Impact of Olive Saplings and Organic Amendments on Soil Microbial Communities and Effects of Mineral Fertilization.

Plant communities and fertilization may have an impact on soil microbiome. Most commercial olive trees are minerally fertilized, while this practice is being replaced by the use of organic amendments. Organic amendments can both fertilize and promote plant growth-promoting organisms. Our aims were (i) to describe the changes in soil bacterial and fungal communities induced by the presence of young olive trees and their interaction with organic amendments and (ii) to compare the effects of mineral and organic fertilization. We set up two parallel experiments in pots using a previously homogenized soil collected from a commercial olive orchard: in the first one, we grew olive saplings in unamended and organically amended soils with two distinct composts and compared these two soils incubated without a plant, while in the second experiment, we comparatively tested the effects of organic and mineral fertilization. OTUs and the relative abundances of bacterial and fungal genera and phyla were analyzed by 16S rRNA and ITS1 gene amplicon using high-throughput sequencing. Basal respiration and substrate-induced respiration were measured by MicroRespTM. The effects of the different treatments were analyzed in all phyla and in the 100 most abundant genera. The presence of olive saplings increased substrate-induced respiration and bacterial and fungal richness and diversity. Organic amendments greatly affected both bacterial and fungal phyla and increased bacterial richness while not affecting fungal richness. Mineral fertilization increased the relative abundance of the less metabolically active bacterial phyla (Actinobacteria and Firmicutes), while it reduced the most metabolically active phylum, Bacteroidetes. Mineral fertilization increased the relative abundance of three N2-fixing Actinobacteria genera, while organic fertilization only increased one genus of Proteobacteria. In organically and minerally fertilized soils, high basal respiration rates were associated with low fungal diversity. Basidiomycota and Chytridiomycota relative abundances positively correlated with basal respiration and substrate-induced respiration, while Ascomycota correlated negatively. Indeed, the Ascomycota phyla comprised most of the fungal genera decreased by organic amendments. The symbiotrophic phylum Glomeromycota did not correlate with any of the C sources. The relative abundance of this phylum was promoted by the presence of plants but decreased when amending soils with composts.

Rhizosphere microorganisms enhance in vitro root and plantlet development of Pyrus and Prunus rootstocks.

MAIN CONCLUSION: The in vitro application of rhizosphere microorganisms led to a higher rooting percentage in Pyrus Py12 rootstocks and increased plant growth of Pyrus Py170 and Prunus RP-20. The rooting of fruit tree rootstocks is the most challenging step of the in vitro propagation process. The use of rhizosphere microorganisms to promote in vitro rooting and plant growth as an alternative to the addition of chemical hormones to culture media is proposed in the present study. Explants from two Pyrus (Py170 and Py12) rootstocks and the Prunus RP-20 rootstock were inoculated with Pseudomonas oryzihabitans PGP01, Cladosporium ramotenellum PGP02 and Phoma sp. PGP03 following two different methods to determine their effects on in vitro rooting and plantlet growth. The effects of the microorganisms on the growth of fully developed Py170 and RP-20 plantlets were also studied in vitro. All experiments were conducted using vermiculite to simulate a soil system in vitro. When applied to Py12 shoots, which is a hard-to-root plant material, both C. ramotenellum PGP02 and Phoma sp. PGP03 fungi were able to increase the rooting percentage from 56.25% to 100% following auxin indole-3-butyric acid (IBA) treatment. Thus, the presence of these microorganisms clearly improved root development, inducing a higher number of roots and causing shorter roots. Better overall growth and improved stem growth of treated plants was observed when auxin treatment was replaced by co-culture with microorganisms. A root growth-promoting effect was observed on RP-20 plantlets after inoculation with C. ramotenellum PGP02, while P. oryzihabitans PGP01 increased root numbers for both Py170 and RP-20 and increased root growth over stem growth for RP-20. It was also shown that the three microorganisms P. oryzihabitans PGP01, C. ramotenellum PGP02 and Phoma sp. PGP03 were able to naturally produce auxin, including indole-3-acetic acid (IAA), at different levels. Overall, our results demonstrate that the microorganisms P. oryzihabitans PGP01 and C. ramotenellum PGP02 had beneficial effects on in vitro rooting and plantlet growth and could be applied to in vitro tissue culture as a substitute for IBA.

Monitoring Lysobacter capsici AZ78 using strain specific qPCR reveals the importance of the formulation for its survival in vineyards.

Survival in the phyllosphere is a critical feature for biofungicides based on non-spore forming bacteria. Moreover, knowledge of their persistence on plants is important to design effective formulations and application techniques. With this scope, the aim of this work was to develop a specific method to monitor the fate in the environment of Lysobacter capsici AZ78, a biocontrol agent of Plasmopara viticola, and to evaluate the contribution of formulation in its persistence on grapevine leaves. A strain-specific primer pair derived from REP-PCR fingerprinting was used in quantitative PCR experiments to track the evolution of L. capsici AZ78 population in vineyards. The population reached between 5 and 6 log10 cells gram of leaf(-1) after application and decreased by more than 100 times in one week. Multiple regression analysis showed that unfavourable temperature was the main environmental factor correlating with the decrease of L. capsici AZ78 persistence on grapevine leaves. Importantly, the use of formulation additives protected L. capsici AZ78 against environmental factors and improved its persistence on the leaves by more than 10 times compared to nude cells. Formulation and the knowledge about the persistence of L. capsici AZ78 in vineyards will be useful to develop commercial biofungicides for foliar application.

Stepwise flow diagram for the development of formulations of non spore-forming bacteria against foliar pathogens: The case of Lysobacter capsici AZ78.

The formulation is a significant step in biopesticide development and is an efficient way to obtain consistency in terms of biological control under field conditions. Nonetheless, there is still a lack of information regarding the processes needed to achieve efficient formulation of non spore-forming bacterial biological control agents. In response to this, we propose a flow diagram made up of six steps including selection of growth parameters, checking of minimum shelf life, selection of protective additives, checking that the additives have no adverse effects, validation of the additive mix under field conditions and choosing whether to use additives as co-formulants or tank mix additives. This diagram is intended to provide guidance and decision-making criteria for the formulation of non spore-forming bacterial biological control agents against foliar pathogens. The diagram was then validated by designing an efficient formulation for a Gram-negative bacterium, Lysobacter capsici AZ78, to control grapevine downy mildew caused by Plasmopara viticola. A harvest of 10(10)L. capsici AZ78cellsml(-1) was obtained in a bench top fermenter. The viability of cells decreased by only one order of magnitude after one year of storage at 4°C. The use of a combination of corn steep liquor, lignosulfonate, and polyethyleneglycol in the formulation improved the survival of L. capsici AZ78 cells living on grapevine leaves under field conditions by one order of magnitude. Furthermore, the use of these additives also guaranteed a reduction of 71% in P. viticola attacks. In conclusion, this work presents a straightforward stepwise flow diagram to help researchers develop formulations for biological control agents that are easy to prepare, stable, not phytotoxic and able to protect the microorganims under field conditions.

Enhanced Botrytis cinerea resistance of Arabidopsis plants grown in compost may be explained by increased expression of defense-related genes, as revealed by microarray analysis.

Composts are the products obtained after the aerobic degradation of different types of organic matter waste and can be used as substrates or substrate/soil amendments for plant cultivation. There is a small but increasing number of reports that suggest that foliar diseases may be reduced when using compost, rather than standard substrates, as growing medium. The purpose of this study was to examine the gene expression alteration produced by the compost to gain knowledge of the mechanisms involved in compost-induced systemic resistance. A compost from olive marc and olive tree leaves was able to induce resistance against Botrytis cinerea in Arabidopsis, unlike the standard substrate, perlite. Microarray analyses revealed that 178 genes were differently expressed, with a fold change cut-off of 1, of which 155 were up-regulated and 23 were down-regulated in compost-grown, as against perlite-grown plants. A functional enrichment study of up-regulated genes revealed that 38 Gene Ontology terms were significantly enriched. Response to stress, biotic stimulus, other organism, bacterium, fungus, chemical and abiotic stimulus, SA and ABA stimulus, oxidative stress, water, temperature and cold were significantly enriched, as were immune and defense responses, systemic acquired resistance, secondary metabolic process and oxireductase activity. Interestingly, PR1 expression, which was equally enhanced by growing the plants in compost and by B. cinerea inoculation, was further boosted in compost-grown pathogen-inoculated plants. Compost triggered a plant response that shares similarities with both systemic acquired resistance and ABA-dependent/independent abiotic stress responses.

Trichoderma asperellum strain T34 controls Fusarium wilt disease in tomato plants in soilless culture through competition for iron.

Trichoderma asperellum strain T34 has been reported to control the disease caused by Fusarium oxysporum f.sp. lycopersici (Fol) on tomato plants. To study the importance of iron concentration in the growth media for the activity and competitiveness of T34 and the pathogen, we tested four iron concentrations in the nutrient solution [1, 10, 100, and 1000 microM provided as EDTA/Fe(III)] in a biological control experiment with T34 and Fol in tomato plants. The reduction of the Fusarium-infected shoot by T34 was only significant at 10 microM Fe. We hypothesized that Fe competition is one of the key factors in the biocontrol activity exerted by T34 against Fol, as an increase in Fe concentration over 10 microM would lead to the suppression of T34 siderophore synthesis and thus inhibition of Fe competition with Fol. T34 significantly reduced the populations of Fol at all the doses of Fe assayed. In contrast, Fol enhanced the populations of T34 at 1 and 10 microM Fe. Nevertheless, several plant physiological parameters like net CO(2) assimilation (A), stomatal conductance (g(s)), relative quantum efficiency of PSII (Phi(PSII)), and efficiency of excitation energy capture by open PSII reactive centers (Fv'/Fm') demonstrated the protection against Fol damage by treatment with T34 at 100 microM Fe. The first physiological parameter affected by the disease progression was g(s). Plant dry weight was decreased by Fe toxicity at 100 and 1,000 microM. T34-treated plants had significantly greater heights and dry weights than control plants at 1,000 microM Fe, even though T34 did not reduce the Fe content in leaves or stems. Furthermore, T34 enhanced plant height even at the optimal Fe concentration (10 microM) compared to control plants. In conclusion, T. asperellum strain T34 protected tomato plants from both biotic (Fusarium wilt disease) and abiotic stress [Fe(III) toxic effects].

Proteome, salicylic acid, and jasmonic acid changes in cucumber plants inoculated with Trichoderma asperellum strain T34.

Trichoderma spp. is one of the most commonly used biological control agents against plant pathogens. This fungus produces changes in plant metabolism, thus increasing growth and enhancing resistance to biotic and abiotic stresses. However, its modes of action remain to be defined. In the first hours of interaction between cucumber plant roots and Trichoderma asperellum strain T34, salicylic and jasmonic acid levels and typical antipathogenic peroxidase activity increase in the cotyledons to different degrees depending on the applied concentration of the fungi. The use of 2-DE protein profiling and MS analysis allowed us to identify 28 proteins whose expression was affected in cotyledons after cucumber root colonization by Trichoderma applied at high concentrations: 17 were found to be up-regulated while 11 were down-regulated. Proteins involved in ROS scavenging, stress response, isoprenoid and ethylene biosynthesis, and in photosynthesis, photorespiration, and carbohydrate metabolism were differentially regulated by Trichoderma. The proteome changes found in this study help to give an understanding of how Trichoderma-treated plants become more resistant to pathogen attacks through the changes in expression of a set of defence-oriented proteins which can directly protect the plant or switch the metabolism to a defensive, nonassimilatory state.

Simultaneous quantitative LC-ESI-MS/MS analyses of salicylic acid and jasmonic acid in crude extracts of Cucumis sativus under biotic stress.

Salicylic acid (SA) and jasmonic acid (JA) are plant hormones involved in basal resistance against plant pathogens and also in induced resistance. The aim of this study is to develop a fast and sensitive method to determine simultaneously the levels of both these hormones. The present paper proposes a method that includes hormone extraction with MeOH-H(2)O-HOAc (90:9:1, v/v), evaporation of the extracts, and injection into the liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) system in multiple reaction monitoring (MRM). Endogenous SA and JA levels in noninfested control cucumber cotyledons were 30.96 and 0.73ngg(-1) fresh weight, respectively. In roots, the levels were 8.31 and 15.82ngg(-1) FW, respectively. In plants treated with the biological control agent Trichoderma asperellum strain T-34, the levels of SA and JA did not differ from control plants. Rhizoctonia solani-diseased cucumber plants showed higher levels of SA and JA compared to noninfested controls (up to 2 and 13-fold higher, respectively). Detection limits for SA and JA were 0.45 and 0.47ngg(-1) fresh weight, respectively. The results of our research include the development of a method that is both fast and highly sensitive in the simultaneous quantitation of SA and JA from crude cucumber plant extracts, avoiding any purification and derivatization steps.