Jessica Vallance

Influence of Pythium oligandrum biocontrol on fungal and oomycete population dynamics in the rhizosphere.

ABSTRACT Fungal and oomycete populations and their dynamics were investigated following the introduction of the biocontrol agent Pythium oligandrum into the rhizosphere of tomato plants grown in soilless culture. Three strains of P. oligandrum were selected on the basis of their ability to form oospores (resting structures) and to produce tryptamine (an auxin-like compound) and oligandrin (a glycoprotein elicitor). Real-time PCR and plate counting demonstrated the persistence of large amounts of the antagonistic oomycete in the rhizosphere throughout the cropping season (April to September). Inter-simple-sequence-repeat analysis of the P. oligandrum strains collected from root samples at the end of the cropping season showed that among the three strains used for inoculation, the one producing the smallest amount of oospores was detected at 90%. Single-strand conformational polymorphism analysis revealed increases in the number of members and the complexity of the fungal community over time. There were no significant differences between the microbial ecosystems inoculated with P. oligandrum and those that were not treated, except for a reduction of Pythium dissotocum (ubiquitous tomato root minor pathogen) populations in inoculated systems during the last 3 months of culture. These findings raise interesting issues concerning the use of P. oligandrum strains producing elicitor and auxin molecules for plant protection and the development of biocontrol.

Biological control of plant pathogens: advantages and limitations seen through the case study of Pythium oligandrum

The management of certain plant beneficial microorganisms [biological control agents (BCAs)] seems to be a promising and environmental friendly method to control plant pathogens. However, applications are still limited because of the lack of consistency of BCAs when they are applied in the field. In the present paper, the advantages and limitations of BCAs are seen through the example of Pythium oligandrum, an oomycete that has received much attention in the last decade. The biological control exerted by P. oligandrum is the result of a complex process, which includes direct effects through the control of pathogens and/or indirect effects mediated by P. oligandrum, i.e. induction of resistance and growth promotion. P. oligandrum antagonism is a multifaceted and target fungus-dependent process. Interestingly, it does not seem to disrupt microflora biodiversity on the roots. P. oligandrum has an atypical relationship with the plant because it rapidly penetrates into the root tissues but it cannot stay alive in planta. After root colonisation, because of the elicitation by P. oligandrum of the plant-defence system, plants are protected from a range of pathogens. The management of BCAs, here P. oligandrum, is discussed with regard to its interactions with the incredibly complex agrosystems.

Influence of the farming system on the epiphytic yeasts and yeast-like fungi colonizing grape berries during the ripening process

Grape berries are colonized by a wide array of epiphytic microorganisms such as yeast and filamentous fungi. This microbiota plays a major role in crop health and also interferes with the winemaking process. In this study, culture-dependent and -independent methods were used to investigate the dynamics and diversity of the yeast and yeast-like microorganisms on the grape berry surface during maturation and the influence of cropping systems in this microflora. The results showed a significant impact of both the farming system and the maturity stage on the epiphytic yeast and yeast-like community. A quantitative approach based on counting cultivable populations indicated an increase in the yeast and yeast-like population during the grape ripening process, reaching a maximum when the berries became overripe. The cultivable yeast and yeast-like population also varied significantly depending on the farming system. Microorganism counts were significantly higher for organically- than conventionally-farmed grapes. The yeast and yeast-like community structures were analysed by culture independent methods, using CE-SSCP. The results revealed changes in the genetic structure of the yeast and yeast-like community throughout the ripening process, as well as the impact of the farming system. Copper-based fungicide treatments were revealed as the main factor responsible for the differences in microbial population densities between samples of different farming systems. The results showed a negative correlation between copper levels and yeast and yeast-like populations, providing evidence that copper inhibited this epiphytic community. Taken together, our results showed that shifts in the microbial community were related to changes in the composition of the grape-berry surface, particularly sugar exudation and the occurrence of copper residues from pesticide treatments.

Pythium oligandrum: an example of opportunistic success.

Pythium oligandrum, a non-pathogenic soil-inhabiting oomycete, colonizes the root ecosystem of many crop species. Whereas most members in the genus Pythium are plant pathogens, P. oligandrum distinguishes itself from the pathogenic species by its ability to protect plants from biotic stresses in addition to promoting plant growth. The success of P. oligandrum at controlling soilborne pathogens is partly associated with direct antagonism mediated by mycoparasitism and antimicrobial compounds. Interestingly, P. oligandrum has evolved with specific mechanisms to attack its prey even when these belong to closely related species. Of particular relevance is the question of how P. oligandrum distinguishes between self- and non-self cell wall degradation during the mycoparasitic process of pathogenic oomycete species. The ability of P. oligandrum to enter and colonize the root system before rapidly degenerating is one of the most striking features that differentiate it from all other known biocontrol fungal agents. In spite of this atypical behaviour, P. oligandrum sensitizes the plant to defend itself through the production of at least two types of microbe-associated molecular patterns, including oligandrin and cell wall protein fractions, which appear to be closely involved in the early events preceding activation of the jasmonic acid- and ethylene-dependent signalling pathways and subsequent localized and systemic induced resistance. The aim of this review is to highlight the expanding knowledge of the mechanisms by which P. oligandrum provides beneficial effects to plants and to explore the potential use of this oomycete or its metabolites as new disease management strategies.

Analyses of the Temporal Dynamics of Fungal Communities Colonizing the Healthy Wood Tissues of Esca Leaf-Symptomatic and Asymptomatic Vines

Esca, a Grapevine Trunk Disease (GTD), is of major concern for viticulture worldwide. Our study compares the fungal communities that inhabit the wood tissues of vines that expressed or not foliar esca-symptoms. The trunk and rootstock tissues were apparently healthy, whether the 10 year-old plants were symptomatic or not. The only difference was in the cordon, which contained white rot, a typical form of esca, in 79% of symptomatic plants. Observations over a period of one year using a fingerprint method, Single Strand Conformation Polymorphism (SSCP), and the ITS-DNA sequencing of cultivable fungi, showed that shifts occurred in the fungal communities colonizing the healthy wood tissues. However, whatever the sampling time, spring, summer, autumn or winter, the fungi colonizing the healthy tissues of asymptomatic or symptomatic plants were not significantly different. Forty-eight genera were isolated, with species of Hypocreaceae and Botryosphaeriaceae being the most abundant species. Diverse fungal assemblages, made up of potentially plant-pathogenic and -protective fungi, colonized these non-necrotic tissues. Some fungi, possibly involved in GTD, inhabited the non-necrotic wood of young plants, but no increase in necrosis areas was observed over the one-year period.

Pathogenic and beneficial microorganisms in soilless cultures

Soilless cultures were originally developed to control soilborne diseases. Soilless cultures provide several advantages for growers such as greater production of crops, reduced energy consumption, better control of growth and independence of soil quality. However, diseases specific to hydroponics have been reported. For instance, zoospore-producing microorganisms such as Pythium and Phytophthora spp. are particularly well adapted to aquatic environments. Their growth in soilless substrates is favoured by the recirculation of the nutrient solution. These pathogenic microorganisms are usually controlled by disinfection methods but such methods are only effective as a preventive measure. Contrary to biofiltration, active treatments such as UV, heat and ozonisation have the disadvantage of eliminating not only the harmful microorganisms but also the beneficial indigenous microorganisms. Here, we review microbial populations that colonise ecological niches of hydroponic greenhouse systems. Three topics are discussed: (1) the general microflora; (2) the pathogenic microflora that are typical to hydroponic systems; and (3) the non-pathogenic and possibly beneficial microflora, and their use in the control of plant diseases in soilless greenhouse systems. Technical, economic and environmental concerns are forcing the adoption of new sustainable methods such as the use of microbial antagonists. Thus, increased attention is now focused on the role of natural microflora in suppressing certain diseases. Managing disease suppression in hydroponics represents a promising way of controlling pathogens. Three main strategies can be used: (1) increasing the level of suppressiveness by the addition of antagonistic microorganisms; (2) using a mix of microorganisms with complementary ecological traits and antagonistic abilities, combined with disinfection techniques; and (3) amending substrates to favour the development of a suppressive microflora. Increasing our knowledge on beneficial microflora, their ecology and treatments that influence their composition will help to commercialise new, ready-to-use substrates microbiologically optimised to protect plants in sustainable management systems.

Phyllosphere Fungal Communities Differentiate More Thoroughly than Bacterial Communities Along an Elevation Gradient.

The distance-decay pattern of similarity in ecological communities has been recognized by ecologists for decades [1]. Such a pattern has been shown for diverse macroorganisms and microorganisms [2–5], including phyllosphere bacterial communities [6]. It results from four eco-evolutionary processes: selection, drift, dispersal and mutation [2, 7]. Selection and drift generate the decay in compositional similarity with geographical distance, while dispersal counteracts it [2]. Mutation decreases the similarity among locations, regardless of the distance between them [2]. The shape of a distance-decay curve is therefore expected to differ between taxonomic or functional groups of species [8]. For instance, groups of smaller species such as bacteria are expected to have a slower distance-decay because of their higher dispersal abilities. They are also expected to have a lower compositional similarity among samples of a given location, because of their higher rates of mutation [9]. Here, we compared the distance-decay curves of fungal and bacterial communities along an elevation gradient. Since bacterial cells (with a diameter of a couple of micrometers) are smaller than the smallest fungal individuals (unicellular yeasts, measuring tens of micrometers across) [9], we hypothesized that (i) like fungi, bacterial communities differentiate along the elevation gradient, but (ii) they differentiate less thoroughly (i.e., they have a slower distance-decay), and (iii) they display lower levels of compositional similarity among samples of each elevation. To test these hypotheses, we compared the distancedecay curves of fungal and bacterial communities inhabiting the European beech (Fagus sylvatica L.) phyllosphere along an elevation gradient extending from 488 to 1533 m over a distance of 23 km. The sampling design was composed of 12 samples, corresponding to 4 elevation sites with 3 forest plots per site [10]. Previous analyses showed considerable variation of the fungal community composition along the elevation gradient, suggesting that selection by climatic conditions (temperature, in particular) drives variations in the composition of phyllosphere fungal communities [10]. Here, we characterized the bacterial communities along the same elevation gradient, by 454 pyrosequencing the bacterial 16S rDNA in the same samples that had been used previously for sequencing the fungal internal transcribed spacer region [10]. Bioinformatic analyses are detailed in Table S1. To avoid biases due to differences in sequence variability between both barcode regions, sequence clustering into operational taxonomic units (OTUs) was performed using four identity cutoff thresholds (90, 95, 97, and 99 %). Electronic supplementary material The online version of this article (doi:10.1007/s00248-016-0742-8) contains supplementary material, which is available to authorized users.

Bacteria in a wood fungal disease: characterization of bacterial communities in wood tissues of esca-foliar symptomatic and asymptomatic grapevines

Esca is a grapevine trunk disease (GTD) associated with different pathogenic fungi inhabiting the woody tissues. Bacteria can also be found in such tissues and they may interact with these fungal colonizers. Although such types of microbial interactions have been observed for wood diseases in many trees, this has never been studied for grapevine. In this study, the bacterial microflora of different vine status (esca-symptomatic and asymptomatic), different anatomical part (trunk and cordon) and different type of tissues (necrotic or not) have been studied. Based on Single Strand Conformation Polymorphism (SSCP) analyses, data showed that (i) specific complexes of bacterial microflora colonize the wood of both necrotic and non-necrotic tissues of esca-foliar symptomatic and asymptomatic vines, and also that (ii) depending on the anatomical part of the plant, cordon or trunk, differences could be observed between the bacterial communities. Such differences were also revealed through the community-level physiological profiling (CLPP) with Biolog EcoplatesTM. Two hundred seventeen bacterial strains were also isolated from plant samples and then assigned to bacterial species based on the 16S rRNA genes. Although Bacillus sp. and Pantoea agglomerans were the two most commonly isolated species from all kinds of tissues, various other taxa were also isolated. Inoculation of vine cuttings with 14 different bacterial species, and one GTD fungus, Neofusicoccum parvum, showed no impact of these bacteria on the size of the wood necroses caused by N. parvum. This study showed, therefore, that bacterial communities differ according to the anatomical part (trunk or cordon) and/or the type of tissue (necrotic or non-necrotic) of wood of grapevine plants showing external symptoms of esca disease. However, research into bacteria having a role in GTD development needs further studies.

Phytoextraction of nickel and rhizosphere microbial communities under mono- or multispecies hyperaccumulator plant cover in a serpentine soil

The efficiency of nickel (Ni) phytoextraction by hyperaccumulating Brassicaceae was compared in two types of covers, namely, monoculture or mixed culture. The selected species were from the Pindus Mountains (Greece), including Alyssum murale, Noccaea tymphaea, Leptoplax emarginata and Bornmuellera tymphaea. After 4 months of culture in mesocosms using ultramafic soil (Ni = 1480 mg kg–1), plant biomass yield and Ni concentrations in shoots and roots were recorded for each of six treatments (mixed-culture cover, four monoculture covers and unplanted soil). Microbial biomass carbon, the size of the cultivable rhizosphere bacterial community and its phenotypic structure (Biolog EcoPlates™), bacterial and fungal genetic structure (SSCP), as well as the potential production of auxin compounds, were also evaluated. Moreover, measurements of various microbial enzymes were performed. The biomass and shoot Ni concentration (albeit not significant) of B. tymphaea increased in co-cropping system. A slight acidification of the soil occurred and a strong correlation between pH and the size of the bacterial community was also observed. No significant change in enzyme activity was observed among the cover types, except in the case of arylsulfatase. The phenotypic structure of the bacterial communities and the bacterial and fungal genetic structures appeared to be specific to the type of cover, although the size of the culturable bacterial community did not show variation among treatments. Therefore, on the basis of the bioaccumulation coefficient and the translocation factor, our results showed that B. tympheae, and to a lesser extent N. tympheae, were the two species with the greatest Ni phytoextraction potential in co-culture systems.

Bio-suppression of Sclerotinia Stem Rot of Tomato and Biostimulation ofPlant Growth Using Tomato-associated Rhizobacteria

A collection of 25 rhizobacterial strains, recovered from rhizospheric soils around healthy tomato plants grown in Rhizoctonia-infested fields, belonging to Bacillus amyloliquefaciens, B. thuringiensis, B. megaterium, B. subtilis, Enterobacter cloacae, Chryseobacterium jejuense, and Klebsiella pneumoniae was screened for its suppressive effects of Sclerotinia Stem Rot of tomato caused by Sclerotinia sclerotiorum and plant growth-promoting ability. The inhibitory effects of diffusible and volatile metabolites from these rhizobacteria against pathogen mycelial growth depended significantly upon strains tested. Growth inhibition caused by diffusible and volatile compounds was of about 37-57% and 24-54%, respectively. All strains tested had totally suppressed myceliogenic germination of sclerotia and improved germination of bacterized tomato seeds as compared to the untreated controls. The screening of their disease-suppressive and plant growth-promoting abilities revealed 72-100% decrease in Sclerotinia Stem Rot severity and significant increments in plant height by 52-67%, roots fresh weight by about 66-88% and aerial part weight by 47-75%, compared to S. sclerotiorum-inoculated and untreated control. The most promising strains combining disease-suppressive and growth-promoting abilities were B. subtilis B10 (KT921327) and B14 (KU161090), B. thuringiensis B2 (KU158884), B. amyloliquefaciens B13 (KT951658) and B15 (KT923051), and E. cloacae B16 (KT921429).