The Volatile Organic Compounds of Streptomyces spp.: An In-Depth Analysis of Their Antifungal Properties

[EN] The study of volatile organic compounds (VOCs) has expanded because of the growing need to search for new bioactive compounds that could be used as therapeutic alternatives. These small molecules serve as signals to establish interactions with other nearby organisms in the environment. In this work, we evaluated the antifungal effect of VOCs produced by different Streptomyces spp. This study was performed using VOC chamber devices that allow for the free exchange of VOCs without physical contact between microorganisms or the diffusible compounds they produce. Antifungal activity was tested against Escovopsis weberi, a fungal pathogen that affects ant nest stability, and the results showed that Streptomyces spp. CS014, CS057, CS131, CS147, CS159, CS207, and CS227 inhibit or reduce the fungal growth with their emitted VOCs. A GS-MS analysis of volatiles produced and captured by activated charcoal suggested that these Streptomyces strains synthesize several antifungal VOCs, many of them produced because of the presence of E. weberi, with the accumulation of various VOCs determining the growth inhibition effect.SIThis research was funded by the Spanish Ministry of Science, Innovation, and Universities, project numbers MCIU-19-RTI2018-093562-B-I00 and PRE2019-089448 (to J.A.S and C.O.) and MCI- 21-PID2020-113062RB-100 (to C.M.)

Involvement of Trichoderma Trichothecenes in the Biocontrol Activity and Induction of Plant Defense-Related Genes

[EN] Trichoderma species produce trichothecenes, most notably trichodermin and harzianum A (HA), by a biosynthetic pathway in which several of the involved proteins have significant differences in functionality compared to their Fusarium orthologues. In addition, the genes encoding these proteins show a genomic organization differing from that of the Fusarium tri clusters. Here we describe the isolation of Trichoderma arundinaceum IBT 40837 transformants which have a disrupted or silenced tri4 gene, a gene encoding a cytochrome P450 monooxygenase that oxygenates trichodiene to give rise to isotrichodiol, and the effect of tri4 gene disruption and silencing on the expression of other tri genes. Our results indicate that the tri4 gene disruption resulted in a reduced antifungal activity against Botrytis cinerea and Rhizoctonia solani and also in a reduced ability to induce the expression of tomato plant defense-related genes belonging to the salicylic acid (SA) and jasmonate (JA) pathways against B. cinerea, in comparison to the wild-type strain, indicating that HA plays an important function in the sensitization of Trichoderma-pretreated plants against this fungal pathogen. Additionally, the effect of the interaction of T. arundinaceum with B. cinerea or R. solani and with tomato seedlings on the expressions of the tri genes was studiedSIResearch project funding was obtained from Junta de Castilla y León (GR67) and the Spanish Ministry of Science and Innovation (AGL2006-05660, AGL2009-13431-C01, and AGL2009-13431-C02). M. G. Malmierca was granted an FPU fellowship by the Spanish Ministry of Science and Innovation (AP2007-02835

Effects of Trichothecene Production on the Plant Defense Response and Fungal Physiology: Overexpression of the Trichoderma arundinaceum tri4 Gene in T. harzianum

[EN] Trichothecenes are fungal sesquiterpenoid compounds, the majority of which have phytotoxic activity. They contaminate food and feed stocks, resulting in potential harm to animals and human beings. Trichoderma brevicompactum and T. arundinaceum produce trichodermin and harzianum A (HA), respectively, two trichothecenes that show different bioactive properties. Both compounds have remarkable antibiotic and cytotoxic activities, but in addition, trichodermin is highly phytotoxic, whileHAlacks this activity when analyzed in vivo. Analysis of Fusarium trichothecene intermediates led to the conclusion that most of them, with the exception of the hydrocarbon precursor trichodiene (TD), have a detectable phytotoxic activity which is not directly related to the structural complexity of the intermediate. In the present work, theHAintermediate 12,13-epoxytrichothec-9-ene (EPT) was produced by expression of the T. arundinaceum tri4 gene in a transgenic T. harzianum strain that already produces TD after transformation with the T. arundinaceum tri5 gene. Purified EPT did not show antifungal or phytotoxic activity, while purified HA showed both antifungal and phytotoxic activities. However, the use of the transgenic T. harzianum tri4 strain induced a downregulation of defense-related genes in tomato plants and also downregulated plant genes involved in fungal root colonization. The production of EPT by the transgenic tri4 strain raised levels of erg1 expression and reduced squalene accumulation while not affecting levels of ergosterol. Together, these results indicate the complex interactions among trichothecene intermediates, fungal antagonists, and host plantsSIThis research was supported by grants from MICINN and MINECO (grants AGL2009-13431-C02 and AGL2012-40041-C02-02) and from the Junta de Castilla y León (grant LE125A12-2). M. G. Malmierca was granted an FPU fellowship by the Spanish Ministry of Science and Innovation (grant AP2007-02835

Influence of Rhizoctonia solani and Trichoderma spp. in growth of bean (Phaseolus vulgaris L.) and in the induction of plant defense-related genes

[EN] Many Trichoderma species are well-known for their ability to promote plant growth and defense. We study how the interaction of bean plants with R. solani and/or Trichoderma affect the plants growth and the level of expression of defense-related genes. Trichoderma isolates were evaluated in vitro for their potential to antagonize R. solani. Bioassays were performed in climatic chambers and development of the plants was evaluated. The effect of Trichoderma treatment and/or R. solani infection on the expression of bean defense-related genes was analyzed by real-time PCR and the production of ergosterol and squalene was quantified. In vitro growth inhibition of R. solani was between 86 and 58%. In in vivo assays, the bean plants treated with Trichoderma harzianum T019 always had an increased size respect to control and the plants treated with this isolate did not decrease their size in presence of R. solani. The interaction of plants with R. solani and/or Trichoderma affects the level of expression of seven defense-related genes. Squalene and ergosterol production differences were found among the Trichoderma isolates, T019 showing the highest values for both compounds. T. harzianum T019 shows a positive effect on the level of resistance of bean plants to R. solani. This strain induces the expression of plant defense-related genes and produces a higher level of ergosterol, indicating its ability to grow at a higher rate in the soil, which would explain its positive effects on plant growth and defense in the presence of the pathogen.SIThe grant awarded to Sara Mayo (FPU12/00677) by the Ministry of Education, Culture and Sport (Spain) according the Resolution of April 25, 2012 (BOE of 10 May), as amended by Resolution of May 18, 2012 (BOE of 22 May), and by Resolution of October 31, 2012 (BOE of 12 November) and granted in the Resolution of December 20, 2012. Ministry of Economy and Competitiveness for National project “Farnesol as self-regulatory molecule in Trichoderma. Tyrosol and farnesol signaling in Trichoderma-bean interaction” (AGL2012-40041-C02-02). Junta de Castilla y León, Consejería de Educación for the project “Effect of terpenes and physiologically related compounds produced by Trichoderma parareesei in the development of common bean (Phaseolus vulgaris L.) and in defensive responses of this plant” (LE228U14). J. Alvarez for his technical support

Overexpression of the Trichoderma brevicompactum tri5 Gene: Effect on the Expression of the Trichodermin Biosynthetic Genes and on Tomato Seedlings

[EN] Trichoderma brevicompactum IBT 40841 produces trichodermin, a trichothecene-type toxin that shares most of the steps of its biosynthesis with harzianum A, another trichothecene produced by several Trichoderma species. The first specific step in the trichothecene biosynthesis is carried out by a terpene cylcase, trichodiene synthase, that catalyzes the conversion of farnesyl pyrophosphate to trichodiene and that is encoded by the tri5 gene. Overexpression of tri5 resulted in increased levels of trichodermin production, but also in an increase in tyrosol and hydroxytyrosol production, two antioxidant compounds that may play a regulatory role in trichothecene biosynthesis, and also in a higher expression of three trichothecene genes, tri4, tri6 and tri10, and of the erg1 gene, which participates in the synthesis of triterpenes. The effect of tri5 overexpression on tomato seedling disease response was also studiedSIResearch project funding was from Junta de Castilla y León (GR67) and the Spanish Ministry of Science and Innovation (AGL2008-0512/AGR, AGL2009-13431-C01 and AGL2009-13431-C02). AT was granted by a Spanish Foreing Office AECID award. MGM was granted with a FPU fellowship by the Spanish Ministry of Science and Innovation (AP2007-02835

Identification of Loci and Functional Characterization of Trichothecene Biosynthesis Genes in Filamentous Fungi of the Genus Trichoderma

[EN] Trichothecenes are mycotoxins produced by Trichoderma, Fusarium, and at least four other genera in the fungal order Hypocreales. Fusarium has a trichothecene biosynthetic gene (TRI) cluster that encodes transport and regulatory proteins as well as most enzymes required for the formation of the mycotoxins. However, little is known about trichothecene biosynthesis in the other genera. Here, we identify and characterize TRI gene orthologues (tri) in Trichoderma arundinaceum and Trichoderma brevicompactum. Our results indicate that both Trichoderma species have a tri cluster that consists of orthologues of seven genes present in the Fusarium TRI cluster. Organization of genes in the cluster is the same in the two Trichoderma species but differs from the organization in Fusarium. Sequence and functional analysis revealed that the gene (tri5) responsible for the first committed step in trichothecene biosynthesis is located outside the cluster in both Trichoderma species rather than inside the cluster as it is in Fusarium. Heterologous expression analysis revealed that two T. arundinaceum cluster genes (tri4 and tri11) differ in function from their Fusarium orthologues. The Tatri4-encoded enzyme catalyzes only three of the four oxygenation reactions catalyzed by the orthologous enzyme in Fusarium. The Tatri11-encoded enzyme catalyzes a completely different reaction (trichothecene C-4 hydroxylation) than the Fusarium orthologue (trichothecene C-15 hydroxylation). The results of this study indicate that although some characteristics of the tri/TRI cluster have been conserved during evolution of Trichoderma and Fusarium, the cluster has undergone marked changes, including gene loss and/or gain, gene rearrangement, and divergence of gene functionSIResearch project funding was from Junta de Castilla y León (GR67) and the Spanish Ministry of Science and Innovation (AGL2006-05660, AGL2009-13431-C01 and AGL2009-13431-C02). M. Gómez was awarded an FPU fellowship by the Spanish Ministry of Science and Innovation (AP2007-02835

Botrydial and botcinins produced by Botrytis cinerea regulate the expression of Trichoderma arundinaceum genes involved in trichothecene biosynthesis

Trichoderma arundinaceum IBT 40837 (Ta37) and Botrytis cinerea produce the sesquiterpenes harzianum A (HA) and botrydial (BOT), respectively, and also the polyketides aspinolides and botcinins (Botcs), respectively. We analysed the role of BOT and Botcs in the Ta37-B. cinerea interaction, including the transcriptomic changes in the genes involved in HA (tri) and ergosterol biosynthesis, as well as changes in the level of HA and squalene-ergosterol. We found that, when confronted with B. cinerea, the tri biosynthetic genes were up-regulated in all dual cultures analysed, but at higher levels when Ta37 was confronted with the BOT non-producer mutant bcbot2Δ. The production of HA was also higher in the interaction area with this mutant. In Ta37-bcbot2Δ confrontation experiments, the expression of the hmgR gene, encoding the 3-hydroxy-3-methylglutaryl coenzyme A reductase, which is the first enzyme of the terpene biosynthetic pathway, was also up-regulated, resulting in an increase in squalene production compared with the confrontation with B. cinerea B05.10. Botcs had an up-regulatory effect on the tri biosynthetic genes, with BotcA having a stronger effect than BotcB. The results indicate that the interaction between Ta37 and B. cinerea exerts a stimulatory effect on the expression of the tri biosynthetic genes, which, in the interaction zone, can be attenuated by BOT produced by B. cinerea B05.10. The present work provides evidence for a metabolic dialogue between T. arundinaceum and B. cinerea that is mediated by sesquiterpenes and polyketides, and that affects the outcome of the interaction of these fungi with each other and their environmentSIFunding was obtained from the Junta de Castilla y Leon (SA260A11-2,LE125A12-2 and LE228U14) and Spanish Government grants MICINN-AGL2009-13431-C02-02, MINECO-AGL2012-40041-C02-01, AGL2012-40041-C02-02 and AGL2012-39798-C02-01. MGM and II-B were granted fellowships from the Spanish Ministry of Science and Innovation(AP2007-02835, BES-2013-063411

Searching for glycosylated natural products in actinomycetes and identification of novel macrolactams and angucyclines

Many bioactive natural products are glycosylated compounds in which the sugar components usually participate in interaction and molecular recognition of the cellular target. Therefore, the presence of sugar moieties is important, in some cases essential, for bioactivity. Searching for novel glycosylated bioactive compounds is an important aim in the field of the research for natural products from actinomycetes. A great majority of these sugar moieties belong to the 6-deoxyhexoses and share two common biosynthetic steps catalyzed by a NDP-D-glucose synthase (GS) and a NDP-D-glucose 4,6-dehydratase (DH). Based on this fact, seventy one Streptomyces strains isolated from the integument of ants of the Tribe Attini were screened for the presence of biosynthetic gene clusters (BGCs) for glycosylated compounds. Total DNAs were analyzed by PCR amplification using oligo primers for GSs and DHs and also for a NDP-D-glucose-2,3-dehydratases. Amplicons were used in gene disruption experiments to generate non-producing mutants in the corresponding clusters. Eleven mutants were obtained and comparative dereplication analyses between the wild type strains and the corresponding mutants allowed in some cases the identification of the compound coded by the corresponding cluster (lobophorins, vicenistatin, chromomycins and benzanthrins) and that of two novel macrolactams (sipanmycin A and B). Several strains did not show UPLC differential peaks between the wild type strain and mutant profiles. However, after genome sequencing of these strains, the activation of the expression of two clusters was achieved by using nutritional and genetic approaches leading to the identification of compounds of the cervimycins family and two novel members of the warkmycins family. Our work defines a useful strategy for the identification new glycosylated compounds by a combination of genome mining, gene inactivation experiments and the activation of silent biosynthetic clusters in Streptomyces strains

Biosíntesis del harzianum A y su papel en la fisiología y en la actividad de biocontrol ejercida por "Trichoderma arundinaceum" = Biosynthesis of hazianum A and its role in the physiology and in the biocontrol activity of "Trichoderma aurundicaceum"

267 p.Trichoderma es un género de hongos filamentosos muy conocido por su aplicación en agricultura como agente de biocontrol, sobre todo frente a otros hongos. Existen varios mecanismos mediante los cuales Trichoderma spp. ejerce este efecto beneficioso sobre las plantas: - Antagonismo de fitopatógenos. Este antagonismo se debe a la suma de 3 procesos: competición por espacio y nutrientes en la rizosfera, micoparasitismo y antibiosis. - Biofertilización del suelo e inducción de las defensas de la planta. - Aumento de la resistencia de la planta a diferentes estreses abiótico

Trichodiene Production in aTrichoderma harzianum erg1-Silenced Strain Provides Evidence of the Importance of the Sterol Biosynthetic Pathway in Inducing Plant Defense-Related Gene Expression

[EN] Trichoderma species are often used as biocontrol agents against plant-pathogenic fungi. A complex molecular interaction occurs among the biocontrol agent, the antagonistic fungus, and the plant. Terpenes and sterols produced by the biocontrol fungus have been found to affect gene expression in both the antagonistic fungus and the plant. The terpene trichodiene (TD) elicits the expression of genes related to tomato defense and to Botrytis virulence. We show here that TD itself is able to induce the expression of Botrytis genes involved in the synthesis of botrydial (BOT) and also induces terpene gene expression in Trichoderma spp. The terpene ergosterol, in addition to its role as a structural component of the fungal cell membranes, acts as an elicitor of defense response in plants. In the present work, using a transformant of T. harzianum, which is silenced in the erg1 gene and accumulates high levels of squalene, we show that this ergosterol precursor also acts as an important elicitor molecule of tomato defense-related genes and induces Botrytis genes involved in BOT biosynthesis, in both cases, in a concentration-dependent manner. Our data emphasize the importance of a balance of squalene and ergosterol in fungal interactions as well as in the biocontrol activity of Trichoderma sppSIThis research was supported by grants from MICINN and MINECO (AGL2009-13431-C02, AGL2012-40041-C02-01, and AGL2012-40041-C02-02) and from Junta de Castilla y León (LE125A12-2). M. Gómez was granted a FPU fellowship by the Spanish Ministry of Science and Innovation (AP2007-02835