The invasive bacterial disease fire blight, caused by Erwinia amylovora has the potential to destroy fruit tree orchards all over Europe. Effective plant protection methods are lacking in many countries, highlighting the increasing importance placed on identification of germplasm with heritable disease resistance. Recent l y. a promising QTL (quantitative trait locus) was identified on linkage group 7 in the apple cultivar 'Fiesta· which is derived from ·cox's Orange Pippin' . I n the present study, 144 Swedish and foreign apple cultivars were analysed with the SCAR markers AE I 0-375 and GE-8019. which flank-. this QTL. Twenty-nine of the analysed cultivars had both markers. 78 had either AE I 0-375 or GE-8019, and 37 cultivars did not carry an) of the two markers. Seventeen cultivars. 7 with both markers and I 0 not having either of the two markers, were then inocoluted with the bacterium in a 4uaran1i ne greenhouse test. Cultivars carrying both DNA markers were significantly less susceptible than cultivars lacking the markers, P<0.001. Cultivars that were most resistant had both markers and had 'Cox· in their pedigree. Unrelated cultivars with the markers may still lack the QTL.
The aim of the Hungarian apple breeding program started in the Department of Fruit Science was to find resistant apple cultivars against major diseases (scab, powdery mildew, fire blight). The outbreak of fire blight (Erwinia amylovora) in 1996 motivated us to search new resistant sources principally from old traditional apple cultivars. First of all, cultivars have been gathered since 1997 from Carpathia (Visk) and evaluated between 2001 and 2003. In this recent study evaluation of resistance of old Hungarian genotypes to fire blight collected from the English National Fruit Collection (Brogdale, Faversham) is presented.
13 old Hungarian apple cultivars in 2002, and 38 genotypes in 2005 have been evaluated. We used `Idared' and 'Jonathan M41' as susceptible controls and 'Liberty' and 'Remo' as resistant ones. Shoots of two-year-old potted plants were inoculated with a mixture of virulent E. amylovora isolates (Ea2, Ea60, Ea67) at a concentration of 5 x 108 cells/ml. Resistance of apple cultivars was evaluated weekly, four times after inoculation by disease severity of symptoms. Numbers of bacterial colonies in 1 cm length shoot were determined in the fourth week after infection.
8 cultivars in 2002 and 9 cultivars in 2005 displayed notable resistance to fire blight based on one-year data. Based on the coincident data of both years, out of the cultivars collected also from Carpathia ‘Pónyik', and `Sikulai' were found to be resistant and gene sources additional old Hungarian valuable apple genotypes could be selected: `Szabadkai szercsika' and `Tordai piros !davit'. The cultivar `Szemes alma' originated from Visk has proved to be recurrently resistant.
The number of bacterial cells in shoots of the investigated cultivars correlated with the severity of symptoms. With this method, which was introduced by us earlier, we can screen cultivars displaying weak visible symptoms, which cannot be proposed as a source of resistance because of their latent infection.
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 introduced 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).
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.
Two apple cultivars that display enhanced resistance to fire blight (causal agent: Erwinia amylovora) were selected. The aim of the present study was to characterize the peroxidase (POD) enzyme activity of `Szemes alma' (a historical cultivar) and MR-03, (a Hungarian multiresistant hybrid of 'Prima') and compare them to susceptible 'Jonathan M 40' and resistant 'Remo' controls. Peroxides enzyme activity during E. amylovora infections was investigated in artificially infected apple shoots. Increases in enzyme activities were observed in a `Jonathan M40' and in 'Remo', MR-03, `Szemes alma' cultivars. There was a consistent relationship between total enzyme activity and fire blight disease severity. High activity of the peroxidase was positively correlated with the degree of resistance to fire blight. A general hypothesis that POD activity is related to fire blight susceptibility/resistance is supported by our results.
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.
Following the first outbreak of fire blight caused by the bacterium Erwinia amylovora (Burrill) Winslow et al. in Hungary, we have started research with the aim to screen domestic gene sources, mostly historical Hungarian apple cultivars, for disease resistance as part of an apple breeding program for multiple resistance. The present study was conducted with the aim to choose the most tolerant historical apple cultivars among 25 selected cultivars by screening their behavior towards fire blight under controlled conditions. Six cultivars were shown to be the most disease tolerant: `Batul', 'London pepin', Nemes Sóvári Alma', `Sikulai', `Szemes alma', Wilmos renet'. We evaluated these cultivars by investigating both morphological- characteristics under original environmental circumstances and fruit quality. The cultivars had a remarkable degree of fire blight resistance compared to the control cultivars. These were not competitive with the commercially grown 'Jonathan M40' during cultivar tests but on the basis of certain characteristics they could serve as genetic sources for breeding new varieties.
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 scattered 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.
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.
The ecological farming and an increasing demand for healthy fruits free from chemical residuals necessitated the reevaluation of the existing cultivars and breeding suitable cultivars for the new requirements. We have gathered the old genotypes known in the Carpathian Basin, and we have selected the most suitable ones according to fruit quality and resistance to diseases in the last fifteen years. The main aim was the selection of resistant genotypes against fire blight and powdery mildew as gene sources for our breeding program. On the other hand, we consider the best quality traditional cultivars as extensive landscape elements (wayside tree rows or extensive squash producing plantations).
During the pathological evaluation 'Szemes alma', 'Pónyik', 'Sikulai', 'Tordai piros 'cavil' and 'Szabadkai szercsika' cultivars proved to be good resistance sources (due to their outstanding resistance to fire blight). The latter four cultivars are not susceptible even to fungal diseases, and their quality is also acceptable so those are commendable for ecological farming or for renewing the sparse orchards. According to fruit quality and low susceptibility to diseases 'Batul' and 'Vilmos renet' can also be offered for extensive organic production.
We have made original or renewed pomological descriptions of the selected cultivars, and we also publish their characters according to the terms of UPOV TG/14/8 based on to our evaluations.
Fire blight, a disease caused by the bacterium Erwinia amylovora (Burrill) Winslow et al., has been causing serious damage in Hungarian pear plantations since 1996. A prospective control measure could be the use of resistant cultivars. For that purpose ten pear cultivars have been tested under laboratory and greenhouse conditions for resistance to Erwinia amylovora strains collected in Hungary. Six of these cultivars are Hungarian ones of unknown origin, while four are traditional old varieties. Resistant cultivars should serve as germplasm for future breeding. Inoculations were made with a mixture of different pear isolates of the bacteria collected from various growing regions of Hungary (Ea 21, 23), at a density of 5x108 cells/ml. Susceptibility/resistance has been assessed on the basis of intensity of blight symptoms observed on shoots, flower parts and fruits. Cultivars were assigned to three susceptibility groups (low, moderate and high). Complete resistance was not found among the cultivars tested. The highest level of resistance was found in cultivar 'Kieffer', while the other cultivars displayed either moderate or high susceptibility to infection.
An apple breeding program has been carried out at the Department of Fruit Science for more than a decade. Several apple selections have been released from the progenies of crosses in 1992 and 1993. Six candidates were submitted for national recognition out of the hybrids examined for more than a decade. The six selections are resistant against all the three most important apple diseases (apple scab, powdery mildew and fire blight). Scab resistance is controlled by the Vf gene originating from the species Malus floribunda 821 and transmitted by cultivar Prima. Heterozygote Vfvf genotype of the six cultivar candidates was proved by molecular genetic examinations of Dept. Genetics and Plant Breeding. Characteristics of these selections from 'Prima' progenies are shown on the base of our own observations.
Research project has been initiated in 1999 with the aim of evaluating the degree of susceptibility/resistance of pear cultivars grown in Hungary to fire blight disease caused by Erwinia amylovora. The recently selected promising cultivars were also examined. Inoculation experiments were conducted in controlled greenhouse conditions because of quarantine regulations in Hungary. Following the disease process, development of symptoms of plant organs (shoots, flower parts, fruits) was observed. Suspension of two E. amylovora strains (Ea 21, Ea 23) isolated from pear was used in a mixture (5x108 cells x m1-1) for the inoculation. Twenty-six pear cultivars were examined and grouped into four categories: low susceptibility, moderately susceptible, susceptible and very susceptible. Most of the cultivars were susceptible or very susceptible while some promising 'Eldorado', 'Harrow Delight' and `Hosui' showed low susceptibility.
In the frame of the apple breeding program at the Department of Fruit Science old cultivars found in the Carpathian basin were involved in examination as well. Beside testing susceptibility to fire blight (Erwinia amylovora of old genotypes (historical, local and regional cultivars) originating from scattered orchards of Visk (foot of the Carpathian Mountains), fruit quality was also evaluated and other open-field observations were made.
According to the results of inner content examinations, 'Szemes alma' and 'Sikulai alma', chosen as gene sources for breeding to resistance against fire blight, had inner content values worthy to mention in some aspects, and competing with those of the control cultivar 'Jonathan'. The quality of 'Pónyik alma', also recommended as a gene source is confirmed by the data of earlier literature. Further three cultivars ('Batul', 'Vilmos renet', 'Zöld sóvári') also proved to be good based on resistance against Erwinia amylovora, the first two cultivars are suitable from the point of view of fruit quality, too.
In our field observations, susceptibility to apple scab and powdery mildew was evaluated. Among cultivars selected from the foot of the Carpathian Mountains on the base of resistance against Erwinia amylovora, the production value of 'Szemes alma' is worsened by its high susceptibility to apple scab. Further selected cultivars ('Sikulai', 'Pónyik alma', 'Batul', 'Vilmos renet') are not too susceptible to fungal diseases and their fruit quality is also suitable, therefore they can be recommended for direct planting in ecological farming or in restoring scattered orchards.
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) determination 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.
The aim of the first Hungarian apple breeding program for multiple resistance started in the beginning of the nineties is to widen Hungarian apple assortment by good quality, resistant apple cultivars with excellent productivity and ecological capability to the most important fruit growing areas in Hungary. In the first years of seedling production we made early selection for susceptibility to apple scab in greenhouse. Alter this, field observation of susceptibility to powdery mildew, scab and canker and a yearly negative selection was carried on. From 1997, fruit quality was evaluated as well, and from 2001 the resistance of shoots to Erwinia ainylovora (Burrill/Winslow et al.) was examined using inoculations in greenhouse conditions. From the progenies of crosses in 1992 and 1993, six candidates were announced to national recognition out of hybrids examined for more than a decade. Descriptions of these selections from 'Prima' progenies and the most important data of their resistance, growing habit, morphological characteristics and fruit quality are shown in this article.
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'.
A useful method was improved to test and to evaluate the susceptibility of plants to fire blight and the virulence of E. amylovora strains. Six Hungarian strains from different host plants were tested on in vitro cultured apple rootstocks. Disease rating was used for the characterization of the process of disease development. The different strains had different capacity to cause disease, mainly in the first period of incubation. There were significant differences between the virulence of the strains.