Antifungal activity of volatile organic compounds from essential oils against the postharvest pathogens Botrytis cinerea, Monilinia fructicola, Monilinia fructigena, and Monilinia laxa.

Gray mold and brown rot, caused respectively by Botrytis cinerea and Monilinia spp., are fungal diseases responsible for significant losses during the storage of fruit and vegetables. Nowadays, the control of postharvest diseases is shifting towards more sustainable strategies, including the use of plant secondary metabolites. In this study, the antifungal activity of Origanum vulgare, Thymus vulgaris, Thymus serpyllum, Melaleuca alternifolia, Lavandula officinalis, Lavandula hybrida, Citrus bergamia, Rosmarinus officinalis, Cinnamomum zeylanicum essential oils (EOs) in vapor phase was tested in vitro against B. cinerea, Monilinia fructicola, Monilinia fructigena, and Monilinia laxa. For the experiments, a protocol using a volatile organic compounds (VOC) chamber was designed. Results indicate a dose-dependent inhibitory activity of all the tested EOs, with O. vulgare, T. vulgaris, and T. serpyllum being the most active ones, with minimum inhibitory concentrations (MIC) of 22.73, 45.45, and 22.73 µl/L, respectively, against B. cinerea and a range between 5.64 and 22.73 µl/L against the three Monilinia spp. Overall, B. cinerea presented lower sensitivity to vapor-phase EOs than any of the Monilinia strains, except for the C. zeylanicum EO, which consistently showed higher inhibition against B. cinerea. Among the three Monilinia spp., M. fructicola was the least sensitive, while M. fructigena was the most sensitive. The use of VOC chambers proved to be a reliable protocol for the assessment of antimicrobial activities of EOs. These results suggest that the VOC emitted by the tested EOs are effective towards important decay-causing fungi, and that they could be used for the control of gray mold and brown rot in in vivo trials.

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

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.

Use of VOC Chambers to evaluate the impact of microbial volatile compounds on dry grain insect pests.

Although increasing focus is being given to the control of insect pests with natural products, the study of volatile mediated microbe-insect interactions has been largely neglected so far. Currently, no standardized method is available for the in vitro evaluation of the effects produced on insects by Volatile Organic Compounds (VOCs) directly emitted by actively growing microbial strains. This manuscript presents a reliable protocol using VOC Chambers (Álvarez-García et al., 2021) to assess these effects and specifically those exerted on dry grain insect pests. The methodology also serves to evaluate the impact of these volatile interactions on the grain damage produced by the insects. It allows to vary the level of gas exchange with the environment, thus providing different experimental conditions to emulate diverse natural ones in which these interactions may occur. Our method adapts the use of VOC Chambers for the evaluation of microbe-insect volatile interactions and the in vitro screening of microbial strains that produce potentially bioactive VOCs against insects. It allows assessing the effects of these volatile interactions on the damage produced by insect pests on stored dry grains. Sealed and unsealed setups can be used to determine the impact of ventilation and gas exchange on these interactions.

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.

Volatile Organic Compound Chamber: A Novel Technology for Microbiological Volatile Interaction Assays.

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.

Self-Inhibitory Activity of Trichoderma Soluble Metabolites and Their Antifungal Effects on Fusarium oxysporum.

Self-inhibitory processes are a common feature shared by different organisms. One of the main mechanisms involved in these interactions regarding microorganisms is the release of toxic diffusible substances into the environment. These metabolites can exert both antimicrobial effects against other organisms as well as self-inhibitory ones. The in vitro evaluation of these effects against other organisms has been widely used to identify potential biocontrol agents against phytopathogenic microorganisms. In the present study, we performed membrane assays to compare the self-inhibitory effects of soluble metabolites produced by several Trichoderma isolates and their antifungal activity against a phytopathogenic strain of Fusarium oxysporum. The results demonstrated that Trichoderma spp. present a high self-inhibitory activity in vitro, being affected in both their growth rate and the macroscopic structure of their colonies. These effects were highly similar to those exerted against F. oxysporum in the same conditions, showing no significant differences in most cases. Consequently, membrane assays may not be very informative by themselves to assess putative biocontrol capabilities. Therefore, different methods, or a combination of antifungal and self-inhibitory experiments, could be a better approach to evaluate the potential biocontrol activity of microbial strains in order to pre-select them for further in vivo trials.

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

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 plant.

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

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 effects 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 µL/sample. The number of holes per bean did not differ between the doses of basil essential oil, not even at the dose of 60 µL, while it was higher at 120 µL, probably due to a lower capacity of movement of the insects treated with this dose and/or the oil's direct or indirect effects 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 affected 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.