Pectobacterium carotovorum subsp. carotovorum – prouzrokovač vlažne truleži biljaka kale u Srbiji i Crnoj Gori

Bacterial strains were isolated from above- and underground parts of diseased calla plants originating from different localities in Serbia and one locality in Montenegro. They were characterized by studying their pathogenic, cultural, biochemical and physiological characteristics. All investigated strains caused soft rot of calla leaf stalks, potato slices and aloe leaves, and induced hypersensitive reaction on tobacco. Bacteriological properties of the strains indicated that symptoms on calla plants were caused by Gram-negative, nonfluorescent, oxidase negative, catalase positive and facultatively anaerobic bacterium belonging to the genus Pectobacterium. The investigated strains grew at 37ºC and in 5% NaCl, utilised lactose and trechalose, and produced neither indol nor lecitinase. These results, as well as the characteristic growth on Logan’s differential medium indicated that soft rot of tuber and stem base of calla plants was caused by Pectobacterium carotovorum subsp. carotovorum. This is the first report of this pathogen affecting calla plants in Serbia.Proučene su patogene, odgajivačke i biohemijsko-fiziološke odlike sojeva izolovanih iz nadzemnih i podzemnih delova obolelih biljaka kale gajene u različitim lokalitetima u Srbiji i jednom lokalitetu u Crnoj Gori. Svi proučavani sojevi ispoljili su izraženu pektolitičku aktivnost prouzrokujući vlažnu trulež lisnih drški kale, kriški krompira, liski aloje ili sanseverije, a prouzrokovali su i hipersenzitivnu reakciju duvana. Proučavanjem bakterioloških karakteristika utvrđeno je da promene na biljkama kale prouzrokuju Gram-negativni, nefluorescentni, oksidaza-negativni, katalaza-pozitivni i fakultativno-anaerobni sojevi bakterije, koja prema navedenim karakteristikama pripada rodu Pectobacterium. Proučavani sojevi se razvijaju pri 37ºC i u prisustvu 5% NaCl, razlažu laktozu i trehalozu, ne stvaraju indol i lecitinazu. Navedeni rezultati, kao i karakterističan razvoj na Loganovoj diferencijalnoj podlozi ukazuju da je vlažnu trulež korena i prizemnog dela biljaka kale prouzrokovala bakterija Pectobacterium carotovorum subsp. carotovorum. Ovo je prvo saopštenje o pojavi P. c. ssp. carotovorum kao patogena kale u Srbiji

Impact of climatic conditions on outbreaks of bacterial spot on tomato and pepper caused by Xanthomonas vesicatoria and Xanthomonas euvesicatoria

Seed-borne bacterial pathogens of tomato and pepper are of major concern worldwide. Xanthomonas vesicatoria (Xv) and Xanthomonas euvesicatoria (Xe), the causal agents of bacterial leaf spot, and Clavibacter michiganensis subsp. michiganensis (Cmm), the causal agent of tomato bacterial canker, are worldwide distributed, but the occurrence of the latter is usually erratic. In order to evaluate the risk of seed transmission and the relationship between seed contamination and disease outbreak, an extensive field trial has been put in place in 2013 for each pathosystem. Three artificially contamination levels were considered, composed of 100 seedlings each. Disease outbreaks were weekly monitored during the growing season until harvesting and disease was quantified by means of AUDPC. Seeds were produced from each plot and analysed in order to assess their contamination level. Preliminary results of our studies showed that disease quantity caused by Xv, Cmm or Xe was directly correlated to the percentage of initial infection, according to AUDPC values obtained. Contamination rate of seed produced in diseased fields was not always correlated with disease quantity observed. A microbial consortium, a bacterial antagonist and a plant polyphenol were assayed to assess their potential efficacy in seed disinfection: naturally contaminated tomato and pepper seeds were treated and sown. Pepper and tomato seedlings were inspected and analysed for the presence of bacterial spot. Preliminary results obtained show that none of the above mentioned treatments was able to eradicate the pathogen from seeds

Diferencijacija fitopatogenih vrsta roda Agrobacterium

Due to the difficulties in differentiation of phytopathogenic Agrobacterium spp. and lack of a standardized protocol, we carried out selection and evaluation of suitable methods based on the bacterial physiological, genetic and pathogenic properties. Strains of Agrobacterium tumefaciens, A. rhizogenes and A. vitis were differentiated using standard bacteriological and molecular methods. The biochemical and physiological tests confirmed authenticity of the strains. Two duplex PCR methods were conducted with four different primer pairs. In all strains, presence of plasmid virD2 and virC pathogenicity genes was detected. Chromosomal pehA gene was determined in A. vitis strain. Pathogenicity was confirmed on carrot slices and young plants of tomato and sunflower. Strains of A. tumefaciens and A. vitis were pathogenic on all test plants, while strain of A. rhizogenes induced characteristic symptoms only on carrot slices. The tests used in this study provided reliable discrimination between the three species and confirmed their identity as tumorigenic (Ti) Agrobacterium tumefaciens and A. vitis, and rhizogenic (Ri) A. rhizogenes.Usled poteškoća u razlikovanju vrsta roda Agrobacterium i nedostatka standardizovanog protokola izvršena je procena i odabir pogodnih metoda u cilju njihove diferencijacije na osnovu fizioloških, genetskih i patogenih odlika. Diferencirani su sojevi Agrobacterium tumefaciens, A. rhizogenes i A. vitis primenom standardnih bakterioloških i molekularnih metoda. Primenom diferencijalnih testova sojevi su ispoljili očekivane biohemijsko-fiziološke karakteristike. Izvedene su dve „duplex“ PCR metode sa 4 različita tipa prajmera. Kod proučavanih sojeva detektovano je prisustvo virD2 i virC gena patogenosti, koji se nalaze na plazmidnoj DNK bakterije. Prisustvo hromozomskog gena pehA, odgovornog za sintezu enzima poligalakturonaze, utvrđeno je kod soja A. vitis. Patogenost je proverena na kriškama mrkve i mladim biljkama paradajza i suncokreta. Sojevi A. tumefaciens i A. vitis bili su tumorogeni na svim test biljkama, dok je soj A. rhizogenes ispoljio patogenost jedino na kriškama mrkve. Na osnovu dobijenih rezultata, proučavani sojevi diferencirani su kao tumorogeni (Ti) Agrobacterium tumefaciens i A. vitis, i kao rizogeni (Ri) A. rhizogenes

61 Analiza masnih kiselina sojeva Erwinia amylovora iz Srbije i Crne Gore

Automated method of fatty acid analysis was used to identify and study heterogeneity of 41 Erwinia amylovora strains, originating from 8 plant species grown in 13 locations in Serbia and one in Montenegro. All strains contained 14:0 3OH fatty acid, characteristic for the “amylovora” group. According to fatty acid composition 39 strains were identified as E. amylovora as the first choice from the database. Due to their specific fatty acid composition, two strains were identified as E. amylovora, but as a second choice. Fatty acid analysis also showed that E. amylovora population from Serbia could be differentiated in three groups, designated in this study as α, β and γ. All strains originating from central or south Serbia, as well as four strains from north Serbia clustered into group α. Group β and γ contained only strains isolated in northern Serbia (Vojvodina). The results show that E. amylovora population in this area is heterogeneous and indicate pathogen introduction from different directions. Fatty acid analysis enabled identification at species level, as well as new insights of heterogeneity of E. amylovora population.Automatizovana metoda analize masnih kiselina primenjena je za identifikaciju i proučavanje heterogenosti Erwinia amylovora. Kao materijal za analizu prikupljen je 41 soj E. amylovora izolovan iz 8 različitih vrsta domaćina gajenih u 13 lokaliteta u Srbiji i jednom lokalitetu u Crnoj Gori. Rezultati ukazuju da svi proučavani sojevi poseduju 14:0 3OH masnu kiselinu, koja je karakteristična za „amylovora“ grupu. Na osnovu sastava masnih kiselina 39 sojeva je identifikovano kao E. amylovora, kao prvi izbor iz baze podataka. Dva soja su identifikovana kao E. amylovora, ali tek kao drugi izbor iz baze podataka, što je najverovatnije posledica specifičnosti u sastavu njihovih masnih kiselina. Rezultati analize masnih kiselina takođe pokazuju da populacija E. amylovora poreklom iz Srbije nije homogena i da među sojevima postoje tri grupe ili profila, koji su u ovom radu obeleženi sa α, β i γ. Svi sojevi koji su izolovani na prostoru centralne ili južne Srbije pripadaju grupi α, kao i četiri soja izolovana na području Vojvodine. Grupama β i γ pripadaju samo sojevi izolovani na području Vojvodine. Dobijeni rezultati predstavljaju dokaz heterogenosti populacije E. amylovora na ovim prostorima i ukazuju na mogućnost prodora patogena u naše područje iz različitih pravaca. Analiza masnih kiselina omogućila je ne samo identifikaciju do nivoa vrste, već i nova saznanja o heterogenosti populacije E. amylovora na ovim prostorima

Karakterizacija sojeva Xanthomonas euvesicatoria, patogena paprike u Srbiji

During spring and summer of 2008, 101 bacterial strains was isolated from the diseased pepper leaves collected from different pepper growing areas in the Republic of Serbia. The aim of this research was to characterize the isolated strains and determine their taxonomic position according to the most recent nomenclature. Pathogenic, biochemical and physiological characteristics of isolated bacteria were tested using standard bacteriological tests. The pathogen races were determined according to the reaction of differential varieties of Early Calwonder (ECW), their isogenic lines (ECW-10R, ECW-20R, ECW-30R) and Capsicum pubescens. The sensitivity of strains to bactericides was studied in vitro by culturing bacteria on sucrose pepton agar (SPA) plates, amended with filter-sterilized aqueous solution of streptomycin and kasugamycin (50, 100, 200 ppm) or copper-sulphate (100, 200 ppm). Based on pathogenic, biochemical and physiological characteristics, the investigated strains belonged to Xanthomonas euvesicatoria. The reaction of pepper differential varieties indicated that these strains belonged to pepper races P1, P3, P7, P8. Streptomycin resistant strains were not detected, but 6 strains were resistant to kasugamycin (50 ppm) and 13 strains to copper-sulphate (200 ppm), indicating bacterial resistance development.Tokom 2008. godine prikupljeni su uzorci obolelog lišća paprike sa simptomima bakteriozne pegavosti iz različitih lokaliteta Republike Srbije. Izolacijom iz zaraženih listova dobijen je 101 soj bakterija. Cilj ovog rada je proučavanje odlika i identifikacija izolovanih sojeva bakterije u skladu sa najnovijom nomenklaturom. Patogene i biohemijsko-fiziološke karakteristike sojeva proučene su korišćenjem standardnih bakterioloških testova. Određivanje fizioloških rasa bakterijske populacije izvršeno je na osnovu reakcije diferencijalnih sorti paprike Early Calwonder (ECW), njenih izogenih linija (ECW-10, ECW-20 ECW-30) i reakcijom sorte Capsicum pubescens. Proučena je osetljivost sojeva u in vitro uslovima na streptomicin, kasugamicin i bakar-sulfat. Rezultati proučavanja biohemijsko-fizioloških odlika sojeva ukazuju da naši sojevi pripadaju genetičkoj grupi „A“, odnosno vrsti Xanthomonas euvesicatoria. Proučavani sojevi, takođe, predstavljaju heterogenu populaciju u kojoj su zastupljene četiri fiziološke rase bakterije X. euvesicatoria (P1, P3, P7, P8). Sojevi rezistentni na streptomicin nisu detektovani ovim istraživanjima. Otpornost na 50 ppm kasugamicina utvrđena je kod 6 sojeva, a 13 sojeva je bilo otporno na 200 ppm bakar-sulfata. Prikazani rezultati ukazuju na opasnost od razvoja rezistentnosti bakterija na ova jedinjenja

The impact of resistance training program on the muscle strength and bone density in adolescent athletes

© 2018 Saša Bubanj et al., published by Sciendo 2018. Strength training and other modes of physical activity may be beneficial in osteoporosis prevention by maximizing bone mineral accrual in childhood and adolescence. This study focuses on the impact of the nine-month long program of resistant exercises with different level of external loads (low, middle and high) on the lower limbs explosive strength and bone tissue density in athletes adolescents aged 17 to 18 years. Sixty healthy, male athletes and non-athletes, divided into experimental (ES, sprinters, N = 45) and control sub-sample (CS, non-athletes, N = 15), were included in study. ES examinees (EG1, EG2 and EG3) were subjected to the program of resistance exercises with low level (60% of the One Repetition Maximum-1RM), middle level (70% 1RM), and high level (85% 1RM) of external loads, respectively. Bone Density values were determined by the use of a clinical sonometer.,Sahara (Hologic, Inc., MA 02154, USA). Explosive strength values of hip extensors and flexors, knee extensors and flexors, and ankle plantar and dorsiflexors were determined by the use of accelerometer.,Myotest (Sion, Switzerland) and the means of Counter Movement Jump without arms swing (CMJ) and half squat. ANOVA method for repeated measures and ANCOVA method were used to determine significant differences and resistance program effects on the lower limbs explosive strength and bone tissue density. Resistance exercise does impact the explosive strength and bone parameters in a way to increase half squat 1RM values, but decreases CMJ values, and increases speed of sound (SOS), broadband ultrasound attenuation (BUA) and bone mineral density (BMD) values in athletes-adolescents, aged 17-18 years

Diferencijacija Pseudomonas syringae patogenih varijeteta poreklom iz koštičavih voćaka

Due to an overlapping host range, similar symptomatology and many common characteristics, Pseudomonas syringae pathovars originating from stone fruits can easily be misidentified. In order to select tests for rapid and efficient differentiation of P. s. pvs. syringae, morsprunorum and persicae, we studied the suitability and differentiating potential of some standard bacteriological and molecular methods. Differentiation of the strains was performed using LOPAT, GATTa and ice nucleation tests, nutrient sucrose broth growth and utilization of various carbon sources. PCR method enabled the detection of toxin-producing genes: syrB and syrD in P. s. pv. syringae, and cfl gene in P. s. pv. morsprunorum race 1. Syringomycin production by pv. syringae was confirmed in bioassay using Geotrichum candidum, Saccharomyces cerevisiae and Rhodotorula pilimanae as indicator organisms. Pathogenicity test on lemon and immature nectarine fruits, as well as on string bean pods, showed different intensity of reaction of the inoculated material which could separate pv. syringae from the other two pathovars. PCR-based repetitive sequences, Rep-PCR with REP, ERIC and BOX primers revealed different genetic profiles within P. syringae pathovars.Patogeni varijeteti Pseudomonas syringae poreklom sa koštičavih voćaka poseduju brojne zajedničke karakteristike u pogledu kruga domaćina, simptomatologije i biohemijskofizioloških osobina, što otežava njihovu identifikaciju. U cilju odabira testova pogodnih za brzu i pouzdanu identifikaciju P. s. pv. syringae, morsprunorum i persicae, primenjeni su standardni bakteriološki i molekularni testovi. Diferencijacija sojeva izvršena je LOPAT i GATTa testovima, posmatranjem razvoja u hranljivom rastvoru sa saharozom, sposobnošću sojeva da formiraju čestice leda, kao i mogućnošću korišćenja različitih ugljenikovih jedinjenja. PCR metod korišćen je u detekciji gena odgovornih za proizvodnju toksina siringomicina kod soja P. s. pv. syringae (syrB i syrD geni) i koronatina kod soja P. s. pv. morsprunorum rase 1 (cfl gen). Proizvodnja siringomicina potvrđena je i biotestom, korišćenjem gljiva Geotrichum candidum, Saccharomyces cerevisiae i Rhodotorula pilimanae kao indikatora. Proverom patogenosti sojeva na plodovima limuna, nesazrelim plodovima nektarine i mahunama boranije, došlo je do ispoljavanja simptoma različitog intenziteta, na osnovu kojih se može izdvojiti pv. syringae od ostala dva patovara. Primenom Rep-PCR metode, uz korišćenje REP, ERIC i BOX prajmera, ustanovljene su razlike u genetskim profilima proučavanih P. syringae patogenih varijeteta

Differentiation of Pseudomonas syringae Pathovars Originating from Stone Fruits

Due to an overlapping host range, similar symptomatology and many common characteristics,Pseudomonas syringae pathovars originating from stone fruits can easily be misidentified.In order to select tests for rapid and efficient differentiation of P. s. pvs. syringae,morsprunorum and persicae, we studied the suitability and differentiating potential ofsome standard bacteriological and molecular methods. Differentiation of the strains wasperformed using LOPAT, GATTa and ice nucleation tests, nutrient sucrose broth growthand utilization of various carbon sources. PCR method enabled the detection of toxin-producinggenes: syrB and syrD in P. s. pv. syringae, and cfl gene in P. s. pv. morsprunorum race1. Syringomycin production by pv. syringae was confirmed in bioassay using Geotrichumcandidum, Saccharomyces cerevisiae and Rhodotorula pilimanae as indicator organisms.Pathogenicity test on lemon and immature nectarine fruits, as well as on string bean pods,showed different intensity of reaction of the inoculated material which could separate pv.syringae from the other two pathovars. PCR-based repetitive sequences, Rep-PCR withREP, ERIC and BOX primers revealed different genetic profiles within P. syringae pathovars

Pectobacterium carotovorum subsp. carotovorum prouzrokovač vlažne truleži brokolija u Srbiji

Soft rot symptoms were observed on broccoli plants in several commercial fields in the western part of Serbia. Six strains of bacteria were isolated from diseased tissues and identified as Pectobacterium carotovorum subsp. carotovorum using conventional bacteriological and molecular methods. All strains were non-fluorescent, gram-negative, facultative anaerobes, oxidase-negative and catalase-positive, causing soft rot on potato and carrot slices and did not induce hypersensitive reaction on tobacco leaves. They grew in 5% NaCl and at 37°C, did not produce acid from á-methyl glucoside, sorbitol and maltose, nor reducing substances from sucrose, but utilized lactose and trehalose, and did not produce indole or lecithinase. The investigated strains showed characteristic growth on Logan’s medium and did not produce blue pigmented indigoidine on GYCA medium nor “fried egg” colonies on PDA. The identity of strains was confirmed by ITS-PCR and ITS-RFLP analyses and by sequence analysis of the 16S rRNA gene. In a pathogenicity assay, all strains caused tissue discoloration and soft rot development on inoculated broccoli head tissue fragments.Simptomi vlažne truleži brokolija uočeni su u nekoliko komercijalnih zasada u zapadnoj Srbiji. Sojevi bakterija izolovani su iz obolelog biljnog tkiva i identifikovani kao Pectobacterium carotovorum subsp. carotovorum korišćenjem konvencionalnih bakterioloških i novijih molekularnih metoda. Svi proučavani sojevi bili su nefluorescentni, Gram-negativni, fakultativno-anaerobni, oksidaza i lecitinaza negativni, katalaza pozitivni, prouzrokujući vlažnu trulež cvetnih drški brokolija, kriški krompira i kriški mrkve, ali nisu indukovali hipersenzitivnu reakciju duvana. Svi sojevi razvijaju se pri 37ºC i u prisustvu 5% NaCl, ne proizvode kiselinu iz α-metil glukozida, sorbitola i maloze, niti redukujuće supstance iz saharoze, ali razlažu laktozu i trehalozu, i ne stvaraju indol. Proučavani sojevi ispoljili su karakterističan razvoj na Loganovoj diferencijalnoj podlozi, i nisu proizvodili plavi pigment na GYCA podlozi niti “fried egg” kolonije na PDA podlozi. Identifikacija sojeva potvrđena je korišćenjem ITS-PCR i ITS-RFLP, kao i analizom sekvenci 16S rRNA gena. Prilikom izvođenja testa patogenosti, svi sojevi prouzrokovali su obezbojavanje tkiva i vlažnu trulež cvetnih drški brokolija

Colletotrichum orbiculare on watermelon: Identification and in vitro inhibition by antagonistic fungi

Anthracnose caused by the fungus Colletotrichum orbiculare is one of the most significant diseases of Cucurbitaceae. In Serbia watermelon fruits with typical anthracnose lesions were collected during the year of 2015. Affected fruits showed sunken, dark brown to black lesions with orange conidial masses produced in black acervuli. In an attempt to identify the causal organism, small pieces of necrotic tissue were surface sterilized and placed on potato dextrose agar (PDA). Macroscopic and microscopic morphologicaly characteristics of three isolates were observed after growth on PDA for 7 days at 25 ºC under a 12 h light/dark cycle. Fungal colonies developed white, grey to black dense aerial mycelium. Conidia were hyaline, aseptate, straight and cylindrical to clavate, 9–12.5 μm × 4–5.5 μm. Fungal isolates were also characterized by sequencing of the internal transcribed spacer (ITS) rDNA region using ITS1F/ITS4 primers and β-tubuline 2 gene using T1/Bt2b primers. The nucleotide sequences were deposited in GenBank (ITS Acc. No. KT454386, KT454387 and KT454388; β-tubuline 2 gene Acc. No. KT581236, KT581237 and KT581238). BLAST analysis of ITS and β-tubuline 2 gene sequences showed that our isolates were 100% identical to other C. orbiculare in NCBI GenBank. Pathogenicity test was conducted on symptomless, detached watermelon fruits. All tested isolates caused anthracnose lesions on watermelon fruits after 10 days of incubation. Trichoderma harzianum (DSM 63059) and Gliocladium roseum (DSM 62726) were evaluated in vitro for their antagonistic potential against C. orbiculare. The results of this study identify T. harzianum and G. roseum as promising biological control agents (BCAs) for further testing against anthracnose disease on watermelon fruits