Sequential Inoculation Research Articles

Impact of Pure, Co-, and Sequential Fermentations with Hanseniaspora sp. and Saccharomyces cerevisiae on the Volatile Compounds of Ciders

Pure, co-, and sequential fermentations of Hanseniaspora uvarum, H. guilliermondii, and Saccharomyces cerevisiae strains were evaluated to improve the aromatic quality of ciders. In sequential fermentations, Hanseniaspora strains were used as starter, followed by S. cerevisiae inoculation succeeding one, two, and three days of fermentation. Kinetics, physicochemical parameters, and volatile compounds were assessed during 10 days of fermentation. The headspace technique was used to capture the volatile compounds from the ciders obtained in each experiment and analyzed by gas chromatography. Fermentations with pure strains of Hansenisaspora sp. showed a high population (>1010 CFU/mL) but had a low fermentation rate (2.3–3.8 CO2 g/L/d), low consumption of amino acids (20–40 mg/L) with a high residual content, high sugar consumption (80–90 g/L), and low alcohol content (<2.0% v/v). The H. uvarum strain produced a notably high ester content (245 mg/L). In the co-fermentations, H. guilliermondii with S. cerevisiae highlighted a significant production of higher alcohols, similar to that produced by S. cerevisiae alone (152–165 mg/L). In general, the maximum fermentation rate of the sequential inoculations was lower than co-fermentations but showed low residual nitrogen content (<69 mg/L) and good conversion of sugars into ethanol (4.3–5.7% v/v). The highest concentrations of volatile compounds were observed in treatments involving the two non-conventional strains: H. uvarum with S. cerevisiae inoculation after three days (564 mg/L) and H. guilliermondii after just one day (531 mg/L) of fermentation. These differences stemmed from the metabolic activity of the strains. H. uvarum was influenced by the presence of Saccharomyces, whereas H. guilliermondii did not exhibit this effect. Thus, a pure H. uvarum inoculum has the potential to produce a demi-sec cider with low alcohol content and high content of esters, contributing to a fruity aroma. In addition, ciders with sequential inoculation were the most promising for dry cider processing concerning fermentation parameters and bioaroma enrichment.

A consortium of different Saccharomyces species enhances the content of bioactive tryptophan-derived compounds in wine fermentations

In recent years, the presence of molecules derived from aromatic amino acids in wines has been increasingly demonstrated to have a significant influence on wine quality and stability. In addition, interactions between different yeast species have been observed to influence these final properties. In this study, a screening of 81 yeast strains from different environments was carried out to establish a consortium that would promote the improvement of indolic compound levels in wine. Two strains, Saccharomyces uvarum and Saccharomyces eubayanus, with robust fermentative capacity were selected to be combined with a Saccharomyces cerevisiae strain with a predisposition towards the production of indolic compounds. Fermentation dynamics were studied in pure cultures, co-inoculations and sequential inoculations, analysing strain interactions and end-of-fermentation characteristics. Fermentations showing significant interactions were further analyzed for the resulting indolic compounds and aroma profile, with the aim of observing potential interactions and synergies resulting from the combination of different strains in the final wine. Sequential inoculation of S. cerevisiae after S. uvarum or S. eubayanus was observed to increase indolic compound levels, particularly serotonin and 3-indoleacetic acid. This study is the first to demonstrate how the formation of microbial consortia can serve as a useful strategy to enhance compounds with interesting properties in wine, paving the way for future studies and combinations.

Fermentation of coffee fruit with sequential inoculation of Lactiplantibacillus plantarum and Saccharomyces cerevisiae: effect on sensory attributes and chemical composition of the beans

This study has innovative aspects related to the use of sequential inoculation technique in the coffee bean fermentation process: the inoculation of Lactiplantibacillus plantarum followed by Saccharomyces cerevisiae, in the fermentation of coffee fruit for the production of specialty natural coffees. The objective was to evaluate the effect of this technique and of the total fermentation time on the sensory attributes of the coffee beverage and on the organic acid profile, bioactive compounds, and fatty acid profile of the beans. The fermentation of coffee fruit with sequential inoculation resulted in greater acidity of the beverage and contributed to increases of up to 2 points in coffee fermented. The total fermentation time was directly related to the organic acid content, and the longer the total fermentation time was, the greater the organic acid content. The fatty acid content and bioactive compound content showed little variation among treatments.

Impact of phenylalanine on Hanseniaspora vineae aroma metabolism during wine fermentation

Hanseniaspora vineae exhibits extraordinary positive oenological characteristics contributing to the aroma and texture of wines, especially by its ability to produce great concentrations of benzenoid and phenylpropanoid compounds compared with conventional Saccharomyces yeasts. Consequently, in practice, sequential inoculation of H. vineae and Saccharomyces cerevisiae allows to improve the aromatic quality of wines. In this work, we evaluated the impact on wine aroma produced by increasing the concentration of phenylalanine, the main amino acid precursor of phenylpropanoids and benzenoids. Fermentations were carried out using a Chardonnay grape juice containing 150 mg N/L yeast assimilable nitrogen. Fermentations were performed adding 60 mg/L of phenylalanine without any supplementary addition to the juice. Musts were inoculated sequentially using three different H. vineae strains isolated from Uruguayan vineyards and, after 96 h, S. cerevisiae was inoculated to complete the process. At the end of the fermentation, wine aromas were analysed by both gas chromatography–mass spectrometry and sensory evaluation through a panel of experts. Aromas derived from aromatic amino acids were differentially produced depending on the treatments. Sensory analysis revealed more floral character and greater aromatic complexity when compared with control fermentations without phenylalanine added. Moreover, fermentations performed in synthetic must with pure H. vineae revealed that even tyrosine can be used in absence of phenylalanine, and phenylalanine is not used by this yeast for the synthesis of tyrosine derivatives.

Fermentation of coffee fruit with sequential inoculation of Lactiplantibacillus plantarum and Saccharomyces cerevisiae: Effects on volatile composition and sensory characteristics

Mixed starter cultures of lactic acid bacteria and yeasts used in the production of fermented foods, including coffee, can improve the sensory quality and food safety. The objective of this study was to evaluate the effects of fermentation of coffee with inoculation of Lactiplantibacillus plantarum followed by Saccharomyces cerevisiae and the effects of fermentation time on the aroma and flavor of the coffee beverage and on the volatile composition of the roasted coffee beans. The coffee was fermented for 48 h or 96 h after inoculation of Lactiplantibacillus plantarum followed by inoculation of Saccharomyces cerevisiae or the respective controls. The aroma and flavor of the coffee beverage fermented with sequential inoculation showed complexity, with a predominance of fruity and fermented sensory notes. Forty-seven volatile compounds were identified. In addition, the sequentially inoculated coffees had greater formation of volatiles and led to greater perception of fruity and fermented flavor and aroma.

Enhancing bioremediation of petroleum sludge-polluted soil: The impact of fungal-bacterial consortium's application strategy

Many microbial consortia have demonstrated superior capabilities in petrochemical sludge bioremediation. However, their vast potential remains underutilized due to the lack of application strategies. In this study, different fungal-bacterial consortia and inoculation strategies were evaluated in biopiles of oily-sludge-contaminated soil with a total petrochemical hydrocarbons (TPH) concentration of 124,000 mg/kg. Strategies using sequential inoculation, one dose administered at the beginning and another at day 30, of a consortium comprising either Bacillus sp. and Acremonium sp. (treatment 1) or Bacillus sp. and Phanerochaete sp. (treatment 3), resulted in higher removal efficiency compared to their corresponding batch inoculation treatments (i.e., treatments 2 & 4) with the same total inoculation dose over a period of two months. Notably, treatment 5, involving sequential inoculation of the consortium of Bacillus sp., Acremonium sp., and Phanerochaete sp., achieved an impressive 91.2 ± 1.6 % TPH removal efficiency, one of the highest reported levels for this level of contamination. Furthermore, sequential inoculation of the same consortium led to enhanced removal of hydrocarbon fractions compared to batch inoculations. The sequential inoculation approach also increased the microbial population and dehydrogenase activity in the soil more effectively than batch inoculation. These findings demonstrate that sequential inoculation allows microbial consortia to function more efficiently than batch inoculation in the bioremediation process. Consequently, the application of sequential inoculation with fungal-bacterial consortia holds potential for large-scale soil bioremediation, contributing to improved soil fertility and sustainability in an effective manner.

Effects of Fermentation with Tetragenococcus halophilus and Zygosaccharomyces rouxii on the Volatile Profiles of Soybean Protein Hydrolysates.

The effects of fermentation with lactic acid bacteria (LAB) and yeast on the aroma of samples were analyzed in this work. The volatile features of different soybean hydrolysates were investigated using both GC-MS and GC-IMS. Only 47 volatile flavor compounds (VFCs) were detected when using GC-IMS, while a combination of GC-MS and GC-IMS resulted in the identification of 150 compounds. LAB-yeast fermentation could significantly increase the diversity and concentrations of VFCs (p < 0.05), including alcohols, acids, esters, and sulfurs, while reduce the contents of aldehydes and ketones. Hierarchical clustering and orthogonal partial least squares analyses confirmed the impact of fermentation on the VFCs of the hydrolysates. Seven compounds were identified as significant compounds distinguishing the aromas of different groups. The partial least squares regression analysis of the 25 key VFCs (ROAV > 1) and sensory results revealed that the treatment groups positively correlated with aromatic, caramel, sour, overall aroma, and most of the key VFCs. In summary, fermentation effectively reduced the fatty and bean-like flavors of soybean hydrolysates, enhancing the overall flavor quality, with sequential inoculation proving to be more effective than simultaneous inoculation. These findings provided a theoretical basis for improving and assessing the flavor of soybean protein hydrolysates.

Impact of fermentation through Wickerhamomyces anomalus and Saccharomyces cerevisiae on aroma and quality of mulberry wine

SummaryWickerhamomyces anomalus D1‐4, a non‐Saccharomyces yeast known for its aroma production, offers a novel approach to improving the aroma and quality of mulberry wine. We employed various inoculation methods for fermenting mulberry wine and assessed its physicochemical, bioactive compounds, antioxidant activities, colour and aroma components. The results revealed that the addition of Wickerhamomyces anomalus D1‐4 to the fermentation led to a significant increase in the levels of alcohols, terpenes, nitrogen oxides and methyl in mulberry wine, especially, produced the characteristic isobutyl acetate and isopentyl hexanoate, and greatly increased the relative content of isopentyl acetate, isopentyl alcohol and phenylethyl alcohol. The sequential inoculation of Saccharomyces cerevisiae for 24 h followed by Wickerhamomyces anomalus D1‐4 fermented for 9 days was the best fermentation method for mulberry wine. Regarding colour, bioactive compounds and antioxidant activities, the fermentation was superior to other fermentations, and it was also able to maximise the aroma of mulberry wine and give it a strong floral and fruity aroma, especially higher ethyl hexanoate and ethyl octanoate. These findings demonstrate the unique fermentation characteristics of non‐Saccharomyces yeasts in enhancing the flavour and quality of mulberry wine.

Sequential inoculation of Torulaspora delbrueckii and Saccharomyces cerevisiae in rosé wines enhances malolactic fermentation and potentially improves colour stability

In rosé wines, colour influences consumer preferences and oenological trends. In this study, we propose an alternative approach employing Torulaspora delbrueckii (Td) as a biotechnological tool for colour modulation, coupled with its potential to enhance malolactic fermentation (MLF), necessary in certain rosé wines. Sequential alcoholic fermentations (AF) were conducted using Cabernet Sauvignon grape must, with inoculations of Torulaspora delbrueckii (Td) and Saccharomyces cerevisiae (Sc). Subsequently, different strains of Oenococcus oeni were employed to carry out MLF on the resulting rosé wines, allowing us to assess the impact of Td strains under these conditions. The formation of anthocyanins and pyranoanthocyanins depended on the specific strains of Td used, resulting in wines with varying hues, ranging from a more yellowish tone (Td + ScQ) to a bluish tone (Td + ScK1). Additionally, the performance of MLF displayed a positive synergy between Td and O. oeni, with outcomes varying depending on the strain combinations employed. In conclusion, this research highlights the potential of reducing total anthocyanins while maintaining a higher proportion of pyranoanthocyanins, offering an interesting technique for rosé wines with a lighter hue. This approach aligns well with evolving oenological trends, as certain regions prefer such rosé wine type

Modulation of the Aromatic Profile of Verdejo Wine through Sequential Inoculation of Wickerhamomyces anomalus and Saccharomyces cerevisiae

Two strains of Saccharomyces cerevisiae (Sc01 and Sc02) and one strain of Wickerhamomyces anomalus (Wa) were isolated from organic Verdejo spontaneous fermentations and used for the development of experimental winemaking. Sc01 and Sc02 represented 52.7% of the population of the Saccharomyces strains isolated throughout the fermentation process. W. anomalus appeared as the predominant species among the non-Saccharomyces yeasts. Wa turned out to be the strain of this species with the shortest lag phase and positive enzymatic activities, and it was selected for white wine production. Fermentations with unique inoculation of S. cerevisiae strains were compared with sequential inoculation with W. anomalus. The results showed that the sequential inoculations did not affect the fermentation kinetics or physicochemical characteristics of the wines compared with the unique inoculations. However, this study identified a significant impact on the aromatic profiles of the produced wines due to the sequential inoculations. This modification resulted in a similar new aromatic profile in both sequential inoculations, demonstrating common characteristics related to the contribution of W. anomalus. In general, the sequential fermentations were mainly characterized by lower levels of acetate esters and an increase in ethyl acetate levels, whereas lower levels of ethyl octanoate and ethyl dec-9-enoate were detected. Propan-1-ol and butan-1-ol showed an increase in the sequential fermentations, while 4-methylpentan-1-ol and 2-phenylethanol were found in lower concentrations. These results highlight the great influence that the presence of specific strains of native non-Saccharomyces yeasts exerts on the characteristics of elaborate wines.

Effects of simultaneous and sequential mixed fermentation of non‐Saccharomyces strains and a Saccharomyces cerevisiae strain on the fermentation process and volatile compounds of Mijiu

SummaryThe combined use of non‐Saccharomyces yeasts and Saccharomyces cerevisiae in mixed fermentation is considering as a potential way to improve the flavour characteristics of Mijiu (a traditional rice wine in China). In this study, we investigated the effects of mixed fermentation, including simultaneous and sequential inoculation fermentation, of typical non‐Saccharomyces yeast strains (Saccharomycopsis fibuligera; Candida glabrata) and an S. cerevisiae strain on the fermentation process and volatile compounds of Mijiu. The results showed that the fermentation rate of Mijiu was improved by the simultaneous inoculation fermentation of different non‐Saccharomyces yeasts and S. cerevisiae. However, the increase of volatile compounds in the simultaneous inoculation fermentation depended on non‐Saccharomyces yeast species, although the positive contribution of non‐Saccharomyces yeasts to volatile compounds of Mijiu was confirmed in their pure fermentation. The sequential inoculation fermentation significantly enhanced the content or complexity of volatile compounds of Mijiu but with low‐fermentation rate. This could be attributed to the change of the number of non‐Saccharomyces yeast cells during the mixed fermentation possibly caused by the inhibition of S. cerevisiae with high concentration on non‐Saccharomyces yeasts. Our work provided useful information on the mixed inoculation fermentation strategy combining suitable non‐Saccharomyces yeasts and S. cerevisiae for improving the fermentation rate and the aroma quality of Mijiu.

Protective and curative effect of an experimental biocomplex against Flavescence dorée phytoplasma infection and transmission by Scaphoideus titanus under laboratory conditions

Flavescence dorée (FD) is one of the most devastating diseases threatening grapevine cultivation in Europe. The causal agents are phytoplasmas, which reside in phloem vessels of infected plants and in the body of leafhopper vectors, the most important being Scaphoideus titanus. Current control mainly relies on vector management, which is often insufficient to prevent from disease outbreaks. Suitable methods to interfere with phytoplasmas in planta are constantly sought to support insecticide sprays. We tested a biocomplex containing trace elements and soluble sugars, with potential systemic spread and antimicrobial activity, with the aim to verify its capability to penetrate plant phloem and to interfere with phytoplasma survival in vector-inoculated plants, using two FD isolates (FD-C and FD-D). The penetration into the plant phloem, as well as its acquisition by phloem-feeding vectors, were tested using an iridium-marked biocomplex. Phytoplasma infection was detected by real Time PCR and compared in plants exposed to biocomplex treatment before inoculation (short-time and mid-time preventive application), and to treatment after inoculation (curative application). We also assessed if the biocomplex interferes with the vector competence of S. titanus, by conducting phytoplasma transmission trials with sequential inoculation steps, where untreated and biocomplex-treated grapevine plantlets were sequentially offered to infectious leafhoppers. Foliar treatment with the biocomplex supported the penetration of phloem tissues, and the product was successfully acquired by the experimental phytoplasma vector Euscelidius variegatus after feeding on the phloem of treated plants. When the biocomplex was applied on grapevines, it induced a significant reduction of phytoplasma infection regardless of FD isolate, both as a preventive and curative treatment, indicating an antibacterial activity in the plant. Moreover, sequential inoculation experiments showed a decrease of S. titanus inoculation rates after vector exposure to a biocomplex-treated plant, suggesting a reduction of transmission efficiency that was higher using the FD-C rather than FD-D isolate. Our results indicate the tested biocomplex as a promising tool for the creation of anti-phytoplasma formulations to support vector management in FD containment.

Effect of Sequential Inoculation of Tetragenococcus halophilus and Wickerhamomyces anomalus on the Flavour Formation of Early-Stage Moromi Fermented at a Lower Temperature.

Microbial inoculation in moromi fermentation has a great influence on the physicochemical and flavour properties of soy sauces. This work investigated the effect of inoculating Tetragenococcus halophilus and Wickerhamomyces anomalus on the flavour formation of early-stage moromi (30 days) fermented at a lower temperature (22 °C) by determining their physicochemical and aroma changes. The results showed that single yeast or LAB inoculation increased the production of amino nitrogen, lactic acid and acetic acid, as well as free amino acids and key flavour components. Particularly, the sequential inoculation of T. halophilus and W. anomalus produced more free amino acids and aromatic compounds, and there might be synergistic effects between these two strains. More characteristic soy sauce flavour compounds, such as benzaldehyde, HEMF, guaiacol and methyl maltol were detected in the sequentially inoculated moromi, and this sample showed higher scores in savoury, roasted and caramel intensities. These results confirmed that sequential inoculation of T. halophilus and W. anomalus could be a choice for the future production of moromi with good flavour and quality under a lower temperature.

Sequential inoculation of flocculent Torulaspora delbrueckii with Saccharomyces cerevisiae increases color density of Pinot Noir wines.

Pinot noir grapes require careful management in the winery to prevent loss of color density and promote aging stability. Winemaking with flocculent yeast has been shown to increase color density, which is desirable to consumers. This research explored interspecies sequential inoculation and co-flocculation of commercial yeast on Pinot noir wine color. Sedimentation rates of six non-Saccharomyces species and two Saccharomyces cerevisiae strains were assayed individually and in combination. The most flocculent pairings, Torulaspora delbrueckii BIODIVA with S. cerevisiae RC212 or VL3, were used to ferment 20 L Pinot noir must. Sequential fermentations produced wines with greater color density at 420 + 520 nm, confirmed by sensory panel. Total and monomeric anthocyanin concentrations were decreased in sequentially fermented wines, despite being the main source of red wine color. BIODIVA adsorbed more anthocyanins than S. cerevisiae, indicating a greater number of cell wall mannoproteins in flocculent yeast, that could then result in a later release of anthocyanins and enhance copigment formation in red wines.

The World's First Acne Dysbiosis-like Model of Human 3D Ex Vivo Sebaceous Gland Colonized with Cutibacterium acnes and Staphylococcus epidermidis.

Acne-prone skin is associated with dysbiosis involving Cutibacterium acnes (C. acnes) and Staphylococcus epidermidis (S. epidermidis) causing increased seborrhea in sebaceous glands (SG) and inflammation. Human primary sebocytes were cultivated using 1.106 UFC/mL C. acnes Type IA (facial acne, ATCC6919) and/or 1.105 UFC/mL S. epidermidis (unknown origin, ATCC12228) for 48 h in our SEB4GLN-optimized media without antibiotics. Bacteria and sebocytes were enumerated and assessed to determine their viability. Lipid production was imaged and quantified via Nile Red staining. SG with hair follicles were microdissected from healthy skin and cultured using 1.105 UFC/mL C. acnes Type 1A and/or 1.104 UFC/mL S. epidermidis (wild-type facial skin strain) through prior fixation and immunostaining for MC5R, C. acnes and nuclei (DAPI) via Z-stack confocal microscopy bioimaging (Leica SP5X & FIJI software, Version 2.9.0). C. acnes growth was not impacted when co-cultivated with sebocytes (2D) or SG (3D) models. Phylotype IA stimulated sebocyte lipid production, which had no impact on viability. The S. epidermidis reference strain overproliferated, inducing sebocyte mortality. For 3D SG model, culture conditions were optimized using a wild-type facial skin strain at a lower concentration, 1:10 ratio to C. acnes, reduced contact time, sequential inoculation and rinsing step. Bioimaging revealed strong C. acnes labeling in the active areas of the pilosebaceous unit. S. epidermidis formed biofilm, which was distributed across the SG via non-specific fluorescence imaging. We developed an innovative model of a sebaceous gland that mimics acne-prone skin with lipid overproduction and virulent phylotype IA C. acnes inoculation.

Sequential Inoculation of Metschnikowia pulcherrima and Saccharomyces cerevisiae as a Biotechnological Tool to Increase the Terpenes Content of Pecorino White Wines

The aim of this study was the evaluation of the effect of the sequential inoculation of Metschnikowia pulcherrima and Saccharomyces cerevisiae for the production of Pecorino wine on the winery scale. Sequential inoculation allowed for wines with interesting oenological characteristics to be obtained. In particular, these wines showed a lower ethanol content than those obtained with S. cerevisiae alone, and the presence of M. pulcherrima induced a reduction in the wine titratable and volatile acidity of about 30%. The effect of M. pulcherrima was relevant also in terms of the volatile profile of the wines. In fact, an increase in the esters content of 60% and a reduction of 53% of the acetic acid was observed. Moreover, the sequential inoculation resulted in an increase in the terpenes content of about 80%. The wines also showed a different sensory profile; in fact, the wines obtained with the sequential inoculation showed higher scores than the other wines for the following descriptors: floral, tropical fruit, citrusy, balsamic, and freshness.

Impact of the Interaction between Meloidogyne javanica and Fusarium oxysporum on the Okra Plantation

The development of diseases in cultivated crops depends on the complex interrelationship between the host, pathogen, and prevailing environmental conditions. The interaction between the root knot nematode Meloidogyne javanica and the fungus Fusarium oxysporum was studied for the okra cultivar Pusa Sawani. Sequential and simultaneous inoculation of the nematode and fungus was performed to observe the disease severity in the plants due to the individual and interactive effects of the tested nematode and fungus. The results indicated that plant growth was adversely affected in all cases where the plant was inoculated with M. javanica and F. oxysporum, compared to the uninoculated control. The study clearly showed that nematodes act as predisposing factors in the spread of secondary fungal pathogens.

Impact of Candida railenensis during fermentation on the aromatic profile of Vidal blanc icewine.

Mixed culture fermentation with non-Saccharomyces yeasts and Saccharomyces cerevisiae as multi-starters has more advantages than spontaneous fermentation, and wine products with distinctive and pleasant aromas can fulfill the diverse demands of consumers. This study was carried out to illuminate the effect of sequential inoculation of indigenous Candida railenensis and S. cerevisiae on alcoholic fermentation behavior and chemical and aromatic characteristics of Vidal blanc icewine. During the mixed culture fermentation, C. railenensis was present in the initial and middle stages but was absent after 14 days. The results of basic chemical parameters showed that the glycerol content in the mixed culture-fermented icewine was higher than that of the pure fermented icewine, but the acetic acid content was the opposite. In terms of volatile aroma compounds, C. railenensis in the mixed culture fermentation reduced some metabolites such as lower alcohols, 1-hexanol, 3-methylthiopropanol, and their unpleasant notes and increased the production of some desired volatile aroma compounds such as benzaldehyde, β-damascenone, 2-furanmethanol, and 5-methyl furfural associated with rose, honey, nut, and caramel characteristics. Furthermore, C. railenensis also changed the sensory performance of icewine by participating in the fermentation of S. cerevisiae. These findings suggest that C. railenensis with positive enological properties has the ability to be used in icewine production, which has never been reported before.

Sequential application of inoculation methods improves mycorrhization of Quercus ilex seedlings by Tuber melanosporum

The use of mycorrhized seedlings has been critical to the success of modern truffle cultivation, which nowadays supplies most European black truffles (Tuber melanosporum) to markets. Ascospore inoculation has been traditionally used to produce these seedlings, but little scientific information is publicly available on the inoculation methods applied or on the possibility of combining them. We evaluated the potential of sequential inoculation for the controlled colonization of holm oak fine roots by T. melanosporum, with two different nursery assays and a full factorial design. Three inoculation methods were sequentially applied: radicle inoculation, inoculation of the substrate in seedling trays and inoculation of the substrate in the final pot. Despite the differences in the results of the two assays, which suggest that cultivation conditions and/or the timing of nursery operations may influence the relative effectiveness of inoculation methods, the sequential application appeared as an effective and realistic alternative for commercial inoculation of holm oak seedlings with T. melanosporum. The increase in the amount of inoculum applied with each inoculation method improved the mycorrhizal colonization of seedlings, whereas separately none of the inoculation methods appeared clearly superior to the other ones. The depth distribution of truffle mycorrhizae pointed that the inoculation in the final pot was more effective than other methods in lower parts of the root system, whereas the early inoculation appeared more effective to reduce the occurrence of the opportunist ectomycorrhizal fungus Sphaerosporella brunnea.

Asymmetric interactions between barley yellow dwarf virus -PAV and wheat dwarf virus in wheat.

The deciphering of the epidemiology of a plant virus has long been focused on the study of interactions between partners of one pathosystem. However, plants are exposed to numerous viruses which lead to frequent co-infection scenarios. This can change characteristics of virus-vector-host interactions and could impact the epidemiology of viral diseases. Barley yellow dwarf virus-PAV (BYDV-PAV; species: Luteovirus pavhordei; genus Luteovirus), wheat dwarf virus (WDV; genus Mastrevirus) and their respective vectors (BYDV-PAV: e.g. Rhopalosiphum padi and WDV: Psammotettix alienus) are commonly found in cereal fields. Wheat plants co-infected with BYDV-PAV and WDV have been reported from field surveys, although epidemiological outcomes of BYDV-PAV - WDV interactions in planta have not yet been studied. Experiments were carried out to evaluate and compare, through different competition scenarios (i.e. single- and co- (simultaneous and sequential) inoculations), the efficiency of BYDV-PAV and WDV to infect, to accumulate in and to be spread between wheat plants. Moreover, the impact of competition scenarios on the biological parameters of these two viruses was evaluated at different stages of the infection and with plants at different ages at inoculation. Results showed i) that these viruses achieve their infection cycle and their plant-to-plant transmission with different efficiencies and ii) BYDV-PAV - WDV interactions lead to different phenotypes ranging from antagonism to synergism. Finally, when these two viruses share a host, the nature and strength of virus-virus interactions varied depending on the order of virus arrival, stages of the infection cycle and plant age at inoculation. Precisely, the introduction (i.e. co- and sequential inoculation) and infection process (i.e. virus accumulation) of BYDV-PAV in a wheat benefit from the presence of WDV. For the latter, the sympatry with BYDV-PAV exerts opposite pressure on parameters involved in virus introduction (i.e. benefit during sequential inoculation) and spread (i.e. lower transmission efficiency and virus accumulation in co-infected plants). In the context of increased potential exposure of crops to insect vectors, this study participates in a better understanding of the impact of BYDV-PAV and WDV co-infections on biological and ecological parameters of the diseases induced by these viruses.