A solid literature base exists that indicates that plant host resistance is down-regulated in micro-g and includes studies that describe decreased cell wall rigidity, cell wall thickness, cellulose and matrix polysaccharides, lignin, and altered host-resistance gene pathways in micro-g. An equally solid literature base indicates that microbial virulence may be up-regulated in microgravity and includes up-regulation of virulence in microbe/microbe, microbe/insect, and pathogen/plant interactions.

However, no data exists on the interactions of a foliar phytopathogen on a plant host with concomitant host-resistance transcriptomics data. The alternative hypothesis (Ha) for the International Space Station (ISS)-flight experiment is: Microgravity Can Down-Regulate Host Resistance and thus May Up-Regulate Plant Disease Development in Space. Results will fill key knowledge gaps into how plant diseases and host resistance are affected by micro-g.

Proposed here is a novel flight experiment that will study the development of a foliar plant pathogen (i.e., phytopathogen) on the well-studied, Arabidopsis thaliana (At) host. The phytopathogen – Golovinomyces cichoracearum (Gc), a powdery mildew fungus - on A. thaliana is a well-studied pathosystem. The Gc/At pathosystem is chosen here because (i) both Gc and At are sequenced and annotated, (ii) extensive literature is available on host-resistance in At, (iii) diverse At cultivars with differential expression of easily measured host resistance mechanisms are available, (iv) most stages of the Gc life-cycle are on external surfaces of leaves and can be easily observed, (v) the expected ease of sanitizing flight hardware, and (vi) maximized crew safety on the ISS because Gc has no established interaction with humans.

We will use the Multi-Use Variable-Gravity Platform (MVP) facilities built by Redwire, Inc. (previously Techshot, Inc.) (2 units are in orbit on the ISS) to investigate the development of Gc on leaves of At. Each MVP has two independently controlled centrifuge rotors fitted with up to 4 Phytofuge plant-growth modules that will be rotated at 1g or left stationary in micro-g. Each Phytofuge unit has three separate petri dishes with light-emitting diode (LED) illumination and an internal camera.

Seed of two cultivars of At will be (1) sown onto growth media in independent petri dishes, (2) held dormant for up to 30 days, and (3) once in orbit, one-half of the petri dishes will be inoculated with the powdery mildew phytopathogen Gc. The aerial mycelia, conidiophores, and spores of Gc will be allowed to develop for 10-12 days and then leaves harvested for two separate research pipelines. First, half of the healthy and half of the infected At plants will be fixed in 3% buffered glutaraldehyde and stored at 4C until processed on the ground for fluorescent staining, Scanning Electron Microscopy (SEM), and Transmission Electron Microscopy (TEM) studies into the process of host infection. Second, the remaining healthy and infected plants will be fixed in RNAlater, frozen at -80C, and later processed for transcriptomics of host-resistance genes.

Results will inform future horticulturists, space engineers, and technologists of the risks of maintaining plant-host resistance in space when challenged by an airborne phytopathogen. The results will also assist in the design of future plant-growth modules for crewed missions to the Moon and Mars.

The hypothesis being tested here is (Ha): Microgravity can down-regulate plant host resistance, and thus, may up-regulate plant disease development in space.

The primary benefit to Earth-based agriculture will be to identify how disease resistance mechanisms operate under the unusual conditions of microgravity. Results may identify how to improve disease resistance in field crops on Earth.

1) We successfully completed two Phytofuge Experiment Module (PEM) tests in late 2023 and Jan/Feb 2024. Results indicate that both plants and disease developed nominally within the Multi-Use Variable-g Platform (MVP) Phytofuge Experiment Modules (PEM) Petri dishes.

2) A spore inoculation device called a Conidia Application Device (CAD) unit (nicknamed the Conidiator) was developed and tested. Initial results are very positive, and we successfully injected visibly observable quantities of Gc conidia into Petri dishes designed for the Gc/At flight experiment.

3) An Experiment Requirements Document (ERD) for the MVP-Plant-02 International Space Station (ISS) flight experiment was developed and completed. A Science Verification Test (SVT) Readiness Review (SVT RR) is scheduled for 10-Dec-2024.

Other Accomplishments for 2024 are:

1) A flight-rated Petri dish was designed and manufactured for the SVT. The Phytofuge Experiment Module (PEM) can accommodate up to three Petri dishes. The new Petri dishes have been modified to allow the injection of Gc conidia during the flight.

2) Nine fluorescent (FL) stains were tested on Gc-infected At leaves. We have chosen three FL stains for the SVT, Experiment Verification Test (EVT) and ISS flight experiments. These are: (a) Coomassie Brilliant Blue (CBB; stains Gc mycelia), (b) Aniline Blue (AB; stains plant callose), and (c) 3,3’-dihxyloxacarbocyanine iodide (DCI; used for whole canopy staining of aerial mycelia).

3) Electron microscopy (EM) fixation and processing protocols are 90% completed. A final test of the scanning electron microscopy (SEM) and transmission electron microscopy (TEM) fixation and handling process will be completed during the SVT (now scheduled for Jan/Feb 2025).

4) A leaf canopy protocol was developed using the program, ImageJ, to precisely estimate the leaf areas of plants growing within the PEM Petri dishes. The ImageJ protocol will be used in the SVT, EVT, and ISS flight experiment.

5) While working on testing SEM fixation protocols for infected leaves, we conducted a small scale experiment to determine if simulated microgravity could promote the development of the fungus, G. cichoracearum on At. Results indicated that germ tube lengths (i.e., a measure of early fungal growth rates) were significantly longer for conidia germinated on squash and At leaves incubated on 2D or 3D clinostats compared to 1-g controls.

6) We have scheduled the SVT for mid-January to mid-February 2025.

A solid literature base exists that indicates that plant host resistance is down-regulated in micro-g and includes studies that describe decreased cell wall rigidity, cell wall thickness, cellulose and matrix polysaccharides, lignin, and altered host-resistance gene pathways in micro-g. An equally solid literature base indicates that microbial virulence may be up-regulated in microgravity and includes up-regulation of virulence in microbe/microbe, microbe/insect, and pathogen/plant interactions.

However, no data exists on the interactions of a foliar phytopathogen on a plant host with concomitant host-resistance transcriptomics data. The alternative hypothesis (Ha) for the International Space Station (ISS)-flight experiment is: Microgravity Can Down-Regulate Host Resistance and thus May Up-Regulate Plant Disease Development in Space. Results will fill key knowledge gaps into how plant diseases and host resistance are affected by micro-g.

Proposed here is a novel flight experiment that will study the development of a foliar plant pathogen (i.e., phytopathogen) on the well-studied, Arabidopsis thaliana (At) host. The phytopathogen – Golovinomyces cichoracearum (Gc), a powdery mildew fungus - on A. thaliana is a well-studied pathosystem. The Gc/At pathosystem is chosen here because (i) both Gc and At are sequenced and annotated, (ii) extensive literature is available on host-resistance in At, (iii) diverse At cultivars with differential expression of easily measured host resistance mechanisms are available, (iv) most stages of the Gc life-cycle are on external surfaces of leaves and can be easily observed, (v) the expected ease of sanitizing flight hardware, and (vi) maximized crew safety on the ISS because Gc has no established interaction with humans.

We will use the Multi-Use Variable-Gravity Platform (MVP) facilities built by Techshot, Inc. (2 units are in orbit on the ISS) to investigate the development of Gc on leaves of At. [Ed. Note: Techshot, Inc. was acquired by Redwire Corporation in November, 2021.] Each MVP has two independently controlled centrifuge rotors fitted with up to 4 Phytofuge plant-growth modules that will be rotated at 1g or left stationary in micro-g. Each Phytofuge unit has three separate petri dishes with light-emitting diode (LED) illumination and an internal camera.

Seed of three cultivars of At will be (1) sown onto growth media in independent petri dishes, (2) held dormant for up to 30 days, and (3) once in orbit, one-half of the petri dishes will be inoculated with the powdery mildew phytopathogen Gc. The aerial mycelia, conidiophores, and spores of Gc will be allowed to develop for 5-7 days and then leaves harvested for two separate research pipelines. First, half of the healthy and half of the infected At plants will be fixed in glutaraldehyde and stored at 4C until processed on the ground for fluorescent staining, Scanning Electron Microscopy (SEM), and Transmission Electron Microscopy (TEM) studies into the process of host infection. Second, the remaining healthy and infected plants will be frozen at -80C and later processed for transcriptomics of host-resistance genes.

Results will inform future horticulturists, space engineers, and technologists of the risks of maintaining plant-host resistance in space when challenged by an airborne phytopathogen. The results will also assist in the design of future plant-growth modules for crewed missions to the Moon and Mars.

The hypothesis being tested here is: Microgravity can down-regulate plant host resistance, and thus, may up-regulate plant disease development in space.

The primary benefit to Earth-based agriculture will be to identify how disease resistance mechanisms operate under the unusual conditions of microgravity. Results may identify how to improve disease resistance in field crops on Earth.

The three criticality-1 activities that must be successfully completed before developing an Experiment Requirements Document (ERD) document for the SVT are:

1) Complete a successful grow out of Col-0 and pmr4 At lines in the Phytofuge modules. As of this writing, the Phytofuge #1 test was started on 11-Oct-2023.

2) Develop a spore applicator that successfully delivers adequate conidia to the leaves of both At lines within the Phytofuge petri dishes.

3) Develop a 4°C storage protocol for Gc conidia that will retain high viability for up to 28 days for maximum flexibility in conducting the ISS flight experiment.

The Gc/At Team is in a good position to complete a series of ongoing protocol development experiments that will permit us to create the ERD no later than 31-Jan-2024. We have two working flight-rated Phytofuge units in the University of Florida Space Life Science Lab (SLSL) that will be used to verify that the At lines of choice – Col-0 (susceptible) and pmr4 (resistant) – can grow nominally in the flight hardware and allow nominal development of the Gc phytopathogen on inoculated leaves. The Phytofuge tests [ongoing] and other protocol development assays will be completed before 15-Dec-2023.

We are on track to move forward with SVT planning no later than 02-Jan-2024.

A solid literature base exists that indicates that plant host resistance is down-regulated in micro-g and includes studies that describe decreased cell wall rigidity, cell wall thickness, cellulose and matrix polysaccharides, lignin, and altered host-resistance gene pathways in micro-g. An equally solid literature base indicates that microbial virulence may be up-regulated in microgravity and includes up-regulation of virulence in microbe/microbe, microbe/insect, and pathogen/plant interactions.

However, no data exists on the interactions of a foliar phytopathogen on a plant host with concomitant host-resistance transcriptomics data. The alternative hypothesis (Ha) for the International Space Station (ISS)-flight experiment is: Microgravity Can Down-Regulate Host Resistance and thus May Up-Regulate Plant Disease Development in Space. Results will fill key knowledge gaps into how plant diseases and host resistance are affected by micro-g.

Proposed here is a novel flight experiment that will study the development of a foliar plant pathogen (i.e., phytopathogen) on the well-studied, Arabidopsis thaliana (At) host. The phytopathogen – Golovinomyces cichoracearum (Gc), a powdery mildew fungus - on A. thaliana is a well-studied pathosystem. The Gc/At pathosystem is chosen here because (i) both Gc and At are sequenced and annotated, (ii) extensive literature is available on host-resistance in At, (iii) diverse At cultivars with differential expression of easily measured host resistance mechanisms are available, (iv) most stages of the Gc life-cycle are on external surfaces of leaves and can be easily observed, (v) the expected ease of sanitizing flight hardware, and (vi) maximized crew safety on the ISS because Gc has no established interaction with humans.

We will use the Multi-Use Variable-Gravity Platform (MVP) facilities built by Techshot, Inc. (2 units are in orbit on the ISS) to investigate the development of Gc on leaves of At. [Ed. Note: Techshot, Inc. was acquired by Redwire Corporation in November, 2021.] Each MVP has two independently controlled centrifuge rotors fitted with up to 4 Phytofuge plant-growth modules that will be rotated at 1g or left stationary in micro-g. Each Phytofuge unit has three separate petri dishes with light-emitting diode (LED) illumination and an internal camera.

Seed of three cultivars of At will be (1) sown onto growth media in independent petri dishes, (2) held dormant for up to 30 days, and (3) once in orbit, one-half of the petri dishes will be inoculated with the powdery mildew phytopathogen Gc. The aerial mycelia, conidiophores, and spores of Gc will be allowed to develop for 5-7 days and then leaves harvested for two separate research pipelines. First, half of the healthy and half of the infected At plants will be fixed in glutaraldehyde and stored at 4C until processed on the ground for fluorescent staining, Scanning Electron Microscopy (SEM), and Transmission Electron Microscopy (TEM) studies into the process of host infection. Second, the remaining healthy and infected plants will be frozen at -80C and later processed for transcriptomics of host-resistance genes.

Results will inform future horticulturists, space engineers, and technologists of the risks of maintaining plant-host resistance in space when challenged by an airborne phytopathogen. The results will also assist in the design of future plant-growth modules for crewed missions to the Moon and Mars.

The hypothesis being tested here is: Microgravity can down-regulate plant host resistance, and thus, may up-regulate plant disease development in space.

The primary benefit to Earth-based agriculture will be to identify how disease resistance mechanisms operate under the unusual conditions of microgravity. Results may identify how to improve disease resistance in field crops on Earth.

1) Viable cultures of the plant pathogen, Golovinomyces cichoracearum (Gc), were obtained from colleagues at the University of Maryland. Cultures of Gc are being maintained on several squash cultivars on agar. 2) The production methods for Arabidopsis thaliana (At) and squash plants were developed in 2022. Plants are grown under white light-emitting diode (LED) arrays, at 22 C, and a 12:12 day/night cycle. However, plant growth has not yet been fully optimized for agar-based media proposed for the space experiment. 3) We have also been working on the protocol to apply spores of Gc to the host plant, Arabidopsis thaliana. Applications of spores of Gc to At leaves do not always yield uniformly infected leaves. It is unknown whether this is an environmental issue, a spore-application issue, or a host-resistance issue in the various assays. However, new assays in late 2022 will evaluate the infection process for the Gc/At pathosystem. 5) Fluorescent stains, scanning electron microscopy, and the study of gene expression in the plants will be used during the flight experiment to evaluate if host resistance has been altered by microgravity. These protocols are now being studied for their utility in the ground and flight research.

A solid literature base exists that indicates that plant host resistance is down-regulated in micro-g and includes studies that describe decreased cell wall rigidity, cell wall thickness, cellulose and matrix polysaccharides, lignin, and altered host-resistance gene pathways in micro-g. An equally solid literature base indicates that microbial virulence may be up-regulated in microgravity and includes up-regulation of virulence in microbe/microbe, microbe/insect, and pathogen/plant interactions.

However, no data exists on the interactions of a foliar phytopathogen on a plant host with concomitant host-resistance transcriptomics data. The alternative hypothesis (Ha) for the International Space Station (ISS)-flight experiment is: Microgravity Can Down-Regulate Host Resistance and thus May Up-Regulate Plant Disease Development in Space. Results will fill key knowledge gaps into how plant diseases and host resistance are affected by micro-g.

Proposed here is a novel flight experiment that will study the development of a foliar plant pathogen (i.e., phytopathogen) on the well-studied, Arabidopsis thaliana (At) host. The phytopathogen – Golovinomyces cichoracearum (Gc), a powdery mildew fungus - on A. thaliana is a well-studied pathosystem. The Gc/At pathosystem is chosen here because (i) both Gc and At are sequenced and annotated, (ii) extensive literature is available on host-resistance in At, (iii) diverse At cultivars with differential expression of easily measured host resistance mechanisms are available, (iv) most stages of the Gc life-cycle are on external surfaces of leaves and can be easily observed, (v) the expected ease of sanitizing flight hardware, and (vi) maximized crew safety on the ISS because Gc has no established interaction with humans.

We will use the Multi-Use Variable-Gravity Platform (MVP) facilities built by Techshot, Inc. (2 units are in orbit on the ISS) to investigate the development of Gc on leaves of At. [Ed. Note: Techshot, Inc. was acquired by Redwire Corporation in November, 2021.] Each MVP has two independently controlled centrifuge rotors fitted with up to 4 Phytofuge plant-growth modules that will be rotated at 1g or left stationary in micro-g. Each Phytofuge unit has three separate petri dishes with light-emitting diode (LED) illumination and an internal camera.

Seed of three cultivars of At will be (1) sown onto growth media in independent petri dishes, (2) held dormant for up to 30 days, and (3) once in orbit, one-half of the petri dishes will be inoculated with the powdery mildew phytopathogen Gc. The aerial mycelia, conidiophores, and spores of Gc will be allowed to develop for 5-7 days and then leaves harvested for two separate research pipelines. First, half of the healthy and half of the infected At plants will be fixed in glutaraldehyde and stored at 4C until processed on the ground for fluorescent staining, Scanning Electron Microscopy (SEM), and Transmission Electron Microscopy (TEM) studies into the process of host infection. Second, the remaining healthy and infected plants will be frozen at -80C and later processed for transcriptomics of host-resistance genes.

Results will inform future horticulturists, space engineers, and technologists of the risks of maintaining plant-host resistance in space when challenged by an airborne phytopathogen. The results will also assist in the design of future plant-growth modules for crewed missions to the Moon and Mars.