In Situ Evaluation of Paenibacillus alvei in Reducing Carriage of Salmonella enterica Serovar Newport on Whole Tomato Plants

Recently, tomatoes have been implicated as a primary vehicle in food-borne outbreaks of Salmonella enterica serovar Newport and other Salmonella serovars. Long-term intervention measures to reduce Salmonella prevalence on tomatoes remain elusive for growing and postharvest environments. A naturally occurring bacterium identified by 16S rRNA gene sequencing as Paenibacillus alvei was isolated epiphytically from plants native to the Virginia Eastern Shore tomato-growing region. After initial antimicrobial activity screening against Salmonella and 10 other bacterial pathogens associated with the human food supply, strain TS-15 was further used to challenge an attenuated strain of S. Newport on inoculated fruits, leaves, and blossoms of tomato plants in an insect-screened high tunnel with a split-plot design. Survival of Salmonella after inoculation was measured for groups with and those without the antagonist at days 0, 1, 2, and 3 and either day 5 for blossoms or day 6 for fruits and leaves. Strain TS-15 exhibited broad-range antimicrobial activity against both major food-borne pathogens and major bacterial phytopathogens of tomato. After P. alvei strain TS-15 was applied onto the fruits, leaves, and blossoms of tomato plants, the concentration of S. Newport declined significantly (P ≤ 0.05) compared with controls. Astonishingly, >90% of the plants had no detectable levels of Salmonella by day 5 for blossoms. The naturally occurring antagonist strain TS-15 is highly effective in reducing the carriage of Salmonella Newport on whole tomato plants. The application of P. alvei strain TS-15 is a promising approach for reducing the risk of Salmonella contamination during tomato production.     

Survival of Salmonellae on and in Tomato Plants from the Time of Inoculation at Flowering and Early Stages of Fruit Development through Fruit Ripening

The fate of salmonellae applied to tomato plants was investigated. Five Salmonella serotypes were used to inoculate tomato plants before and after fruits set, either by injecting stems with inoculum or brushing flowers with it. Ripe tomato fruits were subjected to microbiological analysis. Peptone wash water, homogenates of stem scar tissues, and homogenates of fruit pulp were serially diluted and plated on bismuth sulfite agar before and after enrichment. Presumptive Salmonella colonies were confirmed by serological tests, PCR assay using HILA2 primers, and enterobacterial repetitive intergenic consensus PCR. Of 30 tomatoes harvested from inoculated plants, 11 (37%) were positive for Salmonella. Of the Salmonella-positive tomatoes, 43 and 40%, respectively, were from plants receiving stem inoculation before and after flower set. Two of eight tomatoes produced from inoculated flowers contained Salmonella. Higher percentages of surface (82%) and stem scar tissue (73%) samples, compared to pulp of Salmonella-positive tomatoes (55%), harbored the pathogen. Of the five serotypes in the inoculum, Montevideo was the most persistent, being isolated from tomatoes 49 days after inoculation, and Poona was the most dominant, being present in 5 of 11 Salmonella-positive tomatoes. Results suggest that Salmonella cells survive in or on tomato fruits from the time of inoculation at flowering through fruit ripening. Tomato stems and flowers are possible sites at which Salmonella may attach and remain viable during fruit development, thus serving as routes or reservoirs for contaminating ripened fruit.     

A New Huanglongbing Species, “Candidatus Liberibacter psyllaurous,” Found To Infect Tomato and Potato, Is Vectored by the Psyllid Bactericera cockerelli (Sulc)

A new huanglongbing (HLB) “Candidatus Liberibacter” species is genetically characterized, and the bacterium is designated “Candidatus Liberibacter psyllaurous.” This bacterium infects the psyllid Bactericera cockerelli and its solanaceous host plants potato and tomato, potentially resulting in “psyllid yellowing.” Host plant-dependent HLB transmission and variation in psyllid infection frequencies are found.     

A Multi-Omic View of Host-Pathogen-Commensal Interplay in Salmonella-Mediated Intestinal Infection

The potential for commensal microorganisms indigenous to a host (the ‘microbiome’ or ‘microbiota’) to alter infection outcome by influencing host-pathogen interplay is largely unknown. We used a multi-omics “systems” approach, incorporating proteomics, metabolomics, glycomics, and metagenomics, to explore the molecular interplay between the murine host, the pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), and commensal gut microorganisms during intestinal infection with S. Typhimurium. We find proteomic evidence that S. Typhimurium thrives within the infected 129/SvJ mouse gut without antibiotic pre-treatment, inducing inflammation and disrupting the intestinal microbiome (e.g., suppressing Bacteroidetes and Firmicutes while promoting growth of Salmonella and Enterococcus). Alteration of the host microbiome population structure was highly correlated with gut environmental changes, including the accumulation of metabolites normally consumed by commensal microbiota. Finally, the less characterized phase of S. Typhimurium’s lifecycle was investigated, and both proteomic and glycomic evidence suggests S. Typhimurium may take advantage of increased fucose moieties to metabolize fucose while growing in the gut. The application of multiple omics measurements to Salmonella-induced intestinal inflammation provides insights into complex molecular strategies employed during pathogenesis between host, pathogen, and the microbiome.     

Infection Density Dynamics of the Citrus Greening Bacterium “Candidatus Liberibacter asiaticus” in Field Populations of the Psyllid Diaphorina citri and Its Relevance to the Efficiency of Pathogen Transmission to Citrus Plants

Huanglongbing, or citrus greening, is a devastating disease of citrus plants recently spreading worldwide, which is caused by an uncultivable bacterial pathogen, “Candidatus Liberibacter asiaticus,” and vectored by a phloem-sucking insect, Diaphorina citri. We investigated the infection density dynamics of “Ca. Liberibacter asiaticus” in field populations of D. citri with experiments using field-collected insects to address how “Ca. Liberibacter asiaticus” infection density in the vector insect is relevant to pathogen transmission to citrus plants. Of 500 insects continuously collected from “Ca. Liberibacter asiaticus”-infected citrus trees with pathological symptoms in the spring and autumn of 2009, 497 (99.4%) were “Ca. Liberibacter asiaticus” positive. The infections were systemic across head-thorax and abdomen, ranging from 10³ to 10⁷ bacteria per insect. In spring, the infection densities were low in March, at ∼10³ bacteria per insect, increasing up to 10⁶ to 10⁷ bacteria per insect in April and May, and decreasing to 10⁵ to 10⁶ bacteria per insect in late May, whereas the infection densities were constantly ∼10⁶ to 10⁷ bacteria per insect in autumn. Statistical analysis suggested that several factors, such as insect sex, host trees, and collection dates, may be correlated with “Ca. Liberibacter asiaticus” infection densities in field D. citri populations. Inoculation experiments with citrus seedlings using field-collected “Ca. Liberibacter asiaticus”-infected insects suggested that (i) “Ca. Liberibacter asiaticus”-transmitting insects tend to exhibit higher infection densities than do nontransmitting insects, (ii) a threshold level (∼10⁶ bacteria per insect) of “Ca. Liberibacter asiaticus” density in D. citri is required for successful transmission to citrus plants, and (iii) D. citri attaining the threshold infection level transmits “Ca. Liberibacter asiaticus” to citrus plants in a stochastic manner. These findings provide valuable insights into understanding, predicting, and controlling this notorious citrus pathogen.     

Soil compaction. A role for ethylene in regulating leaf expansion and shoot growth in tomato?

The role of ethylene in regulating growth in tomato (Lycopersicon esculentum Mill.) during compaction stress was examined using wild-type (cv Ailsa Craig) and transgenic (ACO1_AS) genotypes; the latter has a reduced capacity to produce ethylene. Ethephon or silver ions were applied to increase ethylene production or block its action. Shoot growth in both genotypes was comparable in uncompacted (1.1 g cm⁻³) and uniformly compacted soil (1.5 g cm⁻³). However, a 1.1/1.5-g cm⁻³ split-pot treatment invoked marked genotypic differences: growth was reduced in cv Ailsa Craig but was comparable to uncompacted control plants in ACO1_AS. As xylem sap abscisic acid levels were similar, abscisic acid was not responsible for inhibiting growth in cv Ailsa Craig. These genotypic differences in growth were accompanied by increased ethylene evolution in cv Ailsa Craig, suggesting that the ability of ACO1_AS to maintain growth in the split-pot treatment reflected its lower ethylene levels, a view supported by the observation that excising the roots in the compacted compartment reduced ethylene evolution and restored shoot growth in cv Ailsa Craig. Treatment with silver restored shoot growth in cv Ailsa Craig, whereas treatment with ethephon reduced growth in ACO1_AS. Thus, ethylene apparently has a key role in determining growth when tomato plants encounter differential soil compaction.     

Growth, Phosphate Pools, and Phosphate Mobilization of Salt-stressed Sesame and Pepper

The growth and phosphate mobilization of control and salt-stressed sesame (Sesamum indicum L.) and pepper (Capsicum annuum L.) plants were examined to ascertain whether or not translocation limits growth of salt-stressed plants. Plants were grown in a complete nutrient solution with and without excess salt. One-half of the control and salt-stressed plants were later transferred to phosphate-free culture solution (“−P” plants). Measurements of growth and phosphate pools in leaves indicated that with or without salinity “−P” plants utilized their phosphate reserves to support growth for a time at rates equaling those of plants supplied with phosphate. The results indicate that mobilization was not limiting for growth of salt-stressed plants.

Defoliation experiments were performed at a developmental stage when the import of assimilates by the youngest expanding leaves could be changed by removing certain source or sink leaves. These experiments also indicated that phloem transport was not limiting for leaf growth on salt-stressed plants.

     

Enrichment Medium for Selection of Salmonella from Fish Homogenate

A new liquid medium, called “dulcitol selenite enrichment,” has been developed for the detection and enumeration of Salmonella in foods. The medium is not only highly selective in enriching Salmonella and inhibiting completely or appreciably other extraneous organisms commonly found in seafoods, but is also highly sensitive in recovering as low as 2 to 7 cells of Salmonella, even in the presence of large numbers (10⁴ to 10⁶ cells) of mixed flora common to these foods. The addition of seafood material does not seem to interfere with the sensitivity, selectivity, or productivity of the medium. Even physiologically debilitated cells of Salmonella were enriched well enough in this medium to be detected easily.     

Spatial Distribution of Photosynthesis during Drought in Field-Grown and Acclimated and Nonacclimated Growth Chamber-Grown Cotton

Inhomogeneous photosynthetic activity has been reported to occur in drought-stressed leaves. In addition, it has been suggested that these water stress-induced nonuniformities in photosynthesis are caused by “patchy” stomatal closure and that the phenomenon may have created the illusion of a nonstomatal component to the inhibition of photosynthesis. Because these earlier studies were performed with nonacclimated growth chamber-grown plants, we sought to determine whether such “patches” existed in drought-treated, field-grown plants or in chamber-grown plants that had been acclimated to low leaf water potentials (ψ_leaf). Cotton (Gossypium hirsutum L.) was grown in the field and subjected to drought by withholding irrigation and rain from 24 d after planting. The distribution of photosynthesis, which may reflect the stomatal aperture distribution in a heterobaric species such as cotton, was assayed by autoradiography after briefly exposing attached leaves of field-grown plants to ¹⁴CO₂. A homogeneous distribution of radioactive photosynthate was evident even at the lowest ψ_leaf of −1.34 MPa. “Patchiness” could, however, be induced by uprooting the plant and allowing the shoot to air dry for 6 to 8 min. In parallel studies, growth chamber-grown plants were acclimated to drought by withholding irrigation for three 5-d drought cycles interspersed with irrigation. This drought acclimation lowered the ψ_leaf value at which control rates of photosynthesis could be sustained by approximately 0.7 MPa and was accompanied by a similar decline in the ψ_leaf at which patchiness first appeared. Photosynthetic inhomogeneities in chamber-grown plants that were visible during moderate water stress and ambient levels of CO₂ could be largely removed with elevated CO₂ levels (3000 μL L⁻¹), suggesting that they were stomatal in nature. However, advanced dehydration (less than approximately 2.0 MPa) resulted in “patches” that could not be so removed and were probably caused by nonstomatal factors. The demonstration that patches do not exist in drought-treated, field-grown cotton and that the presence of patches in chamber-grown plants can be altered by treatments that cause an acclimation of photosynthesis leads us to conclude that spatial heterogeneities in photosynthesis probably do not occur frequently under natural drought conditions.     

Characterization of the RpoS Status of Clinical Isolates of Salmonella enterica

The stationary-phase-inducible sigma factor, σ^S (RpoS), is the master regulator of the general stress response in Salmonella and is required for virulence in mice. rpoS mutants can frequently be isolated from highly passaged laboratory strains of Salmonella. We examined the rpoS status of 116 human clinical isolates of Salmonella, including 41 Salmonella enterica serotype Typhi strains isolated from blood, 38 S. enterica serotype Typhimurium strains isolated from blood, and 37 Salmonella serotype Typhimurium strains isolated from feces. We examined the abilities of these strains to produce the σ^S protein, to express RpoS-dependent catalase activity, and to resist to oxidative stress in the stationary phase of growth. We also carried out complementation experiments with a cloned wild-type rpoS gene. Our results showed that 15 of the 41 Salmonella serotype Typhi isolates were defective in RpoS. We sequenced the rpoS allele of 12 strains. This led to identification of small insertions, deletions, and point mutations resulting in premature stop codons or affecting regions 1 and 2 of σ^S, showing that the rpoS mutations are not clonal. Thus, mutant rpoS alleles can be found in freshly isolated clinical strains of Salmonella serotype Typhi, and they may affect virulence properties. Interestingly however, no rpoS mutants were found among the 75 Salmonella serotype Typhimurium isolates. Strains that differed in catalase activity and resistance to hydrogen peroxide were found, but the differences were not linked to the rpoS status. This suggests that Salmonella serotype Typhimurium rpoS mutants are counterselected because rpoS plays a role in the pathogenesis of Salmonella serotype Typhimurium in humans or in the transmission cycle of the disease.     

In-Feed Use of Heavy Metal Micronutrients in U.S. Swine Production Systems and Its Role in Persistence of Multidrug-Resistant Salmonellae

The study aimed to characterize the role of heavy metal micronutrients in swine feed in emergence of heavy-metal-tolerant and multidrug-resistant Salmonella organisms. We conducted a longitudinal study in 36 swine barns over a 2-year period. The feed and fecal levels of Cu²⁺ and Zn²⁺ were measured. Salmonella was isolated at early and late finishing. MICs of copper sulfate and zinc chloride were measured using agar dilution. Antimicrobial susceptibility was tested using the Kirby-Bauer method, and 283 isolates were serotyped. We amplified pcoA and czcD genes that encode Cu²⁺ and Zn²⁺ tolerance, respectively. Of the 283 isolates, 113 (48%) showed Cu²⁺ tolerance at 24 mM and 164 (58%) showed Zn²⁺ tolerance at 8 mM. In multivariate analysis, serotype and source of isolates were significantly associated with Cu²⁺ tolerance (P < 0.001). Fecal isolates were more likely to be Cu²⁺ tolerant than those of feed origin (odds ratio [OR], 27.0; 95% confidence interval [CI], 2.8 to 250; P = 0.0042) or environmental origin (OR, 5.8), implying the significance of gastrointestinal selective pressure. Salmonella enterica serotypes Typhimurium and Heidelberg, highly significant for public health, had higher odds of having >20 mM MICs of Cu²⁺ than did “other” serotypes. More than 60% of Salmonella isolates with resistance type (R-type) AmStTeKm (32 of 53) carried pcoA; only 5% with R-type AmClStSuTe carried this gene. czcD gene carriage was significantly associated with a higher Zn²⁺ MIC (P < 0.05). The odds of having a high Zn²⁺ MIC (≥8 mM) were 14.66 times higher in isolates with R-type AmClStSuTe than in those with R-type AmStTeKm (P < 0.05). The findings demonstrate strong association between heavy metal tolerance and antimicrobial resistance, particularly among Salmonella serotypes important in public health.     

Toll-like Receptor 11 (TLR11) Prevents Salmonella Penetration into the Murine Peyer Patches

Toll-like receptors (TLRs) are key molecular sensors used by the mammalian innate immune system to detect microorganisms. Although TLR functions in colonic immune homeostasis and tolerance to commensal bacteria have been intensively researched, the precise roles of different TLRs in response to pathogen infection in the gut remain elusive. Peyer patches are the major entrance of Salmonella infection and antigen transportation in intestine. Here, we report that, in contrast to TLR5 as a “carrier of Salmonella,” TLR11 works as a “blocker of Salmonella” to prevent highly invasive Salmonella from penetrating into the murine Peyer patches and spreading systemically. TLR11 plays an important role in mediating TNF-α induction and systemic inflammation in response to Salmonella infection. Remarkably, in mice lacking TLR11, apparent hemorrhages at Peyer patches are induced by highly invasive Salmonella, a phenotype resembling human Salmonella infection. Therefore, our results indicate a potentially important role for TLR11 in preventing murine intestinal infection and modulating antigen transportation in the gut and imply an important role for various TLRs in cooperation with tight control of pathogens penetrating into Peyer patches. The TLR11 knock-out mouse can serve as a good animal model to study Salmonella infection.     

Trichomes as sensors: Detecting activity on the leaf surface

The dramatic movements of some carnivorous plants species are triggered by sensory structures derived from trichomes. While unusual plant species such as the Venus fly trap and sundews may be expected to have elaborate sensors to capture their insect prey, more modest plant species might not be expected to have similar sensory capabilities. Our recent work, however, has revealed that glandular trichomes on tomato (Solanum lycopersicum) appear to have a function similar to trigger hairs of carnivorous species, acting as “early warning” sensors. Using a combination of behavioral, molecular, and biochemical techniques, we determined that caterpillars, moths and mechanical disruption upregulate signaling molecules and defensive genes found in glandular trichomes. Importantly, we discovered that plants whose trichomes have been broken respond more vigorously when their defenses were induced. Taken together, our results suggest that glandular trichomes can act as sensors that detect activity on the leaf surface, and ready plants for herbivore attack.Key words: glandular trichome, induced responses, jasmonic acid, plant-insect interactions, sensor, Solanum lycopersicum, tomatoCertain plant species are renowned for their ability to respond to contact. The Venus fly trap (Dionaea muscipula) and sundew (Drosera) species come to mind quickly as obviously thigmotropic species. When an insect lands on these carnivorous plant species, dramatic movements ensue once the prey is detected. Some Drosera species respond to contact by bending their “tentacles” toward their trapped prey to further ensnare the victim and begin the process of digestion. These dramatic plant species have captured the attention of many scientists, including Darwin, who remarked on the “extraordinary sensitiveness of [their] glands to slight pressure” and surmised that the tentacles of sundew plants “existed primordially as glandular hairs.”¹ As is often the case, Darwin appears to have been quite right. Indeed, morphological and molecular work supports the notion that sundew tentacles and the trigger hairs of the Venus fly trap are homologous sensory structures likely derived from trichomes.^2,3Given Darwin’s appreciation of these trichome-derived sensory organs, he perhaps would not have been surprised by mounting evidence that suggests that trichomes may play even a broader sensory role for plants. We have recently found evidence that glandular trichomes can act as early detection sensors for some plant species.⁴ These trichomes can be disrupted by the footsteps of walking moths and caterpillars (and other forms of light touching), and this apparently minor plant damage leads to a state of defensive readiness that allows plants to respond to herbivory more quickly than undamaged plants. While this level of trichome-mediated detection does not result in the conspicuous responses of some carnivorous plant species, it still results in significant physiological changes that prepare plants for attack.In our recent effort, we worked with tomato (Solanum lycopersicum), using a combination of behavioral, molecular, and biochemical techniques to understand the role of trichomes in detecting activity on the leaf surface.⁴ Defense signaling has been well studied in tomato and there exists a variety of mutants whose defensive responses have been compromised. Moreover, it has been known that tomatoes have a variety of trichome types, including two types of glandular trichomes that burst upon contact with insects, releasing their cellular contents and physically impeding insects (Fig. 1).^5,6Open in a separate windowFigure 1Surface of a tomato leaf showing (A) intact rounded heads of glandular trichomes (black arrows) and (B) trichomes disrupted with a gloved hand (absence of rounded heads except for a few in the upper left corner [black arrows]). Images were captured at 36x magnification and were taken from different parts of the same leaf.To determine if plant defense pathways were induced by insect contact, we allowed three species of caterpillar (Manduca sexta, Heliothis virescens and Helicoverpa zea) and one species of moth (H. zea) to crawl over tomato leaves for ten minutes. As a positive control, we also lightly rubbed leaves with a gloved hand or a metal rod. Within time frames ranging from three to twenty-four hours all treatments, insect and otherwise, significantly induced defensive genes as measured by qRT-PCR. Using a combination of RT-PCR and in situ hybridization, we confirmed that JA-signaling and defensive genes are expressed in trichomes. A GC-MS-based technique then confirmed that JA was present in trichomes of undamaged plants and DAB staining, in combination with catalase treatment, provided evidence that hydrogren peroxide and JA are key signals mediating defensegene induction. These conclusions were further reinforced by experiments with def1 mutants, a line of tomato impaired in JA signaling, and accession LA3610, a tomato variety with reduced numbers of trichomes. Lastly, we conducted a factorial experiment both disrupting trichomes and treating tomato plants with methyl jasmonate (MeJA), which induces plant defenses and increases densities of trichomes.⁷ Results of this final experiment indicated that plants that received both treatments (i.e., MeJA and disruption) had greater defensive gene induction than plants that were only treated with MeJA or plants whose trichomes remained intact, suggesting that increases in trichomes may contribute to greater sensitivity to touch-induced responses.Taken together, our results are highly suggestive that trichomes can act as “early warning” detectors for plants. Moths seeking to lay eggs on tomato are likely to break trichomes as they explore leaves, upregulating plant defenses in anticipation of egg hatch and feeding by neonate caterpillars. Similarly, herbivores colonizing a new host plant and breaking trichomes on their way across a leaf also appear to “tip the plant off” to impending attack. Considering the drastic response of carnivorous plants to touch, perhaps it should not be surprising that trichomes can function more broadly as sensors. In an evolutionary context, it seems logical that trichomes took on this role. For many plant species, “hairy” varieties receive less herbivory,⁸ so within a population there could have been a fitness advantage in having more trichomes. Once established, this hairy phenotype could then have been refined via mutation and selection for trichome varieties that had functions adaptive for the plant, perhaps driving the evolution of glandular trichomes and their role as sensors.Granted, the generalized nature of our results would appear to indicate that plants could be “primed” by nearly any arthropod species that crosses one of their leaves. This would, of course, include natural enemies, which are capable of decreasing herbivore pressure and improving plant fitness.^9,10 However, it has been hypothesized that priming evolved due to high fitness costs associated with defensive induction following threats of only minor severity.¹¹ Priming provides an advantage by settling plants into an intermediate “ready” state that allows them to deploy strong defense responses more quickly and the fitness cost associated with being “primed” are lower than full defensive induction.¹² Presumably, fitness costs following priming due to natural enemyinduced trichome disruption would also be less than the cost incurred from a bout of unanticipated herbivory and, over the life of the plant, it would be worth the effort to prepare for attack even if the perceived risk is from a natural enemy and not a foe.Our results build on previously reported priming mechanisms that prepare plants for attack.^13,14 And they reveal an additional level of sophistication in the sensory capabilities of plants, which have already been shown to be able to detect nearby threats of herbivory and increase their defenses in response.^15,16 It seems that trichomes may have played a much wider role in shaping the nature of plant-animal interaction than previously recognized and we look forward to further work elaborating their function.     

Flagella facilitate escape of Salmonella from oncotic macrophages

The intracellular parasite Salmonella enterica serovar Typhimurium causes a typhoid-like systemic disease in mice. Whereas the survival of Salmonella in phagocytes is well understood, little has been documented about the exit of intracellular Salmonella from host cells. Here we report that in a population of infected macrophages Salmonella induces “oncosis,” an irreversible progression to eukaryotic cell death characterized by swelling of the entire cell body. Oncotic macrophages (OnMs) are terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling negative and lack actin filaments (F-actin). The plasma membrane of OnMs filled with bacilli remains impermeable, and intracellular Salmonella bacilli move vigorously using flagella. Eventually, intracellular Salmonella bacilli intermittently exit host cells in a flagellum-dependent manner. These results suggest that induction of macrophage oncosis and intracellular accumulation of flagellated bacilli constitute a strategy whereby Salmonella escapes from host macrophages.     

Survival and Transmission of Salmonella enterica Serovar Typhimurium in an Outdoor Organic Pig Farming Environment

It was investigated how organic rearing conditions influence the Salmonella enterica infection dynamics in pigs and whether Salmonella persists in the paddock environment. Pigs inoculated with S. enterica serovar Typhimurium were grouped with Salmonella-negative tracer pigs. Bacteriological and serological testing indicated that organic pigs were susceptible to Salmonella infections, as 26 of 46 (56%) tracer pigs turned culture positive. An intermittent and mainly low-level excretion of Salmonella (<100 cells g⁻¹) partly explains why the bacteriological prevalence appeared lower than the seroprevalence. Salmonella persisted in the paddock environment, as Salmonella was isolated from 46% of soil and water samples (n = 294). After removal of pigs, Salmonella was found in soil samples for up to 5 weeks and in shelter huts during the entire test period (7 weeks). Subsequent introduction of Salmonella-negative pigs into four naturally Salmonella-contaminated paddocks caused Salmonella infections of pigs in two paddocks. In one of these paddocks, all tracer pigs (n = 10) became infected, coinciding with a previous high Salmonella infection rate and high Salmonella excretion level. Our results showed that pigs reared under organic conditions were susceptible to Salmonella infections (just like conventional pigs) and that Salmonella persisting in the paddock environment could pose an infection risk. A driving force for these infections seemed to be pigs with a high Salmonella excretion level, which caused substantial contamination of the environment. This suggests that isolation of animals as soon as a Salmonella infection is indicated by clinical symptoms of diarrhea could be a means of reducing and controlling the spread and persistence of Salmonella in outdoor organic pig production environments.     

A cellular gene as a double surveillance agent for plant to combat pathogen

The tomato extreme resistance R-gene encodes Tm2/Tm2² protein that interacts with the tobamovirus movement protein (MP) to induce hypersensitive response (HR) resulting in local resistance. R-gene mediated local resistance requires a functional RbCS that interacts with MP, restricting virus local infection. RbCS-MP interaction is also required for tobamovirus systemic infection. “Loss-of-function” RbCS allows local but not systemic infection. Thus, RbCS, a cellular gene, acts as a double surveillance agent to protect plant from pathogenic attack, suggesting a previously un-recognized defense strategy in plants.     

Ethylene Production and Respiratory Behavior of the rin Tomato Mutant

Little or no change in ethylene or CO₂ production occurred in rin tomato mutant fruits monitored for up to 120 days after harvest. Of the abnormally ripening tomatoes investigated, including “Never ripe” (Nr Y a h, Nr c l₂ r), “Evergreen” (gf r) and “Green Flesh” (gf), only rin did not show a typical climacteric and ethylene rise.     

Ion Homeostasis in Chloroplasts under Salinity and Mineral Deficiency : I. Solute Concentrations in Leaves and Chloroplasts from Spinach Plants under NaCl or NaNO(3) Salinity

Spinach (Spinacia oleracea var “Yates”) plants in hydroponic culture were exposed to stepwise increased concentrations of NaCl or NaNO₃ up to a final concentration of 300 millimoles per liter, at constant Ca²⁺-concentration. Leaf cell sap and extracts from aqueously isolated spinach chloroplasts were analyzed for mineral cations, anions, amino acids, sugars, and quarternary ammonium compounds. Total osmolality of leaf sap and photosynthetic capacity of leaves were also measured. For comparison, leaf sap from salt-treated pea plants was also analyzed. Spinach plants under NaCl or NaNO₃ salinity took up large amounts of sodium (up to 400 millimoles per liter); nitrate as the accompanying anion was taken up less (up to 90 millimoles per liter) than chloride (up to 450 millimoles per liter). Under chloride salinity, nitrate content in leaves decreased drastically, but total amino acid concentrations remained constant. This response was much more pronounced (and occurred at lower salt concentrations) in leaves from the glycophyte (pea, Pisum sativum var “Kleine Rheinländerin”) than from moderately salt-tolerant spinach. In spinach, sodium chloride or nitrate taken up into leaves was largely sequestered in the vacuoles; both salts induced synthesis of quarternary ammonium compounds, which were accumulated mainly in chloroplasts (and cytosol). This prevented impairment of metabolism, as indicated by an unchanged photosynthetic capacity of leaves.