Persistence of Escherichia coli in the microbiomes of red Romaine lettuce (Lactuca sativa cv. 'Outredgeous') and mizuna mustard (Brassica rapa var. japonica) - does seed sanitization matter?

BACKGROUND: Seed sanitization via chemical processes removes/reduces microbes from the external surfaces of the seed and thereby could have an impact on the plants' health or productivity. To determine the impact of seed sanitization on the plants' microbiome and pathogen persistence, sanitized and unsanitized seeds from two leafy green crops, red Romaine lettuce (Lactuca sativa cv. 'Outredgeous') and mizuna mustard (Brassica rapa var. japonica) were exposed to Escherichia coli and grown in controlled environment growth chambers simulating environmental conditions aboard the International Space Station. Plants were harvested at four intervals from 7 days post-germination to maturity. The bacterial communities of leaf and root were investigated using the 16S rRNA sequencing while quantitative polymerase chain reaction (qPCR) and heterotrophic plate counts were used to reveal the persistence of E. coli. RESULT: E. coli was detectable for longer periods of time in plants from sanitized versus unsanitized seeds and was identified in root tissue more frequently than in leaf tissue. 16S rRNA sequencing showed dynamic changes in the abundance of members of the phylum Proteobacteria, Firmicutes, and Bacteroidetes in leaf and root samples of both leafy crops. We observed minimal changes in the microbial diversity of lettuce or mizuna leaf tissue with time or between sanitized and unsanitized seeds. Beta-diversity showed that time had more of an influence on all samples versus the E. coli treatment. CONCLUSION: Our results indicated that the seed surface sanitization, a current requirement for sending seeds to space, could influence the microbiome. Insight into the changes in the crop microbiomes could lead to healthier plants and safer food supplementation.

Leaf yield and mineral content of mizuna in response to cut-and-come-again harvest, substrate particle size, and fertilizer formulation in a simulated spaceflight environment.

The Veggie plant-growth unit deployed onboard the International Space Station (ISS) grows leafy vegetables to supplement crew diets. "Cut-and-come-again" harvests are tested to maximize vegetative yield while minimizing crew time. Water, oxygen, and fertilizer delivery to roots of leafy greens growing in microgravity have become the center of attention for Veggie. Current Veggie technology wicks water into particulate root substrates incorporating controlled-release fertilizer (CRF). Mizuna mustard (Brassica rapa) was grown under ISS-like environments in ground-based Veggie-analogue units comparing crop response to combinations of two different substrate particle sizes, two different fertilizer formulations, and three leaf-harvest times from each plant. Fine-particle porous ceramic substrate (Profile©) was compared with a 40:60 mix of fine-particle porous ceramic Profile© + coarse porous ceramic Turface© substrate. Identical 18-6-8 (NPK) CRF formulations consisting of [50% fast-release (T70) + 50% intermediate-release (T100) prills] vs. [50% fast-release (T70) + 50% slow-release (T180) prills] were incorporated into each substrate, and leaf tissues were harvested from each treatment combination at 28, 48, and 56 days after sowing. The combination of T100 CRF in 100% Profile© substrate gave the highest fresh mass (FM) and leaf area (LA) across harvests, whereas T180 CRF in 40% Profile© gave the lowest. Dry-mass (DM) yields varied with effects on leaf area. Tissue nitrogen (N) and potassium (K) specific contents declined across harvests for all treatment combinations but tended to be highest for T100 CRF/100% Profile©, and lowest for T180 CRF/40% Profile©. These major macronutrients were taken up faster by roots growing in small particle-size substrate incorporating intermediate-rate CRF, but also were depleted faster from the same treatment combination, suggesting it may not continue to be the best combination for additional harvests. Micronutrients did not decline in tissue specific content across treatment combinations, but manganese (Mn) accumulated in leaf tissue across treatments and apparently comes mainly from the ceramic substrate, regardless of particle size. Substrate leachate analysis following final harvest indicated that pH remained in the range for nominal availability of mineral nutrients for root uptake, but electro-conductivity measurements suggested depletion of fertilizer salts from root zones, especially from the treatment combination supporting the highest yields and major macronutrient contents. Although 100% Profile© was the better growth substrate for mizuna in combination with intermediate-rate CRF and three cut-and-come-again harvests in ground-based studies, mixed-particle-size substrates may be a better choice for plant growth under microgravity conditions, where capillary forces predominant and tend to cause saturation of a fine medium with water. Since there were no statistically significant interactions between substrate and fertilizer in this study, our ground-based findings for CRF choice should translate to the best substrate choice for microgravity, but if NASA wants to consider additional cut-and-come-again harvests from the same mizuna plants, more complex CRF formulations likely will have to be investigated.

Comparison of two controlled-release fertilizer formulations for cut-and-come-again harvest yield and mineral content of Lactuca sativa L. cv. Outredgeous grown under International Space Station environmental conditions.

Red Romaine leaf lettuce (Lactuca sativa L. cv. Outredgeous) was grown in ground-based analogues of the Veggie plant-growth units used to grow salad vegetables for astronauts on the International Space Station (ISS). Plants were grown for 56 days with three "cut-and-come again" leaf harvests from the same plants. Six Biomass-Production-Systems-for-Education (BPSe) units were used to grow 'Outredgeous' ('OR') lettuce in a walk-in growth chamber under temperature, humidity, and LED-lighting conditions similar to those occurring in Veggie on ISS. Because of the ISS micro-gravity environment, both Veggie and ground-based BPSe units utilize one-way capillary wicking of water into an arcillite clay root substrate. In the present study, two different controlled-release fertilizer (CRF) formulations incorporated into the arcillite were compared for effects on 'OR' growth rate, overall yield, and mineral content of leaves harvested from the same plants 28, 48, and 56 days after planting. Both CRF treatments had a rapid-releasing T70 component that kept growth rate equivalent over the first two harvests. Growth rate for both CRF treatments increased from the first to the second harvest, but then declined from the second to the third harvest, more so for the slower-releasing T180 CRF than for the moderately-releasing T100 CRF. Tissue content of the macro-nutrients N, P, and K declined at each harvest for both CRFs, while content of the micro-nutrients B, Zn, and Mn increased. Although pH did not go out of the nominal range for availability of mineral nutrients to roots over the cropping cycle, and electrical-conductivity of rootzone salts was neither excessive nor depleted, tissue macronutrient depletion and micro-nutrient accumulation may have contributed to yield declines. Although either CRF formulation can support adequate yield of 'OR' lettuce over a 56-day period, the moderately-releasing T100 formulation tends to give slightly higher yield, especially during the last growth increment, and with non-deficient mineral content.

Response of Arabidopsis thaliana and Mizuna Mustard Seeds to Simulated Space Radiation Exposures.

One of the major concerns for long-term exploration missions beyond the Earth's magnetosphere is consequences from exposures to solar particle event (SPE) protons and galactic cosmic rays (GCR). For long-term crewed Lunar and Mars explorations, the production of fresh food in space will provide both nutritional supplements and psychological benefits to the astronauts. However, the effects of space radiation on plants and plant propagules have not been sufficiently investigated and characterized. In this study, we evaluated the effect of two different compositions of charged particles-simulated GCR, and simulated SPE protons on dry and hydrated seeds of the model plant Arabidopsis thaliana and the crop plant Mizuna mustard [Brassica rapa var. japonica]. Exposures to charged particles, simulated GCRs (up to 80 cGy) or SPEs (up to 200 cGy), were performed either acutely or at a low dose rate using the NASA Space Radiation Laboratory (NSRL) facility at Brookhaven National Lab (BNL). Control and irradiated seeds were planted in a solid phytogel and grown in a controlled environment. Five to seven days after planting, morphological parameters were measured to evaluate radiation-induced damage in the seedlings. After exposure to single types of charged particles, as well as to simulated GCR, the hydrated Arabidopsis seeds showed dose- and quality-dependent responses, with heavier ions causing more severe defects. Seeds exposed to simulated GCR (dry seeds) and SPE (hydrated seeds) had significant, although much less damage than seeds exposed to heavier and higher linear energy transfer (LET) particles. In general, the extent of damage depends on the seed type.

Evaluating the lettuce metatranscriptome with MinION sequencing for future spaceflight food production applications.

Healthy plants are vital for successful, long-duration missions in space, as they provide the crew with life support, food production, and psychological benefits. The microorganisms that associate with plant tissues play a critical role in improving plant health and production. To that end, we developed a methodology to investigate the transcriptional activities of the microbiome of red romaine lettuce, a key salad crop that was grown under International Space Station (ISS)-like conditions. Microbial transcripts enriched from host-microbe total RNA were sequenced using the Oxford Nanopore MinION sequencing platform. Results show that this enrichment approach was highly reproducible and could be an effective approach for the on-site detection of microbial transcriptional activity. Our results demonstrate the feasibility of using metatranscriptomics of enriched microbial RNA as a potential method for on-site monitoring of the transcriptional activity of crop microbiomes, thereby helping to facilitate and maintain plant health for on-orbit space food production.

Fusarium oxysporum as an Opportunistic Fungal Pathogen on Zinnia hybrida Plants Grown on board the International Space Station.

A plant production system called Veggie was launched to the International Space Station (ISS) in 2014. In late 2015, during the growth of Zinnia hybrida cv. 'Profusion' in the Veggie hardware, plants developed chlorosis, leaf curling, fungal growth that damaged leaves and stems, and eventually necrosis. The development of symptoms was correlated to reduced air flow leading to a significant buildup of water enveloping the leaves and stems in microgravity. Symptomatic tissues were returned to Earth on 18 May 2016 and were immediately processed to determine the primary causal agent of the disease. The presumptive pathogen was identified as Fusarium oxysporum by morphological features of microconidia and conidiophores on symptomatic tissues; that is, by epifluorescent microscopy (EFM), scanning electron microscopy (SEM), metabolic microarrays, and ITS sequencing. Both EFM and SEM imaging of infected tissues showed that germinating conidia were capable of stomatal penetration and thus acted as the primary method for infecting host tissues. A series of ground-based pathogenicity assays were conducted with healthy Z. hybrida plants that were exposed to reduced-airflow and high-water stress (i.e., encased in sealed bags) or were kept in an unstressed configuration. Koch's postulates were successfully completed with Z. hybrida plants in the lab, but symptoms only matched ISS-flown symptomatic tissues when the plants were stressed with high-water exposure. Unstressed plants grown under similar lab conditions failed to develop the symptoms observed with plants on board the ISS. The overall results of the pathogenicity tests imply that F. oxysporum acted as an opportunistic pathogen on severely high-water stressed plants. The source of the opportunistic pathogen is not known, but virulent strains of F. oxysporum were not recovered from unused materials in the Veggie plant pillow growth units assayed after the flight.

Spatial Characterization of Microbial Communities on Multi-Species Leafy Greens Grown Simultaneously in the Vegetable Production Systems on the International Space Station.

The establishment of steady-state continuous crop production during long-term deep space missions is critical for providing consistent nutritional and psychological benefits for the crew, potentially improving their health and performance. Three technology demonstrations were completed achieving simultaneous multi-species plant growth and the concurrent use of two Veggie units on the International Space Station (ISS). Microbiological characterization using molecular and culture-based methods was performed on leaves and roots from two harvests of three leafy greens, red romaine lettuce (Lactuca sativa cv. 'Outredgeous'); mizuna mustard, (Brassica rapa var japonica); and green leaf lettuce, (Lactuca sativa cv. Waldmann's) and associated rooting pillow components and Veggie chamber surfaces. Culture based enumeration and pathogen screening indicated the leafy greens were safe for consumption. Surface samples of the Veggie facility and plant pillows revealed low counts of bacteria and fungi and are commonly isolated on ISS. Community analysis was completed with 16S rRNA amplicon sequencing. Comparisons between pillow components, and plant tissue types from VEG-03D, E, and F revealed higher diversity in roots and rooting substrate than the leaves and wick. This work provides valuable information for food production-related research on the ISS and the impact of the plant microbiome on this unique closed environment.

Growth and photosynthetic responses of Chinese cabbage (Brassica rapa L. cv. Tokyo Bekana) to continuously elevated carbon dioxide in a simulated Space Station "Veggie" crop-production environment.

Among candidate leafy vegetable species initially considered for astronauts to pick and eat from the Veggie plant-growth unit on the International Space Station (ISS), Chinese cabbage (Brassica rapa L. cv. Tokyo Bekana) ranked high in ground-based screening studies. However, subsequent attempts to optimize growth within rigorous ISS-like growth environments on the ground were frustrated by development of leaf chlorosis, necrosis, and uneven growth. 'Tokyo Bekana' ('TB') grown on ISS during the VEG-03B and C flights developed similar stress symptoms. After lengthy troubleshooting efforts to identify causes of sub-par growth in highly controlled environments, the super-elevated CO2 concentrations that plants on ISS are exposed to continuously (average of 2,800 µmol/mol) emerged as a candidate environmental condition responsible for the observed plant-stress symptoms. Subsequent ground-based studies found continuous exposure to ISS levels of CO2 under Veggie environmental and cultural conditions to significantly inhibit growth of 'TB' compared to near-Earth-normal CO2 controls. The present study investigated growth and gas-exchange responses of 'TB' to sub-ISS but still elevated CO2 levels (900 or 1,350 µmol/mol) in combination with other potential stressors related to ISS/Veggie compared to 450 µmol/mol CO2 controls. Shoot dry mass of plants grown at 450 µmol•mol-1 CO2 for 28 days was 96% and 80% higher than that of plants grown at 900 µmol•mol-1 CO2 and 1,350 µmol•mol-1 CO2, respectively. Leaf number and leaf area of controls were significantly higher than those of plants grown at 1,350 µmol•mol-1 CO2. Photosynthetic rate measured using a leaf cuvette was significantly lower for plants grown at 900 µmol•mol-1 CO2 than for controls. The ratio of leaf internal CO2 concentration (Ci) to cuvette ambient CO2 concentration (Ca) was significantly lower for plants grown at 450 µmol•mol-1 CO2 than for plants grown at elevated CO2. Thus, continuously elevated CO2 in combination with a Veggie cultivation system decreased growth, leaf area, and photosynthetic efficiency of Chinese cabbage 'Tokyo Bekana'. The results of this study suggest that 'Tokyo Bekana' is very sensitive to continuously elevated CO2 in such a growth environment, and indicate the need for improved environmental control of CO2 and possibly root-zone factors for successful crop production in the ISS spaceflight environment. Differential sensitivity of other salad crops to an ISS/Veggie growth environment also is possible, so it is important to mimic controllable ISS-like environmental conditions as precisely as possible during ground-based screening.

Microbiological and Nutritional Analysis of Lettuce Crops Grown on the International Space Station.

The ability to grow safe, fresh food to supplement packaged foods of astronauts in space has been an important goal for NASA. Food crops grown in space experience different environmental conditions than plants grown on Earth (e.g., reduced gravity, elevated radiation levels). To study the effects of space conditions, red romaine lettuce, Lactuca sativa cv 'Outredgeous,' plants were grown in Veggie plant growth chambers on the International Space Station (ISS) and compared with ground-grown plants. Multiple plantings were grown on ISS and harvested using either a single, final harvest, or sequential harvests in which several mature leaves were removed from the plants at weekly intervals. Ground controls were grown simultaneously with a 24-72 h delay using ISS environmental data. Food safety of the plants was determined by heterotrophic plate counts for bacteria and fungi, as well as isolate identification using samples taken from the leaves and roots. Molecular characterization was conducted using Next Generation Sequencing (NGS) to provide taxonomic composition and phylogenetic structure of the community. Leaves were also analyzed for elemental composition, as well as levels of phenolics, anthocyanins, and Oxygen Radical Absorbance Capacity (ORAC). Comparison of flight and ground tissues showed some differences in total counts for bacteria and yeast/molds (2.14 - 4.86 log10 CFU/g), while screening for select human pathogens yielded negative results. Bacterial and fungal isolate identification and community characterization indicated variation in the diversity of genera between leaf and root tissue with diversity being higher in root tissue, and included differences in the dominant genera. The only difference between ground and flight experiments was seen in the third experiment, VEG-03A, with significant differences in the genera from leaf tissue. Flight and ground tissue showed differences in Fe, K, Na, P, S, and Zn content and total phenolic levels, but no differences in anthocyanin and ORAC levels. This study indicated that leafy vegetable crops can produce safe, edible, fresh food to supplement to the astronauts' diet, and provide baseline data for continual operation of the Veggie plant growth units on ISS.

Temperature affects long-term productivity and quality attributes of day-neutral strawberry for a space life-support system.

Strawberry (Fragaria x ananassa L.) is a promising candidate crop for space life-support systems with desirable sensory quality and health attributes. Day-neutral cultivars such as 'Seascape' are adaptable to a range of photoperiods, including short days that would save considerable energy for crop lighting without reductions in productivity or yield. Since photoperiod and temperature interact to affect strawberry growth and development, several diurnal temperature regimes were tested under a short photoperiod of 10 h per day for effects on yield and quality attributes of 'Seascape' strawberry during production cycles longer than 270 days. The coolest day/night temperature regime, 16°/8 °C, tended to produce smaller numbers of larger fruit than did the intermediate temperature range of 18°/10 °C or the warmest regime, 20°/12 °C, both of which produced similar larger numbers of smaller fruit. The intermediate temperature regime produced the highest total fresh mass of berries over an entire production cycle. Independent experiments examined either organoleptic or physicochemical quality attributes. Organoleptic evaluation indicated that fruit grown under the coolest temperature regime tended to score the highest for both hedonic preference and descriptive evaluation of sensory attributes related to sweetness, texture, aftertaste, and overall approval. The physicochemical quality attributes Brix, pH, and sugar/acid ratio were highest for fruits harvested from the coolest temperature regime and lower for those from the warmer temperature regimes. The cool-regime fruits also were lowest in titratable acidity. The yield parameters fruit number and size oscillated over the course of a production cycle, with a gradual decline in fruit size under all three temperature regimes. Brix and titratable acidity both decreased over time for all three temperature treatments, but sugar/acid ratio remained highest for the cool temperature regime over the entire production period. Periodic rejuvenation or replacement of strawberry propagules may be needed to maintain both quality and quantity of strawberry yield in space.

Ionic signaling in plant gravity and touch responses.

Plant roots are optimized to exploit resources from the soil and as each root explores this environment it will encounter a range of biotic and abiotic stimuli to which it must respond. Therefore, each root must possess a sensory array capable of monitoring and integrating these diverse stimuli to direct the appropriate growth response. Touch and gravity represent two of the biophysical stimuli that plants must integrate. As sensing both of these signals requires mechano-transduction of biophysical forces to biochemical signaling events, it is likely that they share signal transduction elements. These common signaling components may allow for cross-talk and so integration of thigmotropic and gravitropic responses. Indeed, signal transduction events in both plant touch and gravity sensing are thought to include Ca(2+)- and pH-dependent events. Additionally, it seems clear that the systems responsible for root touch and gravity response interact to generate an integrated growth response. Thus, primary and lateral roots of Arabidopsis respond to mechanical stimuli by eliciting tropic growth that is likely part of a growth strategy employed by the root to circumvent obstacles in the soil. Also, the mechano-signaling induced by encountering an obstacle apparently down-regulates the graviperception machinery to allow this kind of avoidance response. The challenge for future research will be to define how the cellular signaling events in the root cap facilitate this signal integration and growth regulation. In addition, whether other stimuli are likewise integrated with the graviresponse via signal transduction system cross-talk is an important question that remains to be answered.

Touch modulates gravity sensing to regulate the growth of primary roots of Arabidopsis thaliana.

Plants must sense and respond to diverse stimuli to optimize the architecture of their root system for water and nutrient scavenging and anchorage. We have therefore analyzed how information from two of these stimuli, touch and gravity, are integrated to direct root growth. In Arabidopsis thaliana, touch stimulation provided by a glass barrier placed across the direction of growth caused the root to form a step-like growth habit with bends forming in the central and later the distal elongation zones. This response led to the main root axis growing parallel to, but not touching the obstacle, whilst the root cap maintained contact with the barrier. Removal of the graviperceptive columella cells of the root cap using laser ablation reduced the bending response of the distal elongation zone. Similarly, although the roots of the gravisensing impaired pgm1-1 mutant grew along the barrier at the same average angle as wild-type, this angle became more variable with time. These observations imply a constant gravitropic re-setting of the root tip response to touch stimulation from the barrier. In wild-type plants, transient touch stimulation of root cap cells, but not other regions of the root, inhibited both subsequent gravitropic growth and amyloplast sedimentation in the columella. Taken together, these results suggest that the cells of the root cap sense touch stimuli and their subsequent signaling acts on the columella cells to modulate their graviresponse. This interaction of touch and gravity signaling would then direct root growth to avoid obstacles in the soil while generally maintaining downward growth.

Ionic signaling in plant responses to gravity and touch.

Touch and gravity are two of the many stimuli that plants must integrate to generate an appropriate growth response. Due to the mechanical nature of both of these signals, shared signal transduction elements could well form the basis of the cross-talk between these two sensory systems. However, touch stimulation must elicit signaling events across the plasma membrane whereas gravity sensing is thought to represent transformation of an internal force, amyloplast sedimentation, to signal transduction events. In addition, factors such as turgor pressure and presence of the cell wall may also place unique constraints on these plant mechanosensory systems. Even so, the candidate signal transduction elements in both plant touch and gravity sensing, changes in Ca2+, pH and membrane potential, do mirror the known ionic basis of signaling in animal mechanosensory cells. Distinct spatial and temporal signatures of Ca2+ ions may encode information about the different mechanosignaling stimuli. Signals such as Ca2+ waves or action potentials may also rapidly transfer information perceived in one cell throughout a tissue or organ leading to the systemic reactions characteristic of plant touch and gravity responses. Longer-term growth responses are likely sustained via changes in gene expression and asymmetries in compounds such as inositol-1,4,5-triphosphate (IP3) and calmodulin. Thus, it seems likely that plant mechanoperception involves both spatial and temporal encoding of information at all levels, from the cell to the whole plant. Defining this patterning will be a critical step towards understanding how plants integrate information from multiple mechanical stimuli to an appropriate growth response.