Bakteriozno sušenje trešnje (Prunus avium L.) / Bacterial die back of sweet cherry (Prunus avium L.)

Bacterial die back (canker) of sweet cherry (Prunus avium L.) in young orchards and sweet cherry plantations in the past few years has been a significant problem in the production of this fruit species. Symptoms of the disease were manifested in the form of drying branches, twigs or whole trees, which were mainly observed in places of pruning or around the buds, bark changes a color, cracks and cankers has formed. In the period 2012 - 2015 monitoring of the health status of sweet cherries was carried out covering several plantations and smaller orchards of sweet cherries in several localities in Vojvodina and central Serbia (Ritopek). Young fruit trees are obviously the most susceptible, based on monitoring of the health status in many localities and plantations of different ages, the occurrence of bacterial canker in a stronger or weaker intensity was found only in young plantations (up to 3 years old - Selenča, Gornji Tavankut, Donji Tavankut, Ljutovo, Mikićevo and Kanjiža). From collected diseased samples of sweet cherries, as well as healthy buds and leaves of sweet cherry (epiphytic population) isolations on standard nutrient medium, were obtained numerous isolates of P. syringae pvs. and for further investigations was selected 155 isolates. Identification of isolates was performed on the basis of phenotypic and genotypic methods. Based on LOPAT tests isolates belonging to Ia group fluorescent Pseudomonas syringae. According to GATTa tests two groups of P. syringae isolates were identified, I group (G+A +T -Ta- ) and II group (G-A -T +Ta+ ). Additional tests confirmed the GATT tests, on the basis which it was concluded that the drying of young sweet cherry trees caused P. s. pv. syringae (I group) and P. s. pv. morsprunorum race 1 (II group). Among the tested isolates was not exceptions in phenotypic characteristics within the same group, except for the ability to produced syringomycine for some isolates of I groups (pv. syringae). In pathogenicity tests on various plants and host plant were observed differences, but also and some certain similarity between isolates of I and II groups. Clear differences between the groups of isolates were determined in the inoculations of green fruit of sweet cherry, sour cherry, cherry plum and pears, tomatoes, peppers and green bean pods. In the case of inoculation of separate lilac leaves isolates of I group (pv. syringae) and most isolates of II group (pv. morsprunorum race 1) reactions were positive, what indicating the heterogeneity of the population of P. s. pv. morsprunorum race 1. In the inoculation of fruit rootstock seedlings (wild cherry, Magriva, wild plum, wild pear) all isolates pv. syringae caused the characteristic pathological changes on the all fruit species, isolates of pv. morsprunorum race 1 also except on the seedlings of wild plum. These results suggest that the spreading of bacteria is possibly through the rootstock that can also be infected. Inoculations of two – years old branches of sweet cherry during dormancy, was concluded that all isolates pv. syringae and morprunorum race 1 were equally pathogenic in all sweet cherry cultivars (Burlat, Summit, Hedelfigen and Germersdorf). The longest length of necrosis usually was observed on the cultivars Burlat and Summit in combination with isolates of I groups (pv. syringae), in some cases with isolates of II group (pv. morsprunorum race 1), and the lowest mainly in cultivars Germersdorf and Hedelfigen with isolates of II group (pv. morsprunorum race 1). Identification of isolates KBNS71 - 84 (GornjiTavankut) and KBNS85 - 94 (Selenča) based on MLST using genes gyrB, rpoD, gapA and gltA genes clearly showed the presence of two patovars P. s. pv. syringae and P. s. pv. morsprunorum race 1. Comparison with strains H - 1, V - 85 V - 88 (sour cherry) and V - 109 (sweet cherry) showed significant differences and the existence of genetic diversity in the population of these pathogens. Simultaneous detection of syrB and syrD gene was found in 70 isolates of I group (pv. syringae) and only syrB gene in 9 isolates of the same group (pv. syringae). The gene for coronatine synthesis was detected in all 76 isolates of II group (pv. morsprunorum race 1). Rep - PCR method detected significant differences (58%) between isolates of I and II groups (pv. syringae and pv. morsprunorum race 1). The tested isolates from sweet cherry within pv. syringae did not show differences between them, but they were different from the strains from other locations and previously isolated from the same host (V - 109 and T6), as well as strains from other hosts - cherry (V - 85) and pumpkin (Tk21) to 37 %. The differences between isolates pv. morsprunorum race 1 were less than 5% and 24% compared to the same pathovar strain CFBP2119. Rep - PCR analysis indicated a low level of heterogeneity of isolates within the same pathovar. RAPD method using a large number of primers were more successful to compare isolates than rep - PCR. Among 11 tested primers, 4 (SPH1, DJP17, DJ15, DJ16) were selected for further work on the basis of the difference between isolates within same pathovar. Cumulative RAPD analysis showed up to 24% differences among tested isolates of pv. syringae and 41% compared to the strain KFB0103, while among isolates pv. morsprunorum race 1 differences were 15% and 36% compared to the strain CFBP2119. The results of RAPD analysis indicate that a certain heterogeneity 7 exists in the population of both tested groups of isolates, but genetic diversity is more pronounced among isolates of pv. syringae. Studying the epidemiology of this pathogen in field conditions, by inoculating one – year old branches / or shoots sweet cherry cultivars Burlat, Germersdorf, Hedelfigen and Droganova žuta, it was concluded that the sweet cherry in our agroecological conditions becoming sensitive (October) to P. s. pv. morsprunorum race 1 before in relation to the pv. syringae. The first positive results of inoculations with strains pv. syringae were determined in November. Regarding the length of necrosis most successful were inoculation in the November (necrosis longest; 2.17 to 3.35 cm), inoculations also were successful in the January and the March, but the length of necrosis was smaller, respectively. Generally longest necrosis were observed in the cultivar Burlat, and the shortest in cultivar Germersdorf. All inoculations carried out in the period of vegetation were negative. Inoculations of two – three – years old branches of the cultivar Summit, first successful inoculations (for both pathovar) were observed only in November (October was negative), when a greater aggressiveness of pathovar syringae were determined. In inoculations in January length of necrosis was smaller, and in March was negative. All inoculations carried out in the period from buds swelling to leaf falling were also negative. Investigation susceptibility of sweet cherry and some sour cherry cultivars was concluded that against to both pathovars (syringae and morsprunorum race 1) the most susceptible were cultivars of sweet cherry Katalin, Linda, Summit, New Star and Burlat, medium susceptible were cultivar of sour cherry Erdi Botermo and sweet cherry cultivars Droganova žuta, Carmen, Germersdorf and Rana od Noara and low susceptible cultivars of sour cherry Španska and Ujfeheti firtoš and cultivar of sweet cherry Rita.