Comparison of Xanthomonas arboricola pv . juglandis isolates from walnut trees grown in Romania and Hungary

The Persian (English) walnut (Juglans regia L.) is very sensitive to a number of abiotic and biotic factors. Two of the most important abiotic factors are the autumn frost, sometimes leading to tree death, and the late spring frost, having an effect on stem form. The main biotic damage factor is Xanthomonas arboricola pv. juglandis (Pierce 1901) Vauterin et al., 1995 (Xaj), damages leaves and young shoots in humid and mild climate and after several rainy summers some trees might even die (Fernandez-Lopez and Pereira, 1997). The bacterial blight disease is one of the most important diseases of Persian walnut (Loreti, 2001) and is widespread in walnut growing areas. The disease severity depends on the weather, which may be different in each year. It causes severe damage to leaves, twigs, buds, petioles, rachides, male and female catkins, nutlets and kernels, and it is considered a major cause of reduction in fruit yield and tree vigour (Belisario et al., 1999) (Figure 1.). The inoculum is spread by the action of wind and rain, and infection requires the presence of moisture (Miller and Bollen, 1946, Olson et al., 1997). The damage produced by this pathogen is favoured by wet springs, with high humidity. Rainy springs, dew, and continual high humidity conditions are favourable for the development of severe blight, resulting in significant crop loss (Belisario, 1997). Especially if this happens just before and after the flowering time, it may cause losses up to 80% of the crop (Charlot and Radix, 1993; Miller, 1934). The control of the disease is difficult, since large walnut trees are not easy to treat and because more and more copperresistant Xaj strains are developing (Solar et al., 2007; Giovanardi et al., 2010). To date, no Xaj resistance has been found in walnut. Differences have been detected only in the severity of symptoms shown by cultivars planted in the same environment (Frutos and López, 2012). One of the main objectives of breeding and production improvement activities is to produce resistant cultivars. The production and introduction into production of resistant cultivars is one of the possibilities for preventive measures against the walnuts xanthomonas infection. In several walnut producing areas of the world plant breeding is practiced, although the resistance of resistant cultivars improved in distant places is questionable against bacterias from walnut population of other production lands. Many bacterial species show considerable variability in biochemical characters. Hayward has distinguished isolates of Pseudomonas (Ralstonia) solanacearum on the basis of utilization of three hexose alcohols and three sugars and proposed subdivision of the species to biovars (biotypes) (Hayward, 1964). Scortichini has concluded that genetic diversity exists among Xaj strains from different geographical areas of the Comparison of Xanthomonas arboricola pv. juglandis isolates from walnut trees grown in Romania and Hungary


Introduction
The Persian (English) walnut (Juglans regia L.) is very sensitive to a number of abiotic and biotic factors.Two of the most important abiotic factors are the autumn frost, sometimes leading to tree death, and the late spring frost, having an effect on stem form.The main biotic damage factor is Xanthomonas arboricola pv.juglandis (Pierce 1901) Vauterin et al., 1995 (Xaj), damages leaves and young shoots in humid and mild climate and after several rainy summers some trees might even die (Fernandez-Lopez and Pereira, 1997).The bacterial blight disease is one of the most important diseases of Persian walnut (Loreti, 2001) and is widespread in walnut growing areas.The disease severity depends on the weather, which may be different in each year.It causes severe damage to leaves, twigs, buds, petioles, rachides, male and female catkins, nutlets and kernels, and it is considered a major cause of reduction in fruit yield and tree vigour (Belisario et al., 1999) The inoculum is spread by the action of wind and rain, and infection requires the presence of moisture (Miller andBollen, 1946, Olson et al., 1997).The damage produced by this pathogen is favoured by wet springs, with high humidity.Rainy springs, dew, and continual high humidity conditions are favourable for the development of severe blight, resulting in significant crop loss (Belisario, 1997).Especially if this happens just before and after the flowering time, it may cause losses up to 80% of the crop (Charlot and Radix, 1993;Miller, 1934).The control of the disease is difficult, since large walnut trees are not easy to treat and because more and more copperresistant Xaj strains are developing (Solar et al., 2007;Giovanardi et al., 2010).To date, no Xaj resistance has been found in walnut.Differences have been detected only in the severity of symptoms shown by cultivars planted in the same environment (Frutos and López, 2012).One of the main objectives of breeding and production improvement activities is to produce resistant cultivars.The production and introduction into production of resistant cultivars is one of the possibilities for preventive measures against the walnuts xanthomonas infection.In several walnut producing areas of the world plant breeding is practiced, although the resistance of resistant cultivars improved in distant places is questionable against bacterias from walnut population of other production lands.Many bacterial species show considerable variability in biochemical characters.Hayward has distinguished isolates of Pseudomonas (Ralstonia) solanacearum on the basis of utilization of three hexose alcohols and three sugars and proposed subdivision of the species to biovars (biotypes) (Hayward, 1964).Scortichini has concluded that genetic diversity exists among Xaj strains from different geographical areas of the

Comparison of Xanthomonas arboricola pv. juglandis isolates from walnut trees grown in Romania and Hungary
world,for instance from Europa and California (Scortichini et al., 2001).Consequently a breeding programme for a long-lasting resistance should take into account different or similar potential virulence of the pathogen according to their geographical areas.We considered the comparative analyses of the Xaj strains isolated in Hungary and Romania by means of biochemical methods necessary because supposedly walnut varieties may differ in the susceptibility to the bacterial pathogen in Hungary and Romania, and the potential of virulence of Xaj could also be different according to the biotype and race.

Parts of the plants showing symptoms of xanthomonas infection have been collected from different walnut producing areas of Hungary and Transylvania.
In order to carry out the identification of the pathogenic agent, the examination of its morphological, biochemical and physiological characteristics, pathogenicity tests were conducted in the bacteriology laboratory of the Department of Pomology of Corvinus University of Budapest.61 Xaj isolates were examined.These were chosen based on the colony growth characteristics (yellow pigment production, smooth, mucoid, convex colony type, oxidative breakdown of glucose, hypersensitive reaction on tobacco leaves) Once they were identified, they were placed in the DNA database of the NATIONAL COLLECTION OF AGRICULTURAL AND INDUSTRIAL Microorganisms, Corvinus University of Budapest Hungary.The ability to induce hypersensitive reaction was examined on tobacco leave (Nicotiana tabacum L.) (Klement, 1963) and bean pod (Phaseolus vulgaris L), the pathogenicity test was controlled on unripe walnut fruits, similarly we used an isolate from the husk of the walnut fruit from Hungary B02489 (HU) and one from Transylvania B02490 (RO) showing a similar degree of infection (virulence).
As a reference to the biochemical analyses of the two isolates (henceforth strains) we used the NCPPB 411 strain, brought from the National Collection of Plant Pathogenic Bacteria, United Kingdom (NCPPB), deposited also in the NCAIM, Budapest, Hungary (http://ncaim.uni-corvinus.hu).During the biochemical evaluations, the exploitation of the substrates were made on API rapid diagnostic kits (API 20NE és API 50CH -bioMérieux, France), following the instructions of the manufacturer.The evaluations were made after 24 and 48 hours (Figure 2).The API 50CH kit is based on observing the colour change: according to their ability to utilize or oxidize disaccharides and hexose alcohols, if that particular bacteria utilizes the carbohydrate, the original red coloured solution is changing to yellow (acid production), while in the case of positive test result, during gelatin breakdown the gelatin is liquefied (hydrolysis of gelatin) and a black colour reaction appears.For inoculation we prepared a bacteria suspension with a concentration of 6×10 8 cells/ml of 24 h-old cultures grown on Nutrient agar substrate.The incubation temperature was 28 o C. Evaluations, based on the colour changes were realised daily for six days, following the degree of carbohydrate utilization (Figure 3).The resistancy tests of the isolates were conducted and the starch hydrolysis capacity was checked.2).
Until the end of 72-96h, and 144 h time of reaction three (D-Glucose, D-Melibiose, D-Lyxose) and four (Amygdalin, D-Cellobiose) types of carbohydrates were utilized in equal proportion.The B02490 and B02489 strains utilized totally identical carbohydrates, with the difference that B02489 utilized them more quickly, while B02490 -more slowly.The control strain from the USA utilized five carbohydrates (N-Acetyl Glucosamine, D-Saccharose, D-melezitose, D-raffinose, Glycogen) less, than the B02490 (HU) and B02489 (RO) strains.The difference of the carbohydrate utilization between the USA and EU strains could be important in the resistance breeding, if in the case of cultivars is combined with different virulence (specific host change).
According to this difference we have to choose cultivars resistant to the races present on the particular growing area, or during the breeding it is advisable to take those phenotypes from the population, which are resistant against the local varieties, using them as parents in crossbreeding.
Considering the identical carbohydrate-utilization of the isolates from Hungary and Transylvania, we can draw the conclusion that during the resistance/susceptibility test it is sufficient to make the evaluation of cultivar with a mix of isolates.As a result we consider it is possible to accomplish the breeding of a cultivar which is intended to be xanthomonas-resistant.Due to the identical carbohydrate utilization of the two strains from Transylvania B02489 (RO) and Hungary B02490 (HU), it is assumed that these belong to the same biotype, and they have been subjected to the same stress factors.At the same time the strain originated from the USA (B01395), because of its different carbohydrate utilizing makes us reach the conclusion that we have to be precautious with the naturalization of cultivars from remote places, and resistance against local varieties has to be analysed.

Table 1 .
Results from API 20NE system

Table 2 .
Carbohydrates utilized by Xaj isolates using API 50CH system