The wild chili pepper Capsicum chacoense produces the spicy defense compounds known as capsaicinoids, including capsaicin and dihydrocapsaicin, which are antagonistic to the growth of fungal pathogens. Compared to other microbes, fungi isolated from infected seeds of C. chacoense possess much higher levels of tolerance of these spicy compounds, having their growth slowed but not entirely inhibited. Previous research has shown capsaicinoids inhibit microbes by disrupting ATP production by binding NADH dehydrogenase in the electron transport chain (ETC) and, thus, throttling oxidative phosphorylation (OXPHOS). Capsaicinoids may also disrupt cell membranes. Here, we investigate capsaicinoid tolerance in fungal seed pathogens isolated from C. chacoense We selected 16 fungal isolates from four ascomycete genera (Alternaria, Colletotrichum, Fusarium, and Phomopsis). Using relative growth rate as a readout for tolerance, fungi were challenged with ETC inhibitors to infer whether fungi possess alternative respiratory enzymes and whether effects on the ETC fully explained inhibition by capsaicinoids. In all isolates, we found evidence for at least one alternative NADH dehydrogenase. In many isolates, we also found evidence for an alternative oxidase. These data suggest that wild-plant pathogens may be a rich source of alternative respiratory enzymes. We further demonstrate that these fungal isolates are capable of the breakdown of capsaicinoids. Finally, we determine that the OXPHOS theory may describe a weak primary mechanism by which dihydrocapsaicin, but not capsaicin, slows fungal growth. Our findings suggest that capsaicinoids likely disrupt membranes, in addition to energy poisoning, with implications for microbiology and human health.IMPORTANCE Plants make chemical compounds to protect themselves. For example, chili peppers produce the spicy compound capsaicin to inhibit pathogen damage and animal feeding. In humans, capsaicin binds to a membrane channel protein, creating the sensation of heat, while in microbes, capsaicin limits energy production by binding respiratory enzymes. However, some data suggest that capsaicin also disrupts membranes. Here, we studied fungal pathogens (Alternaria, Colletotrichum, Fusarium, and Phomopsis) isolated from a wild chili pepper, Capsicum chacoense By measuring growth rates in the presence of antibiotics with known respiratory targets, we inferred that wild-plant pathogens might be rich in alternative respiratory enzymes. A zone of clearance around the colonies, as well as liquid chromatography-mass spectrometry data, further indicated that these fungi can break down capsaicin. Finally, the total inhibitory effect of capsaicin was not fully explained by its effect on respiratory enzymes. Our findings lend credence to studies proposing that capsaicin may disrupt cell membranes, with implications for microbiology, as well as human health.

Within the landscape of globally distributed pathogens, populations differentiate via both adaptive and nonadaptive forces. Individual populations are likely to show unique trends of genetic diversity, host-pathogen interaction, and ecological adaptation. In plant pathogens, allopatric divergence may occur particularly rapidly within simplified agricultural monoculture landscapes. As such, the study of plant pathogen populations in monocultures can highlight the distinct evolutionary mechanisms that lead to local genetic differentiation. Xylella fastidiosa is a plant pathogen known to infect and damage multiple monocultures worldwide. One subspecies, Xylella fastidiosa subsp. fastidiosa, was first introduced to the United States ∼150 years ago, where it was found to infect and cause disease in grapevines (Pierce's disease of grapevines, or PD). Here, we studied PD-causing subsp. fastidiosa populations, with an emphasis on those found in the United States. Our study shows that following their establishment in the United States, PD-causing strains likely split into populations on the East and West Coasts. This diversification has occurred via both changes in gene content (gene gain/loss events) and variations in nucleotide sequence (mutation and recombination). In addition, we reinforce the notion that PD-causing populations within the United States acted as the source for subsequent subsp. fastidiosa outbreaks in Europe and Asia.IMPORTANCE Compared to natural environments, the reduced diversity of monoculture agricultural landscapes can lead bacterial plant pathogens to quickly adapt to local biological and ecological conditions. Because of this, accidental introductions of microbial pathogens into naive regions represents a significant economic and environmental threat. Xylella fastidiosa is a plant pathogen with an expanding host and geographic range due to multiple intra- and intercontinental introductions. X. fastidiosa subsp. fastidiosa infects and causes disease in grapevines (Pierce's disease of grapevines [PD]). This study focused on PD-causing X. fastidiosa populations, particularly those found in the United States but also invasions into Taiwan and Spain. The analysis shows that PD-causing X. fastidiosa has diversified via multiple cooccurring evolutionary forces acting at an intra- and interpopulation level. This analysis enables a better understanding of the mechanisms leading to the local adaptation of X. fastidiosa and how a plant pathogen diverges allopatrically after multiple and sequential introduction events.

Agricultural management practices affect bulk soil microbial communities and the functions they carry out, but it remains unclear how these effects extend to the rhizosphere in different agroecosystem contexts. Given close linkages between rhizosphere processes and plant nutrition and productivity, understanding how management practices impact this critical zone is of great importance to optimize plant-soil interactions for agricultural sustainability. A comparison of six paired conventional-organic processing tomato farms was conducted to investigate relationships between management, soil physicochemical parameters, and rhizosphere microbial community composition and functions. Organically managed fields were higher in soil total N and NO₃-N, total and labile C, plant Ca, S, and Cu, and other essential nutrients, while soil pH was higher in conventionally managed fields. Differential abundance, indicator species, and random forest analyses of rhizosphere communities revealed compositional differences between organic and conventional systems and identified management-specific microbial taxa. Phylogeny-based trait prediction showed that these differences translated into more abundant pathogenesis-related gene functions in conventional systems. Structural equation modeling revealed a greater effect of soil biological communities than physicochemical parameters on plant outcomes. These results highlight the importance of rhizosphere-specific studies, as plant selection likely interacts with management in regulating microbial communities and functions that impact agricultural productivity.IMPORTANCE Agriculture relies, in part, on close linkages between plants and the microorganisms that live in association with plant roots. These rhizosphere bacteria and fungi are distinct from microbial communities found in the rest of the soil and are even more important to plant nutrient uptake and health. Evidence from field studies shows that agricultural management practices such as fertilization and tillage shape microbial communities in bulk soil, but little is known about how these practices affect the rhizosphere. We investigated how agricultural management affects plant-soil-microbe interactions by comparing soil physical and chemical properties, plant nutrients, and rhizosphere microbial communities from paired fields under organic and conventional management. Our results show that human management effects extend even to microorganisms living in close association with plant roots and highlight the importance of these bacteria and fungi to crop nutrition and productivity.

Filamentous fungi are intensively used for producing industrial enzymes, including lignocellulases. Employing insoluble cellulose to induce the production of lignocellulases causes some drawbacks, e.g., a complex fermentation operation, which can be overcome by using soluble inducers such as cellobiose. Here, a triple β-glucosidase mutant of Neurospora crassa, which prevents rapid turnover of cellobiose and thus allows the disaccharide to induce lignocellulases, was applied to profile the proteome responses to cellobiose and cellulose (Avicel). Our results revealed a shared proteomic response to cellobiose and Avicel, whose elements included lignocellulases and cellulolytic product transporters. While the cellulolytic proteins showed a correlated increase in protein and mRNA levels, only a moderate correlation was observed on a proteomic scale between protein and mRNA levels (R² = 0.31). Ribosome biogenesis and rRNA processing were significantly overrepresented in the protein set with increased protein but unchanged mRNA abundances in response to Avicel. Ribosome biogenesis, as well as protein processing and protein export, was also enriched in the protein set that showed increased abundance in response to cellobiose. NCU05895, a homolog of yeast CWH43, is potentially involved in transferring a glycosylphosphatidylinositol (GPI) anchor to nascent proteins. This protein showed increased abundance but no significant change in mRNA levels. Disruption of CWH43 resulted in a significant decrease in cellulase activities and secreted protein levels in cultures grown on Avicel, suggesting a positive regulatory role for CWH43 in cellulase production. The findings should have an impact on a systems engineering approach for strain improvement for the production of lignocellulases.IMPORTANCE Lignocellulases are important industrial enzymes for sustainable production of biofuels and bio-products. Insoluble cellulose has been commonly used to induce the production of lignocellulases in filamentous fungi, which causes a difficult fermentation operation and enzyme loss due to adsorption to cellulose. The disadvantages can be overcome by using soluble inducers, such as the disaccharide cellobiose. Quantitative proteome profiling of the model filamentous fungus Neurospora crassa revealed cellobiose-dependent pathways for cellulase production, including protein processing and export. A protein (CWH43) potentially involved in protein processing was found to be a positive regulator of lignocellulase production. The cellobiose-dependent mechanisms provide new opportunities to improve the production of lignocellulases in filamentous fungi.

Geothermal systems emit substantial amounts of aqueous, gaseous, and methylated mercury, but little is known about microbial influences on mercury speciation. Here, we report results from genome-resolved metagenomics and mercury speciation analysis of acidic warm springs in the Ngawha Geothermal Field (<55°C, pH <4.5), Northland Region, Aotearoa New Zealand. Our aim was to identify the microorganisms genetically equipped for mercury methylation, demethylation, or Hg(II) reduction to volatile Hg(0) in these springs. Dissolved total and methylated mercury concentrations in two adjacent springs with different mercury speciation ranked among the highest reported from natural sources (250 to 16,000 ng liter-1 and 0.5 to 13.9 ng liter-1, respectively). Total solid mercury concentrations in spring sediments ranged from 1,274 to 7,000 μg g-1 In the context of such ultrahigh mercury levels, the geothermal microbiome was unexpectedly diverse and dominated by acidophilic and mesophilic sulfur- and iron-cycling bacteria, mercury- and arsenic-resistant bacteria, and thermophilic and acidophilic archaea. By integrating microbiome structure and metagenomic potential with geochemical constraints, we constructed a conceptual model for biogeochemical mercury cycling in geothermal springs. The model includes abiotic and biotic controls on mercury speciation and illustrates how geothermal mercury cycling may couple to microbial community dynamics and sulfur and iron biogeochemistry.IMPORTANCE Little is currently known about biogeochemical mercury cycling in geothermal systems. The manuscript presents a new conceptual model, supported by genome-resolved metagenomic analysis and detailed geochemical measurements. The model illustrates environmental factors that influence mercury cycling in acidic springs, including transitions between solid (mineral) and aqueous phases of mercury, as well as the interconnections among mercury, sulfur, and iron cycles. This work provides a framework for studying natural geothermal mercury emissions globally. Specifically, our findings have implications for mercury speciation in wastewaters from geothermal power plants and the potential environmental impacts of microbially and abiotically formed mercury species, particularly where they are mobilized in spring waters that mix with surface or groundwaters. Furthermore, in the context of thermophilic origins for microbial mercury volatilization, this report yields new insights into how such processes may have evolved alongside microbial mercury methylation/demethylation and the environmental constraints imposed by the geochemistry and mineralogy of geothermal systems.

Filamentous fungi are intensively used for producing industrial enzymes, including lignocellulases. Employing insoluble cellulose to induce the production of lignocellulases causes some drawbacks, e.g., a complex fermentation operation, which can be overcome by using soluble inducers such as cellobiose. Here, a triple β-glucosidase mutant of Neurospora crassa, which prevents rapid turnover of cellobiose and thus allows the disaccharide to induce lignocellulases, was applied to profile the proteome responses to cellobiose and cellulose (Avicel). Our results revealed a shared proteomic response to cellobiose and Avicel, whose elements included lignocellulases and cellulolytic product transporters. While the cellulolytic proteins showed a correlated increase in protein and mRNA levels, only a moderate correlation was observed on a proteomic scale between protein and mRNA levels (R² = 0.31). Ribosome biogenesis and rRNA processing were significantly overrepresented in the protein set with increased protein but unchanged mRNA abundances in response to Avicel. Ribosome biogenesis, as well as protein processing and protein export, was also enriched in the protein set that showed increased abundance in response to cellobiose. NCU05895, a homolog of yeast CWH43, is potentially involved in transferring a glycosylphosphatidylinositol (GPI) anchor to nascent proteins. This protein showed increased abundance but no significant change in mRNA levels. Disruption of CWH43 resulted in a significant decrease in cellulase activities and secreted protein levels in cultures grown on Avicel, suggesting a positive regulatory role for CWH43 in cellulase production. The findings should have an impact on a systems engineering approach for strain improvement for the production of lignocellulases.IMPORTANCE Lignocellulases are important industrial enzymes for sustainable production of biofuels and bio-products. Insoluble cellulose has been commonly used to induce the production of lignocellulases in filamentous fungi, which causes a difficult fermentation operation and enzyme loss due to adsorption to cellulose. The disadvantages can be overcome by using soluble inducers, such as the disaccharide cellobiose. Quantitative proteome profiling of the model filamentous fungus Neurospora crassa revealed cellobiose-dependent pathways for cellulase production, including protein processing and export. A protein (CWH43) potentially involved in protein processing was found to be a positive regulator of lignocellulase production. The cellobiose-dependent mechanisms provide new opportunities to improve the production of lignocellulases in filamentous fungi.