Antagonism between Trichoderma harzianum ETS 323 and Botrytis cinerea associated with L-phenylalanine oxidase-induced reactive oxygen species generation
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Nitric oxide (NO) and mitogen-activated protein kinases (MAPKs) are signal molecules involved in the disease resistance of plants. To investigate the role of tomato MAPKs in the NO-mediated defense response, mature green tomatoes (Lycopersicon esculentum Mill. cv. Qian-xi) were treated with a MAPKs inhibitor (1,4-diamino-2,3-dicyano-1,4-bis(o-amino-phenylmercapto) butadiene (U0126)), NO donor sodium nitroprusside (SNP), and SNP plus U0126. Treatment with U0126 increased the incidence of disease and size of lesion areas in the tomato fruits after being inoculated with Botrytis cinerea. NO enhanced the resistance of the tomato fruits against Botrytis cinerea invasion and the activities of nitric oxide synthase, Chitinase, β-1,3-glucanase, polyphenol oxidase, and phenylalanine ammonia-lyase. However, the effects of NO on disease resistance were weakened by the MAPKs inhibitor. Meanwhile, the relative expression of LeMAPK1, LeMAPK2, and LeMAPK3 in the (SNP + U0126)-treated fruits was lower than that in the SNP-treated fruits. The results suggest that LeMAPK1/2/3 are involved in NO-induced disease resistance of tomato fruits against Botrytis cinerea.
Lycopersicon
Phenylalanine ammonia-lyase
Chitinase
Botrytis
Catechol oxidase
Sodium nitroprusside
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Trichoderma harzianum is a filamentous fungus well adapted to different ecological niches. Owing to its ability to antagonize a wide range of plant pathogens, it is used as a biological control agent in agriculture. Selected strains of T. harzianum are also able to increase the tolerance of plants to biotic and abiotic stresses. However, little is known about the regulatory elements of the T. harzianum transcriptional machinery and their role in the biocontrol by this species. We had previously reported the involvement of the transcription factor THCTF1 in the T. harzianum production of the secondary metabolite 6-pentyl-pyrone, an important volatile compound related to interspecies cross-talk. Here, we performed a subtractive hybridization to explore the genes regulated by THCTF1, allowing us to identify a multiprotein bridging factor 1 (mbf1) homolog. The gene from T. harzianum T34 was isolated and characterized, and the generated Thmbf1 overexpressing transformants were used to investigate the role of this gene in the biocontrol abilities of the fungus against two plant pathogens. The transformants showed a reduced antifungal activity against Fusarium oxysporum f. sp. lycopersici race 2 (FO) and Botrytis cinerea (BC) in confrontation assays on discontinuous medium, indicating that the Thmbf1 gene could affect T. harzianum production of volatile organic compounds (VOC) with antifungal activity. Moreover, cellophane and dialysis membrane assays indicated that Thmbf1 overexpression affected the production of low molecular weight secreted compounds with antifungal activity against FO. Intriguingly, no correlation in the expression profiles, either in rich or minimal medium, was observed between Thmbf1 and the master regulator gene cross-pathway control (cpc1). Greenhouse assays allowed us to evaluate the biocontrol potential of T. harzianum strains against BC and FO on susceptible tomato plants. The wild type strain T34 significantly reduced the necrotic leaf lesions caused by BC while plants treated with the Thmbf1-overexpressing transformants exhibited an increased susceptibility to this pathogen. The percentages of Fusarium wilt disease incidence and values of aboveground dry weight showed that T34 did not have biocontrol activity against FO, at least in the 'Moneymaker' tomato variety, and that Thmbf1 overexpression increased the incidence of this disease. Our results show that the Thmbf1 overexpression in T34 negatively affects its biocontrol mechanisms.
Trichoderma harzianum
Trichoderma
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Trichoderma
Trichoderma harzianum
Leptosphaeria maculans
Secondary metabolism
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The biocontrol strain P1 of Trichoderma harzianum was genetically modified by targeted disruption of the single-copy ech42 gene encoding for the secreted 42-kDa endochitinase (CHIT42). Stable mutants in which ech42 was interrupted, and unable to produce CHIT42, were obtained and characterized. These mutants lacked the ech42 transcript, the protein, and endochitinase activity in culture filtrates, and they were unable to clear a medium containing colloidal chitin. Other chitinolytic and glucanolytic enzymes expressed during mycoparasitism were not affected by the disruption of ech42. The disrupted mutant D11 grew and sporulated similarly to the wild type. In vitro antifungal activity of the ech42 disruptant culture filtrates against Botrytis cinerea and Rhizoctonia solani was reduced about 40%, compared with wild type; antifungal activity was fully restored by adding an equivalent amount of CHIT42 as secreted by P1. The mutant exhibited the same biocontrol effect against Pythium ultimum as strain P1, but the antagonism against B. cinerea on bean leaves by the mutant was significantly reduced (33% less biocontrol), compared with strain P1. Conversely, the endochitinase-deficient mutant performed better than the wild type (16% improvement of survival) in biocontrol experiments in soil infested with the soilborne fungus R. solani. These results indicate that the antagonistic interaction between the T. harzianum strain and various fungal hosts is based on different mechanisms.
Trichoderma harzianum
Chitinase
Trichoderma
Pythium ultimum
Wild type
Rhizoctonia
Botrytis
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Plant survival in the terrestrial ecosystem is influenced by both beneficial and harmful microbes. Trichoderma spp. are a group of filamentous fungi that promote plant growth and resistance to harmful microbes. Previously, we showed that the genus Trichoderma could effectively suppress Fusarium wilt in cucumber. However, the mechanisms that underlie the effects of the genus Trichoderma on plant defense have not been fully substantiated. Two essential metabolic pathways, such as the ascorbate (AsA)-glutathione (GSH) cycle and the oxidative pentose phosphate pathway (OPPP), have been shown to participate in plant tolerance to biotic stressors; nevertheless, the involvement of these pathways in Trichoderma-induced enhanced defense remains elusive. Here, we show that Trichoderma harzianum could alleviate oxidative and nitrostative stress by minimizing reactive oxygen species (ROS; hydrogen peroxide and superoxide) and reactive nitrogen species (nitric oxide [NO]) accumulation, respectively, under Fusarium oxysporum infection in cucumber roots. The genus Trichoderma enhanced antioxidant potential to counterbalance the overproduced ROS and attenuated the transcript and activity of NO synthase and nitrate reductase. The genus Trichoderma also stimulated S-nitrosylated glutathione reductase activity and reduced S-nitrosothiol and S-nitrosylated glutathione content. Furthermore, the genus Trichoderma enhanced AsA and GSH concentrations and activated their biosynthetic enzymes, γ-GCS and l-galactono-1,4-lactone dehydrogenase. Interestingly, the genus Trichoderma alleviated Fusarium-inhibited activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, enzymes involved in the OPPP. Such positive regulation of the key enzymes indicates the adequate maintenance of the AsA-GSH pathway and the OPPP, which potentially contributed to improve redox balance, energy flow, and defense response. Our study advances the current knowledge of Trichoderma-induced enhanced defense against F. oxysporum in cucumber.
Trichoderma
Trichoderma harzianum
Glutathione reductase
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Trichoderma T21 and its four REMI transformants were studied.Their antagonistic ability against Botrytis cinerea,biocontrol efficiency in greenhouse and three extracellular enzymes(chitinase,β-1,3-glucanase and cellulose) were examined.The results showed that five Trichoderma strains had different inhibitory effect to Botrytis cinerea,and Ttrm68 was the best.Biocontrol efficiency of Ttrm68 was the best too.The activity of chitinase and β-1,3-glucanase of Trichoderma with better inhibitory effect were higher,nor was the activity of cellulose.
Chitinase
Trichoderma
Glucanase
Botrytis
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The synthesis of reactive oxygen species (ROS) is one of the first events following pathogenic interactions in eukaryotic cells, and NADPH oxidases are involved in the formation of such ROS. The nox1 gene of Trichoderma harzianum was cloned, and its role in antagonism against phytopathogens was analyzed in nox1-overexpressed transformants. The increased levels of nox1 expression in these transformants were accompanied by an increase in ROS production during their direct confrontation with Pythium ultimum. The transformants displayed an increased hydrolytic pattern, as determined by comparing protease, cellulase, and chitinase activities with those for the wild type. In confrontation assays against P. ultimum the nox1-overexpressed transformants were more effective than the wild type, but not in assays against Botrytis cinerea or Rhizoctonia solani. A transcriptomic analysis using a Trichoderma high-density oligonucleotide (HDO) microarray also showed that, compared to gene expression for the interaction of wild-type T. harzianum and P. ultimum, genes related to protease, cellulase, and chitinase activities were differentially upregulated in the interaction of a nox1-overexpressed transformant with this pathogen. Our results show that nox1 is involved in T. harzianum ROS production and antagonism against P. ultimum.
Pythium ultimum
Trichoderma harzianum
NOX1
Chitinase
Trichoderma
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Botrytis
Trichoderma
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To investigate the responses to Botrytis cinerea (B. cinerea) infection and the effects of exogenous Ca2+ on calmodulin (CaM), phenylalanine ammonia-lyase (PAL), and salicylic acid (SA) biosynthesis and concentrations, we investigated the role of exogenous calcium in defense and resistance to the necrotrophic fungus B. cinerea in tomato (Lycopersicon esculentum Mill.). Specifically, 7.5 mM Ca2+ was the best treatment for plant resistance, resulting in stable and permanent resistance to the pathogen. This treatment improved plant resistance and the results also confirmed that the concentrations of CaM and SA are negatively correlated with the resistance response.
Phenylalanine ammonia-lyase
Lycopersicon
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Antagonistic activity of Trichoderma harzianum isolated from the rhizosphere of groundnut was determined in vitro. T. harzianum decreased the root rot incidence in vivo. Potential of T. harzianum to induce systemic resistance was tested in groundnut against Macrophomina phaseolina . Biochemical changes in T. harzianum treated plants, M. phaseolina inoculated plants and healthy plants were assayed at different stages of infection. Treatment with T. harzianum and challenge inoculation of M. phaseolina enhanced induction of defense enzymes such as peroxidase (PO) and polyphenol oxidase (PPO) and defense compounds like total phenol and ortho-dihydric phenol. Total phenols, ortho-dihydric phenols, peroxidase and polyphenol oxidase activities increased at different stages of infection. T. harizanum treatment along with challenge inoculation of the pathogen significantly increased the activity of peroxidase and polyphenol oxidase by about 28.2 % and 95.5%, respectively, in roots at stage 2 compared to untreated plants. Increased levels of peroxidase and polyphenol oxidase were induced in root and shoot of treated plants indicating the systemic protection offered to groundnut by T. harzianum . The observations revealed that T. harzianum was capable of inducing systemic resistance against M. phaseolina by eliciting the production of defense enzymes.
Macrophomina phaseolina
Trichoderma harzianum
Catechol oxidase
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