Irrigation Targeted to Provoke Ejection of Ascospores of Venturia inaequalis Shortens the Season for Ascospore Release and Results in Less Apple Scab.

Trials were carried out in apple orchards of Emilia-Romagna and Trentino-Alto Adige in northern Italy to investigate the effects of sprinkler irrigation on possible reduction in inoculum and subsequent disease pressure of Venturia inaequalis, the ascomycete causing apple scab. In spring, volumetric spore traps were placed above apple leaf litter containing pseudothecia with ascospores of the fungus. Pseudothecia matured more rapidly in irrigated plots, and 95% of the total number of spores trapped in a season was reached on average 164 degree days (base temperature 0°C) earlier in irrigated compared to non-irrigated plots. On average for seven location/year combinations, more than 50% of the ascospores were trapped following irrigations carried out for two hours on sunny days before a forecasted rainfall. Subsequently, a much lower number of spores were trapped on rainy days following irrigation. Field trials with scab susceptible apple cultivars were carried out in the two regions to evaluate the efficacy of sprinkler irrigation on disease. Irrigated and non-irrigated plots were either treated with different fungicide control strategies or not treated. Irrigation significantly reduced the incidence of apple scab at both sites, and the overall number of infected leaves and fruit was reduced by more than 50%. Mid-day sprinkler irrigation can significantly reduce the inoculum pressure of V. inaequalis in apple orchards. This may be a sustainable management strategy, especially in areas with extended dry periods.

Biogenic volatile organic compounds in the grapevine response to pathogens, beneficial microorganisms, resistance inducers, and abiotic factors.

The synthesis of volatile organic compounds (VOCs) in plants is triggered in response to external stimuli, and these compounds can migrate to distal tissues and neighbouring receivers. Although grapevine VOCs responsible for wine aroma and plant-insect communications are well characterized, functional properties of VOCs produced in response to phytopathogens, beneficial microorganisms, resistance inducers, and abiotic factors have been less studied. In this review, we focused on the emission patterns and potential biological functions of VOCs produced by grapevines in response to stimuli. Specific grapevine VOCs are emitted in response to the exogenous stimulus, suggesting their precise involvement in plant defence response. VOCs with inhibitory activities against pathogens and responsible for plant resistance induction are reported, and some of them can also be used as biomarkers of grapevine resistance. Likewise, VOCs produced in response to beneficial microorganisms and environmental factors are possible mediators of grapevine-microbe communications and abiotic stress tolerance. Although further functional studies may improve our knowledge, the existing literature suggests that VOCs have an underestimated potential application as pathogen inhibitors, resistance inducers against biotic or abiotic stresses, signalling molecules, membrane stabilizers, and modulators of reactive oxygen species. VOC patterns could also be used to screen for resistant traits or to monitor the plant physiological status.

Trichoderma spp. volatile organic compounds protect grapevine plants by activating defense-related processes against downy mildew.

Volatile organic compounds (VOCs) are produced by soil-borne microorganisms and play crucial roles in fungal interactions with plants and phytopathogens. Although VOCs have been characterized in Trichoderma spp., the mechanisms against phytopathogens strongly differ according to the strain and pathosystem. This study aimed at characterizing VOCs produced by three Trichoderma strains used as biofungicides and to investigate their effects against grapevine downy mildew (caused by Plasmopara viticola). A VOC-mediated reduction of downy mildew severity was found in leaf disks treated with Trichoderma asperellum T34 (T34), T. harzianum T39 (T39), and T. atroviride SC1 (SC1) and 31 compounds were detected by head space-solid phase microextraction gas chromatography-mass spectrometry. Among the Trichoderma VOCs annotated, α-farnesene, cadinene, 1,3-octadiene, 2-pentylfuran, and 6-pentyl-2H-pyran-2-one reduced downy mildew severity 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, 6-pentyl-2H-pyran-2-one activated the hypersensitive response after P. viticola inoculation, possibly to reinforce the grapevine defense reaction. These results indicate that Trichoderma VOCs can induce grapevine resistance, and these molecules could be further applied to control grapevine downy mildew.

Tissue age, orchard location and disease management influence the composition of fungal and bacterial communities present on the bark of apple trees.

Plants host microbial communities that can be affected by environmental conditions and agronomic practices. Despite the role of bark as a reservoir of plant pathogens and beneficial microorganisms, no information is available on the effects of disease management on the taxonomic composition of the bark-associated communities of apple trees. We assessed the impact of disease management strategies on fungal and bacterial communities on the bark of a scab-resistant apple cultivar in two orchard locations and for two consecutive seasons. The amplicon sequencing revealed that bark age and orchard location strongly affected fungal and bacterial diversity. Microbiota dissimilarity between orchards evolved during the growing season and showed specific temporal series for fungal and bacterial populations in old and young bark. Disease management did not induce global changes in the microbial populations across locations and seasons, but specifically affected the abundance of some taxa according to bark age, orchard location and sampling time. Therefore, the disease management applied to scab-resistant cultivars, which is based on a limited use of fungicides, partially changed the taxonomic composition of bark-associated fungal and bacterial communities, suggesting the need for a more accurate risk assessment regarding possible pathogen outbreaks.

Volatiles of Grape Inoculated with Microorganisms: Modulation of Grapevine Moth Oviposition and Field Attraction.

Semiochemicals released by plant-microbe associations are used by herbivorous insects to access and evaluate food resources and oviposition sites. Adult insects may utilize microbial-derived nutrients to prolong their lifespan, promote egg development, and offer a high nutritional substrate to their offspring. Here, we examined the behavioral role of semiochemicals from grape-microbe interactions on oviposition and field attraction of the grapevine moth Lobesia botrana (Denis & Schiffermüller). The volatile constituents released by grape inoculated with yeasts (Hanseniaspora uvarum (Niehaus), Metschnikowia pulcherrima (Pitt.) M.W. Miller, Pichia anomala, Saccharomyces cerevisiae Meyen ex E.C. Hansen, and Zygosaccharomyces rouxii (Boutroux) Yarrow), sour rot bacteria (Acetobacter aceti (Pasteur) Beijerinck and Gluconobacter oxydans (Henneberg) De Ley), and a fungal pathogen (Botrytis cinerea Pers.) all endemic of the vineyard were sampled by solid-phase microextraction and analyzed by gas-chromatography coupled with mass spectrometry. Ethanol, 3-methyl-1-butanol, and ethyl acetate were the most common volatiles released from all microbe-inoculated grapes. In addition, acetic acid was released at a substantial amount following bacteria inoculation and in a three-way inoculation with yeasts and the fungus. 2-phenylethanol, a compound reported to attract tortricid moths when used in combination with acetic acid, was found at a relatively low level in all microbial combinations as well as in the control grape. While grapes inoculated with a consortium of yeasts stimulated oviposition in comparison with uninoculated berries, the phytopathogenic fungus deterred egg-laying. Nonetheless, the highest preference to lay eggs was measured when the yeasts were co-inoculated with the fungus. The lowest preference was obtained when grapes were inoculated with sour rot bacteria and their binary co-inoculation with yeasts and the fungus. Interestingly, oviposition on berries simultaneously inoculated with all the three microbial groups was unaffected. Lures loaded with either acetic acid or 2-phenylethanol were not attractive when placed in traps as single component in vineyards, but a binary blend attracted both sexes of grapevine moth in significant numbers. Further addition of the three most common volatiles released by infected berries (ethanol, 3-methyl-1-butanol, and ethyl acetate) did not significantly increase moth catch with this binary blend. The ecological implications of the grape-microorganism and grapevine moth interaction as well as the possibility to develop a pest monitoring system based on microbial volatiles are discussed.

Dual RNA-Seq of Lysobacter capsici AZ78 - Phytophthora infestans interaction shows the implementation of attack strategies by the bacterium and unsuccessful oomycete defense responses.

Biological interactions in the microbial communities of the rhizosphere continuously shape the gene expression patterns of each individual microorganism. A dual RNA-Seq approach was applied to obtain a comprehensive overview of the molecular mechanisms activated during the interaction between the biocontrol rhizobacterium Lysobacter capsici AZ78 and the soilborne phytopathogenic oomycete Phytophthora infestans. The RNA-Seq transcriptional profile of L. capsici AZ78 was characterized by up-regulation of genes concerned in the biogenesis of type 4 pilus and lytic enzymes, involved, respectively, in host colonization and subsequent attack of the P. infestans cell wall. The activation of detoxification processes allowed L. capsici AZ78 to overcome the attempted defense processes of P. infestans. Moreover, the genes involved in antibiotic biosynthesis were up-regulated in L. capsici AZ78 and caused cell death in P. infestans, with the activation of putative apoptotic processes. The consequences of P. infestans cell death resulted in the down-regulation of primary metabolic pathways, such as carbohydrates, nucleic acids and protein metabolisms. Overall, the mechanism of action of L. capsici AZ78 was related to parasitism and predatory activities that cause the death of P. infestans.

Temperature drives the assembly of endophytic communities' seasonal succession.

Endophytic microorganisms asymptomatically colonise plant tissues. Exploring the assembly dynamics of bacterial endophytic communities is essential to understand the functioning of the plant holobiont and to optimise their possible use as biopesticides or plant biostimulants. The variation in endophytic communities in above and below-ground organs in Vitis vinifera in the field were studied. To understand the specific effect of temperature on endophytic communities, a separate experiment was set up where grapevine cuttings were grown under controlled conditions at three different temperatures. The findings revealed the succession of endophytic communities over the year. Endophytic communities of roots and stems differ in terms of composition and dynamic response to temperature. Noticeably, compositional differences during the seasons affected bacterial taxa more in stems than in roots, suggesting that roots offer a more stable and less easily perturbed environment. Correlation abundance networks showed that the presence of several taxa (including Bradyrhizobium, Burkholderia, Dyella, Mesorhizobium, Propionibacterium and Ralstonia) is linked in both the field and the greenhouse.

Transcriptomic responses of a simplified soil microcosm to a plant pathogen and its biocontrol agent reveal a complex reaction to harsh habitat.

BACKGROUND: Soil microorganisms are key determinants of soil fertility and plant health. Soil phytopathogenic fungi are one of the most important causes of crop losses worldwide. Microbial biocontrol agents have been extensively studied as alternatives for controlling phytopathogenic soil microorganisms, but molecular interactions between them have mainly been characterised in dual cultures, without taking into account the soil microbial community. We used an RNA sequencing approach to elucidate the molecular interplay of a soil microbial community in response to a plant pathogen and its biocontrol agent, in order to examine the molecular patterns activated by the microorganisms. RESULTS: A simplified soil microcosm containing 11 soil microorganisms was incubated with a plant root pathogen (Armillaria mellea) and its biocontrol agent (Trichoderma atroviride) for 24 h under controlled conditions. More than 46 million paired-end reads were obtained for each replicate and 28,309 differentially expressed genes were identified in total. Pathway analysis revealed complex adaptations of soil microorganisms to the harsh conditions of the soil matrix and to reciprocal microbial competition/cooperation relationships. Both the phytopathogen and its biocontrol agent were specifically recognised by the simplified soil microcosm: defence reaction mechanisms and neutral adaptation processes were activated in response to competitive (T. atroviride) or non-competitive (A. mellea) microorganisms, respectively. Moreover, activation of resistance mechanisms dominated in the simplified soil microcosm in the presence of both A. mellea and T. atroviride. Biocontrol processes of T. atroviride were already activated during incubation in the simplified soil microcosm, possibly to occupy niches in a competitive ecosystem, and they were not further enhanced by the introduction of A. mellea. CONCLUSIONS: This work represents an additional step towards understanding molecular interactions between plant pathogens and biocontrol agents within a soil ecosystem. Global transcriptional analysis of the simplified soil microcosm revealed complex metabolic adaptation in the soil environment and specific responses to antagonistic or neutral intruders.

Laser Microdissection of Grapevine Leaves Reveals Site-Specific Regulation of Transcriptional Response to Plasmopara viticola.

Grapevine is one of the most important fruit crops in the world, and it is highly susceptible to downy mildew caused by the biotrophic oomycete Plasmopara viticola. Gene expression profiling has been used extensively to investigate the regulation processes of grapevine-P. viticola interaction, but all studies to date have involved the use of whole leaves. However, only a small fraction of host cells is in contact with the pathogen, so highly localized transcriptional changes of infected cells may be masked by the large portion of non-infected cells when analyzing the whole leaf. In order to understand the transcriptional regulation of the plant reaction at the sites of pathogen infection, we optimized a laser microdissection protocol and analyzed the transcriptional changes in stomata cells and surrounding areas of grapevine leaves at early stages of P. viticola infection. The results indicate that the expression levels of seven P. viticola-responsive genes were greater in microdissected cells than in whole leaves, highlighting the site-specific transcriptional regulation of the host response. The gene modulation was restricted to the stomata cells and to the surrounding areas of infected tissues, indicating that the host response is mainly located at the infection sites and that short-distance signals are implicated. In addition, due to the high sensitivity of the laser microdissection technique, significant modulations of three genes that were completely masked in the whole tissue analysis were detected. The protocol validated in this study could greatly increase the sensitivity of further transcriptomic studies of the grapevine-P. viticola interaction.

Genetic Diversity and Biocontrol of Rosellinia necatrix Infecting Apple in Northern Italy.

The soilborne fungus Rosellinia necatrix is the causal agent of white root rot disease on numerous plant species, including apple, which, together with the ability to survive in soil for long periods, makes this pathogen difficult to control. To understand the origins of pathogen infestation, a survey of diseased apple orchards in the northeast of Italy was conducted and 35 isolates of R. necatrix were characterized with intersimple sequence repeat markers. High genetic heterogeneity among the collected isolates suggested multiple preexisting sources of inoculum and not movement of infected soil or plant material from a single source. Greenhouse trials confirmed that, as with some other crops, soil water content and temperature were the main factors influencing infection of apple plants, while organic fertilizers and the incorporation of apple wood residues were less important. The efficacy of Trichoderma atroviride SC1 as a biocontrol agent against R. necatrix greatly depended on the timing of application. It reduced white root rot incidence on apple seedlings only if treatment was applied at least 1 week before planting.

Fungal biodiversity in the periglacial soil of Dosdè Glacier (Valtellina, Northern Italy).

Periglacial areas are one of the least studied habitats on Earth, especially in terms of their fungal communities. In this work, both molecular and culture-dependent methods have been used to analyse the microfungi in soils sampled on the front of the East Dosdè Glacier (Valtellina, Northern Italy). Although this survey revealed a community that was rich in fungal species, a distinct group of psychrophilic microfungi has not been detected. Most of the isolated microfungi were mesophiles, which are well adapted to the sensitive climatic changes that occur in this alpine environment. A discrepancy in the results that were obtained by means of the two diagnostic approaches suggests that the used molecular methods cannot entirely replace traditional culture-dependent methods, and vice versa.

Interkingdom transfer of the acne-causing agent, Propionibacterium acnes, from human to grapevine.

Here, we report the surprising and, to our knowledge, unique example of horizontal interkingdom transfer of a human opportunistic pathogen (Propionibacterium acnes) to a crop plant (the domesticated grapevine Vitis vinifera L.). Humans, like most organisms, have established a long-lasting cohabitation with a variety of microbes, including pathogens and gut-associated bacteria. Studies which have investigated the dynamics of such associations revealed numerous cases of bacterial host switches from domestic animals to humans. Much less is, however, known about the exchange of microbial symbionts between humans and plants. Fluorescent in situ hybridization localized P. acnes in the bark, in xylem fibers, and, more interestingly, inside pith tissues. Phylogenetic and population genetic analyses suggest that the establishment of the grapevine-associated P. acnes as obligate endophyte is compatible with a recent transfer event, likely during the Neolithic, when grapevine was domesticated.

Transcriptional Reprogramming of the Mycoparasitic Fungus Ampelomyces quisqualis During the Powdery Mildew Host-Induced Germination.

Ampelomyces quisqualis is a mycoparasite of a diverse range of phytopathogenic fungi associated with the powdery mildew disease. Among them are several Erysiphaceae species with great economic impact on high-value crops such as grape. Due to its ability to parasitize and prevent the spread of powdery mildews, A. quisqualis has received considerable attention for its biocontrol potential. However, and in sharp contrast to the extensively studied biocontrol species belonging to the genus Trichoderma, little is known about the biology of A. quisqualis at the molecular and genetic levels. We present the first genome-wide transcription profiling in A. quisqualis during host-induced germination. A total of 1,536 putative genes showed significant changes in transcription during the germination of A. quisqualis. This finding denotes an extensive transcriptional reprogramming of A. quisqualis induced by the presence of the host. Several upregulated genes were predicted to encode for putative mycoparasitism-related proteins such as secreted proteases, virulence factors, and proteins related to toxin biosynthesis. Our data provide the most comprehensive sequence resource currently available for A. quisqualis in addition to offering valuable insights into the biology of A. quisqualis and its mycoparasitic lifestyle. Eventually, this may improve the biocontrol capacity of this mycoparasite.

Resilience of the natural phyllosphere microbiota of the grapevine to chemical and biological pesticides.

The phyllosphere is colonized by complex microbial communities, which are adapted to the harsh habitat. Although the role and ecology of nonpathogenic microorganisms of the phyllosphere are only partially understood, leaf microbiota could have a beneficial role in plant growth and health. Pesticides and biocontrol agents are frequently applied to grapevines, but the impact on nontarget microorganisms of the phyllosphere has been marginally considered. In this study, we investigated the effect of a chemical fungicide (penconazole) and a biological control agent (Lysobacter capsici AZ78) on the leaf microbiota of the grapevine at three locations. Amplicons of the 16S rRNA gene and of the internal transcribed spacer were sequenced for bacterial and fungal identification, respectively. Pyrosequencing analysis revealed that the richness and diversity of bacterial and fungal populations were only minimally affected by the chemical and biological treatments tested, and they mainly differed according to grapevine locations. Indigenous microbial communities of the phyllosphere are adapted to environmental and biotic factors in the areas where the grapevines are grown, and they are resilient to the treatments tested. The biocontrol properties of phyllosphere communities against downy mildew differed among grapevine locations and were not affected by treatments, suggesting that biocontrol communities could be improved with agronomic practices to enrich beneficial populations in vineyards.

Moderate warming in microcosm experiment does not affect microbial communities in temperate vineyard soils.

Changes in the soil microbial community structure can lead to dramatic changes in the soil ecosystem. Temperature, which is projected to increase with climate change, is commonly assumed to affect microbial communities, but its effects on agricultural soils are not fully understood. We collected soil samples from six vineyards characterised by a difference of about 2 °C in daily soil temperature over the year and simulated in a microcosm experiment different temperature regimes over a period of 1 year: seasonal fluctuations in soil temperature based on the average daily soil temperature measured in the field; soil temperature warming (2 °C above the normal seasonal temperatures); and constant temperatures normally registered in these temperate soils in winter (3 °C) and in summer (20 °C). Changes in the soil bacterial and fungal community structures were analysed by automated ribosomal intergenic spacer analysis (ARISA). We did not find any effect of warming on soil bacterial and fungal communities, while stable temperatures affected the fungal more than the bacterial communities, although this effect was soil dependent. The soil bacterial community exhibited soil-dependent seasonal fluctuations, while the fungal community was mainly stable. Each soil harbours different microbial communities that respond differently to seasonal temperature fluctuations; therefore, any generalization regarding the effect of climate change on soil communities should be made carefully.

Abiotic stresses affect Trichoderma harzianum T39-induced resistance to downy mildew in grapevine.

Enhancement of plant defense through the application of resistance inducers seems a promising alternative to chemical fungicides for controlling crop diseases but the efficacy can be affected by abiotic factors in the field. Plants respond to abiotic stresses with hormonal signals that may interfere with the mechanisms of induced systemic resistance (ISR) to pathogens. In this study, we exposed grapevines to heat, drought, or both to investigate the effects of abiotic stresses on grapevine resistance induced by Trichoderma harzianum T39 (T39) to downy mildew. Whereas the efficacy of T39-induced resistance was not affected by exposure to heat or drought, it was significantly reduced by combined abiotic stresses. Decrease of leaf water potential and upregulation of heat-stress markers confirmed that plants reacted to abiotic stresses. Basal expression of defense-related genes and their upregulation during T39-induced resistance were attenuated by abiotic stresses, in agreement with the reduced efficacy of T39. The evidence reported here suggests that exposure of crops to abiotic stress should be carefully considered to optimize the use of resistance inducers, especially in view of future global climate changes. Expression analysis of ISR marker genes could be helpful to identify when plants are responding to abiotic stresses, in order to optimize treatments with resistance inducers in field.

Downy mildew resistance induced by Trichoderma harzianum T39 in susceptible grapevines partially mimics transcriptional changes of resistant genotypes.

BACKGROUND: Downy mildew, caused by Plasmopara viticola, is one of the most severe diseases of grapevine and is commonly controlled by fungicide treatments. The beneficial microorganism Trichoderma harzianum T39 (T39) can induce resistance to downy mildew, although the molecular events associated with this process have not yet been elucidated in grapevine. A next generation RNA sequencing (RNA-Seq) approach was used to study global transcriptional changes associated with resistance induced by T39 in Vitis vinifera Pinot Noir leaves. The long-term aim was to develop strategies to optimize the use of this agent for downy mildew control. RESULTS: More than 14.8 million paired-end reads were obtained for each biological replicate of T39-treated and control leaf samples collected before and 24 h after P. viticola inoculation. RNA-Seq analysis resulted in the identification of 7,024 differentially expressed genes, highlighting the complex transcriptional reprogramming of grapevine leaves during resistance induction and in response to pathogen inoculation. Our data show that T39 has a dual effect: it directly modulates genes related to the microbial recognition machinery, and it enhances the expression of defence-related processes after pathogen inoculation. Whereas several genes were commonly affected by P. viticola in control and T39-treated plants, opposing modulation of genes related to responses to stress and protein metabolism was found. T39-induced resistance partially inhibited some disease-related processes and specifically activated defence responses after P. viticola inoculation, causing a significant reduction of downy mildew symptoms. CONCLUSIONS: The global transcriptional analysis revealed that defence processes known to be implicated in the reaction of resistant genotypes to downy mildew were partially activated by T39-induced resistance in susceptible grapevines. Genes identified in this work are an important source of markers for selecting novel resistance inducers and for the analysis of environmental conditions that might affect induced resistance mechanisms.

Fungal endophytic communities in grapevines (Vitis vinifera L.) respond to crop management.

We studied the distribution of fungal endophytes of grapevine (Vitis vinifera L.) plants in a subalpine area of northern Italy, where viticulture is of high economic relevance. We adopted both cultivation-based and cultivation-independent approaches to address how various anthropic and nonanthropic factors shape microbial communities. Grapevine stems were harvested from several locations considering organic and integrated pest management (IPM) and from the cultivars Merlot and Chardonnay. Cultivable fungi were isolated and identified by internal-transcribed-spacer sequence analysis, using a novel colony-PCR method, to amplify DNA from fungal specimens. The composition of fungal communities was assessed using a cultivation-independent approach, automated ribosomal intergenic spacer analysis (ARISA). Multivariate statistical analysis of both culture-dependent and culture-independent data sets was convergent and indicated that fungal endophytic communities in grapevines from organically managed farms were different from those from farms utilizing IPM. Fungal communities in plants of cv. Merlot and cv. Chardonnay overlapped when analyzed using culture-dependent approaches but could be partially resolved using ARISA fingerprinting.

Proteomic analysis of grapevine resistance induced by Trichoderma harzianum T39 reveals specific defence pathways activated against downy mildew.

Downy mildew is caused by the oomycete Plasmopara viticola and is one of the most serious diseases of grapevine. The beneficial microorganism Trichoderma harzianum T39 (T39) has previously been shown to induce plant-mediated resistance and to reduce the severity of downy mildew in susceptible grapevines. In order to better understand the cellular processes associated with T39-induced resistance, the proteomic and histochemical changes activated by T39 in grapevine were investigated before and 1 day after P. viticola inoculation. A comprehensive proteomic analysis of T39-induced resistance in grapevine was performed using an eight-plex iTRAQ protocol, resulting in the identification and quantification of a total of 800 proteins. Most of the proteins directly affected by T39 were found to be involved in signal transduction, indicating activation of a complete microbial recognition machinery. Moreover, T39-induced resistance was associated with rapid accumulation of reactive oxygen species and callose at infection sites, as well as changes in abundance of proteins involved in response to stress and redox balance, indicating an active defence response to downy mildew. On the other hand, proteins affected by P. viticola in control plants mainly decreased in abundance, possibly reflecting the establishment of a compatible interaction. Finally, the high-throughput iTRAQ protocol allowed de novo peptide sequencing, which will be used to improve annotation of the Vitis vinifera cv. Pinot Noir proteome.

Studying trophic interactions between a plant pathogen and two different antagonistic microorganisms using a 13C-labeled compound and isotope ratio mass spectrometry.

RATIONALE: The study of the interactions among microorganisms, especially between pathogens and other microorganisms, is a very useful way to identify possible biocontrol agents (BCAs). In this study we verified the capability of δ(13)C analysis using isotope ratio mass spectrometry (IRMS) to detect active parasitism or metabolic assimilation of (13)C-labeled Armillaria mellea (plant pathogen) by Trichoderma atroviride and Pseudomonas fluorescens (two BCAs). METHODS: The three microorganisms were labeled in pure-culture using a specific medium to which D-glucose (13)C was added. The δ(13)C analysis of mycelia/cells and DNA was undertaken using IRMS at different times, to study the uptake kinetics of (13)C. The mechanisms of interaction were studied by implementing dual-culture tests and measuring the δ(13)C values of the two BCAs after 29 days of contact with the labeled pathogen. RESULTS: A. mellea absorbed (13)C more slowly (plateau at 21 days) than T. atroviride and P. fluorescens (3 and 1 day, respectively) in pure-culture. The maximum δ(13)C values were higher in A. mellea and T. atroviride mycelia (8,019.9‰ and 10,383.7‰, respectively) than in P. fluorescens (953.4‰ in cells). In dual-culture the mycelia of T. atroviride which remained in direct contact with labeled A. mellea showed an increased δ(13)C value with respect to the unlabeled treatment (66.4‰ and -26.6‰, respectively), due to active interaction. Lower assimilation of (13)C was detected in P. fluorescens. CONCLUSIONS: This work demonstrates that IRMS can be used for the in-depth study of direct parasitism and interaction process between biocontrol agents and labeled pathogens, allowing the screening of potential new BCAs.