Romaine Lettuce Plants Research Articles

Respon Pertumbuhan dan Produksi Tanaman Selada Romaine terhadap Otomasi Aerasi pada Sistem Hidroponik Rakit Apung

Romaine lettuce is a nutritious leaf vegetable with a high selling price and its production can be increased using a hydroponic system. Floating raft hydroponics is a hydroponic system with plants floating on styrofoam and their roots submerged in a nutrient solution. Providing aeration using an aerator connected to a timer switch is necessary to increase dissolved oxygen in the nutrient solution to optimize plant growth and production. This research aims to analyze the effect of aeration automation and the best aeration duration on the growth and production of romaine lettuce plants in a floating raft hydroponic system. The research was conducted at Greenhouse UG Technopark in May-June 2023. This experiment used a one-factor Completely Randomized Design with 4 aeration duration treatments: no aeration (control), 6 hours aeration/day, 15 hours/day aeration, and 24 hours/day aeration. Each treatment was repeated 5 times so that there were 20 experimental units and consisted of 5 samples for a total of 100 samples. The results of this research show that aeration 24 hours/day has a real influence on the parameters of plant height in the 3rd week, leaf length, leaf width, leaf wet weight, and root shoot ratio (P<0,05). Aeration 24 hours/day provides the best results for the growth and production of romaine lettuce plants. Aeration automation of 15 hours/day can be used as an alternative from the producer's side to reduce electricity use.

Growth and Production of Romaine Lettuce (Lactuca sativa L.) on Several Dosage of Rabbit Urine

Romaine lettuce is a vegetable with high economic value. Romaine lettuce needs to be grown organically. This research aimed to study the effect of various doses of rabbit urine on romaine lettuce. This research was conducted from June to August 2022 at the Smart and Urban Farming Laboratory Greenhouse, Campus F7, Gunadarma University, East Jakarta, Indonesia. The study used a randomized block design (RBD) with five replications, consisting of 5 treatments, namely: P0: without rabbit urine (control); P1: Concentration of rabbit urine 25%; P2: Concentration of rabbit urine 50%; P3: Concentration of rabbit urine 75%; P4: Concentration of rabbit urine 100%. The results showed that the administration of rabbit urine had no significant effect on the growth and yield of romaine lettuce plants, and the best concentration of rabbit urine for romaine lettuce plants in this study was 80 ml (P2).

Cultivar was more influential than bacterial strain and other experimental factors in recovery of Escherichia coli O157:H7 populations from inoculated live Romaine lettuce plants.

The varied choice of bacterial strain, plant cultivar, and method used to inoculate, retrieve, and enumerate Escherichia coli O157:H7 from live plants could affect comparability among studies evaluating lettuce-enterobacterial interactions. Cultivar, bacterial strain, incubation time, leaf side inoculated, and sample processing method were assessed for their influence in recovering and quantifying E. coli O157:H7 from live Romaine lettuce. Cultivar exerted the strongest effect on E. coli O157:H7 counts, which held up even when cultivar was considered in interactions with other factors. Recovery from the popularly grown green Romaine "Rio Bravo" was higher than from the red variety "Outredgeous." Other modulating variables were incubation time, strain, and leaf side inoculated. Sample processing method was not significant. Incubation for 24 hours post-lettuce inoculation yielded greater counts than 48 hours, but was affected by lettuce cultivar, bacterial strain, and leaf side inoculated. Higher counts obtained for strain EDL933 compared to a lettuce outbreak strain 2705C emphasized the importance of selecting relevant strains for the system being studied. Inoculating the abaxial side of leaves gave higher counts than adaxial surface inoculation, although this factor interacted with strain and incubation period. Our findings highlight the importance of studying interactions between appropriate bacterial strains and plant cultivars for more relevant research results, and of standardizing inoculation and incubation procedures. The strong effect of cultivar exerted on the E. coli O157:H7-lettuce association supports the need to start reporting cultivar information for illness outbreaks to facilitate the identification and study of plant traits that impact food safety risk.IMPORTANCEThe contamination of Romaine lettuce with Escherichia coli O157:H7 has been linked to multiple foodborne disease outbreaks, but variability in the methods used to evaluate E. coli O157:H7 association with live lettuce plants complicates the comparability of different studies. In this study, various experimental variables and sample processing methods for recovering and quantifying E. coli O157:H7 from live Romaine lettuce were assessed. Cultivar was found to exert the strongest influence on E. coli O157:H7 retrieval from lettuce. Other modulating factors were bacterial incubation time on plants, strain, and leaf side inoculated, while sample processing method had no impact. Our findings highlight the importance of selecting relevant cultivars and strains, and of standardizing inoculation and incubation procedures, in these types of assessments. Moreover, results support the need to start reporting cultivars implicated in foodborne illness outbreaks to facilitate the identification and study of plant traits that impact food safety risk.

Rhizoctonia solani AG1-IB, AG1-IC, and AG4-HGII cause bottom rot of field lettuce in Vermont.

Members of Rhizoctonia solani (teleomorph: Thanatephorus cucumeris) species complex cause bottom rot on lettuce (Latuca sativa) and yield losses up to 70% (Subbarao et al. 2017). Severe symptoms include necrosis, stem rot, and/or discoloration especially on the leaf midrib. In Vermont, vegetable farms are small (0.5-30 acres) and grow lettuce concurrently with other vegetable crops in the same field but the AG(s) that causes the disease in Vermont has not been determined. Isolates (n = 157) were collected from 31 fields with reported history of bottom rot between July 10 and October 8, 2019, across Addison, Caledonia, Chittenden, Franklin, Lamoille, and Orleans counties. Isolates were collected from lettuce tissue or potato (Solanum tuberosum), a common rotation crop, or uncropped soil baited using radish (Raphanus sativus). Pieces of tissue (5-10 mm) were cut from the leading margin of lesions, surface disinfested with 0.1% NaClO for 1 min followed by 2 rinses with sterile water, blotted dry, and plated onto acidified 2% water agar (0.085% lactic acid, pH 4.8). After incubation for 48 to 72 h, mycelia resembling Rhizoctonia were examined for morphological characteristics including hyphal branching at ca. 90o angles, a septum near the branching point, multiple nuclei per cell, and lack of both clamp connections and conidia (Sneh et al. 1991). Colonies were white to dark brown, and some produced small sclerotia. Koch's postulates were performed by inoculating nine 8-week-old (9 leaf pairs) romaine lettuce plants (Johnny's Seeds, Winslow, ME, cv. Monte Carlo) per isolate. Isolates were grown on 2% potato dextrose agar for 1 week, from which a 5-mm agar plug was placed on the adaxial leaf surface at the base of a petiole. Plants were enclosed in a plastic bag to maintain high humidity and grown under a 16-hour photoperiod at 24 °C. Disease severity was rated 4 days after inoculation (0: healthy, 1: isolated lesions, 2: lesions across multiple petioles, and 3: systemic disease). Putative AG were determined by Sanger sequencing of the internal transcribed spacer (ITS) region using the ITS1F and ITS4B primer pair (758 bp) (Gardes and Bruns 1993). Contigs were assembled using CAP3 software (Huang and Madan 1999). Taxonomy was assigned to each OTU via the NCBI BLASTn database with criteria as 0.0 E and nucleotide match of at least 97%. Of the 10 isolates sequenced with sufficient coverage (735 to 784 bp alignment length) and definitive resolution (96.7 to 99.9% identity), 5 were putative AG 1-IB (Genbank Accession HG934430.1), 2 AG 1-IC (Genbank Accession AF354058.1), 2 AG 3 (Genbank Accession AF354064.1), and 1 AG 4-HGII (Genbank Accession AF354074.1). Fasta files and metadata are archived at 10.6084/m9.figshare.20301324, 10.6084/m9.figshare.20301375. Putative AG 1-IB was highly virulent on lettuce plants whether it originated from potato (mean 2.6) or lettuce (mean 1.3 to 3). AG 4-HGII and AG 1-IC isolated from lettuce and radish, respectively, were moderately severe (mean 1.4 to 2.2) on lettuce with identical symptoms. The two potato isolates (AG3) were not pathogenic on lettuce. Similarly, higher incidence of AG 1-IB is reported on lettuce in Quebec (Wallon et al. 2021), Ohio (Herr 1993), and Germany (Grosch et al. 2004). Because AG vary in their host range (Sneh et al. 1991), knowing the AG will inform management decisions such as crop rotation and weed control. This is the first report of the causal agent of bottom rot of lettuce or any AG of R. solani in Vermont.

Predictive Population Dynamics of Escherichia coli O157:H7 and Salmonella enterica on Plants: a Mechanistic Mathematical Model Based on Weather Parameters and Bacterial State.

Weather affects key aspects of bacterial behavior on plants but has not been extensively investigated as a tool to assess risk of crop contamination with human foodborne pathogens. A novel mechanistic model informed by weather factors and bacterial state was developed to predict population dynamics on leafy vegetables and tested against published data tracking Escherichia coli O157:H7 (EcO157) and Salmonella enterica populations on lettuce and cilantro plants. The model utilizes temperature, radiation, and dew point depression to characterize pathogen growth and decay rates. Additionally, the model incorporates the population level effect of bacterial physiological state dynamics in the phyllosphere in terms of the duration and frequency of specific weather parameters. The model accurately predicted EcO157 and S. enterica population sizes on lettuce and cilantro leaves in the laboratory under various conditions of temperature, relative humidity, light intensity, and cycles of leaf wetness and dryness. Importantly, the model successfully predicted EcO157 population dynamics on 4-week-old romaine lettuce plants under variable weather conditions in nearly all field trials. Prediction of initial EcO157 population decay rates after inoculation of 6-week-old romaine plants in the same field study was better than that of long-term survival. This suggests that future augmentation of the model should consider plant age and species morphology by including additional physical parameters. Our results highlight the potential of a comprehensive weather-based model in predicting contamination risk in the field. Such a modeling approach would additionally be valuable for timing field sampling in quality control to ensure the microbial safety of produce. IMPORTANCE Fruits and vegetables are important sources of foodborne disease. Novel approaches to improve the microbial safety of produce are greatly lacking. Given that bacterial behavior on plant surfaces is highly dependent on weather factors, risk assessment informed by meteorological data may be an effective tool to integrate into strategies to prevent crop contamination. A mathematical model was developed to predict the population trends of pathogenic E. coli and S. enterica, two major causal agents of foodborne disease associated with produce, on leaves. Our model is based on weather parameters and rates of switching between the active (growing) and inactive (nongrowing) bacterial state resulting from prevailing environmental conditions on leaf surfaces. We demonstrate that the model has the ability to accurately predict dynamics of enteric pathogens on leaves and, notably, sizes of populations of pathogenic E. coli over time after inoculation onto the leaves of young lettuce plants in the field.

The Influence of Consortia of Beneficial Microorganisms on the Growth and Yield of Aquaponically Grown Romaine Lettuce

This study evaluated the effects of fish farm wastewater from the production of hybrid sturgeon (Acipenser gueldenstaedtii Brandt × Acipenser baeri Brandt) on the growth and quality parameters of romaine lettuce (Lactuca sativa var. longifolium cv. “Elizium”). The tested combinations were fish farm wastewater, fish farm wastewater enriched with one of the three microbiological consortia, and fish farm wastewater supplemented with minerals. The best growth parameters of romaine lettuce plants were obtained in the combination of wastewater from fish farming supplemented with mineral nutrients. The application of fish farm wastewater and beneficial microbiological consortia positively influenced the fresh weight of lettuce leaves and the number of leaves per plant. However, plants fed with wastewater supplemented with minerals were characterized by the strongest symptoms of leaf tip-burn and the lowest commercial value. By comparison, plants fed only with fish farm wastewater or wastewater with microorganisms were characterized by a high, similar commercial value. After the application of increased doses of minerals, there was evidence of greater activity of microorganisms involved in nutrient cycling in aquaponic lettuce cultivation. The application of the microbiological consortia and minerals significantly increased the numbers and activity of the bacteria in the culture liquids 7, 14, and 21 days after inoculation.

Rice Husk Ash Incorporation in Container Substrates Effect on Romaine Lettuce Plant Growth

Primary substrate components include peat moss, vermiculite, perlite, bark, and compost are commonly-used substrate in horticultural crop production.These substrate components are high cost due to the extreme cost of transportation, fuel for extraction, and processing. Local rice husk ash (RHA) as an alternative substrate component was used to evaluatethe growth of Romaine Lettuce plants in response to the application of different incorporations of RHA in container substrates. The test was conducted with a completely randomized design of four RHA treatment, rates at 0%, 10%, 30%, and 50% (by vol.), with 10 replicates. The results showed that plant stem elongation increased with decreased incorporations of RHA application. Although the fresh weight of plants(shoot or root)and number of leaves increased with the RHA application, no significant difference for the plant biomass produced (dry weight of root and shoot) betweenthe 30% and 50% RHA treatments. The RHA can be used as a local and low cost substrate component; and the incorporation of 30% (by vol.) RHA in container substrates is a certain amount for lettuce plant growth.

Towards Nutrition-Sensitive Agriculture: An evaluation of biocontrol effects, nutritional value, and ecological impact of bacterial inoculants

Nutrition-Sensitive Agriculture (NSA) is a novel concept in agriculture that considers not only yield, but also nutritional value of produce, sustainability of production, and ecological impact of agriculture. In accordance with its goals, NSA would benefit from applying microbial-based products as they are deemed more sustainable than their synthetic counterparts.This study characterized 3 plant-beneficial bacterial strains (Paenibacillus pasadenensis strain R16, Pseudomonas syringae strain 260-02, Bacillus amyloliquefaciens strain CC2) on their biocontrol activity and effect on nutritional and texture quality of romaine lettuce plants (Lactuca sativa) in greenhouse. The pathogens used in the trials are Rhizoctonia solani and Pythium ultimum.The obtained results indicate that strain R16 had a significant ability to cause a statistically significant reduction in the symptoms caused by both P. ultimum (reduction of 32%) and R. solani (reduction of 42%), while the other two strains showed a less efficient biocontrol ability.Indices of the nutritional quality (content in phenols, carotenoids and chlorophyll) were unaffected by the treatments, indicating that the product was equivalent to that obtained without using the bacteria, while the texture of the leaves benefits from the biocontrol treatments. In particular, the mechanical resistance of the leaves was significantly higher in non-treated plants affected by R. solani but was restored to the values of healthy plants when the bacterial inoculants were present as well.The ecological impact was evaluated by characterizing the bacterial microbiota in bulk soil, rhizosphere, and root in the presence or absence of the inoculants.The composition of the microbiota, analyzed with a Unifrac model to describe beta-diversity, was radically different in the rhizosphere and the root endosphere among treatments, while the bulk soil formed a single cluster regardless of treatment, indicating that the use of these treatments did not have an ecological impact outside of the plant.

Conditions at the time of inoculation influence survival of attenuated Escherichia coli O157:H7 on field-inoculated lettuce

The impact of plant development, environmental conditions at the time of inoculation, and inoculum concentration on survival of attenuated BSL1 Escherichia coli O157:H7 strain ATCC 700728 on field-grown romaine lettuce was evaluated over 3 years. E. coli 700728 was inoculated onto 4- and 6-week-old romaine lettuce plants in the Salinas Valley, CA, at night or the next morning with either low (5 log) or high (7 log) cell numbers per plant to simulate a single aqueous contamination event. At night, when leaf wetness and humidity levels were high, E. coli cell numbers declined by 0.5 log CFU/plant over the first 8–10 h. When applied in the morning, E. coli populations declined up to 2 log CFU/plant within 2 h. However, similar numbers of E. coli were retrieved from lettuce plants at 2 and 7 days. E. coli cell numbers per plant were significantly lower (P < 0.05) 7 days after application onto 4-week-old compared to 6-week-old plants. E. coli 700728 could be recovered by plating or enrichment from a greater proportion of plants for longer times when inoculated at high compared with low initial concentrations and after inoculation of 6-week-old plants compared with 4-week-old plants, even at the low initial inoculum. A contamination event near harvest or when leaf wetness and humidity levels are high may enhance survivability, even when low numbers of E. coli are introduced.

Effects of Two Doses of Organic Extract-Based Biostimulant on Greenhouse Lettuce Grown Under Increasing NaCl Concentrations.

The enhancement of plant tolerance toward abiotic stresses is increasingly being supported by the application of biostimulants. Salinity represents a serious problem in the Mediterranean region. To verify the effects deriving from the application of biostimulants, trials on Romaine lettuce plants under salt exposure were performed, in greenhouse. Plants were subjected to three NaCl solutions with 0.8, 1.3, and 1.8 dS/m of electrical conductivity. The volume of the solution was 200 mL/plant and delivered every 3 days. Biostimulant treatments started after crop establishment and were: control (water) and two doses (0.1 or 0.2 mL/plant) of the commercial biostimulant Retrosal® (Valagro S.p.A), containing calcium, zinc, and specific active ingredients. Four Retrosal® treatments were applied, every 7 days, directly to the substrate. Non-destructive analyses were conducted to assess the effects on leaf photosynthetic efficiency. At harvest, plants fresh weight (FW) and dry weight were determined, as well as the concentration of chlorophylls, carotenoids, total sugars, nitrate, proline, and abscisic acid (ABA). The biostimulant tested increased significantly the FW of lettuce (+65% in the highest dose) compared to controls. Results indicate that treatments positively affected the chlorophyll content measured in vivo (+45% in the highest dose) and that a general positive effect was observable on net photosynthesis rate. Retrosal® seems to improve the gas exchanges under our experimental conditions. The total sugars levels were not affected by treatments. Biostimulant allowed maintaining nitrate concentration similar to the untreated and unstressed controls. The increasing levels of water salinity caused a raise in proline concentration in control plants (+85%); biostimulant treatments at 0.2 mL/plant dose kept lower the proline levels. All plants treated with the biostimulant showed lower value of ABA (-34%) compared to controls. Results revealed that Retrosal® is able to stimulate plant growth independently from the salinity exposure. However, treated plants reached faster the commercial maturity stage. The fresh biomass of control at the end of experiment, after 30 days, ranged from 15 to 42 g/head, while in biostimulant treated plants ranged from 45 to 94 g/head. The product applied at maximum dose seems to be the most effective in our experimental conditions.

Internalization and dissemination of human norovirus and Tulane virus in fresh produce is plant dependent

Human norovirus (NoV) is a leading cause of fresh produce associated outbreaks. Previous research indicates that the roots of growing leafy greens and berries internalize human NoV. However the effect of plant type and inoculum level on internalization rates has not been directly compared. In this study we compared the internalization and dissemination rates of human NoV and its surrogate, Tulane virus (TV) in green onion, radishes, and Romaine lettuce. We also evaluated the effect inoculum level and plant growth matrix on the rate of viral internalization. In the hydroponic growth system, we detected internalization and dissemination of human NoV RNA in green onions. In hydroponically growing green onions inoculated with high titer TV, we found higher rates of internalization and dissemination compared to green onions inoculated with low titer TV. In soil growth systems, no infectious TV was detected in either green onion or radishes. However, in Romaine lettuce plants grown in soil approximately 4 log10 PFU/g was recovered from all tissues on day 14 p.i. Overall, we found that the type of plant, growth matrix, and the inoculum level influences the internalization and dissemination of human NoV and TV.

Listeria monocytogenes Internalizes in Romaine Lettuce Grown in Greenhouse Conditions

Listeria monocytogenes has been implicated in a number of outbreaks involving fresh produce, including an outbreak in 2016 resulting from contaminated packaged salads. The persistence and internalization potential of L. monocytogenes in romaine lettuce was evaluated, and the persistence of two L. monocytogenes strains was assessed on three romaine lettuce cultivars. Seeds were germinated, and plants grown in three soil types (i.e., standard potting mix, autoclaved potting mix, and top soil) and sterile soft-top agar for up to 21 days. Average CFU per gram of L. monocytogenes on seeds and plants was calculated from five replicates per harvest day. Up to 8.2 log CFU/g L. monocytogenes persisted on romaine lettuce plants (Braveheart cultivar) grown in soft-top agar, while those grown in commercial potting mix (initial soil aerobic plate count of 4.0 × 104 CFU/g) had a final concentration of 5.4 log CFU/g, and autoclaved commercial potting mix had a final concentration of 3.8 ± 0.2 log CFU/g after a 21-day period. Pathogen levels dropped below the limit of detection (2 log CFU/g) by day 18 in 75% topsoil (initial soil aerobic plate count of 4.0 × 101 CFU/g); this did not occur in sterile media. Although L. monocytogenes strain differences and presence of a clay coating on seeds did not affect persistence, differences were observed in L. monocytogenes growth and survival among cultivars. To assess internalization, seeds were inoculated with L. monocytogenes expressing green fluorescent protein. Three plants were fixed, paraffin embedded, and sectioned; localization was studied by using standard immunohistochemistry techniques. A total of 539 internalized L. monocytogenes cells were visualized among three 20-day seedlings. L. monocytogenes cells were located in all major tissue types (pith followed by cortex, xylem, phloem, and epidermis). The presence of L. monocytogenes in the plant vasculature suggests potential for transport throughout the plant into edible tissue.

RNA-Based Detection Does not Accurately Enumerate Living Escherichia coli O157:H7 Cells on Plants.

The capacity to distinguish between living and dead cells is an important, but often unrealized, attribute of rapid detection methods for foodborne pathogens. In this study, the numbers of enterohemorrhagic Escherichia coli O157:H7 after inoculation onto Romaine lettuce plants and on plastic (abiotic) surfaces were measured over time by culturing, and quantitative PCR (qPCR), propidium monoazide (PMA)-qPCR, and reverse transcriptase (RT)-qPCR targeting E. coli O157:H7 gapA, rfbE, eae, and lpfA genes and gene transcripts. On Romaine lettuce plants incubated at low relative humidity, E. coli O157:H7 cell numbers declined 107-fold within 96 h according to culture-based assessments. In contrast, there were no reductions in E. coli levels according to qPCR and only 100- and 1000-fold lower numbers per leaf by RT-qPCR and PMA-qPCR, respectively. Similar results were obtained upon exposure of E. coli O157:H7 to desiccation conditions on a sterile plastic surface. Subsequent investigation of mixtures of living and dead E. coli O157:H7 cells strongly indicated that PMA-qPCR detection was subject to false-positive enumerations of viable targets when in the presence of 100-fold higher numbers of dead cells. RT-qPCR measurements of killed E. coli O157:H7 as well as for RNaseA-treated E. coli RNA confirmed that transcripts from dead cells and highly degraded RNA were also amplified by RT-qPCR. These findings show that neither PMA-qPCR nor RT-qPCR provide accurate estimates of bacterial viability in environments where growth and survival is limited.

Influence of mycorrhizal fungi on fate of E. coli O157:H7 and Salmonella in soil and internalization into Romaine lettuce plants

The objectives of this study were to determine the influence of a symbiotic arbuscular mycorrhizal (AM) fungus on persistence of Salmonella and enterohemorrhagic Escherichia coli O157:H7 (EHEC) within soil, and survival within Romaine lettuce. Romaine seedlings were grown with or without AM fungi. Soil surrounding plants was inoculated with ca. 8log CFU/plant of either Salmonella enterica or E. coli EHEC composites. Samples (soil, root, and shoot) were analyzed on days 1, 8, 15 and 22 for Salmonella and EHEC by direct plating and selective enrichment. Twenty-four hours after inoculation, populations of Salmonella and EHEC, respectively, were 4.20 and 3.24log CFU/root, 2.52 and 1.17log CFU/shoot, and 5.46 and 5.17log CFU/g soil. By selective enrichment, samples tested positive for Salmonella or EHEC at day 22 at rates of 94 and 68% (shoot), 97 and 56% (root), and 100 and 75% (soil), respectively, suggesting that Salmonella has a greater propensity for survival than EHEC. Salmonella populations in soil remained as high as 4.35log CFU/g by day 22, while EHEC populations dropped to 1.12log CFU/g in the same amount of time. Ninety-two percent of all Romaine leaves in our study were positive for internalized Salmonella from days 8 to 22 and remained as high as 1.26log CFU/shoot on day 22 in AM fungi+Romaine plants. There were no differences (P>0.05) between the survival of either pathogen based on the presence or absence of mycorrhizal fungi. Results of this study suggest that AM fungi do not affect the internalization and/or survival of either S. enterica or E. coli O157:H7 in Romaine lettuce seedlings. Our results should provide Romaine lettuce farmers confidence that the presence and/or application of AM fungi to crop soil is not a contributing factor to the internalization and survival of Salmonella or E. coli O157:H7 within Romaine lettuce plants.

Phyllosphere microbiota composition and microbial community transplantation on lettuce plants grown indoors.

ABSTRACTThe aerial surfaces of plants, or phyllosphere, are microbial habitats important to plant and human health. In order to accurately investigate microbial interactions in the phyllosphere under laboratory conditions, the composition of the phyllosphere microbiota should be representative of the diversity of microorganisms residing on plants in nature. We found that Romaine lettuce grown in the laboratory contained 10- to 100-fold lower numbers of bacteria than age-matched, field-grown lettuce. The bacterial diversity on laboratory-grown plants was also significantly lower and contained relatively higher proportions of Betaproteobacteria as opposed to the Gammaproteobacteria-enriched communities on field lettuce. Incubation of field-grown Romaine lettuce plants in environmental growth chambers for 2 weeks resulted in bacterial cell densities and taxa similar to those on plants in the field but with less diverse bacterial populations overall. In comparison, the inoculation of laboratory-grown Romaine lettuce plants with either freshly collected or cryopreserved microorganisms recovered from field lettuce resulted in the development of a field-like microbiota on the lettuce within 2 days of application. The survival of an inoculated strain of Escherichia coli O157:H7 was unchanged by microbial community transfer; however, the inoculation of E. coli O157:H7 onto those plants resulted in significant shifts in the abundance of certain taxa. This finding was strictly dependent on the presence of a field-associated as opposed to a laboratory-associated microbiota on the plants. Phyllosphere microbiota transplantation in the laboratory will be useful for elucidating microbial interactions on plants that are important to agriculture and microbial food safety.

First Report of White Mold Caused by Sclerotinia minor on Mexican Sunflower in California.

Mexican sunflower (Tithonia rotundifolia) is a plant in the Asteraceae that is grown commercially as a cutflower commodity and also as a beneficial insectary plant. In June 2012 in coastal California (Santa Cruz County), several fields of organic lettuce (Lactuca sativa) were interplanted with direct-seeded rows of Mexican sunflower (cv. Torch) in order to attract beneficial insects. When approximately 2 to 3 weeks from harvest, lettuce plants began to wilt and collapse. Lettuce crowns were decayed and covered with white mycelium and small (0.5 to 3 mm diameter), irregularly shaped, black sclerotia. These plants were confirmed to have lettuce drop disease caused by Sclerotinia minor (2). In addition, Mexican sunflower plants began to wilt and eventually died. Initial symptoms on crowns and bases of the main stems in contact with soil consisted of a light tan discoloration. These discolored areas turned darker brown, became necrotic, and later were covered with white mycelium and sclerotia that were identical to those found on lettuce. Symptomatic sunflower stems were surface disinfested and small pieces from the margins of necrotic areas were placed into petri plates containing acidified potato dextrose agar. Resulting fungal colonies were white, produced profuse numbers (approx. 39 sclerotia/cm2) of small black sclerotia, and were identified as S. minor. Six-week-old Mexican sunflower plants grown in a peat moss-based rooting medium in 5-cm square pots were used to test the pathogenicity of four isolates. Isolates were grown on cubed and autoclaved potato pieces and resulting sclerotia were recovered and dried (1). For each isolate, 12 plants for each of three cultivars (cvs. Fiesta del Sol, Torch, and Yellow Torch) were inoculated by placing 3 to 5 sclerotia 1 cm below the soil level and adjacent to the plant crowns/stem bases. Sterile sand was placed next to crowns of the control plants. Plants were maintained in a greenhouse at 22 to 24°C. Symptom development was rapid and after 6 to 7 days, inoculated Tithonia plants exhibited brown necrosis at inoculated areas. After 10 days, Tithonia crowns were decayed and plants wilted. S. minor was reisolated from selected necrotic crown and stem tissues. Diseased plants that were not used for reisolations later supported the growth of the characteristic white mycelium and black sclerotia. There were no significant differences between the Tithonia cultivars, and overall disease incidence ranged from 74 to 100%. Non-inoculated plants were asymptomatic. The experiment was repeated and results were similar. In addition, the sclerotia of the four Tithonia isolates were similarly inoculated onto sets of 12 romaine lettuce plants (cv. Green Towers). After 5 to 6 days, all plants developed lettuce drop disease and the pathogen was reisolated. To my knowledge, this is the first report of Mexican sunflower as a host of S. minor. These findings indicate that Mexican sunflower and lettuce are susceptible to the same lettuce drop pathogen, and that this beneficial insectary plant could increase soilborne inoculum of S. minor. Growers should therefore be aware of the host status of beneficial insectary and other plants interplanted with crops.

Season, irrigation, leaf age, and Escherichia coli inoculation influence the bacterial diversity in the lettuce phyllosphere.

The developmental and temporal succession patterns and disturbance responses of phyllosphere bacterial communities are largely unknown. These factors might influence the capacity of human pathogens to persist in association with those communities on agriculturally-relevant plants. In this study, the phyllosphere microbiota was identified for Romaine lettuce plants grown in the Salinas Valley, CA, USA from four plantings performed over 2 years and including two irrigation methods and inoculations with an attenuated strain of Escherichia coli O157:H7. High-throughput DNA pyrosequencing of the V5 to V9 variable regions of bacterial 16S rRNA genes recovered in lettuce leaf washes revealed that the bacterial diversity in the phyllosphere was distinct for each field trial but was also strongly correlated with the season of planting. Firmicutes were generally most abundant in early season (June) plantings and Proteobacteria comprised the majority of bacteria recovered later in the year (August and October). Comparisons within individual field trials showed that bacterial diversity differed between sprinkler (overhead) and drip (surface) irrigated lettuce and increased over time as the plants grew. The microbiota were also distinct between control and E. coli O157:H7-inoculated plants and between E. coli O157:H7-inoculated plants with and without surviving pathogen cells. The bacterial inhabitants of the phyllosphere therefore appear to be affected by seasonal, irrigation, and biological factors in ways that are relevant for assessments of fresh produce food safety.

Impact of preinoculation culture conditions on the behavior of Escherichia coli O157:H7 inoculated onto romaine lettuce (Lactuca sativa) plants and cut leaf surfaces.

Inoculum preparation methods can impact growth or survival of organisms inoculated into foods, thus complicating direct comparison of results among studies. The objective of this study was to evaluate preinoculation culture preparation for impact on Escherichia coli O157:H7 inoculated onto leaves of romaine lettuce plants and cut leaf surfaces. E. coli O157:H7 was grown quiescently or shaken at 15, 25, or 37 degrees C to different growth phases in tryptic soy or M9 minimal salts broth or agar. Cells were harvested, washed, and suspended in 0.1% peptone, Milli Q water, or well water and refrigerated for 0 or 18 h. Prepared inoculum was spotted onto cut romaine lettuce (10 microl; 3 x 10(4) CFU/10 g) or onto romaine lettuce plants (20 microl; 3 x 10(6) CFU per leaf). Cut lettuce was sealed in 100-cm2 bags (made from a commercial polymer film) and incubated at 5 or 20 degrees C. Lettuce plants were held at 23 degrees C for 24 h. For all tested conditions, levels of E. coli O157:H7 increased at 20 degrees C on cut lettuce and decreased on cut lettuce stored at 5 degrees C or on leaves of lettuce plants. At 20 degrees C, preinoculation culture conditions had little impact on growth of E. coli O157:H7 on cut lettuce. However, survival at 5 degrees C was significantly better (P < 0.05) for cultures grown at 15 or 37 degrees C in minimal medium and to late stationary phase. Impact of preinoculation handling on survival on lettuce plants was less clear due to relatively high standard deviations observed among samples.

Leaf age as a risk factor in contamination of lettuce with Escherichia coli O157:H7 and Salmonella enterica.

Outbreaks of Escherichia coli O157:H7 infections have been linked increasingly to leafy greens, particularly to lettuce. We present here the first evidence that this enteric pathogen can multiply on the leaves of romaine lettuce plants. The increases in population size of E. coli O157:H7 in the phyllosphere of young lettuce plants ranged from 16- to 100-fold under conditions of warm temperature and the presence of free water on the leaves and varied significantly with leaf age. The population size was consistently ca. 10-fold higher on the young (inner) leaves than on the middle leaves. The growth rates of Salmonella enterica and of the natural bacterial microflora were similarly leaf age dependent. Both enteric pathogens also achieved higher population sizes on young leaves than on middle leaves harvested from mature lettuce heads, suggesting that leaf age affects preharvest as well as postharvest colonization. Elemental analysis of the exudates collected from the surfaces of leaves of different ages revealed that young-leaf exudates were 2.9 and 1.5 times richer in total nitrogen and carbon, respectively, than middle-leaf exudates. This trend mirrored the nitrogen and carbon content of the leaf tissue. Application of ammonium nitrate, but not glucose, to middle leaves enhanced the growth of E. coli O157:H7 significantly, suggesting that low nitrogen limits its growth on these leaves. Our results indicate that leaf age and nitrogen content contribute to shaping the bacterial communities of preharvest and postharvest lettuce and that young lettuce leaves may be associated with a greater risk of contamination with E. coli O157:H7.

Manganese toxicity and marginal chlorosis of lettuce

Romaine lettuce plants were grown in 15-liter tanks containing one-fifth strength Hoagland's solution over a range of Mn concentrations from zero to 10 ppm. This covered a span of Mn from deficiency to toxicity. Maximum yields were obtained at 0.1 and 0.5 ppm Mn supplied in the nutrient solution. A marginal chlorosis appeared at the highest Mn rates and was more pronounced in the older leaves. The plants were divided into young and old leaves and roots. The yellow borders of the old leaves were separated for analysis from the interior green tissues. The Mn content of the chlorotic borders averaged three times higher than that in the interior green tissues and reached a maximum of 5430 ppm Mn as opposed to 1940 ppm. The Fe content of the chlorotic bands was more than twice as high as that found in the green parts of the blades. The maximum content of Mn found in the roots was 3660 ppm. re]19720118