Invasion and colonization of mature apple fruit by Erwinia amylovora tagged with bioluminescence genes

Invasion and colonization of mature apple fruit by a transformant of Erwinia amylovora tagged with bioluminescence genes from Vibrio fischeri was examined. The transformant was deposited on cut surfaces of fruit stems, wounds on the shoulders and calyces, injured fruit-bearing twigs of harvested apple fruit, and cut fruit flesh. After incubation in closed stainless steel or plastic boxes at 25°C, fruit were periodically observed with a two-dimensional luminometer. The presence of the transformant in luminous areas was confirmed by isolating it on selective media. E. amylovora, when deposited in fruit stems: (1) can invade mature as well as immature apple fruit; (2) vertically and horizontally spreads and colonizes along vascular bundles, increasing its population; (3) reaches the calyx end and the flesh just under the exocarp within 3–4 days after inoculation; (4) when deposited on cut fruit flesh, irrespective of its maturity, can easily increase its population and survive 2–4 weeks or more at 25°C; and (5) even at the time of fruit maturation, can migrate within twigs rapidly and reaches the abscission layers between fruit-bearing twigs and fruit stems.     

Infection frequency of mature apple fruit with Erwinia amylovora deposited on pedicels and its survival in the fruit stored at low temperature

The infection frequency of mature apple fruit by Erwinia amylovora and the survival of E. amylovora in the fruit stored at low temperature were investigated. The fruit stems (pedicels) of 460 mature apple fruit were inoculated with 10⁵ or 10⁴ cfu of bioluminescent E. amylovora, tagged with lux genes. Nine days after inoculation, 43% and 27% of the fruit inoculated with 10⁵ and 10⁴ cfu, respectively, were infected. All infected fruit looked healthy. After 6 months of storage at 5°C, almost all of the 142 infected fruit had viable E. amylovora. Of the fruit containing E. amylovora internally, 19.5% had latent infections and the rest had blight symptoms. E. amylovora was not uniformly distributed in the fruit flesh, and internal brown lesions were observed where E. amylovora was densely distributed. These findings showed that mature apple fruit may be infected with E. amylovora, especially as latent infections, and act as a source for long-range dissemination.     

Assessment of <Emphasis Type="Italic">Pichia anomala</Emphasis> (strain K) efficacy against blue mould of apples when applied pre- or post-harvest under laboratory conditions and in orchard trials

The yeast Pichia anomala strain K was selected in Belgium from the apple surface for its antagonistic activity against post-harvest diseases of apples. The efficacy of this strain against P. expansum was evaluated in the laboratory in three scenarios designed to mimic practical conditions, with different periods of incubation between biological treatment, wounding of fruit surface, and pathogen inoculation. Higher protection levels and higher final yeast densities were obtained when the applied initial concentration was 1 × 10⁸ cfu ml⁻¹ than when it was only 1 × 10⁵ cfu ml⁻¹. The protection level correlated positively with the yeast density determined in wounds and was influenced by apple surface wetness. In orchard trials spanning two successive years, biological treatment against P. expansum, based on a powder of P. anomala strain K (1 × 10⁷ cfu ml⁻¹), β-1,3-glucans (YGT 2 g l⁻¹), and CaCl₂.2H₂0 (20 g l⁻¹), was applied to apples pre- or post-harvest under practical conditions and its effect compared with standard chemical treatments. The first year, the highest reduction (95.2%) against blue decay was obtained by means of four successive fungicide treatments and the next-highest level (87.6%) with pre-harvest high-volume spraying of the three-component mixture 12 days before harvest. The second year, the best results were obtained with post-harvest Sumico (carbendazim 25% and diethofencarb 25%) treatment and post-harvest biological treatment, both by dipping the apples, 88.3 and 56.3% respectively. A density threshold of 1 × 10⁴ cfu cm⁻² of strain K on the apple surface seemed to be required just after harvest for high protective activity, whatever the method and time of application. In the case of pre-harvest biological treatments, variations in meteorological conditions between the 2 years may have considerably affected strain K population density and its efficacies.     

Alternative inoculum sources for fire blight: the potential role of fruit mummies and non‐host plants

Fire blight is the most damaging bacterial disease in apple production worldwide. Cankers and symptomless infected shoots are known as sites for the overwintering of Erwinia amylovora, subsequently providing primary inoculum for infection in the spring. In the present work, further potential sources of inoculum were investigated. Real‐time PCR assays covering a 3‐year‐period classified 19·9% of samples taken from fruit mummies as positive. Bacterial abundance in fruit mummies during autumn, winter and spring was up to 10⁹ cells per gram of tissue and correlated well with later infection rates of blossoms. Blossoms of non‐host plants growing close to infected trees were also shown to be colonized by E. amylovora and to enable epiphytic survival and propagation of bacteria. The results indicate a potential role of fruit mummies and buds in overwintering and as a source of primary inoculum for dissemination of the pathogen early in the growing season. Non‐host blossoms may also serve as an inoculum source in the build‐up of the pathogen population. Both aspects may contribute significantly to the epidemiology of E. amylovora. The significance of infected rootstocks as an inoculum source is also discussed. Fruit mummies might be used to determine pathogen pressure in an orchard before the beginning of the blooming period.     

Blue EaLAMP—a specific and sensitive method for visual detection of genomic Erwinia amylovora DNA

Fire blight caused by the gram-negative bacterium Erwinia amylovora is a major disease of pome fruit like apple (Malus domestica) or pear (Pyrus communis). Detection of the pathogen is hampered by low titres usually being present during initial flower infection and the brief time frame for analysis to decide upon subsequent countermeasures. Here we describe a loop-mediated isothermal amplification of DNA (LAMP) assay for genomic DNA of Erwinia amylovora, which relies on a highly specific primer design that excludes amplification from typical DNA sources of the orchard biological system and from sample handling. The assay enables the fast detection of down to approximately 20?cfu of pure Erwinia amylovora or 100?fg genomic DNA (corresponding to approximately 25 E. amyolovora equivalents) per reaction within 45?min. Fast and reliable detection of E. amylovora in orchard samples by naked eye is achieved through a visual colour change indication with hydroxynaphthol blue. The assay avoids use of complex technical devices and is thus suitable for field testing.     

Distribution of streptomycin-resistant strainsof Erwinia amylovora in Israel and occurrence of blossom blight in the autumn

Following failure in control of fire blight with streptomycin, the distribution of streptomycin-resistant strains ofErwinia amylovora in Israel was surveyed. During 1994–1997 109 pear, apple, loquat and quince orchards were monitored. Streptomycin-resistant strains ofE. amylovora were recovered from flowers and from infected branches collected at 18 locations in the Sharon, Galilee and Golan Heights regions. In the Sharon region all the isolated strains ofE. amylovora were streptomycin-resistant, whereas in the Galilee and Golan Heights, resistant as well as sensitiveE. amylovora strains were recovered at different locations. In the southern coastal plain no resistance could be detected. Streptomycin-resistant strains ofE. amylovora did not hybridize with the DNA probe SMP3, and resistance could not be transferred by mating to a sensitive strain, suggesting that streptomycin resistance in Israel is not plasmid-mediated. Fire blight symptoms were observed, for the first time, on pear blossoms during the autumn of 1994. A high population of 2x 10⁶-6x 10⁷ CFU/flower in the autumn of 1995 and of 1996 was correlated with the appearance of blossom blight symptoms.     

Protective Effects Against Erwinia amylovora Induced by hrp Mutants in the Whole Plant are Only Partially Mimicked in Cultivated Cells

A virulent strain of Erwinia amylovora, the causal agent of fire blight of Maloideae, and two of its non-virulent hrp mutants (a secretory and a regulatory mutant) were inoculated into apple cell suspensions either alone or in mixed inoculations. In single inoculations, death of 4- to 5-day-old apple cells occurred only when the concentration of the virulent strain of E. amylovora reached a threshold inoculum concentration of 10⁴CFUml^–1, while high concentrations of the hrp mutants were unable to kill apple cells. When mixed inoculated with the virulent parental strain, both hrp mutants protected apple cells from death caused by the virulent strain. The protective effect was associated with a decrease in the population level of the virulent strain and it was dependent on the non-virulent to virulent inoculum concentration suggesting a competition between the non-virulent mutant and the virulent strain. However, no differential protective ability between the two types of mutants could be noticed, contrary to previous results obtained with apple seedlings or apple flowers in which the regulatory mutant was significantly more effective than the secretory mutant. In conclusion, inoculation of apple cell cultures with E. amylovora does not seem to be a model suitable for investigating mechanisms leading to protection.     

Necrotrophic behaviour of Erwinia amylovora in apple and tobacco leaf tissue

An important issue related to the epidemiology of fire blight, a devastating disease of apples and pears, is how its causal agent, the bacterium Erwinia amylovora, survives and disseminates in the environment. Almost no information is available on the possibility of this pathogen overwintering as a necrotroph. In this study, bacterial survival in dead apple and tobacco (a non‐host) leaf tissues was addressed. In necrotized leaves collected 5, 6, 7 and 8 months following shoot inoculation of apple trees, viable E. amylovora cells were present in over 50% of samples from the midrib and in over 10% of samples from lateral veins, but were never found in parenchyma. Using a PCR‐based method, pathogen DNA was detected in more than 50% of samples that were found to be free of viable cells by conventional plating out. However, PCR analysis was insufficient to distinguish between the DNA of viable and dead bacteria. Sugars appropriate for bacterial growth were found in dead apple leaves. In spot‐inoculated attached apple and tobacco leaves, a remarkable increase in the bacterial population was observed in lesions that developed as a hypersensitive response (HR). As in other necrotrophic interactions, bacterial proliferation was associated with massive hydrogen peroxide production and progression toward plant cell death. The results indicate that E. amylovora has an ability to survive as a semi‐necrotroph or necrotroph, which allows for overwintering in dead apple leaves.     

Influence of Stigmatic Morphology on Flower Colonization by <Emphasis Type="Italic">Erwinia amylovora</Emphasis> and <Emphasis Type="Italic">Pantoea agglomerans</Emphasis>

The morphology of apple and pear stigma was investigated with confocal laser scanning microscopy and scanning electron microscopy. The floral colonization process by Erwinia amylovora was studied with gfp-labelled bacteria and confocal laser scanning microscopy to allow the in vivo observation of the pathogen colonization on intact, viable plant tissues without any kind of staining of the specimens. The interaction on the stigma between Erwinia amylovora and Pantoea agglomerans, both labelled with genes encoding for fluorescent proteins (DsRed-GFP), was also investigated. A stylar groove, covered by papillae and dwelling from the stigma along the style, was visualized. In laboratory conditions, this groove was shown to be an important way for E. amylovora migration towards the nectarthodes. Due to its anatomical structure the groove can sustain bacterial multiplication and thus may play an important role on the interactions between the pathogen and the bacterial antagonist P. agglomerans.     

Detection of the fire blight biocontrol agent <Emphasis Type="Italic">Bacillus subtilis</Emphasis> BD170 (Biopro<Superscript>®</Superscript>) in a Swiss apple orchard

Fire blight, caused by Erwinia amylovora, is a major disease threat to apple, pear and other pome fruit worldwide. The disease is widespread in Europe and has recently become established in Switzerland. Antibiotics are the most effective controls used in North America but these are not permitted for agricultural use in most European countries. A newly registered biological control product Biopro^®, based on the antagonist Bacillus subtilis strain BD170, is being used as an alternative strategy for fire blight management. A specific molecular marker was developed for monitoring the spread of this agent on blossoms after Biopro^® spray application in a Swiss apple orchard throughout the bloom period for 2years. Direct spraying resulted in efficient primary colonisation of pistils in flowers that were open at the time of treatment. Subsequent bacterial dissemination (secondary colonisation) of flowers that were closed or at bud stage at the time of treatment was observed but was found to be dependent on the timing of treatments relative to bloom stage in the orchard. Foraging honeybees were shown to be disseminators of Biopro^®. We also report detection of the biocontrol agent in honey collected from hives where bees were exposed by placing Biopro^® at the entrance or in the hatching nest and from hives that were simply placed in sprayed orchards.     

Use of a diagnostic medium for<Emphasis Type="Italic">in situ</Emphasis> determination of the response of<Emphasis Type="Italic">Erwinia amylovora</Emphasis> strains to bactericides

Erwinia amylovora, the causal agent of fire blight, is managed by application of bactericides to protect fruit tree blossoms from infection. Monitoring the response ofE. amylovora strains to bactericides is crucial for adequate disease management. The coliform agar medium produced by Merck was recently reported as an effective tool for rapid diagnosis ofE. amylovora (RD-medium). The objective of the present study was to examine the possibility of using the RD-medium forin situ determination of the response ofE. amylovora strains to oxolinic acid and streptomycin. The phenotypic response of 48E. amylovora strains isolated in 2002 to both bactericides was determined with the RD-medium and, for comparison, by a routine laboratory test. The results of 45 samples (93.7%) were in agreement with the findings of the routine laboratory test. Aχ ² test rejected the null hypothesis that the phenotypic characteristics as determined by the two respective methods differed significantly (P=0.389). Thein situ test was implemented on a national scale in 2003 and the results were in agreement with those obtained in laboratory tests, which suggests that this medium can be usedin situ for monitoring the appearance of resistance inE. amylovora populations. http://www.phytoparasitica.org posting Feb. 11, 2004.     

Levels of fire blight (<Emphasis Type="Italic">Erwinia amylovora</Emphasis>) susceptibility of native apple,pear and quince germplasm from Lake Van Basin,Turkey

Fire blight resistance of apple, pear and quince genetic resources from Lake Van Basin (eastern Turkey) was tested using Erwinia amylovora strain Ea Van. Shoot tips of 92 native accessions (48 accessions for apple, 38 accessions for pear and 6 accessions for quince) were wounded for inoculation, and artificially inoculated with pathogenic bacteria under greenhouse conditions. The levels of resistance of accessions were classified in comparison with control varieties according to the genotype susceptibility index (GSI%) scores based on the lesion length on shoots of each genotype. Fire blight resistance of accessions consisted of five classes: resistant (R), moderately resistant (MR), moderately susceptible (MS), susceptible (S) and highly susceptible (HS). GSI% scores differed significantly among accessions from each fruit species (p < 0.01). GSI values ranged from 12.4% to 64.1% for apple genotypes, from 17.2% to 55.1% for pear genotypes, and from 17.8% to 43.4% for quince genotypes. No resistant genotypes of apple, pear and quince were observed. Seven accessions of apple, two accessions of pear and one accession of quince were MR. 25 accessions of apple, 14 accessions of pear and one accession of quince were MS. These findings indicate a considerable variation in fire blight resistance and could contribute to breeding efforts regarding fire blight resistance in apple, pear and quince.     

Erwinia isolates from the bacterial shoot blight of pear in Japan are closely related to Erwinia pyrifoliae based on phylogenetic analyses of gyrB and rpoD genes

The phylogenetic relationships among Erwinia amylovora biovar 4 (the pathogen of bacterial shoot blight of pear in Japan), other biovars of E. amylovora, and Erwinia pyrifoliae were investigated using the sequences of 16S rRNA, gyrB, and rpoD genes. The tested isolates formed two distinct monophyletic groups in the phylogenetic trees constructed based on the gyrB gene, rpoD gene, or a combination of the three genes: group 1 contained E. amylovora biovars 1, 2, and 3; group 2 contained E. amylovora bv. 4 and E. pyrifoliae. This phylogenetic analysis showed that E. amylovora bv. 4 was more closely related to E. pyrifoliae than to other biovars of E. amylovora. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB242876 to AB242925.     

Viability and release of Neonectria ditissima ascospores on apple fruit in Brazil

During European canker monitoring in an apple experimental orchard, 14 mummified fruit (two and three trees with 10 and four positive records in 2018 and 2019, respectively) showed perithecia. Perithecium production on apple fruit, confirmation of pathogenicity of Neonectria ditissima isolated from mummified fruit, and ascospore release from fruit tissues has rarely been reported, and their role in the epidemiology of European canker has been largely overlooked. Thus, the objectives of our study were to (a) prove the presence of both conidia and ascospores of N. ditissima in mummified fruit in an experimental field, confirming pathogenesis in different apple cultivars, and (b) monitor production of the two types of inoculum in infected apple fruit over time. Canker incidence in this orchard was 47% of trees with symptoms in 2018 and 48% in 2019. Molecular and morphological tests confirmed that the fungus detected in the mummified apple fruit was N. ditissima. Apple fruit with sporodochia and perithecia washed immediately after collection from the orchard showed conidia but no ascospores of N. ditissima. However, after 4 days’ incubation, perithecia on mummified fruit showed many ascospore cirri. Koch's postulates were fulfilled on apple plants and mature fruit. Fruit inoculated with N. ditissima released spores for over a year under Brazilian field conditions. The release of both spore types peaked in May (Brazilian leaf fall) and October (spring); release of conidia also peaked in February (early harvest). These results support our hypothesis that fruit can serve as primary inoculum for European canker in Brazilian apple orchards.     

Prohexadione‐Ca induces resistance to fireblight and other diseases1

Prohexadione‐Ca is primarily used for the control of shoot growth in pome and other fruit trees. During its development, treated apple and pear trees were found to be significantly less affected by fireblight (Erwinia amylovora) and other pathogens, although prohexadione‐Ca is inactive as a bactericide or fungicide. Prohexadione is a structural mimic of 2‐oxoglutaric acid, and the distinct dioxygenases involved in gibberellin biosynthesis that require this compound as a cosubstrate are blocked. As a result, less growth‐active gibberellins are formed and treated plants remain compact. 2‐Oxoglutaric acid‐dependent dioxygenases are also involved in flavonoid metabolism. In shoots of apples and pears, prohexadione‐Ca causes considerable changes in the formation of flavonoids and their phenolic precursors by inhibiting flavanone 3‐hydroxylase. Convincing evidence is available that prohexadione‐Ca triggers pathogen resistance primarily by inducing the formation of 3‐deoxyflavonoids, in particular luteoforol, with phytoalexin‐like properties. Morphoregulatory effects caused by prohexadione‐Ca are only of secondary relevance. The simultaneous control of excessive shoot growth and shoot infections by fireblight is seen as a major advantage of using prohexadione‐Ca in pome fruit trees.     

Inoculation of Malus genotypes with a set of Erwinia amylovora strains indicates a gene‐for‐gene relationship between the effector gene eop1 and both Malus floribunda 821 and Malus ‘Evereste’

The Gram‐negative bacterium Erwinia amylovora, causal agent of fire blight disease in pome fruit trees, encodes a type three secretion system (T3SS) that translocates effector proteins into plant cells that collectively function to suppress host defences and enable pathogenesis. Until now, there has only been limited knowledge about the interaction of effector proteins and host resistance presented in several wild Malus species. This study tested disease responses in several Malus wild species with a set of effector deletion mutant strains and several highly virulent E. amylovora strains, which are assumed to influence the host resistance response of fire blight‐resistant Malus species. The findings confirm earlier studies that deletion of the T3SS abolished virulence of the pathogen. Furthermore, a new gene‐for‐gene relationship was established between the effector protein Eop1 and the fire blight resistant ornamental apple cultivar Evereste and the wild species Malus floribunda 821. The results presented here provide new insights into the host–pathogen interactions between Malus sp. and E. amylovora.     

基于MaxEnt模型预测梨火疫病菌的潜在地理分布

为探究梨火疫病菌解淀粉欧文氏菌Erwinia amylovora在全球的潜在地理分布,基于其全球分布数据和筛选得到的环境变量,利用MaxEnt模型对其在当前气候和未来气候条件下的潜在地理分布进行预测,并利用刀切法和皮尔逊相关性分析法筛选对梨火疫病菌分布有重要影响的环境变量。结果显示,对梨火疫病菌分布有重要影响的环境变量包括2月平均最高温度、1月平均降水量、7月平均最低温度、温度变化方差、昼夜温差月均值和7月平均降水量,表明春季和夏季的温度和降水对梨火疫病菌的分布有较大影响。在当前气候条件下,梨火疫病菌在全球的适生区分布较广,适生区总面积达到5.58×10⁷ km²,且高适生区主要分布在北美洲沿海地区、地中海沿岸和亚洲中部及东部的部分地区;梨火疫病菌在我国的适生区总面积为7.36×10⁶ km²,占全国陆地总面积的76.70%;在未来气候SSP126和SSP585情景下,梨火疫病菌在全球的适生区总面积分别为5.52×10⁷ km²和5.24×10⁷ km²。表明梨火疫病菌对我国大部分地区有潜在威胁,应加强监测与防控。     

A brief review of the status of fireblight in UK1

Fireblight (due to Erwinia amylovora) has continued to spread northwards and westwards in UK. In SW England, the disease has been particularly severe in perry pear orchards, causing the loss of many thousands of mature trees, and in cider apple orchards. Cider apple trees are not rapidly killed but crop losses have been severe in some years. The disease has become established in the NW midland counties of England and also in Wales. Outbreaks have occurred as far north as Lancashire just above the administrative line which separates the affected area from the disease-free (clear) area. Spread has occurred through infections in Crataegus hedges, especially along motorways, and also through movement of infected ornamental species. New legislation is envisaged which will redefine the clear area and increase the protection given.     

Recent Progress in Breeding for Fireblight Resistance in Apples and Pears in North America1

Resistance in apple is evaluated by needle–inoculation of succulent shoot tips with 10⁶–10⁷ cells of a virulent isolate of Erwinia amylovora (Burr.) Winslow et al. (the incitant of fireblight) and measurement of the resulting cortical lesion when extension is complete. Data are now available on practically all commercial cultivars, some of which have a useful level of resistance. Some newer cultivars, particularly those with resistance to scab (Venluria inaequalis [Cooke] Wint.) derived from Malus floribunda, have good resistance to E. amylovora. A very high level of resistance is present in Asiatic Malus species, including M. x robusta, M. x sublo–bata, M. x atrosanguinea, and M. prunifolia, and in the North American species M. fusca. This type of resistance appears to be inherited polygenically, and because of its detectability in young seedlings can be used conveniently in breeding. Objectives of pear breeding programs are aimed at developing superior fruit quality combined with resistance to fireblight, psylla, and Fabraea leaf spot. Many high quality cultivars of Pyrus communis are extremely susceptible to E. amylovora and sensitivity appears to be controlled by a dominant gene Se. A high level of resistance is present in P. ussuriensis but varies considerably between clonal selections of other Pyrus species. Pear seedlings from controlled pollinations are artificially inoculated in the glasshouse with a similar bacterial suspension as used for apples, and only the most resistant ones are selected and planted in the field for future evaluation. In Beltsville, heritability studies of crosses between non–sensitive parents have indicated that selection for resistance within progenies results in a high degree of genetic gain. Interspecific hybridization has an advantage over P. communis crosses mainly when insect or fungus resistance is required.     

Extended longevity of Erwinia amylovora vectored by honeybees under in vitro conditions and its capacity for dissemination

Fire blight outbreaks in Korea were first reported in 2015. Regular outbreaks have occurred since, indicating a continuous cycle of the fire blight pathogen in Korea. We determined the role of Apis mellifera (honeybee) as a vector of Erwinia amylovora by verifying the following: (a) E. amylovora longevity in/on honeybees; (b) the most common body parts that carry the bacteria; (c) the rate of bacterial spread to healthy host organs; and (d) the relationship between dispersal of viable but nonculturable (VBNC) and virulent bacterial cells. E. amylovora survived for 15 days on the exterior of honeybee bodies and was most abundant on the abdomen in comparison to other areas such as the labellum, wings, and hind legs. In the digestive system of honeybees, E. amylovora survived for 7 days, and bacteria were found in faeces for 3 days after exposure. The bacteria are likely to be VBNC on honeybees. Honeybees that were contaminated with bacteria transferred E. amylovora to healthy immature apple fruit, shoots, and flowers for 10 days after exposure. E. amylovora was also transferred from inoculated plant parts to uncontaminated honeybees. In addition, bacteria moved from inoculated plant tissues to unexposed honeybees and then from these honeybees to healthy plant tissues. Therefore, E. amylovora can survive in/on honeybees for extended amounts of time, which contradicts previous reports. The bacteria moved to host tissues via honeybees, suggesting that honeybees are the vectors of E. amylovora and play a role in the development of new outbreaks of fire blight disease in the central regions of Korea.