Mogućnost zaštite pasulja od Xanthomonas campestris pv. phaseoli primenom bakarnih preparata i aktivatora otpornosti

The efficacy of several new formulations of copper compounds, namely Cuprozin 35 WP (copper-oxychloride), Cuproxat (copper-sulphate), Funguran OH (copper-hydroxide) and the plant activator Bion (acibenzolar-S-methyl), and their combinations with dithiocarbamates (Dithane M-70) was estimated in controlling Xanthomonas campestris pv. phaseoli (artificial inoculation) in field conditions in two localities during 2006. In the locality Zemun, the efficacy of copper compounds ranged from 92.7% to 98.5%. The plant activator Bion 50 WG exhibited similar efficacy (94.4-97.1%). Combinations of Funguran OH and Dithane M-70, applied at different concentrations, also showed high efficacy (98.3-99.3%), as well as the combinations of Bion 50 WG and the other bactericides (95.5-96.8%). There was no significant difference between the efficacies achieved by the compounds applied individually and their combinations, except Cuproxat, which exhibited decreased efficacy at lower concentration. In the locality Smederevska Palanka, the efficacy of copper compounds was 95.0-98.2%, while Bion achieved 96.8-97.7% efficacy. Combinations of copper-hydroxide (Funguran OH) and dithiocarbamates (Dithane M-70) also showed high efficacy (98.1- 99.4%) but without a significant difference. The efficacy of combinations of Bion and copper- hydroxide, and Bion and mancozeb was 97.9-98.9%. There was no significant difference in the efficacies of the bactericides tested or the efficacies of their combinations in that locality. Our investigation confirmed high efficacy of acibenzolar-S-methyl, which was equal to the efficacy of standard bactericide treatment. This compound therefore offers a very good alternative to conventional chemicals used for controlling bacterial diseases in beans.U radu je ispitivana efikasnost novijih formulacija bakarnih jedinjenja; Cuprozin 35 WP (bakar-oksihlorid), Cuproxat (bakar-sulfat), Funguran OH (bakar-hidroksid) i aktivatora otpornosti Bion-a (acibenzolar-S-metil), i njihovih kombinacija sa ditiokarbamatima (Dithane M-70), u suzbijanju Xanthomonas campestris pv. phaseoli (veštačka inokulacija). Ispitivanja su obavljena tokom 2006. godine u poljskim uslovima na dva lokaliteta. Na lokalitetu Zemun, efikasnost bakarnih preparata kretala se 92.7-98.5%. Sličnu efikasnost ispoljio je i activator otpornosti Bion 50 WG (94.4-97.1%). Zadovoljavajuću efikasnost (98.3-99.3%) imale su I kombinacije preparata Funguran OH i Dithane M-70 pri različitim koncentracijama primene, kao i kombinacije Bion-a sa drugim preparatima (95.5-96.8%). Statistički značajne razlike u efikasnosti primenjenih preparata i njihovih mešavina nisu zabeležene, izuzev kod preparata Cuproxat, koji je ispoljio slabiju efikasnost pri nižoj koncentraciji primene. Na lokalitetu Smederevska Palanka efikasnost bakarnih jedinjenja iznosila je 95.0-98.2%, a za Bion 96.8-97.7%. Kombinacije bakar-hidroksida (Funguran OH) i ditiokarbamata (Dithane M-70) ispoljile su nešto veću efikasnost (98.1-99.4%), ali razlike nisu bile statistički značajne. Efikasnost Bion-a u mešavini sa bakar-hidroksidom i mankozebom iznosila je 97.9-98.9%. Na ovom lokalitetu nisu konstatovane statistički značajne razlike između samostalno primenjenih preparata i njihovih kombinacija. U ovim ispitivanjima potvrđena je i visoka efikasnost acibenzolar-S-metil-a (preparat Bion), koja je bila na nivou efikasnosti standardnih baktericida. Zbog toga ovo jedinjenje predstavlja dobru alternativu konvencionalnom hemijskom suzbijanju bakterioza pasulja

Razrada metoda za dokazivanje Pseudomonas savastanoi pv. phaseolicola na semenu pasulja

Pseudomonas savastanoi pv. phaseolicola detection on artificially inoculated bean seeds was investigated. The method of the International Seed Federation – ISF (2006) was used. It includes bacteria extraction from seeds, isolation on semiselective media and checking the pathogenicity of investigated isolates. For verification of results, quick new methods of investigation were used (ELISA test and PCR). The results show that semiselective media MT (Milk Tween Agar) and MSP (Modified Sucrose Peptone Agar) can be appropriate for isolation of this bacterium. Pathogenicity of the investigated isolates was confirmed on cotyledon leaves of bean. ELISA test and PCR confirmed that all investigated isolates and reisolates belong to the bacterium P. s. pv. phaseolicola.U radu je ispitivana mogućnost utvrđivanja prisustva bakterije Pseudomonas savastanoi pv. phaseolicola sa veštački inokulisanog semena pasulja. Korišćena je metoda International Seed Federation (ISF), koja uključuje ekstrakciju bakterija iz semena, izolaciju na poluselektivne podloge i proveru patogenosti dobijenih izolata. U cilju potvrde rezultata korišćene su brze savremene metode identifikacije (ELISA test i PCR). Rezultati dobijeni tokom rada pokazuju da su poluselektivne podloge MT (Milk Tween Agar) i MSP (Modified Sucrose Peptone Agar) pogodne za izolaciju ove bakterije. Patogenost odabranih izolata dokazana je na kotiledonim listovima pasulja. Primenom ELISA testova i PCR, potvrđeno je da svi ispitivani izolati i reizolati pripadaju bakteriji P. s. pv. phaseolicola

Razrada metoda za dokazivanje Xanthomonas axonopodis pv. phaseoli na semenu pasulja

Xanthomonas axonopodis pv. phaseoli detection on artificially inoculated bean seeds was investigated. The method of the International Seed Federation – ISF (2006) was used. It included bacteria extraction from seeds and isolation on semiselective media with pathogenicity test of the investigated isolates. ELISA and PCR were used for verification of results. The results showed that the semiselective media MT (Milk Tween Agar) and XCP1 (Xanthomonas campestris pv. phaseoli Agar) were very suitable for isolation of X. a. pv. phaseoli. Pathogenicity was confirmed on young bean plants. ELISA test and PCR confirmed that all investigated isolates and reisolates belong to the bacterium X. a. pv. phaseoli.U radu je ispitivana mogućnost utvrđivanja prisustva bakterije Xanthomonas axonopodis pv. phaseoli na veštački inokulisanom semenu pasulja. Ispitivanja su vršena po metodi International Seed Federation (ISF), ekstrakcijom bakterija iz semena i izolacijom na poluselektivne podloge uz proveru patogenosti dobijenih izolata. Za potvrdu dobijenih rezultata korišćen je ELISA test i PCR. Prema dobijenim rezultatima ova bakterija se može uspešno izolovati na poluselektivne podloge MT (Milk Tween Agar) i XCP1 (Xanthomonas campestris pv. phaseoli Agar). Patogenost je dokazana na mladim biljčicama pasulja. ELISA testovi i PCR su potvrdili da svi ispitivani izolati i reizolati pripadaju bakteriji X. a. pv. phaseoli

Ispitivanje mogućnosti suzbijanja Venturia inaequalis ekološki prihvatljivim preparatima

Possibility of V. inaequalis control on apple variety Idared was evaluated by ecologically acceptable products such as inorganic fungicides and acidified clays. The obtained results were compared with common, chemical protection. Year 2009 was one of the unfavorable for V. inaequalis, and 2010 was extremely favorable for apple scab development. Accordingly, in 2009 intensity of infection on apple leaves and fruits in control variant was low (leaves 15,4-34,9%; fruits 0-23,8%). In 2010 infection intensity on leaves and fruits was high (leaves 81,2%; fruits 100%). During period 2009-2010, in these different environmental conditions, common chemical protection showed high efficacy (leaves 96,2-100%; fruits 97,5-100%). High efficacy level in control of V. inaequalis on leaves of 87,4-99,7% was achieved also in variants in which copper products were applied in low concentration of 0,05%. It should be noted that in the most critical period, in April and May of 2010, the treatments were carried out in 5-6 days interval. Copper products showed differing efficacy on fruits (48,5-90,2%). In 2009 which is considered as one of the unfavorable for V. inaequalis development, product Ulmasud B applied alone on leaves was less efficiacy (51,8-58,6%), than on fruits (87,8%). In the same year, efficacy of this product in combination with sulfur, was better (leaves 71,4-76,6%; fruits 86,6). In 2010, which was especially rainy and favourable for spreading the disease, efficiacy of mention combination of products didn,t satisfy (leaves 44,8%; fruits 41,0%). In the system of organic production, during vegetation period, program of apple protection in control of V. inaequalis should be supplemented by use of low concentrations of copper products (0,05%) and by various alternative products of different action mechanisms, such as mineral clays, sulfur products and etc. These researches should be continued.Ispitivana je mogućnost suzbijanja V. inaequalis na jabuci (ajdared) ekološki prihvatljivim preparatima (neorganski fungicidi i kisela glina). Dobijeni rezultati su poređeni sa uobičajenom, hemijskom zaštitom. Godina 2009. je bila jedna od najnepovoljnijih za V. inaequalis, a 2010. jedna od najpovoljnih za širenje ovog patogena na jabuci. Shodno tome, u 2009. godini (tri lokaliteta) intenzitet zaraze u kontroli na listovima i plodovima jabuke bio je nizak (listovi 15,4-34,9%; plodovi 0-23,8%). U 2010. godini intenzitet zaraze na listovima i plodovima je bio visok (listovi 81,2%; plodovi 100%). Pri navedenim različitim uslovima zaraze, efikasnost uobičajene, hemijske zaštite, u obe godine je bila na visokom nivou (listovi 96,2-100%; plodovi 97,5-100%). Visoka efikasnost u suzbijanju V. inaequalis na listovima (87,4-99,7%), postignuta je i u varijantama sa primenom bakarnih preparata u niskoj koncentraciji (0,05%), uz napomenu da su u 2010. godini tretmani u najkritičnijem periodu izvođeni u intervalu 5-6 dana. Efikasnost bakarnih preparata na plodovima je bila različta (48,5-90,2%). U 2009. godini, koja je bila jedna od nepovoljnijih za širenje crne pegavosti na jabuci, preparat Ulmasud B primenjen samostalno, ispoljio je znatno slabiju efikasnost na listovima (51,9-58,6%), u odnosu na plodove (87,8%). Iste godine, ovaj preparat u kombinaciji sa sumpornim preparatima, obezbedio je bolju efiksanost (listovi 71,4-76,6%; plodovi 86,6%). Suprotno tome, u 2010. godini, koja je bila izrazito povoljna za širenje ovog patogena, efikasnost navedene kombinacije preparata nije bila zadovoljavajuća (listovi 44,8%; plodovi 41,0%). U sistemu organske proizvodnje, program zaštite jabuke od V. inaequalis tokom vegetacije treba dopuniti korišćenjem preparata na bazi bakra (u niskoj koncentraciji) i raznih alternativnih preparata (na bazi mineralnih glina, sumpornih preparata i sl.). Ova ispitivanja bi trebalo nastaviti

Pojava Xanthomonas campestris pv. campestris (Pammel, 1895) Dowson 1939, na kupusnjačama u Crnoj Gori

Brassicas form the most important group of vegetable crops in Montenegro. The cabbage (Brassica oleracea var. capitata) is most commonly grown, although other brassicas, particularly kale, Brussels sprout, cauliflower and broccoli, have been increasingly produced since recently. One of the specialties of vegetable production in Montenegro is growing of collard (Brassica oleracea var. acephala), which is the simplest variety of the Brassica oleracea species and in the nearest relation with their wild ancestor – the sylvestris variety. Diseases are the main restrictive factors for successful production of these vegetables. Susceptibility of the cultivars and inadequate control often result in more or less damaged crops in some plots. Causal agents of brassica diseases, especially bacterial, have not been investigated in Montenegro until 2009. Since the symptoms observed in 2009 were „V” shaped leaf edge necrosis and black rot of vascular tissue, it was assumed that they were caused by plant pathogenic bacterium Xanthomonas campestris pv. campestris. Samples of the infected plants were collected from different localities in Montenegro. Isolation and identification of the bacterium were performed using laboratory methods according to Schaad (1980), Lelliott and Stead (1987) and Arsenijeviæ (1997). Examination of chosen bacterial isolates was conducted using both, classical bacteriological methods (examination of their pathogenic, morphological, cultivation and biochemical and physiological characteristics), and ELISA test. The obtained results confirmed the presence of X.campestris pv. campestris (Pammel, 1895) Dowson 1939, on cabbage, kale, broccoli and collard in Montenegro. This is the first experimental evidence that collard is the host of X. campestris pv. campestris in Montenegro.Kupusnjače predstavljaju grupu najznačajnijih povrtarskih kultura u Crnoj Gori. Najviše se gaji kupus glavičar (Brassica oleracea var. capitata), a u novije vreme uvode se u proizvodnju i ostale kupusnjače, u prvom redu kelj, kelj pupčar, karfiol i brokola. Jedna od specifičnosti povrtarske proizvodnje u Crnoj Gori je gajenje raštana (Brassica oleracea var. acephala), najprostijeg varijeteta vrste B. oleracea, najsrodnijeg divljem pretku – varijetetu sylvestris. Bolesti predstavljaju glavni ograničavajući faktor uspešne proizvodnje ovih biljaka. Zbog osetljivosti sortimenta i neadekvatne zaštite, često se dešava da usev na pojedinim parcelama bude u manjoj ili većoj meri oštećen. U Crnoj Gori do sada nisu sprovedena istraživanja o prouzrokovačima bolesti na kupusnjačama, a posebno ne istraživanja bakterioznih oboljenja. S obzirom da su tokom 2009. godine na biljkama konstatovani simptomi nekroze ivice lista u obliku latiničnog slova V i crna trulež sudovnog sistema, pretpostavka je bila da je prouzrokovač ovih patoloških promena fitopatogena bakterija Xanthomonas campestris pv. campestris. Uzorci sa obolelih biljaka prikupljeni su sa različitih lokaliteta u Crnoj Gori. Izolacija i identifikacija bakterije urađena je laboratorijskim metodama prema Schaad-u (1980), Lelliott i Stead-u (1987) i Arsenijeviću (1997). Ispitivanja odabranih bakterijskih izolata obuhvatala su primenu klasičnih bakterioloških metoda (ispitivanje patogenih, morfoloških, odgajivačkih i biohemijsko-fizioloških osobina dobijenih izolata) i ELISA test. Dobijeni rezultati potvrdili su prisustvo i pojavu X.campestris pv. campestris (Pammel 1895) Dowson 1939 u Crnoj Gori, na kupusu, kelju, brokoliju i raštanu. Ovo je prvi eksperimentalni podatak da je raštan domaćin X. campestris pv. campestris u Crnoj Gori

Clavibacter michiganensis subsp. michiganensis, bakteriozni rak paradajza: 1. Konvencionalna i molekularna identifikacija

In May 2006, tomato leaves with watersoaked areas between leaf veins were detected in glasshouses in Padinska Skela. The early-stage symptoms were followed by rapid desiccation to white and pale brown necrosis and subsequently by wilting of entire tomato plants. In June 2006, symptoms of bacterial canker and wilt were also recorded in many greenhouses in the Jablanica County in southern Serbia. Samples of diseased tomato plants were collected from several tomato-growing regions in Serbia in order to identify the causal agent using conventional identification methods (isolation, pathogenicity and bacteriological determinative tests). Another objective of this study was to confirm the identity of isolated bacterial strains by polymerase chain reaction (PCR method). According to the morphological characteristics observed on NA and NBY media, biochemical characteristics, hypersensitive response in four-o’clock plant leaves and pathogenicity test on tomato seedlings, the investigated strains were identified as C. michiganensis subsp. michiganensis. To confirm the identity of isolated strains two PCR protocols were used. Amplification of expected length DNA fragments, 614 bp and 270 bp, respectively, confirmed that the investigated strains belonged to C. michiganensis subsp. michiganensis.Tokom maja 2006. godine zapažene su vlažne pege na lišću paradajza gajenom u staklenicima u Padinskoj Skeli. Ovi početni simptomi praćeni su brzim sušenjem lisnog tkiva, pojavom bele i svetlomrke nekroze i uvenućem celih biljaka. U junu 2006. godine, simptomi bakterioznog raka i uvenuća paradajza takođe su zapaženi i u mnogim plastenicima u Jablaničkom okrugu. Uzorci obolelih biljaka paradajza sakupljeni su sa nekoliko lokaliteta u Srbiji, u cilju identifikacije prouzrokovača bolesti korišćenjem konvencionalnih metoda za identifikaciju (izolacija, patogenost i bakteriološki testovi). Drugi cilj istraživanja bio je da potvrdimo identitet izolovanih sojeva bakterija primenom lančane reakcije polimeraze (PCR metod). Prema morfološkim karakteristikama na mesopeptonskoj (MPA) i NBY podlozi, biohemijsko-fiziološkim odlikama, hipersenzitivnoj reakciji na noćurku (Mirabilis jalapa) i patogenim odlikama na rasadu paradajza, izolovani sojevi su identifikovani kao C. michiganensis subsp. michiganensis. Identifikacija izolovanih sojeva bakterija potvrđena je korišćenjem dva PCR protokola. Amplifikovani su DNA fragmenti očekivane dužine, 614 bp i 270 bp, čime je po tvrđeno da testirani sojevi pripadaju bakteriji C. michiganen sis subsp. michiganensis

Status Erwinia amylovora na teritoriji bivše Jugoslavije tokom dve poslednje decenije

Erwinia amylovora, the causal agent of fire blight (FB) on fruit trees and ornamental plants, rapidly spread across eastern Mediterranean countries in the early 1980s. This quarantine bacterium probably arrived in the southern parts of the former Yugoslavia (now FYR Macedonia) from Greece. Based on symptoms, and isolation and identification data, it was concluded that Erwinia amylovora was the causal agent of pear drying in Macedonia (1989). It was the first experimental confirmation of a presence of E. amylovora in the territory of the former Yugoslavia. The presence of E. amylovora was also proved in Serbia that same year. In Bosnia and Herzegovina, FB was detected during 1990. Based on an official report filed with the Federal Ministry of Agriculture in Belgrade, the presence of E. amylovora in Yugoslavia was confirmed (EPPO – Reporting Service, 1991). Therefore, the presence of the bacterium E. amylovora in the territory of Yugoslavia was officially confirmed in 1990. In Croatia, FB was first observed in villages near the border on Serbia in 1995. In Montenegro, FB was first detected in 1996. In Slovenia, FB appeared as late as in 2001. E. amylovora is now present on 10 hosts (pear, wild pear, apple, quince, medlar, mountainash, hawthorn, firethorn, cotoneaster and Japanese quince) in the territory of the former Yugoslav republics. Based on literature data, losses caused by FB in the former Yugoslav republics in the period 1989-1992 were estimated at about 12,000,000 DEM (mostly in Macedonia) and in the period 1992–1996 at 6,000,000 DEM. Total damage in a more recent epiphytotic year in Slovenia (2003) was estimated at about 474,200 EUR. Conventional and up-to-date rapid methods (PCR, ELISA and IF, BIOLOG and API System, FAME and SDS-PAGE) have been used to identify E. amylovora. Mainly preventive measures have been used to control E. amylovora in the former Yugoslav republics. Spraying with copper products has been practiced during the dormant period and in early spring. In rare cases, spraying has been done at the flowering stage (MARYBLYT), but the problem is that no synthetic bactericides are available on the market. When symptoms occur, only mechanical measures are being applied.Početkom 1980-ih Erwinia amylovora se brzo širila na teritoriji zemalja istočnog Mediterana. Iz Grčke (1986) ova karantinska bakterija je dospela u Makedoniju (južni deo Jugoslavije). Na osnovu simptoma, izvršenih izolacija i identifikacije dobijenih izolata, zaključeno je da je E. amylovora prouzrokovač sušenja kruške u Makedoniji (1989). To je bio prvi eksperimentalni dokaz o prisustvu E. amylovora na teritoriji bivše Jugoslavije. E. amylovora je iste godine dokazana i u Srbiji. U Bosni i Hercegovini je zapažena tokom 1990. Na osnovu zvaničnog dopisa Saveznom ministarstvu poljoprivrede u Beogradu, zvanično je potvrđeno prisustvo E. amylovora na teritoriji bivše Jugoslavije (EPPO – Reporting Service, 1991). Tako je prisustvo E. amylovora na teritoriji bivše Jugoslavije zvanično potvrđeno 1990. U Hrvatskoj je bakteriozna plamenjača prvi put zapažena 1995. u selima koja se nalaze u graničnom području prema Srbiji. U Crnoj Gori bakteriozna plamenjača je prvi put zapažena 1996. U Sloveniji se bakteriozna plamenjača pojavila tek 2001. Prisustvo E. amylovora je do sada utvrđeno na 10 domaćina (kruška, divlja kruška, jabuka, dunja, mušmula, oskoruša, glog, vatreni trn, kotonester i japanska dunja) na teritoriji bivše Jugoslavije. Na osnovu literaturnih podataka (1996) gubici prouzrokovani bakterioznom plamenjačom na teritoriji bivše Jugoslavije su procenjeni na oko 12,000,000 DM. Ukupni gubici u godini epifitocije u Sloveniji (2003) su procenjeni na oko 474,200 EUR. Za identifikaciju E. amylovora korišćene su klasične i savremene metode identifikacije (PCR, ELISA, IF, BIOLOG i API sistem, FAME i SDS-PAGE). Za suzbijanje E. amylovora na teritoriji bivše Jugoslavije su uglavnom korišćene preventivne mere zaštite. Prskanje u periodu mirovanja i u rano proleće se izvodi bakarnim preparatima. U retkim slučajevima prskanje se primenjuje i u fazi cvetanja (MARYBLYT), ali je problem što na tržištu nemamo sintetičkih baktericida. Ukoliko se simptomi ipak pojave, primenjuju se mehaničke mere zaštite

Evaluation of possibilities of Venturia inaequlis control by ecologically acceptable products

Possibility of V. inaequalis control on apple variety Idared was evaluated by ecologically acceptable products such as inorganic fungicides and acidified clays. The obtained results were compared with common, chemical protection. Year 2009 was one of the unfavorable for V. inaequalis, and 2010 was extremely favorable for apple scab development. Accordingly, in 2009 intensity of infection on apple leaves and fruits in control variant was low (leaves 15,4-34,9%; fruits 0-23,8%). In 2010 infection intensity on leaves and fruits was high (leaves 81,2%; fruits 100%). During period 2009-2010, in these different environmental conditions, common chemical protection showed high efficacy (leaves 96,2-100%; fruits 97,5-100%). High efficacy level in control of V. inaequalis on leaves of 87,4-99,7% was achieved also in variants in which copper products were applied in low concentration of 0,05%. It should be noted that in the most critical period, in April and May of 2010, the treatmentswere carried out in 5-6 days interval. Copper products showed differing efficacy on fruits (48,5-90,2%). In 2009 which is considered as one of the unfavorable for V. inaequalis development, product Ulmasud B applied alone on leaves was less efficiacy (51,8-58,6%), than on fruits (87,8%). In the same year, efficacy of this product in combination with sulfur, was better (leaves 71,4-76,6%; fruits 86,6). In 2010, which was especially rainy and favourablefor spreading the disease, efficiacy of mention combination of products didn,t satisfy (leaves 44,8%; fruits 41,0%). In the system of organic production, during vegetation period, program of apple protection in control of V. inaequalis should be supplemented by use of low concentrations of copper products (0,05%) and by various alternative products of different action mechanisms,such as mineral clays, sulfur products etc. These researches should be continued

Clavibacter michiganensis subsp. michiganensis, bakteriozni rak paradajza: 2. Poređenje efikasnosti procedura za ekstrakciju i osetljivosti metoda za detekciju na semenu paradajza

Two seed extraction procedures, used for detection of Clavibacter michiganensis subsp. michiganensis (Cmm) in artifficially infested tomato seed lots, were evaluated. A comparison of the efficiency of pathogen detection by using different extraction methods showed that a grinding procedure was more effective than soaking seed samples. The extraction by grinding resulted in a higher number of samples with Cmm colonies than did the method that included soaking. The detection threshold of Cmm in relation to seed sample size was evaluated by adding different numbers of artificially infested seeds to uninfected samples of 2000 or 5000 seeds. Four detection methods were simultaneously compared for their sensitivity in Cmm detection in seeds: isolation on semiselective media (mSCM, D2ANX, mCNS), direct PCR from seed material, Bio-PCR with initial culturing of bacteria on NBY agar prior to PCR, and Enrichment PCR. The pathogen was detected in samples of 2000 seeds containing one, five and ten infested seeds, in at least two out of three replicates by three detection methods (selective plating, direct PCR and Bio-PCR), using the grinding extraction method with an addition of centrifugation step. In samples of 5000 seeds, five infested seeds were detected in all replicates by the same detection methods. Similar results were obtained by the soaking extraction method. In Enrichment PCR, positive results were obtained only in samples of 2000 seeds containing five and ten infested seeds regardless of the extraction method.Ocenjivane su dve procedure ekstrakcije koje se primenjuju u detekciji Clavibacter michiganensis subsp. michiganensis (Cmm) u uslovima veštačke inokulacije uzoraka semena paradajza. Poređenje efikasnosti detekcije patogena korišćenjem metoda mlevenja i potapanja semena pokazalo je da je metod mlevenja bio efikasniji. Ekstrakcija mlevenjem semena rezultirala je neznatno veæim brojem uzoraka sa kolonijama Cmm nego procedura potapanja semena. Prag detekcije Cmm u zavisnosti od velièine uzorka ocenjivan je dodavanjem različitog broja veštački inokulisanih semena u nezaražene uzorke semena veličine 2000 i 5000 semena. Istovremeno je poređena osetljivost četiri metode za detekciju Cmm na semenu: izolacija na poluselektivne podloge (mSCM, D2ANX, mCNS), direktni PCR iz uzoraka semena, Bio-PCR koji uključuje gajenje bakterija na NBY podlozi pre PCR reakcije i Enrichment PCR. Patogen je detektovan u uzorcima veličine 2000 semena u koje su dodati jedno, pet i deset veštački zaraženih semena, u najmanje dva od tri ponavljanja korišćenjem tri metode za detekciju (izolacija na poluselektivne podloge, direktni PCR i Bio-PCR), nakon ekstrakcije mlevenjem uzoraka. Istim metodama, u uzorcima velièine 5000 semena, pet zaraženih semena je detektovano u svim ponavljanjima. Slični rezultati su dobijeni i nakon ekstrakcije potapanjem semena. U Enrichment PCR reakciji, pozitivni rezultati su dobijeni samo u uzorcima veličine 2000 semena u koje je dodato pet i deset zaraženih semena nezavisno od toga koja je procedura ekstrakcije korišćena

Morfološka i molekularna identifikacija Colletotrichum acutatum sa ploda paradajza

Colletotrichum gloeosporioides, Colletotrichum acutatum, Colletotrichum coccodes, and Colletotrichum dematium are the four main species of Colletotrichum that cause tomato anthracnose. In Serbia, the occurrence of anthracnose on tomato fruit has been recorded during the last several years. Typical fruit symptoms include dark, sunken, and circular lesion with orange conidial masses. Pathogen isolates were obtained from a diseased tomato fruits, on PDA medium forming a white to gray colonies. The cultures developed black acervuli around the center of the colony. Conidia were hyaline, aseptate, and fusiform or rarely cylindrical. Appressoria were smooth, simple, clavate to ovate, and varied from light to dark brown. Pathogenicity tests with representative isolates were conducted on symptomless, detached tomato fruits. All tested isolates caused anthracnose lesions on tomato fruit after 7 days of incubation. Koch’s postulates were fulfilled by reisolation from inoculated tomato fruits. PCR analysis (using species-specific primer pair, CaInt2/ ITS4) of genomic DNA from tomato isolates resulted in an amplification product of 490 bp, specific for C. acutatum, further confirming the identity of the pathogen. Based on morphological and molecular characteristics, the isolates from tomato fruit were determined as C. acutatum.Četiri glavna prouzrokovača antraknoze paradajza iz roda Colletotrichum su: Colletotrichum gloeosporioides, Colletotrichum acutatum, Colletotrichum coccodes i Colletotrichum dematium. U Srbiji je tokom poslednjih godina zabeležena pojava antraknoze ploda paradajza. Tipični simptomi na plodu su tamne, ulegnute, kružne lezije sa masom narandžastih konidija. Sa obolelih plodova paradajza dobijeni su izolati patogena koji na KDA podlozi obrazuju bele do sivo obojene kolonije. Acervuli crne boje formiraju se u kulturi oko centra kolonije. Konidije su hialinske, neseptirane, fusiformne ili ređe cilindrične. Apresorije su glatke, jednostavne, okruglastog ili oblika izdužene palice, svetlo do tamno braon boje. Test patogenosti je obavljen sa reprezentativnim izolatima, na odabranim, zdravim plodovima. Svi ispitivani izolati prouzrokuju antraknozne lezije na plodu paradajza 7 dana nakon inokulacije. Kohovi postulati su zadovoljeni reizolacijama sa inokulisanih plodova paradajza. PCR analiza (korišćenjem para prajmera specifičinog za vrstu, CaInt2/ITS4) iz genoma DNA izolata sa paradajza rezultirala je amplifikacijom produkta od 490 bp, specifičnog za C. acutatum, što je potvrdilo identitet patogena. Na osnovu morfoloških i molekularnih karakteristika izolati sa ploda paradajza determinisani su kao C. acutatum