Survival Studies with the Fire Blight Pathogen Erwinia amylovora in Soil and in a Soil-inhabiting Insect

The survival of Erwinia amylovora in soil and in the soil-inhabiting insect Folsomia candida was investigated. Although E. amylovora is an air-borne pathogen, it may reach the soil with rain and other dissemination events. The pathogen was selected on agar plates with two antibiotics and identified by its characteristic colony morphology on minimal agar with copper ions and confirmed by polymerase chain reaction assays. Rates of bacterial decline were much higher in non-sterile than in sterile soil, where the number of living bacteria was stable over 11 weeks. It declined rapidly in untreated soil from a field, and the pathogen was no longer detected 5 weeks after inoculation. Soil-living microarthropods such as F. candida (Collembola) contain large amounts of bacteria in their gut. When fed with gfp -labelled E. amylovora , the pathogen was digested in the gut within a few days, contributing to the decrease of the bacterial population in the soil and thus preventing bacterial distribution by insects.     

Conventional and Real-Time PCRs for Detection of Erwinia piriflorinigrans Allow Its Distinction from the Fire Blight Pathogen,Erwinia amylovora

Erwinia piriflorinigrans is a new pathogenic species of the bacterial genus Erwinia that has been described recently in Spain. Accurate detection and identification of E. piriflorinigrans are challenging because its symptoms on pear blossoms are similar to those caused by Erwinia amylovora, the causal agent of fire blight. Moreover, these two species share phenotypic and molecular characteristics. Two specific and sensitive conventional and real-time PCR protocols were developed to identify and detect E. piriflorinigrans and to differentiate it from E. amylovora and other species of this genus. These protocols were based on sequences from plasmid pEPIR37, which is present in all strains of E. piriflorinigrans analyzed. After the stability of the plasmid was demonstrated, the specificities of the protocols were confirmed by the amplification of all E. piriflorinigrans strains tested, whereas 304 closely related pathogenic and nonpathogenic Erwinia strains and microbiota from pear trees were not amplified. In sensitivity assays, 10³ cells/ml extract were detected in spiked plant material by conventional or real-time PCR, and 10² cells/ml were detected in DNA extracted from spiked plant material by real-time PCR. The protocols developed here succeeded in detecting E. piriflorinigrans in 102 out of 564 symptomatic and asymptomatic naturally infected pear samples (flowers, cortex stem tissue, leaves, shoots, and fruitlets), in necrotic Pyracantha sp. blossoms, and in necrotic pear and apple tissues infected with both E. amylovora and E. piriflorinigrans. Therefore, these new tools can be used in epidemiological studies that will enhance our understanding of the life cycle of E. piriflorinigrans in different hosts and plant tissues and its interaction with E. amylovora.     

First Report of Erwinia amylovora Fire Blight in Belarus

Erwinia amylovora is a phytopathogenic bacterium that causes fire blight, an economically important disease of Rosaceae . Several isolates from pears and apples with fire blight symptoms from Belarus were identified as E. amylovora . All tested isolates were yellow and mucoid on MM2Cu medium, positive in levan production and showed pathogenicity in immature pear fruits. These isolates have identical total protein patterns with E. amylovora 1/79. The PCR with specific primers for E. amylovora harpin gene also gave positive results.     

Occurrence of Erwinia amylovora on Insects in a Fire Blight Orchard

In order to screen for natural dissemination of fire blight under field conditions, insects were caught in an experimental orchard and assayed on semi-selective agar plates and by BIO-polymerase chain reaction for their contamination with Erwinia amylovora , the causative agent of the bacterial disease. The pathogen was detected on 15 out of 348 insects collected (4.3%). The insects contaminated with E. amylovora have different living habits and belong to four different orders (Homoptera, Coleoptera, Hymenoptera and Diptera) and at least eight insect families (Aphididae, Psyllidae, Coccinellidae, Curculionidae, Ichneumonidae, Formicidae, Apidae, and Muscidae) and to other Brachycera. After contact of insects with E. amylovora , the pathogen could survive at least 5 days in/on the green lacewing ( Chrysoperla carnea ) and 12 days on aphids ( Aphis pomi ).     

Characterization of Serracin P, a Phage-Tail-Like Bacteriocin, and Its Activity against Erwinia amylovora, the Fire Blight Pathogen

Serratia plymithicum J7 culture supernatant displayed activity against many pathogenic strains of Erwinia amylovora, the causal agent of the most serious bacterial disease of apple and pear trees, fire blight, and against Klebsiella pneumoniae, Serratia liquefaciens, Serratia marcescens, and Pseudomonas fluorescens. This activity increased significantly upon induction with mitomycin C. A phage-tail-like bacteriocin, named serracin P, was purified from an induced culture supernatant of S. plymithicum J7. It was found to be the only compound involved in the antibacterial activity against sensitive strains. The N-terminal amino acid sequence analysis of the two major subunits (23 and 43 kDa) of serracin P revealed high homology with the Fels-2 prophage of Salmonella enterica, the coliphages P2 and 168, the CTX prophage of Pseudomonas aeruginosa, and a prophage of Yersinia pestis. This strongly suggests a common ancestry for serracin P and these bacteriophages.     

Discovery of Plant Phenolic Compounds That Act as Type III Secretion System Inhibitors or Inducers of the Fire Blight Pathogen,Erwinia amylovora

Erwinia amylovora causes a devastating disease called fire blight in rosaceous plants. The type III secretion system (T3SS) is one of the important virulence factors utilized by E. amylovora in order to successfully infect its hosts. By using a green fluorescent protein (GFP) reporter construct combined with a high-throughput flow cytometry assay, a library of phenolic compounds and their derivatives was studied for their ability to alter the expression of the T3SS. Based on the effectiveness of the compounds on the expression of the T3SS pilus, the T3SS inhibitors 4-methoxy-cinnamic acid (TMCA) and benzoic acid (BA) and one T3SS inducer, trans-2-(4-hydroxyphenyl)-ethenylsulfonate (EHPES), were chosen for further study. Both the T3SS inhibitors (TMCA and BA) and the T3SS inducer (EHPES) were found to alter the expression of T3SS through the HrpS-HrpL pathway. Additionally, TMCA altered T3SS expression through the rsmB_Ea-RsmA_Ea system. Finally, we found that TMCA and BA weakened the hypersensitive response (HR) in tobacco by suppressing the T3SS of E. amylovora. In our study, we identified phenolic compounds that specifically targeted the T3SS. The T3SS inhibitor may offer an alternative approach to antimicrobial therapy by targeting virulence factors of bacterial pathogens.     

Copper-Based Compounds against Erwinia amylovora: Response Parameter Analysis and Suppression of Fire Blight in Apple

Fire blight, caused by Erwinia amylovora, is one of the major bacterial disease of apple and pear, causing enormous economic losses worldwide. Several control measures against E. amylovora have been reported till date, however, none of them have proved to be effective significantly against the pathogen. In this study, mechanisms of the copper-based control agents (CBCAs): copper oxychloride (COCHL), copper oxide (COX), copper hydroxide (CHY), copper sulfate basic (CSB), and tribasic copper sulfate (TCS) and their disease severity reduction efficacy against E. amylovora were analyzed. Bis-1,3-dibutylbarbituric acid trimethine oxonol, carboxyl fluorescein diacetate succinimidyl ester, and 5-cyano-2,3-ditolyl tetrazolium chloride staining were used to check the damage of membrane potential, cytoplasmic pH_in, and respiration of CBCAs-treated E. amylovora, respectively. High disturbance in the membrane potential of E. amylovora was found under COX and COCHL treatments. Similarly, higher significant changes in the inner cytoplasmic pH_in were observed under COX, COCHL, and TCS treatment. CHY and COCHL-treated E. amylovora showed a significant reduction in respiration. In vitro bioassay results revealed that CHY, CSB, and TCS at 2,000 ppm reduced the severity of fire blight both in pre- and post-treatment of CBCAs in immature apple fruits and seedlings. Overall, the most effective CBCAs against E. amylovora could be CHY at 2,000 ppm as its showed inhibition mechanisms and disease severity reduction.     

Methyl palmitate: a novel product of the Medfly (Ceratitis capitata) corpus allatum

The corpus allatum (CA) of adult female Ceratitis capitata produces methyl palmitate (MP) in vitro, in addition to JHB₃ and JH III. Biosynthesized MP migrates on TLC and co-elutes from RP-18 HPLC with synthetic MP. Its identity is verified herein by GCMS. MP production is up-regulated twofold by mevastatin, an inhibitor of mevalonic acid-dependent isoprene biosynthesis. Fosmidomycin, an inhibitor of mevalonic acid-independent isoprene synthesis in graminaceous plants, up-regulates MP synthesis by about fourfold. However, it does not depress JHB₃ biosynthesis concurrently. This suggests that the initial enzyme(s) in the conversion of 1-deoxy-xylulose 5-phosphate to isoprene is presumably present in C. capitata, but is inhibited by fosmidomycin, and this inhibition diverts precursors to MP synthesis. Phytol, an acyclic diterpene, might be suppressing isoprene biosynthesis by CA, thereby resulting in a fourfold increase in the MP biosynthesis. Linolenic acid is an end-product and its presence in incubation media up-regulates MP biosynthesis by twofold, presumably due to the feedback diversion to biosynthesis of C_16:0 and its methyl ester. Biosynthesis of MP is markedly depressed after mating, while otherwise maintained at significantly higher levels in virgin females. MP biosynthesis is significantly reduced in virgin females by direct axonal control but is less consistent after mating.     

Bacteria Associated with the Oesophageal Bulb of the Medfly Ceratitis capitata (Diptera:Tephritidae)

Extracellular Gram negative bacteria were found to be commonly associated to the oesophageal bulb of Ceratitis capitata with Klebsiella oxytoca and Enterobacter agglomerans as the most common species. All the isolates tested in vitro, except one, were sensitive to the antibacterial material present on the medfly laid egg surface. Received: 3 May 2001 / Accepted: 7 June 2001     

Presence and titer of methyl palmitate in the Medfly (Ceratitis capitata) during reproductive maturation

The relative amounts of methyl palmitate (MP) during the first 10 days post-eclosion were determined in whole-body extracts of adult female Ceratitis capitata by SIM monitoring of the 74 m/z fragment. MP peaks in receptive 3-day-old virgin females coincide with previously reported production of Juvenile Hormone (JH) by the corpus allatum (CA). Mating in the Medfly induces female non-receptivity. Indirect evidence suggests that the mevalonate pathway to sesquiterpene biosynthesis is underdeveloped in newly eclosed females. We propose that the pathway leading to synthesis of JH is markedly diverted in non-receptive virgin females to fatty acid synthesis, and partly so-in non-receptive mated females, leading to production of palmitic acid, presumably methylated thereafter. MP is depressed and remains marginal thereafter for the 7 days examined in the virgin female but goes through an apparent second cycle in the mated female. This contrasts with the consistent increase of allatal biosynthesis of MP of virgin and mated females previously reported and suggests additional control mechanisms in vivo. During the period of reduced receptivity following the first mating a second apparent peak of MP is observed. MP is a metabolic default metabolite of reproductively immature females whose putative role in reproductive physiology remains to be defined.     

Effect of Dietary Components on Larval Life History Characteristics in the Medfly (Ceratitis capitata: Diptera,Tephritidae)

Background

The ability to respond to heterogenous nutritional resources is an important factor in the adaptive radiation of insects such as the highly polyphagous Medfly. Here we examined the breadth of the Medfly’s capacity to respond to different developmental conditions, by experimentally altering diet components as a proxy for host quality and novelty.

Methodology/Principal Findings

We tested responses of larval life history to diets containing protein and carbohydrate components found in and outside the natural host range of this species. A 40% reduction in the quantity of protein caused a significant increase in egg to adult mortality by 26.5%±6% in comparison to the standard baseline diet. Proteins and carbohydrates had differential effects on larval versus pupal development and survival. Addition of a novel protein source, casein (i.e. milk protein), to the diet increased larval mortality by 19.4%±3% and also lengthened the duration of larval development by 1.93±0.5 days in comparison to the standard diet. Alteration of dietary carbohydrate, by replacing the baseline starch with simple sugars, increased mortality specifically within the pupal stage (by 28.2%±8% and 26.2%±9% for glucose and maltose diets, respectively). Development in the presence of the novel carbohydrate lactose (milk sugar) was successful, though on this diet there was a decrease of 29.8±1.6 µg in mean pupal weight in comparison to pupae reared on the baseline diet.

Conclusions

The results confirm that laboratory reared Medfly retain the ability to survive development through a wide range of fluctuations in the nutritional environment. We highlight new facets of the responses of different stages of holometabolous life histories to key dietary components. The results are relevant to colonisation scenarios and key to the biology of this highly invasive species.     

Survival Strategy of Erwinia amylovora against Copper: Induction of the Viable-but-Nonculturable State

Copper compounds, widely used to control plant-pathogenic bacteria, have traditionally been employed against fire blight, caused by Erwinia amylovora. However, recent studies have shown that some phytopathogenic bacteria enter into the viable-but-nonculturable (VBNC) state in the presence of copper. To determine whether copper kills E. amylovora or induces the VBNC state, a mineral medium without copper or supplemented with 0.005, 0.01, or 0.05 mM Cu²⁺ was inoculated with 10⁷ CFU/ml of this bacterium and monitored over 9 months. Total and viable cell counts were determined by epifluorescence microscopy using the LIVE/DEAD kit and by flow cytometry with 5-cyano-2,3-ditolyl tetrazolium chloride and SYTO 13. Culturable cells were counted on King's B nonselective solid medium. Changes in the bacterial morphology in the presence of copper were observed by scanning electron microscopy. E. amylovora entered into the VBNC state at all three copper concentrations assayed, much faster when the copper concentration increased. The addition of different agents which complex copper allowed the resuscitation (restoration of culturability) of copper-induced VBNC cells. Finally, copper-induced VBNC cells were virulent only for the first 5 days, while resuscitated cells always regained their pathogenicity on immature fruits over 9 months. These results have shown, for the first time, the induction of the VBNC state in E. amylovora as a survival strategy against copper.     

Survival strategy of Erwinia amylovora against copper: induction of the viable-but-nonculturable state

Copper compounds, widely used to control plant-pathogenic bacteria, have traditionally been employed against fire blight, caused by Erwinia amylovora. However, recent studies have shown that some phytopathogenic bacteria enter into the viable-but-nonculturable (VBNC) state in the presence of copper. To determine whether copper kills E. amylovora or induces the VBNC state, a mineral medium without copper or supplemented with 0.005, 0.01, or 0.05 mM Cu(2+) was inoculated with 10(7) CFU/ml of this bacterium and monitored over 9 months. Total and viable cell counts were determined by epifluorescence microscopy using the LIVE/DEAD kit and by flow cytometry with 5-cyano-2,3-ditolyl tetrazolium chloride and SYTO 13. Culturable cells were counted on King's B nonselective solid medium. Changes in the bacterial morphology in the presence of copper were observed by scanning electron microscopy. E. amylovora entered into the VBNC state at all three copper concentrations assayed, much faster when the copper concentration increased. The addition of different agents which complex copper allowed the resuscitation (restoration of culturability) of copper-induced VBNC cells. Finally, copper-induced VBNC cells were virulent only for the first 5 days, while resuscitated cells always regained their pathogenicity on immature fruits over 9 months. These results have shown, for the first time, the induction of the VBNC state in E. amylovora as a survival strategy against copper.     

Survival and Possible Spread of Erwinia amylovora and Related Plant-Pathogenic Bacteria Exposed to Environmental Stress Conditions

The fire blight pathogen Erwinia amylovora was assayed for survival under unfavourable conditions such as on nitrocellulose filters, in non‐host plants as well as in inoculated mature apples and in infested apple stem sections. In a sterile dry environment, an E. amylovora EPS (exopolysaccharide) mutant, and to a lesser extent its parental wild‐type strain decreased within 3 weeks to a low titre. However, under moist conditions the decrease of viable cells occurred only partially for both strains. Very low cell titres were recovered after application of E. amylovora onto the surface of tobacco leaves, whereas infiltration into the leaves produced lesions (hypersensitive response, HR), in which the bacteria survived in significant amounts. A similar effect was found for the necrotic zones of HR in tobacco leaves caused by E. pyrifoliae, by Pseudomonas syringae pathovars and HR‐deficient E. amylovora mutants or mutants deficient in EPS synthesis and disease‐specific genes. During 7 years of storage, the viability of E. amylovora in wood sections from fire blight‐infested apple trees declined to a low titre. In tissue of mature apples, E. amylovora cells slowly dispersed and could still be recovered after several weeks of storage at room temperature. A minimal risk of accidental dissemination of E. amylovora apart from infested host plants can experimentally not be excluded, but other data confirm a very low incidence of any long distance distribution.     

Potential for host range expansion in Ceratitis capitata flies: impact of proximity of adult food to egg-laying sites

Abstract.

• 1 In field cage and field tests, female Mediterranean fruit flies, Ceratitis capitata (Wiedemann), in trees alighted in significantly greater numbers upon sticky-coated (non-odour-emitting) kumquats, Fortunella japonica, that were in the vicinity of odorous natural proteinaceous food (bird faeces) or synthetic food odour than on similar kumquats distant from bird faeces or synthetic food odour.
• 2 In field cage tests, oviposition in non-sticky kumquats nearby bird faeces was significantly greater than in non-sticky kumquats distant from bird faeces.
• 3 In field tests, medflies laid significantly more eggs in host kumquat and non-host hawthorn, Crataegus mollis, fruit adjacent to bird faeces and synthetic food odour than in fruit of these types distant from food-type stimuli.
• 4 These findings suggest that odour of natural food of medflies could lure flies to plants whose fruit emit little or no attractive odour and are not permanent hosts but which are nonetheless susceptible to egg-laying and larval development, resulting in temporary expansion of host range.

     

The Survival of Erwinia amylovora in Airborne Particles: Tests in the Laboratory and in the Open Air

The causative organism of fire-blight, Erwinia amylovora , survives well when contained in small airborne particles held in the laboratory at relative humidities between 40 and 90 %. Although greater loss of viability occurred when simulated aerosols were exposed to the open air, there were still significant numbers of organisms viable after 2 h.     

Chemosterilants as control agents of Ceratitis capitata (Diptera: Tephritidae) in field trials

Lufenuron is a chitin synthesis inhibitor, which is able to impede Mediterranean fruit fly, Ceratitis capitata (Wiedemann), reproduction. In laboratory trials, following ingestion of lufenuron, the eggs laid by female Ceratitis capitata were prevented from hatching. In field trials in Valencia, Spain, lufenuron showed its effectiveness by reducing C. capitata wild populations and its continuous application to several generations of fruit fly resulted in increased pest control. This field trial was conducted in an isolated valley some 80 ha in size, over a continuous four-year period. In order to maintain the sterilizing effect in the field throughout the whole year, a new lufenuron bait gel was developed. This bait gel was introduced in to delta traps suspended in trees at a density of 24 traps ha-1, and these traps were replaced once a year during the field trial. Monitoring of the adult C. capitata population was conducted to assess the effects of the chemosterilant treatment. In the first year of treatment with sterilizing traps, a reduction of the C. capitata population was observed, indicating that the traps reduce the population right from the first generation. In the second, third and fourth years, a continuous and progressive reduction of the adult Mediterranean fruit fly population was observed. Therefore, the successive application of chemosterilization treatment has a cumulative effect on reducing the fly population year after year. Aerial treatment using malathion does not produce this cumulative effect, and consequently every year it is necessary to start again with the same number of flies as the year before. The possibility of using the chemosterilant method alone or combined with the sterile insect technique is discussed.