Using plant growth‑promoting microorganisms (PGPMs) to improve plant development under in vitro culture conditions

Plant in vitro culture techniques are highly useful to obtain signifcant amounts of true-to-type and disease-free plant materials. One of these techniques is clonal micropropagation which consists on the establishment of shoot tip cultures, shoot multiplication, in vitro rooting and acclimatization to ex vitro conditions. However, in some cases, the existence of recalcitrant genotypes, with a compromised multiplication and rooting ability, or the difculties to overcome the overgrowth of endophytic contaminations might seriously limit its efciency. In this sense, the establishment of benefcial interactions between plants and plant growth-promoting microorganisms (PGPMs) under in vitro culture conditions might represent a valuable approach to efciently solve those restrictions. During the last years, signifcant evidence reporting the use of benefcial microorganisms to improve the yield of in vitro multiplication or rooting as well as their acclimatization to greenhouse or soil conditions have been provided. Most of these positive efects are strongly linked to the ability of these microorganisms to provide in vitro plants with nutrients such as nitrogen or phosphorous, to produce plant growth regulators, to control the growth of pathogens or to mitigate stress conditions. The culture of A. thaliana under aseptic conditions has provided high-quality knowledge on the root development signaling pathways, involving hormones, triggered in the presence of PGPMs. Overall, the present article ofers a brief overview of the use of microorganisms to improve in vitro plant performance during the in vitro micropropagation stages, as well as the main mechanisms of plant growth promotion associated with these microorganismsinfo:eu-repo/semantics/acceptedVersio

Verifying the biocontrol activity of novel film‐forming formulations of Candida sake CPA‐1: resilience in relation to environmental factors, rainfall episodes, and control of Botrytis cinerea on different hosts

BACKGROUND The efficacy of Candida sake CPA‐1 as a biocontrol agent against several diseases has been studied since it was isolated 20 years ago. However, it was only recently that two suitable and effective film‐forming formulations based on potato starch and maltodextrins were developed using the fluidized‐bed spray‐drying system. The present work aimed to confirm the capability of both novel formulations by testing their resilience on grapes at different temperatures (0, 22, and 30 °C), relative humidities (40% and 85%), and simulated rainfall levels. Another objective was to examine the control of Botrytis cinerea in different hosts. RESULTS The CPA‐1 cells from both dried formulations survived better than the liquid formulation on grapes stored at 0 and 22 °C regardless of the relative humidity. After simulated rainfall, potato starch formulation achieved significantly higher populations than maltodextrin formulation, although the highest reduction was −1.6 log N N0−1. A positive effect of cell establishment prior to the simulated rainfall was shown, and recovered cells from the potato starch formulation were significantly higher after 72 h of cell establishment. Finally, both formulations reduced the incidence and severity of B. cinerea on pears, apples, and tomatoes. CONCLUSION The potential of these novel film‐forming formulations of C. sake CPA‐1 was verified. The resilience of formulated C. sake was better than the commercialized liquid formulation, the adherence of the formulations to the grapes improved after an establishment period prior to rain exposure, and the control of B. cinerea was verified in a wider range of hosts. © 2019 Society of Chemical Industryinfo:eu-repo/semantics/acceptedVersio

An Agronomic Approach to Pine Nut Production by Grafting Stone Pine on Two Rootstocks

Stone pine (Pinus pinea) grows in natural stands within the Mediterranean basin and its nut is highly appreciated for its nutritional profile. Nevertheless, a decline in this species due to biotic and abiotic damages is currently being clearly observed. This situation has led to its development as a nut crop, to try to save its production and obtain regular harvests over the years. Under this agronomic scenario, the aim of this work was to compare the behavior of the stone pine grafted onto two rootstocks, P. pinea (PP) and P. halepensis (PH), by evaluating cone productivity, tree growth response, mast seeding patterns and pine nut composition. The field test was composed of 14 PH and 14 PP, randomly distributed into groups of 4–5 trees/rootstock. Data were from seven productive growing seasons. The results show higher growth and ripe cone production on PP rather than PH, although the productivity (cones/m2 canopy) was similar. Any effect of rootstock was observed on the mast seeding pattern and weight of cones, while the pine nut composition showed differences in the fatty acids content. The global quality of production was similar in PH and PP.info:eu-repo/semantics/publishedVersio

Novel film‐forming formulations of the biocontrol agent Candida sake CPA‐1: biocontrol efficacy and performance at field conditions in organic wine grapes

BACKGROUND: The biocontrol agent (BCA)Candida sakeCPA-1 has previously effectively reducedBotrytisbunch rot (BBR) andit was also suggested as a promising strategy to control sour rot in grapes under field conditions. However, biocontrol efficacyof solid formulations of CPA-1 has never been tested in field trials. The present study aims to confirm the efficacy against BBRand sour rot in grapes under field conditions of two novel formulations recently developed by the addition of biodegradablecoatings using a fluidized-bed spray-drying system.RESULTS: Novel film-forming formulations of the BCAC. sakeCPA-1 controlledB. cinereaas well as liquid formulation. Sour rotcontrol resulted better in the second season and severity reductions were more satisfactory than incidence control. Visual andcryoSEM observations revealed that film-forming treatments were uniformly distributed on plant surfaces. CPA-1 coating couldbe observed on grapes at harvest time.CONCLUSION: The results of this work suggest that solid formulations would be a competitive alternative to conventionalfungicides because they were easy to package and transport, and cell viability could be maintained for a long period of time.© 2018 Society of Chemical Industryinfo:eu-repo/semantics/acceptedVersio

Inoculation of in vitro cultures with rhizosphere microorganisms improve plant development and acclimatization during immature embryo rescue in nectarine and pear breeding programs

In the present study, two fungi Cladosporium ramotenellum strain PGP02 and Phoma spp. strain PGP03 and the bacterium Pseudomonas oryzihabitans PGP01 were isolated from Pyrus and Prunus in vitro rescued embryos, whose plantlets showed a better growth than non-contaminated cultures. Upon identification, concentrated solutions of the three microorganisms were applied to pear (Pyrus communis L.) in vitro rooted plantlets, increasing in different ways biometric parameters such as plant fresh weight (FW), stem length and root length. Then, these microorganisms were tested in embryos derived from three directed crosses between early ripening nectarine varieties (Prunus persica cv. Nectarina). In a first cross, in vitro cultured embryos were inoculated with both fungi, C. ramotenellum PGP02 and Phoma spp. PGP03, at 2 × 107 esp mL−1, and the bacterium P. oryzihabitans PGP01, at 2 × 108 CFU mL−1. In the following crosses, only the bacterium P. oryzihabitans PGP01, at 2 × 108 CFU mL−1, was employed. The effects on number of germinated embryos, development of the subsequent plants, after 24 weeks of in vitro culture, as well as their ex vitro acclimatization performance were analysed. These microorganisms had no effect on the germination efficiency of nectarine embryos. However, the presence of the bacterium P. oryzihabitans PGP01 modified root system architecture in the three crosses, increasing root volume and thickness, which in consequence enhanced the acclimatization efficiency to soil in those crosses with poor acclimation efficiencies. These results enforce a breakthrough in the use of microorganisms along the in vitro embryo rescue used in early ripening peaches and nectarines breeding programmes, and the production of plants more resistant to the stressful conditions imposed by the acclimatization to soil.info:eu-repo/semantics/acceptedVersio

Deciphering the Effect of Light Wavelengths in Monilinia spp. DHN-Melanin Production and Their Interplay with ROS Metabolism in M. fructicola

Pathogenic fungi are influenced by many biotic and abiotic factors. Among them, light is a source of information for fungi and also a stress factor that triggers multiple biological responses, including the activation of secondary metabolites, such as the production of melanin pigments. In this study, we analyzed the melanin-like production in in vitro conditions, as well as the expression of all biosynthetic and regulatory genes of the DHN–melanin pathway in the three main Monilinia species upon exposure to light conditions (white, black, blue, red, and far-red wavelengths). On the other hand, we analyzed, for the first time, the metabolism related to ROS in M. fructicola, through the production of hydrogen peroxide (H2O2) and the expression of stress-related genes under different light conditions. In general, the results indicated a clear importance of black light on melanin production and expression in M. laxa and M. fructicola, but not in M. fructigena. Regarding ROS-related metabolism in M. fructicola, blue light highlighted by inhibiting the expression of many antioxidant genes. Overall, it represents a global description of the effect of light on the regulation of two important secondary mechanisms, essential for the adaptation of the fungus to the environment and its survival.This research was funded by the national projects AGL2017-84389-C2-1-R and PID2020-1157AGL2017 from the Ministry of Science, Innovation and Universities (MCIU, Spain) and from the CERCA Program/Generalitat de Catalunya. L. Verde-Yáñez received a Ph.D. fellowship PRE2018-085428 from Agencia Estatal de Investigación (AEI, Spain).info:eu-repo/semantics/publishedVersio

Light Intensity Alters the Behavior of Monilinia spp. in vitro and the Disease Development on Stone Fruit-Pathogen Interaction

The development of brown rot caused by the necrotrophic fungi Monilinia spp. in stone fruit under field and postharvest conditions depends, among others, on environmental factors. The effect of temperature and humidity are well studied but there is little information on the role of light in disease development. Herein, we studied the effect of two lighting treatments and a control condition (darkness) on: (i) several growth parameters of two Monilinia spp. (M. laxa and M. fructicola) grown in vitro and (ii) the light effect in their capacity to rot the fruit (nectarines) when exposed to the different lighting treatments. We also assessed the effect of such abiotic factors in the development of the disease on inoculated nectarines during postharvest storage. Evaluations also included testing the effect of fruit bagging on disease development as well as on ethylene production. Under in vitro conditions, lighting treatments altered colony morphology and conidiation of M. laxa but this effect was less acute in M. fructicola. Such light-induced changes under in vitro development also altered the capacity of M. laxa and M. fructicola to infect nectarines, with M. laxa becoming less virulent. The performance of Monilinia spp. exposed to treatments was also determined in vivo by inoculating four bagged or unbagged nectarine cultivars, indicating an impaired disease progression. Incidence and lesion diameter of fruit exposed to the different lighting treatments during postharvest showed that the effect of the light was intrinsic to the nectarine cultivar but also Monilinia spp. dependent. While lighting treatments reduced M. laxa incidence, they enhanced M. fructicola development. Preharvest conditions such as fruit bagging also impaired the ethylene production of inoculated fruit, which was mainly altered by M. laxa and M. fructicola, while the bag and light effects were meaningless. Thus, we provide several indications of how lighting treatments significantly alter Monilinia spp. behavior both in vitro and during the interaction with stone fruit. This study highlights the importance of modulating the lighting environment as a potential strategy to minimize brown rot development on stone fruit and to extent the shelf-life period of fruit in postharvest, market, and consumer’s house.info:eu-repo/semantics/publishedVersio

Dehydration of Ampelomyces quisqualis CPA-9 conidia by adding biodegradable coatings: Biocontrol activity against powdery mildew and physical characterization of the formulated product

Ampelomyces quisqualis CPA-9 was reported as a promising biocontrol fungus against powdery mildew on cucurbits, although its formulation had not been developed. The present work aimed to develop a fluidised-bed spray-dried formulation of CPA-9 that included film-forming compounds, which could improve the behaviour of the microorganism under practical conditions. Film forming compounds, efficacy, shelf life, physical stability as a function of aw, and solubility in water were determined. A film-forming formulation based on native potato starch and pregelatinised potato starch as carrier and binder was obtained, and sucrose plus skimmed milk were used as protective compounds. Dehydrated conidia maintained the efficacy of fresh cells against Podosphaera xanthii on zucchini leaves, and powdery mildew was significantly reduced compared with control treatment. Despite of results obtained for the glassy state guaranteed the stability of the powder against sticking and caking processes, the shelf life of the product was limited to 4 months of storage at 4 °C. On the other hand, the formulation was mainly dispersible, due to the high concentration of native potato starch which reduces the solubility of the product. Results obtained in this work, such as the high efficacy of the developed product, the low cost of the employed substances, the film-forming ability of the rehydrated powder, and finally the effectiveness of the dehydration process, confirmed the potential of the product. Further studies to verify the improvement of CPA-9 behaviour under practical conditions or to improve the storage conditions to enlarge the shelf life of the BCA should be done to consider the developed product as a biocontrol-based product for cucurbits against powdery mildew.info:eu-repo/semantics/acceptedVersio

Efficacy of Candida sake CPA-1 Formulation for Controlling Penicillium expansum Decay on Pome Fruit from Different Mediterranean Regions

The effectiveness of a formulated product of the yeast Candida sake CPA-1 for controlling postharvest diseases on pome fruits was demonstrated in laboratory, semicommercial, and commercial trials carried out in the major pome fruit producing region of the European Union. First, one wettable powder and seven liquid formulations were tested in laboratory trials that involved two varieties of apples and two varieties of pears. In all cases, an efficacy similar to that of fresh cells was demonstrated in the control of artificial Penicillium expansum infection. After these trials, the formulated product chosen for semicommercial and commercial trials was LF1, a liquid formulation that is particularly suitable for commercial applications. In semicommercial trials, LF1 showed a performance similar to fresh cells in most trials, and the population dynamics of both fresh and formulated cells were quite stable throughout the storage period. This indicates the high viability of C. sake CPA-1 in this formulation and the absence of adverse effects during the formulation of the product, which may significantly affect both its ability to grow on fruit and its antagonistic activity. We evaluated the control of natural infection after applying the formulated product in a commercial drencher in different packinghouses. A significant reduction in the incidence of diseases was observed with a recommended dose of around 10(7) CFU/ml when natural infections were greater than 1%. In general, large quantities of yeast were observed on the surface of unwounded fruits of different pome fruit cultivars. Moreover, populations of this biocontrol agent increased rapidly on fruit surfaces and remained quite stable for a long time under commercial storage conditions. Commercial practices used in packinghouses were therefore successfully applied for this formulated product