Novel culture chamber to evaluate in vitro plant-microbe volatile interactions: effects of Trichoderma harzianum volatiles on wheat plantlets

.The field of plant-microbe interactions mediated by Biogenic Volatile Organic Compounds (BVOCs) still faces several limitations due to the lack of reliable equipment. We present a novel device designed to evaluate in vitro plant-microbe volatile interactions, the plant-microbe VOC Chamber. It was tested by evaluating the effects exerted on wheat development by volatiles from three Trichoderma harzianum strains, a wild type and two genetically modified strains; one expressing the tri5 gene, which leads to the synthesis and emission of the volatile trichodiene, and the other by silencing the erg1 gene, impairing ergosterol production. The wild type and the erg1-silenced strain enhanced fresh weight and length of the aerial part, but reduced root dry weight. Interestingly, no differences were found between them. Conversely, the tri5-transformant strain reduced root and aerial growth compared to the control and the other strains. No differences were observed regarding chlorophyll fluorescence quantum yield and leaf chlorophyll content, suggesting that the released BVOCs do not interfere with photosynthesis. The plant-microbe VOC Chamber proved to be a simple and reliable method to evaluate the in vitro effects of microbial BVOCs on plant development, perfect for the screening of microorganisms with interesting volatile traits. Availability of data and materials The data that support the findings of this study are available from the corresponding authors (Antonio Encina and Samuel Álvarez-García) upon reasonable request. All microbiological strains used in this study will be made available to researchers upon reasonable request. VOC Chambers will be made available to researchers upon reasonable request, unless commercial agreements reached with third parties regarding the patent exploitation prohibit it (in which case the VOC Chambers should be available in the market).S

Volatile-mediated interactions between Trichoderma harzianum and Acanthoscelides obtectus: A novel in vitro methodology to evaluate the impact of microbial volatile compounds on dry grain storage pests

.Biological interactions mediated by Biogenic Volatile Organic Compounds (BVOCs) is a well-established field that has been researched for decades. Although extensive focus is currently given to the control of insect pests using natural molecules, the study of volatile interactions between microorganisms and insects has been largely neglected and has only begun to attract more attention in recent years. In this work, we developed a novel protocol to assess the effects of microbial BVOCs directly produced by growing microbial strains on dry grain insect pests and the seed damage they cause, using VOC Chambers to evaluate both sealed and unsealed conditions. Four Trichoderma harzianum strains were tested against Acanthoscelides obtectus, a wild type and three of its transformants. These had been previously obtained by introducing the tri5 gene and thus overproducing the volatile trichodiene, or by silencing the erg1 gene, which encodes for a squalene epoxidase, therefore reducing ergosterol levels and increasing squalene ones in the fungus. Results demonstrated that ventilation plays a key role in these interactions. All fungal strains significantly increased adult mortality in sealed conditions, while this effect was barely noticeable in unsealed ones. Nevertheless, subsequent insect emergence from bean seeds and bean damage were still significantly reduced in both conditions. The erg1 silenced strains caused significantly higher levels of adult mortality than the rest in sealed conditions and lower insect emergence in both sealed and unsealed ones. Bean damage produced by insects was lower also when exposed to BVOCs from these strains in sealed conditions. Conversely, trichodiene overproduction did not show enhanced toxicity or significant reduction of insect emergence and bean damage in the tested conditions. Therefore, T. harzianum BVOCs, especially those from erg1 silenced strains, should be further researched for their potential use in the biological control of A. obtectus infestation in dry grain storing facilities. VOC Chambers have shown themselves to be a reliable method in the screening of in vitro volatile mediated interactions between growing microbial strains and insect pests.S

Germination and Agronomic Traits of Phaseolus vulgaris L. Beans Sprayed with Trichoderma Strains and Attacked by Acanthoscelides obtectus

[EN] Acanthoscelides obtectus, one of the world’s most important post-harvest pests, attacks wild and cultivated common beans (Phaseolus vulgaris L.). Four Trichoderma strains, Trichoderma arundinaceum IBT 40,837 (=Ta37), a wild-type strain producer of trichothecene harzianum A (HA); two transformed strains of Ta37, Ta37-17.139 (∆tri17) and Ta37-23.74 (∆tri23); and T. brevicompactum IBT 40,841 (=Tb41), a wild-type strain producer of the trichothecene trichodermin, were evaluated to determine the effect of these compounds on the virulence of A. obtectus and the effect of these strains on the seed’s capacity of germination and on the agronomic traits of the plants grown from these seeds. Treatments of bean seeds with different Trichoderma strains provided varying survival rates in A. obtectus adults, so life survival of insects after Tb41 strain application was reduced to 15 days. ∆tri17 and Tb41 strains sprayed on P. vulgaris beans resulted in low weight losses (1.21 and 1.55%, respectively). In spite of the low germination percentage of beans treated with ∆tri23 strain (lower than the germination percentages of the rest of the fungal strains applied), this treatment encouraged a greater Wet Weight of Aerial Part of the plants grown from both damaged and undamaged beans. High germination rates of Ta37 and ∆tri17 strains (higher than with the rest of treatments), did not turn into a greater Wet Weight Aerial Part and Wet Weight of Root System in the future plants developed. Linear regression between the number of exit holes and the wet weight aerial part on the one hand, and between the number of exit holes and the wet weight root system on the other, showed interaction, so ∆tri23 and Tb41 strains behaved differently in comparison to their respective control treatments. The number of exit holes of beans treated with ∆tri23 or Tb41 was negatively correlated with both the wet weight aerial part and the wet weight root system in P. vulgaris plants. ∆tri23 sprayed on undamaged beans caused the greatest Wet Weight Aerial Part and wet weight root system in plants. Due to the good results obtained by ∆tri23 and Tb41 strains in this work, more studies for A. obtectus control, P. vulgaris plant growth and trichothecenes production by these strains should be explored, in order to advance in the knowledge of how these fungi could be used in the field crop, together with the application of management strategies to mitigate risks for farmers and to minimize environmental contamination

Insecticidal Properties of Ocimum basilicum and Cymbopogon winterianus against Acanthoscelides obtectus, Insect Pest of the Common Bean (Phaseolus vulgaris, L.)

[EN] The bean weevil, Acanthoscelides obtectus Say (Coleoptera: Chrysomelidae: Bruchinae), causes severe post-harvest losses in the common bean, Phaseolus vulgaris L. The control of this insect is still poor and involves the use of conventional insecticides. There is an increasing demand in the search for new active substances and products for pest control towards reduction of adverse e ects on human health and the environment. The protection of grains with alternative products, such as essential oils, is a possible alternative to meet the needs described above. Therefore, this investigation evaluated the applications of basil, Ocimum basilicum, and citronella, Cymbopogon winterianus, essential oils for A. obtectus control. These essential oils significantly reduced the bean weight losses and the number of beans damaged by A. obtectus at higher doses than 60 or 120 L/sample. The number of holes per bean did not di er between the doses of basil essential oil, not even at the dose of 60 L, while it was higher at 120 L, probably due to a lower capacity of movement of the insects treated with this dose and/or the oil’s direct or indirect e ects on the insects. Basil and citronella oils exhibited similar patterns of insecticidal activity over the insect, both directly in adult insects or indirectly over bean seeds. These essential oils a ected the development of A. obtectus since the greatest doses applied on beans decreased the emergence of the bean weevil. The results prove the insecticidal capacity of the tested essential oils and hence their potential as active substances against A. obtectus in environmentally low risk pest control strategies. Supplementary trials should be conducted under real storage conditions.SIThis work was funded by the Ministry of Science, Innovation and Universities (Government of Spain), according to the Resolution of 27 July 2018, (BOE No. 184, of July 31) through the grant awarded to Álvaro Rodríguez González (PTA2017–14403-I). The authors are grateful to the Federal University of Reconcavo de Bahia and especially to Franceli Da Silva for providing us with their knowledge about essential oils

Genetic Response of Common Bean to the Inoculation with Indigenous Fusarium Isolates

[EN] Fungal species from the genus Fusarium are important soil-borne pathogens worldwide, causing significant economic losses in diverse crops. The need to find sustainable solutions against this disease has led to the development of new strategies—for instance, the use of biocontrol agents. In this regard, non-pathogenic Fusarium isolates have demonstrated their ability to help other plants withstand subsequent pathogen attacks. In the present work, several Fusarium isolates were evaluated in climatic chambers to identify those presenting low or non-pathogenic behavior. The inoculation with a low-pathogenic isolate of the fungus did not affect the development of the plant, contrary to the results observed in plants inoculated with pathogenic isolates. The expression of defense-related genes was evaluated and compared between plants inoculated with pathogenic and low-pathogenic Fusarium isolates. Low-pathogenic isolates caused a general downregulation of several plant defense-related genes, while pathogenic ones produced an upregulation of these genes. This kind of response to low-pathogenic fungal isolates has been already described for other plant species and fungal pathogens, being related to enhanced tolerance to later pathogen attacks. The results here presented suggest that low-pathogenic F. oxysporum and F. solani isolates may have potential biocontrol activity against bean pathogens via induced and systemic responses in the plantSIThis research was funded by Junta de Castilla y León, Consejería de Educación for the project “Application of Trichoderma strains in sustainable quality bean production” (LE251P18), the grant to SA-G of the Junta de Castilla y León according to ORDER EDU/529/2017, of June 26, co-financed by the European Social Fund, and the grant to AP-A (FPU19/03650) of Ministerio de Ciencia, Innovacion y Universidades (Spain)

Organic and Conventional Bean Pesticides in Development of Autochthonous Trichoderma Strains

[EN] 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.SIThis research was funded by Junta de Castilla y León, Consejería de Educación for the project “Application of Trichoderma strains in sustainable quality bean production” (LE251P18), co-financed by the European Social Fund, and the grants to Alejandra Porteous Álvarez (FPU19/03650) of Ministerio de Ciencia, Innovación y Universidades (Spain) and to Guzmán Carro Huerga (FPU 15/04681) of Ministerio de Educación, Cultura y Deporte (Spain)

Identification of plant genes putatively involved in the perception of fungal ergosterol‐squalene

[EN] Trichoderma biocontrol strains establish a complex network of interactions with plants, in which diverse fungal molecules are involved in the recognition of these fungi as nonpathogenic organisms. These molecules act as microbial-associated molecular patterns that trigger plant responses. Previous studies have reported the importance of ergosterol produced by Trichoderma spp. for the ability of these fungi to induce plant growth and defenses. In addition, squalene, a sterol biosynthetic intermediate, seems to play an important role in these interactions. Here, we analyzed the effect of different concentrations of ergosterol and squalene on tomato (Solanum lycopersicum) growth and on the transcription level of defense- and growth-related genes. We used an RNA-seq strategy to identify several tomato genes encoding predicted pattern recognition receptor proteins or WRKY transcription factors, both of which are putatively involved in the perception and response to ergosterol and squalene. Finally, an analysis of Arabidopsis thaliana mutants lacking the genes homologous to these tomato candidates led to the identification of a WRKY40 transcription factor that negatively regulates salicylic acid-related genes and positively regulates ethylene- and jasmonate-related genes in the presence of ergosterol and squaleneSIThis work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO-AGL2015–70671-C2-2-R and MICINN-RTI2018–099600-B-I00 to S.G.), and also by the Junta de Castilla y León (Spain) (LE251P18). L. Lindo was granted a fellowship by the University of León (Spain

Failure under stress of grapevine wood: The effects of the cerambycid Xylotrechus arvicola on the biomechanics properties of Vitis vinifera

[EN] Xylotrechus arvicola is an insect pest on Vitis vinifera in the main wine-producing regions of Spain. X. arvicola larvae bore inside grapevine wood, which cause structural damages in the plants´ biomechanical properties. Grapevine wood affected and unaffected by larvae, were collected from vineyards. Compression and flexural tests were used to quantify biomechanical wood properties. Affected wood broke more quickly and endured a lower supported force than unaffected wood in both varieties and moisture states. Tempranillo was the most resistant variety on trunks, while Cabernet-Sauvignon was the most resistant variety on branches, where all infested varieties showed a lower rate of bending. Grapevine wood affected by X. arvicola larvae shows an important decrease in its resistance in both moisture states - dry and wet wood - and it is observed due to the faster break in time and a lower supported force. These damages give the affected wood greater sensitivity to external mechanical factors in the vineyards, such as strong winds, harvest weight and the vibration exerted by harvesting machines. The aspect of stress-time curves in all cases follow similar patterns, so in future studies might be possible to stablish relationships between both wet and dry samples and different infestation levels

Colonization of Vitis vinifera L. by the Endophyte Trichoderma sp. Strain T154: Biocontrol Activity Against Phaeoacremonium minimum

[EN] 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.SIThe grant awarded to GC-H (FPU15/04681) comes from the Ministry of Education, Culture, and Sport (Spain). We thank Pago de Carraovejas winery for the project “GLOBALVITI IDI- 20120746” “Solució n global para mejorar la producció n vitivinı́ cola frente al cambio climá tico basada en robó tica, tecnologı́ a IT y en estrategias biotecnoló gicas y de manejo del viñedo” (Global solution for enhancing viticulture production against: climatic change based on: robotics, IT technology, biotechnological strategies, and vineyard management) that was granted by the Centro para el Desarrollo Tecnoló gico Industrial –CDTI-. SC and MG received funding via DaFNE Project Nr. 101384 from the Austrian Federal Ministry for Sustainability and Tourism (BMNT)

The Influence of Temperature on the Growth, Sporulation, Colonization, and Survival of Trichoderma spp. in Grapevine Pruning Wounds

[EN] 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 vineyardSIThis research was supported by project GLOBALVITI ‘Solución global para mejorar la This research was funded by the Centro para el Desarrollo Tecnológico Industrial–CDTI—(Madrid, Spain) for the project GLOBALVITI project (CIEN Program) IDI-20160746, and the project LowpHwine (CIEN Program) IDI ‘Estudio de nuevos factores relacionados con el suelo, la planta y la microbiota enológica que influyen en el equilibrio de la acidez de los vinos y en su garantía de calidad y estabilidad en climas cálidos’—and the grant awarded to GC-H comes from the Ministry of Education, Culture, and Sport (SPAIN), grant number (FPU15/04681) and Bodega Pago de Carraovejas S.L.U. We thank technical assistance of Javier Saiz Gade