Tõrjemeetmete tegevuskava ohtlikele kultiveerimismaterjali kahjustajatele punavöötaudile ja pruunvöötaudile : kokkuvõttev aruanne

Invasiivsete patogeenide koloniseerimisprotsessi on vallandanud peamiselt kliima soojenemine ja kasvav majanduse globaliseerumine. Viimasel aastakümnel on Balti- ja Põhjamaades dokumenteeritud ligi tosin invasiivset puude haiguse tekitajat, sealhulgas ka männi okkahaigused pruunvöötaud (Lecanosticta acicola (Thümen) A. Sydow) ja punavöötaud (tekitajad Dothistroma septosporum (Dorog.) M. Morelet ja Dothistroma pini Hulbary). Käesoleva töö eesmärk on saada tegevusjuhised kultiveerimismaterjali ohtlike kahjustajate punavöötaudi ja pruunvöötaudi ennetamiseks või siis juhuks, kui nimetatud kahjustajad on avastatud kultiveerimismaterjali tootvas ja importivas taimlas. Punavöötaudi tekitaja kottseen Dothistroma septosporum (varasemad nimed Mycosphaerella pini, Scirrhia pini) ja D. pini ning pruunvöötaudi tekitaja kottseen Lecanosticta acicola (varasemad nimed Mycosphaerella dearnessii, Scirrhia acicola, Lecanosticta pini) on ohtlike kahjustajate nimekirjas juhul, kui need esinevad männi Pinus L. istutamiseks ette nähtud taimedel, välja arvatud seemned (Nõukogu direktiivi 2000/29/EÜ II lisa). Ohtlike kahjustajate levitamine on keelatud ning nende leviku piiramiseks tuleb rakendada tõrjeabinõusid. Töös antakse põhjalik Eesti teadlaste-patoloogide poolt tehtud tööde baasil ning asjakohasel teaduskirjandusel baseeruv ülevaade puna- ja pruunvöötaudide tekitajate bioloogiast, ökoloogiast, levikust nende sümptomitest ja diagnostikast ning võimalikest tõrjeabinõudest üldiselt ja siis kui taimlas on avastatud eelpoolnimetatud ohtlik kahjustaja. Kirjeldatud on riske importiva taimla ja ainult kodumist materjali kasutava taimla vahel. Lisaks on analüüsitud majanduslikku kahju ning kahju minimeerimise võimalusi taimlates

First report of Lecanosticta acicola on pine and non-pine hosts in Turkey

Acknowledgements We would like to thank İstanbul Bahçeköy Forestry Enterprise for allowing us to sample in the Arboretum, and to Merve Kartaloğlu Sönmez the manager of the Arboretum, for her hospitality. This study was partly supported by the Estonian Science Foundation grant PSG136, the Ministry of Rural Affairs of Estonia, and the Euphresco projects; Lecanosticta - Brown spot disease of pines – spread in European forest ecosystems: impact on pines, predisposing and contributing factors, control (BROWNSPOTRISK) and the International Plant Sentinel Network as an early warning system; research on future pest threats (IPSN II).Peer reviewedPostprin

Keskkonnainvesteeringute Keskuse (leping nr. 3-2_8/5671-5/2016) 2016.a. metsanduse programmi projekti nr. 12391

Vastavalt Eesti Maaülikooli (EMÜ) ja SA Keskkonnainvesteeringute Keskuse (KIK) vahel 30. novembril 2016. aastal sõlmitud lepingule nr. 3-2_8/5671-5/2016 teostati 2016.-2018. aastal olulisimate juuremädanike tekitajate leviku ja kahjustuse uuringud okaspuupuistutes. Projekti vastutavaks täitjaks: Eesti Maaülikooli metsandus- ja maaehitusinstituudi metsakasvatuse- ja metsaökoloogia õppetooli teadur Tiia Drenkhan ning projekti täitjateks Eesti Maaülikooli metsakasvatuse- ja metasökoloogia õppetooli laborant Katrin Jürimaa, doktorant Marili Laas, teadur Kalev Adamson ja dotsent Rein Drenkhan. Juurepessu kahjustuse modelleerimise mudelid koostas metsakorralduse- ja metsatööstuse osakonna teadur Allar Padari. Käesolev projekt on jätkuprojekt KIKi projektile nr. 3698 (2012-2014) „Olulisemate juuremädanike tekitajate leviku ja kahjustuste uuring Eestis“, mille tulemustes toodi välja võimalik kahjude hinnang sõltuvalt puistu keskmisest vanusest viljakate kuuse- ja männienamusega puistutes ning esitati mudelid juuremädanike ja eelkõige juurepessu kahjustuste (leviku) modelleerimiseks. Projekti esimeses järgus koguti proove vaid uuendusraie puistutest, seetõttu oli mudeli prognoosi täpsuse parandamiseks vajalik koguda lisaproove ka noorendike ja harvendusraie puistutest. Seda eesmärki silmas pidades koguti käesoleva projekti raames proove erineva vanusega männipuistutest: noorendikud, harvendusraie ja uuendusraie alad. Aruandes esitatakse hariliku männi andmestik, hariliku kuuse erinevates vanusevahemikus (1-20; 21-40; 41-60; 61-80; 81-100 ja üle 100) kogutud proovide tulemusi analüüsitakse ning esitatakse vastavad andmed RMK projekti „Kuusikute raieaja ja raieviiside mõju patogeenide levikule ja arvukusele ning puistu elurikkusele viljakates kasvukohatüüpides“ (kestvus 01.09.2016-31.08.2019) raames, mille põhitäitjate hulgas on ka käesoleva aruande autorid (R. Drenkhan, A. Padari, T. Drenkhan). Käesoleva projekti eesmärgiks on olulisemate juuremädanike tekitajate (juurepess ja külmaseen) leviku ulatuse ja liigilise koosseisu ning esinemise sageduse uurimine viljakates kasvukohatüüpides ning juurepessu kahjustuste modelleerimise mudeli täiendamine hariliku männi puistutest kogutud andmete põhjal. Okaspuupuistutes, mis moodustavad enamuse meie metsadest ja annavad olulisema osa saadavast tarbepuistust, põhjustavad juuremädanikud enim kahjustusi. Hinnanguliselt kaotab üksnes juurepessu tõttu metsaomanik kuni kolmandiku potentsiaalsest metsatulust ning kahju kogu Euroopas ulatub ca 790 miljoni euroni aastas (Woodward et al., 1998). Lisaks tuleb arvestada patogeenide võimalikke kahjutusi kliimamuutuste kontekstis, sest ilmastikus toimuvad muutused soodustavad patogeenide levikut. Projekti tulemused on olulised rahvusvahelises koostöös, näiteks juurepessu geneetilise struktuuri uuringute osas. Juurepessu geneetilise struktuuri tundmine on oluline efektiivsema tõrjestrateegia välja töötamiseks, see annab informatsiooni seene geneetilisest mitmekesisusest ning aitab hinnata patogeeni levikupotentsiaali ja elujõulisust. Tulemuste analüüsimisel on oluline taustainfona silmas pidada eelmise metsapõlve kändude nakkuskoormust noorendikes (so. mädaniku olemasolu kändudel) ning mädaniku ulatust raiekändudel (harvendusraie, uuendusraie alad). Käesoleva projekti tulemuste põhjal ei esitata konkreetseid numbreid juuremädanike poolt tekitatava majandusliku kahju suuruse kohta. Küll aga antakse hinnang sõltuvalt puistu vanusest ja metsa arenguklassist ning raieviisist männienamusega puistutes

Diversity, migration routes, and worldwide population genetic structure of Lecanosticta acicola, the causal agent of brown spot needle blight

Lecanosticta acicola is a pine needle pathogen causing brown spot needle blight that results in premature needle shedding with considerable damage described in North America, Europe, and Asia. Microsatellite and mating type markers were used to study the population genetics, migration history, and reproduction mode of the pathogen, based on a collection of 650 isolates from 27 countries and 26 hosts across the range of L. acicola. The presence of L. acicola in Georgia was confirmed in this study. Migration analyses indicate there have been several introduction events from North America into Europe. However, some of the source populations still appear to remain unknown. The populations in Croatia and western Asia appear to originate from genetically similar populations in North America. Intercontinental movement of the pathogen was reflected in an identical haplotype occurring on two continents, in North America (Canada) and Europe (Germany). Several shared haplotypes between European populations further suggests more local pathogen movement between countries. Moreover, migration analyses indicate that the populations in northern Europe originate from more established populations in central Europe. Overall, the highest genetic diversity was observed in south-eastern USA. In Europe, the highest diversity was observed in France, where the presence of both known pathogen lineages was recorded. Less than half of the observed populations contained mating types in equal proportions. Although there is evidence of some sexual reproduction taking place, the pathogen spreads predominantly asexually and through anthropogenic activity

Population genetic analysis of invasive disease agent Lecanosticta acicola

Magistritöö Metsamajanduse õppekavalL. acicola on Euroopas karantiinne patogeen, mis põhjustab paljudel männiliikidel varajast okaste varisemist. Ligi kümme aastat peale esmaleidu Eestist on see invasiivne patogeen levinud hajusalt kogu riigis, nakatades peamiselt mägimändi (Pinus mugo), mis on populaarne liik haljastuses. Seni on teadmata patogeeni geneetiline mitmekesisus Eestis, mis võimaldaks hinnata selle populatsiooni elujõulisust, patogeensust ning levikuteid. Magistritöö eesmärkideks on (1) dokumenteerida patogeeni geneetiline mitmekesisus Eestis, (2) hinnata sugulise paljunemise toimumise võimalikkust, (3) uurida patogeeni levikut Eestis ja analüüsida võimalikku antropogeenset mõju sellele ja (4) analüüsida patogeeni populatsioonigeneetilisi erinevusi kahel peremeestaimel: P. mugo ja P. sylvestris. Perioodil juuli 2015 kuni jaanuar 2017 koguti sümptomaatilisi okkaproove, millest patogeen isoleeriti puhaskultuuri molekulaarseteks uuringuteks. Lisaks kasutati isolaate varasematest L. acicola leidudest Eestis. Isolaadid jaotati populatsioonidesse nende geograafilise asukoha järgi. Kokku kasutati populatsioonianalüüsiks 104 isolaati, mida analüüsiti kasutades paarumistüüpide- ja mikrosatelliitide markereid. Andmeanalüüsiks kasutati programme GenAlex, GeneMapper ja Structure. Eesti L. acicola populatsiooni iseloomustab vähene geneetiline mitmekesisus ja suur kloonide osakaal. 104-lt isolaadilt tuvastati 51 erinevat haplotüüpi ning 11 analüüsitud lookuses 43 erinevat alleeli. Uurimuse tulemused viitavad patogeeni mitmekordsele sissetoomisele Eestisse ning inimese mõjule patogeeni levitamisel riigis. Esmakordselt Eestis ja PõhjaEuroopas tuvastati L. acicola harilikult männilt. Uue peremeestaimena maailma jaoks registreeriti Pinus x rhaetica. Eestis on levinum paarumistüüp MAT1-1 ning domineerib patogeeni suguta paljunemine. Kuid kuna populatsioonides esineb samaaegselt mõlemat paarumistüüpi, siis suguline paljunemine on võimalik. Edasise seire, populatsioonide analüüsi ja nakatamiskatsetega on oluline välja selgitada, kas patogeenist kujuneb tõsine oht harilikule männile Põhja-Euroopas ning jälgida patogeeni geneetilise mitmekesisuse muutuseid Eestis, mis viitaksid uute tüvede sissetoomisele või sugulisele paljunemisele.In Europe, L. acicola is a quarantine listed pathogen, which causes serious needle loss in many pine (Pinus) species. Approximately ten years after the first detection of the pathogen in Estonia, it has spread across the country, infecting and affecting mainly mountain pine (Pinus mugo), which is a popular species for greeneries and urban space. So far, there has been no data on the pathogens genetic diversity in Estonia, which would allow for the evaluation of its populations viability, pathogenicity and pathways. The aims of the thesis is (1) to document the pathogens genetic diversity in Estonia, (2) to evaluate the contingency of sexual reproduction in the population, (3) to investigate the spread of the pathogen in Estonia and to analyse the possible anthropogenic activity on it and (4) to analyse the genetic differences of the pathogen in two host species P. mugo and P. sylvestris. During the period of July 2015 to January 2017, samples of symptomatic needles were collected, from which the pathogen was isolated for molecular study. In addition, some earlier isolates of L. acicola in Estonia were used. Isolates were divided into populations according to their geographic locations in Estonia. All together, 104 isolates were used for the population analysis, which were analysed using mating type and microsatellite markers. Data analysis were done using programs GenAlex, GeneMapper and Structure. Estonian L. acicola population is characterised by low genetic variance and high number of clones. From the 104 isolates, 51 different haplotypes and 43 alleles on 11 loci were identified. Results indicate to multiple introduction events in Estonia and it supports human activity in the spread of the pathogen in the country. The first record of the L. acicola in Estonia and northern Europe, was identified on Scots pine (Pinus sylvestris). Pinus x rhaetica was registered as a new host for the pathogen in the world. In Estonia mating type idiomorph MAT1-1 is more common and it indicates that asexual reproduction dominates. However in populations, both mating types occur simultaneously, which means that sexual reproduction is possible. During future monitoring, population genetic analyses and inoculation tests, it is important to determine if the pathogen will develope into a serious threat to the Scots pine in northern Europe and to monitor changes in the pathogens genetic diversity, which would indicate to the introduction of new strain or sexual recombination

Early diagnostics of Lophodermium needle cast (Lophodermium seditiosum)

Männi-pudetõbi on patogeense kottseene tõve-pigihuul (Lophodermium seditiosum) tekitatud okkahaigus harilikul männil (Pinus sylvestris L.). Pika latentse perioodi ning hariliku männi okastel leiduvate pigihuule perekonna liikide (L. seditiosum, L. conigenum, L. pinastri) viljakehade ja puhaskultuuride morfoloogilise sarnasuse tõttu on keeruline tuvastada okaste nakatumist tõve-pigihuulega. Lõputöö eesmärkideks oli (1) testida tõve-pigihuule varajaseks tuvastamiseks disainitud liigispetsiifilisi PCR praimereid ja hinnata nende sobivust kasutamiseks labortöödel patogeeni kiireks ja usaldusväärseks tuvastamiseks, (2) testida L. conigenum’i ja L. pinastri’i liigispetsiifilisi DNA praimereid ja välja selgitada nende sobivus lähedaste liikide täpseks eristamiseks, (3) edasisteks uuringuteks isoleerida L. seditiosum puhaskultuuri 2013. aasta haiguspuhangust nakatunud okastest. 2013. aastal kahest männinoorendikust kokku 41 puult kogutud okkaproovidest isoleeriti puhaskultuuri seened, mis vastasid sihtmärkliikide tunnustele. Praimerite testimiseks eraldati valitud kultuuridest DNA, valmistati PCR segu, viidi läbi PCR reaktsioonid ja geelelektroforees tulemuste kontrollimiseks. Katsetati kahte L. seditiosum’i, ühte L. conigenum’i ja kahte L. pinastri tuvastamiseks disainitud praimerite paari. L. seditiosum’i praimeritega testiti ka okastest eraldatud DNA-d. Töö tulemustest selgus, et praimereid kasutades on võimalik tuvastada kiirelt ja täpselt patogeen tõve-pigihuul ning eristada seda lähedastest liikidest. Kõik kasutatud praimerid on kõrge spetsiifilisusega ehk nad amplifitseerivad ainult sihtmärkliigi DNA, kuid erinevused esinevad katsetatud DNA praimerite tundlikkuses. Edasisteks uuringuteks isoleeriti kokku 66 tõve-pigihuule puhaskultuuri. Kiire analüüsi teostamiseks on vajalik täpselt tuvastada okastest latentne L. seditiosum’i nakkus ning eristada omavahel erineva patogeensusega lähedasi liike. Antud töö tulemuste põhjal saab otsustada, millised praimereid kasutada laborites patogeeni tuvastamiseks. Tõve-pigihuule isolaate 2013. aasta epideemiast on võimalik kasutada edasistes uurimustöödes, et hinnata muutuseid liigi populatsioonides ja välja selgitada võimalikud muutused patogeeni virulentsuses erinevate puhangute vahel.Lophodermium needle cast is a needle disease of Scots pine (Pinus sylvestris L.) caused by pathogenic ascomycete Lophodermium seditiosum. It is difficult to identify if the needles are infected by L. seditiosum because of the long latent phase of the infection and morphologically similar fruiting bodies and cultures of all Lophodermium species found in Scots pine needles (L. seditiosum, L. conigenum, L. pinastri). The aim of this thesis was (1) to test species specific PCR primers designed for early detection of Lophodermium seditiosum and to assess their suitability for fast and accurate detection of the pathogen in the laboratory work, (2) to test specific DNA primers designed for the detection of other Lophodermium species found on Scots pine needles (L. conigenum and L. pinastri) and to ascertain their suitability in accurately differentiating related species, (3) to isolate Lophodermium seditiosum from epidemic of 2013 for further research. In 2013 needle samples were collected from two young Scots pine stands from a total of 41 trees. Fungi that matched the culture descriptions of the target species were isolated from needle samples. In order to test the primers, DNA was extracted from a selection of cultures, PCR mix was preparated, PCR reactions were carried out and the results were visualised by gelelectroforesis. Two primer pairs designed for the detection of L. seditiosum, one for L. conigenum and two for L. pinastri were tested with DNA extracted from the cultures. With L. seditiosum primers DNA extracted from needles was also analysed. The results showed that it is possible to use primers for fast and accurate detection of the pathogen L. seditiosum and to distinguish it from closely related species. All tested primers are highly specific and they amplify only the DNA of the target-species. The sensitivity of named primers is different. For further research 66 cultures of L. seditiosum were obtained. For a quick analysis, it is important to accurately detect Lophodermium seditiosum infection in pine needles and to distinguish closely related species which have different pathogenicy. The results of this work help to decide which primers to use in laboratory work for the identification of the pathogen. The isolates of the pathogen from the epidemic of 2013 can be used for further research in order to assess changes in the population genetics of the pathogen and to find out possible changes in the pathogens virulence between disease epidemics

Diversity, migration routes, and worldwide population genetic structure of Lecanosticta acicola, the causal agent of brown spot needle blight

Lecanosticta acicola is a pine needle pathogen causing brown spot needle blight that results in premature needle shedding with considerable damage described in North America, Europe, and Asia. Microsatellite and mating type markers were used to study the population genetics, migration history, and reproduction mode of the pathogen, based on a collection of 650 isolates from 27 countries and 26 hosts across the range of L. acicola. The presence of L. acicola in Georgia was confirmed in this study. Migration analyses indicate there have been several introduction events from North America into Europe. However, some of the source populations still appear to remain unknown. The populations in Croatia and western Asia appear to originate from genetically similar populations in North America. Intercontinental movement of the pathogen was reflected in an identical haplotype occurring on two continents, in North America (Canada) and Europe (Germany). Several shared haplotypes between European populations further suggests more local pathogen movement between countries. Moreover, migration analyses indicate that the populations in northern Europe originate from more established populations in central Europe. Overall, the highest genetic diversity was observed in south-eastern USA. In Europe, the highest diversity was observed in France, where the presence of both known pathogen lineages was recorded. Less than half of the observed populations contained mating types in equal proportions. Although there is evidence of some sexual reproduction taking place, the pathogen spreads predominantly asexually and through anthropogenic activity

Highly Clonal Structure and Abundance of One Haplotype Characterise the Diplodia sapinea Populations in Europe and Western Asia

Diplodia sapinea is a cosmopolitan endophyte and opportunistic pathogen having occurred on several conifer species in Europe for at least 200 years. In Europe, disease outbreaks have increased on several Pinus spp. in the last few decades. In this study, the genetic structure of the European and western Asian D. sapinea population were investigated using 13 microsatellite markers. In total, 425 isolates from 15 countries were analysed. A high clonal fraction and low genetic distance between most subpopulations was found. One single haplotype dominates the European population, being represented by 45.3% of all isolates and found in nearly all investigated countries. Three genetically distinct subpopulations were found: Central/North European, Italian and Georgian. The recently detected subpopulations of D. sapinea in northern Europe (Estonia) share several haplotypes with the German subpopulation. The northern European subpopulations (Latvia, Estonia and Finland) show relatively high genetic diversity compared to those in central Europe suggesting either that the fungus has existed in the North in an asymptomatic/endophytic mode for a long time or that it has spread recently by multiple introductions. Considerable genetic diversity was found even among isolates of a single tree as 16 isolates from a single tree resulted in lower clonal fraction index than most subpopulations in Europe, which might reflect cryptic sexual proliferation. According to currently published allelic patterns, D. sapinea most likely originates from North America or from some unsampled population in Asia or central America. In order to enable the detection of endophytic or latent infections of planting stock by D. sapinea, new species-specific PCR primers (DiSapi-F and Diplo-R) were designed. During the search for Diplodia isolates across the world for species specific primer development, we identified D. africana in California, USA, and in the Canary Islands, which are the first records of this species in North America and in Spain.publishedVersio

Highly Clonal Structure and Abundance of One Haplotype Characterise the Diplodia sapinea Populations in Europe and Western Asia

Diplodia sapinea is a cosmopolitan endophyte and opportunistic pathogen having occurred on several conifer species in Europe for at least 200 years. In Europe, disease outbreaks have increased on several Pinus spp. in the last few decades. In this study, the genetic structure of the European and western Asian D. sapinea population were investigated using 13 microsatellite markers. In total, 425 isolates from 15 countries were analysed. A high clonal fraction and low genetic distance between most subpopulations was found. One single haplotype dominates the European population, being represented by 45.3% of all isolates and found in nearly all investigated countries. Three genetically distinct subpopulations were found: Central/North European, Italian and Georgian. The recently detected subpopulations of D. sapinea in northern Europe (Estonia) share several haplotypes with the German subpopulation. The northern European subpopulations (Latvia, Estonia and Finland) show relatively high genetic diversity compared to those in central Europe suggesting either that the fungus has existed in the North in an asymptomatic/endophytic mode for a long time or that it has spread recently by multiple introductions. Considerable genetic diversity was found even among isolates of a single tree as 16 isolates from a single tree resulted in lower clonal fraction index than most subpopulations in Europe, which might reflect cryptic sexual proliferation. According to currently published allelic patterns, D. sapinea most likely originates from North America or from some unsampled population in Asia or central America. In order to enable the detection of endophytic or latent infections of planting stock by D. sapinea, new species-specific PCR primers (DiSapi-F and Diplo-R) were designed. During the search for Diplodia isolates across the world for species specific primer development, we identified D. africana in California, USA, and in the Canary Islands, which are the first records of this species in North America and in Spain

Diversity, migration routes, and worldwide population genetic structure of Lecanosticta acicola, the causal agent of brown spot needle blight

Lecanosticta acicola is a pine needle pathogen causing brown spot needle blight that results in premature needle shedding with considerable damage described in North America, Europe, and Asia. Microsatellite and mating type markers were used to study the population genetics, migration history, and reproduction mode of the pathogen, based on a collection of 650 isolates from 27 countries and 26 hosts across the range of L. acicola. The presence of L. acicola in Georgia was confirmed in this study. Migration analyses indicate there have been several introduction events from North America into Europe. However, some of the source populations still appear to remain unknown. The populations in Croatia and western Asia appear to originate from genetically similar populations in North America. Intercontinental movement of the pathogen was reflected in an identical haplotype occurring on two continents, in North America (Canada) and Europe (Germany). Several shared haplotypes between European populations further suggests more local pathogen movement between countries. Moreover, migration analyses indicate that the populations in northern Europe originate from more established populations in central Europe. Overall, the highest genetic diversity was observed in south-eastern USA. In Europe, the highest diversity was observed in France, where the presence of both known pathogen lineages was recorded. Less than half of the observed populations contained mating types in equal proportions. Although there is evidence of some sexual reproduction taking place, the pathogen spreads predominantly asexually and through anthropogenic activity