Phytophthora root and crown rot on apples in Bulgaria

Abstract

Phytophthora is a genus of Oomycota responsible for some of the most serious diseases with great economic impact (Judelson and Blanco, 2005). While 54 species were found in the 20th century (Erwin and Ribeiro, 1996) another 51-54 new species have been identified (Brasier, 2008) since the year 2000. They are spread worldwide and have broad range of host plants - fruit trees, citrus, forest and park species. Phytophthora can cause serious damages in orchards and nurseries of apples, cherries, etc. In Bulgaria they have been found first on young apples and cherries (1998-1999) in Plovdiv region (Nakova, 2003). Surveys have been done for discovering disease symptoms in Plovdiv and Kjustendil regions. Isolates have been obtained from infected plant material (roots and stem bases) applying baiting bioassay (green apples, variety Granny Smith) and/or PARP 10 selective media. Phytophthora strains were identified based on standard morphology methods - types of colonies on PDA, CMA, V 8, type and size of sporangia, oogonia and antheridia, and oospores. Cardial temperatures for their growth were tested on CMA and PDA. For molecular studies, DNA was extracted from mycelium using the DNA extraction kit. DNA was amplified using universal primers ITS 6 and ITS 4. Amplification products concentrations were estimated by comparison with the standard DNA. Sequencing was done at the Scottish Crop Research Institute (SCRI, Dundee, Scotland). Phytophthora root and crown rot symptoms first appear in early spring. Infected trees show bud break delay, have small chlorotic leaves, and branches die all of a sudden. Later symptoms are found in August-September. Leaves of the infected trees show reddish discoloration and drop down. Both symptoms are connected with lesions (wet, necrotic in appearance) at stem bases of the trees. Disease spread was 2-3% in most gardens, only in an apple orchard in Bjaga (Plovdiv region) it was up to 8-10%. Morphologically, the isolates acquired from the apple trees were identified as Phytophthora cactorum, P. citrophthora and P. cryptogea. Cardial temperatures for their growth were tested on CMA and PDA. PCR tests with ITS primers 4 and 6 generated a band at about 800 bP. Consequent sequencing showed that 2 strains, Bg 1/1 and Bg ?, belong to Phytophthora cryptogea.Phytophthora je rod Oomycota odgovoran za neke od najozbiljnijih bolesti sa velikim ekonomskim posledicama (Judelson i Blanco, 2005). Tokom 20. veka identifikovane su 54 vrste (Erwin i Ribeiro, 1996), a posle 2000. godine jos 51-54 (Brasier, 2008). Ove vrste su rasirene po celom svetu na velikom broju biljaka domacina - stablima razlicitih vocaka, citrusnih, sumskih i parkovskih vrsta. Phytophthora moze da prouzrokuje ozbiljnu stetu u vocnjacima i rasadnicima jabuke, tresnje i drugih vocaka. U Bugarskoj je bolest najpre pronadjena na mladicama jabuke i tresnje (1998-1999) u regionu Plovdiva (Nakova, 2003). Sprovedena su posmatranja kako bi se otkrili simptomi bolesti u podrucju Plovdiva i Kjustendila. Izolati su dobijeni sa zarazenog biljnog materijala (korena i korenovog vrata) 'metodom zamke' u biotestu (zelena jabuka, sorta greni smit) i/ili na selektivnom medijumu PARP 10. Sojevi Phytophthora su identifikovani standardnim morfoloskim metodama na osnovu: vrsta kolonija na PDA, CMA i V 8 medijumima, tipa i velicine sporangija, oogonija, anteridija, kao i oospora. Kardijalna temperatura rasta ispitivana je na CMA i PDA medijumima. U molekularnim ispitivanjima DNK je ekstrahovana iz micelija pomocu DNK ekstrakcionog kita. DNK je umnozena pomocu univerzalnih prajmera ITS 6 i ITS 4. Koncentracije produkata amplifikacije ocenjivane su poredjenjem sa standardnom DNK. Sekvencioniranje je uradjeno u Scottish Crop Research Institute (SCRI, Dundee, SkotskaPrvi simptomi plamenjace korena i krosnje uzrokovani rodom Phytophthora pojavljuju se u rano prolece. Kod zarazenih stabala javlja se okasnelo pucanje pupoljaka i mali hloroticni listovi, a grane iznenada odumiru. Kasniji simptomi javljaju se u avgustu i septembru. Listovi zarazenog drveca imaju crvenkastu boju i opadaju. Oba simptoma su u vezi sa lezijama (vlaznog, nekroticnog izgleda) u vratu korena drveca. U vecini vocnjaka, rasprostranjenost bolesti je bila 2-3%, osim u jednom vocnjaku u Bjagi (Plovdiv) gde je rasirenost bila 8-10%. Izolati dobijeni sa stabala jabuke morfoloski su identifikovani kao Phytophthora cactorum, P. citrophthora i P. cryptogea. Kardijalne temperature za njihov rast ispitivane su na CMA i PDA medijumima. PCR testovi sa ITS prajmerima 4 i 6 dali su traku od oko 800 bP. Sekvencioniranje je pokazalo da dva soja, Bg 1/1 i Bg 1/2, pripadaju vrsti Phytophthora cryptogea.