Effect of Trichoderma spp. on Growth Promotion and Antioxidative Activity of Pepper Seedlings

Abstract One of the main challenges in pepper production is to enhance seed germination energy and germination, and to grow healthy nursery plants with strong root system. Trichoderma species colonize roots as they grow and provide season-long benefits to plants, which is why Trichoderma species are widely used as plant growth promoter agents and promoters of plant defence mechanisms. This study evaluated the effectiveness of seed biopriming with Trichoderma isolates for growth promotion of pepper plants in early stage and their effects on seedling physiology. Nine out of ten Trichoderma isolates positively affected root weight of pepper seedlings, while three out of ten positively affected shoot weight. Root and shoot lengths were mainly unaffected. Germination energy was positively affected by five isolates with up to 40% increase compared to the control, while germination was significantly enhanced by two isolates with up to 22% increase. Considering seedling physiology, two different strain-dependent modes of actions were expressed. Promising Trichoderma isolates induced formation and accumulation of reactive oxygen species (ROS) which acted as signal molecules that increased germination energy and germination. Positive correlation was found between pyrogallol peroxidase, superoxide dismutase, catalase activity and germination in plants treated with these isolates.

Degradation of keratin substrates by keratinolytic fungi

Background: The hydrolysis of keratin wastes by microorganisms is considered a biotechnological alternative for recycling and valorization through keratinolytic microorganisms. Despite their resistant structure, keratin wastes can be efficiently degraded by various microorganisms through the secretion of keratinases, which are promising enzymes for several applications, including detergents, fertilizers, and leather and textile industry. In an attempt to isolate keratinolytic microorganisms that can reach commercial exploitation as keratinase producers, the current work assesses the dynamics of keratin biodegradation by several keratinolytic fungal strains isolated from soil. The activity of fungal strains to degrade keratin substrates was evaluated by SEM, FTRIR-ATR spectra and TGA analysis. Results: SEM observations offered relevant information on interactions between microorganism and structural elements of hair strands. FTIR spectra of the bands at 1035­1075 cm-1 assigned to sulfoxide bond appeared because of S­S bond breaking, which demonstrated the initiation of keratin biodegradation. According to TGA, in the second zone of thermal denaturation, where keratin degradation occurs, the highest weight loss of 71.10% was obtained for sample incubated with Fusarium sp. 1A. Conclusions: Among the tested strains, Fusarium sp. 1A was the most active organism in the degradation process with the strongest denaturation of polypeptide chains. Because keratinolytic microorganisms and their enzymes keratinases represent a subject of scientific and economic interest because of their capability to hydrolyze keratin, Fusarium sp. 1A was selected for further studies.

Improving the thermostability of Trichoderma reesei xylanase 2 by introducing disulfide bonds

Background: Xylanases are considered one of the most important enzymes in many industries. However, their low thermostability hampers their applications in feed pelleting, pulp bleaching, and so on. The main aim of this work was to improve the thermostability of Trichoderma ressei xylanase 2 (Xyn2) by introducing disulfide bonds between the N-terminal and α-helix and the ß-sheet core. Results: In this work, two disulfide bonds were separately introduced in the Xyn2 to connect the N-terminal and α-helix to the ß-sheet core of Xyn2. The two disulfide bonds were introduced by site-directed mutagenesis of the corresponding residues. The half-life of the mutants Xyn2C14­52 (disulfide bond between ß-sheets B2 and B3) and Xyn2C59­149 (disulfide bond between ß-sheets A5 and A6) at 60°C was improved by approximately 2.5- and 1.8-fold compared to that of the wild type Xyn2. In addition, the enzyme's resistance to alkali and acid was enhanced. Conclusion: Our results indicated that the connection of the N-terminal and α-helix to the ß-sheet core is due to the stable structure of the entire protein.

Screening of Trichoderma isolates for their potential of biosorption of nickel and cadmium

Abstract Fourteen Trichoderma isolates were evaluated for their tolerance to two heavy metals, nickel and cadmium. Three isolates, MT-4, UBT-18, and IBT-I, showed high levels of nickel tolerance, whereas MT-4, UBT-18, and IBT-II showed better tolerance of cadmium than the other isolates. Under nickel stress, biomass production increased up to a Ni concentration of 60 ppm in all strains but then decreased as the concentrations of nickel were further increased. Among the nickel-tolerant isolates, UBT-18 produced significantly higher biomass upon exposure to nickel (up to 150 ppm); however, the minimum concentration of nickel required to inhibit 50% of growth (MIC50) was highest in IBT-I. Among the cadmium-tolerant isolates, IBT-II showed both maximum biomass production and a maximum MIC50 value in cadmium stress. As the biomass of the Trichoderma isolates increased, a higher percentage of nickel removal was observed up to a concentration of 40 ppm, followed by an increase in residual nickel and a decrease in biomass production at higher nickel concentrations in the medium. The increase in cadmium concentrations resulted in a decrease in biomass production and positively correlated with an increase in residual cadmium in the culture broth. Nickel and cadmium stress also influenced the sensitivity of the Trichoderma isolates to soil fungistasis. Isolates IBT-I and UBT-18 were most tolerant to fungistasis under nickel and cadmium stress, respectively.

Determination of lytic enzyme activities of indigenous Trichoderma isolates from Pakistan

Abstract This study investigated lytic enzyme activities in three indigenous Trichoderma strains namely, Trichoderma asperellum, Trichoderma harzianum and Trichoderma sp. Native Trichoderma strains and a virulent strain of Rhizoctonia solani isolated from infected bean plants were also included in the study. Enzyme activities were determined by measuring sugar reduction by dinitrosalicylic acid (DNS) method using suitable substrates. The antagonists were cultured in minimal salt medium with the following modifications: medium A (1 g of glucose), medium B (0.5 g of glucose + 0.5 g of deactivated R. solani mycelia), medium C (1.0 g of deactivated respective antagonist mycelium) and medium D (1 g of deactivated R. solani mycelia). T asperellum showed presence of higher amounts of chitinases, β-1, 3-glucanases and xylanases in extracellular protein extracts from medium D as compared to medium A. While, the higher activities of glucosidases and endoglucanses were shown in medium D extracts by T. harzianum. β-glucosidase activities were lower compared with other enzymes; however, activities of the extracts of medium D were significantly different. T. asperellum exhibited maximum inhibition (97.7%). On the other hand, Trichoderma sp. did not show any effect on mycelia growth of R. solani on crude extract.

Morphological changes and growth of filamentous fungi in the presence of high concentrations of PAHs

In this study, we evaluated the effect of low and high molecular weight polycyclic aromatic hydrocarbons (PAHs), i.e., Phenanthrene, Pyrene and Benzo[a]pyrene, on the radial growth and morphology of the PAH-degrading fungal strains Aspergillus nomius H7 and Trichoderma asperellum H15. The presence of PAHs in solid medium produced significant detrimental effects on the radial growth of A. nomius H7 at 4,000 and 6,000 mg L⁻¹ and changes in mycelium pigmentation, abundance and sporulation ability at 1,000–6,000 mg L⁻¹. In contrast, the radial growth of T. asperellum H15 was not affected at any of the doses tested, although sporulation was observed only up to 4,000 mg L⁻¹ and as with the H7 strain, some visible changes in sporulation patterns and mycelium pigmentation were observed. Our results suggest that fungal strains exposed to high doses of PAHs significantly vary in their growth rates and sporulation characteristics in response to the physiological and defense mechanisms that affect both pigment production and conidiation processes. This finding is relevant for obtaining a better understanding of fungal adaptation in PAH-polluted environments and for developing and implementing adequate strategies for the remediation of contaminated soils.

Hydroxylation of 1,8-cineole by Mucor ramannianus and Aspergillus niger

The monoterpenoid 1,8-cineole is obtained from the leaves of Eucalyptus globulus and it has important biological activities. It is a cheap natural substrate because it is a by-product of the Eucalyptus cultivation for wood and pulp production. In this study, it was evaluated the potential of three filamentous fungi in the biotransformation of 1,8-cineole. The study was divided in two steps: first, reactions were carried out with 1,8-cineole at 1 g/L for 24 h; afterwards, reactions were carried out with substrate at 5 g/L for 5 days. The substrate was hydroxylated into 2-exo-hydroxy-1,8-cineole and 3-exo-hydroxy-1,8-cineole by fungi Mucor ramannianus and Aspergillus niger with high stereoselectivity. Trichoderma harzianum was also tested but no transformation was detected. M. ramannianus led to higher than 99% of conversion within 24 h with a starting high substrate concentration (1 g/L). When substrate was added at 5 g/L, only M. ramannianus was able to catalyze the reaction, but the conversion level was 21.7% after 5 days. Both products have defined stereochemistry and could be used as chiral synthons. Furthermore, biological activity has been described for 3-exo-hydroxy-1,8-cineol. To the best of our knowledge, this is the first report on the use of M. ramannianus in this reaction.

Characterization and evaluation of coconut aroma produced by Trichoderma viride EMCC-107 in solid state fermentation on sugarcane bagasse

Background Sugarcane bagasse was shown to be an adequate substrate for the growth and aroma production by Trichoderma species. In the present work the ability of Trichoderma viride EMCC-107 to produce high yield of coconut aroma in solid state fermentation (SSF) by using sugarcane bagasse as solid substrate was evaluated. The produced aroma was characterized. Results Total carbohydrates comprised the highest content (43.9% w/w) compared with the other constituents in sugarcane bagasse. The sensory and gas chromatography-mass spectrometric (GC-MS) analysis revealed that the highest odor intensity and maximum yield of volatiles were perceived at the 5th d of induction period. The unsaturated lactone, 6-pentyl-α-pyrone (6-PP), was the major identified volatile compound. Saturated lactones, δ-octalactone, γ-nonalactone, γ-undecalactone, γ-dodecalactone and δ-dodecalactone, were also identified in the coconut aroma produced during the induction period (12 d). A quite correlation was found between the composition and odor profile of the produced aroma. The effect of varying the concentration of sugarcane bagasse on 6-PP production and biomass growth was evaluated. The results revealed high 6-PP production at 4.5 g sugarcane bagasse whereas the biomass showed significant (P < 0.05) increase by increasing the concentration of sugarcane bagasse. Conclusion The concentration of 6-PP, the most contribution of coconut aroma, produced in present study (3.62 mg/g DM) was higher than that reported in previous studies conducted under the same fermentation conditions. The significant increase in biomass with increasing the concentration of sugarcane bagasse may be attributed to the increase in sugar content that acts as carbon and energy source.

Biocontrol capacity of wild and mutant Trichoderma harzianum (Rifai) strains on Rhizoctonia solani 618: effect of temperature and soil type during storage

Wild (Th11, Th12 and Th650) and mutant (Th11A80.1, Th12A40.1, Th12C40.1 and Th650-NG7) Trichoderma harzianum strains were stored for 180 days at 5ºC or at 22ºC, in two types of soils. Strains recovered at 90, 120 and 180 days from the two types of soils, retained their full capacity to biocontrol Rhizoctonia solani 618, that produces crown and root rot of tomatoes. Recovery, estimated as colony forming units (cfu) of the wild and mutant strains, showed that all increased their cfu after storage independently of the type of soil and temperature, although kinetic behavior differed among strains. Ratios of recovery after storage in type B soil/ type A soil or at 22ºC/5ºC, higher or lower than one respectively, allowed to establish that Th11 and Th12 were the most appropriate strains for the biocontrol of R. solani in conditions where growth of the phytopathogen is optimal.

Influence of buffer systems on Trichoderma reesei Rut C-30 morphology and cellulase production

The cellulase enzyme production is a key issue in the enzymatic hydrolysis of lignocellulosic materials. Since fungal morphology influences the productivity of fungal fermentations, it is of major importance to well know the fungal behavior during culture for cellulase production. In this work, the influence of medium supplementation, with different buffer systems at two different concentrations and pH conditions, on the morphology of T. reesei Rut C-30 and cellulase production, was investigated. A medium without buffer was used as control. The results suggest that fungal morphology is significantly dependent on the addition of different buffer systems to the nutrient broth. The mycelial morphology shows a clear transition from clumped to pelleted forms in cultures with variation of buffer systems and concentration. The higher filter paper activity was obtained using 100 mM succinate buffer, at pH 4.8, in the medium supplementation, corresponding to a dispersed mycelial morphology.

Biocontrol of root and crown rot in tomatoes under greenhouse conditions using Trichoderma harzianum and Paenibacillus lentimorbus: additional effect of solarization

Trichoderma harzianum 650 (Th650) and Paenebacillus lentimorbus 629 (Pl629) selected earlier for their ability to control Rhizoctonia solani, Fusarium solani and F. oxysporum in vitro, were applied alone or combined with solarization (summer assay) and/or with methyl bromide (MeBr) (summer and winter assays) to a soil with a high inoculum level, for the control of tomato root rot caused by the complex F. oxysporum f. sp. lycopersici - Pyrenochaeta lycopersici - Rhizoctonia solani. Evaluations were also performed independently for root damage caused by P. lycopersici, and also for R. solani in the summer assay. MeBr decreased tomato root damage caused by the complex from 88.7 percent to 21.2 percent and from 78.4 percent to 35.7 percent in the summer and in the winter assay, respectively. None of the bio-controllers could replace MeBr in the winter assay, but Th650 and Pl629 reduced root damage caused by this complex in the summer assay. Treatments with bio-controllers were improved by their combination with solarization in this season. Independent evaluations showed that the positive control of Th650 towards R. solani and the lack of effect on P. lycopersici correlates well with the endochitinase pattern expressed by Th650 in response to these phytopathogens. Root damage caused by R. solani can be controlled at a similar level as it does MeBr in summer assays, thus representing an alternative to the use of this chemical fungicide for the control of this phytopathogen.

Carbohydrate-hydrolysing enzyme activity production by solid-state cultures of Trichoderma harzianum strains

Linhagens de Trichoderma harzianum produzem, quando crescidas em meio sólido contendo farelo de trigo como fonte de carbono, atividades enzimáticas que hidrolisam diferentes tipos de carboidratos. As maiores atividades enzimáticas foram de ß-glicosidase, ß-xilosidase e alfa-amilase, enquanto que foram observados níveis muito baixos de atividades de ß-manosidase, alfa-glucuronidase, carboximetil celulase e FPase. As linhagens 4 (CNP 17) e 8 (SS13) foram os melhores produtores de atividade enzimática. O método de extraçäo enzimática na presença de Tween 80, tampäo acetato de sódio, 25mM, pH 5.0 e homogenizaçäo em liquidificador foi menos eficiente que o procedimento que utiliza apenas a soluçäo tampäo descrita acima. O conteúdo de proteína obtido pelo método de extraçäo na ausência de tween 80 foi muito baixo. A atividade de carboximetil celulase foi detectada somente através da extraçäo com Teen 80

Transcriptional control of the cellulase genes in Trichoderma reesei

The expression of the cellulase transcripts of Trichoderma reesei is controlled by the nature of the energy carbon sources used in the culture medium. Cellulose and the soluble disaccharide sophorose, but not glycerol or glucose, act as inducers. Evidence is presented suggesting that a low constitutive extracellular cellulolytic system catalyzes the formation of a soluble inducer from cellulose, and this inducer triggers the expression of the cellulase transcripts. This basal and cellulose-induced expression of the cellobiohydrolase I mRNAs (cbh1), the major member of the cellulase system, is transcriptionally controlled by two independent cis-acting DNA regions. In addition, expression of the cbh1 transcript is influenced by the physiological state of the mitochondria and this sensitivity is controlled through the 5,-flanking DNA sequence of this gene.

Cellulolytic activities of Humicola sp

O fungo termo-celulolítico Humicola sp apresentou alta atividade de exo-ß-D-glucanase (C1) ligada a célula e baixa atividade de endo ß-Glucanase (Cx) ligada a célula e no sobrenadante. Celulose microcristalina foi melhor indutor de celulase e melhor fonte de carbono do que carboximetil celulose. A atividade de ß-glucosidase foi medida somente so sobrenadante da cultura e seu valor foi maior quando carboximetil celulose foi usada como fonte de carbono em pH 7.0

Ethanol fermentation of enzymatically hydrolysed leached beet cosette using Trichoderma harzian um cellulases and Saccharomyces cerevisiae

Se hidrolizó coseta agotada de remolacha utilizando celulasas provenientes de una especie nativa de Trichoderma hazianum. Se obtuvo un 52% de conversión de los carbohidratos presentes en la coseta a azúcares reductores. La fermentación de los azúcares liberados a etanol mediante un proceso de hidrólisis y fermentación combinadas dio como resultado un 66% de conversión del component celulósico de la coseta en eatanol, con un rendimiento de 4,4 g/L de etanol. Al realizar la hidrólisis y fermentación en forma simultánea los rendimientos fueron similares, lográndose un 62% de conversión del componente celulósico de la coseta en etanol. Estos resultados se comparan con los obtenidos utilizando celulosa microcristalina como sustrato