Phytophthora root and crown rot on apples in Bulgaria

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.