Plant-Pathogenic Ralstonia Phylotypes Evolved Divergent Respiratory Strategies and Behaviors To Thrive in Xylem.

Bacterial pathogens in the Ralstonia solanacearum species complex (RSSC) infect the water-transporting xylem vessels of plants, causing bacterial wilt disease. Strains in RSSC phylotypes I and III can reduce nitrate to dinitrogen via complete denitrification. The four-step denitrification pathway enables bacteria to use inorganic nitrogen species as terminal electron acceptors, supporting their growth in oxygen-limited environments such as biofilms or plant xylem. Reduction of nitrate, nitrite, and nitric oxide all contribute to the virulence of a model phylotype I strain. However, little is known about the physiological role of the last denitrification step, the reduction of nitrous oxide to dinitrogen by NosZ. We found that phylotypes I and III need NosZ for full virulence. However, strains in phylotypes II and IV are highly virulent despite lacking NosZ. The ability to respire by reducing nitrate to nitrous oxide does not greatly enhance the growth of phylotype II and IV strains. These partial denitrifying strains reach high cell densities during plant infection and cause typical wilt disease. However, unlike phylotype I and III strains, partial denitrifiers cannot grow well under anaerobic conditions or form thick biofilms in culture or in tomato xylem vessels. Furthermore, aerotaxis assays show that strains from different phylotypes have different oxygen and nitrate preferences. Together, these results indicate that the RSSC contains two subgroups that occupy the same habitat but have evolved divergent energy metabolism strategies to exploit distinct metabolic niches in the xylem. IMPORTANCE Plant-pathogenic Ralstonia spp. are a heterogeneous globally distributed group of bacteria that colonize plant xylem vessels. Ralstonia cells multiply rapidly in plants and obstruct water transport, causing fatal wilting and serious economic losses of many key food security crops. The virulence of these pathogens depends on their ability to grow to high cell densities in the low-oxygen xylem environment. Plant-pathogenic Ralstonia can use denitrifying respiration to generate ATP. The last denitrification step, nitrous oxide reduction by NosZ, contributes to energy production and virulence for only one of the three phytopathogenic Ralstonia species. These complete denitrifiers form thicker biofilms in culture and in tomato xylem, suggesting they are better adapted to hypoxic niches. Strains with partial denitrification physiology form less biofilm and are more often planktonic. They are nonetheless highly virulent. Thus, these closely related bacteria have adapted their core metabolic functions to exploit distinct microniches in the same habitat.

Rapid Pivot of CURE Wet Lab to Online with the Help of Instructor Communities.

The pivot to remote and hybrid learning during the Covid-19 pandemic presented a challenge for many in academia. Most institutions were not prepared to support this rapid change, and instructors were left with the burden of converting a traditional face-to-face course into multiple modalities with very limited preparation time. When institutional support is lacking, we posit that instructor communities of practice can help provide the resources needed to meet the instructional demands. Tiny Earth, a course-based-undergraduate research experience (CURE) and international network of instructors and students, responded to the instructional challenges of the pandemic by leveraging its large community of instructors to create several smaller working groups to form focused communities of practice. Using the pedagogical principles of backward design and scientific teaching, one working group, the Tiny Earth Pivot Group (Pivot Group) generated a course map of remote learning activities and simulated learning resources to fulfill the Tiny Earth learning objectives and maintain the essential tenets of a CURE. Additional working groups were created to disseminate the resources collated and created by the Pivot Group to the greater community. In terms of Tiny Earth, the community structure provided the means for instructors to rapidly pivot their course materials to multiple modalities while upholding the student CURE experience. Harnessing the hallmarks of communities of practice-collective workpower toward common purpose, diversity of perspectives, and ongoing evolution-coupled with high-structured course design allows instructors flexibility and adaptability in meeting the changing modalities of higher education.

Temporal Responses of Microbial Communities to Anaerobic Soil Disinfestation.

Anaerobic soil disinfestation (ASD) is an organic amendment-based management tool for controlling soil-borne plant diseases and is increasingly used in a variety of crops. ASD results in a marked decrease in soil redox potential and other physicochemical changes, and a turnover in the composition of the soil microbiome. Mechanisms of ASD-mediated pathogen control are not fully understood, but appear to depend on the carbon source used to initiate the process and involve a combination of biological (i.e., release of volatile organic compounds) and abiotic (i.e., lowered pH, release of metal ions) factors. In this study, we examined how the soil microbiome changes over time in response to ASD initiated with rice bran, tomato pomace, or red grape pomace as amendments using growth chamber mesocosms that replicate ASD-induced field soil redox conditions. Within 2 days, the soil microbiome rapidly shifted from a diverse assemblage of taxa to being dominated by members of the Firmicutes for all ASD treatments, whereas control mesocosms maintained diverse and more evenly distributed communities. Rice bran and tomato pomace amendments resulted in microbial communities with similar compositions and trajectories that were different from red grape pomace communities. Quantitative PCR showed nitrogenase gene abundances were higher in ASD communities and tended to increase over time, suggesting the potential for altering soil nitrogen availability. These results highlight the need for temporal and functional studies to understand how pathogen suppressive microbial communities assemble and function in ASD-treated soils.

Ralstonia solanacearum uses inorganic nitrogen metabolism for virulence, ATP production, and detoxification in the oxygen-limited host xylem environment.

UNLABELLED: Genomic data predict that, in addition to oxygen, the bacterial plant pathogen Ralstonia solanacearum can use nitrate (NO3(-)), nitrite (NO2(-)), nitric oxide (NO), and nitrous oxide (N2O) as terminal electron acceptors (TEAs). Genes encoding inorganic nitrogen reduction were highly expressed during tomato bacterial wilt disease, when the pathogen grows in xylem vessels. Direct measurements found that tomato xylem fluid was low in oxygen, especially in plants infected by R. solanacearum. Xylem fluid contained ~25 mM NO3(-), corresponding to R. solanacearum's optimal NO3(-) concentration for anaerobic growth in vitro. We tested the hypothesis that R. solanacearum uses inorganic nitrogen species to respire and grow during pathogenesis by making deletion mutants that each lacked a step in nitrate respiration (ΔnarG), denitrification (ΔaniA, ΔnorB, and ΔnosZ), or NO detoxification (ΔhmpX). The ΔnarG, ΔaniA, and ΔnorB mutants grew poorly on NO3(-) compared to the wild type, and they had reduced adenylate energy charge levels under anaerobiosis. While NarG-dependent NO3(-) respiration directly enhanced growth, AniA-dependent NO2(-) reduction did not. NO2(-) and NO inhibited growth in culture, and their removal depended on denitrification and NO detoxification. Thus, NO3(-) acts as a TEA, but the resulting NO2(-) and NO likely do not. None of the mutants grew as well as the wild type in planta, and strains lacking AniA (NO2(-) reductase) or HmpX (NO detoxification) had reduced virulence on tomato. Thus, R. solanacearum exploits host NO3(-) to respire, grow, and cause disease. Degradation of NO2(-) and NO is also important for successful infection and depends on denitrification and NO detoxification systems. IMPORTANCE: The plant-pathogenic bacterium Ralstonia solanacearum causes bacterial wilt, one of the world's most destructive crop diseases. This pathogen's explosive growth in plant vascular xylem is poorly understood. We used biochemical and genetic approaches to show that R. solanacearum rapidly depletes oxygen in host xylem but can then respire using host nitrate as a terminal electron acceptor. The microbe uses its denitrification pathway to detoxify the reactive nitrogen species nitrite (a product of nitrate respiration) and nitric oxide (a plant defense signal). Detoxification may play synergistic roles in bacterial wilt virulence by converting the host's chemical weapon into an energy source. Mutant bacterial strains lacking elements of the denitrification pathway could not grow as well as the wild type in tomato plants, and some mutants were also reduced in virulence. Our results show how a pathogen's metabolic activity can alter the host environment in ways that increase pathogen success.