Effect of Plant Systemic Resistance Elicited by Biological and Chemical Inducers on the Colonization of the Lettuce and Basil Leaf Apoplast by Salmonella enterica.

Mitigation strategies to prevent microbial contamination of crops are lacking. We tested the hypothesis that induction of plant systemic resistance by biological (induced systemic resistance [ISR]) and chemical (systemic acquired resistance [SAR]) elicitors reduces endophytic colonization of leaves by Salmonella enterica serovars Senftenberg and Typhimurium. S. Senftenberg had greater endophytic fitness than S. Typhimurium in basil and lettuce. The apoplastic population sizes of serovars Senftenberg and Typhimurium in basil and lettuce, respectively, were significantly reduced approximately 10- to 100-fold by root treatment with microbial inducers of systemic resistance compared to H2O treatment. Rhodotorula glutinis effected the lowest population increases of S. Typhimurium in lettuce and S. Senftenberg in basil leaves, respectively 120- and 60-fold lower than those seen with the H2O treatment over 10 days postinoculation. Trichoderma harzianum and Pichia guilliermondii did not have any significant effect on S. Senftenberg in the basil apoplast. The chemical elicitors acidobenzolar-S-methyl and dl-ß-amino-butyric acid inhibited S. Typhimurium multiplication in the lettuce apoplast 10- and 2-fold, respectively, compared to H2O-treated plants. All ISR and SAR inducers applied to lettuce roots in this study increased leaf expression of the defense gene PR1, as did Salmonella apoplastic colonization in H2O-treated lettuce plants. Remarkably, both acidobenzolar-S-methyl upregulation and R. glutinis upregulation of PR1 were repressed by the presence of Salmonella in the leaves. However, enhanced PR1 expression was sustained longer and at greater levels upon elicitor treatment than by Salmonella induction alone. These results serve as a proof of concept that priming of plant immunity may provide an intrinsic hurdle against the endophytic establishment of enteric pathogens in leafy vegetables. IMPORTANCE Fruit and vegetables consumed raw have become an important vehicle of foodborne illness despite a continuous effort to improve their microbial safety. Salmonella enterica has caused numerous recalls and outbreaks of infection associated with contaminated leafy vegetables. Evidence is increasing that enteric pathogens can reach the leaf apoplast, where they confront plant innate immunity. Plants may be triggered for induction of their defense signaling pathways by exposure to chemical or microbial elicitors. This priming for recognition of microbes by plant defense pathways has been used to inhibit plant pathogens and limit disease. Given that current mitigation strategies are insufficient in preventing microbial contamination of produce and associated outbreaks, we investigated the effect of plant-induced resistance on S. enterica colonization of the lettuce and basil leaf apoplast in order to gain a proof of concept for the use of such an intrinsic approach to inhibit human pathogens in leafy vegetables.

Polyethylene mulch modifies greenhouse microclimate and reduces infection of phytophthora infestans in tomato and Pseudoperonospora cubensis in cucumber.

The individual and joint effects of covering the soil with polyethylene mulch before planting and fungicides commonly used by organic growers on tomato late blight (caused by Phytophthora infestans) were studied in three experiments conducted from 2002 to 2005. Application of fungicides resulted in inconsistent and insufficient late blight suppression (control efficacy +/- standard error of 34.5 +/- 14.3%) but the polyethylene mulch resulted in consistent, effective, and highly significant suppression (control efficacy of 83.6 +/- 5.5%) of the disease. The combined effect of the two measures was additive. In a second set of three experiments carried out between 2004 and 2006, it was found that the type of polyethylene mulch used (bicolor aluminized, clear, or black) did not affect the efficacy of late blight suppression (control efficacy of 60.1 to 95.8%) and the differences in the effects among the different polyethylene mulches used were insignificant. Next, the ability of the mulch to suppress cucumber downy mildew (caused by Pseudoperonospora cubensis) was studied in four experiments carried out between 2006 and 2008. The mulch effectively suppressed cucumber downy mildew but the effect was less substantial (control efficacy of 34.9 +/- 4.8%) than that achieved for tomato late blight. The disease-suppressing effect of mulch appeared to come from a reduction in leaf wetness duration, because mulching led to reductions in both the frequency of nights when dew formed and the number of dew hours per night when it formed. Mulching also reduced relative humidity in the canopy, which may have reduced sporulation.

Evaluating the survival and environmental fate of the biocontrol agent Trichoderma atroviride SC1 in vineyards in northern Italy.

AIMS: To study the survival in the soil and the dispersion in the environment of Trichoderma atroviride SC1 after soil applications in a vineyard. METHODS AND RESULTS: Trichoderma atroviride SC1 was introduced into soil in two consecutive years. The levels of T. atroviride populations at different spatial and temporal points following inoculation were assessed by counting the colony-forming units and by a specific quantitative real-time PCR. A high concentration of T. atroviride SC1 was still observed at the 18th week after inoculation. The vertical migration of the fungus to a soil depth of 0.4 m was already noticeable during the first week after inoculation. The fungus spread up to 4 m (horizontally) from the point of inoculation and its concentration decreased with the increasing distance (horizontal and vertical). It was able to colonize the rhizosphere and was also found on grapevine leaves. One year after soil inoculation, T. atroviride SC1 could still be recovered in the treated areas. CONCLUSIONS: Trichoderma atroviride SC1 survived and dispersed becoming an integrant part of the local microbial community under the tested conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The persistence and rapid spread of T. atroviride SC1 represent good qualities for its future use as biocontrol agent against soilborne pathogens.

A generic theoretical model for biological control of foliar plant diseases.

We have developed a generic modelling framework to understand the dynamics of foliar pathogen and biocontrol agent (BCA) populations in order to predict the likelihood of successful biocontrol in relation to the mechanisms involved. The model considers biocontrol systems for foliar pathogens only and, although it is most applicable to fungal BCA systems, does not address a specific biocontrol system. Four biocontrol mechanisms (competition, antibiosis, mycoparasitism and induced resistance) were included within the model rubric. Because of the wide range of mechanisms involved we use Trichoderma/Botrytis as an exemplar system. Qualitative analysis of the model showed that the rates of a BCA colonising diseased and/or healthy plant tissues and the time that the BCA remains active are two of the more important factors in determining the final outcome of a biocontrol system. Further evaluation of the model indicated that the dynamic path to the steady-state population levels also depends critically on other parameters such as the host-pathogen infection rate. In principle, the model can be extended to include other potential mechanisms, including spatio-temporal heterogeneity, fungicide effects, non-fungal BCA and strategies for BCA application, although with a cost in model tractability and ease of interpretation.

Conditions for development of powdery mildew of tomato caused by Oidium neolycopersici.

Oidium neolycopersici causes severe powdery mildew on all aerial parts of tomato, excluding the fruit. The objective of the present work was to examine factors that influence the development of O. neolycopersici on tomato and to identify potential methods for managing tomato powdery mildew. Under controlled conditions, the highest rates of conidial germination were observed at 25 degrees C, 99% relative humidity (RH) and minimal light, and the lowest on leaves adjacent to fruits. Optimal conditions for appressoria formation were 25 degrees C, RH ranging from 33 to 99%, and 1,750 lux light intensity. More conidia were formed at 20 degrees C, 70 to 85% RH, and 5,150 lux light intensity than at 16 and 26 degrees C, 99% RH, and 480 to 1,750 lux, respectively. Conidia survived and remained capable of germination for over four months when initially incubated at lower temperatures and higher RH, as compared with their fast decline under more extreme summer shade conditions. In growth chamber experiments, disease did not develop at 28 degrees C. Within the range of 70 to 99% RH, disease was less severe under the higher RH than the drier conditions. Disease was also less severe at lower light intensities. Data collected in three commercial-like greenhouse experiments involving various climate regimes were used to draw correlations regarding the effects of temperature and RH on the development of epidemics. Severity of powdery mildew was positively correlated with the duration of the range 15 to 25 degrees C, 1 to 4 weeks before disease evaluation (BDE), RH levels of 60 to 90% at 2 to 4 weeks BDE, and RH of 50 to 60% during the week BDE. Conversely, disease was negatively correlated with the duration of temperatures in the low and high ranges (5 to 15 degrees C and 35 to 40 degrees C) at 1 to 4 weeks BDE, with the duration of RH levels of 40% and below at 1 to 4 weeks BDE, and with 50 to 60% RH during the third week BDE. High (90 to 100%) RH was also negatively correlated with disease severity. These results suggest that the combination of high temperatures and low RH may help reduce O. neolycopersici powdery mildew severity in greenhouse tomatoes.

Use of Selenate-Resistant Strains as Markers for the Spread and Survival of Botrytis cinerea Under Greenhouse Conditions.

ABSTRACT Botrytis cinerea marked strains combining traits of fungicide resistance or sensitivity (carbendazim, iprodione) with resistance to selenate were created and assessed for use in studying the dispersal of B. cinerea and its survival inside plant tissue under greenhouse conditions. Marked strains differed in their ability to cause lesions and to disperse in the greenhouse. A strain that was the most aggressive in infecting plants was also the most successful in spreading across the greenhouse. Following 7 to 14 days of exposure to marked inoculum, about 90% of plants showed quiescent B. cinerea infection with no significant difference between hosts or seasons. However, in a warm season, most of the plants were infected with wild-type B. cinerea, whereas most of the winter-recovered B. cinerea strains were of the marked phenotype, showing the importance of local inoculum from within the glasshouse in winter. The air of the greenhouse contained the same population of marked B. cinerea in warm and in cold periods, whereas the total population was significantly higher in summer. In the warm season, mycelium of B. cinerea inside plant debris lost viability within 3 to 4 months, whereas it stayed viable for 4 months in the winter (December to March) and started to lose viability in April.

Biocontrol of foliar pathogens: mechanisms and application.

Biocontrol offers attractive alternatives or supplements to the use of conventional methods for plant disease management. Vast experience has been gained in the biocontrol of plant diseases. Prevention of infection by biocontrol agents or suppression of disease is based on various modes of action. Pathogens are typically affected by certain modes of actions and not by others according to their nature (i.e. biotrophs vs. necrotrophs). Resistance in the host plant may be induced locally or systemically by either live or dead cells of the biocontrol agent and may affect pathogens of various groups. As some pathogens are negatively affected by lake of nutrients in the infection court, competition for nutrients and space was long recognized as antagonism trait. Antibiosis and hyperparasitism affect pathogens of various groups. Other valid mechanisms are reduction of the saprophytic ability and reducing spore dissemination. Recently it was revealed that restraining of pathogenicity factors of the pathogens, i.e. host hydrolyzing proteins or reactive oxygen species takes place when biocontrol is used. It is likely that several modes of action concomitantly participate in pathogens suppression but the relative importance of each one of them is not clear. Examples of effective prevention of infection in the phyllosphere that rely on multiple modes of action will be demonstrated with Trichodermo harzianum T39 (TRICHODEX), Bacillus mycoides and Pichia guilermondii, a filamentous fungus, bacterium and yeast biocontrol agents, respectively. Several commercial products based on microorganisms have been developed and are starting to penetrate the market. However, large-scale use is still limited because of variability and inconsistency of biocontrol activity. In some cases this may be caused by sensitivity of the biocontrol agents to environmental influences. Ways to overcome biocontrol limitations and to improve its efficacy are i. integration of biocontrol with chemical fungicides on a calendar basis or according to ecological requirements of the biocontrol agents relying on the advise of a decision support system; ii. introduction of two or more biocontrol agents in a mixture, assuming that each one of them has different ecological requirements and/or different modes of action. Implementation of one (or more) of these approaches, using biocontrol preparations mentioned above lowered the variability and increased the consistency of disease suppression. The expected long-term result of the implementation of these suggested strategies is reduced risk of uncontrolled epidemics and increase of confidence of growers in using this non-chemical control measure on a large scale.

Improving biological control by combining biocontrol agents each with several mechanisms of disease suppression.

ABSTRACT Two biocontrol agents, a yeast (Pichia guilermondii) and a bacterium (Bacillus mycoides), were tested separately and together for suppression of Botrytis cinerea on strawberry leaves and plants. Scanning electron microscopy revealed significant inhibition of Botrytis cinerea conidial germination in the presence of Pichia guilermondii, whereas Bacillus mycoides caused breakage and destruction of conidia. When both biocontrol agents were applied in a mixture, conidial destruction was more severe. The modes of action of each of the biocontrol agents were elucidated and the relative quantitative contribution of each mechanism to suppression of Botrytis cinerea was estimated using multiple regression with dummy variables. The improvement in control efficacy achieved by introducing one or more mechanisms at a time was calculated. Pichia guilermondii competed with Botrytis cinerea for glucose, sucrose, adenine, histidine, and folic acid. Viability of the yeast cells played a crucial role in suppression of Botrytis cinerea and they secreted an inhibitory compound that had an acropetal effect and was not volatile. Bacillus mycoides did not compete for any of the sugars, amino acids, or vitamins examined at a level that would affect Botrytis cinerea development. Viable cells and the compounds secreted by them contributed similarly to Botrytis cinerea suppression. The bacteria secreted volatile and non-volatile inhibitory compounds and activated the defense systems of the host. The nonvolatile compounds had both acropetal and basipetal effects. Mixture of Pichia guilermondii and Bacillus mycoides resulted in additive activity compared with their separate application. The combined activity was due to the summation of biocontrol mechanisms of both agents. This work provides a theoretical explanation for our previous findings of reduced disease control variability with a mixture of Pichia guilermondii and Bacillus mycoides.

Combining biocontrol agents to reduce the variability of biological control.

ABSTRACT Two biocontrol agents, a yeast (Pichia guilermondii) and a bacterium (Bacillus mycoides), were tested separately and together for suppression of Botrytis cinerea on strawberry leaves. The aims of the research were to determine whether the use of their combination would broaden the environmental conditions under which biological control is effective, and to test the hypothesis that it would reduce the variability of control efficacy under diverse conditions. Applied separately, the biocontrol agents significantly inhibited spore germination, lesion formation, and lesion development at most temperatures, relative humidities, and spray-timing combinations (temperatures: 10, 15, 20, 23, 25, and 30 degrees C; relative humidities: 78, 85, 96, and 100%; and spray-timings: 0, 4, and 7 days before inoculation). However, control efficacy was highly variable, and under certain combinations it was not adequate. Control efficacy achieved by the biocontrol agents applied separately ranged between 38 and 98% (mean 74%) and the coefficient of variation ranged from 9.7 to 75%. The mixture of Bacillus mycoides and Pichia guilermondii suppressed Botrytis cinerea effectively (80 to 99.8% control) under all conditions, and the coefficients of variation were as low as 0.4 to 9% in all cases. Thus, application of both biocontrol agents resulted in better suppression of Botrytis cinerea, and also reduced the variability of disease control. Application of more than one biocontrol agent is suggested as a reliable means of reducing the variability and increasing the reliability of biological control.

Mycorrhiza-induced changes in disease severity and PR protein expression in tobacco leaves

The development of leaf disease symptoms and the accumulation of pathogenesis-related (PR) proteins were monitored in leaves of tobacco (Nicotiana tabacum cv. Xanthinc) plants colonized by the arbuscular mycorrhizal fungus Glomus intraradices. Leaves of mycorrhizal plants infected with the leaf pathogens Botrytis cinerea or tobacco mosaic virus showed a higher incidence and severity of necrotic lesions than those of nonmycorrhizal controls. Similar plant responses were obtained at both low (0.1 mM) and high (1.0 mM) nutritional P levels and with mutant plants (NahG) that are unable to accumulate salicylic acid. Application of PR-protein activators induced PR-1 and PR-3 expression in leaves of both nonmycorrhizal and mycorrhizal plants; however, accumulation and mRNA steady-site levels of these proteins were lower, and their appearance delayed, in leaves of the mycorrhizal plants. Application of 0.3 mM phosphate to the plants did not mimic the delay in PR expression observed in the mycorrhizal tobacco. Together, these data strongly support the existence of regulatory processes, initiated in the roots of mycorrhizal plants, that modify disease-symptom development and gene expression in their leaves.

Epidemiology of Botrytis cinerea in Sweet Basil and Implications for Disease Management.

Sweet basil (Ocimum basilicum) is a herbaceous annual plant that is highly susceptible to gray mold, caused by Botrytis cinerea. Infections are initiated on the surface of stem wounds that are caused during harvest; the pathogen then progresses on the stem, killing leaves and secondary buds. When the infection reaches the main stem, the entire plant dies. A study of the epidemiology of the disease and of host-pathogen interactions led to the development of rules for effective disease management. The research was conducted in three steps. (i) Natural epidemics in commercial crops were monitored during the 1993 to 1994 and 1994 to 1995 growing seasons. Disease outbreaks were found to coincide with harvests during rainy days, whereas disease intensity did not change much when harvests were completed during rainless days. (ii) Studies under controlled environmental conditions revealed that cut ends of stems were highly susceptible soon after harvest, susceptibility diminished gradually, and stem cuts inoculated 48 h after harvest were rarely diseased. Observations under a scanning electron microscope showed that an opaque layer had appeared over the cut surface and, as the entire wound surface became covered, penetration of the fungus into the tissue was prevented. Based on these findings, it was hypothesized that avoidance of harvesting during rain events and application of one fungicidal spray, soon after harvest, would result in adequate disease suppression. (iii) These hypotheses were tested and corroborated in greenhouses in two experiments, in 1994 to 1995 and 1995 to 1996. Management of basil crops according to these concepts would also lower the risks for contamination of the marketable product with pesticide residues.

Incorporation of Weather Forecasting in Integrated, Biological-Chemical Management of Botrytis cinerea.

ABSTRACT A strategy for integrated biological and chemical control of Botrytis cinerea in nonheated greenhouse vegetables was developed. The biocontrol agent used was a commercial preparation developed from an isolate of Trichoderma harzianum, T39 (Trichodex). Decisions concerning whether to spray the biocontrol agent or a fungicide were made based on a weather-based disease warning system. The integrated strategy (BOTMAN [short for Botrytis manager]) was implemented as follows: when slow or no disease progress was expected, no spraying was needed; when an outbreak of epidemics was expected, use of a chemical fungicide was recommended; in all other cases, application of T. harzianum T39 was recommended. Future weather information (a 4-day weather forecast provided by the Israel Weather Forecast Service) was more useful for disease warnings than immediate past weather. The integrated strategy was compared with weekly applications of fungicide in 11 experiments conducted over 3 years in greenhouse-grown tomato and cucumber. Disease reduction in the integrated strategy (63.9 +/- 3.0%) did not differ significantly (P < 0.05) from the fungicide-only treatment (70.1 +/- 3.6%). The number of fungicide sprays in the integrated strategy ranged from 2 to 7 (mean 4.2) compared to 7 to 13 (mean 10.5) in the fungicide treatment. The integrated strategy averaged 5.9 sprays of T. harzianum T39. For the integrated strategy, one treatment omitted use of T. harzianum T39 to estimate the contribution of this agent to disease control. Disease reduction in that treatment (49.1 +/- 4.8%) was significantly (P < 0.05) inferior to the combined chemical and biological strategy, indicating that the T. harzianum T39 sprays had a measurable effect on disease control.

Effect of Some Host and Microclimate Factors on Infection of Tomato Stems by Botrytis cinerea.

The susceptibility of tomato stems to infection by Botrytis cinerea and the influence of temperature and humidity on disease development were investigated with stem pieces and whole plants. Stem rotting resulted after inoculation of wounded stems with a conidial suspension in water or with dry conidia; no symptoms developed following inoculation of unwounded stems. The proportion of inoculated stems developing Botrytis rot increased as the inoculum concentration was increased from 10 to 10,000 conidia per stem. Stem susceptibility to infection declined from 60 to 8% as wound age increased from 0 to 24 h before inoculation. Wounded stem pieces maintained in a low vapor pressure deficit (VPD) environment (<0.2 kPa) remained susceptible for a longer period than those maintained at a high VPD. Infection and stem rotting occurred at temperatures of 5 to 26°C, with disease development most rapid at 15°C. Sporulation was optimal at 15°C and did not occur within 20 days of incubation at 5 or 26°C. Incubation at high humidity following inoculation of fresh wounds (VPD <0.2 kPa) did not increase infection incidence or tissue rotting, compared with incubation at a VPD >1.3 kPa; however, incubation at the lower VPD did increase the intensity of sporulation.

Effects of elevated intranasal temperature on subjective and objective findings in perennial rhinitis.

The effects of elevated intranasal temperature on symptoms and signs of perennial rhinitis were studied in 78 patients by a double-blind, randomized, placebo-controlled clinical trial. Patients were subjected to two treatments separated by a 1-week interval. Each treatment consisted of three 30-minute sessions, during which the patient's intranasal temperature was raised by inhalation of saturated hot air (42 degrees C to 44 degrees C). Subjective response was recorded on a daily symptom score card during the week following treatment. Nasal patency was determined before and after treatment by measuring maximal nasal expiratory and inspiratory airflow and by measuring the area covered with vapor formed by the exhaled air on a plate. Highly reproducible results were obtained by using these three objective methods. Elevation of intranasal temperature resulted in amelioration of rhinitis symptoms and in objective evidence of increased nasal patency in a significant percentage of patients compared to the placebo-treated group.

Effects of inhaled humidified warm air on nasal patency and nasal symptoms in allergic rhinitis.

The effects of inhaled warm air on nasal patency and on allergic rhinitic symptoms were studied in 102 patients. Treatment consisted of two consecutive 30-minute sessions, during which the patient inhaled saturated, hot (42-44 degrees C) air through the nose. The treatment was repeated 1 week later. During the week following each treatment, every patient recorded his or her subjective response on a daily symptom score card. Nasal patency was determined before and after each treatment by measuring peak nasal inspiratory and expiratory air flow and by measuring the area covered with vapor formed by the exhaled air on a plate. Highly reproducible results were obtained by using these three objective methods. Inhalation of humidified warm air resulted in amelioration of allergic rhinitis symptoms and in increased nasal patency in a high proportion of patients. There were no adverse side effects. This form of therapy seemed effective in the 2-week period in which it was used and would be an additional form of therapy available for those suffering discomfort from allergic rhinitis.

Effects of steam inhalation on nasal patency and nasal symptoms in patients with the common cold.

The effects of steam inhalation on nasal patency and on nasal symptoms were studied in 62 patients with the common cold by a double-blind, randomized, placebo-controlled clinical trial. Treatment consisted of two 20-minute sessions, during which the patient inhaled saturated, hot (42 degrees to 44 degrees C) air through the nose. The subjective response was recorded by each patient during the week following treatment on a daily symptom score card. Nasal patency was determined before treatment, the following day, and 1 week later by measuring peak nasal expiratory and inspiratory air flow. Highly reproducible results were obtained by using these objective methods. Steam inhalation resulted in alleviation of cold symptoms and increased nasal patency in a significantly higher percentage of patients in the actively treated group than in the placebo-treated group. Possible explanations for the effectiveness of treatment are discussed.

Possible role of lectins in mycoparasitism.

Lectin activity in a host-mycoparasite relationship was demonstrated with Rhizoctonia solani and Trichoderma harzianum. Attachment of O but not A and B erythrocytes to hyphae occurred on R. solani but not on its mycoparasite. This phenomenon, which was Ca2+ and Mn2+ dependent, was prevented by galactose, present in T. harzianum cell walls, and by fucose.