Efficacy of Ozonated Water Against Erwinia carotovora subsp. carotovora in Brassica campestris ssp. chinensis

Erwinia carotovora subsp. carotovora (Ecc) is a bacterial pathogen that causes decay of vegetables and is found in a wide range of Brassica crops in the Shanghai area, particularly in non-heading Chinese cabbage (NHCC). In this study, aqueous ozone was used as an antimicrobial agent to prevent the growth of Ecc bacterial colonies. Ecc that were treated with aqueous ozone were completely inactivated after the bacteria had been exposed to 0.5 mg/L ozone at 28 °C. Furthermore, a susceptible NHCC cultivar was directly sprayed with different concentrations of dissolved ozonated water as a pesticide substitute. The effects of the treatments on morphology were analyzed, and some treatments were characterized by induction of visible symptoms of senescence. No negative effects were observed after treatment by spraying ozonated water compared with the control at concentrations below 8 mg/L. However, visible damage to leaves was observed after the plants were exposed to 10 mg/L ozonated water via spraying for 15 days during the plant reproductive stage. Additionally, enzyme activities and antioxidant responses gradually increased to a certain degree and then decreased in the untreated and ozonated water-treated plants. These results showed that ozonated water was effective in restraining pathogens and potentially defending against disease in growing NHCC plants within a certain concentration range. These results provide a theoretical basis for preventing disease by applying ozonated water to vegetables as an alternative to pesticides.     

Research on the Feasibility of Spraying Micro/Nano Bubble Ozonated Water for Airborne Disease Prevention

This research combined micro/nano bubble techniques with ozone disinfection to determine feasibility of applying micro/nano bubble ozonated water in preventing tomato airborne disease. Results indicated that dissolving ozone in micro/nano bubbles is more efficient than using a mixing pump. In our in vitro studies, when dissolved ozone concentration was 1.6 mg/L, a 5.2 to 3.3 log reduction in Alternaria solani Sorauer conidia was observed; with concentration of 1.8 mg/L, there was a 5.0 to 3.7 log reduction in Cladosporium fulvum conidia. Furthermore, spraying ozonated water in a certain concentration range (0.6–1.8 mg/L) had no significant negative effects on tomato growth.     

Nutrient Solution Prepared with Ozonated Water does not Damage Early Growth of Hydroponically Grown Tomatoes

Ozonated water as a water source for nutrient solution was investigated in hydroponically grown tomatoes. Nutrient solution was prepared by diluting concentrated nutrient solutions with ozonated water with dissolved ozone (DO₃) concentrations of 0 to 10 mg L^?1. Manganese concentration in the nutrient solution decreased with increasing DO₃ concentrations. Initial growth of tomato plants supplied with nutrient solution prepared with ozonated water with a DO₃ concentration of 1.5 mg L^?1 was greater than that with a DO₃ concentration of 0 mg L^?1. These results indicate that ozonated water can be used as a disinfectant for source water in hydroponics during the early growth stage of tomatoes.     

Ozonated Nutrient Solution Treatment as an Alternative Method for the Control of Root-Knot Nematodes in Soilless Cultivation

ABSTRACT

Root-knot nematodes (Meloidogyne incognita) are plant parasitic worms that widely influence the agricultural production including soilless cultivation. In this study, we investigated the inactivating effect of ozonated nutrient solution on the root-knot nematode juveniles in both nutrient solution and substrate, as well as the in vitro egg hatching. The results showed that twice ozonation treatments with dissolved ozone concentration (DOC) at 0.5 mg/L resulted in a mortality at 90.8% for in vitro study. DOC of 4.0 mg/L with three treatment repeats in the substrate experiment resulted in a mortality at 88.3%. Our results imply that the application of ozonated nutrient solution might be a feasible method in soilless cultivation for the control of root-knot nematodes. In practical application, ozonation treatment should be conducted with appropriate DOC and treatment repeats.     

Effects of Spraying Ozonated Water on the Severity of Powdery Mildew Infection on Cucumber Leaves

Effects of spraying ozonated water on the severity of powdery mildew infection, visible disorder/injury occurrence, and net photosynthesis in cucumbers were investigated. The severity in the ozonated water treatment was contained to almost the same level throughout the 14-day period of the experiment, while the severity steadily increased in the non-treated control and distilled water treatment. Neither visible disorder/injury on the leaves nor a large difference in net photosynthesis between before and after spraying the ozonated water was observed. The results indicate that ozonated water can be at least a partial alternative to agricultural chemical fungicides for powdery mildew on cucumber leaves.     

Effects of Ozonated Water Application on the Population Density of Fusarium oxysporum f. sp. lycopersici in Soil Columns

The potential of ozonated water was investigated as an alternative to soil disinfectants in reducing the population densities of Fusarium oxysporum f. sp. lycopersici in artificially infested quartz sand and Kuroboku soil columns. Significant reductions in the population density were observed with dissolved ozone concentrations (DOCs) of 6 and 12 mg L^?1 in infested quartz sand but not in infested Kuroboku soil. The results indicate that repetitive application of ozonated water with higher DOCs should be required for reducing the population density of F. oxysporum in soils with high content of organic substances and large surface area.     

Survival of Bacteria After Ozonation

The change of the bacterial population after each water treatment process was examined in a full-scale water treatment plant that uses ozone as a primary disinfectant. The fluctuation of heterotrophic bacterial number along the water treatment processes was determined. After ozonation, the bacterial number decreased to 13 CFU/mL. The surviving bacteria after each water treatment process were identified to genus or species level. The significant finding was the predominance of double-layered gram-positives (75%) among the surviving bacteria after ozonation. It included Mycobacterium spp., Bacillus spp., Corynebacterium spp., and Micrococcus spp. On the other hand, the dominance of gram-negatives was observed in most other water samples but each treatment process exerted different selection on dominant bacterial groups. The proportion of opportunistic pathogens was the lowest in the ozonated water.     

Tolerance of the rotifer Brachionus plicatilis to ozone and total oxidative residuals

Live prey items, such as the rotifer Brachionus plicatilis, are an essential component of many marine larval production systems. However, in intensive larval rearing systems live food items are also a potential source of pathogens which could adversely affect survival of the larvae. Consequently, it would be advantageous to reduce or eliminate potential pathogens associated with rotifers and their culture water prior to feeding. Ozone is a strong oxidizing agent which can be utilized as a disinfectant. Ozone is utilized extensively by commercial operators for disinfection and treatment of culture water, but there is limited data on tolerance of rotifers to ozone, residual oxidants associated with using ozone in seawater and the efficacy of an ozone wash. This study sought to evaluate the effects of exposing rotifers to ozone and/or residual oxidants associated with the use of ozone in seawater. Under the described conditions, the rotifer was found to be sensitive to ozonated seawater, both as total residual oxidants (TRO) consisting of ozone, chloramines and bromamines and residual oxidants (RO) excluding ozone. A “no observable effect concentration” (NOEC) of 0.22 mg/L TRO was determined for maximum survival of the rotifers. High levels of ozone (≥ 1.63 mg/L) were found to inactivate rotifer eggs. Based on published values of the inactivation of bacterial and viral pathogens, a NOEC of 0.22 mg/L TRO would appear to be sufficient to inactivate a variety of surficial potential pathogens. Consequently, the utilization of an ozone bath is suitable for the surficial disinfection of rotifers prior to feeding and hence may help reduce the vertical transmission of potential pathogens. Although RO did not appear as toxic as TRO, rotifers were sensitive to RO remaining in seawater after the dissipation of ozone. A NOEC of 0.3 mg/L for a 1 min exposure or 0.5 mg/L for a 5 or 10 min exposure time was observed for RO. Due to potential adverse effects of RO, it is recommended that RO be neutralized if ozone‐treated water is to be used to culture rotifers.     

Ozone Application for Postharvest Disinfection of Tomatoes

Ozone is an effective alternative for the postharvest treatment of fruits and vegetables. The purpose of this study was to evaluate the ozone application in gaseous or aqueous phases for postharvest disinfection of tomatoes (Lycopersicon esculentum Mill) cultivar FA-180. Fruits harvested at the breaker stage were exposed to ozone concentrations of 25 and 45 mg m^?3 for 2 h per day during 16 days, at non-controlled temperature and relative humidity. Exposure to ozone during storage extends the shelf-life of tomatoes, besides preserving its sensory attributes. Mature tomatoes, inoculated with Escherichia coli ATCC 25922, were washed with ozonated water containing 0.5 to 1.0 mg L^?1 during 15 to 30 min. To achieve an adequate disinfection, 1 mg L^?1 and 15 min are recommended. The disinfection with ozonated water was effective.     

Allelopathic Suppression of Pseudomonas solanacearum Infection of Tomato (Lycopersicon esculentum) in a Tomato-Chinese Chive (Allium tuberosum) Intercropping System

Tomato plants were grown alone or intercropped with Chinese chive plants, with or without the inoculation of Pseudomonas solanacearum. Chinese chive plants had no detrimental effects on the growth of tomato plants but significantly delayed and suppressed the occurrence of bacterial wilt of tomato. P. solanacearum population decreased faster in the soil grown with tomato alone than that in the soil grown with both tomato and Chinese chive. However, P. solanacearum population in bare soil was higher than that grown with Chinese chive. Root exudates of Chinese chive collected with a continuously trapping system were inhibitory to multiplication of P. solanacearum.     

Cell-Wall-Degrading Enzymes-Related Genes Originating from Rhizoctonia solani Increase Sugar Beet Root Damage in the Presence of Leuconostoc mesenteroides

Sugar beet crown and root rot caused by Rhizoctonia solani is a major yield constraint. Root rot is highly increased when R. solani and Leuconostoc mesenteroides co-infect roots. We hypothesized that the absence of plant cell-wall-degrading enzymes in L. mesenteroides and their supply by R. solani during close contact, causes increased damage. In planta root inoculation with or without cell-wall-degrading enzymes showed greater rot when L. mesenteroides was combined with cellulase (22 mm rot), polygalacturonase (47 mm), and pectin lyase (57 mm) versus these enzymes (0–26 mm), R. solani (20 mm), and L. mesenteroides (13 mm) individually. Carbohydrate analysis revealed increased simpler carbohydrates (namely glucose + galactose, and fructose) in the infected roots versus mock control, possibly due to the degradation of complex cell wall carbohydrates. Expression of R. solani cellulase, polygalacturonase, and pectin lyase genes during root infection corroborated well with the enzyme data. Global mRNAseq analysis identified candidate genes and highly co-expressed gene modules in all three organisms that might be critical in host plant defense and pathogenesis. Targeting R. solani cell-wall-degrading enzymes in the future could be an effective strategy to mitigate root damage during its interaction with L. mesenteroides.     

A Volatile Relationship: Profiling an Inter-Kingdom Dialogue Between two Plant Pathogens,Ralstonia Solanacearum and Aspergillus Flavus

Microbes in the rhizosphere have a suite of extracellular compounds, both primary and secondary, that communicate with other organisms in their immediate environment. Here, we describe a two-way volatile interaction between two widespread and economically important soil-borne pathogens of peanut, Aspergillus flavus and Ralstonia solanacearum, a fungus and bacterium, respectively. In response to A. flavus volatiles, R. solanacearum reduced production of the major virulence factor extracellular polysaccharide (EPS). In parallel, A. flavus responded to R. solanacearum volatiles by reducing conidia production, both on plates and on peanut seeds and by increasing aflatoxin production on peanut. Volatile profiling of these organisms using solid-phase micro-extraction gas chromatography mass spectroscopy (SPME-GCMS) provided a first glimpse at the compounds that may drive these interactions.     

Ralfuranone Thioether Production by the Plant Pathogen Ralstonia solanacearum

Ralfuranones are aryl‐substituted furanone secondary metabolites of the Gram‐negative plant pathogen Ralstonia solanacearum. New sulfur‐containing ralfuranone derivatives were identified, including the methyl thioether‐containing ralfuranone D. Isotopic labeling in vivo, as well as headspace analyses of volatiles from R. solanacearum liquid cultures, established a mechanism for the transfer of an intact methylthio group from L ‐methionine or α‐keto‐γ‐methylthiobutyric acid. The methylthio acceptor molecule ralfuranone I, a previously postulated biosynthetic intermediate in ralfuranone biosynthesis, was isolated and characterized by NMR. The highly reactive Michael acceptor system of this intermediate readily reacts with various thiols, including glutathione.     

Analysis of Damage on the Streptococcus mutans Immersed in Ozonated Water: Preliminary Study for Application as Mouth Rinse

Ozonated water has been demonstrated to induce significant results in terms of the elimination of microorganisms. The present study assessed the damage to Streptococcus mutans after exposure to ozonated water; the ozone generator was adjusted to provide an outlet concentration of 60 mg/L, the samples were submitted to different ozonation times 1, 2, 4, 6, and 10 mi. Scanning electron microscopy and atomic force images were obtained to identify damage to the bacteria, followed by reactive oxygen species (ROS) evaluation and microbial viability. The results showed a significant reduction in viability and the images evidenced the generation of gaps on the microbial wall and surface layer alterations. Ozone can induce significant damage to S. mutans, thus suggesting that the use of ozonated water to prevent carious lesion formation is extremely promising.     

Ozone-induced changes in host-plant suitability: Interactions ofKeiferia lycopersicella andLycopersicon esculentum

Tomato pinworms,Keiferia lycopersicella (Walsingham), survived better and developed faster on tomato plants,Lycopersicon esculentum Mill., damaged by ozone than on plants not subjected to ozone fumigation. Other measures of fitness, including survival during pupation, sex ratio of adults, female longevity, and fecundity, were not affected. Analyses of ozonated foliage at zero, two, and seven days following fumigation demonstrated a transient but significant increase (18–24%) in soluble protein concentration. Although the concentration of the total free amino acids in ozonated foliage did not increase significantly, significant changes were observed in at least 10 specific amino acids, some of which are critical for either insect development or the production of plant defensive chemicals. A reduction in total nitrogen in ozonated foliage at seven days postfumigation indicated that nitrogen was being translocated to other portions of the plant. The implications of increases in assimilable forms of nitrogen in ozonated foliage, which lead to improved host-plant suitability for insect herbivores, are discussed both in relation to some current ecological theories and in regard to pest-management strategies.     

Methyl 3‐Hydroxymyristate,a Diffusible Signal Mediating phc Quorum Sensing in Ralstonia solanacearum

Ralstonia solanacearum, a plant pathogenic bacterium causing “bacterial wilt” on crops, uses a quorum sensing (QS) system consisting of phc regulatory elements to control its virulence. Methyl 3‐hydroxypalmitate (3‐OH PAME) was previously identified as the QS signal in strain AW1. However, 3‐OH PAME has not been reportedly detected from any other strains, and this suggests that they produce another unknown QS signal. Here we identify (R)‐methyl 3‐hydroxymyristate [(R)‐3‐OH MAME] as a new QS signal that regulates the production of virulence factors and secondary metabolites. (R)‐3‐OH MAME was synthesized by the methyltransferase PhcB and sensed by the histidine kinase PhcS. The phylogenetic trees of these proteins from R. solanacearum strains were divided into two groups, according to their QS signal types—(R)‐3‐OH MAME or (R)‐3‐OH PAME. These results demonstrate that (R)‐3‐OH MAME is another crucial QS signal and highlight the unique evolution of QS systems in R. solanacearum.     

Inactivation of Baculovirus by Isoflavonoids on Chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis>) Leaf Surfaces Reduces the Efficacy of Nucleopolyhedrovirus Against <Emphasis Type="Italic">Helicoverpa armigera</Emphasis>

Biological pesticides based on nucleopolyhedroviruses (NPVs) can provide an effective and environmentally benign alternative to synthetic chemicals. On some crops, however, the efficacy and persistence of NPVs is known to be reduced by plant specific factors. The present study investigated the efficacy of Helicoverpa armigera NPV (HearNPV) for control of H. armigera larvae, and showed that chickpea reduced the infectivity of virus occlusion bodies (OBs) exposed to the leaf surface of chickpea for at least 1 h. The degree of inactivation was greater on chickpea than that previously reported on cotton, and the mode of action is different from that of cotton. The effect was observed for larvae that consumed OBs on chickpea leaves, but it also occurred when OBs were removed after exposure to plants and inoculated onto artificial diet, indicating that inhibition was leaf surface-related and permanent. Despite their profuse exudation from trichomes on chickpea leaves and their low pH, organic acids—primarily oxalic and malic acid—caused no inhibition. When HearNPV was incubated with biochanin A and sissotrin, however, two minor constituents of chickpea leaf extracts, OB activity was reduced significantly. These two isoflavonoids increased in concentration by up to 3 times within 1 h of spraying the virus suspension onto the plants and also when spraying only the carrier, indicating induction was in response to spraying and not a specific response to the HearNPV. Although inactivation by the isoflavonoids did not account completely for the level of effect recorded on whole plants, this work constitutes evidence for a novel mechanism of NPV inactivation in legumes. Expanding the use of biological pesticides on legume crops will be dependent upon the development of suitable formulations for OBs to overcome plant secondary chemical effects.     

Elicitors of Plant Defensive Systems Reduce Insect Densities and Disease Incidence

Some elicitors of plant defensive systems can induce biochemical changes that enable the plant to reduce disease incidence; however, little is known about the effect of these induced responses on insect herbivores. We approached this problem using exogenous field applications of several abiotic elicitors of defensive systems in tomatoes (Lycopersicon esculentum), and evaluated the ability of the elicitors [benzo(1,2,3)thiadiazole-7-carbothioic acid (S)-methyl ester (BTH, Actigard); Probenazole; chitosan; salicylic acid; KeyPlex 350; KeyPlex DP2; and KeyPlex DP3] to reduce pest densities and to provide cross-resistance against various insect herbivores and pathogens. Only BTH provided cross-resistance and significantly reduced the incidence of bacterial spot (Xanthomonas campestris pv. vesicatoria), early blight (Alternaria solani), leaf mold (Fulvia fulva), and leafminer larval densities (Liriomyza spp.). The effects on leafminer larval densities were more pronounced during the early stages of plant development. A trend of reduced densities of whiteflies (Bemisia argentifolii) and powdery mildew (Oidium sp.), although not significant, was also found on the BTH-treated plants. Other elicitors had no significant effect on insect populations, but Probenazole and KeyPlex 350 significantly reduced bacterial spot and early blight incidence. The antiherbivore effects of BTH on leafminers was confirmed in a laboratory two-choice experiment. Adult leafminers preferred untreated plants to the BTH-treated tomatoes as ovipositioning host plants, generally corresponding with larval performance. BTH induced high levels of pathogenesis-related proteins in tomato plants including peroxidase, lysozymes, chitinase, and -1,3-glucanases. The possible cross-resistance role of these proteins is discussed. The demonstration that exogenous induction of plant defensive systems in the field can result in lower damage caused by various pathogens and insects, supports the hypothesis that plant defensive systems may be general.