Analysis of the effect of Bacillus velezensis culture filtrate on the growth and proteome of Cladobotryum mycophilum

peer-reviewedCladobotryum mycophilum, the causative agent of cobweb disease on Agaricus bisporus results in significant crop losses for mushroom growers worldwide. Cobweb disease is treated through strict hygiene control methods and the application of chemical fungicides but an increase in fungicide resistant Cladobotryum strains has resulted in a need to develop alternative biocontrol treatment methods. The aim of the work presented here was to evaluate the response of C. mycophilum to a Bacillus velezensis isolate to assess its potential as a novel biocontrol agent. Exposure of 48 hr C. mycophilum cultures to 25% v/v 96 hr B. velezensis culture filtrate resulted in a 57% reduction in biomass (P < 0.0002), a disruption in hyphal structure and morphology, and the appearance of aurofusarin, a secondary metabolite which is a known indicator of oxidative stress, in culture medium. Proteomic analysis of B. velezensis culture filtrate revealed the presence of peptidase 8 (subtilisin), peptide deformylase and probable cytosol aminopeptidase which are known to induce catalytic activity. Characterisation of the proteomic response of C. mycophilum following exposure to B. velezensis culture filtrate revealed an increase in the abundance of a variety of proteins associated with stress response (ISWI chromatin-remodelling complex ATPase ISW2 (+24 fold), carboxypeptidase Y precursor (+3 fold) and calmodulin (+2 fold). There was also a decrease in the abundance of proteins associated with transcription (40 S ribosomal protein S30 (−26 fold), 40 S ribosomal protein S21 (−3 fold) and carbohydrate metabolism (l-xylulose reductase (−10 fold). The results presented here indicate that B. velezensis culture filtrate is capable of inhibiting the growth of C. mycophilum and inducing a stress response, thus indicating its potential to control this important pathogen of mushrooms

Analysis of the effect of Bacillus velezensis culture filtrate on the growth and proteome of Cladobotryum mycophilum

Cladobotryum mycophilum, the causative agent of cobweb disease on Agaricus bisporus results in significant crop losses for mushroom growers worldwide. Cobweb disease is treated through strict hygiene control methods and the application of chemical fungicides but an increase in fungicide resistant Cladobotryum strains has resulted in a need to develop alternative biocontrol treatment methods. The aim of the work presented here was to evaluate the response of C. mycophilum to a Bacillus velezensis isolate to assess its potential as a novel biocontrol agent. Exposure of 48 hr C. mycophilum cultures to 25% v/v 96 hr B. velezensis culture filtrate resulted in a 57% reduction in biomass (P < 0.0002), a disruption in hyphal structure and morphology, and the appearance of aurofusarin, a secondary metabolite which is a known indicator of oxidative stress, in culture medium. Proteomic analysis of B. velezensis culture filtrate revealed the presence of peptidase 8 (subtilisin), peptide deformylase and probable cytosol aminopeptidase which are known to induce catalytic activity. Characterisation of the proteomic response of C. mycophilum following exposure to B. velezensis culture filtrate revealed an increase in the abundance of a variety of proteins associated with stress response (ISWI chromatin-remodelling complex ATPase ISW2 (+24 fold), carboxypeptidase Y precursor (+3 fold) and calmodulin (+2 fold). There was also a decrease in the abundance of proteins associated with transcription (40 S ribosomal protein S30 (−26 fold), 40 S ribosomal protein S21 (−3 fold) and carbohydrate metabolism (l-xylulose reductase (−10 fold). The results presented here indicate that B. velezensis culture filtrate is capable of inhibiting the growth of C. mycophilum and inducing a stress response, thus indicating its potential to control this important pathogen of mushrooms

Characterising the proteomic response of mushroom pathogen Lecanicillium fungicola to Bacillus velezensis QST 713 and Kos biocontrol agent

The fungal pathogen Lecanicillium fungicola causes dry bubble disease in Agaricus bisporus cultivation and affected mushrooms significantly reduce the yield and revenue for mushroom growers. Biocontrol agents may represent an alternative and more environmentally friendly treatment option to help control dry bubble on mushroom farms. Serenade ® is a commercially available biocontrol product used for disease treatment in plant crops. In this work, the in vitro response of L. fungicola to the bacterial strain active in Serenade, Bacillus velezensis (QST 713) and a newly isolated B. velezensis strain (Kos) was assessed. B. velezensis (QST713 and Kos) both produced zones of inhibition on plate cultures of L. fungicola, reduced the mycelium growth in liquid cultures and damaged the morphology and structure of L. fungicola hyphae. The proteomic response of the pathogen against these biocontrol strains was also investigated. Proteins involved in growth and translation such as 60S ribosomal protein L21-A (−32- fold) and 40S ribosomal protein S30 (−17-fold) were reduced in abundance in B. velezensis QST 713 treated samples, while proteins involved in a stress response were increased (norsolorinic acid reductase B (47-fold), isocitrate lyase (11-fold) and isovaleryl-CoA dehydrogenase (8-fold). L. fungicola was found to have a similar proteomic response when exposed to B. velezensis (Kos). This work provides information on the response of L. fungicola to B. velezensis (QST 713) and indicates the potential of B. velezensis Kos as a novel biocontrol agent

Effects of two neuromuscular training programs on running biomechanics with load carriage: a study protocol for a randomised controlled trial

Background In recent years, athletes have ventured into ultra-endurance and adventure racing events, which tests their ability to race, navigate, and survive. These events often require race participants to carry some form of load, to bear equipment for navigation and survival purposes. Previous studies have reported specific alterations in biomechanics when running with load which potentially influence running performance and injury risk. We hypothesize that a biomechanically informed neuromuscular training program would optimize running mechanics during load carriage to a greater extent than a generic strength training program. Methods This will be a two group, parallel randomized controlled trial design, with single assessor blinding. Thirty healthy runners will be recruited to participate in a six weeks neuromuscular training program. Participants will be randomized into either a generic training group, or a biomechanically informed training group. Primary outcomes include self-determined running velocity with a 20 % body weight load, jump power, hopping leg stiffness, knee extensor and triceps-surae strength. Secondary outcomes include running kinetics and kinematics. Assessments will occur at baseline and post-training. Discussion To our knowledge, no training programs are available that specifically targets a runner’s ability to carry load while running. This will provide sport scientists and coaches with a foundation to base their exercise prescription on

Biomass offsets little or none of permafrost carbon release from soils, streams, and wildfire : an expert assessment

As the permafrost region warms, its large organic carbon pool will be increasingly vulnerable to decomposition, combustion, and hydrologic export. Models predict that some portion of this release will be offset by increased production of Arctic and boreal biomass; however, the lack of robust estimates of net carbon balance increases the risk of further overshooting international emissions targets. Precise empirical or model-based assessments of the critical factors driving carbon balance are unlikely in the near future, so to address this gap, we present estimates from 98 permafrost-region experts of the response of biomass, wildfire, and hydrologic carbon flux to climate change. Results suggest that contrary to model projections, total permafrost-region biomass could decrease due to water stress and disturbance, factors that are not adequately incorporated in current models. Assessments indicate that end-of-the-century organic carbon release from Arctic rivers and collapsing coastlines could increase by 75% while carbon loss via burning could increase four-fold. Experts identified water balance, shifts in vegetation community, and permafrost degradation as the key sources of uncertainty in predicting future system response. In combination with previous findings, results suggest the permafrost region will become a carbon source to the atmosphere by 2100 regardless of warming scenario but that 65%-85% of permafrost carbon release can still be avoided if human emissions are actively reduced.Peer reviewe

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Building advisory relationships with farmers to foster innovation

European Seminar of Extension and Education, Acireale, Sicily, Italy, 18-21 June, 2019Increasing attention is being given to evaluating the impact of advisory services in terms of their effectiveness in providing farmers with knowledge and networks for innovation as well as understanding the factors that influence this effectiveness (Prager et al, 2017). The demand and uptake of advisory services is one factor and Klerkx et al (2017) comment on the variation in farmers’ demand and the influences of variables such as farm size, asset status and education as well as stability or turbulence in the regulatory environment. Some research has started to look at the quality of the service provided by advisors and debates in the literature have paid some attention to the new skills and profiles to be developed by agricultural advisors (Faure et al, 2012) including the importance of credibility and relational trust in the advisor - farmer relationship (Sutherland et al, 2013). Despite general agreement on the importance of social expertise, there are still wide differences in the conceptual understanding of the advisory relationship and how trust and confidence work to provide opportunities for information acquisition and knowledge exchange. Ingram (2008) has shown that farmers can be proactive or reactive in their relationship with advisors and how this relationship may be steered by either party or be more equal. The relationship between advisors and the farmers with whom they work is often one of the most influential in the transfer of knowledge and learning (Kuehne et al., 2015) yet it remains something of an enigma, often more art than science. Despite the diversity in public and private advisory service providers, there is increasing attention being given to the professional formation and continuing professional development of extension professionals (Gorman, 2018). The ProAKIS project called for the introduction and development of certification and accreditation schemes with could establish or strengthen the profession of agricultural advisor with curricula that include methodological emphasis as well as technical know-how. CECRA (European Certificate for Consultants in Rural Areas) is an example of an accreditation scheme for advisors that is focusing on such communication and methodological competencies.Teagas

Detection of Trichoderma aggressivum in bulk phase III substrate and the effect of T. aggressivum inoculum, supplementation and substrate-mixing on Agaricus bisporus yields

Trichoderma aggressivum is an aggressive contaminant mould in the cultivation of Agaricus bisporus leading to severe reductions in mushroom yields. Production of fully colonised A. bisporus substrate in Europe is commonly carried out in large tunnels (Phase III), after which the substrate undergoes several bulk handling (mixing) operations before ending up on shelves in mushroom growing facilities. The work presented here studied the effect of Trichoderma aggressivum inoculum, substrate mixing and supplementation on Agaricus bisporus yields and evaluated four methods to detect T. aggressivum in bulk handled substrate. Inoculum dilution level was shown to correlate well with mushroom yield (P < 0.0001) with reductions of 2–6 % at the most dilute level (10−4) and 60–100 % at the most concentrated level (10−1), depending on the experiment. Supplementation, with or without T. aggressivum, had no significant effect on mushroom yield (P ≥ 0.85) but a high degree of substrate mixing was shown to significantly increase (P < 0.0001) T. aggressivum-associated crop losses. Four T. aggressivum detection methods were evaluated and a quantitative polymerase chain reaction (qPCR) method gave the most consistent and least variable results. Cycle threshold (CT) values ranged from 24 to 40, depending on the experiment and the inoculum dilution level, and false negatives (CT = 40) were reported on one occasion with the most dilute samples. The results indicate that Phase III mushroom substrate is vulnerable to infection by T. aggressivum when the fully colonised substrate is broken up and mixed during bulk handling operations, identifying a previously unidentified risk for Phase III substrate producers