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Xylotrechus arvicola Olivier 1795 (Coleoptera: Cerambycidae) is an insect pest that affects Vitis vinifera L. 1753 (Vitales: Vitaceae) plants in the main wine-producing regions of Spain. X. arvicola larvae bore into grapevine wood, causing both direct damage (ingestion of vascular tissues) and indirect damages (introduction of wood fungi) to the plant. The aim of research was to evaluate the effective section of wood damaged by larvae and assess its resistance capacity through compression tests and loading and breaking times. Compressive tests (on trunks) and flexural tests (on branches) were performed to evaluate the effective section. Trunk samples exhibited a higher effective section than branches samples, with effective section percentages ranging from 91.49 % to 93.53 % in trunks and decreasing from 84.91 % to 86.95 % in branches. Both loading times (Time 1) and breakage times (Time 2) increased with the effective section of the wood, although these times were lower in damaged wood samples of both trunks and branches. Additionally, significant differences were observed in the interactions between loading time x effective section and breakage time x effective section in dry trunks. This indicates a stronger relationship between the effective section and increased resistance in trunks. The results suggest that, in ‘Tempranillo’ variety, branches with a lower effective section are more prone to breakage when affected by X. arvicola larvae, whereas trunks, with a greater effective section, maintain better stability. This research should be continued with the evaluation of other vine varieties and different years of X. arvicola attacks, as the current findings are based on a single variety (‘Tempranillo’) over a period of ten years.

Trichoderma strains used in vineyards for the control of grapevine trunk diseases (GTDs) present a promising alternative to chemical products. Therefore, the isolation and characterization of new indigenous Trichoderma strains for these purposes is a valuable strategy to favor the adaptation of these strains to the environment, thus improving their efficacy in the field. In this research, a new Trichoderma species, Trichoderma carraovejensis, isolated from vineyards in Ribera de Duero (Spain) area, has been identified and phylogenetically analyzed using 20 housekeeping genes isolated from the genome of 24 Trichoderma species. A morphological description and comparison of the new species has also been carried out. In order to corroborate the potential of T. carraovejensis as a biological control agent (BCA), confrontation tests against pathogenic fungi, causing various GTDs, have been performed in the laboratory. The compatibility of T. carraovejensis with different pesticides and biostimulants has also been assessed. This new Trichoderma species demonstrates the ability to control pathogens such as Diplodia seriata, as well as high compatibility with powdered sulfur-based pesticides. In conclusion, the autochthonous species T. carraovejensis can be an effective alternative to complement the currently used strategies for the control of wood diseases in its region of origin.

Xylotrechus arvicola represents a significant insect pest impacting Vitis vinifera within the principal wine-producing territories of the Iberian Peninsula. The larvae of this species bore into grapevine wood, resulting in significant structural and biomechanical deterioration to the plant. Compressive and flexural tests were conducted to assess the mechanical properties of wood affected by X. arvicola. Compressive and flexural strength exhibited a decline with the escalation of the Total Damaged Surface Area (TDSA) of the samples, ranging from 0.31% to 0.73% in trunks and from 0.04 to 0.76% in branches, irrespective of the wood moisture content (fresh and dry). The most significant reduction in resistance occurred in affected dry trunks and branches. Notably, the deflection at break for dry samples was lower compared to fresh samples (65.00 and 97.85 mm, respectively). Moreover, the deflection at break for affected fresh samples (164.37 mm) significantly surpassed that of unaffected fresh samples (72.58 mm) and affected dry samples (37.50 mm). It is noteworthy that a higher percentage of TDSA coincided with diminished wood resistance. The percentage of fungal growth symptoms observed in affected wood samples was 66.66% for dry trunks, 75.00% for fresh branches, and 60.00% for dry branches. The damage inflicted by larvae facilitated the spread of grapevine diseases via emergence of holes created by insects upon exiting the wood and through the larval galleries connected to them. This damage also altered the mechanical properties of grapevine plants, with fresh branches exhibiting the most pronounced effects.

The trichothecene toxin-producing fungus Trichoderma arundinaceum has potential as a biological control agent. However, most biocontrol studies have focused only on one strain, IBT 40837. In the current study, three Trichoderma isolates recovered from bean-field soils produced the trichothecene harzianum A (HA) and trichodermol, the latter being an intermediate in the HA biosynthesis. Based on phylogenetic analysis, the three isolates were assigned to the species T. arundinaceum. Their genome sequences had a high degree of similarity to the reference IBT 40837 strain, in terms of total genome size, number of predicted genes, and diversity of putative secondary metabolite biosynthetic gene clusters. HA production by these bean-field isolates conferred significant in vitro antifungal activity against Rhizoctonia solani and Sclerotinia sclerotiorum, which are some of the most important bean pathogens. Furthermore, the bean-field isolates stimulated germination of bean seeds and subsequent growth of above ground parts of the bean plant. Transcriptomic analysis of bean plants inoculated with these T. arundinaceum bean-field soil isolates indicated that HA production significantly affected expression of plant defense-related genes; this effect was particularly significant in the expression of chitinase-encoding genes. Together, these results indicate that Trichoderma species producing non-phytotoxic trichothecenes can induce defenses in plants without negatively affecting germination and development

Native strains of Trichoderma in vineyard soil represent an opportunity for reducing the incidence of grapevine trunk diseases (GTDs) in vineyards. Moreover, its relationship with the environment (physicochemical soil characteristics and farming management practices) remains unclear. In the current study, a survey was carried out on farming management used by viticulturists, and soil samples were studied to analyze their physicochemical properties and to isolate Trichoderma strains. Later, statistical analyses were performed to identify possible correlations between Trichoderma populations, soil management and soil characteristics. In addition, in vitro tests, including antibiosis and mycoparasitism, were performed to select those Trichoderma strains able to antagonize Phaeoacremonium minimum. In this study a positive correlation was found between the iron content and pH in the soil, and a lower pH increases Trichoderma populations in soils. Vineyard management also affects Trichoderma populations in the soil, negatively in the case of fertilization and tillage and positively in the case of herbicide spraying. Two Trichoderma native strains were selected as potential biocontrol agents (Trichoderma gamsii T065 and Trichoderma harzianum T087) using antibiosis and mycoparasitism as mechanisms of action. These results led to the conclusion that native Trichoderma strains hold great potential as biological control agents and as producers of secondary metabolites.

Trichothecenes are terpenoid toxins produced by species in 10 fungal genera, including species of Trichoderma. The trichothecene biosynthetic gene (tri) cluster typically includes the tri5 gene, which encodes a terpene synthase that catalyzes formation of trichodiene, the parent compound of all trichothecenes. The two Trichoderma species, Trichoderma arundinaceum and T. brevicompactum, that have been examined are unique in that tri5 is located outside the tri cluster in a genomic region that does not include other known tri genes. In the current study, analysis of 35 species representing a wide range of the phylogenetic diversity of Trichoderma revealed that 22 species had tri5, but only 13 species had both tri5 and the tri cluster. tri5 was not located in the cluster in any species. Using complementation analysis of a T. arundinaceum tri5 deletion mutant, we demonstrated that some tri5 homologs from species that lack a tri cluster are functional, but others are not. Phylogenetic analyses suggest that Trichoderma tri5 was under positive selection following its divergence from homologs in other fungi but before Trichoderma species began diverging from one another. We propose two models to explain these diverse observations. One model proposes that the location of tri5 outside the tri cluster resulted from loss of tri5 from the cluster in an ancestral species followed by reacquisition via horizontal transfer. The other model proposes that in species that have a functional tri5 but lack the tri cluster, trichodiene production provides a competitive advantage.

Acanthoscelides obtectus is an insect pest that attacks wild and cultivated common beans (Phaseolus vulgaris L). Four Trichoderma strains, the T. arundinaceum IBT 40837 wild-type strain (=Ta37), a producer of trichothecene harzianum A (HA), two transformants of T. arundinaceum strain, Ta37-17.139 (=Δtri17) and Ta37-23.74 (=Δtri23), and the T. brevicompactum IBT 40841 wild-type strain (=Tb41), which produces the trichothecene trichodermin, were assessed to establish their direct effect on insect attacks and their indirect effect on the plants grown from the beans treated with those fungal strains and exposed to insect attacks. Treatments of bean seeds with different Trichoderma strains led to different survival rates in the insects, and the Tb41 strain caused the lowest survival rate of all. An 86.10% of the insect cadavers (in contact with Δtri23) showed growth of this strain. This was the treatment that attracted the greatest number of insects. The daily emergence was reduced in beans treated with the Ta37, Tb41, and Δtri17 strains. The undamaged beans treated with Ta37 and Δtri23 showed a high capacity of germination (80.00% and 75.00%, respectively), whereas the Δtri17 and Tb41 treatments increased the capacity of germination in the damaged beans (66.67%). The undamaged beans treated with Δtri23 had the greatest dry weights for the aerial part (4.22 g) and root system in the plants (0.62 g). More studies on the mechanisms of insect control, plant growth promotion, and trichodermol and trichodermin production by Δtri23 and Tb41, respectively, should be explored in order to commercialize these fungal species on a large scale.

Farnesol is an isoprenoid intermediate in the mevalonate (MVA) pathway and is produced by the dephosphorylation of farnesyl diphosphate. Farnesol plays a central role in cell growth and differentiation, controls production of ubiquinone and ergosterol, and participates in the regulation of filamentation and biofilm formation. Despite these important functions, studies of farnesol in filamentous fungi are limited, and information on its effects on antifungal and/or biocontrol activity is scarce. In the present article, we identified the Trichoderma harzianum gene dpp1, encoding a diacylglycerol pyrophosphatase that catalyzes production of farnesol from farnesol diphosphate. We analyzed the function of dpp1 to address the importance of farnesol in Trichoderma physiology and ecology. Overexpression of dpp1 in T. harzianum caused an expected increase in farnesol production as well as a marked change in squalene and ergosterol levels, but overexpression did not affect antifungal activity. In interaction with plants, a dpp1-overexpressing transformant acted as a sensitizing agent in that it up-regulated expression of plant defense salicylate-related genes in the presence of a fungal plant pathogen. In addition, toxicity of farnesol on Trichoderma and plants was examined. Finally, a phylogenetic study of dpp1 was performed to understand its evolutionary history as a primary metabolite gene. This article represents a step forward in the acquisition of knowledge on the role of farnesol in fungal physiology and in fungus-environment interactions.

Pesticides of chemical synthesis have mainly been used to control pests, diseases and adventitious plants up until now. However, it has been shown that some pesticides can remain in the soil for long periods of time, thus affecting the development of organisms in the rhizosphere as well as human health, which are two of the most noteworthy side effects. The aim of this research was to analyze the compatibility of autochthonous Trichoderma strains with different synthetic fungicides, acaricides, insecticides (including an entomopathogenic fungus) and herbicides. Sulfur encouraged the growth of all autochthonous strains assayed, and the combination Trichoderma-B. bassiana did not disturb their growth. So, the combination of the autochthonous Trichoderma strains with these organic pesticides will be a positive strategy to apply in the field to control pests and some diseases. Conventional pesticides modified the development of all autochthonous Trichoderma strains, demonstrating that not only do they affect weeds, fungus or pests but also rhizosphere microorganisms. In conclusion, conventional pesticides indiscriminately used to control pests, diseases and weeds could reduce the development of autochthonous Trichoderma strains, especially fungicides and herbicides.

Trichoderma strains used in biological control products usually exhibit high efficiency in the control of plant diseases. However, their behavior under field conditions is difficult to predict. In addition, the potential of indigenous strains has been poorly assayed as well as their possible behavior as endophytes. Hence, niche colonization is a key feature for an effective protection. In this study, we aimed to: (i) explore the possibility of using a new Trichoderma strain isolated from vine to control pathogens, (ii) study the in planta interaction with the pathogen Phaeoacremonium minimum W. Gams, Crous, M.J. Wingf. & L. Mugnai (formerly Phaeoacremonium aleophilum), a pioneer fungus involved in Grapevine Trunk Diseases (GTDs) such as esca. For this purpose, fluorescently tagged Trichoderma sp. T154 and a P. minimum strain were used for scanning electron microscopy and confocal scanning laser microscopy analyses. Data showed that the Trichoderma strain is able to colonize plants up to 12 weeks post inoculation and is located in xylem, fibers, as well as in parenchymatic tissues inside the wood. The beneficial fungus reduced colonization of the esca-related pathogen colonizing the same niches. The main observed mechanism involved in biocontrol of Trichoderma against the esca pathogen was spore adhesion, niche exclusion and only few typical hypha coiling was found between Trichoderma and the pathogen. These results suggest that the Trichoderma strain has potential for reducing the colonization of Phaeoacremonium minimum and thus, an inoculation of this biological control agent can protect the plant by limiting the development of GTD, and the strain can behave as an endophyte.

Trichoderma is a genus of fungi used for the biological control of plant diseases and a large number of its bio-formulates are available in the market. However, its efficacy under field conditions remains unclear, especially for the protection of grapevine plants against Grapevine Trunk Diseases (GTDs). These diseases are caused by a complex of fungal pathogens whose main point of entrance into the affected plants is through pruning wounds. In this research, different Trichoderma native strains have been evaluated according to their ability to grow at different temperatures and their capacity to colonize pruning wounds in adverse climatic conditions. Strains from section Trichoderma have adapted to cooler conditions. On the other hand, strains from clade Harzianum/Virens grow at higher temperatures. However, differences can also be found between strains inside the same clade/section. Native strains were able to colonize more than 70% of vine pruning wounds in winter conditions. The Trichoderma strain T154 showed a significantly higher re-isolation degree from vine plants and its concentration was optimized for spraying onto vine plants. In conclusion, Trichoderma native strains are better adapted to survive in a changing environment, and they could give better protection to grapevine plants in co-evolution with each specific vineyard.

The interest in the study of microbiological interactions mediated by volatile organic compounds (VOCs) has steadily increased in the last few years. Nevertheless, most assays still rely on the use of non-specific materials. We present a new tool, the volatile organic compound chamber (VOC chamber), specifically designed to perform these experiments. The novel devices were tested using four Trichoderma strains against Fusarium oxysporum and Rhizoctonia solani. We demonstrate that VOC chambers provide higher sensitivity and selectivity between treatments and higher homogeneity of results than the traditional method. VOC chambers are also able to test both vented and non-vented conditions. We prove that ventilation plays a very important role regarding volatile interactions, up to the point that some growth-inhibitory effects observed in closed environments switch to promoting ones when tested in vented conditions. This promoting activity seems to be related to the accumulation of squalene by T. harzianum. The VOC chambers proved to be an easy, homogeneous, flexible, and repeatable method, able to better select microorganisms with high biocontrol activity and to guide the future identification of new bioactive VOCs and their role in microbial interactions.

Spain has ranked 6th on the harvested bean area and 8th in bean production in the European Union (EU). The soils of this area have mixed silt loam and sandy loam texture, with moderate clay content, neutral or acidic pH, rich in organic matter and low carbonate levels, providing beans with high water absorption capacity and better organoleptic qualities after cooking. Similar to other crops, it is attacked by some phytopathogens. Hitherto, chemical methods have been used to control these organisms. However, with the Reform of the Community Agrarian Policy in the EU, the number of authorized plant protection products has been reduced to prevail food security, as well as to be sustainable in the long term, giving priority to the non-chemical methods that use biological agents, such as Trichoderma. This study aimed to investigate the relative importance of various crop soil parameters in the adaptation of Trichoderma spp. autoclaved soils (AS) and natural soils (NS) from the Protected Geographical Indication (PGI) “Alubia La Bañeza—León” that were inoculated with Trichoderma velutinum T029 and T. harzianum T059 and incubated in a culture chamber at 25 °C for 15 days. Their development was determined by quantitative PCR. Twelve soil samples were selected and analyzed from the productive zones of Astorga, La Bañeza, La Cabrera, Esla-Campos and Páramo. Their physicochemical characteristics were different by zone, as the texture of soils ranged between sandy loam and silt loam and the pH between strongly acid and slightly alkaline, as well as the organic matter (OM) concentration between low and remarkably high. Total C and N concentrations and their ratio were between medium and high in most of the soils and the rest of the micronutrients had an acceptable concentration except for Paramo’s soil. Both Trichoderma species developed better in AS than in NS, T. velutinum T029 grew better with high levels of OM, total C, ratio C:N, P, K, Fe, and Zn than T. harzianum T059 in clay soils, with the highest values of cation exchange capacity (CEC), pH, Ca, Mg and Mn. These effects were validated by Canonical Correlation Analysis (CCA), texture, particularly clay concentration, OM, electrical conductivity (EC), and pH (physical parameters) and B and Cu (soil elements) are the main factors explaining the influence in the Trichoderma development. OM, EC, C:N ratio and Cu are the main soil characteristics that influence in T. velutinum T029 development and pH in the development of T. harzianum T059.

Fungi from the genus Trichoderma have a worldwide distribution and are commonly found in agricultural lands. Further, it has been described as a non-virulent and symbiont microorganism that can contribute to minimize the pernicious effects of pathogens. In the present work we have isolated Trichoderma spp. from bark of grapevine in different orchards in order to determine the influence of fungicide application and vine age on Trichoderma diversity in plant. An opposite correlation between the number of fungicides sprayed per campaign and the diversity of Trichoderma spp. isolates was found. Moreover, the older are grapevine plants the higher is the diversity of Trichoderma spp. isolates. The different Trichoderma strains isolated were tested against Phaeoacremonium minimum, a grapevine trunk pathogen, to evaluate their biocontrol capacity. Three Trichoderma strains shown a significant capacity to control P. minimum and were selected as candidates to be used as biological control agents. In addition, a rapid and easy method for isolating Trichoderma spp. from grapevine plants has been developed, which allowed to determine that the reduction in the amount of pesticide use, together with the preservation of old vineyards, lead to healthier agroecosystems containing higher levels of beneficial microorganisms.