Suchergebnisse

peer-reviewed ; The large pine weevil Hylobius abietis L. is a major forestry pest in 15 European countries, where it is a threat to 3.4 million hectares of forest. A cellular and proteomic analysis of the effect of culture filtrate of three entomopathogenic fungi (EPF) species on the immune system of H. abietis was performed. Injection with Metarhizium brunneum or Beauvaria bassiana culture filtrate facilitated a significantly increased yeast cell proliferation in larvae. Larvae co-injected with either Beauvaria caledonica or B. bassiana culture filtrate and Candida albicans showed significantly increased mortality. Together these results suggest that EPF culture filtrate has the potential to modulate the insect immune system allowing a subsequent pathogen to proliferate. Injection with EPF culture filtrate was shown to alter the abundance of protease inhibitors, detoxifing enzymes, antimicrobial peptides and proteins involved in reception/detection and development in H. abietis larvae. Larvae injected with B. caledonica culture filtrate displayed significant alterations in abundance of proteins involved in cellulolytic and other metabolic processes in their haemolymph proteome. Screening EPF for their ability to modulate the insect immune response represents a means of assessing EPF for use as biocontrol agents, particularly if the goal is to use them in combination with other control agents. ; This research was funded by the Irish Government (Department of Agriculture, Food and the Marine) (10/RD/MCOP/NUIM/720) under the National Development Plan 2007–2013 and through the MU Department of Biology Contingency Fund. The Q-Exactive quantitative mass spectrometer was funded under the SFI Research Infrastructure Call 2012; Grant Number: 12/RI/2346 (3) to Prof. S. Doyle.

Resumen del trabajo presentado en la XXIV Reunión de la Sociedad Española de Biología de Plantas - XVII Congreso Hispano-Luso de Biología de Plantas, celebrado online del 7 al 9 de julio de 2021 ; Microbes synthesize a multitude of substances including carbohydrates, proteins, lipids, amino acids, hormones, volatiles, etc., which regulate plant growth and morphogenesis. Irrigation with culture filtrates (CFs) of some plant beneficial microorganisms promotes plant growth and enhances yield. However, nothing is known about the response of plants to soil application of CFs of microbial phytopathogens. In this work we explored the response of two pepper (Capsicum annuum L.) cultivars (i.e. Sweet Italian and Piquillo) and soil microbiota to CFs of the beneficial fungus (i.e. Trichoderma harzianum) and two fungal phytopathogens (i.e. Alternaria alternata and Penicilliun aurantiogriseum). We found that soil application of CFs of the three fungal species similarly enhanced root growth (40-70% increase under greenhouse conditions) and commercial fruit yield (50-112% and 20-28% increase under greenhouse and open field conditions, respectively). High-throughput amplicon sequencing of bacterial 16S and fungal ITS rRNA genes of the soil microbiota revealed that the CF treatments led to strong enrichment of the populations of the same beneficial bacterial and fungal species. Our findings show that the application of microbial CFs to the soil represents a potentially very useful strategy for increasing the yield of horticultural crops in a sustainable and environmentally friendly way ; This work was supported by the Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (Spain) (grants BIO2013-49125-C2-1- P, BIO2016-78747-P and PID2019-104685GB-100), the Government of Navarra (refs. P1044 AGROESTI, P1004 PROMEBIO and P1046 MICROBIOME), and the Ministry of Education, Youth and Sport of the Czech Republic and the ERDF project "Plants as a tool for sustainable global development" (No. CZ.02.1.01/0.0/0.0/16_019/0000827).

Plants communicate with microorganisms by exchanging chemical signals throughout the phytosphere. Such interactions are important not only for plant productivity and fitness, but also for terrestrial ecosystem functioning. It is known that beneficial microorganisms emit diffusible substances including volatile organic compounds (VOCs) that promote growth. Consistently, soil application of cell-free culture filtrates (CF) of beneficial soil and plant-associated microorganisms enhances plant growth and yield. However, how this treatment acts in plants and whether it alters the resident soil microbiota, are largely unknown. In this work we characterized the responses of pepper (Capsicum annuum L.) plants cultured under both greenhouse and open field conditions and of soil microbiota to soil application of CFs of beneficial and phytopathogenic fungi. To evaluate the contribution of VOCs occurring in the CFs to these responses, we characterized the responses of plants and of soil microbiota to application of distillates (DE) of the fungal CFs. CFs and their respective DEs contained the same potentially biogenic VOCs, and application of these extracts enhanced root growth and fruit yield, and altered the nutritional characteristics of fruits. High-throughput amplicon sequencing of bacterial 16S and fungal ITS rRNA genes of the soil microbiota revealed that the CF and DE treatments altered the microbial community compositions, and led to strong enrichment of the populations of the same beneficial bacterial and fungal taxa. Our findings show that CFs of both beneficial and phytopathogenic fungi can be used as biostimulants, and provide evidence that VOCs occurring in the fungal CFs act as mediators of the plants' responses to soil application of fungal CFs through stimulation of the beneficial soil microbiota. ; This work was supported by the Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (Spain) (grants BIO2013-49125-C2-1-P, BIO2016-78747-P, and PID2019-104685GB-100), the Government of Navarra (refs. P1044 AGROESTI, P1004 PROMEBIO, and P1046 MICROBIOME), and the project "Plants as a tool for sustainable global development" (registration number: CZ.02.1.01/0.0/0.0/16_019/0000827) within the program Research, Development and Education (OP RDE). ; Peer reviewed

Plants communicate with microorganisms by exchanging chemical signals throughout the phytosphere. Such interactions are important not only for plant productivity and fitness, but also for terrestrial ecosystem functioning. It is known that beneficial microorganisms emit diffusible substances including volatile organic compounds (VOCs) that promote growth. Consistently, soil application of cell-free culture filtrates (CF) of beneficial soil and plant-associated microorganisms enhances plant growth and yield. However, how this treatment acts in plants and whether it alters the resident soil microbiota, are largely unknown. In this work we characterized the responses of pepper (Capsicum annuum L.) plants cultured under both greenhouse and open field conditions and of soil microbiota to soil application of CFs of beneficial and phytopathogenic fungi. To evaluate the contribution of VOCs occurring in the CFs to these responses, we characterized the responses of plants and of soil microbiota to application of distillates (DE) of the fungal CFs. CFs and their respective DEs contained the same potentially biogenic VOCs, and application of these extracts enhanced root growth and fruit yield, and altered the nutritional characteristics of fruits. High-throughput amplicon sequencing of bacterial 16S and fungal ITS rRNA genes of the soil microbiota revealed that the CF and DE treatments altered the microbial community compositions, and led to strong enrichment of the populations of the same beneficial bacterial and fungal taxa. Our findings show that CFs of both beneficial and phytopathogenic fungi can be used as biostimulants, and provide evidence that VOCs occurring in the fungal CFs act as mediators of the plants' responses to soil application of fungal CFs through stimulation of the beneficial soil microbiota. ; This work was supported by the Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (Spain) (grants BIO2013-49125-C2-1-P, BIO2016-78747-P, and PID2019-104685GB-100), the Government of Navarra (refs. P1044 AGROESTI, P1004 PROMEBIO, and P1046 MICROBIOME), and the project "Plants as a tool for sustainable global development" (registration number: CZ.02.1.01/0.0/0.0/16_019/0000827) within the program Research, Development and Education (OP RDE).

Fusarium langsethiae is amongst the most recently discovered pathogens of small grains cereals. F. langsethiae is the main producer, in Europe, of T2 and HT-toxins in small grain cereals, albeit often asymptomatic; this makes its control challenging. The European Union (EU) is pushing hard on the use of biocontrol agents to minimize the use of fungicides and pesticides, which are detrimental to the environment and responsible for serious pollution of the soil and superficial water. In line with EU directives (e.g., 128/2009), here we report the use of protein fractions, purified from the culture filtrate of the basidiomycete Trametes versicolor, for controlling F. langsethiae. T. versicolor, a so-called medicinal mushroom which is applied as a co-adjuvant in oncology and other pathologies as a producer of biological response modifiers. In this study, the exo-proteome of T. versicolor proved highly efficient in inhibiting the growth of F. langsethiae and the biosynthesis of the T2 toxin. Results are promising for its future use as a sustainable product to control F. langsethiae infection in cereals under field conditions.

Fusarium langsethiae is amongst the most recently discovered pathogens of small grains cereals. F. langsethiae is the main producer, in Europe, of T2 and HT-toxins in small grain cereals, albeit often asymptomatic ; this makes its control challenging. The European Union (EU) is pushing hard on the use of biocontrol agents to minimize the use of fungicides and pesticides, which are detrimental to the environment and responsible for serious pollution of the soil and superficial water. In line with EU directives (e.g., 128/2009), here we report the use of protein fractions, purified from the culture filtrate of the basidiomycete Trametes versicolor, for controlling F. langsethiae. T. versicolor, a so-called medicinal mushroom which is applied as a co-adjuvant in oncology and other pathologies as a producer of biological response modifiers. In this study, the exo-proteome of T. versicolor proved highly efficient in inhibiting the growth of F. langsethiae and the biosynthesis of the T2 toxin. Results are promising for its future use as a sustainable product to control F. langsethiae infection in cereals under field conditions.

The symbiotic effect of acetic acid bacteria and yeast on bacterial cellulose (BC) synthesis in kombucha was explored. Firstly, the optimal culture ratio of acetic acid bacteria and yeast was optimised through single factor and orthogonal test. The results showed that when Komagataeibacter intermedius:Brettanomyces bruxellensis:Zygosaccharomyces bisporus ratio was 1:10:10, and the inoculation amounts of K. intermedius, B. bruxellensis, and Z. bisporus were 104, 105, and 105 CFU/mL, respectively, the yield of BC was the highest, and the dry basis was 5.51 g/L. It was determined that the metabolites of B. bruxellensis and Z. bisporus could promote the synthesis of BC by K. intermedius. In addition, the composition of yeast filtrate was analysed by amino acid analyser, gas chromatography-mass spectrometry (GC-MS), and high performance liquid chromatograph (HPLC). Results showed that 16 amino acids were detected in yeast filtrate, and cysteine was only detected in yeast filtrate. The increase in isoleucine before and after fermentation was the highest, which was 11.64 times that of the control group. The increase in aspartic acid and glycine were second and third, accounting for 60.00 and 41.67%, respectively. The main volatile substances were alcohols, accounting for 84.89%, of which the relative content of ethanol was the highest at 77.35%. The relative contents of 3-methyl-1-butanol and phenylethanol were also high, accounting for 4.13 and 3.14%, respectively. Tartaric, citric, and succinic acids were detected in the yeast filtrate. The chemical species did not change before and after fermentation, but the content decreased. Vitamins B2 and B6 were detected in yeast filtrate, and the species and content did not change significantly before and after fermentation. A theoretical basis for kombucha fermentation and BC synthesis was provided.

Bacterial spot of tomato, caused by Xanthomonas spp., is a common disease in tomato fields that causes significant economic losses. Due to the difficulty with control of bacterial spot by conventional methods, new techniques such as biological control and induction of resistance are gaining prominence. This study aimed to select saprobe fungi from semi-arid regions of the Brazilian Northeast for the biological control of bacterial spot of tomato. To select the best isolates to control bacterial spot, a greenhouse experiment was initially conducted. Tomato plants ('Santa Cruz Kada') were treated with filtrates of 25 saprobe fungi and inoculated three days later with Xanthomonas euvesicatoria. Filtrates of Memnoniella levispora, Periconia hispidula, Zygosporium echinosporum, and Chloridium virescens var. virescens were selected as the most effective. Filtrates and volatile compounds from these four isolates were tested for their antibacterial activity in cultures of X. euvesicatoria and in tomato plants ('Santa Cruz Kada') inoculated with X. euvesicatoria. In vitro, the addition of nonvolatile fungal metabolites into the culture medium at 5% and 50% (v/v) inhibited bacterial growth by 28.9% and 53.8%, respectively. The volatile compounds produced by C. virescens var. virescens reduced the number of colony-forming units of X. euvesicatoria by 25.9%. In vivo, all treatments reduced from 62.4 to 71.3% the area under bacterial spot progress curve, showing the same control efficacy as the commercial resistance inducer used as a positive control (acibenzolar-S-methyl). Systemicity of the fungal filtrates was confirmed in a separate experiment, where application of the treatments exclusively to the third leaf decreased the severity of the disease on the fourth leaf (except for C. virescens var. virescens). These results show that M. levispora, P. hispidula, Z. echinosporum, and C. virescens var. virescens are potential biocontrol agents against bacterial spot of tomato. Further studies are necessary to elucidate the disease control mechanisms of saprobe fungi.

Résumé. — Des bactéries alginolytiques isolées de thalles de Fucus spp. ont été cultivées en milieu liquide où elles libèrent des exo-alginases. Les filtrats de ces cultures ont été utilisés pour mettre au point une méthode viscosimétrique permettant le dosage quantitatif et spécifique des alginases. Nous avons défini une unité alginoly tique proportionnelle à la valeur du coëfficiënt angulaire de la droite représentant les variations de fluidité spécifique du milieu réactionnel en fonction de la durée de l'incubation.

In aquaculture, vaccination is one of the approaches for disease prevention and control. The aim of the present study was to determine the efficacy of a VP28 double stranded RNA (VP28 dsRNA) and recombinant VP28 protein (rVP28) administered together as an antiviral treatment against WSSV. Double-stranded RNA was produced in RNAsedeficient Escherichia coli HT115 following published methods. To determine the appropriate dose, different concentrations of dsRNA ranging between 0.2 μg and 20 μg, were either injected intramuscularly or delivered orally to the shrimp via the feed ration. Thereafter, the shrimp were challenged with WSSV either by injection (LD 50 =10 -7 dilution of the gill tissue filtrate) or bath immersion (LD 50 =10 -4 dilution of the filtrate) in glass aquaria and transferred to fiberglass tanks for daily monitoring and recording of mortalities. Results showed significant differences in survival between PBS and the 0.2, 10, and 20 μg dsRNA/shrimp doses. Time to 100 % mortality significantly differed among the treatments with the control reaching mortality earlier (day 4) while shrimp injected with 0.2 and 10 μg dsRNA succumbed to WSSV much later on days 9–12. Different frequencies of dsRNA administration were also tested. The best result obtained was a dose of 20 μg/shrimp administered 4 times over 28 days (2 times before and 2 times during challenge for a total 80 μg/shrimp). Finally, VP28 dsRNA was combined with rVP28 at ratios of 1:1, 1:2, 1:3, 3:1, and 2:1, entrapped in chitosan microparticles and delivered per os via the feed according to the dose and frequency as previously determined. Following bath exposure challenge with WSSV, the best survival obtained in trials 1 and 2 was 40 % and 43 % at 1:3 VP28 dsRNA to rVP28 ratio. ; We are indebted to the Government of Japan-Trust Fund for the grant funds (study code: FH-03-C2015T) and SEAFDEC/AQD management for the financial and moral support. We are also thankful to the Fish Health Section for assistance in various analyses.

In aquaculture, vaccination is one of the approaches for disease prevention and control. The aim of the present study was to determine the efficacy of a VP28 double stranded RNA (VP28 dsRNA) and recombinant VP28 protein (rVP28) administered together as an antiviral treatment against WSSV. Double-stranded RNA was produced in RNAsedeficient Escherichia coli HT115 following published methods. To determine the appropriate dose, different concentrations of dsRNA ranging between 0.2 μg and 20 μg, were either injected intramuscularly or delivered orally to the shrimp via the feed ration. Thereafter, the shrimp were challenged with WSSV either by injection (LD 50 =10 -7 dilution of the gill tissue filtrate) or bath immersion (LD 50 =10 -4 dilution of the filtrate) in glass aquaria and transferred to fiberglass tanks for daily monitoring and recording of mortalities. Results showed significant differences in survival between PBS and the 0.2, 10, and 20 μg dsRNA/shrimp doses. Time to 100 % mortality significantly differed among the treatments with the control reaching mortality earlier (day 4) while shrimp injected with 0.2 and 10 μg dsRNA succumbed to WSSV much later on days 9–12. Different frequencies of dsRNA administration were also tested. The best result obtained was a dose of 20 μg/shrimp administered 4 times over 28 days (2 times before and 2 times during challenge for a total 80 μg/shrimp). Finally, VP28 dsRNA was combined with rVP28 at ratios of 1:1, 1:2, 1:3, 3:1, and 2:1, entrapped in chitosan microparticles and delivered per os via the feed according to the dose and frequency as previously determined. Following bath exposure challenge with WSSV, the best survival obtained in trials 1 and 2 was 40 % and 43 % at 1:3 VP28 dsRNA to rVP28 ratio. ; We are indebted to the Government of Japan-Trust Fund for the grant funds (study code: FH-03-C2015T) and SEAFDEC/AQD management for the financial and moral support. We are also thankful to the Fish Health Section for assistance in various analyses.

Objetivou-se avaliar a eficácia in vitro do filtrado e extrato etanólico de isolados de Aspergillus terreus, na inibição da eclodibilidade (IE) e do desenvolvimento larval (IDL) de Haemonchus contortus. Para a avaliação da IE, após 72 horas de incubação com filtrado (1, 2, 3,01 e 4.02 mg/ mℓ), e extrato etanólico (17,31, 34,62, 51,93 e 69,25 mg/ mℓ), foram quantificados ovos não embrionados, larvados e larvas infectantes de primeiro estágio (L1). Utilizou-se água destilada estéril como controle negativo e 15 mg/mℓ de fosfato de levamisol como controle positivo. Para a IDL foi utilizado o método adaptado de coprocultura quantitativa. Após sete dias de incubação das fezes com diferentes concentrações dos filtrados dos fungos, as larvas infectantes foram coletadas e quantificadas, obtendo-se o número de larvas desenvolvidas por grama de fezes (LDGP). Foram utilizados como controles 15 mg/mℓ de ivermectina ou água destilada. Na IE, o extrato etanólico de Aspergillus terreus, na concentração de 69,25 mg/mℓ, apresentou eficácia de 100%. Quanto ao IDL, concentrações iguais ou superiores a 34,62 mg/ mℓ apresentaram resultados semelhantes ao controle positivo (levamisol). Em bovinos, o extrato etanólico apresentou eficiência de 100% em todos os níveis testados sobre diversos gêneros de parasitas gastrointestinais. Conclui-se que os filtrados e extratos etanólicos dos fungos avaliados neste trabalho apresentam potencial para o controle alternativo de parasitos intestinais.

Palavras-chave: Aspergillus terréus. Controle Alternativo. Ovinocultura. Bovinocultura.

Abstract
The objective of this study was to evaluate the in vitro efficacy of the filtrate and ethanol extract of Aspergillus terreus isolates in inhibiting the hatchability (IE) and larval development (IDL) of Haemonchus contortus. For the evaluation of IE, after 72 hours of incubation with filtrate (1, 2, 3.01 and 4.02 mg/ mℓ) and ethanol extract (17.31, 34.62, 51.93 and 69.25 mg/ mℓ), non-embryonic eggs, larvae and infective first stage larvae (L1) were quantified. Sterile distilled water was used as a negative control and 15 mg/mℓ of levamisole phosphate as a positive control. For IDL, the adapted method of quantitative stool culture was used. After seven days of incubation of feces with different concentrations of fungal filtrates, the infecting larvae were collected and quantified, obtaining the number of larvae developed per gram of feces (LDGP). 15 mg/mℓ of ivermectin or distilled water were used as controls. In IE, the ethanol extract of Aspergillus terreus, at a concentration of 69.25 mg/mℓ, showed 100% efficacy. As for the IDL, concentrations equal to or greater than 34.62 mg/mℓ presented results similar to the positive control (levamisol). In cattle, the ethanol extract showed 100% efficiency at all levels tested on different genera of gastrointestinal parasites. It is concluded that the ethanol filtrates and extracts of the fungi evaluated in this paper have potential for the alternative control of intestinal parasites.

Keywords: Aspergillus terreus. Alternative Control. Sheep Farming. Cattle Farming.