Elevated CO2 stimulates plant growth and affects quantity and composition of root exudates, followed by response of its microbiome. Three scenarios representing nitrate fertilization regimes: limited (30 ppm), moderate (70 ppm) and excess nitrate (100 ppm) were compared under ambient and elevated CO2 (eCO(2), 850 ppm) to elucidate their combined effects on root-surface-associated bacterial community abundance, structure and function. Wheat root-surface-associated microbiome structure and function, as well as soil and plant properties, were highly influenced by interactions between CO2 and nitrate levels. Relative abundance of total bacteria per plant increased at eCO(2) under excess nitrate. Elevated CO2 significantly influenced the abundance of genes encoding enzymes, transporters and secretion systems. Proteobacteria, the largest taxonomic group in wheat roots (similar to 75%), is the most influenced group by eCO(2) under all nitrate levels. Rhizobiales, Burkholderiales and Pseudomonadales are responsible for most of these functional changes. A correlation was observed among the five gene-groups whose abundance was significantly changed (secretion systems, particularly type VI secretion system, biofilm formation, pyruvate, fructose and mannose metabolism). These changes in bacterial abundance and gene functions may be the result of alteration in root exudation at eCO(2), leading to changes in bacteria colonization patterns and influencing their fitness and proliferation.

Root selection of their associated microbiome composition and activities is determined by the plant's developmental stage and distance from the root. Total gene abundance, structure and functions of root-associated and rhizospheric microbiomes were studied throughout wheat growth season under field conditions. On the root surface, abundance of the well-known wheat colonizers Proteobacteria and Actinobacteria decreased and increased, respectively, during spike formation, whereas abundance of Bacteroidetes was independent of spike formation. Metagenomic analysis combined with functional co-occurrence networks revealed a significant impact of plant developmental stage on its microbiome during the transition from vegetative growth to spike formation. For example, gene functions related to biofilm and sensorial movement, antibiotic production and resistance and carbons and amino acids and their transporters. Genes associated with these functions were also in higher abundance in root vs. the rhizosphere microbiome. We propose that abundance of transporter-encoding genes related to carbon and amino acid, may mirror the availability and utilization of root exudates. Genes related to antibiotic resistance mechanisms were abundant during vegetative growth, while after spike formation, genes related to the biosynthesis of various antibiotics were enriched. This observation suggests that during root colonization and biofilm formation, bacteria cope with competitor's antibiotics, whereas in the mature biofilm stage, they invest in inhibiting new colonizers. Additionally, there is higher abundance of genes related to denitrification in rhizosphere compared to root-associated microbiome during wheat growth, possibly due to competition with the plant over nitrogen in the root vicinity. We demonstrated functional and phylogenetic division in wheat root zone microbiome in both time and space: pre- and post-spike formation, and root-associated vs. rhizospheric niches. These findings shed light on the dynamics of plant-microbe and microbe-microbe interactions in the developing root zone.

Bacteria are social organisms that interact extensively within and between species while responding to external stimuli from their environments. Designing synthetic microbial communities can enable efficient and beneficial microbiome implementation in many areas. However, in order to design an efficient community, one must consider the interactions between their members. Using a reductionist approach, we examined pairwise interactions of three related Pseudomonas species in various microenvironments including plant roots and inert surfaces. Our results show that the step between monoculture and co-culture is already very complex. Monoculture root colonization patterns demonstrate that each isolate occupied a particular location on wheat roots, such as root tip, distance from the tip, or scattered along the root. However, pairwise colonization outcomes on the root did not follow the bacterial behavior in monoculture, suggesting various interaction patterns. In addition, we show that interspecies interactions on a microscale on inert surface take part in co-culture colonization and that the interactions are affected by the presence of root extracts and depend on its source. The understanding of interrelationships on the root may contribute to future attempts to manipulate and improve bacterial colonization and to intervene with root microbiomes to construct and design effective synthetic microbial consortia.

Elevated CO2 (eCO(2)) stimulates plant growth and photosynthesis, which affect root deposition, leading to altered structure and function of the root microbiome. We studied the effect of eCO(2) on wheat-root microbiome composition and plant development, with an emphasis on denitrifying communities. Wheat plants were grown in a greenhouse with continuous fertigation for 6 weeks under ambient CO2 (400 ppm) or eCO(2) (850 ppm). The total bacterial community was quantified using qPCR with universal 16S rRNA gene primers, and denitrifying genes (nirK, nirS, nosZ) were measured. In addition, total (16S-based) and N2O-reducing (nosZ-based) bacterial community compositions in the soil and roots were analyzed by amplicon sequencing during plant growth. eCO(2) had a significant impact on abundance of the studied denitrifying genes, particularly during the late stages of wheat growth before spike formation. Moreover, eCO(2) had a significant impact on N2O-reducing community structure in roots. This effect was more pronounced on Burkholderiales and Rhizobiales, with a minor effect on Pseudomonadales. In addition, as expected, bacterial community structure (total and N2O-reducing bacteria), and denitrifying gene abundance, were primary influenced by habitat (soil vs. roots), and secondarily by plant developmental stage. In summary, it is suggested that eCO(2) may change root microbiome, enhance wheat development and N demand without an increase in N2O emission.

Small secreted proteins (SSPs) comprise 40-60% of the total fungal secretome and are present in fungi of all phylogenetic groups, representing the entire spectrum of lifestyles. They are characteristically shorter than 300 amino acids in length and have a signal peptide. The majority of SSPs are coded by orphan genes, which lack known domains or similarities to known protein sequences. Effectors are a group of SSPs that have been investigated extensively in fungi that interact with living hosts, either pathogens or mutualistic systems. They are involved in suppressing the host defense response and altering its physiology. Here, we aim to delineate some of the potential roles of SSPs in saprotrophic fungi, that have been bioinformatically predicted as effectors, and termed in this mini-review as ``effector-like'' proteins. The effector-like Ssp1 from the white-rot fungus Pleurotus ostreatus is presented as a case study, and its potential role in regulating the ligninolytic system, secondary metabolism, development, and fruiting body initiation are discussed. We propose that deciphering the nature of effector-like SSPs will contribute to our understanding of development and communication in saprophytic fungi, as well as help, to elucidate the origin, regulation, and mechanisms of fungal-host, fungal-fungal, and fungal-bacterial interactions.

Fungi have the ability to transform organic materials into a rich and diverse set of useful products and provide distinct opportunities for tackling the urgent challenges before all humans. Fungal biotechnology can advance the transition from our petroleum-based economy into a bio-based circular economy and has the ability to sustainably produce resilient sources of food, feed, chemicals, fuels, textiles, and materials for construction, automotive and transportation industries, for furniture and beyond. Fungal biotechnology offers solutions for securing, stabilizing and enhancing the food supply for a growing human population, while simultaneously lowering greenhouse gas emissions. Fungal biotechnology has, thus, the potential to make a significant contribution to climate change mitigation and meeting the United Nation’s sustainable development goals through the rational improvement of new and established fungal cell factories. The White Paper presented here is the result of the 2nd Think Tank meeting held by the EUROFUNG consortium in Berlin in October 2019. This paper highlights discussions on current opportunities and research challenges in fungal biotechnology and aims to inform scientists, educators, the general public, industrial stakeholders and policymakers about the current fungal biotech revolution.

The function of small secreted proteins (SSPs) in saprotrophic fungi is, for the most part, unknown. The white-rot mushroom Pleurotus ostreatus produces considerable amounts of SSPs at the onset of secondary metabolism, during colony development, and in response to chemical compounds such as 5-hydroxymethylfurfural and aryl alcohols. Genetic manipulation of Ssp1, by knockdown (KDssp1) or overexpression (OEssp1), indicated that they are, in fact, involved in the regulation of the ligninolytic system. To elucidate their potential involvement in fungal development, quantitative secretome analysis was performed during the trophophase and the idiophase and at a transition point between the two growth phases. The mutations conferred a time shift in the secretion and expression patterns: OEssp1 preceded the entrance to idiophase and secondary metabolism, while KDssp1 was delayed. This was also correlated with expression patterns of selected genes. The KDssp1 colony aged at a slower pace, accompanied by a slower decline in biomass over time. In contrast, the OEssp1 strain exhibited severe lysis and aging of the colony at the same time point. These phenomena were accompanied by variations in yellow pigment production, characteristic of entrance of the wild type into idiophase. The pigment was produced earlier and in a larger amount in the OEssp1 strain and was absent from the KDssp1 strain. Furthermore, the dikaryon harboring OEssp1 exhibited a delay in the initiation of fruiting body formation as well as earlier aging. We propose that Ssp1 might function as a part of the fungal communication network and regulate the pattern of fungal development and metabolism in P. ostreatus.IMPORTANCE Small secreted proteins (SSPs) are common in fungal saprotrophs, but their roles remain elusive. As such, they comprise part of a gene pool which may be involved in governing fungal lifestyles not limited to symbiosis and pathogenicity, in which they are commonly referred to as “effectors.” We propose that Ssp1 in the white-rot fungus Pleurotus ostreatus regulates the transition from primary to secondary metabolism, development, aging, and fruiting body initiation. Our observations uncover a novel regulatory role of effector-like SSPs in a saprotroph, suggesting that they may act in fungal communication as well as in response to environmental cues. The presence of Ssp1 homologues in other fungal species supports a common potential role in environmental sensing and fungal development.

β-Glucosidases are key enzymes in the process of cellulose utilization. It is the last enzyme in the cellulose hydrolysis chain, which converts cellobiose to glucose. Since cellobiose is known to have a feedback inhibitory effect on a variety of cellulases, β-glucosidase can prevent this inhibition by hydrolyzing cellobiose to non-inhibitory glucose. While the optimal temperature of the Clostridium thermocellum cellulosome is 70 °C, C. thermocellum β-glucosidase A is almost inactive at such high temperatures. Thus, in the current study, a random mutagenesis directed evolutionary approach was conducted to produce a thermostable mutant with Kcat and Km, similar to those of the wild-type enzyme. The resultant mutant contained two mutations, A17S and K268N, but only the former was found to affect thermostability, whereby the inflection temperature (Ti) was increased by 6.4 °C. A17 is located near the central cavity of the native enzyme. Interestingly, multiple alignments revealed that position 17 is relatively conserved, whereby alanine is replaced only by serine. Upon the addition of the thermostable mutant to the C. thermocellum secretome for subsequent hydrolysis of microcrystalline cellulose at 70 °C, a higher soluble glucose yield (243%) was obtained compared to the activity of the secretome supplemented with the wild-type enzyme. © 2019 by the authors. Licensee MDPI, Basel, Switzerland.

Plant hosts recruit and maintain a distinct root-associated microbiota based on host and bacterium traits. However, past studies disregarded microbial strain-host specificity and spatial micro-heterogeneity of the root compartment. Using genetic manipulation, confocal laser scanning microscopy, real-time quantitative PCR, and genome sequencing we characterized the colonization patterns of three Pseudomonas spp. isolates native to wheat roots, on the micro-scale. Namely, isolates P. fluorescens NT0133, P. stutzeri NT124, and P. stutzeri NT128. All three isolates preferentially colonized wheat over cucumber roots that served as control for host specificity. Furthermore, not only had the isolates strong host specificity but each isolate had a distinct spatial distribution on the root, all within a few millimeters. Isolate P. stutzeri-NT0124 preferentially colonized root tips, whereas P. fluorescens-NT0133 showed a preference for zones distant from the tip. In contrast, isolate P. stutzeri-NT0128 had no preference for a specific niche on the root. While all isolates maintained genetic potential for motility and biofilm formation their phenotype varied significantly and corresponded to their niche preference. These results demonstrate the importance of spatial colonization patterns, governed by both niche and bacterial characteristics which will have great importance in future attempts to manipulate the plant microbiome by constructing synthetic microbial consortia. © 2019 Frontiers Media S.A. All Rights Reserved.

We have recently demonstrated that we could enhance glucan content in Pleurotus eryngii following cultivation of the mushrooms on a substrate containing different concentrations of olive mill solid waste (OMSW). These changes are directly related to the content of OMSW in the growing substrate. Using dextran sulfate sodium (DSS)-inflammatory bowel disease (IBD) mice model, we measured the colonic inflammatory response to the different glucan preparations. We found that the histology damaging score (HDS) resulting from DSS treatment reach a value of 11.8 ± 2.3 were efficiently downregulated by treatment with the fungal extracted glucans. Glucans extracted from stalks cultivated at 20% OMSW downregulated to a HDS value of 6.4 ± 0.5 whereas those cultivated at 80% OMSW showed the strongest effects (5.5 ± 0.6). Similar downregulatory effects were obtained for expression of various intestinal cytokines. All tested glucans were equally effective in regulating the number of CD14/CD16 monocytes from 18.2 ± 2.7% for DSS to 6.4 ± 2.0 for DSS + glucans extracted from stalks cultivated at 50% OMSW. We tested the effect of glucans on lipopolysaccharide- induced production of TNF-α, which demonstrated that stalk-derived glucans were more effective than caps-derived glucans. Isolated glucans competed with anti-Dectin-1 and anti-CR3 antibodies, indicating that they contain β-glucans recognized by these receptors. In conclusion, the most effective glucans in ameliorating IBD-associated symptoms induced by DSS treatment in mice were glucan extracts prepared from the stalk of P. eryngii grown at higher concentrations of OMSW. We conclude that these stress-induced growing conditions may be helpful in selecting more effective glucans derived from edible mushrooms. © 2019 by Begell House, Inc.

Background: Biofuels derived from lignocellulosic biomass are a viable alternative to fossil fuels required for transportation. Following plant biomass pretreatment, the furan derivative furfural is present at concentrations which are inhibitory to yeasts. Detoxification of furfural is thus important for efficient fermentation. Here, we searched for new genetic attributes in the fungus Neurospora crassa that may be linked to furfural tolerance. The fact that furfural is involved in the natural process of sexual spore germination of N. crassa and that this fungus is highly amenable to genetic manipulations makes it a rational candidate for this study. Results: Both hypothesis-based and unbiased (random promotor mutagenesis) approaches were performed to identify N. crassa genes associated with the response to furfural. Changes in the transcriptional profile following exposure to furfural revealed that the affected processes were, overall, similar to those observed in Saccharomyces cerevisiae. N. crassa was more tolerant (by ∼ 30%) to furfural when carboxymethyl cellulose was the main carbon source as opposed to sucrose, indicative of a link between carbohydrate metabolism and furfural tolerance. We also observed increased tolerance in a Δcre-1 mutant (CRE-1 is a key transcription factor that regulates the ability of fungi to utilize non-preferred carbon sources). In addition, analysis of aldehyde dehydrogenase mutants showed that ahd-2 (NCU00378) was involved in tolerance to furfural as well as the predicted membrane transporter NCU05580 (flr-1), a homolog of FLR1 in S. cerevisiae. Further to the rational screening, an unbiased approach revealed additional genes whose inactivation conferred increased tolerance to furfural: (i) NCU02488, which affected the abundance of the non-anchored cell wall protein NCW-1 (NCU05137), and (ii) the zinc finger protein NCU01407. Conclusions: We identified attributes in N. crassa associated with tolerance or degradation of furfural, using complementary research approaches. The manipulation of the genes involved in furan sensitivity can provide a means for improving the production of biofuel producing strains. Similar research approaches can be utilized in N. crassa and other filamentous fungi to identify additional attributes relevant to other furans or toxic chemicals. © 2019 The Author(s).

One of the most persistent pharmaceutical compounds commonly found in treated wastewater is lamotrigine (LTG). It has also been detected in soils and crops irrigated with treated wastewater. Here we focused on the ability of the white-rot edible mushroom Pleurotus ostreatus to remove and transform LTG in liquid cultures. At concentrations of environmental relevance (1 and 10 μg L−1) LTG was almost completely removed from the culture medium within 20 days. To elucidate the mechanism of LTG removal and transformation, we applied a physiological-based approach using inhibitors and a competing agent. These experiments were conducted at a higher concentration for metabolites detection. Based on identification of sulfur-containing metabolites and LTG N2-oxide and the effect of specific inhibitors, cytochrome P450 oxidation is suggested as one of the reaction mechanisms leading to LTG transformation. The variety and number of transformation products (i.e., conjugates) found in the current study were larger than reported in mammals. Moreover, known conjugates with glucuronide, glutathione, or cysteine/glycine, were not found in our system. Since the majority of the identified transformation products were conjugates of LTG, this study highlights the persistence of LTG as an organic pollutant in ecosystems exposed to wastewater.

Nitrous oxide (N2O) is a greenhouse gas and a potent ozone-depleting substance in the stratosphere. Agricultural soils are one of the main global sources of N2O emissions, particularly from cereal fields due to their high areal coverage. The aim of this study was to isolate N2O-reducing bacteria able to mitigate N2O emissions from the soil after inoculation. We isolated several bacteria from wheat roots that were capable of N2O reduction in vitro and studied their genetic potential and activity under different environmental conditions. Three of these isolates- all carrying the nitrous oxide reductase-encoding clade I nosZ, able to reduce N2O in vitro, and efficient colonizers of wheat roots- presented different N2O-reduction strategies when growing in the root zone, possibly due to the different conditions in situ and their metabolic preferences. Each isolate seemed to prefer to operate at different altered oxygen levels. Isolate AU243 (related to Agrobacterium/Rhizobium) could reduce both nitrate and N2O and operated better at lower oxygen levels. Isolate AU14 (related to Alcaligenes faecalis), lacking nitrate reductases, operated better under less anoxic conditions. Isolate NT128 (related to Pseudomonas stutzeri) caused slightly increased N2O emissions under both anoxic and ambient conditions. These results therefore emphasize the importance of a deep understanding of soil–plant–microbe interactions when environmental application is being considered.

With increasing fresh water (FW) scarcity, the use of treated wastewater (TWW) for crop irrigation is expanding globally. Besides clear benefits, some undesired long-term effects of irrigation with this low quality water on plant performance have been reported. As the rhizosphere microbiome can mediate plant-soil interactions, an examination of the response of these organisms to TWW is necessary to understand the full effects of water quality. In the current study, the effects of irrigation water quality on the microbial community structure of soil and roots as well as edaphic properties and plant performance were evaluated. We compared soil and roots microbiomes of two different plant species (tomato and lettuce), each grown in two distinct soils, and irrigated with either FW or TWW. Irrigation with TWW significantly increase soil pH, EC, K, Na and DOC, and decrease plant fruit and shoot weight, relatively to samples irrigated with FW. We calculated the effect size of plant species, soil type, and irrigation water quality on microbial community structure in soil and root. In the roots, plant species and irrigation water were the dominant factors in shaping both total (DNA based) and active (RNA based) microbial communities, with both factors contributing similarly to the observed microbial population. Soil type and irrigation water were the dominant factors shaping the total microbial community in the soil and were of similar magnitude. Irrigation water quality is demonstrated to be a major force in shaping root-associated microbiome, leading to altered microbial community structure in the critical juncture between plant and soil.

We compared the immune-modulating activity of glucans extracted from P. ostreatus and P. eryngii on phagocytosis of peripheral blood neutrophils, and superoxide release from HL-60 cells. The results suggest that the anti-inflammatory properties of these glucans are partially mediated through modulation of neutrophil effector functions (P. eryngii was more effective). Additionally, both glucans dose-dependently competed for the anti-Dectin-1 and anti-CR3 antibody binding. We then tested the putative anti-inflammatory effects of the extracted glucans in inflammatory bowel disease (IBD) using the dextran sulfate sodium (DSS)–induced model in mice. The clinical symptoms of IBD were efficiently relieved by the treatment with two different doses of the glucan from both fungi. Glucan fractions, from either P. ostreatus or P. eryngii, markedly prevented TNF-α mediated inflammation in the DSS–induced inflamed intestine. These results suggest that there are variations in glucan preparations from different fungi in their anti-inflammatory ability.

During the process of bioethanol production, cellulose is hydrolyzed into its monomeric soluble units. For efficient hydrolysis, a chemical and/or mechanical pretreatment step is required. Such pretreatment is designed to increase enzymatic digestibility of the cellulose chains inter alia by de-crystallization of the cellulose chains and by removing barriers, such as lignin from the plant cell wall. Biological pretreatment, in which lignin is decomposed or modified by white-rot fungi, has also been considered. One disadvantage in biological pretreatment, however, is the consumption of the cellulose by the fungus. Thus, fungal species that attack lignin with only minimal cellulose loss are advantageous. The secretomes of white-rot fungi contain carbohydrate-active enzymes (CAZymes) including lignin-modifying enzymes. Thus, modification of secretome composition can alter the ratio of lignin/cellulose degradation.

: Pleurotus eryngii is recognized for its prominent nutritional and medicinal value. In our study, we tested the effect of glucans on lipopolysaccharide (LPS)-induced production of TNF-α. We demonstrated that glucan extracts are more effective than mill mushroom preparations. Additionally, the effectiveness of stalk-derived glucans were slightly more pronounced than of caps. Cap and stalk glucans from mill or isolated glucan competed dose-dependently with anti-Dectin-and anti-CR-3 antibodies, indicating that they contain β-glucans recognized by these receptors. Using the dextran sulfate sodium (DSS)-inflammatory bowel disease mice model, intestinal inflammatory response to the mill preparations was measured and compared to extracted glucan fractions from caps and stalks. We found that mill and glucan extracts were very effective in downregulating IFN-γ and MIP-2 levels and that stalk-derived preparations were more effective than from caps. The tested glucans were equally effective in regulating the number of CD14/CD16 monocytes and upregulating the levels of fecal-released IgA to almost normal levels. In conclusion, the most effective glucans in ameliorating some IBD-inflammatory associated symptoms induced by DSS treatment in mice were glucan extracts prepared from the stalk of P. eryngii. These spatial distinctions may be helpful in selecting more effective specific anti-inflammatory mushrooms-derived glucans.

ABSTRACTSeveral composts were tested for their capacity to moderate the effect of Verticillium dahliae Kleb. (VCG B4, VD) on eggplant (Solanum melongena) under greenhouse conditions. Eggplants plantlets were inoculated by immersing their roots in conidial suspension and then planted in pots filled with mixtures of compost or peat moss, mixed with perlite. Six composts and peat moss mixtures were tested, of which tomato waste compost suppressed V. dahliae, and turkey litter compost partially suppressed it. Reduced levels of symptoms and lower fungal colonization were detected in the xylem of eggplants planted in tomato waste compost, and these plants accumulated more dry matter and had higher chlorophyll content compared to other media. However, survival of conidia in tomato waste compost showed only a moderate decrease compared with a sharp decrease in other media, suggesting that conidial eradication cannot be proposed as the suppressiveness mechanism. ? irradiation of tomato waste compost and peat at 2.5 Mrad reduced microorganism density by four orders of magnitude, but irradiation of tomato waste compost did not reduce its suppressiveness of V. dahliae. Composts properties affected progress rate of VD in the xylem tissue of eggplant seedling. These properties could indicate both biotic and abiotic factors affecting the process.

Anthropogenic activities alter the structure and function of a bacterial community. Furthermore, bacterial communities structured by the conditions the anthropogenic activities present may consequently reduce their stability in response to an unpredicted acute disturbance. The present mesocosm-scale study exposed soil bacterial communities to different irrigation water types, including freshwater, fertilized freshwater, treated wastewater, and artificial wastewater, and evaluated their response to a disturbance caused by heat. These effectors may be considered deterministic and stochastic forces common in agricultural operations of arid and semiarid regions. Bacterial communities under conditions of high mineral and organic carbon availability (artificial wastewater) differed from the native bacterial community and showed a proteobacterial dominance. These bacterial communities had a lower resistance to the heat treatment disturbance than soils under conditions of low resource availability (high-quality treated wastewater or freshwater). The latter soil bacterial communities showed a higher abundance of operational taxonomic units (OTUs) classified as Bacilli. These results were elucidated by soil under conditions of high resource availability, which lost higher degrees of functional potential and had a greater bacterial community composition change. However, the functional resilience, after the disturbance ended, was higher under a condition of high resource availability despite the bacterial community composition shift and the decrease in species richness. The functional resilience was directly connected to the high growth rates of certain Bacteroidetes and proteobacterial groups. A high stability was found in samples that supported the coexistence of both resistant OTUs and fast-growing OTUs.IMPORTANCE This report presents the results of a study employing a hypothesis-based experimental approach to reveal the forces involved in determining the stability of a soil bacterial community to disturbance. The resultant postdisturbance bacterial community composition dynamics and functionality were analyzed. The paper demonstrates the relatedness of community structure and stability under cultivation conditions prevalent in an arid area under irrigation with water of different qualities. The use of common agricultural practices to demonstrate these features has not been described before. The combination of a fundamental theoretical issue in ecology with common and concerning disturbances caused by agricultural practice makes this study unique. Furthermore, the results of the present study have applicable importance regarding soil conservation, as it enables a better characterization and monitoring of stressed soil bacterial communities and possible intervention to reduce the stress. It will also be of valued interest in coming years, as fresh water scarcity and the use of alternative water sources are expected to rise globally.