs, with emphasis on the production of molecules related to antagonism (Hassan, 2014;Singh et al., 2016) and salinity tolerance (Mohamed and Haggag, 2006), mainly using chemical and physical mutagens and single protoplast fusion approaches. ...
Use of biopesticides results in a reduction of the worldwide dependence on chemical pesticides, ending in a more sustainable crop protection. For this reason, biological control industry invests in continued research for more effective biocontrol agents (BCAs) using basically two strategies: isolation of new strains from nature or conventional genetic improvement. However, both are highly onerous and time-consuming. Here we show a pipeline for the genetic improvement of fungal BCAs, based on genome shuffling, the most recent and promising non-recombinant DNA technology for the rapid phenotype improvement of microbial strains. The method consisted of the construction of a parent library using mutagenic agents, followed by genome shuffling and high-throughput screening. Sarocladium oryzae BRM 6461, a known cerulenin antifungal producer strain and high biocontrol potential, served as the model fungal BCA. The pipeline aimed to select mutant enhanced strains at least one of these three desirable characteristics: antagonism, UV-B irradiation tolerance and high temperature tolerance. The experiments were conducted in laboratory and green house. After four cycles of genome shuffling we selected the superior S. oryzae GS4-03 strain, showing cerulenin production of 203.3 ± 1.4 µg/mL, 42% higher than the wild-type strain BRM 6461. The GS4-03 strain exhibited good genetic stability for at least five successive subcultivation assays. Antagonism assay showed an increased micelial inhibition of Rhizoctonia solani and Sclerotinia sclerotiorum when using GS4-03 strain compared to the BRM 6461 strain. The bioassays, showed that the superior strain GS4-03 have an increased ability to control root rot (23.4%) and white mold (8.1 times) disease compared to the wild type strain BRM 6461. Assessment of virulence of the superior strain GS4-03 against rice plants showed that improvement procedures did not change the original strain behavior in this subject. Hydrolytic activity of enzymes related to the biocontrol action as chitinase, protease and β-1,3-glucanase did not present statistical difference between GS4-03 and BRM 6461 strains. Results of dual assay culture between GS4-03 and BRM 6461 strains showed that both have different genetic backgrounds, although RAPD and ITS-rDNA tests were not fully efficient to distinguish genetically the strains. Moreover, mutations resulted in S. oryzae strains more tolerant to UV-B irradiation, but with low genetic stability. Our results report for the first time an efficient pipeline for the genetic improvement of fungal BCAs based on genome shuffling.
