Vol 10, No 2 (2004)

Published May 10, 2004



Floral biological investigations of apple cultivars in relation to fire blight

Floral activity was studied in two apple cultivars: an Erwinia-tolerant (Treedorn') and a sensitive one (`Sampion'). Since more types of protogyny occur in apples, the period of stigma activity is different. Papillae of the exposed stigma in flowers of 'Freedom' function longer (usually more than a week) than in the delayed homogamous `Sampion'.... Despite of this, cv. 'Freedom' is tolerant to Erwinia amylovora (Burr.) Winslow et al., suggesting no relationship between the floral biological type (including the exposure and longevity of stigma) and the infection by E. amylovora. According to SEM micrographs, nectary stomata in `Freedom' are already open in the flower bud, where nectar secretion starts and continues until the senescence of the stigma. However, the long period of nectar secretion does not create optimal conditions for bacterial growth, since nectar production is scant in the flowers of 'Freedom'. The surface of the nectary, its nectar-retaining capacity, and the amount and concentration of nectar may influence the susceptibility of apple cultivars. It is manifested well by the smooth nectary surface with nectary stomata rising slightly above the epidermis in flowers of cv. 'Freedom', contrasting the wrinkled, striate nectary surface with slightly sunken stomata in the flowers of 'Sampion'.

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Study of Erwinia amylovora colonization and migration on blossoms of susceptible and tolerant apple cultivars

The stigmata of detached flowers of susceptible and tolerant apple cultivars were inoculated with about 104 gfp labeled Erwinia amylovora . There were no apparent differences in the colonization, multiplication and survival of the bacteria on the stigmatic surface of the culivars. Bacteria were washed down to the hy...panthium surface 24 hours after inoculation. The visual symptoms of the infection were the discoloration and shrinkage of the floral parts. The gradual browning associated with the infection appeared first on the surface of the hypanthium followed by the discoloration of the style. The color of the filaments turned into brown only 120 hours after the inoculation. Bacterial cells were not detected in the tissues of the styles and filaments. The traits of the hypanthium surface are of prominent importance in the progression of the infection. The wrinkled surface, the convex shape of the outer epidermal cell walls with thin cuticle and the sunken stomata helped to preserve a water film for a longer period providing medium for the motility of the bacteria in the susceptible cultivar. Bacteria were restricted to small water droplets on the flat and waxy surface of the hypanthium of the tolerant cultivar and only a few were able to enter the tissues.

Large bacterium aggregations were detected in the intercellular spaces of the parenchyma of the susceptible cultivar 48 hours after the inoculation. In the next period the Erwinia amylovora cells gradually invaded the intercellulars of the hypanthium wall, the wall of the ovary and the pedicel. Low level of bacterium aggregation was found in the intercellulars of the tolerant cultivars. It is suggested that the progression of the infection was inhibited also by physiological factors.

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Some structural characteristics of the flowers of apple cultivars with different susceptibilty to fire blight

Several morphological characters of the hypanthium (size and form of the surface, the shape of the hypanthium) and anatomical traits (number and pattern of stomata) of apple cultivars (Malus domestica L.) with different susceptibility to fire blight were studied. The size of hypantium surface was calculated by modelling the hypanthium ...with a truncated cone. Three types of hypanthium surface form have been revealed: straight, convex and a complex "shouldered" one. The angle between the style and the wall of the hypanthium was narrow or wide. The stomata on the hypanthium surface can be arranged in a zone in the middle third of the hypanthium or dispersed more or less evenly. The number of stomata/flower substantially differed among the cultivars examined. The highest stomata number was detected in the flowers of the tolerant cultivar (Freedom)

No single characteristics of the hypanthium could convincingly be correlated with susceptibility to fire blight. We suggest, however, that combination of morphological properties that sustain moist environment in the hypanthium contribute to susceptibility.

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Floral biological studies on pear cultivars in relation to fire blight susceptibility

Floral biological characteristics that may influence cultivar susceptibility to fire blight were studied in 10 pear cultivars in two Hungarian orchards from 1999 to 2003. The receptaculo-ovarial, automorphic nectary is usually bigger in tolerant cultivars than in susceptible ones. Nectary stomata are meso- or xeromorphic. Susceptible cultivars ...tend to have more xeromorphic stomata, where guard cells are located 1-3 cell rows below the epidermis. The size of nectar chambers is usually smaller in susceptible cultivars. Floral nectar, consisting mainly of glucose and fructose, is more abundant and less concentrated if the cultivar is susceptible to fire blight. The amount of chlorogenic acid was higher in the flowers of tolerant cultivars than in susceptible ones.

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Carbohydrate utilization of Erwinia amylovora in vitro

Nectar is a multi-component aqueous solution that promotes bacterial multiplication. The concentration of nectar in plant flowers is not stable since it is under the influence of environmental conditions, especially free moisture and relative humidity. Experiments were conducted with "artificial nectar" and directed along two lines: (1) determi...nation of the optimal concentrations of carbohydrates for the growth of E. amylovora development (2) consumption of different carbohydrates besides basic sugars.

Solutions of "artificial nectar" were prepared in different compositions by changing the dominance of basic sugars (fructose — glucose —sucrose) in proportions of 2:1:1, 1:2:1, 1:1:2 and between concentrations of 10-0.6% (diluted with Basal minimum broth) in order to determine optimal conditions for the development of E. amylovora.

At a basic sugar concentration of 10% bacterial multiplication started and continued until I log degree (from 106 to 107 cfu/ml). At concentrations of 5% and 2,5 % cells developed with nearly the same kinetics (from 106 to 8x107 cfu/ml and from 106 to 9x107 cfu/ml, respectively). Multiplication was more pronounced and nearly the same at concentrations of 1.2 % and 0.6 % (from106 to 2x108 cfu/ml). At a basic sugar concentration 30% total sugars bacterial multiplication did not occur, while at 20 % it was negligible, not measurable photometrically.

At minimal concentrations of F, G, S (between 1-0.1 %) bacterial cells were still able to multiply, producing organic acids from sugars.

Our study showed that E. amylovora requires only a small amount of sugars (0.1%) for multiplication (acid production) while high concentrations inhibit multiplication. There was a negative correlation between sugar content and cell density. The optimal range of sugar concentration was at about 1%.

Effect of "less frequent carbohydrates" to E. amylovora multiplication was also determined using the API 50 CH strip. We could provide information on utilization of 39 carbohydrates by the bacterium at different categories as follows: Not utilized-, Slowly and weakly utilized-, Slowly and completely utilized-, Quickly and completely utilized carbohydrates. We suppose that carbohydrates that belong to the latter two groups could play an important role as nectar components in promoting E. amylovora multiplication in the blossoms of pome fruit trees.

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Flavonoids, chalcones and phenyl-propanoids in apple and pear flowers

The presence of phloretin-glycosides in the hypanthium and pistil of apple and pear flowers can be verified. Thin layer chromatography is a reliable method for detecting phloretin, gained by acidic hydrolysis. The dominance of phloretin was equally characteristic for flowers in apple (`Sampion', 'Freedom') and pear (Tem-re Bosc', 'Co...nference') cultivars treated with various bioregulators (Biomit, Bion 50WG, Regalis), no significant difference could be found visually as compared to control samples. Chlorogenic acid and caffeic acid was detected in all apple and pear samples, rutin was present only in pear, and hyperoside was found only in a few apple samples.

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Prediction infection risk on the basis of weather-related factors and Erwinia amylovora colonization in apple and pear flowers

Current infection risk prediction models utilize environmental parameters and field records, but do not take into account the estimated inoculum potential within the orchard. The object of this study was to survey the accuracy of three simple prediction methods under Hungarian climatic conditions, which could easily be used by the farmers. We a...lso tested whether the accuracy of infection risk predictions can be improved by taking into consideration the incidence and/or rate of flower colonization by Erwinia amylovora.

After preliminary investigations in 1999-2001, data concerning the weather-related infection risk were recorded in 5 apple and 1 pear orchards in 2002, and in 12 apple and I pear orchards in 2003. The weather data were processed by the easy-to-use risk assessment models of the mean temperature prediction line (MTL), Smith's Cougarblight 98C and Billing's integrated system (BIS), and by the MaryblytTM 4.3 computer-assisted model for reference. The population size of E. amylovora in the flower samples was estimated within an order of magnitude by PCR.

For all years and orchards tested, Maryblyt indicated 35 days on which there was an acute infection risk. The same days were indicated by all 3 methods in 23 cases (66%), 8 days were indicated by 2 methods (23%) and 4 days were indicated by 1 method only. A similarly good correlation was found for prediction of the date of the first massive infection risk: in 2003, for instance, there was a perfectly consistent prediction by all 4 models in 9 of the 13 participating orchards. A coincidental forecast was provided by 3 of the 4 models in the other 4 orchards.

The results indicate that any of the risk assessment models could provide an increased accuracy of the actual infection risk prediction if combined with an estimation of the incidence of Erwinia amylovora colonization in the open flowers. We found no convincing differences in the size of the epiphytic population in flowers of cultivars possessing high or low susceptibility to Erwinia amylovora.

We conclude that the easy-to-use methods tested could be used by the fanners to recognize weather-related risks, especially when coupled with an estimation of the proportion of the pathogen-infested flowers. This local prediction would provide rapid information (faster than the regional forecast systems) specifically for a given orchard.

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Field trials with non-bactericide products to control fire blight in apple orchards

Recently, novel strategies and chemical agents for prophylactic protection against the bacterial (Erwinia amylovora) disease fire blight are being sought. Resistance-inducing compounds, such as prohexadione-Ca represent promising alternatives. Prohexadione-Ca is the active ingredient of the bioregulator Regalis, currently being introdu...ced in several European countries and overseas. Another product used in this study was Biomit Plussz, a leaf fertiliser providing harmonic supply of nutrient elements, the complete supply is assumed to improve the tolerance against diseases.

Treatments' effects of both of these products were compared to the effectiveness of treatments with antibiotics repeated twice, three or four times a season.

In the years of 2001 and 2002, the effectiveness of both Regalis and Biomit Plussz in reduction of incidence of shoot blight was similar, or proved to be superior to the check treatments consisting of repeated sprayings of antibiotics. Last year (2003) treatments of streptomycin resulted — although within the same magnitude — in a somewhat better control of shoot blight than sprayings with the other compounds.

As regards severity of blossom blight, inconsistent results were recorded concerning both Regalis and Biomit Plussz.

In general, prohexadione-Ca is less efficient for controlling flower infection by E. amylovora as compared to shoot infections, since successful prophylactic treatments are difficult to carry out early in the season. The highest effectiveness in fire blight management can, therefore, be achieved by using prohexadione-Ca (as preventive protection) in combination with streptomycin or other suitable antibiotics (as curative protection).

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The effect of Regalis® (prohexadione calcium) on the reduction of fire blight (Erwinia amylovora) severity in apple trees

Due to the lack of effective and non-phytotoxic materials for control of the blossom and shoot blight phase of fire blight in pome fruit trees, two novel control strategies have emerged: shoot growth retardation by bioregulators and applying resistance inducer compounds. Prohexadione calcium (ProCa) is the active ingredient of the bioregulator ...Regalis® registered in several European countries. The reduction of shoot growth elongation is the most obvious effect of ProCa. Furthermore, it causes significant changes in the spectrum of flavonoids and their phenolic precursors, leading to the considerable reduction of susceptibility to fire blight.

In Poland, potted one-year-old apple trees of cvs. Gala Must grafted on M.26 and Sampion on M.9 (in 2001) as well as Gala Must on P.60 (in 2002) were treated with Regalis® at a range of concentration of 250, 150 or 150 + 100 ppm, respectively. The inoculation of shoots was made with the strain No.691 of E. amylovora (107 cfu/ml), on the 7th and 21st'day after treatments with Regalis. In Hungary, during the years of 2002 and 2003 one-year-old container grown apple trees of the cvs. Idared/M.9 and Freedom/M.9 were treated with the prohexadione-Ca, the active ingredient of Regalis® 100, 150 or 200 ppm, two weeks before inoculation with the Ea 1 strain of E. amylovora (107 cfu/m1).

In Poland, the suppression of fire blight in shoots reached up to 80%, depending on concentration and application time of Regalis®. In Hungary, the effect of prohexadione-Ca treatments, determined by the length of necrotic lesion developed, proved to be better than that of streptomycin used for comparison.

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Fire blight in Hungary between 1996 and 2003

Shoot blight of pome fruits caused by Erwinia amylovora, i.e. fire blight, is present in numerous countries of Europe. The disease must have entered into Hungary in the middle of the 1990's and it was first noted and, respectively, identified in 1996 (Bacs-Kiskun county). The losses caused by the pathogen appeared — in orchards and s...cattered sites of production — in four counties, namely Bacs-Kiskun, Baranya, Bekes and Csongrad at the beginning. From June 1996, a process of eliminating infected parts started in the course of a large action performed under the control of the Department of Plant Protection and Agro-Environmental Economy of the Ministry of Agriculture, under the direction of the plant protection inspectors of the then existing Stations of Plant Health and Soil Conservation. The 'operation' against the disease commenced by cutting back out the infected parts of the canopy and, grubbing them out, respectively. As for the spread of the pathogen (1996-1998) it could be observed that the disease entered into Hungary from the south, south-east and then it also spread into the middle part of the country. As a result of adequate official action and efforts as well as of adequate chemical and antibiotic treatments, moreover because of the introduction of more modern technologies of plant cultivation and those of plant protection it can be reported on that the pathogen hardly appears or does not occur at all on the northern, north-western part of the country. The infection also appears mainly on the parts east of the Danube. Cultivars less susceptible or non-susceptible to the disease are planted in recently established orchards what is also a considerable factor in respect of preventing spread of the pathogen.

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