Characterization of Моnilinia species pathogens of stone fruits in Serbia and sensitivity to fungicides

Prikupljanje obolelih delova koštičavih voćaka sa simptomima sušenja cvetova, grana i grančica i mrke truleži plodova, koje prouzrokuju vrste roda Monilinia, obavljeno je tokom trogodišnjeg perioda od 2010. do 2012. godine. Pregledom 119 lokaliteta iz 15 okruga u Srbiji, prikupljen je 321 uzorak obolelih biljnih delova kako iz komercijalnih zasada, tako i iz okućnica i sa zelenih pijaca. Dobijeno je ukupno 246 monosporijalnih izolata sa morfološkim odlikama vrsta roda Monilinia. Sa ciljem utvrđivanja rasprostranjenosti vrsta roda Monilinia i rasvetljavanja etiologije oboljenja u našoj zemlji, izvršena je identifikacija dobijenih izolata do nivoa vrste na osnovu patogenih, morfoloških, odgajivačkih i ekoloških karakteristika, sa jedne strane, i molekularnih karakteristika, sa druge strane. Na osnovu svih proučenih karakteristika utvrđeno je prisustvo tri različite vrste: M. laxa, M. fructigena i M. fructicola. Dominantan prouzrokovač mrke truleži plodova i sušenja cvetova, grana i grančica koštičavih voćaka u Srbiji je M. laxa (prisutna u 96,34% uzoraka), dok je zastupljenost ostalih vrsta ovog roda znatno manja – M. fructigena (2,44% uzoraka) i M. fructicola (1,22% uzoraka). Po prvi put u Srbiji, detektovano je prisustvo M. fructicola, patogena sa IA dela I Liste karantinskih štetnih organizama, na koštičavim voćkama u našoj zemlji. Analiza SSR markera pokazala je da izolati M. fructicola iz Srbije pripadaju haplotipu iz Italije, što ukazuje na mogući put introdukcije ovog karantinskog patogena u Srbiju. Tri vrste roda Monilinia mogu se jasno razdvojiti na osnovu kompleksa morfoloških karaktera koji obuhvata: izgled, oblik i boju kolonije, prisustvo spora i koncentričnog prstena spora u kulturi, brzinu porasta micelije, kao i veličinu konidija, načina klijanja konidija i dužinu kličine cevčice pre prvog grananja. Međutim, za pouzdanu i brzu detekciju može se preporučiti Multiplex PCR metoda koja je prilagođena i uspešno primenjena za specifično dokazivanje prisustva Monilinia spp. na koštičavim voćkama u Srbiji...The diseased plant parts of stone fruits expressing symptoms of blossom, branch and twig blight and fruit brown rot, caused by the species from the genus Monilinia, were collected during a three-year period (from 2010 to 2012). Screening of 119 localities within 15 districts in Serbia was completed; 321 samples of diseased plant parts from commercial orchards, as well as from house yards and green markets, were collected. In total, 246 monosporial isolates with morphological characteristics resembling to those of Monilinia spp. were obtained. These isolates were identified at the species level based on their pathogenic, morphological, cultural and ecological characteristics; however, identification based on their molecular characteristics was also performed. This two-level identification was conducted in order to determine the distribution of the Monilinia species in Serbia and to clarify etiology of the disease they cause. According to their characteristics, the presence of three different species was determined: M. laxa, M. fructigena and M. fructicola. The dominant causal agent of fruit brown rot and blossom, branch and twig blight of stone fruits in Serbia was M. laxa (it was present in 96.34% samples) while the presence of M. fructigena (2.44% samples) and M. fructicola (1.22% samples) was smaller. It was the first time in Serbia that M. fructicola, a pathogen from IA part of the List I of quarantine pest organisms on stone fruit species in our country, was registered. Moreover, the analysis of SSR markers showed that M. fructicola isolates originating from Serbia belonged to the same haplotype as the isolates originating from Italy, which indicated a possible path of introduction of this species to Serbia. Three species of Monilinia genus could be clearly distinguished on the basis of the complexity of morphological characters (colony appearance, shape and colour, presence of spores and concentric ring of spores in a culture, mycelial growth rate, conidia size, mode of conidia germination and length of germ tube). However, Multiplex PCR method adjasted and successfully applied for specific attestation of presence of Monilinia spp. on stone fruit species in Serbia, can be recommended for reliable and rapid detection..

Rod Botrytis i vrsta Botrytis cinerea: Patogene, morfološke i epidemiološke karakteristike

Species of the genus Botrytis occur wherever their hosts are grown, ranging from cold areas of Alaska to warm and dry areas in Israel. They have a necrotrophic life style which is often associated with phenology of the host plant. The genus comprises 22 species, most of which have a narrow host range. Polifagous species Botritys cinerea, a causal agent of grey mould disease, is the most important and the most extensively studied representative of this genus. More than 350 papers related to all aspects of the research of this necrotrophic pathogen are published each year. In this paper up-to-date knowledge about pathogenic, morphological and epidemic characteristics of the genus Botrytis and, particularly, species B. cinerea are summarized. Symptoms caused by B. cinerea on various plant species and various plant parts are shown. Morphological and genetic variability of the species is described. The possible mechanisms of variability, as well as the attempts to divide the species into Group I (B. 'pseudocinerea') and Group II (B. cinerea 'sensu-stricto') are pointed out.Vrste roda Botrytis rasprostranjene su svuda gde se gaje ili su prisutne njihove biljke-domaćini - od hladnih zona Aljaske do toplih i suvih područja u Izraelu. Imaju nekrotrofni životni ciklus koji je najčešće tesno povezan sa fenologijom domaćina. Rod obuhvata 22 vrste od kojih većina ima uzak krug domaćina - parazitira biljke jednog roda ili jedne familije. Polifagna vrsta Botrytis cinerea, prouzrokovač sive truleži, najznačajniji je i najproučavaniji predstavnik ovog roda. Godišnje se objavi više od 350 radova koji se odnose na sve aspekte istraživanja ovog nekrotrofnog patogena. U ovom radu je dat pregled dosadašnjih saznanja o patogenim, morfološkim i epidemiološkim karakteristikama vrsta roda Botrytis, a posebno vrste B. cinerea. Detaljno su obrađeni simptomi oboljenja koje prouzrokuje B. cinerea na različitim biljnim vrstama i različitim delovima biljaka, morfološka i genetička varijabilnost vrste. Takođe, ukazano je i na moguće izvore varijabilnosti, kao i na pokušaje podele vrste na dve grupe - Grupu I (B. 'pseudocinerea') i Grupu II (B. cinerea 'sensu-stricto')

Botrytis squamosa - prouzrokovač lisne pegavosti luka u Bosni i Herzegovini

Over the past several decades, necrotic spots, lesions and blight symptoms have been observed on onion leaves in several locations in Bosnia and Herzegovina, where the crop is grown intensively. The type of symptoms indicated a possible infection with Botrytis squamosa, a widespread pathogen of onion. As symptoms of leaf spots and necrotic lesions can also be caused by some other biotic and abiotic factors, our research focused on identifying the causal agent of the observed symptoms. The pathogen was isolated from diseased tissue using standard phytopathological procedure and identified based on pathogenic and morphological features. Identification was confirmed by amplification and sequencing of the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA). The influence of temperature and growth medium on mycelial growth rate of the isolates was also studied.U poslednjih nekoliko godina, na nekoliko lokaliteta u području intenzivne proizvodnje crnog luka u Bosni i Hercegovini uočena je pojava nekrotičnih pega i sušenja lišća. Simptomi oboljenja ukazali su na moguće prisustvo široko rasprostranjenog patogena luka, vrste Botrytis squamosa. S obzirom da pegavost i nekrotične lezije na listu luka mogu biti prouzrokovane različitim biotičkim i abiotičkim faktorima, cilj ovog istraživanja bio je da se precizno identifikuje prouzrokovač oboljenja. Patogen je izolovan primenom standardnih fitopatoloških metoda i identifikovan na osnovu proučenih patogenih i morfoloških karakteristika dobijenih izolata. Identifikacija je potvrđena amplifikacijom i sekvenciranjem ITS rDNA genomnog regiona. Takođe, proučen je uticaj temperature i hranljive podloge na porast izolata

Integralni pristup u suzbijanju najvažnijih prouzrokovača bolesti cveća u zaštićenom prostoru

Disease causal agents are serious treat for the production of ornamentals under both open fi led and protected cultivation. Success in the disease prevention depends mainly on the level of knowledge on the specific requirements of the host plants, biology of the pathogen, disease epidemiology and the relationship between the parasite and the host plant. The selection of effective control measures is based on the comprehensive knowledge on all the factors contributing directly or indirectly to the disease development. The most important disease causal agents of ornamental plants under protected cultivation, required environmental conditions for disease development and cost-effective and environmentally safe control measures are discussed in this paper.Prouzrokovači bolesti predstavljaju veliki problem u proizvodnji cveća, kako na otvorenom polju, tako i u zaštićenom prostoru. Uspeh u sprečavanju pojave i razvoja oboljenja u velikoj meri zavisi od stepena poznavanja specifi čnih zahteva biljke domaćina, biologije patogena, epidemiologije bolesti i odnosa parazit i biljka- domaćin. Samo na osnovu dobrog poznavanja svih činilaca koji direktno ili indirektno utiču na razvoj oboljenja moguće je primeniti kompleks mera kojima se sprečavaju štete. U radu je dat pregled najvažnijih prouzrokovača bolesti cveća u zaštićenom prostoru, uslovi u kojima nanose štete, mera koje je neophodno primeniti sa ciljem njihovog efikasnog, ekološki i ekonomski prihvatljivog suzbijanja, kao i mera koje umanjuju rizik razvoja rezistentnosti na fungicide

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

First Report of Brown Rot Caused by Monilinia fructicola on Nectarine in Serbia

In August 2011, nectarine (Prunus persica (L.) Batsch var. nucipersica (Suckow) C. K. Schneid) fruit originated from Oplenac region with symptoms of fruit rot was collected at a green market in Belgrade. Fruit had large, brown, sunken lesions covered with grayish brown tufts. Symptoms resembled those caused by species of Monilinia including M. laxa, M. fructigena, or M. fructicola (2). In order to isolate the causal organism, small superficial fragments of pericarp were superficially disinfected with commercial bleach and placed on potato dextrose agar (PDA). The majority (32 out of 33) isolates formed rosetted non-sporulating colonies with lobed margins resembling those of M. laxa. However, one isolate (Npgm) produced an abundant, grayish-white colony with even margins and concentric rings of sporogenous mycelium, resembling those described for M. fructicola (2). Conidia were one-celled, hyaline, ellipsoid to lemon shaped, 7.38 to 14.76 × 4.92 to 9.84 μm, and borne in branched monilioid chains. The average daily growth on PDA at 24°C was 10.9 mm. A single-spore isolate of Npgm was identified as M. fructicola based on the morphology of colony and conidia, temperature requirements, and growth rate (2). Morphological identification was confirmed by an amplified product of 535 bp using genomic DNA extracted from the mycelium of pure culture and species-specific PCR for the detection of M. fructicola (2). The ribosomal internal transcribed spacer (ITS) region of rDNA of Npgm was amplified and sequenced using primers ITS1/ITS4. Sequence analysis of ITS region revealed 100% nucleotide identity between the isolate Npgm (GenBank Accession No. JX127303) and 17 isolates of M. fructicola from different parts of the world, including four from Europe (FJ411109, FJ411110, GU967379, JN176564). Pathogenicity of the isolate Npgm was confirmed by inoculating five surface-disinfected mature nectarine and five apple fruits by placing a mycelial plug under the wounded skin of the fruit. Nectarine and apple fruits inoculated with sterile PDA plugs served as a negative controls. After a 3-day incubation at 22°C, inoculated sites developed brown lesions and the pathogen was succesfully reisolated. There were no symptoms on the control nectarine or apple fruits. M. fructicola is commonly present in Asia, North and South America, New Zealand, and Australia, while in the EPPO Region the pathogen is listed as an A2 quarantine organism (3). In Europe, the first discovery of M. fructicola was reported in France and since then, it has been found in Hungary, Switzerland, the Czech Republic, Spain, Slovenia, Italy, Austria, Poland, Romania, Germany, and Slovakia (1). Most recently, M. fructicola was found on stored apple fruits in Serbia (4). To our knowledge, this is the first report of M. fructicola decaying peach fruit in Serbia. These findings suggest that the pathogen is spreading on its principal host plants and causing substantial economic losses in the Serbian fruit production. References: (1) R. Baker et al. European Food Safety Authority. Online publication. www.efsa.europa.eu/efsajournal . EFSA J. 9:2119, 2011. (2) M. J. Côté. Plant Dis. 88:1219, 2004. (3) OEPP/EPPO. EPPO A2 list of pests recommended for regulation as quarantine pests. Version 2009-09. http://www.eppo.org/QUARANTINE/listA2.htm . (4). M. Vasic et al. Plant Dis. 96:456, 2012. </jats:p

Suzbijanje Botrytis cinerea i problem rezistentnosti na fungicide

Botrytis cinerea, the causal agent of grey mould, greatly affects fruit, grapevine, vegetable and ornamental crops production. It is a common causal agent of diseases in plants grown in protected areas, as well as fruit decay during storage and transport. The fungus invades almost all parts of the plant in all developmental stages, and the symptoms are usually described as grey mould, grey mildew, brown rot and seedling blight. The paper reviews the current knowledge on control possibilities of this necrotrophic pathogen. The attention is particularly paid to the mode of action of novel fungicides and to the problem of resistance. It is pointed out that by limiting the number of treatments in the growing season, avoiding the use of only one fungicide with a high risk for resistance development, appropriate application rate and timing, using mixtures of pesticides with different modes of action, as well as by alternative use of pesticides from different resistance groups, a longterm preservation of pesticide efficacy is provided.Botrytis cinerea, prouzrokovač sive truleži, značajno ugrožava proizvodnju voća, grožđa, povrća i ukrasnog bilja. Često izaziva bolesti biljaka gajenih u zaštićenom prostoru, kao i trulež plodova tokom skladištenja i transporta. Gljiva parazitira skoro sve delove biljke u svim fazama razvoja, a simptomi se najčešće opisuju kao siva trulež, siva plesan, palež, mrka trulež, a zapaženo je i poleganje klijanaca. U radu je dat pregled dosadašnjih saznanja o mogućnostima suzbijanja ovog nekrotrofnog patogena. Posebna pažnja je posvećena mehanizmu delovanja fungicida novije generacije kao i problemu rezistentnosti. Istaknuto je da se ograničenjem broja tretiranja u toku jedne vegetacije, izbegavanjem upotrebe samo jednog fungicida visokog rizika za razvoj rezistentnosti, odgovarajućom dozom i vremenom primene, upotrebom mešavina pesticida različitog mehanizma delovanja kao i alternativnom primenom pesticida iz različitih rezistentnih grupa omogućuje dugotrajno očuvanje efikasnosti pesticida

Da li je niska efikasnost fungicida uvek posledica razvoja rezistentnosti u populacijama patogena?

Efficacy of four fungicides with different modes of action (vinclozolin, pyrimethanil, benomyl and fenhexamid) in control of B. cinerea in raspberry, was investigated in the paper. The trials were conducted at two localities in commercial raspberry plantations. In the case of unsatisfactory fungicide efficacy, qualitative and/or quantitative test of the susceptibility of the isolates to particular fungicide was performed, to determine whether the low efficacy is a consequence of resistance development in the pathogen population. At both localities, pyrimethanil and fenhexamid demonstrated the highest efficacy (73.2-89.6%), while the efficacy of vinclozolin was statistically significantly lower (48.7-63.4%) at both localities. However, qualitative and quantitative test of susceptibility to vinclozolin showed that all the isolates were susceptible to vinclozolin and that the reason for unsatisfactory efficacy should be primarily sought in inadequate fungicide application.U radu je ispitivana efikasnost četiri fungicida različitog mehanizma delovanja (vinklozolin, pirimetanil, benomil i fenheksamid) u zaštiti maline od B. cinerea. Ogledi su izvedeni na dva lokaliteta u komercijalnim zasadima maline. U slučajevima nezadovoljavajuće efikasnosti fungicida, kvalitativnim i/ili kvantitativnim testom osetljivosti izolata in vitro, utvrđeno je da li je niska efikasnost posledica razvoja rezistentnosti populacije patogena. Na oba lokaliteta, najveću efikasnost su ispoljili pirimetanil i fenheksamid (73,2-89,6%), dok je efikasnost vinklozolina bila statistički značajno niža (48,7-63,4%). Međutim, kvalitativni i kvantitativni test osetljivosti na vinklozolin je pokazao da su svi izolati u kategoriji osetljivih na vinklozolin, te da razlog za nezadovoljavajuću efikasnost ovog fungicida treba tražiti pre svega u neadekvatnoj primeni fungicida

Botrytis squamosa - patogen crnog luka (Allium cepa)

Leaf blight disease, caused by Botrytis squamosa, is a serious treat for green onion and bulb production in numerous production regions. The paper summarizes current knowledge about the disease causal agent, symptoms that develop on plants, epidemiology of the disease and the possibilities of control. Survival of the pathogen during winter, inoculum sources, conditions for infection establishment and the development of the disease symptoms are described in detail. In addition, production losses caused by the disease, as well as cultural an chemical control measures are presented.Pegavost i sušenje lista luka, koju prouzrokuje Botrytis squamosa, predstavlja veliki problem u proizvodnji mladog luka i crnog luka za proizvodnju glavica u većini proizvodnih regiona sveta. U radu su sistematizovana dosadašnja saznanja o prouzrokovaču oboljenja, simptomima koje prouzrokuje na biljkama, epidemiologiji bolesti i mogućnostima suzbijanja. Detaljno je opisano održavanje patogena tokom zime, izvori inokuluma, uslovi za ostvarenje infekcije, razvoj simptoma oboljenja, štete koje prouzrokuje, kao i agrotehničke i hemijske mere suzbijanja

Effects of 1-MCP and dynamic controlled atmosphere on apple fruit rot caused by Fusarium avenaceum

Fusarium species are increasingly detected as the causal agents of decay of stored apple fruits. Fusarium avenaceum is particularly significant due to its predominant occurrence among Fusarium species in stored apple fruits and its ability to produce mycotoxins. Treatments with 1-methylcyclopropene (1-MCP) and different storage conditions affect the aggressiveness of F. avenaceum and development of fungal-caused decay in stored apple fruits. In this study, apple fruits (cv. 'Granny Smith') were treated with 1-MCP, and artificially inoculated with F. avenaceum. The isolate used for inoculation, originating from apple fruit, was identified based on morphological characteristics and by polymerase chain reaction (PCR) using a species-specific primer pair (FA-ITSF and FA-ITSR) for F. avenaceum. After inoculation, treated and untreated fruits were stored at room temperature and cold-stored under dynamic controlled atmosphere (DCA). Diameters of necrotic lesions were measured after 7, 14 and 21 days of incubation on fruits stored at room temperature, while necrosis diameters on DCA-stored fruits were measured immediately at the end of storage period (143 days), and after 7, 14 and 21 days of additional incubation at room temperature. The results show that treatment with 1-MCP inhibits the development of F. avenaceum on apple fruits during storage under DCA. However, after storage, i.e. during incubation at room temperature, no significant difference between 1-MCP-treated and untreated fruits was observed. On fruits stored at room temperature only, no difference between 1-MCP-treated and untreated fruits was observed. However, 1-MCP-treated fruits stored at room temperature only developed significantly smaller necrosis lesions compared to 1-MCP-treated and DCA stored fruits. It infers that both 1-MCP treatment and DCA storage inhibit fungal decay caused by F. avenaceum on apple fruits. However, the effects do not persist after storage