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New components of the chitinolytic system of Trichoderma harzianum
Abstract
Trichoderma harzianum is a mycoparasite known as a biocontrol agent of several economically important plant pathogenic fungi. Trichoderma spp. utilize chitinolytic enzymes, along with β-1,3-glucanase, to degrade the pathogen's cell walls and thus reduce disease level. We employed a set of three fluorescent substrates to identify chitinolytic activities of proteins renatured following their separation by electrophoresis. The chitinolytic system of T. harzianum was found to be more complex than previously reported, consisting of six distinct enzymes, two of which are described here for the first time. The system is composed of two β-1,4 N-acetylglucosaminidases (CHIT102 and CHIT73), and four endochitinases (CHIT52, CHIT42, CHIT33 and CHIT31). The newly described enzymes are CHIT73 and CHIT52. All the chitinolytic enzymes were induced and excreted during growth of Trichoderma on chitin as the sole carbon source. Only CHIT102 was expressed intracellulary, at a low constitutive level, when Trichoderma was grown on glucose. Polyclonal antibodies raised against a purified 41-kDa endochitinase produced by T. harzianum strain P1, reacted only with our CHIT42, suggesting that serologically all the other chitinolytic enzymes are not closely related to the 41-kDa endochitinase. The complexity and diversity of the chitinolytic system of T. harzianum involves the complementary modes of action of six enzymes, all of which are apparently required for maximum efficiency against a broad spectrum of chitin-containing plant pathogenic fungi.
... Regulation of expression of purified chitinases cloned from Trichoderma spp. such as 42 kDa endochitinase CHIT42 (Cruz et al. 1992;Harman et al. 1993), 52 kDa endochitinase CHIT52 (Haran et al. 1995), 40 kDa endochitinase (Ulhoa and Peberdy 1992), 33 kDa endochitinase chit33 (Limón et al. 1995), CHIT37 and CHIT33 (Cruz et al. 1992), CHIT31 and CHIT33 (Haran et al. 1995), ech42, chit42 and ThEn4 (Carsolio et al. 1994;Garcia et al. 1994;Hayes et al. 1994;Irene et al. 1994) pointed to inducible nature of endochitinase production and that its catalytic function is retained under in vitro conditions. Likewise, a 118 kDa 1,4β-N-acetylglucosaminidases designated as CHIT102 (Ulhoa and Peberdy 1991), 73 kDa (CHIT73) and 102 kDa (CHIT102) (Haran et al. 1995), 102 kDa glucosaminidase (CHIT 102) (Inbar and Chet 1995), 72 kDa gene coding an N-acetyl-β-D-glucosaminidase (nag1) demonstrated that glucosaminidases produced by Trichoderma spp. ...
... Regulation of expression of purified chitinases cloned from Trichoderma spp. such as 42 kDa endochitinase CHIT42 (Cruz et al. 1992;Harman et al. 1993), 52 kDa endochitinase CHIT52 (Haran et al. 1995), 40 kDa endochitinase (Ulhoa and Peberdy 1992), 33 kDa endochitinase chit33 (Limón et al. 1995), CHIT37 and CHIT33 (Cruz et al. 1992), CHIT31 and CHIT33 (Haran et al. 1995), ech42, chit42 and ThEn4 (Carsolio et al. 1994;Garcia et al. 1994;Hayes et al. 1994;Irene et al. 1994) pointed to inducible nature of endochitinase production and that its catalytic function is retained under in vitro conditions. Likewise, a 118 kDa 1,4β-N-acetylglucosaminidases designated as CHIT102 (Ulhoa and Peberdy 1991), 73 kDa (CHIT73) and 102 kDa (CHIT102) (Haran et al. 1995), 102 kDa glucosaminidase (CHIT 102) (Inbar and Chet 1995), 72 kDa gene coding an N-acetyl-β-D-glucosaminidase (nag1) demonstrated that glucosaminidases produced by Trichoderma spp. ...
... such as 42 kDa endochitinase CHIT42 (Cruz et al. 1992;Harman et al. 1993), 52 kDa endochitinase CHIT52 (Haran et al. 1995), 40 kDa endochitinase (Ulhoa and Peberdy 1992), 33 kDa endochitinase chit33 (Limón et al. 1995), CHIT37 and CHIT33 (Cruz et al. 1992), CHIT31 and CHIT33 (Haran et al. 1995), ech42, chit42 and ThEn4 (Carsolio et al. 1994;Garcia et al. 1994;Hayes et al. 1994;Irene et al. 1994) pointed to inducible nature of endochitinase production and that its catalytic function is retained under in vitro conditions. Likewise, a 118 kDa 1,4β-N-acetylglucosaminidases designated as CHIT102 (Ulhoa and Peberdy 1991), 73 kDa (CHIT73) and 102 kDa (CHIT102) (Haran et al. 1995), 102 kDa glucosaminidase (CHIT 102) (Inbar and Chet 1995), 72 kDa gene coding an N-acetyl-β-D-glucosaminidase (nag1) demonstrated that glucosaminidases produced by Trichoderma spp. are inducible in nature. ...
Trichoderma (teleomorph Hypocrea) the soil-borne fungus grows in its natural habitat on plant root surfaces and acts as a mycoparasite. This mycoparasitic fungus adopts several methods of fungal disease control that occur sequentially and the article summarizes the mode of action of extracellular cell wall degrading lytic enzymes, antifungal metabolites, short oligosaccharides and host-derived signals in eliciting mycoparasitism in perspective to the genes encoding these mechanisms. Distinction of this study pertains to classification of Trichoderma spp. genes involved in mycoparasitism according to their mode of action that can lead to improved practical application by desigining efficient expression cassettes for combating plant fungal diseases through transgensis.
... The entomopathogen Trichoderma sp. secretes, while infecting the host, hydrolytic enzymes like chitinases, β-(1,3)-glucanases, proteases and lipase with a potential to act as biocontrol agent against insect pests and plant pathogenic fungi (Haran et al. 1995;Harman et al. 2004). There are four endochitinases and two β-(1,4)-Nacetylglucosaminidases present in Trichoderma and this number varies from species to species. ...
... There are four endochitinases and two β-(1,4)-Nacetylglucosaminidases present in Trichoderma and this number varies from species to species. In T. harzianum its chitinolytic system comprises of five to seven distinct enzymes which work with a complementary mode of action (Haran et al. 1995). In addition, two extracellular enzymes namely β-1,6-glucanase and hydrolases are also produced while growing on chitin. ...
Growing concern and general awareness of the increased use of pesticides and their overall adverse effects on ecosystem and human health, bioaccumulation in food chains, pest resistance and persistence of chemicals in the environment elicited the search for alternate pest controlling strategies. Hydrolytic enzymes including chitinases, proteases, lipases and glucanases, which are key biochemical components of insect metabolism and life cycle, have become new arsenal for controlling pests. Owing to the presence of cuticle covering, hydrolytic enzymes act on and degrade the insectbarriers, and start the body infection process. Various entomopathogenic fungi, bacteria and viruses have been reported to release hydrolytic enzymes against pests. This chapter provides an insight of hydrolytic enzymes and their role in Insect Pest Management (IPM). Potential use of these hydrolytic enzymes, their virulence and mechanism of action could be valuable for producing more potent and safer insecticides. Comprehensive understanding and knowledge-based chemistry and regulation of chitin metabolism, part of the insect pest which is more susceptible to hydrolytic enzymes, life cycle and stages of insects and thorough understanding of metabolic pathways will help in control of pests and achieving IPM goals.
... It has been observed in several reports that Trichoderma spp. inhibits the growth of various soil-borne pathogens by secreting chitinases (Rai et al. 2016a;Almeida et al. 2007;Kashyap et al. 2011), β-glucanases (Haran et al. 1995), and proteinases (Geremia et al. 1993) enzymes. Similarly in the current study, Trichoderma and Hypocrea isolates displayed significant biological control activity toward R. solani on both dual plate confrontation as well as volatile metabolite assays; however, a noticeable decline was observed in their antagonistic activity as salt concentration increased. ...
... Similarly in the current study, Trichoderma and Hypocrea isolates displayed significant biological control activity toward R. solani on both dual plate confrontation as well as volatile metabolite assays; however, a noticeable decline was observed in their antagonistic activity as salt concentration increased. Abd-Alla and Omar (1998) and Haran et al. (1995) also observed a decrease in enzymatic activity of T. harzianum by incorporating NaCl to the cultivation medium. Further, Mohamed and Haggag (2006) demonstrated that halotolerant mutants of T. harzianum significantly surpassed their wild-type strain in growth, sporulation, and antagonistic proficiency against F. oxysporum under saline conditions. ...
The aim of present study was to decipher the effect of salinity stress on growth and antagonistic potential of Trichoderma and Hypocrea isolates against tomato root rot pathogen (Rhizoctonia solani AG-4) under saline soil conditions. In vitro salinity assays, dual plate confrontation, and volatile metabolite assays were employed to establish the antagonistic potential of Trichoderma and Hypocrea isolates against R. solani AG-4 under varied salt gradients. Potential Trichoderma and Hypocrea isolates were evaluated against tomato root rot disease in greenhouse conditions under salt stress condition. Polymerase chain reaction (PCR) assay was performed to confirm the presence of endochitinase gene in salt-tolerant antagonists. Enzyme and biochemical assays were conducted to define the role of compatible solutes and defense-related enzymes in controlling tomato root rot under saline soil conditions. Trichoderma and Hypocrea isolates were capable to grow and sporulate up to 250 mM NaCl and also showed strong antagonism against R. solani. Enzymatic estimation of hydrolytic enzymes and amplification of endochitinase gene suggested that the test isolates are potent antagonistic agents. Under greenhouse evaluation, Trichoderma and Hypocrea fortified tomato plants showed significant reduction in tomato root rot disease in saline soils over untreated control. Significant increase in total phenol, polyphenol oxidase, peroxidase, β-1,3-glucanase, phenylalanine lyase, chitinase, proline, reducing sugar, and total soluble sugar displayed direct association with salt stress tolerance. Application of salt-tolerant Trichoderma and Hypocrea isolates emerged as a simple, safe, and cheap method for the biological management of tomato root rot under saline condition.
... The ability to invade the sclerotium and use the nutrients found in its environment usually relies on the possibility of the existence of enzymes and toxins, which open up the pathways and disengage nutrients. Their presence and role have been examined thoroughly (Aggelaki 1996;Dunlop et al. 1989;Correa et al. 1995;Haran et al. 1995;Lee and Wu. 1984;Madsen and de Neergaard 1999;Machida et al. 2001;McQuilken et al. 2003;Ordentlich et al. 1992;Rodriguez et al. 2006;Wolffhechel and Jensen 1992). ...
... The sclerotial disruption has started since the 20th day, without being at immediate contact with the cells of T. koningii probably due to some enzyme mechanism (Aggelaki 2001). The production and effect of enzymes, antibiotics and toxins are well documented in the relevant bibliography (Correa et al. 1995;de la Cruz et al. 1995a, b;Dunlop et al. 1989;El Hajji et al. 1989;Lee and Wu 1984;Haran et al. 1995;Machida et al. 2001;McQuilken et al. 2003). ...
Mycoparasitism is an important process in microcosm of microorganisms. Understanding the mechanisms taking place may allow to effectively improved biocontrol of phytopathogens. A sequence of events during mycoparasitism process by three mycoparasites of different origin and aggressiveness was studied through an optical microscope. Additionally, both germination and entry procedure of mycoparasites on host’s surface were observed through an electron scanning microscope. The development of sclerotia parasitism shows many common features in all three mycoparasites indicating very likely both common course and mechanisms. G21-3 (Gliocladium spp.) is the faster and more destructive mycoparasite followed by T12-9 (Trichoderma spp.) and FD6-15 (Fusarium spp.). In the present study, it was presented in a novel way both the appearance of hyphae of D6-15 isolation (Fusarium spp.) intra-cellularly and also the formation of chlamydospores (intra-cellularly) from G21-3 isolation (Gliocladium spp.). G21-3 germination on the sclerotial surface was completed within 15 h of incubation, and germ tubes will be strayed enough before entering into sclerotium. Appressoria and germ tube branches formation were not observed.
... and some pathogenic fungi (Haran et al., 1996). The chitinolytic system of T.harzianum consists of five to seven distinct enzymes, depending on the strain (Haran et al., 1995). Adhikari et.al., (2014) also reported that Trichoderma sp can inhibit around 75% growth of S. rolfsii on PDA. ...
... Trichoderma spp. produce extracellular β-(1, 3) -glucanases, chitinases, lipases, and proteases when they are grown on cell walls of pathogenic fungi (Haran et al., 1995). Elad et al. (1980) found that a wheat-bran preparation of T. harzianum applied to fields at rates of 500-1500 kg/ha, reduced the incidence of diseases caused by S. rolfsii. ...
The study was carried out in 28 villages covering131 mango growers of three
Upazilas in Chittagong District with view to examine the adoption status of
BARI mango varieties at farmer’s level. The adoption status of improved mango
varieties was unknown to the region. Results revealed that out of 11 varieties of
BARI mango, the highest 77% farmers adopted BARI Aam-3 followed by BARI
Aam- 4 (22.1%) and BARI Aam-8 15.9%. But the rate of adoption of other
varieties of BARI Aam was found to be lower irrespective of all locations due to
unavailability of sapling and unknown to the variety. The rate of adoption of
individual production technologies of BARI mango varieties was found
unsatisfactory. Majority of the farmers did not adopt recommended practices as
stated in BARI Krishi Projokti Hathboi such as pit size, planting distance,
application of manure and fertilizers, plant growth regulator, insects and
diseases management. Farmers maintained pit size for mango sapling (1.4 ft ×
1.4 ft× 1.3 ft) compared to recommended size of (3ft×3ft×3ft). Similarly,
planting distance was 12.0 x11.7ft as against the recommended distance of
25ft×30ft. About 67.7% farmers adopted the improved practice such as breaking
inflorescence of mango trees and 65.0% of farmers used mulching. But majority
(52%) of farmers did not receive training and practice pruning for mango trees.
Probit regression analysis revealed that yield of mango variety, training,
extension contact, risk taking behavior and willingness to take loan has indeed
helped in contributing to adopt BARI mango varieties significantly. Therefore,
promoting training on BARI mango production technologies; ensuring the
availability of BARI mango saplings, and campaigning about the varieties in
mass media could help to increase the rate of adoption of BARI mango varieties
in the region.
... and some pathogenic fungi (Haran et al., 1996). The chitinolytic system of T.harzianum consists of five to seven distinct enzymes, depending on the strain (Haran et al., 1995). Adhikari et.al., (2014) also reported that Trichoderma sp can inhibit around 75% growth of S. rolfsii on PDA. ...
... Trichoderma spp. produce extracellular β-(1, 3) -glucanases, chitinases, lipases, and proteases when they are grown on cell walls of pathogenic fungi (Haran et al., 1995). Elad et al. (1980) found that a wheat-bran preparation of T. harzianum applied to fields at rates of 500-1500 kg/ha, reduced the incidence of diseases caused by S. rolfsii. ...
p>The released three varieties of bush bean (BARI Jhar Shim-1, 2 and 3) were grown in both Sclerotium rolfsii inoculated and un-inoculated plots to assess the yield loss in the field of Plant Pathology Division, Bangladesh Agricultural Research Institute (BARI), Gazipur during 2013. Sclerotium rolfsii infected the bush bean plant in every growth stage and reduced the germination capacity and lessened plant vigor. The yield loss ranged from 55.43 to 64.94%. A screening study was conducted to observe the resistant level of released varieties and local lines collected from On Farm Research Division (OFRD), BARI, Sylhet during 2013. B0124 and BARI Jhar Shim-3 showed susceptible and the others showed highly susceptible reaction. The antagonistic potential of the local isolates of Trichoderma harzianum against the phytopathogenic S. rolfsii was investigated in dual culture and bioassay on bean plants. Isolated T. harzianum showed antagonism effect against S. rolfsii and restricted around 80% mycelial growth in-vitro. A field experiment was carried out in Plant Pathology Division, BARI, Gazipur during 2014 to observe the efficacy of T. harzianum in controlling foot rot disease induced by S.rolfsii of bush bean in the field. Soil treated with T. harzianum mass plus seed treated with Provax 200 WP showed the lowest disease incidence (28.47%) and the highest germination (98.61%) and yield (873.33 kg/ha) in compared to other treatments with pathogen inoculated soil.
Bangladesh J. Agril. Res. 43(2): 289-299, June 2018</p
... Only the purified 42 kDa chitinase hydrolyzed B. cinerea purified cell walls in vitro, but this effect was heightened in the presence of either of the other two isoenzymes. According to Haran et al. (1995), the chitinolytic system of T. harzianum was more complex, consisting of six distinct enzymes. The system is apparently composed of two ß-(1,4)-N-acetylglucosaminidases of 102 and 73 kDa, respectively, and four endochitinases of 52, 42, 33, and 31 kDa, respectively. ...
... The 1,4-β-N-acetyl-glucosaminidases of 72 kDa have been purified from T. harzianum strain (Lorito et al. 1994). Haran et al. (1995) reported the chitinase isolated from respective T. harzianum had different molecular weights: 73 kDa heat-stable glucosaminidase (CHIT 73), isolated from T. harzianum strain TM, an endochitinase of 52 kDa (CHIT 52), an endochitinase of 42 kDa (CHIT 42), the endochitinases produced by the other strains of T. harzianum which saminamehnaz@fccollege.edu.pk are of 33 kDa (CHIT 33) and 31 kDa (CHIT 31), and two endochitinases, having molecular weights of 37 kDa and 33 kDa, which were expressed by T. harzianum strain CECT 2413. ...
This book describes the contributions of rhizotrophs – microbes associated with the parts of plants below ground – in sustainable agriculture. It covers a broad range of aspects, from plant growth promotion to bioremediation. It highlights the role of bacteria, actinomycetes, mycorrhizal fungi, and most interestingly protists, in the sustainability of agriculture. Further, it addresses in detail the involvement of quorum sensing signals, and the role of hydrolytic enzymes and bacteriocin in combating the phytopathogen.
The book sheds light on the interaction of rhizotrophs in rhizosphere and how these microbes support plants growing under adverse stress conditions such as saline, drought or heavy-metals contamination. Challenges faced in the field application of these microbes, strategies for modifying the rhizosphere to improve crop yield, and the latest advances in rhizobial bioformulations are also discussed. Overall, the book provides comprehensive information on how various microbes can be used to improve the sustainability of agriculture without disturbing the environment.
... Only the purified 42 kDa chitinase hydrolyzed B. cinerea purified cell walls in vitro, but this effect was heightened in the presence of either of the other two isoenzymes. According to Haran et al. (1995), the chitinolytic system of T. harzianum was more complex, consisting of six distinct enzymes. The system is apparently composed of two ß-(1,4)-N-acetylglucosaminidases of 102 and 73 kDa, respectively, and four endochitinases of 52, 42, 33, and 31 kDa, respectively. ...
... The 1,4-β-N-acetyl-glucosaminidases of 72 kDa have been purified from T. harzianum strain (Lorito et al. 1994). Haran et al. (1995) reported the chitinase isolated from respective T. harzianum had different molecular weights: 73 kDa heat-stable glucosaminidase (CHIT 73), isolated from T. harzianum strain TM, an endochitinase of 52 kDa (CHIT 52), an endochitinase of 42 kDa (CHIT 42), the endochitinases produced by the other strains of T. harzianum which are of 33 kDa (CHIT 33) and 31 kDa (CHIT 31), and two endochitinases, having molecular weights of 37 kDa and 33 kDa, which were expressed by T. harzianum strain CECT 2413. ...
Microbial community in the rhizosphere produces a variety of hydrolytic enzymes that are responsible for the degradation of various components of fungal pathogens. The extracellular hydrolytic enzymes excreted by soil rhizobia degrade cell wall components of plant pathogenic microbes. The enzymes of these types are able to breakdown glycosidic linkages present in the polysaccharide of the cell wall of phytopathogens. In this regard, plant growth-promoting rhizobacteria (PGPR) are known to colonize rhizosphere and enhance plant growth through different mechanisms that include (i) plant growth promotion and (ii) biological control of plant disease. Plant growth promotion mechanisms include mineralization of insoluble substances, production of plant growth hormones, biological nitrogen fixation, and promotion of root growth. Biocontrol mechanism involves competition, antibiosis, parasitism, induction of systemic acquired resistance (SAR), induction of systemic resistance (ISR), soil suppressiveness, and production of various antifungal metabolites; hydrolytic enzymes such as chitinase, glucanase, protease, and cellulase; and antibiotics such as 2,4-diacetyl phloroglucinol (DAPG), amphisin, oomycin A, hydrogen cyanide, phenazine, pyoluteorin, pyrrolnitrin, cyclic lipopeptides, oligomycin A, zwittermicin A, kanosamine, and xanthobaccin. Production of hydrolytic enzymes by PGPR is an important mechanism directed against phytopathogens for sustainable plant disease management. These enzymes break down the cell wall of fungal pathogens causing cell death. This review focuses on the different aspects of various hydrolytic enzymes produced by rhizoflora and their role in sustainable biocontrol of phytopathogens.
... In mycoparasites, purification was mainly focused on the chitinolytic system of T. harzianum. Several chitinases, including endochitinases, chitobiosidases and N-acetyl-glucosaminidases, from T. harzianum have been purified [136,137,143]. The purification of chitinases from other mycoparasites, such as A. album and Gliocladium virens [140], Fusarium chlamydosporum [144], Trichothecium roseum [141], Stachybotry elegans [145], and Talaromyces flavus [142] have also been reported. ...
... Chitinase production by fungi is inducible in nature, and chitinase activities are generally detected when the fungi are grown in a chitin-containing medium [136,138,140,143,145]. It has been reported that chitinase gene expression in fungi is controlled by a repressor/inducer system, in which chitin or other products of degradation (such as N-acetyl-b-glucosamine) act as inducers, and glucose or easily metabolised carbon sources act as repressors. ...
Entomopathogenic fungi are well-known biological control agents of insects that have broadly replaced the chemicals used in biopesticides for agricultural purposes. The pathogenicity of entomopathogenic fungi depends on the ability of its enzymatic equipment, consisting of lipases, proteases and chitinases, which degrade the insect's integument. Additionally, the researchers studied the content of β-galactosidase, l-glutaminase, and catalase within entomopathogenic fungi. With highly focused investigations on the use of enzymes for green technology, the group of entomopathogens are slowly gaining applications in these areas, even as phytopathogenic fungi (disease originator). This brief review will serve as a reference of the enzymes derived from entomopathogenic fungi and of their current and potential applications.
... Molecular weight of proteins was estimated as described by Sambrook et al. 10 . Enzymes were reactivated in the replica gel by removing SDS by casein-EDTA procedure developed by McGrew and Green 11 , and modified by Haran et al. 12 . In this procedure, after electrophoresis, the gel was immersed in Buffer A: 1 M Tris HCl, pH 9.0 (40 ml), 20% sodium azide (1 ml), 0.5 M EDTA, pH 8.0 (4 ml) and casein (10 g) per litre. ...
... Cloning of genes encoding for endochitinase from Trichoderma spp. has already been reported 3,12,[27][28][29][30] . A chitinase gene ech42 of 1447 bp was obtained from T. atroviride, which had three introns in the sequence 31 . ...
The saprophytic fungus, Trichoderma harzianum strain T5 antagonistic to the red rot pathogen of sugarcane, Colletotrichum falcatum excreted chitinases, viz. N-acetyl-β-D-glucosaminidase, 1,4-β-D-N-N′ chitobiosidase and 1,4-β-D-N-N′-N″ chitotriase into the culture medium containing pathogenic cell wall or chitin. The protein profile analysis on SDS-PAGE stained proteins is in the range of 20-124 kDa. The fungus produced a 97 kDa N-acetyl glucosaminidase in medium amended with fungal cell wall or colloidal chitin. Chitobiase isoforms of 66, 56, and 50 kDa and 66 and 50 kDa were detected in fungal cell wall and colloidal chitin amended media respectively. The fungus also excreted chitotriase isoforms of 66 and 50 kDa in cell wall and 50 kDa in colloidal chitin amended media respectively. The chitinase enzymes of 66 and 50 kDa, which degraded both chitobiose and chitotriose, are isoforms of both chitobiase and chitotriase that got separated at the same distance in SDS-PAGE. In an attempt to clone the endochitinase gene from T. harzianum T5, a partial cDNA of 246 bp (Genbank accession number AY762230) was obtained through RT-PCR and the deduced sequence showed high level of homology with chitinase sequences of the database.
... Mycoparasitism by T. harzianum is a complex process, involving recognition of the host, attachment to the mycelium, coiling around the hyphae, partial degradation of the cell wall and penetration of the host mycelium (Elad et al., 1983a, b;Benhamou and Chet, 1993). Cell wall degradation is achieved by six chitin-induced chitinolytic enzymes (comprising two B-1, 4-N-acetylglucoseaminidases and four endochitinases), all of which are required for effective parasitism (Haran et al., 1995). Once, the host mycelium has been penetrated, additional extracellular enzymes such as lipases and proteases are produced to induce degradation of the cell contents . ...
... Antagonism by T. harzianum depends on mycoparasitism rather than antibiosis, as evident from the relatively slight inhibition of mycelial growth of R. solani, particularly AG-3; in dual culture (Shternshis, 2002). Besides, being an aggressive mycoparasite (Elad et al., 1983b;Benhamou and Chet, 1993;Haran et al., 1995;Elad et al., 1982;Chet and Baker, 1981), T. harzianum is also known to enhance plant growth in the absence of any pathogens, probably by producing plant growth promoting metabolites in the rhizosphere (Chang et al., 1986;Kleifeld and Chet, 1992). ...
The efficacy of soil application with microbial preparations viz. Trichoderma harzianum, effective microbe (EM) culture and biological potassium fertilizer (BPF) was evaluated for the management of soil-borne inoculum of Rhizoctonia solani the cause of black scurf of potato cv. Desiree. Soil application with three dosages of culture suspension of T. harzianum, effective microbe (EM) culture and biological potassium fertilizer (BPF) were applied in the soil to know the efficacy of these treatments in reducing the disease. Soil application with T. harzianum at the time of sowing followed by two and three dosages at 20 days intervals gave significant protection to eyes with EGI of 30.55%, SK 24.07%, SCI 36.10%, StCI 30.60%, BSDI 26.43% and YR of 35.09% against the fungus which ultimately contributed to better crop stand and increased yield as compared to inoculated control and rest of the treatments.
... These results confirmed that T. asperellum produced a multi-enzymatic cocktail devoted to chitin degradation, as previously described also for other Trichoderma spp. 24,39,41,42 To investigate the substrate specificity of these T. asperellum chitinases, their activity was tested at 25°C on three differentlength fluorogenic analogues of chitooligosaccharides, varying the pH of the assay solutions (at pH values of 5.0, 7.0, and 9.0). Overall, the total chitinase activity was highest at acid pH: only c. 20% of the total activity was detectable at neutral pH, while at basic pH only traces of it were measurable (Fig. 4). ...
BACKGROUND
Despite their known negative effects on ecosystems and human health, synthetic pesticides are still largely used to control crop insect pests. Currently, the biopesticide market for insect biocontrol mainly relies on the entomopathogenic bacterium Bacillus thuringiensis (Bt). New biocontrol tools for crop protection might derive from fungi, in particular from Trichoderma spp., which are known producers of chitinases and other bioactive compounds able to negatively affect insect survival.
RESULTS
In this study, we first developed an environmentally sustainable production process for obtaining chitinases from Trichoderma asperellum ICC012. Then, we investigated the biological effects of this chitinase preparation – alone or in combination with a Bt‐based product – when orally administered to two lepidopteran species. Our results demonstrate that T. asperellum efficiently produces a multi‐enzymatic cocktail able to alter the chitin microfibril network of the insect peritrophic matrix, resulting in delayed development and larval death. The co‐administration of T. asperellum chitinases and sublethal concentrations of Bt toxins increased larval mortality. This synergistic effect was likely due to the higher amount of Bt toxins that passed the damaged peritrophic matrix and reached the target receptors on the midgut cells of chitinase‐treated insects.
CONCLUSION
Our findings may contribute to the development of an integrated pest management technology based on fungal chitinases that increase the efficacy of Bt‐based products, mitigating the risk of Bt‐resistance development. © 2024 Society of Chemical Industry.
... Likewise, the increase in soil chitinase activity with Trichoderma sp. application was in line with Haran et al. [70] who stated that T. harzianum produces two N-acetylglucosaminidases, four endochitinases, and one chitobiosidase. Also, T. saturnisporum produces Chitobiosidase [71]. ...
Abstract Background The increase in the human consumption of fish results in the production of organic fish wastes (FW). For enhanced soil fertility and plant growth at a lower cost and without the negative impacts of chemical fertilizers, these wastes could be employed as a valuable organic fertilizer. To determine the synergistic bio-efficacy of Trichoderma sp. and arbuscular mycorrhizal (AM) fungi in stimulating the morphological and physiological characteristics of FW-fertilized Alium cepa, as well as to investigate their involvement in boosting soil fertility, the current study was carried out. Overall, eight treatments were applied as follows: AM, Trichoderma sp., AM + Trichoderma sp., FW, AM + FW, Trichoderma sp. + FW, AM + Trichoderma sp. + FW, and control. Growth and physiological assessments of onion plants were taken after 8 weeks from FW application. Results Our results showed that FW application combined with AM fungi and Trichoderma sp. inoculations increased aggregate stability of the soil (glomalin content) and soil chitinase activity. Moreover, using the bio-inoculations along with FW amendments significantly (p
... Reports are available on significant large size of "fungal/plant" chitinases to "fungal/bacterial" chitinases. Trichoderma harzianum synthesizes all three chitinases in seven types as one chitobiosidase, two N-acetylglucosaminidases, and four endochitinases [48]. In the sediments of Lake Chełmzynskie, 32%-40% of molds were able to decompose chitin [21]. ...
Microorganisms synthesize an array of glycosyl hydrolytic enzymes quantitatively and chitinase is one among them. In general, the persistence of chitinase form microbial source is availing elemental carbon and nitrogen as precursors or nutrient sources and the insatiability of this enzyme plays a significant role mainly in parasitism against chitinaceous host. Out of different microbial chitinases availed in public domain, soil bacterial chitinases share more than half of genomes and mostly are of actinobacterial origin. The others are from proteobacteria, yeast, moulds, and few viral sources. With the advent of biotechnology, the production of chitinases is increased to many folds. Extracellular chitinases disintegrate chitin polymers to produce oligomers having several applications. The microbial chitinases are being broadly applied in various realms including agricultural, biomedical, pharmaceutical, industrial, and environmental. The wide application is well marked as biopesticides and biocontrol agents. Furthermore, the waste minimization with chitinase application is surplus to the environmental cleanup initiatives. The present chapter is inclusive of production of microbial chitinases and their detail applications in descriptive manner.
... The production of chitinases by Trichoderma spp. is widely known, and several genes encoding chitin-and β-glucan-degrading enzymes have been identified in different species [52,69], and can explain this phenomenon. When quantifying the chitinolytic activities of Xeda, exochitinases were massively detected, but not endochitinases, unlike other species of Trichoderma able to produce N-acetylglucosaminidases, endochitinases, and chitobiosidases [70]. However, other strains of T. asperellum have shown their ability to produce mainly exochitinases but also small amounts of endochitinase, especially in the contact phase but not in the pre-contact or post-contact phase with the pathogen under study [71], which may suggest that chronology plays an important role in the analysis of chitinolytic activities. ...
The aim of this study was to develop a set of experiments to screen and decipher the mechanisms of biocontrol agents (BCAs), isolated from commercial formulation, against two major mycotoxigenic fungi in cereals, Fusarium graminearum and Fusarium verticillioides. These two phyto-pathogens produce mycotoxins harmful to human and animal health and are responsible for the massive use of pesticides, for the protection of cereals. It is therefore essential to better understand the mechanisms of action of alternative control strategies such as the use of BCAs in order to optimize their applications. The early and late stages of interaction between BCAs and pathogens were investigated from germination of spores to the effects on perithecia (survival form of pathogen). The analysis of antagonist activities of BCAs revealed different strategies of biocontrol where chronological , process combination and specialization aspects of interactions are discussed. Streptomyces griseoviridis main strategy is based on antibiosis with the secretion of several compounds with anti-fungal and anti-germination activity, but also a mixture of hydrolytic enzymes to attack pathogens, which compensates for an important deficit in terms of spatial colonization capacity. It has good abilities in terms of nutritional competition. Trichoderma asperellum is capable of activating a very wide range of defenses and attacks combining the synthesis of various antifungal compounds (me-tabolite, enzymes, VOCs), with different targets (spores, mycelium, mycotoxins), and direct action by mycoparasitism and mycophagy. Concerning Pythium oligandrum, its efficiency is mainly due to its strong capacity to colonize the environment, with a direct action via microbial predation, stimulation of its reproduction at the contact of pathogens and the reduction of perithecia formation.
... Mehmood et al. (2010) demonstrated that four chitinases secreted by Aeromonas caviae CB101 are encoded by a single chitinase gene Chi1. Furthermore, the well-known fungus Trichoderma harzianum secretes N-acetylglucosaminidases, endochitinases, and chitobiosidase (Haran et al. 1995). Aggarwal et al. (2015a) reported presence of two isoforms of chitinases in entomopathogenic S. marcescens with molecular weight of ~51 kDa and 32 kDa. ...
Chitin is an important structural component of many plant pathogenic fungi. Similarly, it is also an important part of the insect cuticle and peritrophic matrices, which function as a permeability barrier, enhance digestive processes and protecting the brush border from mechanical disruption as well as from attacks by toxins and pathogens. Chitin degrading lytic enzymes (such as chitinases, and glucanase) produced by bacteria and other microorganisms can impede the growth of many insect pests and fungal phytopathogens that pose a severe risk to global crop production. Pathogenic microorganisms produce a variety of lytic enzymes such as proteases, chitinases, lipases etc. which play an important role in the virulence of entomopathogens. Many chitinolytic bacteria have the potential to control pests and fungal pathogens of crops owing to their ability to disintegrate chitin containing cellular structures. Currently, efforts are being made to discover producers of chitinolytic enzymes in nature. Production of lytic enzymes has been reported in a number of virulent pathogens such as Serratia, Pseudomonas or Bacillus spp. Bioprospecting and exploitation of chitinolytic bacteria will help in developing biocontrol agents, which have the potential to control fungal plant pathogens and insect pests. Thus, these bacteria-based biofungicides and biopesticides may replace or supplement the chemical fungicides and insecticides, reducing the negative impact of chemicals on the environment and supporting the sustainable development of agriculture-based ecosystem. This chapter focuses on the scope and potential of chitinolytic bacterial and fungal organisms in the management of insect pests and fungal pathogens of agricultural crops.
... Therefore, it has been used with varying degrees of success. Although, Geremia et al. (1993) and Haran et al. (1995) reported that this process is further supported by the secretion of extracellular enzymes such as chitinases or βeta glucanases and proteinases, whereas, Harman et al. (2004) referred to the effects of these compounds in phytopathogenic fungi include degradation of the cell walls. ...
... These microbes stimulate the production of auxins, degrade the compounds of phenolic nature released by plants, may become an endophyte, enhance the uptake of water and nutrients, help in solubilization of soil nutrients, promote efficient utilization of nitrogen by plant and increase their vigor and vitality, encourage the growth of roots and other plant organs, help in the formation of root hairs, improve the plant's photosynthetic capacity, they accelerate the germination of seeds, produce secondary metabolites like harzianic acid which encourage plant growth, helps to deal with abiotic stresses, and increase resistance capacity of plants, especially during unsuitable growing conditions (Contreras-Cornejo et al. 2009;Ruocco et al. 2009;Hermosa et al. 2012;Harman 2006;Shoresh et al. 2010;Lorito et al. 2010;Mastouri et al. 2010;Vinale et al. 2009). The chitinolytic system of Trichoderma harzianum consists of five to seven different enzymes (vary from strain to strain) and these enzymes help in killing the pathogenic fungi (Haran et al. 1995). In another study conducted by de Paula et al. (2001), T. harzianum safeguarded the seedlings of bean against preemergence damping-off infection caused by Rhizoctonia solani pathogen, resulting in better growth of bean plants. ...
This book is about the role played by microbes in their community mode in sustaining ecosystems. The descriptions given in its chapters indicate clearly that microbial communities are more effective in delivering multifaceted benefits to the soil-plant system than those offered by microbial monocultures in planktonic modes. The role these communities play in a multitude of microbe-microbe and plant-microbe interactions have not yet been fully exploited to gain benefits in this field as well as to achieve sustainability in agriculture practices. Amply discussed are the beneficial characteristics and metabolic capacities of specific microbial groups and the use of microbial traits for the benefit of plant growth. The book suggests the need to develop new microbial technologies to utilize plant-associated microbes for increased crop productivity and agroecosystem balance in order to ensure sustainability. This also provides an effective guidance to scientists, academics, researchers, students and policy makers of the sphere to achieve the above outcomes.
... These microbes stimulate the production of auxins, degrade the compounds of phenolic nature released by plants, may become an endophyte, enhance the uptake of water and nutrients, help in solubilization of soil nutrients, promote efficient utilization of nitrogen by plant and increase their vigor and vitality, encourage the growth of roots and other plant organs, help in the formation of root hairs, improve the plant's photosynthetic capacity, they accelerate the germination of seeds, produce secondary metabolites like harzianic acid which encourage plant growth, helps to deal with abiotic stresses, and increase resistance capacity of plants, especially during unsuitable growing conditions (Contreras-Cornejo et al. 2009;Ruocco et al. 2009;Hermosa et al. 2012;Harman 2006;Shoresh et al. 2010;Lorito et al. 2010;Mastouri et al. 2010;Vinale et al. 2009). The chitinolytic system of Trichoderma harzianum consists of five to seven different enzymes (vary from strain to strain) and these enzymes help in killing the pathogenic fungi (Haran et al. 1995). In another study conducted by de Paula et al. (2001), T. harzianum safeguarded the seedlings of bean against preemergence damping-off infection caused by Rhizoctonia solani pathogen, resulting in better growth of bean plants. ...
The natural environment contains a diverse range of microorganisms and plants, which interact with each other in a variety of ways. This interaction may range from two-partite symbiosis (formation of single nodule by symbiotic association of legume and rhizobia which assists in nitrogen fixation in environment) to multi-partite epiphytic or endophytic. There are some exudates produced by soil microorganisms which help in recycling of essential nutrients like phosphorus and nitrogen. A fundamental understanding of evolution, molecular biology, genetics, and ecology are required to promote sustainable agricultural practices based on microbes. The recruitment of such fields of compatible researches may help to have a remarkable productive capacity and versatile function as well. Crop production based upon the microbes has the potential to replace the existing harmful chemicals, with biofertilizers, and therefore aid in economic benefit and enrich the quality of agricultural goods obtained.
... Endophytes can colonize healthy plant tissues without causing host symptoms or losses, and have minimal environmental impacts. The mechanisms of action of Trichoderma spp. as BCAs include mycoparasitism (Harman et al., 2004), secretion of mycolytic enzymes (Reino et al., 2007), competition for limiting resources (Harman et al., 1993;Haran et al., 1995;Howell, 2006) and/or production of antibiotic metabolites (Harman and Kubicek, 2002;Vinale et al., 2006;Mutawila et al., 2016b). Trichoderma species also have positive effects on their hosts, including growth enhancement and resistance activation (Harman, 2006;Vinale et al., 2008a;Gallou et al., 2009;Parrilli et al., 2019). ...
Several Trichoderma species can act as biocontrol agents and hold the potential to control soilborne diseases through different modes of action. Little is known about the colonization pattern of Trichoderma atroviride in grapevine roots and activation of induced systemic resistance in planta. A laboratory model was developed to assess root colonization and its impact on grapevine defence activation. Rootstock cuttings from 1-year-old dormant canes were inoculated with conidium suspensions of T. atroviride T-77 or T. atroviride USPP T1, and host and inoculum colonization were assessed after 21 d. The two strains of T. atroviride were re-isolated from the treated plants (from 70% of the roots and 20% of crowns). Colonization rates did not depend on the Trichoderma strain or rootstock cultivar. However, up-regulation of targeted defence genes was dependent on the inoculated Trichoderma strain and rootstock cultivar. Furthermore, in leaves of rootstock cultivars 'US 8-7' and 'Paulsen 1103' , genes were up-regulated which encode for PR proteins involved in plant defence or production of stilbenic phytoalexins. Trichoderma atroviride T-77 was transformed with tdTo-mato fluorescent protein to allow visualization by confocal laser scanning microscopy. These results give new insights into the mechanisms of grapevine-Trichoderma interactions , and allow detection of establishment of potential biocontrol agents within host tissues.
... Our study showed that the tested bacilli produce different chitinolytic enzymes. Haran et al. [31] reported that endochitinases yield fluorescent products from 4MU-(GlcNAc)2. Lindahl and Finlay [18], found that both 4MU-GlcNAc and 4MU-(GlcNAc)2 were degraded by commercial endochitinase, but at lower rate than 4MU-(GlcNAc)3. ...
Plant fungal diseases generate serious losses in the agriculture. The bacteria producing biologically active substances that inhibit the growth of fungal pathogens can be an alternative to the chemicals. The chitinolytic bacteria were isolated from the rhizosphere of wheat (Triticum aestivum L.) and their physiological properties which may be useful in the promotion of plant growth have been investigated. Their chitinases and antifungal activity were studied. The isolates were also tested for indirect growth-promoting traits such as ammonia production, siderophore production, hydrogen cyanide production, and salicylic acid production. Two chitinolytic strains B3 and B5 were identified as Bacillus subtilis and Bacillus sp., respectively. They produced active chitinases on a medium containing shrimp shell powder. The purified chitinases having the molecular weight of 35–45 kDa inhibited the growth of important plant pathogens such as Alternaria alternata, and Fusarium oxysporum. Additionally, the isolates showed the ability to produce a broad range of biological substances promoting the growth of plants.
... Microtitrate assay is also helpful for measurement of endochitinase activity as defined for exochitinase but using p-nitrophenyl-β-D-N, N''acetylchitotriose as substrate. In some studies, 4-methylumbelliferyl-β-D-N', N"-diacetylchitotrioside or 4-methylumbelliferyl-Nacetyl-β-D-glucosamide (4-MU-GlcNAc) a fluorogenic analogue of chitin was used as substrate for hydrolysis [168].when we can get enough information about domains of our gene of interest, by using PCR based techniques to find the sequence from DNA. ...
Chitin stands at second, after cellulose, as the most abundant polysaccharide in the world. Chitin is found naturally in marine environments as it is a crucial structural component of various marine organisms. Vast amounts of waste chitin and chitosan can be discovered in the biosphere. Chitinase producing microbes help to hydrolyze chitin waste to play an essential function for the removal of chitin pollution in the marine atmosphere. Chitin can be converted by using chemical and biological methods into prominent derivate chitosan. Numerous bacteria naturally have chitin degrading ability. Chitin shows promise in terms of biocompatibility, low toxicity, complete biodegradability, nontoxicity, and film-forming capability. The application of these polymers in the different sectors of biomedical, food, agriculture, cosmetics, pharmaceuticals could be lucrative. Also, the most recent achievement in nanotechnology is based on chitin and chitosan-based materials. In this review, we examine chitin in terms of its natural sources and different extraction methods, chitinase producing microbes and chitin, chitosan together with its derivatives for use in biomedical and agricultural applications. Myxobacteria are recognized because of its large size genome beyond 9 Mb and its social behaviors. Myxococcus Fulvus belong from genus Myxococcus in Myxococcaceae family. M.Fulvus UM01 strain was isolated from a soil sample collected in Jiangsu China in the present study, 10 soil samples were collected from different locations and all 10 samples were screened for chitinase production based on hydrolysis, 2 samples were used for further screening. Most potent isolate due to its potency identified as Myxococcus Fulvus was selected for further study. The effects of several media and fermentation conditions for optimization of chitinase enzyme production were studied. The best production rate for chitinase was reported at 7 pH, 35°C temperature and using Colloidal chitin as substrate. Three chitinases related genes were identified by using a degenerative primer. Out of the three genes, one gene named UMCda was cloned in E. coli DH5a by using the pET-28a vector. The molecular mass of the recombinant enzyme was estimated to be 20.2 kDa. Recombinant UMCda was further studied for its antifungal activity against T. reesei and it shows great antagonistic activity.
... Inbar and Chet [8] showed the induction of glucosaminidase in T. harzianum after the initial recognition of S. rolfsii. Simultaneously, Haran et al. [36] reported the expression of β-1, 4 N-acetyl-glucosaminidase (chit102) by T. harzianum strain TM when grown on chitin as the sole carbon source. β-1, 4 N-acetyl-glucosaminidase is one of the predominant enzymes in parasitization and lysis of phytopathogenic fungi. ...
Sclerotium rolfsii, a soil-borne fungal pathogen, infects more than 500 crop species and causes stem rot/collar rot/seed rot/southern blight/wilt in a wide variety of crops which results in significant yield loses. Presently, antagonistic microbes are gaining more importance in managing plant pathogens because they control the pathogen in an environment-friendly manner. Trichoderma is an antagonistic fungi and most popularly used biocontrol agent against phytopathogenic fungi. It is predominantly used to treat soil and seed for the control of Sclerotium rolfsii infestation. In this study, the Trichoderma koningii IABT1252 that performed better in controlling groundnut seed/ seedling rot caused by S. rolfsii in pot experiments were selected to know the molecular basis for the control. Differentially expressed genes in Trichoderma at two different stages of interaction (prior to contact and after contact with S. rolfsii) were identified. In both the stages, some of the differentially expressed genes included ones coding for hydrolytic enzymes, secondary metabolite biosynthesis, transcription factors, signaling proteins, transporter proteins, and proteins involved in mycoparasitic process of Trichoderma.
... Size Determination Interestingly, expression of chitinase in 95 percent of mutants was higher than wild type of T. harzianum. Haran et al. (1995) reported that, depending on the strain, the chitinolytic system of T. harzianum may contain five to seven individual enzymes. In the well-characterized strain T. harzianum TM, this system comprises two β-(1, 4) Nacetyl-glucosaminidases (102 and 73 kDa), four endochitinases (52, 42, 33, and 31 kDa), and one exochitinase (40 kDa) (Lorito et al., 1993). ...
Trichoderma species are known as effective agents used for biological control of plant pathogenic fungi. The Trichoderma harzianum and its mutant isolates were cultured and their traits including, mycelial growth, antagonistic activity and extracellular proteins and enzymes production (Chitinase and Cellulase) were investigated to select the most effective mutant isolates against plant pathogenic fungus Rhizoctonia solani. Also, the purity and composition of enzyme-rich protein samples were evaluated under denaturing gel electrophoresis. This study clearly showed the possibility of improving mycelia growth rate (from 1.18 to 1.33 cm d-1), the antagonistic capability of Trichoderma (from 54.9% growth inhibition of R. solani to 66%), extracellular proteins and enzymes production for biological control of plant diseases through mutation with γ-radiation. Also, compared to wild type strain, protein production in the mutant isolates increased. Moreover, the highest specific chitinase enzyme activities were observed in mutant isolates T. h M8 (42.48 U mg-1) and T. h M15 (38.25 U mg-1). Trichoderma mutant of T. h M8 maintained higher mycelia growth rate and higher ability to inhibit growth of R. solani. The SDS-PAGE profiles had several enzyme protein bands such as CelloBioHydrolases (CBHs), EndoGlucanases (EGs), β-Glucosidases (BGLs), endochitinases, and β-(1, 4)-N-acetyl glucoaminidases. SDS-PAGE analysis indicated the presence of different protein bands in the range of 10.5 to 245 KDa. Interestingly, expression of chitinase in 95 percent of mutants was higher than wild type of T. harzianum. The results showed that gamma mutation could increase the efficiency and amount of enzymes in T. harzianum, while these enzymes are involved in antagonistic properties of T. harzianum.
... Trichoderma harzianum is well-known as mycoparasite and effective biocontrol agent of fruit and vegetable pathogens (fungi and nematode) by its complex system of more than six chitinolytic enzymes [11]. In this study, a GH18 chitinase gene, Chit46 from T. harzianum sensu stricto was cloned and expressed in Pichia pastoris with a high enzyme activity of 31.4 ...
In this study, a chitinase gene, Chit46 from a mycoparasitic fungus Trichoderma harzianum was successfully expressed in Pichia pastoris with a high heterologous chitinase production of 31.4 U/mL, much higher than the previous reports. The active center and substrate binding pocket of the recombinant Chit46 (rChit46)were analyzed and the effects of pH, temperature, metal ions and glycosylation on its activity were tested. rChit46 effectively hydrolyzed colloidal chitin with a high conversion rate of 80.5% in 3 h and the chitin hydrolysates were mainly composed of (GlcNAc) 2 (94.8%), which make it a good candidate for the green recycling of chitinous waste. rChit46 could also significantly inhibit growth of the phytopathogenic fungus Botrytis cinerea, which endowed it with the potential as a biocontrol agent.
... The previous studies indicated a vital role for Trichoderma species in biocontrol of M. phaseolina, Aspergillus species and M. incognita (Al-Hazmi and Javeed, 2016;Khaledi and Taheri, 2016;Mendoza et al., 2015;Shoaib et al., 2018;Gajera et al., 2011;Sharon et al., 2011). As the cell wall of nematodes and pathogenic fungi is mainly composed of chitin, Trichoderma have chitinase enzymes able to degrade the cell wall of these pathogens (Loc et al., 2011;Haran et al., 1995;Ike et al., 2006). Identification of Trichoderma spp. to be applied in the field of biological control is an important issue. ...
Ten Trichoderma isolates were isolated from different locations in Egypt. Amplification and sequencing of internal transcribed spacers (ITS) was employed to identify Trichoderma isolates that exhibited from 99 to 100% identity with three species of Trichoderma: Trichoderma harzianum, Trichoderma asperellum and Trichoderma longibrachiatum. The biocontrol activity of Trichoderma isolates against Macrophomina phaseolina, Aspergillus niger and Meloidogyne incognita was tested in vitro and under greenhouse conditions. The results show that the isolate Th2 (T. harzianum) gave the best antagonism against M. phaseolina and A. niger with inhibition rates of 72.85 and 64.28%, respectively. Moreover, the isolate Ta1 (T. asperellum) was the best efficient isolate in reduction of each second stage juveniles (J2), number of galls, egg masses and females per root system with 90.33, 90.59, 90.06 and 85.50%, respectively. Treatment with Trichoderma isolates improved tomato growth parameters (root length, plant height, roots and shoots fresh weight and shoots dry weight).
... The protein size revealed by SDS-PAGE for T. harzianum BIO10671 was 42 kDA which was larger than the purified chitinase collected by Deane et al. [6] from T. harzianum T198, but similar to the range of endochitinases reported previously by Ulhoa and Peberdy [21], Haran et al. [8] and Matsumiya et al. [14]. The molecular weight of purified chitinase may differ between species and also within species [19] and it is not known whether they are differently processed gene products from the same gene or from separate genes. ...
Chitinase 42 kDa produced by Trichoderma harzianum has been proven as a prime compound to be excreted onto the hyphae of the pathogen causing localised cell wall lysis at the point of interaction. This finally initiate the process of the host cell becomes empty of cytoplasm, disintegrates and shows a rapid collapse. This study investigates the existence of N-glycan linked mannose in chitinase 42 kDa produced by the Malaysian T. harzianum strain BIO10671. The chitinase 42 kDa from T. harzianum BIO10671 was initially purified using anion exchange chromatography prior to a series of experiments such as immunoblotting against the chitinase 42 kDa antibody, lectin staining for detecting any terminal linked mannose, and galactofuranose detection to determine the presence of galatofuranose components in glycoproteins. The enzyme purification harvested about 12-fold of chitinase 42 kDa from T. harzianum BIO10671 with strong indication of the presence chitinase 42 kDa presence on SDS-Page. This was confirmed by immunoblotting with a strong response around 42 kDa after overnight incubation in chitinase 42 kDa antibody suggesting that the gene for chitinase 42 kDa was greatly expressed in this strain. There are no intervation of galatofuranose on any of the terminal mannose in chitinase 42 kDa as shown by negative results on samples treated with or without endoglycosidase-H and lectin staining. Therefore, it can be concludeed that glycosylation occurred in the chitinase 42 kDa from T. harzianum 42 kDa was not in the form of N-glycan linked mannose as expected.
... ChiA activity was also tested with recombinant purified ChiA, as well as purified ChiA mixed with whole-protein extracts of the chiA mutant for 10 min at 60 r.p.m. using a vertical orbital shaker, before being subjected to native electrophoresis. For each condition, chitinolytic activity was visualized under UV light [26]. Each strainmedium-time point combination was replicated three times. ...
Xylella fastidiosa colonizes the xylem network of host plant species as well as the foregut of required insect vectors to ensure its efficient propagation. Disease management strategies remain inefficient due to a limited comprehension of mechanisms governing both insect and plant colonization. It was previously shown that X. fastidiosa has a functional chitinase (ChiA), and that chitin likely serves as a carbon source for this bacterium. We expand on that research showing that a chiA mutant strain is unable to grow on chitin as the sole carbon source. qPCR assays allowed us to detect bacterial cells on the foregut of vectors after pathogen acquisition; populations of the wild type and complemented mutant strain were both significantly larger than the chiA mutant strain 10 days but not 3 days post acquisition. These results indicate that adhesion of the chiA mutant strain to vectors may not be impaired, but cell multiplication is limited. The mutant was also affected in its transmission by vectors to plants. In addition, the chiA mutant strain was unable to colonize host plants, suggesting that the enzyme has other substrates associated with plant colonization. Lastly, ChiA requires other X. fastidiosa protein(s) for its in vitro chitinolytic activity. The observation that the chiA mutant strain is not able to colonize plants warrants future attention to the substrates for this enzyme.
... are active mycoparasites against a range of economically Fig. 1 General procedure of seed biopriming important soilborne plant pathogens and are successfully used as a biocide in greenhouse and field applications (Chet 1987;Papavizas 1985;Nayak et al. 2009). This is well complimented by the secretion of extracellular hydrolytic enzymes such as chitinases (Carsolio et al. 1994;de la Cruz et al. 1992;Harman et al. 1993), β-glucanases (Haran et al. 1995;Lora et al. 1995;Lorito et al. 1994), and proteases (Geremia et al. 1993). The effect of these compounds in phytopathogenic fungi includes degradation of the cell wall (Harman et al. 2004). ...
Seeds are the crucial input in agriculture as most of the world food crops are grown from seeds and they are circulated at large scale in international trade. However, many plant pathogens can be seed transmitted, and seed distribution is an extremely capable way of introducing plant pathogens into fresh areas as well as a means of endurance of the pathogen between growing seasons. In past decades, chemicals are widely used for seed treatment as a potent approach towards disease control; however, rising concern about their negative impact on the environment and human health minimizes their use and promotes biological control for plant pathogens. Biopriming is a currently popular approach of seed treatment which includes inoculation of seed with beneficial microorganisms (biological aspect) and seed hydration (physiological aspect) to protect the seed from various seed- and soilborne diseases. Biopriming treatment is able to incite changes in plant characteristics and facilitate uniform seed germination and growth associated with microorganism inoculation. Seed priming and osmo-priming are commonly being used in many horticultural crops to amplify the growth and uniformity of germination. However, it may be used alone or in combination with biocontrol agents to advance the rate of seed emergence and minimize soilborne diseases. On the other hand, some biocontrol agents are used as seed dressers and are able to colonize the rhizosphere, helping seeds to resist various abiotic stresses such as salinity, drought, low fertility and heavy metal stress, etc. Therefore, biopriming is becoming a viable alternative for inorganic chemicals.
... In the past few years, Trichoderma spp. the most common saprophytic fungi in the rhizosphere, have received considerable attention as potential biocontrol agents (Samuels, 1996;Zhang and Wang 2012). The mechanisms by which Trichoderma isolates control pathogenic populations in the rhizosphere have been extensively studied, the antagonistic process relies on the production of antibiotics ( Ghisalberti and Sivasithamparam, 1991) and/or hydrolytic enzymes (Haran et al., 1995 and) associated with possible competition for nutrients in the rhizosphere (Sivan and Chet, 1993). ...
Abstract
Trichoderma spp. are well-known biological control agents because of their antimicrobial activity against bacterial, fungal and viral phytopathogens. In this study, we found that the filtrate of Trichoderma sp. Shmosa Tri (FJ937359) could induce systemic resistance in squash (Cucurbita pepo L.) against zucchini yellow mosaic potyvirus (ZYMV) infection. The mechanically infected squash plants with ZYMV showed severe symptoms such as mosaic, thread like leaves, green blisters, chlorosis, Leaf malformation and leaf roll. In vitro treatment (fungal filtrate mixed with sap containing virus) led to 90% ZYMV inhibition compared to viral control. The treatment of squash leaves with filtrate of Trichoderma sp. Shmosa Tri (FJ937359) before ZYMV inoculation( Pre-inoculation) greatly reduced the number of infected plants and gave 75% inhibition. The treatment of squash leaves with fungal filtrate after ZYMV inoculation (post-inoculation) led to 55% inhibition in comparison with viral control. However, the soaking of squash seeds in the fungal filtrate led to mild inhibition. This means that the foliar treatments are better than the seed treatment. Moreover shoot length, fresh weight and number of leaves were significantly (p< 0.05) increased compared to viral control in all treatments. Additionally, application of fungal filtrate ( in vitro treatment) increased the production of phenolic compounds, total protein, peroxidase and polyphenoloxidase enzymes in squash compared to viral control.
... are active mycoparasites against a range of economically Fig. 1 General procedure of seed biopriming important soilborne plant pathogens and are successfully used as a biocide in greenhouse and field applications (Chet 1987;Papavizas 1985;Nayak et al. 2009). This is well complimented by the secretion of extracellular hydrolytic enzymes such as chitinases (Carsolio et al. 1994;de la Cruz et al. 1992;Harman et al. 1993), β-glucanases (Haran et al. 1995;Lora et al. 1995;Lorito et al. 1994), and proteases (Geremia et al. 1993). The effect of these compounds in phytopathogenic fungi includes degradation of the cell wall (Harman et al. 2004). ...
... (Sivan and Chet, 1989). N-acetyl-β-glucosaminidase forms part of the chitinase complex: two isoenzymes were described in the most isolated species, T. harzianum (Haran et al. 1995). Chitinase encoding genes are also the most used to improve plant defence against fungal pathogens (Sharma et al., 2012). ...
... Cultivos sumergidos: En cultivos sumergidos se estudió la producción de azúcares reductores (AR) al evaluar dos variables: efecto de sales minerales y disgregación de las partículas. Los lodos papeleros a una concentración final de 2% p/v se adicionaron individualmente a un medio salino propuesto por Okon et al [9] y constituido por: Mg 2 SO 4 (7H 2 O) 0,2 g/L; K 2 HPO 4 0,6 g/L; KCl 0,15 g/L; NH 4 NO 3 1 g/L; FeSO 4 (7H 2 O) 0,005 g/L; MnSO 4 (7H 2 O) 0,006 g/L; ZnSO 4 (7H 2 O) 0,004 g/L; CoCl 2 , 0,002 g/L. Se realizó un control utilizando agua destilada y lodo papelero. ...
Resumen: La producción del papel en Venezuela genera una gran cantidad de desechos de fibras de celulosa no utilizables (lodo papelero), produciéndose problemas ambientales y de almacenamiento. La búsqueda de usos para este lodo papelero disminuiría los problemas generados. En este trabajo, se utilizó al hongo Trichoderma reesei para la realización de fermentaciones líquidas y el estudio del efecto de sales minerales, tamaño de partícula del lodo y pretratamientos ácidos y básicos, sobre la producción de azúcares reductores y la actividad celulasa. También se realizaron fermentaciones sólidas para obtener biomasa (esporas) del hongo. Los resultados indicaron que el uso de lodo higiénico, sales minerales y la reducción del tamaño de partícula, mejoran el rendimiento en azúcares reductores (AR). La máxima actividad celulasa obtenida, medida con carboximetilcelulosa, fue de 2,6 mg AR/L/h. Se determinó que con el pretratamiento ácido se obtienen mayores cantidades de AR (17 mg/L) que con el básico (7,4 mg/L). Se demostró que T. reesei puede crecer en fermentaciones sólidas a expensas del lodo papelero generando hasta 1,24x10 8 esporas por gramo de lodo seco. Se requiere estandarizar y escalar estas técnicas para usar al lodo papelero como materia prima en este tipo de procesos industriales. Palabras clave: fermentación líquida, fermentación sólida, lodo papelero, Trichoderma reesei. Production of cellulase enzymes and biomass of the fungus Trichoderma reesei using paper sludge as carbon source Abstract: Paper production in Venezuela generates large amounts of cellulose fibers (paper sludge), producing environmental and storage problems. Finding uses for this papermaking sludge would decrease the problems caused. In this work the fungus Trichoderma reesei was used on submerged fermentation to study the effect of mineral salts, particle size and acid-alkali pretreatment of sludge on production of reducing sugars (RS) and cellulase activity. Solid fermentations were also conducted to obtain biomass (spores) of the fungus. The results indicated that the use of sanitized sludge, mineral salts and smaller particles, increased the production of reducing sugars (RS). The maximum cellulase activity, measured with carboxymethylcellulose, was RS 2.6 mg/L/h. Acid pretreatment was the best in rendering RS (17 mg/L) in comparison with alkali treatment (7.4 mg/L). It was also demonstrated that T. reesei can grow on paper sludge producing biomass (spores) with a yield of 1,24x10 8 spores/g of dry sludge. It is necessary to standardize and scale range these procedures in order to be able to use paper sludge to industrialize these processes.
... The antagonistic mechanism of Trichoderma is a complex process involving chemotropism (Chet et al. 1981), lectin-mediated recognition (Inbar & Chet 1992, and formation of trapping and penetration structures (Elad et al. 1983a,b). This process is further supported by the secretion of extracellular enzymes such as chitinases (Carsolio et al. 1994;de la Cruz et al. 1992;Harman et al. 1993), β-glucanases (Haran et al. 1995;Lora et al 1995;Lorito et al 1994), and proteinases (Geremia et al. 1993). The effects of these compounds in phytopathogenic fungi include degradation of the cell walls, forming holes (Harman et al. 2004). ...
Stem rot of canola (Brassica napus ) caused by Sclerotinia sclerotiorum is one of the most serious of plant diseases. From 30 Trichoderma isolates, three different species T. harzianum-8, T. atroviride PTCC5220 and T. longibrachiatum PTCC5140, were selected on the basis of their high level of chitinase and/or glucanase activity, along with their rapid growth rate in vitro. These showed high growth inhibition of two phytopathogenic isolates of Sclerotinia sclerotiorum (S1and S2), with T. atroviride the greatest effect, reducing growth by 85-93%. They showed coil formation and penetration structures against the hyphae of the pathogenic isolates. T. atroviride PTCC5220 can be used for assessment of field biocontrol against S. sclerotiorum.
... These could be anticipated from the suggestion that β-HexNAc'ases, as a component of the extracellular binary chitinolytic system effecting the complete degradation of chitin to GlcNAc, is a part of the enzyme inventory necessary for the heterolytic digestion of chitinous materials including chitin and fungal cell wall (Haran et al., 1995;Horsch et al., 1997). Previously, several fungal β-HexNAc'ases have been considered to play an essential role in the lysis of phytopathogenic fungal cell walls during their parasitic entrance into the plants, thus serving as potentially useful biocontrol agents against economically important plant pathogens (Horsch et al., 1997;Brunner et al., 2003;Mamarabadi et al., 2009). ...
N-acetyl-β-D-hexosaminidase was purified from wheat bran and characterized. The purified enzyme showed two protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with molecular mass of 75 and 78 kDa. The enzyme exhibited optimum pH and temperature at 5.0 and 50 o C, respectively. The enzyme was active on the substrates of p-nitrophenyl-N-acetyl-β-D-glucosaminide (pNP-GlcNAc) and p-nitrophenyl-N-acetyl-β-D-galactosaminide (pNP-GalNAc), whereas inactive on pNP-β-D-glucopyranoside, pNP-β-D-galactopyranoside, swollen chitin, and colloidal chitin, suggesting high substrate specificity. The enzyme activity for pNP-GlcNAc was stable at pH 3–6 and under 50 o C. The K m , V max and K cat for pNP-GlcNAc were 0.014 mM, 0.05 µmol/min, and 3.01×10 6 min −1 , respectively. The enzyme could be completely inhibited at 1–10 mM HgCl 2 and AgNO 3, suggesting that the intact thiol group is essential for activity. β-N-Acetylhexosaminidase from wheat bran could inhibit the conidial germination and digest the hyphae of Fusarium solani.
... system, especially T. harzianum and T. atroviride. These fungi contain a complex system of more than six chitinolytic enzymes, two N-acetylhexosaminidases and several endochitinases (Haran et al., 1995;Viterbo et al., 2001) (Table 4). Apart from Trichoderma spp., other fungi such as V. biguttatum, T. flavus, C. rosea, Chaetomium globosum and C. cupreum secrete chitin-degrading enzymes during mycoparasitism (McQuilken and Gemmell, 2004;Duo-Chuan et al., 2005;Liu et al., 2008;Mamarabadi et al., 2008aMamarabadi et al., , 2008bWang and Yang, 2009) (Table 4). ...
Soil suppressiveness to soil-borne diseases relies mainly on microbial interactions. Some of them, e.g. antibiosis and mycoparasitism, are directly deleterious to pathogenic fungi; others indirectly affect microbial populations, pathogens included, when quite active non pathogenic microorganisms intensively exploit trophic or spatial resources. The mechanisms that govern the suppressive nature of the various known suppressive soils are often hypothetical. The objective of this article is to review the fungal proteins and corresponding genes directly or indirectly involved in antagonistic relationships between pathogens and non-pathogens and associated with biocontrol of soil-borne pathogens. The current hypothesis is that they contribute to soil suppressiveness. We assigned the proteins encoded by these genes to five function-based groups. The first group contains the proteins involved in host recognition and signaling pathways and the transcription factors involved in biocontrol activities. Proteins that protect antagonistic fungi against their own toxins and against other microorganisms are also included in this first group. The second group lists enzymes and proteins involved in the biosynthesis pathway of secondary metabolites, such as peptaibols, terpenes, polyketides, and gliotoxins that have antifungal activity towards soil-borne plant pathogens. The third group deals with proteins and molecules involved in competition for nutrients and root colonization. The fourth one contains the fungal cell wall-degrading enzymes secreted by antagonistic fungi during mycoparasitism. They are mainly chitin-degrading enzymes, glucanases and proteases. Finally, the last group gathers fungal proteins and molecules that induce plant defense reactions and prevent infection by plant pathogens. We conclude that the proteins involved or simply associated with the specific suppression of pathogens are not all known yet, but genes encoding a number of them or facilitating their expression are identified. Selecting candidate genes among them may help to understand the underlying mechanisms of soil suppressiveness when using metatranscriptomic analyses to identify functional groups.
Endophytic bacteria is promised solution to suppress basal stem rot disease caused by fungus Ganoderma boninense . The antifungal activities of selected isolate endophytic bacteria from oil palm plantation in South Kalimantan was studied. Bacillus cereus was tested for its antifungal activities of crude chitinase and secondary metabolites against the growth of Ganoderma boninense mycelium with dual cultured method. The results were showed that chitinase had the largest inhibition zone (18.5%) against growth inhibition of fungi Ganoderma boninense’ s mycelium. A series of optimation assays of chitinase activity were conducted at pH 3 to 8 with 0.5 interval, and temperature at 30 to 70°C, with 5°C interval. Specific chitinase activities was measured using the colorimetric method. The highest specific chitinase activity significantly at 1.66393±0.04807 mU/μg (95% confidence level) at pH 5.5 and temperature 45°C.
Methods for developing mathematical models describing the process of growing organic cotton are
given. This takes into account vegetation features such as budding, fruiting, crop formation, as well as the possibility
of their use in the cultivation of cotton.
Changes in mass and viscoelasticity of chitin layers in fungal cell walls during chitinase attack are vital for understanding bacterial invasion of and human defense against fungi. In this work, regenerated chitin (RChitin) thin films mimicked the fungal chitin layers and facilitated studies of degradation by family 18 chitinases from Trichoderma viride (T. viride) and family 19 chitinases from Streptomyces griseus (S. griseus) that possessed chitin-binding domains (CBDs) that were absent in the family 18 chitinases. Degradation was monitored via a quartz crystal microbalance with dissipation monitoring (QCM-D) in real time at various pH and temperatures. Compared to substrates of colloidal chitin or dissolved chitin derivatives and analogues, the degradation of RChitin films was deeply affected by chitinase adsorption. While the family 18 chitinases had greater solution activity on chitin oligosaccharides, the family 19 chitinases exhibited greater surface activity on RChitin films, illustrating the importance of CBDs for insoluble substrates.
Stem canker and black scurf disease is one of the most important
disease of potato and widespread in all potato production areas in Egypt and
the world. This disease is one of the limiting factors to potato production in
the world, which causes yield reduction and reduces the tubers quality. This
disease caused by the fungus Rhizoctonia solani Kuhn. So the present study
aimed to (I) isolate the causal pathogen from different varieties and locations
in Egypt (II) identify pathogen isolates by using morphological
characteristics, anastomosis group and electrophoresis (III) control the
disease by untraditional methods such as biocontrol agents, application of
certain animal manures to the soil and tuber seeds treatment before sowing
with some salts. The results of this study could be summarized as follows:
1- Thirty five isolates of R. solani were isolated from diseased potato plants
from stem and root cankers, tubers and plant rhizosphere from different
localities and cultivars in Egypt.
2- These isolates differed in the colour of culture and number and the size of
sclerotia. The isolates were classified into seven groups according to their
cultural colour, number and the size of sclerotia.
3- Pathogenicity test of thirty five isolates of R. solani on potato Nicola
cultivar proved that all tested isolates were able to infect potato plants
and caused typical symptoms of the disease such as eyes germination
inhibition, dead sprouts, stem canker and black scurf. The isolates varied
in their pathogenic potentialities, as isolate No. 35 caused the highest
percentage of stem canker (95.10%) and black scurf (92.35%). Isolates
No. 15 caused the lowest percentage of infection, while other isolates
gave moderate percent.
Summary 139
4- Fifteen isolates of R. solani which varied in their pathogenic capability
were tested for their ability to anastomose with the tester isolate (AG-3)
by using the clean slide technique. Microscopic examination showed that
all the tested isolates belong to R. solani AG-3 although they came from
different varieties and locations. AG-3 is common on potato in all
production areas in Egypt.
5- Cluster analysis was used to compare between protein banding patterns
obtained by sodium sulfate-polyacrylamide gel electrophoresis (SDSPAGE)
from nine R. solani isolates. A very high level of similarity
among the tested isolates in protein banding patterns (98.32%) indicates
that a low level of genetic variation occurs in the populations of R. solani
AG-3. The tested isolates were almost identical in protein profiles
although came from different governorates and were isolated from
different cultivars. These results suggest that AG-3 of R. solani is a very
homogeneous group in protein profiles.
6- Study has been done to investigate the disease transmission potentiality
by tuber seeds, which were taken from diseased fields. This has been
achieved by infested soil with isolates of pathogen. Then, the diseased
tubers showing symptoms have been left to be sprouted and isolation
from sprouts. Results of this test that the three isolates which were
inoculated in the soil are the same isolates isolated from the sprouts.
Inoculated and reisolated isolates were similar in colour culture and
sclerotia formation. Results show that significant similarity in protein
profiles between inoculated and reisolated isolates (79.17%), which
classified the isolates for two categories. Isolates taken from Upper Egypt
Summary 140
showed highly percent of similarity as the same was shown in case of
isolates taken from Nile Delta.
7- Studying the effect of some Trichoderma isolates (T. harzianum and T.
longibrachiatum) which isolated from the rhizosphere and roots of potato
plants against the pathogen, in vitro and greenhouse conditions showed
that:
a- Six isolates of Trichoderma sp. were isolated from rhizosphere and
roots of potato plants. Three isolates were identified as T. harzianum and
three isolates as T. longibrachiatum.
b- All tested isolates of Trichoderma were able to inhibit the mycelial
growth of the pathogen isolates in vitro (dual culture), but these isolates
varied in their ability according to antagonistic isolates. In general, T.
harzianum isolates were the most effect on the pathogen growth, while T.
longibrachiatum isolates were the least in their antagonistic effect.
c- Culture filtrates of both T. harzianum and T. longibrachiatum which
were sterilized by Seitz filter significantly inhibited the mycelial growth
of all the tested pathogen isolates and completely inhibited the mycelial
growth of the pathogen at concentration 30 and 40% (vol/vol) from both
of them. Generally, culture filtrate of T. longibrachiatum was more
effective on pathogen growth than the culture filtrate of T. harzianum.
d- Under greenhouse conditions, addition of T. harzianum, T.
longibrachiatum and mycorrhiza (G. mosseae) separately to infested soil
with pathogen isolates exhibited high ability for controlling the disease
and reduced the disease severity, such as eyes germination inhibition,
dead sprouts, stem canker and black scurf. In general, treatment with T.
Summary 141
longibrachiatum was the more effective in controlling the disease than
other treatments. At the same time, all treatments improved agronomic
characters of potato plants and increased tuber yield.
8- Studying the effect of certain animal manures (chicken, pigeons and cow)
against the pathogen in vitro and greenhouse conditions showed:
a- In vitro, the effect of manures extract (chicken, pigeons and cow) on
the mycelial growth of R. solani isolates was tested on PDA media at
different concentrations (0, 5, 10, 20, 30, 40 and 50 % vol/vol). Results
showed that all tested extracts significantly inhibited the growth of R.
solani isolates. Generally, pigeons manure extract gave the highest effect
on mycelial growth and inhibition percent of the pathogen (54.44%) at
concentration 50%, followed by chicken manure extract. Cow manure
extract caused the lowest reduction of mycelial growth of the pathogen at
concentration 5 %.
b- Under greenhouse conditions, soil amended with these manures at
concentrations 0.5 and 1% weight of the soil before sowing reduced the
disease severity of either in the form of eyes germination inhibition, dead
sprouts, stem canker or in black scurf symptoms. Generally, cow manure
treatment was the highest effect on controlling the disease followed by
pigeons and chicken manure. At the same time, addition of manures to
the soil was the more effective in increasing eyes germination and
decreased dead sprouts, stem canker and black scurf. Treatment with all
tested manures increased tuber yield, improved agronomic characters and
influenced characters of tuber quality. Application of manures reduced
sclerotia formation on the surface of tubers and hence disease incidence.
Summary 142
9- The effect of certain inorganic salts and boric acid to control the disease
and inhibit the growth of R. solani was studied under both laboratory and
greenhouse conditions:
a- In vitro, evaluated the effect of sodium carbonate, potassium chloride
and boric acid on mycelial growth of R. solani isolates at different
concentrations (0.01, 0.02, 0.05, 0.1 and 0.2 M) on PDA medium. All
tested chemicals inhibited significantly the mycelial growth of the
pathogen isolates with different degrees according to substance and
concentration. The reduction of mycelial growth increased by increasing
chemicals concentration in the media and reached its maximum at
concentration of 2 M. Sodium carbonate and boric acid had the similar
effect on the pathogen isolates at the tested concentrations and
completely inhibited the mycelial growth at concentrations 0.1 and 0.2
M. While, potassium chloride caused the lowest inhibition % of the
pathogen growth.
b- Under greenhouse conditions, tuber seeds treatment with solution of
sodium carbonate, potassium chloride and boric acid at concentrations
0.1 and 0.2 M for 30 minutes before sowing was effective in decreasing
Rhizoctonia disease with all parameters. Generally, all tested chemicals
exhibited high control for stem canker and black scurf disease, also
increased eyes germination and decreased dead sprouts. Concentration
0.2 M of all chemicals was more effective on the disease reduction than
concentration 0.1 M. Data also indicated that sodium carbonate was the
best in controlling the disease, followed by boric acid. Potassium chloride
showed the lowest effect on the disease with all parameters.
Globally, agricultural practices confront several problems such as diseases, drought, pests, declined soil fertility, pollution and health hazards due to the use of toxic chemical pesticides. Phytopathogens are a major constraint to crop production as they cause severe disease outbreaks and yield losses. Vascular wilt caused by soil borne Fusarium oxysporum species complex and Ralstonia solanacearum is a devastating disease threatening the production and yield of several major crops. The failure of traditional disease management practices and chemicals to control these destructive pathogens is the major concern. Therefore, it is imperative to find eco-friendly biopesticides that help combat the wilt pathogens. Biopesticides are disease control agents based on living microorganisms or natural products. Among the different biopesticides, Trichoderma is important and extensively used in the management of plant pathogens in agriculture. Trichoderma strains deploy a complex network of mechanisms such as colonizing the soil and root of the host, inhabiting a physical space and evading the multiplication of the phytopathogens, producing cell wall-degrading enzymes, producing antimicrobial metabolites that kill the pathogens, induction of plant defense mechanisms, promoting plant development and improve plant tolerance to biotic and abiotic stresses. This chapter presents the potential fungal biopesticide Trichoderma spp. for control of wilt diseases of various crops, the mechanism of Trichoderma spp. and their impacts on agriculture.
Chitinases enzymatically hydrolyze chitin, a highly abundant and utilized polymer of N-acetyl-glucosamine. Fungi are a rich source of chitinases; however, the phylogenetic and functional diversity of fungal chitinases are not well understood. We surveyed fungal chitinases from 373 publicly available genomes, characterized domain architecture, and conducted phylogenetic analyses of the glycoside hydrolase (GH18) domain. This large-scale analysis does not support the previous division of fungal chitinases into three major clades (A, B, C) as chitinases previously assigned to the “C” clade are not resolved as distinct from the “A” clade. Fungal chitinase diversity was partly shaped by horizontal gene transfer, and at least one clade of bacterial origin occurs among chitinases previously assigned to the “B” clade. Furthermore, chitin-binding domains (including the LysM domain) do not define specific clades, but instead are found more broadly across clades of chitinases. To gain insight into biological function diversity, we characterized all eight chitinases (Cts) from the thermally dimorphic fungus, Histoplasma capsulatum: six A clade, one B clade, and one formerly classified C clade chitinases. Expression analyses showed variable induction of chitinase genes in the presence of chitin but preferential expression of CTS3 in the mycelial stage. Activity assays demonstrated that Cts1 (B-I), Cts2 (A-V), Cts3 (A-V), Cts4 (A-V) have endochitinase activities with varying degrees of chitobiosidase function. Cts6 (C-I) has activity consistent with N-acetyl-glucosaminidase exochitinase function and Cts8 (A-II) has chitobiase activity. These results suggest chitinase activity is variable even within subclades and that predictions of functionality require more sophisticated models.
Chitinases enzymatically hydrolyze chitin, a highly abundant biomolecule with many potential industrial and medical uses in addition to their natural biological roles. Fungi are a rich source of chitinases, however the phylogenetic and functional diversity of fungal chitinases are not well understood. We surveyed fungal chitinases from 373 publicly available genomes, characterized domain architecture, and conducted phylogenetic analyses of the glycoside hydrolase family 18 (GH18) domain. This large-scale analysis does not support the previous division of fungal chitinases into three major clades (A, B, C). The chitinases previously assigned to the "C" clade are not resolved as distinct from the "A" clade in this larger phylogenetic analysis. Fungal chitinase diversity was partly shaped by horizontal gene transfer, and at least one clade of bacterial origin occurs among chitinases previously assigned to the "B" clade. Furthermore, chitin binding domains (CBD) including the LysM domain do not define specific clades but instead are found more broadly across clades of chitinase enzymes. To gain insight into biological function diversity, we characterized all eight chitinases (Cts) from the thermally dimorphic fungus, Histoplasma capsulatum: six A clade (3 A-V, 1 A-IV, and two A-II), one B clade (B-I), and one formerly classified C clade (C-I) chitinases. Expression analyses showed variable induction of chitinase genes in the presence of chitin but preferential expression of CTS3 in the mycelial stage. Activity assays demonstrated that Cts1 (B-I), Cts2 (A-V), Cts3 (A-V), Cts4 (A-V) have endochitinase activities with varying degrees of chitobiosidase function. Cts6 (C-I) has activity consistent with N-acetyl-glucosaminidase exochitinase function and Cts8 (A-II) has chitobiase activity. This suggests chitinase activity is variable even within sub-clades and that predictions of functionality require more sophisticated models.
Filamentous fungi from the genus Trichoderma are well known for their biocontrol potential and have been used as antagonistic agents as well as plant growth promoters. Chitinases released by Trichoderma spp. have been capable of hydrolyzing chitin by splitting their β-1, 4-glucosidic bonds. The aim of the present study was to isolate and characterize an endochitinase gene from native Trichoderma harzianum isolate which is involved in mycoparasitism. In total, twelve Trichoderma isolates were screened for chitinolytic activity via dual plate method and greenhouse studies. T. harzianum isolate (SVPRT-THLi03) was selected as a target for isolation, cloning, characterization and expression profiling of an endochitinase gene due to its high chitinolytic activity recorded by the degradation of chitin substrates. The genomic DNA of Trichoderma isolates was amplified and cloned in pGEMT cloning vector. The recombinant clones were confirmed through colony PCR and restriction analysis. The sequenced 1223 bp clone nucleotide sequence of putative endochitinase gene, ChitTh showed 99% homology to T. harzianum chit-HAR2 endochitinase (AB041752.1) with 0.0 E-value. The complete nucleotide sequence of ChitTh contained a single ORF of 379 amino acids with 40.7 kDa molecular weight and theoretical pI 8.3. The precursor protein contained 22 amino acids long signal peptide at N terminus. Phylogenetic analysis showed that ChitTh protein was clustered into group V with other Trichoderma spp. Semi-quantitative endochitinase gene expression was analysed for different isolates viz. T. harzianum (SVPRT-THLi03 and SVPRT-47) and T. nigricans (SVPPP-7). Among the three isolates, higher expression was observed in SVPRT-THLi03 and SVPRT-47 whereas SVPPP-7 showed lesser gene expression with respect to the other isolates.
Cell-wall-degrading enzymes are produced by fungal parasites. Parasitism of Rhizoctonia solani was first reported by Weindling (1932), who observed the parasitic nature of Trichoderma lignorum on several plant pathogens. Weindling’s (1932) report on the possible use of mycoparasites for the control of plant pathogens raised a great deal of interest in this area of research, including the role of hydrolytic enzymes in microbial interactions. It prompted reviews and discussions of the subject in numerous articles (Barnett, 1963; Boosalis, 1964; Barnett and Binder, 1973; Ayers and Adams, 1981; Wells, 1981; Baker, 1988; Ghaffar, 1988; Elad & Chet, 1995; see also chapter VI.B5). One of the features required for successful degradation of host fungi is the ability to produce the enzymes which degrade the polymers underlying the cell wall and membranes of the host. Boosalis (1956) estimated the frequency of R. solani parasitism by Penicillium vermiculatum in soil by observing the parasitism of R. solani hyphae in situ. Indeed, living fungal structures are often parasitized in natural soils (Boosalis, 1956; Hunter et al., 1977; Sneh et al., 1977). For this purpose, it may be assumed that the parasites produce the hydrolytic enzymes in natural niches where the pathogen is present. However, even with increasing evidence of natural mycoparasitism, the ecological significance of this phenomenon, at least in soil, is still ambiguous. Griffin (1972) maintained that mycoparasitism is of no ecological significance in soil, while others have suggested that mycoparasitism has considerable impact on soil fungal populations, by reducing the longevity of survival structures (Sneh et al., 1977). In any case, one of the features required for successful degradation of host fungi is the ability to produce enzymes which degrade the polymers underlying the cell wall and membranes of the host. The studies related to these enzymes, as related to degradation of R. solani, will be described in this chapter.
The fungi from genus Trichodemia are well known as biocontrol agents, used worldwide against different plant pathogenic fungi. Trichoderma spp. are free-living fungi that are common in soil and root ecosystems. Seven local isolates from Trichodemia spp. were investigated for their ability to suppress growth of various plant pathogenic fungi and the ability to produce substances with antifungal activity. Fungal hydrolytic enzyme activities, produced by Trichodemia isolates, such as proteolytic and cellulolytic activities, were compared as well. Among all isolates, Trichoderma spp. Tr5 and Tr6 showed a wide range of inhibitory effects against the plant pathogens, good antibiotic and enzyme activity and were selected for further investigation for large scale biocontrol application.
Assessment the efficiency of fungal antagonists and chitosan as foliar application against cucumber grey mold was carried out in greenhouse condition. The evaluated antagonistic fungi were Trichoderma harzianum, Trichoderma hamatum, Trichoderma viride, Gliocladium roseum and Gliocladium vireins. Chitosan was tested at three concentrations 0.1, 0.2 and 0.3%. It was noticed that all treatments significantly reduced grey mold incidence in cucumber plants comparing with untreated plants. Among all tested antagonists, T. harzianum and T. viride better than others were in inhibiting disease incidence and improving plant defense against the pathogen. Chitosan treatments did not show antimicrobial activity against Botrytis cinerea isolates in vitro however, when it used as a foliar spray on greenhouse it showed a highly significant control level of grey mold disease. According to obtained results from the current study, it could be suggested that the application of antagonistic fungi and chitosan might be an easily applied, safely and cost effective alternative control method to chemical control of grey mold of cucumber.
A glucan 1,3-β-glucosidase (EC 3.2.1.58) and an N-acetyl-β-glucosaminidase (EC 3.2.1.30) were purified to homogeneity from the culture filtrate of Tricloderma harzianum strain PI. The molecular masses and the pIs were 78 kDa and 6.2, respectively, for the glucan 1,3-β-glucosidase and 72 kDa and 4.6, respectively, for the N-acetyl-β-glucosaminidase. The glucan 1,3-β-glucosidase and the N-acetyl-β-glucosaminidase were tested against Botrytis cinerea, and their antifungal activity was compared with that obtained for an endochitinase and a chitin 1,4-β-chitobiosidase also purified from T. harzianun strain P1. The four cell wall-degrading enzymes were also tested as mixtures containing two, three, or all four proteins in all possible combinations [...]
Trichoderma harzianum strain P1 produces a variety of chitinolytic enzymes including N-acetyl-β-D-glucosaminidases, chitin 1,4-β-chitobiosidases, and an endochitinase. Chitabiosidases and an endochitinase were purified from dialyzed, concentrated culture filtrates using gel filtration, chromatofocusing, and isoelectric focusing. Three protein bands were evident in the purified chitobiosidase preparation, representing different levels of N-glycosylation of the same protein. The pI of all purified proteins was ∼3.9 []
Two chitinolytic enzymes from Trichoderma harzianum strain P1 were tested for their antifungal activity in bioassays against nine different fungal species. Spore germination (or cell replication) and germ tube elongation were inhibited for all chitin-containing fungi except T. harzianum strain P1. The degree of inhibition was proportional to the level of chitin in the cell wall of the target fungi. For most of the fungi tested, the ED 50 values for the endochitinase and the chitobiosidase were 35-135 μg ml -1 and 62-180 μg ml -1 , respectively. Complete inhibition occurred at 200-300 μg ml -1 []
The production of chitinolytic enzymes by Trichoderma is of interest in relation to their use in biocontrol and as a source of mycolytic enzymes. The chitinolytic system of T. harzianum resembles that of most chitinolytic organisms and consists of two types of hydrolases: chitinase and chitobiase. Both enzymes are produced when T. harzianum is grown on a chitin-containing medium and repressed when the mycelium is provided with an easily metabolized carbon source such as glucose or N-acetylglucosamine (GlcNAc). Chitobiase is also produced in GlcNAc-containing medium.
There has been a considerable amount of recent research aimed at elucidating the roles of chitinase in fungi and plants. In filamentous fungi and yeasts, chitinase is involved integrally in cell wall morphogenesis. Chitinase is also involved in the early events of host-parasite interactions of biotrophic and necrotrophic mycoparasites, entomopathogenic fungi and vesicular arbuscular mycorrhizal fungi. In plants, induction of chitinase and other hydrolytic enzymes is one of a coordinated, often complex and multifaceted defense mechanism triggered in response to phytopathogen attack. Chitinase induction in plants is not considered solely as an antifungal resistance mechanism. Plant chitinases can be induced by various abiotic factors as well and there is some circumstantial evidence to suggest a morphogenetic role despite the apparent absence of the substrate in plant cells. Finally, some chitinases and other chitin-binding proteins including some plant lectins share chitin-binding domains as part of their molecular structure and provide fuel for the so-called ‘lectin-chitinase’ debate and speculation for the origin of chitinase in plants.
Protein synthesis and translocation of l-[14C]leucine, d-[14C]glucose and inorganic 32P were enhanced in cultures of Sclerotium rolfsii Sacc. grown on medium supplemented with 0·5% lactose. Ethanol (2%, v/v) inhibited translocation but not protein synthesis. Neither lactose nor ethanol affected the uptake of radioactive substances by the fungal mycelium. Cycloheximide applied to the colony margins prevented sclerotium formation and protein synthesis without inhibiting translocation. It was concluded that translocation and protein synthesis in S. rolfsii are independent processes and are both essential for sclerotium formation.
When Sclerotium rolfsii was sprayed with partially purified chitinase produced by the cloned gene, rapid and extensive bursting of the hyphal tips was observed. This chitinase preparation was found to be effective in reduction of disease incidence caused by S. rolfsii in beans and Rhizoctonia solani in cotton under greenhouse conditions (62% disease reduction in both diseases). A similar effect was obtained when we used viable cells of E. coli containing the plasmid pLCHIA. However, E. coli carrying the plasmid lacking the pL promotor did not have any effect. These results suggest a role for chitinase in biological control of plant pathogenic fungi
Chitobiase (EC 3.2.1.29), from the culture filtrate ofTrichoderma harzianum, was purified in sequential steps by ammonium sulfate precipitation, ion exchange chromatography, and gel filtration. The physical and biochemical properties of the enzyme have been determined. The native enzyme has a molecular weight of 118 kDa when determined by gel filtration, and 64 kDa by SDS-PAGE. The enzyme catalyzed the hydrolysis of N,N-diacetylchitobiose andp-nitrophenyl--N-acetyl glucosamine with apparent Km of 575 M and 235 M, respectively. The pH optimum for the enzyme was pH 5.5, and maximum activity was obtained at 50C. Glucosamine and N-acetylglyucosamine strongly inhibited the enzyme.
Chitinase (EC 3.2.1.14), from the culture filtrate of Trichoderma harzianum, was successively purified by precipitation with ammonium sulfate followed by ion-exchange chromatography on Q-Sepharose, gel filtration on Sephadex G-100, and hydrophobic interaction on Phenyl-Sepharose CL-4B. A typical procedure provided 76-fold purification with 10% yield. The molecular weight of the purified chitinase was found to be approximately 40,000 Da by SDS-PAGE and 36,000 Da by gel filtration. The pH optimum for the enzyme was pH 4.0–4.5, and maximum activity was obtained at 40°C. The chitinase rapidly hydrolysed swollen chitin and regenerated chitin, but glycol chitin and powdered chitin very slowly. N',N-Diacetylchitobiose was not affected by enzyme.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Three proteins which display chitinase activity were purified from the supernatants of Trichoderma harzianum CECT 2413 grown in minimal medium supplemented with chitin as the sole carbon source. Purification was carried out after protein precipitation with ammonium sulphate, adsorption to colloidal chitin and digestion, and, finally, chromatofocusing. By this procedure, two chitinases of 42 kDa (CHIT42) and 37 kDa (CHIT37) were purified to homogeneity, as judged by SDS/PAGE and gel filtration, whereas a third, of 33 kDa (CHIT33), was highly purified. The isoelectric points for CHIT42, CHIT37 and CHIT33 were 6.2, 4.6 and 7.8, respectively. The three enzymes displayed endochitinase activities and showed different kinetic properties. CHIT33 was able to hydrolyze chitin oligomers of a polymerization degree higher than n = 4, its Km for colloidal chitin being 0.3 mg/ml. CHIT42 and CHIT37 were able to hydrolyze chitin oligomers with a minimal polymerization degree of n = 3, their Km values for colloidal chitin being 1.0 mg/ml and 0.5 mg/ml respectively. With regard to their lytic activity with purified cell walls of the phytopathogenic fungus Botrytis cinerea, a hydrolytic action was observed only when CHIT42 was present. Antibodies against CHIT42 and CHIT37 specifically recognized the proteins and did not display cross-reaction, suggesting that each protein is encoded by a different gene.
The production of chitinase by Trichoderma species is of interest in relation to their use in biocontrol and as a source of mycolytic enzymes. Fourteen isolates of the genus were screened to identify the most effective producer of chitinase. The best strain for chitinase was Trichoderma harzianum 39.1, and this was selected for study of the regulation of enzyme synthesis. Washed mycelium of T. harzianum 39.1 was incubated with a range of carbon sources. Chitinase synthesis was induced on chitin-containing medium, but repressed by glucose and N-acetylglucosamine. Production of the enzyme was optimal at a chitin concentration of 0.5%, at 28 degrees C, pH 6.0 and was independent of the age of the mycelium. The synthesis of chitinase was blocked by both 8-hydroxyquinoline and cycloheximide, inhibitors of RNA and protein synthesis, respectively. The mode of chitinase synthesis in this fungus is discussed.
The inclusion of 1% casein or bovine serum albumin in buffer used to reactivate enzymes subjected to sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis resulted in accelerated removal of SDS and restoration of nuclease and beta-galactosidase enzyme activities. Nuclease and beta-galactosidase activities which are absent from gels after longer wash procedures are detectable with this technique. Enzyme activity in gels prepared with SDS which contained inhibitory contaminants was partially restored by the casein wash procedure. The threshold of detection of two-dimensionally separated deoxyribonuclease I using the casein wash procedure was 1 picogram.
An improved procedure for staining of proteins following separation in polyacrylamide gels is described which utilizes the colloidal properties of Coomassie Brilliant Blue G-250 and R-250. The new method is based on addition of 20% v/v methanol and higher concentrations of ammonium sulfate to the staining solution previously described. The method combines the advantage of much shorter staining time with high sensitivity, a clear background not requiring destaining, stepwise staining, and stable fixation after staining. The method has been applied to staining of polyacrylamide gels after sodium dodecyl sulfate-electrophoresis and isoelectric focusing in carrier ampholyte-generated pH gradients.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
There are no reports of gene sequences coding for extracellular chitinolytic enzymes from filamentous fungi, even though these enzymes are considered critical to the biological control of plant pathogenic fungi. The purpose of this paper was to report the isolation of a gene (ThEn-42) encoding endochitinase (Ech) from Trichoderma harzianum strain P1, describe its sequence, and to determine whether it was related to genes coding for enzymes with similar functions from prokaryotic or other eukaryotic sources. A clone containing a 1096-bp foreign cDNA fragment was isolated from thalli grown under induced conditions. This cDNA molecule was sequenced and found to lack a portion of the 5' terminus. Polymerase chain reaction (PCR) was used to isolate a fragment from the lambda gt11 library which contained the 5' terminus plus an overlap region with the 1096-bp cDNA clone. The full-length cDNA sequence, consisting of 1554 bp, contained an open reading frame (ORF) expressing a protein of 424 amino acids (aa). Southern analysis of genomic DNA indicated that there is only a single gene in strain P1 with sequence identity to the sequence described in this report. One region within the protein, thought to be required for catalytic activity of the enzyme, was highly conserved between genes coding for Ech from Th, Serratia marcescens, Bacillus circulans, Streptomyces plicatus, Vibrio parahemolyticus and Kluyveromyces lactis.
Procedures are described for the direct assay of N-acetyl-beta-glucosaminidase (EC 3.2.1.30), chitobiosidase, and endochitinase (EC 3.2.1.14) after separation on starch or polyacrylamide electrophoresis gels. The enzymes were visualized as fluorescent bands by using an agarose overlay containing 4-methylumbelliferyl derivatives of N-acetyl-beta-D-glucosaminide, beta-D-N,N'-di-acetylchitobioside, or beta-D-N,N',N"-triacetylchitotriose for N-acetyl-beta-glucosaminidase, chitobiosidase, or endochitinase, respectively. For quantitative assay of N-acetyl-beta-glucosaminidase and chitobiosidase in solutions, a rapid technique using nitrophenyl-N-acetyl-beta-D-glucosaminide and nitrophenyl-beta-D-N,N'-diacetyl-chitobiose, respectively, was used. Endochitinase activity was quantitatively measured by determining the percentage reduction in turbidity of a reaction mixture that contained purified colloidal chitin. Trichoderma harzianum strain P1 was shown to produce three kinds of chitinolytic enzymes, and there were multiple forms of some of these.
Genetic engineering of microorganisms for improved biocontrol activity
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