The Amount of the Rare Sugar Tagatose on Tomato Leaves Decreases after Spray Application under Greenhouse Conditions.

Tagatose is a rare sugar that suppresses plant diseases, such as late blight of tomato, caused by Phytophthora infestans. Tagatose can be metabolized by some microorganisms and no information is available on its persistence on tomato leaves. The aim of this study was to assess the persistence of tagatose on tomato leaves under commercial greenhouse conditions. The amount of tagatose on tomato leaves and the inhibitory activity against P. infestans decreased seven days after spray application in the absence of rain wash-off. Potential tagatose-degrading bacteria were isolated from tomato leaves, and they belonged to Acinetobacter sp., Bacillus sp., Comamonas sp., Enterobacter sp., Methylobacterium sp., Microbacterium sp., Pantoea sp., Plantibacter sp., Pseudomonas sp., Ralstonia sp., Rhodococcus sp., Sphingobium sp., and Sphingomonas sp. Thus, indigenous phyllosphere microorganisms could partially metabolize tagatose laid on plant leaves after spray application, reducing the persistence of this fungal inhibitor on tomato leaves.

D-Tagatose-Based Product Triggers Sweet Immunity and Resistance of Grapevine to Downy Mildew, but Not to Gray Mold Disease.

The use of natural bio-based compounds becomes an eco-friendly strategy to control plant diseases. Rare sugars would be promising compounds as inducers of plant "sweet immunity". The present study aimed to investigate the induced resistance of grapevine leaves against Plasmopara viticola and Botrytis cinerea by a rare sugar-based product (IFP48) and its active ingredient D-tagatose (TAG), in order to elucidate molecular mechanism involved in defense-related metabolic regulations before and after pathogen challenge. Data showed that spraying leaves with IFP48 and TAG lead to a significant reduction of downy mildew, but not of gray mold disease. The induced protection against P. viticola relies on IFP48's and to a lesser extent TAG's ability to potentiate the activation of salicylic acid- and jasmonic acid/ethylene-responsive genes and stilbene phytoalexin accumulation. Most of defense responses remained upregulated in IFP48-treated plants after infection with P. viticola, but inconsistent following challenge with B. cinerea. The beneficial effects of IFP48 were associated with an enhanced accumulation of tagatose inside leaf tissues compared to TAG treatment. Meanwhile, the amounts of sugars, glucose, fructose, maltose, galactose and trehalose remained unchanged or decreased in IFP48-treated leaves after P. viticola infection, although only a few genes involved in sugar transport and metabolism showed transcriptional regulation. This suggests a contribution of sugar homeostasis to the IFP48-induced sweet immune response and priming plants for enhanced resistance to P. viticola, but not to B. cinerea.

Wood pellets as carriers of conidia of Trichoderma atroviride SC1 for soil application.

The use of biocontrol agents to control soilborne diseases is a promising alternative to chemical pesticides, however, obtaining a homogeneous distribution and incorporation of conidia of fungal biocontrol agents into the soil is often difficult. Several carriers/formulations have been proposed over time, unfortunately without offering an ultimate solution. We propose the use of wood pellets as a carrier of conidia of a saprophytic fungus that has good biodegradation and biocontrol properties (Trichoderma atroviride SC1). The coating process is based on the direct spraying of wood pellets with a conidial suspension at different rates. Beech, fir, and chestnut wood pellets were compared in terms of relevant physicochemical traits and efficacy in supporting the growth of the fungus. Beech wood pellets displayed the best characteristics in terms of water holding capacity, swelling properties, and disintegration time. T. atroviride SC1 grows best on beech and fir wood pellets and reaches a plateau after nine days of incubation, regardless of the initial coating concentrations. The addition of small quantities of a nitrogen source as tryptone or soy flour, soy proteins, and a mixture of animal proteins used as pet food to the conidial suspension can increase the growth by ten-folds on all types of wood pellets. Our results demonstrate that beech and fir wood pellets could be suitable carriers to deliver and sustain the growth of T. atroviride SC1.

Effect of a Wood-Based Carrier of Trichoderma atroviride SC1 on the Microorganisms of the Soil.

Wood pellets can sustain the growth of Trichoderma spp. in soil; however, little is known about their side effects on the microbiota. The aims of this study were to evaluate the effect of wood pellets on the growth of Trichoderma spp. in bulk soil and on the soil microbial population's composition and diversity. Trichoderma atroviride SC1 coated wood pellets and non-coated pellets were applied at the level of 10 g∙kg-1 of soil and at the final concentration of 5 × 103 conidia∙g-1 of soil and compared to a conidial suspension applied at the same concentration without the wood carrier. Untreated bulk soil served as a control. The non-coated wood pellets increased the total Trichoderma spp. population throughout the experiment (estimated as colony-forming unit g-1 of soil), while wood pellets coated with T. atroviride SC1 did not. The wood carrier increased the richness, and temporarily decreased the diversity, of the bacterial population, with Massilia being the most abundant bacterial genus, while it decreased both the richness and diversity of the fungal community. Wood pellets selectively increased fungal species having biocontrol potential, such as Mortierella, Cladorrhinum, and Stachybotrys, which confirms the suitability of such carriers of Trichoderma spp. for soil application.

The Differential Growth Inhibition of Phytophthora spp. Caused by the Rare Sugar Tagatose Is Associated With Species-Specific Metabolic and Transcriptional Changes.

Tagatose is a rare sugar with no negative impacts on human health and selective inhibitory effects on plant-associated microorganisms. Tagatose inhibited mycelial growth and negatively affected mitochondrial processes in Phytophthora infestans, but not in Phytophthora cinnamomi. The aim of this study was to elucidate metabolic changes and transcriptional reprogramming activated by P. infestans and P. cinnamomi in response to tagatose, in order to clarify the differential inhibitory mechanisms of tagatose and the species-specific reactions to this rare sugar. Phytophthora infestans and P. cinnamomi activated distinct metabolic and transcriptional changes in response to the rare sugar. Tagatose negatively affected mycelial growth, sugar content and amino acid content in P. infestans with a severe transcriptional reprogramming that included the downregulation of genes involved in transport, sugar metabolism, signal transduction, and growth-related process. Conversely, tagatose incubation upregulated genes related to transport, energy metabolism, sugar metabolism and oxidative stress in P. cinnamomi with no negative effects on mycelial growth, sugar content and amino acid content. Differential inhibitory effects of tagatose on Phytophthora spp. were associated with an attempted reaction of P. infestans, which was not sufficient to attenuate the negative impacts of the rare sugar and with an efficient response of P. cinnamomi with the reprogramming of multiple metabolic processes, such as genes related to glucose transport, pentose metabolism, tricarboxylic acid cycle, reactive oxygen species detoxification, mitochondrial and alternative respiration processes. Knowledge on the differential response of Phytophthora spp. to tagatose represent a step forward in the understanding functional roles of rare sugars.

Rare Sugars: Recent Advances and Their Potential Role in Sustainable Crop Protection.

Rare sugars are monosaccharides with a limited availability in the nature and almost unknown biological functions. The use of industrial enzymatic and microbial processes greatly reduced their production costs, making research on these molecules more accessible. Since then, the number of studies on their medical/clinical applications grew and rare sugars emerged as potential candidates to replace conventional sugars in human nutrition thanks to their beneficial health effects. More recently, the potential use of rare sugars in agriculture was also highlighted. However, overviews and critical evaluations on this topic are missing. This review aims to provide the current knowledge about the effects of rare sugars on the organisms of the farming ecosystem, with an emphasis on their mode of action and practical use as an innovative tool for sustainable agriculture. Some rare sugars can impact the plant growth and immune responses by affecting metabolic homeostasis and the hormonal signaling pathways. These properties could be used for the development of new herbicides, plant growth regulators and resistance inducers. Other rare sugars also showed antinutritional properties on some phytopathogens and biocidal activity against some plant pests, highlighting their promising potential for the development of new sustainable pesticides. Their low risk for human health also makes them safe and ecofriendly alternatives to agrochemicals.

Interactions of tagatose with the sugar metabolism are responsible for Phytophthora infestans growth inhibition.

Tagatose is a rare sugar metabolised by a limited number of microorganisms that inhibits a large spectrum of phytopathogens. In particular, tagatose inhibited Phytophthora infestans growth and negatively affected mitochondrial processes. However, the possible effects of tagatose on P. infestans metabolism have not yet been investigated. The aim of this study was to analyse the impact of this rare sugar on the sugar metabolism in P. infestans, in order to better understand its mode of action. Tagatose inhibited the growth of P. infestans with a precise reprogramming of the carbohydrate metabolism that involved a decrease of glucose, glucose-1-phosphate and mannose content and ß-glucosidase activity. The combination of tagatose with common sugars led to three different responses and highlighted antagonistic interactions. In particular, glucose partially attenuated the inhibitory effects of tagatose, while fructose fully impaired tagatose-mediated growth inhibition and metabolite changes. Moreover, sucrose did not attenuate tagatose effects, suggesting that the inhibition of sucrose catabolism and the alteration of glucose-related pathways contributed to the growth inhibition caused by tagatose to P. infestans. The interactions of tagatose with the common sugar metabolism were found to be a key mode of action against P. infestans growth, which may represent the basis for the further development of tagatose as an eco-friendly fungicide.

Volatile-Mediated Inhibitory Activity of Rhizobacteria as a Result of Multiple Factors Interaction: The Case of Lysobacter capsici AZ78.

Plant beneficial rhizobacteria may antagonize soilborne plant pathogens by producing a vast array of volatile organic compounds (VOCs). The production of these compounds depends on the medium composition used for bacterial cell growth. Accordingly, Lysobacter capsici AZ78 (AZ78) grown on a protein-rich medium was previously found to emit volatile pyrazines with toxic activity against soilborne phypathogenic fungi and oomycetes. However, the discrepancy between the quantity of pyrazines in the gaseous phase and the minimum quantity needed to achieve inhibition of plant pathogens observed, lead us to further investigate the volatile-mediated inhibitory activity of AZ78. Here, we show that, besides VOCs, AZ78 cells produce ammonia in increased amounts when a protein-rich medium is used for bacterial growth. The production of this volatile compound caused the alkalinization of the physically separated culture medium where Rhizoctonia solani was inoculated subsequently. Results achieved in this work clearly demonstrate that VOC, ammonia and the growth medium alkalinization contribute to the overall inhibitory activity of AZ78 against R. solani. Thus, our findings suggest that the volatile-mediated inhibitory activity of rhizobacteria in protein-rich substrates can be regarded as a result of multiple factors interaction, rather than exclusively VOCs production.

Volatile Organic Compounds From Lysobacter capsici AZ78 as Potential Candidates for Biological Control of Soilborne Plant Pathogens.

The genus Lysobacter includes several bacterial species which show potential for being used in biological control of plant diseases. It was shown recently that several Lysobacter type strains produce volatile organic compounds (VOCs) which controlled the growth of Phytophthora infestans in vitro when the bacteria were grown on a protein rich medium. In the present study, Lysobacter capsici AZ78 (AZ78) has been tested for its potential to produce VOCs that may contribute to the bioactivity against soilborne plant pathogens. To this end, split Petri dish assays of bacterial cultures have been combined with GC-MS measurements with the aim to reveal the identity of the VOCs which inhibit the growth of Pythium ultimum Rhizoctonia solani, and Sclerotinia minor. While AZ78 completely suppressed the growth of P. ultimum and S. minor, the growth of R. solani was still reduced significantly. The GC-MS analysis revealed 22 VOCs to be produced by AZ78, the majority of which were (putatively) identified as mono- and dialkylated methoxypyrazines. Based on additional cultivation and GC-MS experiments, 2,5-dimethylpyrazine, 2-ethyl-3-methoxypyrazine and 2-isopropyl-3-methoxypyrazine were selected as presumable bioactive compounds. Further bioassays employing indirect exposure to standard solutions (1-10 mg per Petri dish) of the synthetic compounds via the gas phase, revealed that each of these pyrazines was able to suppress the growth of the pathogens under investigation. The results of this study highlight the possible future implementation of pyrazine derivatives in the control of soilborne plant diseases and further support the biocontrol potential of L. capsici AZ78.

The Rare Sugar Tagatose Differentially Inhibits the Growth of Phytophthora infestans and Phytophthora cinnamomi by Interfering With Mitochondrial Processes.

Rare sugars are monosaccharides with limited availability in nature and their biological functions are largely unknown. Among them, tagatose was developed as a low-calorie sweetener and showed beneficial effects on human health. Tagatose is metabolized by only certain microbial taxa and inhibits the growth of important crop pathogens (e.g., Phytophthora infestans), but its mode of action and the microbial responses are unknown. The aim of this study was to understand the tagatose mode of action against Phytophthora spp., with the final aim of developing new plant protection products. Tagatose inhibited P. infestans growth in vitro and caused severe ultrastructural alterations, with the formation of circular and concentric mitochondrial cristae. Decreased ATP content and reduced oxygen consumption rate (OCR) were found in tagatose-incubated P. infestans as compared to the control, with the consequent accumulation of reactive oxygen species (ROS) and induction of genes related to apoptosis and oxidative stress response. On the other hand, tagatose did not, or only slightly, affect the growth, cellular ultrastructure and mitochondrial processes in Phytophthora cinnamomi, indicating a species-specific response to this rare sugar. The mode of action of tagatose against P. infestans was mainly based on the inhibition of mitochondrial processes and this rare sugar seems to be a promising active substance for the further development of eco-friendly fungicides, thanks to its anti-nutritional properties on some phytopathogens and low risk for human health.

Ecological impact of a rare sugar on grapevine phyllosphere microbial communities.

Plants host a complex microbiota inside or outside their tissues, and phyllosphere microorganisms can be influenced by environmental, nutritional and agronomic factors. Rare sugars are defined as monosaccharides with limited availability in nature and they are metabolised by only few certain microbial taxa. Among rare sugars, tagatose (TAG) is a low-calories sweetener that stimulates and inhibits beneficial and pathogenic bacteria in the human gut microbiota, respectively. Based on this differential effect on human-associated microorganisms, we investigated the effect of TAG treatments on the grapevine phyllosphere microorganisms to evaluate whether it can engineer the microbiota and modify the ratio between beneficial and pathogenic plant-associated microorganisms. TAG treatments changed the structure of the leaf microbiota and they successfully reduced leaf infections of downy mildew (caused by Plasmopara viticola) and powdery mildew (caused by Erysiphe necator) under field conditions. TAG increased the relative abundance of indigenous beneficial microorganisms, such as some potential biocontrol agents, which could partially contribute to disease control. The taxonomic composition of fungal and bacterial leaf populations differed according to grapevine locations, therefore TAG effects on the microbial structure were influenced by the composition of the originally residing microbiota. TAG is a promising biopesticide that could shift the balance of pathogenic and beneficial plant-associated microorganisms, suggesting selective nutritional/anti-nutritional properties for some specific taxa. More specifically, TAG displayed possible plant prebiotic effects on the phyllosphere microbiota and this mechanism of action could represent a novel strategy that can be further developed for sustainable plant protection.

Functional components of the bacterial CzcCBA efflux system reduce cadmium uptake and accumulation in transgenic tobacco plants.

Cadmium (Cd) is a toxic trace element released into the environment by industrial and agricultural practices, threatening the health of plants and contaminating the food/feed chain. Biotechnology can be used to develop plant varieties with a higher capacity for Cd accumulation (for use in phytoremediation programs) or a lower capacity for Cd accumulation (to reduce Cd levels in food and feed). Here we generated transgenic tobacco plants expressing components of the Pseudomonas putida CzcCBA efflux system. Plants were transformed with combinations of the CzcC, CzcB and CzcA genes, and the impact on Cd mobilization was analysed. Plants expressing PpCzcC showed no differences in Cd accumulation, whereas those expressing PpCzcB or PpCzcA accumulated less Cd in the shoots, but more Cd in the roots. Plants expressing both PpCzcB and PpCzcA accumulated less Cd in the shoots and roots compared to controls, whereas plants expressing all three genes showed a significant reduction in Cd levels only in shoots. These results show that components of the CzcCBA system can be expressed in plants and may be useful for developing plants with a reduced capacity to accumulate Cd in the shoots, potentially reducing the toxicity of food/feed crops cultivated in Cd-contaminated soils.

Leaf Treatments with a Protein-Based Resistance Inducer Partially Modify Phyllosphere Microbial Communities of Grapevine.

Protein derivatives and carbohydrates can stimulate plant growth, increase stress tolerance, and activate plant defense mechanisms. However, these molecules can also act as a nutritional substrate for microbial communities living on the plant phyllosphere and possibly affect their biocontrol activity against pathogens. We investigated the mechanisms of action of a protein derivative (nutrient broth, NB) against grapevine downy mildew, specifically focusing on the effects of foliar treatments on plant defense stimulation and on the composition and biocontrol features of the phyllosphere microbial populations. NB reduced downy mildew symptoms and induced the expression of defense-related genes in greenhouse- and in vitro-grown plants, indicating the activation of grapevine resistance mechanisms. Furthermore, NB increased the number of culturable phyllosphere bacteria and altered the composition of bacterial and fungal populations on leaves of greenhouse-grown plants. Although, NB-induced changes on microbial populations were affected by the structure of indigenous communities originally residing on grapevine leaves, degrees of disease reduction and defense gene modulation were consistent among the experiments. Thus, modifications in the structure of phyllosphere populations caused by NB application could partially contribute to downy mildew control by competition for space or other biocontrol strategies. Particularly, changes in the abundance of phyllosphere microorganisms may provide a contribution to resistance induction, partially affecting the hormone-mediated signaling pathways involved. Modifying phyllosphere populations by increasing natural biocontrol agents with the application of selected nutritional factors can open new opportunities in terms of sustainable plant protection strategies.

A complex protein derivative acts as biogenic elicitor of grapevine resistance against powdery mildew under field conditions.

Powdery mildew caused by Erysiphe necator is one of the most important grapevine diseases in several viticulture areas, and high fungicide input is required to control it. However, numerous synthetic chemical pesticides are under scrutiny due to concerns about their impact on human health and the environment. Biopesticides, such as biogenic elicitors, are a promising alternative to chemical fungicides. Although several studies have reported on effective elicitors against grapevine diseases, their efficacy under field conditions has not been investigated extensively or has occurred at rather limited levels. Our goal was to examine the efficacy of a protein-based composition, namely nutrient broth (NB), against powdery mildew under field conditions and to characterize its mechanism of action. Weekly treatments with NB was highly effective in controlling powdery mildew on grapevine across seasons with different disease pressures. The level of disease control achieved with NB was comparable to standard fungicide treatments both on leaves and bunches across three different years. NB has no direct toxic effect on the germination of E. necator conidia, and it activates plant resistance with both systemic and translaminar effect in experiments with artificial inoculation under controlled conditions. NB induced the expression of defense-related genes in grapevine, demonstrating stimulation of plant defense mechanisms, prior to and in the early stages of pathogen infection. NB is a natural derivative from meat and yeast, substances that tend not to raise concerns about toxicological and ecotoxicological properties. NB represents a valid control tool for integrated plant protection programs against powdery mildew, to reduce the use of synthetic pesticides on grapevine.