Dieback and Mortality of Pinus radiata Trees in Italy Associated with Phytophthora cryptogea

Abstract

Pinus radiata D. Don is a forest tree species native to the Monterey Baja in California. Due to its rapid growth and desirable lumber and pulp qualities, between 1960 and 1980, about 12,000 ha of P. radiata were planted in Sardinia, Italy. The only disease reported on this conifer species has been Diplodia pinea, which causes tip and branch dieback (3). In January 2012, dieback and mortality of 25-year-old radiata pine trees were observed in a reforestation area of about 20 ha located in northern Sardinia (40°43′N, 9°22′E, 600 m a.s.l.). Symptoms included chlorosis, reddish-brown discoloration of the whole crown or dieback starting in the upper crown and progressing downward through the crown, and necrotic bark tissues at root collar. Approximately 25% of the trees were affected. In a first attempt, a Phytophthora species was consistently isolated from the rhizosphere of 23 symptomatic trees, which included necrotic fine roots using oak leaves as bait (4). Afterwards, it was also isolated from phloem samples taken from the margins of fresh lesions at the stem base and upper roots of affected trees using synthetic mucor agar medium (1). Isolation from soil samples of six healthy pine trees randomly selected in the site did not yield any Phytophthora isolate. On carrot agar (CA), Phytophthora colonies were stellate to slightly radiate with limited aerial mycelium. Sporangia were obpiryform, non-papillate, and non-caducous, measuring 46.9 to 51.2 × 29.1 to 32.6 μm (l:b ratio 1.9). Hyphal swellings were formed in chains or clusters; chlamydospores were not observed. These isolates had cardinal temperatures of <5°C, 25°C, and 35°C, respectively. Their morphological and cultural features were typical of Phytophthora cryptogea Pethybridge & Lafferty. They were heterothallic and produced oogonia with amphyginous antheridia when paired with an A2 mating type tester strain of P. cryptogea. This identity was corroborated by sequence analysis of the internal transcribed spacer (ITS) region of the rDNA. BLAST searches showed 99% homology with sequences of P. cryptogea available in GenBank (DQ479410 and HQ697245). The ITS sequence of a representative isolate (PH101) was submitted to GenBank (Accession Nos. KC603895). The strain PH101 was stored in the culture collection of the Department of Agriculture at the University of Sassari. Pathogenicity of isolate PH101 was verified by inoculating five freshly cut logs of radiata pine (1 m long and 15 cm diam.) with a 5-mm agar plug taken from the margin of 4-day-old culture grown on CA (4). The plug was inserted in a 5-mm hole made through the bark with a cork borer. Five control logs were inoculated with sterile CA. All logs were incubated in a growth chamber at 20°C. Phloem lesion sizes were assessed after 1 month and measured 9.7 ± 5.5 cm2 (average ± standard deviation). Control logs had no lesions. The pathogen was re-isolated from the lesions, thus fulfilling Koch's postulates. P. cryptogea has been previously reported in Australia, causing decline of radiata pine trees in wet and flooded soils (2). To our knowledge, this is the first report of P. cryptogea on P. radiata trees in Europe. References: (1) C. M. Brasier and S. A. Kirk. Plant Pathol. 50:218, 2001. (2) M. Bumbieris. Aust. J. Bot. 24:703, 1976. (3) A. Franceschini et al. Informatore Fitopatologico 1:54, 2006. (4) B. Scanu et al. For. Pathol. 43:340, 2013.