Effects of Trichoderma asperellum BV10 and Bacillus amyloliquefaciens BV03 in Meloidogyne incognita Control Considering Three Different Management Systems

Crop yield decrease is the main concern when a pathogen or plague is identified in an agriculture field. Thus, part of this issue can be attributed to plant-parasitic nematodes (PPNs), such as Meloidogyne species, due to, most of the time, the hard diagnosis, and non-specific symptoms. Its management is mainly based on chemical pesticides, followed by a few potential biological control agents, and the management system. Therefore, this study aimed to evaluate the effects of biological agents in Meloidogyne incognita control in different soil systems. For that, two biological products were chosen, Trichoderma asperellum BV10 and Bacillus amyloliquefaciens BV03, and soils were sampled from three different managements systems: (i) soybean no-tilled system at Goiás state, Brazil; (ii) forest soil at Goiás state, Brazil, and (iii) soybean conventional managed system at Mato Grosso do Sul state, Brazil. Biocontrol and growth promotion effects, volatile organic compounds (VOCs) and soil respiration were determined in vegetation house and laboratory, respectively. As a result, both BV10 and BV03 had significant nematode control activity, comparing to control treatment, in all the three soils systems. Plus, the number of immobile nematodes by potential VOCs had significant increase when BV03 was applied, while the application of BV10 agent raised the soil respiration rate. In conclusion, both biocontrol agents presented great efficiency in control M. incognita, with better performance of BV03. Lastly, more studies must be done to elucidate how the resident soil microbiome can influence on biocontrol agent establishment and performance, as well as the consequence of the application of biological products on soil microbiome network.

Effect of Glucose on Endo-xylanase and β-xylosidase Production by Fungi Isolated in Indonesia

Xylanases are widely produced by fungi, and the production of polysaccharide-degrading enzymes, in general, are usually subjected to carbon catabolite repression. In this work, the ability of several Indonesian indigenous fungi to produce endo-xylanase and β-xylosidase and their responses to glucose as a repressor were determined. Ten fungi were grown in a liquid medium supplemented with glucose as the repressor (0, 1%, 3%, and 5%), and the endo-xylanase and β-xylosidase productions were assayed. Aspergillus aculeatus FIG1 and A. oryzae KKB4 produced 3.85 and 0.70 U/mL of endo-xylanase, respectively, compared with other strains (0.22 U/mL or less). Trichoderma asperellum PK1J2, T. virens MLT2J2, A. aculeatus FIG1, T. asperellum MLT5J1, A. oryzae KKB4, and T. asperellum MLT3J2 produced 0.021–0.065 U/mL of β-xylosidase, whereas the other strains produced 0.013 U/mL or less of β-xylosidase. Adding 1% glucose to the growth medium can partially repress endo-xylanase production in A. aculeatus FIG1, T. asperellum PK1J2, and T. virens MLT4J1 and completely repress other strains. By adding 1% glucose, strains FIG1, PK1J2, and MLT4J1 suffered almost complete repression of β-xylosidase production, although such strains exhibited partial repression of endo-xylanase production. β-Xylosidase produced by the other strains showed complete repression by adding 1% glucose, except for A. aculeatus FIG1, A. tamarii FNCC 6151, and T. asperellum MLT1J1, which showed partial repression. Therefore, adding 3% glucose to the growth medium can result in complete repression of endo-xylanase and β-xylosidase productions in all strains examined.

Effects of Trichoderma asperellum 6S-2 on Apple Tree Growth and Replanted Soil Microbial Environment

Trichoderma asperellum strain 6S-2 with biocontrol effects and potential growth-promoting properties was made into a fungal fertilizer for the prevention of apple replant disease (ARD). 6S-2 fertilizer not only promoted the growth of Malus hupehensis Rehd seedlings in greenhouse and pot experiments, but also increased the branch elongation growth of young apple trees. The soil microbial community structure changed significantly after the application of 6S-2 fertilizer: the relative abundance of Trichoderma increased significantly, the relative abundance of Fusarium (especially the gene copy numbers of four Fusarium species) and Cryptococcus decreased, and the relative abundance of Bacillus and Streptomyces increased. The bacteria/fungi and soil enzyme activities increased significantly after the application of 6S-2 fertilizer. The relative contents of alkenes, ethyl ethers, and citrullines increased in root exudates of M. hupehensis Rehd treated with 6S-2 fertilizer and were positively correlated with the abundance of Trichoderma. The relative contents of aldehydes, nitriles, and naphthalenes decreased, and they were positively correlated with the relative abundance of Fusarium. In addition, levels of ammonium nitrogen (NH4-N), nitrate nitrogen (NO3-N), available phosphorus (AP), available potassium (AK), organic matter (SOM), and pH in rhizosphere soil were also significantly related to changes in the microbial community structure. In summary, the application of 6S-2 fertilizer was effective in alleviating some aspects of ARD by promoting plant growth and optimizing the soil microbial community structure.

OPTIMIZING THE PRODUCTION OF A FUNCTIONAL TYPE A RECOMBINANT ENDOCHITINASE FROM Trichoderma asperellum IN Escherichia coli

Chitinases from the genus Trichoderma fungi are mainly responsible for their anti-fungal activities, which allow them to become the most widely used fungal biocontrol. Therefore, several Trichoderma chitinases have been cloned and expressed to facilitate their production and applications. A previous study of the same authors has characterized an endochitinase from a relatively novel Trichoderma spp., Trichoderma asperellum. To produce this enzyme more economically and efficiently, we reported the synthesis and expression of its synthetic encoding gene in the Escherichia coli M15 strain and established the optimal conditions for preparative scale production of the enzyme in its functional form. By lowering the induction temperatures, we observed substantial improvement in the expression levels of the active enzyme. At 30 oC and 0.5 mM IPTG induction, 1 L of cells yielded approximately 80 - 100 mg of soluble protein, accounting for about 9-11 % of total soluble protein. This figure may be an underestimation of the actual yield, as deduced from the SDS-PAGE data. The recombinant enzyme can be retrieved by simple repeated freezing and thawing cycles and purified to near homogeneity using Ni-NTA chromatography. The purified enzyme showed in vitro colloidal chitin hydrolysis activity. These results could be scaled up to produce soluble 42 kDa chitinase in E. coli. The study demonstrated an economical method to produce chitinases for various agricultural and environmental applications.

The Endophytic Strain Trichoderma asperellum 6S-2: An Efficient Biocontrol Agent against Apple Replant Disease in China and a Potential Plant-Growth-Promoting Fungus

A study was conducted for endophytic antagonistic fungi obtained from the roots of healthy apple trees growing in nine replanted orchards in Shandong Province, China. The fungi were assessed for their ability to inhibit Fusarium proliferatum f. sp. malus domestica MR5, a fungal strain associated with apple replant disease (ARD). An effective endophyte, designated as strain 6S-2, was isolated and identified as Trichoderma asperellum. Strain 6S-2 demonstrated protease, amylase, cellulase, and laccase activities, which are important for the parasitic and antagonistic functions of pathogenic fungi. The inhibition rate of 6S-2 against Fusarium proliferatum f. sp. malus domestica MR5 was 52.41%. Strain 6S-2 also secreted iron carriers, auxin, ammonia and was able to solubilize phosphorus. Its fermentation extract and volatile substances inhibited the growth of MR5, causing its hyphae to twist, shrink, swell, and rupture. The antifungal activity of the 6S-2 fermentation extract increased with increasing concentrations. It promoted the production and elongation of Arabidopsis thaliana lateral roots, and the strongest effects were seen at a concentration of 50 mg/mL. A GC-MS analysis of the 6S-2 fermentation extract and volatile substances showed that they comprised mainly alkanes, alcohols, and furanones, as well as the specific volatile substance 6-PP. The application of 6S-2 spore suspension to replanted apple orchard soils reduced plant oxidative damage and promoted plant growth in a pot experiment. Therefore, the endophytic strain T. asperellum 6S-2 has the potential to serve as an effective biocontrol fungus for the prevention of ARD in China, and appears to promote plant growth.

Enhanced Resistance To Fungal Pathogens in Transgenic Peanut (Arachis Hypogaea L.) Cultivar L14 by Overexpression of Gene encoding Chitinase 42 kDa from Trichoderma Asperellum SH16

This study reports the expression of 42 kDa chitinase genes from Trichoderma asperellum SH16 in peanut (Arachis hypogaea) roots under the regulation of tissue-specific Asy promoter through Agrobacterium tumefaciens-mediated transformation. The 42 kDa chitinase genes, including one wild-type sequence (Chi42) and two synthetic sequences (syncodChi42-1 and syncodChi42-2) which were optimized for codon usage for plant expression, were incorporated into the peanut genome and successfully expressed in their roots. The investigation revealed that the enzyme chitinase from two synthetic genes had higher activity than that from the wild-type gene, about 901 U/mg (140 U/mL) and 1124 U/mg (197 U/mL) vs about 508 U/mg (87 U/mL). Transgenic peanut roots also exhibited extracellular chitinase activity which was driven by signal peptide of rice amylase 3D gene against the pathogenic fungus Sclerotium rolfsii under in vitro conditions. The higher chitinase activity of two synthetic genes in peanut roots promises potential applications in the field of transgenic crops against phytopathogenic fungi.