Männi-pudetõve tekitaja (Lophodermium seditiosum) varajane diagnostika
Laen...
Kuupäev
2015
Kättesaadav alates
Autorid
Ajakirja pealkiri
Ajakirja ISSN
Köite pealkiri
Kirjastaja
Abstrakt
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.
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.
Kirjeldus
Märksõnad
bakalaureusetööd