More than a Virulence Factor: Patulin Is a Non-Host-Specific Toxin that Inhibits Postharvest Phytopathogens and Requires Efflux for Penicillium Tolerance.

Mycotoxin contamination is a leading cause of food spoilage and waste on a global scale. Patulin, a mycotoxin produced by Penicillium spp. during postharvest pome fruit decay, causes acute and chronic effects in humans, withstands pasteurization, and is not eliminated by fermentation. While much is known about the impact of patulin on human health, there are significant knowledge gaps concerning the effect of patulin during postharvest fruit-pathogen interactions. Application of patulin on six apple cultivars reproduced some blue mold symptoms that were cultivar-independent and dose-dependent. Identical symptoms were also observed in pear and mandarin orange. Six Penicillium isolates exposed to exogenous patulin exhibited delayed germination after 24 h, yet all produced viable colonies in 7 days. However, four common postharvest phytopathogenic fungi were completely inhibited by patulin during conidial germination and growth, suggesting the toxin is important for Penicillium to dominate the postharvest niche. Using clorgyline, a broad-spectrum efflux pump inhibitor, we demonstrated that efflux plays a role in Penicillium auto-resistance to patulin during conidial germination. The work presented here contributes new knowledge of patulin auto-resistance, its mode of action, and inhibitory role in fungal-fungal interactions. Our findings provide a solid foundation to develop toxin and decay mitigation approaches.

Effect of Far-Red Light on Biomass Accumulation, Plant Morphology, and Phytonutrient Composition of Ruby Streaks Mustard at Microgreen, Baby Leaf, and Flowering Stages.

Far-red (FR) light influences plant development significantly through shade avoidance response and photosynthetic modulation, but there is limited knowledge on how FR treatments influence the growth and nutrition of vegetables at different maturity stages in controlled environment agriculture (CEA). Here, we comprehensively investigated the impacts of FR on the yield, morphology, and phytonutrients of ruby streaks mustard (RS) at microgreen, baby leaf, and flowering stages. Treatments including white control, white with supplementary FR, white followed by singularly applied FR, and enhanced white (WE) matching the extended daily light integral (eDLI) of FR were designed for separating the effects of light intensity and quality. Results showed that singular and supplemental FR affected plant development and nutrition similarly throughout the growth cycle, with light intensity and quality playing varying roles at different stages. Specifically, FR did not affect the fresh and dry weight of microgreens but increased those values for baby leaves, although not as effectively as WE. Meanwhile, FR caused significant morphological change and accelerated the development of leaves, flowers, and seedpods more dramatically than WE. With regard to phytonutrients, light treatments affected the metabolomic profiles for baby leaves more dramatically than microgreens and flowers. FR decreased the glucosinolate and anthocyanin contents in microgreens and baby leaves, while WE increased the contents of those compounds in baby leaves. This study illustrates the complex impacts of FR on RS and provides valuable information for selecting optimal lighting conditions in CEA.

Differential microbiota shift on whole romaine lettuce subjected to source or forward processing and on fresh-cut products during cold storage.

Romaine lettuce in the U.S. is primarily grown in California or Arizona and either processed near the growing regions (source processing) or transported long distance for processing in facilities serving distant markets (forward processing). Recurring outbreaks of Escherichia coli O157:H7 implicating romaine lettuce in recent years, which sometimes exhibited patterns of case clustering in Northeast and Midwest, have raised industry concerns over the potential impact of forward processing on romaine lettuce food safety and quality. In this study, freshly harvested romaine lettuce from a commercial field destined for both forward and source processing channels was tracked from farm to processing facility in two separate trials. Whole-head romaine lettuce and packaged fresh-cut products were collected from both forward and source facilities for microbiological and product quality analyses. High-throughput amplicon sequencing targeting16S rRNA gene was performed to describe shifts in lettuce microbiota. Total aerobic bacteria and coliform counts on whole-head lettuce and on fresh-cut lettuce at different storage times were significantly (p < 0.05) higher for those from the forward processing facility than those from the source processing facility. Microbiota on whole-head lettuce and on fresh-cut lettuce showed differential shifting after lettuce being subjected to source or forward processing, and after product storage. Consistent with the length of pre-processing delays between harvest and processing, the lettuce quality scores of source-processed romaine lettuce, especially at late stages of 2-week storage, was significantly higher than of forward-processed product (p < 0.05).

Microgreens for Home, Commercial, and Space Farming: A Comprehensive Update of the Most Recent Developments.

Microgreens are edible young plants that have recently attracted interest because of their color and flavor diversity, phytonutrient abundance, short growth cycle, and minimal space and nutrient requirements. They can be cultivated in a variety of systems from simple home gardens to sophisticated vertical farms with automated irrigation, fertilizer delivery, and lighting controls. Microgreens have also attracted attention from space agencies hoping that their sensory qualities can contribute to the diet of astronauts in microgravity and their cultivation might help maintain crew physical and psychological health on long-duration spaceflight missions. However, many technical challenges and data gaps for growing microgreensboth on and off Earth remain unaddressed. This review summarizes recent studies on multiple aspects of microgreens, including nutritional and socioeconomic benefits, cultivation systems, operative conditions, innovative treatments, autonomous facilities, and potential space applications. It also provides the authors' perspectives on the challenges to stimulating more extensive interdisciplinary research.

Mining the Penicillium expansum Genome for Virulence Genes: A Functional-Based Approach to Discover Novel Loci Mediating Blue Mold Decay of Apple Fruit.

Blue mold, a postharvest disease of pome fruits, is caused by the filamentous fungus Penicillium expansum. In addition to the economic losses caused by P. expansum, food safety can be compromised, as this pathogen is mycotoxigenic. In this study, forward and reverse genetic approaches were used to identify genes involved in blue mold infection in apple fruits. For this, we generated a random T-DNA insertional mutant library. A total of 448 transformants were generated and screened for the reduced decay phenotype on apples. Of these mutants, six (T-193, T-275, T-434, T-588, T-625, and T-711) were selected for continued studies and five unique genes were identified of interest. In addition, two deletion mutants (Δt-625 and Δt-588) and a knockdown strain (t-434KD) were generated for three loci. Data show that the ∆t-588 mutant phenocopied the T-DNA insertion mutant and had virulence penalties during apple fruit decay. We hypothesize that this locus encodes a glyoxalase due to bioinformatic predictions, thus contributing to reduced colony diameter when grown in methylglyoxal (MG). This work presents novel members of signaling networks and additional genetic factors that regulate fungal virulence in the blue mold fungus during apple fruit decay.

Avirulent Isolates of Penicillium chrysogenum to Control the Blue Mold of Apple Caused by P. expansum.

Blue mold is an economically significant postharvest disease of pome fruit that is primarily caused by Penicillium expansum. To manage this disease and sustain product quality, novel decay intervention strategies are needed that also maintain long-term efficacy. Biocontrol organisms and natural products are promising tools for managing postharvest diseases. Here, two Penicillium chrysogenum isolates, 404 and 413, were investigated as potential biocontrol agents against P. expansum in apple. Notably, 404 and 413 were non-pathogenic in apple, yet they grew vigorously in vitro when compared to the highly aggressive P. expansum R19 and Pe21 isolates. Whole-genome sequencing and species-specific barcoding identified both strains as P. chrysogenum. Each P. chrysogenum strain was inoculated in apple with the subsequent co-inoculation of R19 or Pe21 simultaneously, 3, or 7 days after prior inoculation with 404 or 413. The co-inoculation of these isolates showed reduced decay incidence and severity, with the most significant reduction from the longer establishment of P. chrysogenum. In vitro growth showed no antagonism between species, further suggesting competitive niche colonization as the mode of action for decay reduction. Both P. chrysogenum isolates had incomplete patulin gene clusters but tolerated patulin treatment. Finally, hygromycin resistance was observed for both P. chrysogenum isolates, yet they are not multiresistant to apple postharvest fungicides. Overall, we demonstrate the translative potential of P. chrysogenum to serve as an effective biocontrol agent against blue mold decay in apples, pending practical optimization and formulation.

Comparative Penicillium spp. Transcriptomics: Conserved Pathways and Processes Revealed in Ungerminated Conidia and during Postharvest Apple Fruit Decay.

Blue mold, caused by Penicillium spp., is an impactful postharvest disease resulting in significant economic losses due to reduced pome fruit quality and mycotoxin contamination. Using two Penicillium species with different levels of aggressiveness, transcriptomics were implemented in order to identify genes expressed during apple fruit decay and loci expressed in ungerminated conidia. Total RNA was isolated from ungerminated conidia and decayed apple fruit infected with P. expansum R19 or P. polonicum RS1. There were 2442 differentially expressed genes (DEGs) between the R19 and RS1 in apple. Comparisons within species between apple and conidia revealed 4404 DEGs for R19 and 2935 for RS1, respectively. Gene ontology (GO) analysis revealed differential regulation in fungal transport and metabolism genes during decay, suggesting a flux in nutrient acquisition and detoxification strategies. In R19, the oxidoreductase GO category comprised 20% of all DEG groups in apple verses conidia. Ungerminated conidia from both species showed DEGs encoding the glyoxylate shunt and beta-oxidation, specifying the earliest metabolic requirements for germination. This is the first study to identify pre-loaded transcripts in conidia from blue mold fungi, reveal unique genes between species expressed during apple decay, and show the expression dynamics of known fungal virulence factors. These findings will enable development of targeted approaches for blue mold abatement strategies.

Agarose hydrogel composite supports microgreen cultivation with enhanced porosity and continuous water supply under terrestrial and microgravitational conditions.

Hydrogels are attractive soilless media for plant cultivation with strong water and nutrient retention. However, pristine hydrogels contain mostly ultra-micro pores and lack air-filled porosity for root zone aeration. Herein we report a porous hydrogel composite comprising an agarose network and porous growing mix particle (GMP) fillers. The agarose backbone allowed the composite to sustain a 12-d growth cycle for red cabbage microgreens without the need for watering or crew interaction. Moreover, the GMP induced greater total pore volume and increased the prevalence of pores >30 µm by 8-fold. Further investigation suggested that the nutrients from GMP accounted for a 54 % increase in microgreen yield over pristine hydrogel, while the porous structure introduced by GMP improved the yield by another 44 %. Increased air-filled porosity accelerated the water transport and loss of hydrogel but maintained favorable water potential levels for plant extraction. Finally, the hydrogel composite supported microgreen growth satisfyingly under simulated microgravity despite some morphological changes. Results of this study reveal a novel growth substrate that is lightweight, convenient, and water-efficient, while effectively sustaining plant growth for multiple applications including indoor farming and space farming.

The Good, the Bad, and the Ugly: Mycotoxin Production During Postharvest Decay and Their Influence on Tritrophic Host-Pathogen-Microbe Interactions.

Mycotoxins are a prevalent problem for stored fruits, grains, and vegetables. Alternariol, aflatoxin, and patulin, produced by Alternaria spp., Aspergillus spp., and Penicillium spp., are the major mycotoxins that negatively affect human and animal health and reduce fruit and produce quality. Control strategies for these toxins are varied, but one method that is increasing in interest is through host microbiome manipulation, mirroring a biocontrol approach. While the majority of mycotoxins and other secondary metabolites (SM) produced by fungi impact host-fungal interactions, there is also an interplay between the various organisms within the host microbiome. In addition to SMs, these interactions involve compounds such as signaling molecules, plant defense and growth hormones, and metabolites produced by both the plants and microbial community. Therefore, studies to understand the impact of the various toxins impacting the beneficial and harmful microorganisms that reside within the microbiome is warranted, and could lead to identification of safe analogs for antimicrobial activity to reduce fruit decay. Additionally, exploring the composition of the microbial carposphere of host plants is likely to shed light on developing a microbial consortium to maintain quality during storage and abate mycotoxin contamination.

Nutrition by Design: Boosting Selenium Content and Fresh Matter Yields of Salad Greens With Preharvest Light Intensity and Selenium Applications.

Selenium (Se) is an essential mineral in multiple human metabolic pathways with immune modulatory effects on viral diseases including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and HIV. Plant-based foods contain Se metabolites with unique functionalities for the human metabolism. In order to assess the value of common salad greens as Se source, we conducted a survey of lettuce commercially grown in 15 locations across the USA and Canada and found a tendency for Se to accumulate higher (up to 10 times) in lettuce grown along the Colorado river basin region, where the highest amount of annual solar radiation of the country is recorded. In the same area, we evaluated the effect of sunlight reduction on the Se content of two species of arugula [Eruca sativa (E. sativa) cv. "Astro" and Diplotaxis tenuifolia (D. tenuifolia) cv. "Sylvetta"]. A 90% light reduction during the 7 days before harvest resulted in over one-third Se decline in D. tenuifolia. The effect of light intensity on yield and Se uptake of arugula microgreens was also examined under indoor controlled conditions. This included high intensity (HI) (160 µ mol-2 s-1 for 12 h/12 h light/dark); low intensity (LI) (70 µ mol m-2 s-1 for 12 h/12 h light/dark); and HI-UVA (12 h light of 160 µ mol m-2 s-1, 2 h UVA of 40 µ mol m-2 s-1, and 10 h dark) treatments in a factorial design with 0, 1, 5, and 10 ppm Se in the growing medium. HI and HI-UVA produced D. tenuifolia plants with 25-100% higher Se content than LI, particularly with the two higher Se doses. The addition of Se produced a marked increase in fresh matter (>35% in E. sativa and >45% in D. tenuifolia). This study (i) identifies evidence to suggest the revision of food composition databases to account for large Se variability, (ii) demonstrates the potential of introducing preharvest Se to optimize microgreen yields, and (iii) provides the controlled environment industry with key information to deliver salad greens with targeted Se contents.

Assessing the Food Safety Risk Posed by Birds Entering Leafy Greens Fields in the US Southwest.

In the US Southwest, it is common to observe birds in leafy green fields, though the risk they contribute to foodborne outbreaks remains unclear. In this study, we investigated and recorded the relationship between birds near leafy green fields and the risk for contaminated irrigation water or leafy green plants. We monitored the presence of birds for over two years and performed cloacal swab analysis for non-pathogenic Escherichia coli, E. coli O157:H7 and Salmonellaenterica, while also monitoring the incidence of other microbial indicators. We also assessed the risks from bird feces by performing observations in a commercial field reported with Salmonella positive samples and by analyzing the survival of foodborne pathogens in bird feces. Our results showed that most of the birds near the crop fields were resident small birds. We did not observe a correlation between the number of birds in sites and the incidence of indicator bacteria (e.g., coliforms, E. coli) in irrigation canal water, with the exception of one out of four sites where water flow was low or stagnant. Using walk-in-traps, 305 birds were captured and placed in short-term captivity to determine the presence of various bacteria. None of the birds tested positive for E. coli O157:H7 or Salmonella. However, nearly 40% of the birds captured were confirmed positive for non-pathogenic E. coli. We found no correlation between age (young, adult, unknown), gender (male, female, unknown) and the incidence of E. coli positive birds, but we observed significantly higher probability of incidence during October-December. The role of relative humidity and temperature on bacterial survival appeared to play a key role in the survival of Salmonella on the leaves of spinach plants in a commercial field. This was also confirmed in laboratory conditions where Salmonella inoculated in bird feces and exposed to 15 °C and 80% RH(Relative humidity) survived beyond 133 days, while at 26 °C and 40% RH, the organism was undetectable after 63 days. Our results suggest that local birds associated with leafy green fields likely pose a minimal impact of risk for food contamination, but also points out the need for increased analysis specifically for E. coli O157:H7. Furthermore, our study suggests the need for expanding research that addresses risks associated with large migratory birds, especially in areas where stagnated water sources would be used for overhead sprinkle irrigation.

Modified Coring Tool Designs Reduce Iceberg Lettuce Cross-Contamination.

Contaminated coring tools may transfer bacteria to iceberg lettuce. The efficiency of coring tool design modifications in reducing bacterial transfer to lettuce heads was evaluated under simulated field operations. The standard coring tool consists of a stainless steel cylindrical tube welded to a tab that is inserted into a plastic handle. Design modifications included removal of the welded portion, incorporation of a shorter front straight bottom edge, or an angled bottom edge toward the front. In the first study, coring tools of four different designs were inoculated by dipping in a tryptic soy broth (TSB) suspension that contained 8.85 Log CFU/mL of Escherichia coli K-12 and then were used to core 100 lettuce heads, consecutively. Use of the standard tool resulted in 91% ± 9% positive lettuce heads. Removing the welded surface from the standard tool resulted in the highest reduction of E. coli transfer (44% ± 11.9% positive lettuce heads, P < 0.05), whereas incorporation of a short front straight edge with no welding resulted in 65.6% ± 5.6% of the cored lettuce heads being positive for E. coli. Removal of the welded surface resulted in a 40% decrease in E. coli contamination among the last 20 cored lettuce heads (81 to 100), which indicates that coring tool design modifications resulted in reduced cross-contamination. In the second study, the transfer of Salmonella to coring tools after their immersion in rinsing solutions was evaluated using imaging. The tools were dip inoculated for 2 min in water, water with lettuce extract, or TSB containing 7 Log CFU/mL bioluminescent Salmonella Newport; they were then imaged to observe spatial distribution of bacteria. There was greater retention and spatial distribution of Salmonella on the surface of tools immersed in water containing lettuce extract than in TSB and water. The results of the second study indicate that rinsing solutions that contain lettuce particulate and organic load could facilitate cross-contamination of Salmonella Newport to tool surfaces.

Salt in irrigation water affects the nutritional and visual properties of romaine lettuce (Lactuca sativa L.).

The influence of salinity stress on the growth, appearance, and nutritional compounds, especially phenolic compounds and carotenoids, of romaine lettuce (Lactuca sativa L.), a low salt tolerant plant, was studied. The dry weight, height, and color of the lettuce plants were significantly changed by long-term irrigation (15 days) with higher NaCl concentration (i.e., >100 mM). However, no significant differences were observed in the growth and appearance among the control, all short-term treatments (2 days; 50, 100, 500, and 1000 mM), and long-term irrigation with low salt concentration. Moreover, in romaine lettuce treated with long-term irrigation with 5 mM NaCl, the total carotenoid content increased without color change, and the contents of major carotenoids in romaine lettuce, lutein and beta-carotene, increased 37 and 80%, respectively. No differences were observed in lutein and beta-carotene contents in short-term-treated lettuce. The phenolic content of the romaine lettuce declined with short-term salt irrigation, whereas there were no significant differences among treatments exposed to long-term irrigation. This research indicates that long-term irrigation with relatively low salt concentration, rather than short-term irrigation with high salt concentration, can increase carotenoid content in romaine lettuce without causing a tradeoff in yield or visual quality.

Effect of methyl jasmonate on phenolic compounds and carotenoids of romaine lettuce (Lactuca sativa L.).

The effect of exogenous methyl jasmonate (MeJA) on antioxidative compounds of romaine lettuce ( Lactuca sativa L.) was investigated. Lettuces were treated with various MeJA solutions (0, 0.05, 0.1, 0.25, and 0.5 mM) before harvest. Total phenolic compounds content and antioxidant capacity of romaine lettuce significantly increased after MeJA treatments (0.1, 0.25, and 0.5 mM). The total content of phenolic compounds of the romaine lettuce treated with 0.5 mM MeJA (31.6 microg of gallic acid equivalents/mg of dry weight) was 35% higher than that of the control. The increase in phenolic compound content was attributed to a caffeic acid derivative and an unknown phenolic compound, which also contributed to increased antioxidant capacity. The induction of phenylalanine ammonia-lyase (PAL) activity by the MeJA treatment indicated that phenolic compounds were altered due to the activation of the phenylpropandoid pathway. Total content of carotenoids, including lutein and beta-carotene, of the MeJA-treated lettuce did not change after 8 days of treatment, whereas the content of the control without MeJA decreased after 8 days. This research indicated that preharvest application of MeJA could increase the nutritional value of romaine lettuce under determined conditions discussed in this work.

Parthenolide and abscisic acid synthesis in feverfew are associated but environmental factors affect them dissimilarly.

The effect of harvest time, shading prior to harvest and water stress on parthenolide (PRT) concentration in feverfew and its possible connection with the abscisic acid (ABA) pathway were investigated. In plants harvested at different times of the day, acetumar the PRT levels were highest during late afternoon while ABA levels were greatest during morning hours. Shading plants during the afternoon prior to harvest caused a two-fold increase in ABA and no significant difference in PRT levels. ABA was higher in water-stressed plants while PRTcontent increased in plants following recovery from a water stress event. ABA inhibitors, norflurazon, sodium tungstate, naproxen and sodium bisulfite, were used to determine the connection between the biosynthesis of PRTand ABA. Norflurazon and naproxen reduced PRT concentration in cut flowers and in 2-month old plants. Sodium bisulfite and sodium tungstate reduced PRT only in cut flowers. Application of 2,4-D, a promoter of ABA synthesis, to potted plants resulted in a 2.5 fold increase in PRT levels. The inhibition of PRT formation in response to ABA inhibitors and the increase in PRT concentration observed with 2,4-D application indicated that PRT is derived from carotenoid synthesis similarly to ABA and not directly from farnesyl pyrosphosphate (FPP) as suggested for other sesquiterpene Lactones. However, PRT and ABA levels are affected dissimilarly by environmental conditions. The overall results of the study indicated that simple agricultural practices, such as harvesting during afternoon and subjecting plants to a single water stress event, can increase PRT concentration in the final feverfew product with no additional costs of production prior to harvest.