Volatile-mediated inhibitory activity of Rhizobacteria as a result of multiple factors interaction: the case of Lysobacter capsici AZ78

Plant beneficial rhizobacteria may antagonize soilborne plant pathogens by producing a vast array of volatile organic compounds (VOCs). The production of these compounds depends on the medium composition used for bacterial cell growth. Accordingly, Lysobacter capsici AZ78 (AZ78) grown on a protein-rich medium was previously found to emit volatile pyrazines with toxic activity against soilborne phypathogenic fungi and oomycetes. However, the discrepancy between the quantity of pyrazines in the gaseous phase and the minimum quantity needed to achieve inhibition of plant pathogens observed, lead us to further investigate the volatile-mediated inhibitory activity of AZ78. Here, we show that, besides VOCs, AZ78 cells produce ammonia in increased amounts when a protein-rich medium is used for bacterial growth. The production of this volatile compound caused the alkalinization of the physically separated culture medium where Rhizoctonia solani was inoculated subsequently. Results achieved in this work clearly demonstrate that VOC, ammonia and the growth medium alkalinization contribute to the overall inhibitory activity of AZ78 against R. solani. Thus, our findings suggest that the volatile-mediated inhibitory activity of rhizobacteria in protein-rich substrates can be regarded as a result of multiple factors interaction, rather than exclusively VOCs productio

Ecological role of volatile organic compounds emitted by Pantoea agglomerans as interspecies and interkingdom signals

Volatile organic compounds (VOCs) play an essential role in microbe–microbe and plant–microbe interactions. We investigated the interaction between two plant growth-promoting rhizobacteria, and their interaction with tomato plants. VOCs produced by Pantoea agglomerans MVC 21 modulates the release of siderophores, the solubilisation of phosphate and potassium by Pseudomonas (Ps.) putida MVC 17. Moreover, VOCs produced by P. agglomerans MVC 21 increased lateral root density (LRD), root and shoot dry weight of tomato seedlings. Among the VOCs released by P. agglomerans MVC 21, only dimethyl disulfide (DMDS) showed effects similar to P. agglomerans MVC 21 VOCs. Because of the effects on plants and bacterial cells, we investigated how P. agglomerans MVC 21 VOCs might influence bacteria–plant interaction. Noteworthy, VOCs produced by P. agglomerans MVC 21 boosted the ability of Ps. putida MVC 17 to increase LRD and root dry weight of tomato seedlings. These results could be explained by the positive effect of DMDS and P. agglomerans MVC 21 VOCs on acid 3-indoleacetic production in Ps. putida MVC 17. Overall, our results clearly indicated that P. agglomerans MVC 21 is able to establish a beneficial interaction with Ps. putida MVC 17 and tomato plants through the emission of DMD

Risks to human and animal health related to the presence of moniliformin in food and feed

The CONTAM Panel wishes to acknowledge all European competent authorities and other stakeholders that provided occurrence data on moniliformin in food and feed, and supported the consumption data collection for the Comprehensive European Food Consumption Database. Adopted: 21 November 2017Peer reviewedPublisher PD

Volatile-mediated inhibitory activity of the biocontrol agent Lysobacter capsici AZ78 as a result of multiple factors interaction

Plant beneficial rhizobacteria are able to inhibit the growth of soilborne phytopathogenic microorganisms through the release of a relevant number of volatile compounds. Based on this, we investigated the ability of the biocontrol agent Lysobacter capsici AZ78 (AZ78) to produce volatile organic compounds (VOCs) that may contribute to its efficacy in controlling soilborne phytopathogenic microorganisms. AZ78 significantly reduced the growth of Pythium ultimum, Rhizoctonia solani and Sclerotinia minor in split Petri dish assays. The GC-MS analysis revealed that AZ78 produce 22 VOCs and most of them were putatively identified as mono- and dialkylated methoxypyrazines. Exposure to 2,5-dimethylpyrazine, 2-ethyl-3-methoxypyrazine and 2-isopropyl-3-methoxypyrazine determined a drastic reduction of Pythium ultimum, Rhizoctonia solani and Sclerotinia minor mycelium growth in split Petri dish assays. However, the discrepancy of the toxicity between the quantity of pyrazines and the AZ78 lead us to further investigate the volatile-mediated inhibitory activity of the biocontrol bacterium. Further experiments revealed the ability of AZ78 cells to produce ammonia that caused the alkalinization of the physically separated culture medium in split Petri dishes assays. Results achieved in this work clearly demonstrated that VOCs, ammonia and the alkalinization of growth medium contribute to the overall inhibitory activity of AZ78 against soilborne phytopathogenic microorganisms

Identification and functional characterization of grapevine volatile organic compounds for the sustainable control of downy mildew

Introduction Grapevine (Vitis vinifera) is one of the most widely cultivated fruit crops and is susceptible to various pathogens, such as Plasmopara viticola that causes downy mildew (Gessler et al., 2011). Wild grapevine species are resistant to P. viticola and breeding programs have introduced resistance traits to susceptible cultivars. Plant defence responses are based on different mechanisms and volatile organic compounds (VOCs) play a crucial role in the communication between plants and other organisms. Although the emission of VOCs upon P. viticola inoculation was shown in resistant grapevine genotypes (Algarra Alarcon et al., 2015), the molecular structure and functional role of these molecules in the grapevine defence was not yet investigated. The aim of this study was to identify and functionally characterize VOCs produced by resistant and susceptible grapevine genotypes in response to P. viticola in order to further develop innovative methods for the sustainable control of downy mildew. Material and methods The susceptible V. vinifera cultivar Pinot noir and four resistant genotypes (Kober 5BB, SO4, BC4 and Solaris) were grown for three months under greenhouse conditions. Plants were inoculated with a suspension of P. viticola sporangia as previously described (Perazzolli et al., 2012). Downy mildew severity was assessed at seven days after inoculation according to the OIV-452 descriptor and scores from 1 (the most susceptible) to 9 (the totally resistant) were assigned (Bellin et al., 2009). Leaf samples were collected before (T0) and six days (T1) after P. viticola inoculation and five replicates (plants) were analysed for each genotype at each time point. The complete experiment was carried out twice. Each sample was frozen in liquid nitrogen and ground to a fine powder. Leaf powder was weighed into 20 mL headspace vials and analysed by headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME/GC-MS; Weingart, et al. 2012). Eight of the identified VOCs were selected according to their emission profiles and pure compounds were tested against P. viticola by leaf disk assays. Downy mildew development was assessed on leaf disks at one, two and six days post inoculation (dpi) by aniline blue staining. Results and discussion VOC profiles measured by HS-SPME/GC-MS analysis were consistent between the two experiments. Terpenes, isoprenoids, aldehydes, alcohols, esters and heterocyclic compounds were found in both experiments in all five tested genotypes . In general, after P. viticola treatment (T1) the abundance of the detected VOCs was higher in resistant genotypes as compared with Pinot noir. Interestingly, the relative induction of VOC levels was found to be significantly higher in resistant genotypes compared to only small changes in Pinot noir. Compared to Pinot noir, treatment with P. viticola resulted in two sesquiterpenes being more abundant in all five resistant genotypes, while three other sesquiterpenes showed a higher abundance in three resistant genotypes (BC4, Kober 5BB and Solaris). At T1, Kober 5BB and Solaris showed also a higher abundance of one heterocyclic compound and one isoprenoid as compared with Pinot noir. Finally, the abundance of a C5 aldehyde was higher in Kober 5BB as compared with Pinot noir at T1. These eight pure VOCs were tested against P. viticola in liquid suspension and in air volume. The eight VOCs impaired the development of downy mildew symptoms at dosages that ranged from 0.1 to 10.0 g/L in liquid suspension. However, five of them also showed severe phytotoxic effects on leaf disks at the dosage of 10.0 g/L. Four pure VOCs (one isoprenoid, one alcohol, one C5 aldehyde and one heterocyclic compound) significantly reduced downy mildew symptoms at the dosage of 20.0 mg/L in air volume, when each VOC was applied to a filter paper disk and placed on the lid of the Petri dish. Microscope observations with aniline blue staining revealed marked morphological changes in VOC-treated leaf disks after P. viticola inoculation. The number of pathogen structures was reduced in leaf disks treated with one isoprenoid, one alcohol and one heterocyclic compound as compared to control disks at one, two and six dpi. Moreover, no P. viticola structures were visible on leaf disks treated with the C5 aldehyde. This aldehyde and one isoprenoid were also able to reduce the diameter of P. viticola sporangia. In conclusion, downy mildew increased the production of VOCs (terpenes, isoprenoid, alcohols, aldehydes and heterocyclic compounds) in resistant but not in the susceptible genotype and these molecules are associated to the activation of grapevine defence mechanisms. Moreover, VOCs of resistant genotypes have the porential to contribute to grapevine resistance and significantly reduced downy mildew symptoms on susceptible leaf disks, indicating that they can be further developed as sustainable control molecules

Characterization of the volatilome of Lysobacter capsici AZ78 and its bioactivity against soilborne plant pathogenic fungi and oomycetes

Recently, it has been shown that volatile organic compounds (VOCs) produced by soil bacterium L. capsici DSM 19286 were highly active against Phytophthora infestans in vitro, when the strain was grown in a protein rich medium [1]. In contrast, the VOC-mediated inhibitory effect was attenuated when the strain was grown in a sugar rich medium. Based on these findings, we studied the effect of medium composition on the inhibition activity of L. capsici AZ78 against plant pathogens (e.g. Rhizoctonia solani). In parallel, GC-MS was combined with dynamic headspace (DHS) extraction and thermodesorption to investigate both type and relative amount of VOCs produced by the bacterium grown in media with crescent sugar (glucose) concentrations. Generally, the emission profiles differed in the relative amount and not in the type of VOCs produced. The chemical group of pyrazines was the most abundant in the volatile profile of L. capsici AZ78 growing in the various media. We additionally conducted experiments using a setup with Petri dishes having two compartments, where we measured the VOCs profile in the one compartment when the bacterium was growing on crescent sugar concentrations in the other. We confirmed the presence of the identified VOCs, thus giving an insight into which compounds may contribute to L. capsici AZ78 bioactivity, exhibited during the pathogen inhibition assay. Currently, we are examining the inhibitory effects of the identified compounds against various plant pathogens in vitro, with the aim to understand the mechanisms of VOC-mediated microbe-microbe communications and to select bioactive VOCs for the further development of novel biopesticides

Characterization of the volatilome of Lysobacter capsici AZ78 and its bioactivity against soilborne plant pathogenic fungi and oomycetes

Antibiotics, toxins and volatile organic compounds (VOCs), are some of the plethora of secondary metabolites produced by soil bacteria. These secondary metabolites have been shown to impact on microbial interactions in the soil. Here, we studied the activity and production of VOCs emitted by Lysobacter capsici AZ78, a soil bacterium, which produces non-volatile secondary metabolites toxic against plant pathogens [1]. Recently, it has been shown that VOCs produced by L. capsici DSM 19286 grown in a protein rich medium were highly active against Phytophthora infestans in vitro [2]. In contrast, the VOC-mediated inhibitory effect was attenuated when the strain was grown in a sugar rich medium. Based on these findings, we studied the effect of medium composition on the inhibition activity of L. capsici AZ78 against plant pathogens (e.g. Rhizoctonia solani). In parallel, GC-MS was combined with dynamic headspace (DHS) extraction and thermodesorption to investigate both type and relative amount of VOCs produced by the bacterium grown in media with crescent sugar (glucose) concentrations. Generally, VOC emission profiles exhibited mainly quantitative and not qualitative differences. The chemical group of pyrazines was the most abundant in the volatile profile of L. capsici AZ78 growing in the various media. We additionally conducted experiments using a setup with Petri dishes having two compartments, where we measured the VOCs profile in the one compartment when the bacterium was growing on crescent sugar concentrations in the other. We confirmed the presence of the identified VOCs, thus giving an insight into which compounds could participate in L. capsici AZ78 bioactivity, exhibited during the pathogen inhibition assay. Currently, we are examining the inhibitory effects of the identified compounds against various plant pathogens in vitro, with the aim to understand the mechanisms of VOC-mediated microbe-microbe communications and to select bioactive VOCs for the further development of novel biopesticides

Moniliformin in Norwegian grain

Norwegian grain samples (73 oats, 75 barley, 83 wheat) from the 2000-02 growing seasons were examined for contamination with moniliformin, and the association between the fungal metabolite and the number of kernels infected with common Fusaria was investigated. Before quantification of moniliformin using ion pairing reversed-phase high-performance liquid chromatography with diode array ultraviolet light detection, all samples were extracted using acetonitrile/water (84/16) and disposable strong anion exchange columns used for clean up. The limit of detection was 40 μg k

Downy mildew symptoms on grapevines can be reduced by volatile organic compounds of resistant genotypes

Volatile organic compounds (VOCs) play a crucial role in the communication of plants with other organisms and are possible mediators of plant defence against phytopathogens. Although the role of non-volatile secondary metabolites has been largely characterised in resistant genotypes, the contribution of VOCs to grapevine defence mechanisms against downy mildew (caused by Plasmopara viticola) has not yet been investigated. In this study, more than 50 VOCs from grapevine leaves were annotated/identifed by headspace-solid-phase microextraction gas chromatography-mass spectrometry analysis. Following P. viticola inoculation, the abundance of most of these VOCs was higher in resistant (BC4, Kober 5BB, SO4 and Solaris) than in susceptible (Pinot noir) genotypes. The post-inoculation mechanism included the accumulation of 2-ethylfuran, 2-phenylethanol, β-caryophyllene, β-cyclocitral, β-selinene and trans-2-pentenal, which all demonstrated inhibitory activities against downy mildew infections in water suspensions. Moreover, the development of downy mildew symptoms was reduced on leaf disks of susceptible grapevines exposed to air treated with 2-ethylfuran, 2-phenylethanol, β-cyclocitral or trans-2-pentenal, indicating the efcacy of these VOCs against P. viticola in receiver plant tissues. Our data suggest that VOCs contribute to the defence mechanisms of resistant grapevines and that they may inhibit the development of downy mildew symptoms on both emitting and receiving tissues

Quantitation of mycotoxins in food and feed from Burkina Faso and Mozambique using a modern LCMS/MS multitoxin method

In this study an LC-MS/MS multitoxin method covering a total of 247 fungal and bacterial metabolites was applied to the analysis of different foods and feedstuffs from Burkina Faso and Mozambique. Overall, 63 metabolites were determined in 122 samples of mainly maize and groundnuts and a few samples of sorghum, millet, rice, wheat, soy, dried fruits, other processed foods and animal feeds. Aflatoxin B1 was observed more frequently in maize (Burkina Faso, 50% incidence, median = 23.6 μg/kg; Mozambique, 46% incidence, median = 69.9 μg/kg) than in groundnuts (Burkina Faso, 22% incidence, median = 10.5 μg/kg; Mozambique, 14% incidence, median = 3.4 μg/kg). Fumonisin B1 concentrations in maize were higher in Mozambique (92% incidence, median = 869 μg/kg) than in Burkina Faso (81% incidence, median = 269 μg/kg). In addition, ochratoxin A, zearalenone, deoxynivalenol, nivalenol, and other less reported mycotoxins such as citrinin, alternariol, cyclopiazonic acid, sterigmatocystin, moniliformin, beauvericin, and enniatins were detected. Up to 28 toxic fungal metabolites were quantitated in a single sample, emphasizing the great variety of mycotoxin coexposure. Most mycotoxins have not been reported before in either country