Biološko suzbijanje Colletotrichum spp. parazitauskladištenih plodova jabuke

Colletotrichum spp. su značajni prouzrokovači bolesti biljaka u svetu i u našoj zemlji, u polju i u skladištu. Na plodovima jabuke prisutne su dve vrste ovog roda, C. acutatum i C. gloeosporioides. Pomenute vrste uzrokuju ekonomski značajne gubitke posle berbe plodova jabuke, tokom skladištenja, transporta i plasmana na tržište. Nedoumice sa kojima se naučna javnost suočava kada je u pitanju ovaj rod jeste precizno utvrđivanje sistematske pozicije i definisanje vrsta i nižih kategorija. Klasične fitopatološke metode ne omogućavaju preciznu iv identifikaciju do nivoa vrste. Otuda su molekularni pristupi sve zastupljeniji u identifikaciji Colletotrichum spp. U suzbijanju skladišnih patogena pretežno se primenjuju hemijski fungicidi. Zbog nepovoljnih toksikoloških svojstava i pojave rezistentnosti, primena hemijskih fungicida se sve više redukuje, a njihova upotreba posle berbe plodova zabranjena je u većini zemalja. Stoga, za održivi razvoj poljoprivredne proizvodnje neophodna su istraživanja usmerena ka otkrivanju mikroorganizama i prirodno sintetisanih materija koje imaju potencijala za primenu u biološkoj zaštiti. Cilj ovog rada je da se utvrdi zastupljenost Colletotrichum spp. na uskladištenim plodovima jabuke u R. Srbiji, kao i pouzdanost klasičnih i molekularnih metoda za njihovu identifikaciju do nivoa vrste i nižih kategorija. Utvrđivanje filogenetske pozicije i genetičke udaljenosti izolata je takođe svrstano u ciljeve istraživanja. Nadalje, cilj je i da se izdvoje mikroorganizami i etarska ulja koja ispoljavaju antifungalno delovanje na Colletotrichum spp. Zatim, da se za mikroorganizam sa najizraženijim antifungalnim delovanjem definišu uslovi kultivacije (sastav hranljive podloge i trajanje) koji maksimizuju njegovu antifungalnu aktivnost i utvrdi način njegovog delovanja. Utvrđeno je da su Colletotrichum spp. redovno prisutni na uskladištenim plodovima jabuke u Vojvodini i delovima zapadne, centralne i jugoistočne Srbije i da je njihova zastupljenost u odnosu na druge fitopatogene gljive 7,8-10%. Zbog osetljivosti C. gloeosporioides na niske temperature, C. acutatum postaje sve dominantnija vrsta ovog roda na uskladištenim plodovima jabuke. Razlikovanje C. acutatum i C. gloeosporioides je moguće na osnovu fenotipskih karakteristika kolonija (u slučaju hromogenih izolata C. acutatum), dužine konidija, rasta kolonija na 5 i 35°C i brzine rasta kolonija na različitim podlogama. Oblik i širina konidija i optimalna temperatura rasta su nedovoljno pouzdani kriterijumi za identifikaciju do nivoa vrste. PCR metodom uz primenu prajmera specifičnih za vrstu uspešno se identifikuju C. acutatum i C. gloeosporioides. Primenom univerzalnih prajmera ITS1 i ITS4 amplifikuju se rDNK-ITS sekvence ovih izolata. Analizom sekvenci izrađuju se filogenetska stabla visoke stabilnosti i jasno razdvajaju C. acutatum i C. gloeosporioides, a u okviru C. acutatum se odvajaju nehromogeni i hromogeni izolati. S. hygroscopicus, S. aureus, B. cereus, P. aeruginosa i B. subtilis sojevi N146, ST 1/III, Č13 i QST 713 ispoljavaju antifungalnu aktivnost na Colletotrichum spp. in vitro i in vivo. S. hygroscopicus obezbeđuje zaštitu plodova na nivou sa hemijskim fungicidima (trifloksistrobin, boskalid+piraklostrobin, pirimetanil+flukvinkonazol, kaptan). Kultivacijom S. hygroscopicus u podlozi sa 15,07 g/l glicerola, 5,28 g/l ekstrakta kvasca i 0,81 g/l fosfata, u v trajanju 3-4 dana, postiže se maksimalno antifungalno delovanje na Colletotrichum spp. Antifungalno delovanje S. hygroscopicus zasnovano je na produkciji ekstracelularnih, termostabilnih metabolita. Dvomesečno skladištenje trtiranih plodova na 2±0,5°C ne slabi antifungalno delovanje S. hygroscopicus. Etarska ulja origana i timijana ispoljavaju snažno inhibitorno delovanje na Colletotrichum spp.Colletotrichum spp. are significant plant pathogens worldwide in field, as well as on stored fruits. Two species of this genera, C. acutatum and C. gloeosporioides, can occur on apple fruits. The species cause significant economic losses on apple fruits after harvest, during storage, transport and marketing. Scientific community faces confusion in defining precise systematic position of this genera, as well as in identification to the species level and lower categories. Conventional viii phytopathological methods do not provide precise identification to the species level. Thus, molecular approaches are taking the lead in Colletotrichum spp. identification. Post-harvest pathogens are mainly managed by chemical fungicides. Due to adverse toxicological properties and resistance occurrence, use of chemical fungicides is being reduced, and their application after harvest is prohibited in most countries. Therefore, studies regarding detection of microorganisms and naturally synthetized substances with a potential for application in biological control are necessary for sustainable development of agriculture. The aim of this study was to determine frequency of Colletotrichum spp. on stored apple fruits in the Republic of Serbia as well as reliability of conventional and molecular methods in their identification to the species level and lower categories. Determination of phylogenetic position and molecular distance of the isolates were also set as investigation goals. The aim was also to determine microorganisms and essential oils with antifungal activity against Colletotrichum spp. Defining of cultivation conditions (nutrient medium composition and duration) for the microorganism with the most pronounced antifungal activity which maximize its activity and defining its mode of action were also set as goals. It was found that Colletotrichum spp. are commonly present on stored apple fruits in Vojvodina Province and in western, central and southeastern parts of Serbia with a share of 7.8-10% among other phytopathogenic fungi. Due to susceptibility of C. gloeosporioides to low temperatures, C. acutatum is becoming dominant species of this genus on stored apple fruits. Discrimination between C. acutatum and C. gloeosporioides is possible on the basis of phenotypic characteristics of colony (in the case of chromogenus isolates of C. acutatum), conidium length, colony growth at 5 and 35°C and colony growth rate on different media. Conidium shape and width and optimal growth temperature are insufficient criteria for identification to the species level. PCR method using species-specific primers is reliable for identification of C. acutatum and C. gloeosporioides. rDNK-ITS sequences of the isolates can be successfully amplified with universal primers, ITS1 and ITS4. Analyses of the sequences alow construction of highly stable phylogenetic trees with distinctively separated C. acutatum and C. gloeosporioides clades, and also additional clades of non-chromogenum and chromogenum C. acutatum isolates. S. hygroscopicus, S. aureus, B. cereus, P. aeruginosa and B. subtilis strains N146, ST 1/III, Č13 and QST ix 713 exhibit antifungal activity against Colletotrichum spp. in vitro and in vivo. S. hygroscopicus ensures fruit protection at the same level as chemical fungicides (trifloxistrobin, boscalid+pyraclostrobine, pyrimethanil+flukvinconazol, captan). Cultivation of S. hygroscopicus in the medium with 15.07 g/l glycerol, 5.28 g/l yeast extract and 0.81 g/l phosphates, for 3-4 days, ensures maximal antifungal activity against Colletotrichum spp. Antifungal activity of S. hygroscopicus is based on production of extracelular, thermostable metabolites. Two-month storage of treated apple fruits at 2±0.5°C does not reduce antifungal activity of S. hygroscopicus. Oregano and thyme essential oils exhibit strong inhibitory effects on Colletotrichu

Monitoring of colorado potato beetle (Leptinotarsa decemlineata Say) sensitivity to insecticides

Sensitivity of two field populations of Colorado potato beetle (Leptinotarsa decemlineata Say.) - CPB (Vilovo and Čurug) adults to insecticides were determined in 2008 and 2012. Bioassay was performed using screening test that allows rapid assessment of sensitivity of over wintered adults to insecticides. Sensitivity to four insecticides commonly used for CPB control in Serbia (chlorpyrifos, cypermethrin, thiamethoxam and fipronil) was assessed. Insecticides were applied at label rates, and two, five and 10 fold higher rates by soaking method. Insect mortality was assessed after 72 h. Sensitivity was assessed using a modified IRAC method (2009) and 1-5 scale. In 2008, both tested CPB populations were resistant to chlorpyrifos label rate, as well as in 2012. Both tested populations were also resistant to cypermethrin in 2008, while in 2012, population from Vilovo was resistant, and population from Čurug was moderately resistant. Significant differences in CPB sensitivity to thiamethoxam between populations tested in 2008 and 2012 were recorded

In vitro potencijal antagonista Bacillus spp. za suzbijanje fitopatogena Xanthomonas euvesicatoria

Bacterial pathogen Xanthomonas euvesicatoria, principal causer of bacterial spot, represents a significant problem in agricultural practice due to high yield losses in the production of pepper and tomato. The development of resistance to copper pesticides has shifted research, in the field of its suppression, towards biopesticides. In this study, several Bacillus strains were tested against Xanthomonas euvesicatoria strains, isolated from pepper leaves with symptoms of bacterial spot, to select a sufficiently effective antagonist. When it comes to the testing of cultivation broth, containing biomass of tested antagonists, the best results were achieved using isolate Bacillus sp3. On the other hand, when biomass-free supernatants, containing produced antimicrobial compounds, were tested, Bacillus sp1 and Bacillus sp2 have shown the highest antimicrobial activity. The results of this study represent a basis for further development of bioprocess solutions for the production of biopesticides based on Bacillus spp. biomass or antimicrobial compounds, showing high efficiency in suppression of pepper bacterial spot.Bakterijski patogen Xanthomonas euvesicatoria predstavlja glavnog uzročnika bakteriozne pegavosti, stoga predstavlja značajan problem u poljoprivrednoj praksi usled izazivanja velikih gubitaka prinosa u proizvodnji paprike i paradajza. Uobičajena sredstva za suzbijanje i kontrolu ovog fitopatogena predstavljaju preparati na bazi bakra. Međutim, razvoj rezistentnosti prema pesticidima na bazi bakra doveo je do zaokreta u polju suzbijanja ovih patogena ka primeni biopesticida, odnosno bioloških kontrolnih agensa. Bakterije roda Bacillus i njihovi metaboliti sa izraženom antimikrobnom aktivnošću protiv ciljanih fitopatogena predstavljaju najperspektivnije aktivne komponente biokontrolnih preparata za zaštitu bilja. U ovom istraživanju nekoliko sojeva roda Bacillus ispitano je sa ciljem odabira antagonista dovoljno efikasnih u suzbijanju sojeva Xanthomonas euvesicatoria, koji su izolovani sa listova paprike sa simptomima bakteriozne pegavosti. Prilikom testiranja antimikrobne aktivnosti uzoraka kultivacionih tečnosti, koji sadrže i biomasu testiranih antagonista, najbolji rezultati u suzbijanju testiranih fitopatogena su postignuti primenom izolata Bacillus sp3. Sa druge strane, prilikom testiranja antimikrobne aktivnosti supernatanata oslobođenih biomase antagonista, koji sadrže samo produkovana antimikrobna jedinjenja, izolati Bacillus sp1 i Bacillus sp2 su pokazali najveću antimikrobnu aktivnost protiv fitopatogena Xanthomonas euvesicatoria. Rezultati ovog istraživanja predstavljaju osnovu za dalji razvoj bioprocesnih rešenja za proizvodnju biopesticida na bazi biomase ili antimikrobnih jedinjenja koja proizvode antagonisti roda Bacillus, a koji pokazuju visoku efikasnost u suzbijanju bakteriozne pegavosti paprike

Screening of Local Wild Xanthomonas Species for Xanthan Production on Crude Glycerol-based Medium

In this study, the effect of cultivation time on xanthan biosynthesis by different Xanthomonas campestris and Xanthomonas euvesicatoria strains, isolated from crucifers and pepper leaves, respectively, was examined. Xanthan was produced by submerged cultivation on crude glycerol-based medium at a laboratory level under aerobic conditions at 30 °C and 150 rpm for 168 h and 240 h. Bioprocess efficacy was estimated based on the xanthan concentration in media at the end of bioprocess and its average molecular weight. According to the obtained results, Xanthomonas strains have statistically significant effect on xanthan concentration in cultivation media when biosynthesis is performed by X. euvesicatoria strains, and cultivation time has significant effect on this parameter only when bioprocess is performed by X.campestris strains. The combination of Xanthomonas strains and cultivation time has a statistically significant effect on xanthan concentration in medium for both groups of isolates. The obtained results show that all applied Xanthomonas strains and cultivation time as well as their combination have statistically significant effect on average molecular weight of xanthan produced in applied experimental conditions. It is found that X. euvesicatoria strains produce higher amount of xanthan in a shorter period of time (168 h) when compared to the X.campestris strains. Xanthan of higher average molecular weight was produced when cultivation of both groups of isolates was performed for 240 h in applied experimental conditions. Results obtained in this research suggest that X. euvesicatoria strains have the greatest potential for application in biotechnological production of xanthan on crude glycerol-based medium

Selection of antagonists for biocontrol of xanthomonas euvesicatoria

Xanthomonas euvesicatoria is a worldwide causer of pepper bacterial spot, a bacterial plant disease responsible for massive losses of fresh pepper fruits. Considering the current problems in management of bacterial plant diseases, biological control using antagonistic microbial strains with high potential for plant pathogens suppression emerges as a possible solution. The aim of this study was to select suitable antagonists for suppression of X. euvesicatoria among the bacteria, yeast and fungi from the genera Pseudomonas, Lactobacillus, Saccharomyces and Trichoderma, based on in vitro antimicrobial activity testing using the diffusion disc method. The results of this study have revealed that cultivation broth samples of the antagonists Lactobacillus MK3 and Trichoderma reseii QM 9414, as well as supernatant samples of the antagonist Pseudomonas aeruginosa I128, have showed significant potential to be applied in biological control of X. euvesicatoria. Further research would be required to formulate suitable cultivation medium and optimize bioprocess conditions for production of the proposed pepper bacterial spot biocontrol agents

biofungicidi

Agricultural production in developed countries undergoes various changes, some of which take place at consumers’ request, while others are of ethical importance. This is especially true of plant protection. A global demand for reducing the use of chemical pesticides, which are regarded as harmful to the consumer, induce the developement of new, less harmful and sustainable strategies of plant protection. Many chemical pesticides have been excluded from further use (e.g. organochlorine insecticides, methyl bromide) due to their potential risk to human health, the environment and non-target organisms, or developement of resistance of harmful organisms to those substances. A need for developing alternative protection systems in the future is beyond doubt and they should be implemented either as an addition or a substitute for conventional pesticides. Well-considered use of biological products in combination with other protection measures would meet the requirements for producing sanitary and health-safe agricultural products, and food in general. In the paper, we discuss the modes of action, formulation types and applicability of different biological fungicides, and list them individually with their advantages and disadvantages, as well as the production and application risks associated with biological products.Poljoprivredna proizvodnja u razvijenim zemljama podleže raznim promenama, neke su posledica zahteva potrošača a neke su etičkog značaja. Ovo se posebno odnosi na zaštitu bilja. Globalni zahtevi za smanjenje upotrebe pesticida hemijske prirode koji se smatraju štetnim za potrošača uslovljavaju razvoj novih, manje opasnih, održivih strategija u zaštiti bilja. Veliki broj hemijskih pesticida je isključen iz upotrebe (organohlorni insekticidi, metil- bromid), ili zbog potencijalnog rizika po ljudsko zdravlje, zagađenja životne sredine, efekata na neciljane organizme, ili razvoja rezistentnosti kod štetnih organizama na pomenuta jedinjenja. Nema sumnje da postoji potreba za razvojem alternativnih sistema zaštite u budućnosti i da se oni moraju implementirati kao dopuna ili zamena konvencionalnoj primeni pesticida. Osmišljena primena biopreparata u kombinaciji sa drugim merama zaštite, zadovoljila bi zahteve za proizvodnju higijenski ispravnih i zdravstveno bezbednih poljoprivrednih proizvoda i hrane uopšte. U radu su prikazani mehanizmi delovanja, oblici formulacija bioloških fungicida i mogućnosti primene, pregled biofungicida, zatim prednosti i nedostaci, kao i rizici pri proizvodnji i primeni bioloških preparata

Insekticidni efekat mešavina insekticida, fungicida, kompleksnog đubriva i okvašivača zavisno od tvrdoće vode

Simultaneous occurrence of different harmful species in agricultural practice necessitates that different plant protection chemicals be applied at the same time (tank mix). Mix components differ in purpose, mode of action and/or formulation, while addition of nonpesticide components (complex fertilizers, adjuvants and wetting agents) is widely practiced today. However, data concerning the effects of water quality used for preparation of working liquids on the biological effects of pesticides is still scarce. Therefore, the objective of this study was to determine insecticidal effects as depending on components used in mixes and water hardness. The effects of mixtures of thiametoxam (Actara 25-WG 0,07kg/ha) with azoxystrobin (Quadris 0.75 l/ha), mancozeb (Dithane M-70 2.5 kg/ha), a complex fertilizer (Mortonijc plus 3 kg/ha) and a wetting agent (Silwet L-77), depending on the components and water hardness (slightly hard (15.4 d°) - tap water from Novi Sad, and very hard (34.7 d°) - well water from Adica, a Novi Sad suburb), were determined in a bioassay based on adult mortality rate of the first generation of Colorado potato beetle (Leptinotarsa decemlineata Say). The mixtures were applied by a flooding method. The trial was set up to include four replications. Insecticidal effects were determined 24 h and 48 h after exposure. Thiametoxam effectiveness 24 h and 48 h after application in slightly hard water was 100% when the insecticide was applied alone and in double and triple mixes with the fungicides, complex fertilizer and wetting agent, showing no dependency on mix components. The tested adult population of Colorado potato beetle demonstrated high susceptibility to thiametoxam, while the other components had no impact in slightly hard water. In very hard water, 24 h after application, the insecticidal effect had the same level of significance to thiametoxam in double and triple mixes, with an exception of thiametoxam+mancozeb+Mortonijc plus and thiametoxam+mancozeb+wetting agent combinations, which showed significantly lower efficacy. After 48 h, substantially lower effectiveness, in comparison with the sole insecticide and other combinations, was only observed in thiametoksam+mancozeb+Mortonjic plus combination. Significantly lower efficacy of that combination is probably due to an incompatibility of the macozeb preparation and the complex fertilizer containing boron (B), which was evidenced only in very hard water.Pojava različitih štetnih vrsta u poljoprivrednoj proizvodnji u isto vreme, uslovljava istovremenu primenu više sredstava za zaštitu bilja (tank-miks). Komponente u smeši su različite po nameni, delovanju i/ili obliku formulacija, međutim aktuelno je i dodavanje nepesticidnih komponenata (kompleksna đubriva, ađuvanti, okvašivači), ali malo je podataka o uticaju kvaliteta vode za pripremu radnih tečnosti na biološke efekte pesticida. Otuda je cilj rada bio da se odredi insekticidni efekat, zavisno od komponenti u smeši i tvrdoće vode. Efekti mešavina tiametoksama (Actara 25-WG 0,07 kg/ha) sa azoksistrobinom (Quadris 0,75 l/ha), mankozebom (Dithane M-70 2,5 kg/ha), kompleksnim đubrivom (Mortonijc plus 3 kg/ha) i okvašivačem (Silwet L-77), zavisno od komponenti i tvrdoće vode (slabo tvrda/15,4 do/ – česmenska voda iz Novog Sada i jako tvrda /34,7 do/ – bunarska voda iz Adica), određeni su biotestom preko smrtnosti imaga prve generacije krompirove zlatice (Leptinotarsa decemlineata Say.). Mešavine su primenjene metodom potapanja. Ogled je postavljen u četiri ponavljanja. Insekticidni efekat je određen posle 24 i 48h ekspozicije. Efikasnost tiametoksama posle 24 i 48h od primene u slabo tvrdoj vodi, iznosila je 100%, kako pojedinačno primenjenog, tako i u dvojnim i trojnim mešavinama sa fungicidima, komleksnim đubrivom i okvašivačem nezavisno od komponenti u smeši. Ispitivana populacija imaga krompirove zlatice ispoljila je visoku osetljivost prema tiametoksamu, a druge komponente nisu uticale na promenu posle primene u slabo tvrdoj vodi. U jako tvrdoj vodi, posle 24h od primene, insekticidni efekat na istom nivou značajnosti sa tiametoksamom, registrovan je i kod dvojnih i trojnih mešavina, izuzimajući smeše tiametoksam+mankozeb+Mortonijc plus i tiametoksam+mankozeb+okvašivač, gde je efikasnost bila značajno niža. Posle 48h, značajno niža efikasnost u odnosu na sam insekticid i ostale mešavine evidentirana je samo kod mešavine tiametoksam+mankozeb+Mortonijc plus. Značajno smanjena efikasnost pomenute mešavine je verovatno posledica inkompatibilnosti preparata na bazi mankozeba i kompleksnog đubriva koje sadrži bor (B), a ispoljena je samo u jako tvrdoj vodi

Comparative evaluation of insecticides in control of bothynoderes punctiventris germ. Under laboratory and field conditions

Beet weevil (Bothynoderes punctiventris Germ.) is the most damaging pest of sugar beet in south-eastern Europe, from seedling emergence and in the first phases of crop development. Efficacy of insecticides (active ingredients of chlorpyriphos + bifenthrin, chlorpyriphos + beta-cyfluthrin and chlorpyriphos + cypermethrin) for the control of beet weevil was tested during 2010 and 2011 under laboratory and field conditions. A wet filter paper method (contact action) was employed in laboratory tests. Simultaneously, field trials were conducted (Curug, Rimski Sancevi, Budisava, Kovilj) (contact and digestive action). The trial was designed according to EPPO method and insecticide efficacy was tested in accordance with the pest biology and phenophase of the crop. Insecticide efficacy was calculated using Abbot's formula, and damage of plants was assessed using 0-5 scale. Commercial insecticide Nurelle D (active ingredient of chlorpyriphos + cypermethrin) maintained high contact and digestive action on beet weevil, although it has been in use for 20 years. Commercial insecticide Pyrinex Super (active ingredient of chlorpyriphos + bifenthrin), although with reduced content of chlorpyriphos by 30% and of bifenthrin by 20%, achieved efficacy that was at the same level of significance with Nurelle-D, regardless of the experimental conditions (laboratory or field). Compared to Nurelle-D, the product MCW 784 (chlorpyriphos + beta-cyfluthrin) had significantly lower initial efficacy 3 h after application in laboratory trial, and also 24 h after application in field trial

Upotreba komorača u organskoj poljoprivredi

Fennel (Foeniculum vulgare Mill.) originated from Mediterranean area. His ap­pearance is very similar to dill, but unlike it has a sweet taste and odor similar to anise. Fennel has two varieties: var. vulgare also called bitter, and var. dulce or sweet fennel. In organic farming has a much greater significance var. vulgare, which is a perennial species that are grown for fruit. The essential oil of the fruit has antifungal, virostatic, insecticidal and nematicidal properties, and is therefore very suitable for the synthesis of potential biopesticides that could have wide application in organic agriculture. Fennel in flowering produces large quantities of pollen and nectar, so it attracts a large number of beneficial insects for which it is often used for joint planting with other plants, but also as a buffer in organic agriculture. This plant has long been known as a healing, both in human medicine and in veterinary medicine and animal nutrition. Mainly used as a natural antibiotic and stimulator of growth, particularly in poultry, but has great significance in beekeeping, fish and sheep breeding.Komorač (Foeniculum vulgare Mill.) potiče iz Sredozemlja. Izgled mu je veo­ma sličan mirođiji, ali za razliku od nje ima sladak ukus i miris sličan anisu. Komorač ima dva varijeteta: var. vulgare koji se još naziva gorki, i var. dulce ili slatki komorač. Za organsku poljoprivredu mnogo veći značaj ima var. vulgare, koji je višegodišnja vrsta koja se gaji zbog plodova. Etarsko ulje plodova poseduje antifungalne, virostatičke, insekticidne i nematocidne osobine, te je stoga veoma pogodano za sintezu potencijalnih biopesticida koji bi mogli da imaju široku primenu u organskoj poljoprivredi. Komorač u cvetanju produkuje veliku količinu polena i nektara, pa privlači veliki broj korisnih insekata, zbog čega se često ko­risti za združenu setvu sa drugim biljkama, ali i kao zaštitni pojas u organskoj poljoprivredi. Ova biljka je od davnina poznata i kao lekovita, kako u humanoj medicini, tako i u veterini i ishrani domaćih životinja. Uglavnom se koristi kao prirodni antibiotik i stimulator rasta, posebno u živinarstvu, ali ima veliki značaj i u pčelarstvu, ribarstvu i ovčarstvu

Molekularna detekcija Monilinia fructigena prouzrokovača truleži ploda dunje

Species of the genus Monilinia are important causal agents of fruit rot on pome and stone fruits in Serbia. The pathogen is very harmful, especially on small properties and cottage plantations where intensive control measures are not applied. Quince is important host for the pathogens of this genus. During spring 2010, intensive occurrence of mummified fruits overwintering on branches of the quince was observed. The pathogen was isolated using standard phytopathological methods. Pathogenicity of eight obtained isolates was tested by artificial inoculation of injured apple fruits. Identification was performed according to pathogenic, morphological and ecological properties, and was confirmed by Multiplex polimeraze chain reaction, PCR. All the isolates studied caused brown rot on inoculated apple fruits. The isolates form light yellow colonies with lobate margins, with single-celled, transparent, elliptical or oval conidia in chains, regardless temperature or light presence. Sclerotia are observed in 14 days old cultures. The highest growth rate of most of the isolates is at 27°C and in dark. Based on studied pathogenic, morphological and ecological characteristics, it was found that the Monilinia fructigena is causal agent of brown rot of quince. Using specific primers (MO368-5, MO368-8R, MO368-10R, Laxa-R2) for detection of Monilinia species in Multiplex PCR reaction, the expected fragment 402 bp in size was amplified, which confirmed that the studied isolates belonged to the species M. fructigena.Vrste roda Monilinia su značajni prouzrokovači truleži ploda jabučastog i koštičavog voća u našoj zemlji. Parazit nanosi velike štete, posebno na malim posedima i u vikend zasadima u kojima se ne primenjuje redovna zaštita. Dunja je značajan domaćin patogena ovog roda. Tokom proleća 2010. godine ustanovljena je intenzivnija pojava mumificiranih plodova prezimelih na granama. Cilj ovog rada bio je da se identifikuje prouzrokovač mumifikacije plodova dunje, prikupljenih sa različitih lokaliteta. Patogen je izolovan primenom standardnih fitopatoloških metoda. Patogenost osam dobijenih izolata proverena je veštačkom inokulacijom povređenih plodova jabuke. Identifikacija je obavljena na osnovu patogenih, morfoloških i ekoloških osobina, a potvrđena je primenom Multiplex PCR. Svi proučavani izolati su na inokulisanim plodovima jabuke prouzrokovali trulež smeđe boje. Proučavani izolati na KDA podlozi formiraju svetlo-žute kolonije režnjevitog oboda. U kulturi gljiva, nezavisno od uticaja temperature i prisustva ili odsustva svetlosti, formira jednoćelijske, providne, eliptične ili ovalne konidije u nizovima. Sklerocije se uočavaju u kulturama starosti 14 dana. Većina izolata ostvaruje najbolji porast na temperaturi od 27°C i u odsustvu svetlosti. Na osnovu proučenih patogenih, morfoloških i odgajivačkih odlika, utvrđeno je da izolati pripadaju vrsti Monilinia fructigena. Korišćenjem specifičnih prajmera (MO368-5, MO368-8R, MO368-10R, Laxa-R2) za detekciju vrsta roda Monilinia u Multiplex PCR reakciji, amplifikovan je očekivani fragment veličine oko 402 bp čime je potvrđeno da proučavani izolati pripadaju vrsti M. fructigena