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Volatile-mediated inhibitory activity of the biocontrol agent Lysobacter capsici AZ78 as a result of multiple factors interaction
No full textPlant 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 of volatile organic compounds emitted by different grapevine genotypes in response to downy mildew infection
No full textVitis vinifera is susceptible to several pathogens including Plasmopara viticola, the causal agent of downy mildew. American grapevine species are resistant or tolerant to P. viticola and breeding programs have introduced resistance traits to susceptible cultivars. Although grapevine resistance to P. viticola has been widely characterized in resistant genotypes, the possible contribute of volatile organic compounds (VOCs) was not yet investigated. The aim of this work was the characterization of VOCs produced by resistant and susceptible genotypes in response to P. viticola inoculation, in order to identify VOCs associated to grapevine resistance. The susceptible V. vinifera cultivar Pinot noir, and the resistant genotypes Kober 5BB, SO4, BC4 and Solaris were grown under greenhouse conditions and they were subsequently inoculated with P. viticola. Leaves were harvested immediately before (0 dpi) and six days (6 dpi) after inoculation, and the lower disease severity in resistant genotypes as compared with Pinot noir was confirmed. A solid-phase microextraction-gas chromatography-mass spectrometry approach (SPME/GC-MS) was used to analyze VOCs emitted by the five genotypes studied. GC-MS chromatograms showed specific VOC emission profiles of the four resistant genotypes as compared with Pinot noir at 6 dpi. VOCs specifically emitted by resistant genotypes were then selected, and pure compounds were tested against P. viticola by leaf discs assays. Particularly, three sesquiterpenes, two aldehydes and one heterocyclic compound significantly reduced downy mildew severity on Pinot noir, demonstrating that VOCs could play an important role in the resistance against downy mildew by direct toxicity against P. viticola
Identification and functional characterization of grapevine volatile organic compounds for the sustainable control of downy mildew
No full textIntroduction
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
The good, the bad and the ugly: how volatile organic compounds (VOCs) produced by Trichoderma spp. protect grapevine plants against downy mildew
No full textTrichoderma spp. are among the most widespread soil microorganisms and have been widely used as biological control agents against phytopathogens. Trichoderma spp. are known to produce volatile organic compounds (VOCs) that play crucial roles in the inter-kingdom communications and biological control mechanisms. Several studies highlighted the importance of Trichoderma spp. as an alternative strategy against grapevine pathogens, but no information is available on the possible biological control mechanisms mediated by Trichoderma VOCs against grapevine downy mildew (caused by Plasmopara viticola). The aim of this study was to characterize VOCs produced by three Trichoderma strains and to investigate their effects against grapevine downy mildew. VOCs produced by Trichoderma asperellum T34, T. harzianum T39, and T. atroviride SC1 reduced downy mildew symptoms on grapevine leaf disks. A head space–solid phase microextraction gas chromatography–mass spectrometry analysis revealed 31 VOCs in two independent experiments, including alkenes, ketones, pyrones, furanes and terpenes. Five reduced downy mildew symptoms on grapevine leaf disks. In particular, 6-pentyl-2H-pyran-2-one and 2-pentylfuran increased the accumulation of callose and enhanced the modulation of defense-related genes after P. viticola inoculation, indicating an induction of grapevine defense mechanisms. Moreover, one 6-pentyl-2H-pyran-2-one activated the hypersensitive response after P. viticola inoculation to reinforce the grapevine defense reaction. These results indicate that Trichoderma VOCs can induce grapevine resistance and these molecules will be further characterized to better clarify the mode of action against grapevine downy mildew
Identification of volatile organic compounds in different grapevine genotypes after inoculation with Plasmopara viticola
No full textThe grapevine Vitis vinifera cv Pinot noir is susceptible to several pathogens including Plasmopara viticola that is the causal agent of downy mildew [1]. Hybrids of V. berlandieri and V. riparia (SO4 and Kober 5BB) and hybrids of Muscadinia rotundifolia and V. vinifera (BC4) and others such as Solaris are resistant or tolerant to downy mildew. It has been demonstrated recently [2] that resistant in vitro hybrids SO4 and Kober 5BB emit volatile organic compounds (VOCs) in response to P. viticola infection. In particular, the most interesting class of VOCs constitutes terpenoids (mono- and sesquiterpenes) emitted by the resistant cultivars, whereas for Pinot noir no terpenes have been detected under the tested conditions.
In the present study we have used gas chromatography coupled with mass spectrometry (GC-MS) to study in more detail the chemical identity of the compounds produced by selected plants of the five genotypes Pinot noir, Kober 5BB, SO4, BC4 and Solaris. All the genotypes were cultured in the greenhouse and leaves were harvested immediately (0 dpi) and six (6 dpi) days after the inoculation with P. viticola. All samples were immediately frozen and homogenized under cooled conditions. VOCs were extracted by using solid phase microextraction (SPME) and analyzed by GC-MS. Mass spectral deconvolution and annotation / identification of volatile compounds was based on comparison of mass spectra and retention indices with reference values and performed by Metabolite Detector software [3].
Preliminary results showed increased levels compared to day zero of sesquiterpenes in resistant cultivars six days after inoculation, demonstrating that terpenes could play an important role in plant resistance against downy mildew in resistant genotypes.
[1] C. Gessler, I. Pertot, M. Perazzolli. (2011). Plasmopara viticola: a review of knowledge on downy mildew of grapevine and effective disease management. Phytopathologia Mediterranea, 50, p. 3-44
[2] A. Algarra Alarcon, V. Lazazzara, L. Cappellin, P. L. Bianchedi, R. Schuhmacher, G. Wohlfahrt, I. Pertot, F. Biasioli and M. Perazzolli. (2015). Emission of volatile sesquiterpenes and monoterpenes in grapevine genotypes following Plasmopara viticola inoculation in vitro. Journal of Mass Spectrometry, 50, p. 1013–1022
[3] K. Hiller, J. Hangebrauk, C. Jäger, J. Spura, K. Schreiber, D. Schomburg. (2009) MetaboliteDetector: comprehensive analysis tool for targeted and nontargeted GC/MS based metabolome analysis. Analytical Chemistry, 81(9), p. 3429-343
Characterization of the volatilome of Lysobacter capsici AZ78 and its bioactivity against soilborne plant pathogenic fungi and oomycetes
No full textAntibiotics, 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
Identification by HS-SPME/GC-MS and activity testing of Trichoderma spp. volatile organic compounds against grapevine downy mildew
No full textDowny mildew, caused by the biotrophic oomycete Plasmopara viticola, is one of the most destructive diseases of the grapevine [1]. Fungi of the genus Trichoderma are economically important biocontrol agents since they play a crucial role in plantgrowth promotion, mycoparasitism of plant pathogens and priming of plant defence. Trichoderma spp. also release a high diversity of volatile organic compounds (VOCs), which play a decisive role against plant pathogens [2]. However, the possible contribution of Trichoderma VOCs in antagonistic processes against grapevine downy mildew has not yet been investigated. In this work, VOC emission profiles of three Trichoderma strains belonging to T. asperellum, T. atroviride and T. harzianum were analysed using headspace-solidphase microextraction gas chromatography-mass spectrometry (HS-SPME/GC-MS). Total ion current chromatograms were processed by an open source software [3], and statistical analysis on the obtained raw data was carried out using an in-house R-script. VOCs emitted by the Trichoderma strains at specific time-points were selected, and pure compounds were tested against downy mildew by leaf disks assays on susceptible grapevines. The development of downy mildew symptoms was reduced on leaf disks exposed to air treated with two sesquiterpenes, one hydrocarbon, or one heterocyclic compound, indicating the efficacy of these VOCs against downy mildew in plant tissues. Our data suggest that VOCs emitted by the Trichoderma strains under study can effectively be detected and identified by HS-SPME/GC-MS, and can inhibit the development of downy mildew symptoms on susceptible grapevine
Identification and activity testing of volatile organic compounds (VOCs) found in different grapevine genotypes in response to downy mildew infection
No full textVitis vinifera is susceptible to several pathogens including Plasmopara viticola, the causal agent of downy mildew (1). American grapevine species are resistant or tolerant to P. viticola and breeding programs have introduced resistance traits to susceptible cultivars. Although grapevine resistance mechanisms against downy mildew have been widely characterized in resistant genotypes (1), the possible contribution of volatile organic compounds (VOCs) was not yet investigated. The aim of this work was the characterization of VOCs produced by resistant and susceptible grapevine genotypes in response to P. viticola inoculation, in order to identify VOCs associated to grapevine resistance against downy mildew. The susceptible V. vinifera cultivar Pinot noir, and the resistant genotypes Kober 5BB, SO4, BC4 and Solaris were grown under greenhouse conditions and they were subsequently inoculated with P. viticola. Leaves were harvested immediately before (T0) and six days (T1) after inoculation, and the lower disease severity in resistant genotypes as compared with Pinot noir was confirmed. A headspace-solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME/GC-MS) approach was used to analyze VOCs from the five studied genotypes. GC-MS chromatograms showed specific VOC emission profiles of the four resistant genotypes as compared with Pinot noir at T1. VOCs specifically found in resistant genotypes were selected, and pure compounds were tested against P. viticola sporangia by leaf disk assays. Particularly, four sesquiterpenes, one C5 aldehyde, one terpenoid, one alcohol and one heterocyclic compound were tested in liquid suspension with P. viticola sporangia and significantly reduced downy mildew symptoms on Pinot noir leaf disks. Moreover, four of these VOCs were tested in air volume and displayed significant reduction of downy mildew symptoms demonstrating that VOCs could play an important role in the resistance against downy mildew by direct toxicity against P. viticola sporangia
Identification of Trichoderma spp. volatile organic compounds (VOCs) by HS-SPME/GC-MS analysis
No full textIntroduction
Trichoderma spp. are among the most widespread soil microorganisms and have been widely used as biological control agents against numerous phytopathogens [1]. Trichoderma biological control mechanisms are based on the production of antimicrobial substances, lytic enzymes, competition with other microorganisms for nutrients and/or space, mycoparasitism, and plant resistance induction [2]. Moreover, Trichoderma spp. are known to produce numerous volatile organic compounds (VOCs) [3] that play crucial roles in the inter-kingdom communications and biological control mechanisms [4]. Three Trichoderma strains, such as T. asperellum T34 (T34), T. atroviride SC1 (SC1), T. harzianum T39 (T39) are well documented for their ability to cope with a broad spectrum of phytopathogens and are commercialised as biofungicides. Several studies highlighted the importance of Trichoderma spp. as an alternative strategy against grapevine pathogens [5], but no information is available on the possible biological control mechanism mediated by Trichoderma VOCs against grapevine downy mildew (caused by Plasmopara viticola). The aim of this study was to identify VOCs produced by T34, SC1 and T39 using headspace-solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME/GC-MS) analysis.
Materials and Methods
Volatile-mediated effects of Trichoderma strains were assessed on grapevine leaf disks incubated with uninoculated potato dextrose agar (PDA) Petri dishes (Control) and dishes with PDA-grown T34, SC1 or T39 colonies without contact with leaf tissues. Leaf disks were inoculated with a suspension of P. viticola sporangia and downy mildew severity was assessed at six days post inoculation (dpi).
For headspace analysis of Trichoderma spp. VOCs, 20 μL of the conidial suspension of the respective Trichoderma strain were inoculated on PDA poured into sterile 20 mL-HS vials. VOCs produced by Trichoderma strains were measured using HS-SPME/GC-MS analysis at 48 or 72 h of incubation at 25°C. For compound identification, deconvoluted mass spectra were compared with an in-house library obtained with authentic reference standards. Compound annotation was achieved imposing a relative deviation of retention index (RI) value lower than 2% from the reference value published in the NIST14 database, and according to the mass spectrum similarity score set higher than 70%. Two independent experiments were carried out. Five of the identified VOCs were selected according to their emission profiles and pure compounds were tested against P. viticola by leaf disk assay.
Results
VOCs produced by T34, SC1 or T39 reduced downy mildew symptoms on grapevine leaf disks. HS-SPME/GC-MS analysis revealed a total of 26 and 21 Trichoderma VOCs found in the first and second experiments, respectively. Trichoderma VOCs included alkenes, ketones, pyrones, furanes and terpenes. Terpenes were successfully identified using manual inspection of extracted ion current (EIC) chromatograms at m/z 136 for monoterpenes, m/z 202 and 204 for sesquiterpenes, and m/z 272 for diterpenes. VOC profiles differed according to the Trichoderma strains and T39 was the highest producer of VOCs and terpenes compared to T34 and SC1. Five metabolite groups were identified according to changes in abundance among the three Trichoderma strains: VOCs with consistent changes in both experiments at both (Group 1), or at one time point (Group 2); VOCs with different changes in abundance in both experiments (Group 3); VOCs detected only in the first (Group 4) or second (Group 5) experiment. Five VOCs (two sesquiterpenes, one alkene, one furan and one pyrone) were tested against P. viticola and each VOC was applied to a filter paper disk on the lid of the Petri dish without contact with leaf tissues. Three VOCs slightly reduced the development of downy mildew symptoms at dosages of 50 mg/L in air volume. Two pure VOCs significantly reduced downy mildew symptoms at the dosage of 10 mg/L in air volume.
Discussion/Conclusions
HS-SPME/GC-MS analysis is a powerful tool to detect and identify Trichoderma VOCs. VOC profiles differed in the three Trichoderma strains and T39 was the highest producer of terpenes, followed by T34 and SC1, indicating genotypic determinants of VOC production. Five VOCs (two sesquiterpenes, one alkene, one furan and one pyrone) reduced downy mildew symptoms on grapevine leaf disks. Preliminary results suggest that two most effective VOCs enhanced the grapevine defence responses against P. viticola. These results indicate that Trichoderma VOCs are promising molecules that could be further developed to naturally control grapevine downy mildew. Further transcriptomic and metabolomic studies are required to shed light on the grapevine defence mechanisms activated by Trichoderma VOCs against downy mildew
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