Outbreak of crown and root rot of walnut caused by Phytophthora cinnamomi in Chile

Walnut industry (Juglans regia L.) has increased in the last decade in Chile from 9,734 ha to 27,941 ha (www.odepa.cl, 2016) and many walnut orchards have been established in lands predisposed to crown and root rot development in walnut trees. The main variety is Chandler followed by Serr, both grafted on J. regia rootstock. During the 2015-16 growing season, a survey was conducted in 49 orchards in central Chile (UTM 30°36′S to 37°40′S). Disease prevalence averaged 15.7%. Symptoms were characterized by decay, chlorosis, and leaf fall, and in some cases, presence of dark brown cankers at the crown were observed. In all cases, the trees presented feeder root rot. Three samples of rotted roots, cankered cortex tissue, and soil from symptomatic trees were collected, and three trees per orchard were sampled. Necrotic tissues from root rot and cankered samples (3 to 4 mm long) obtained from the margin of the lesions were plated on P5ARP semiselected medium (Jeffers and Martin 1986) and incubated at 25°C for 7 days. Soil samples (500 g) were introduced into a pot (500 ml), saturated with sterile distilled water (SDW), and rhododendron leaves were used as bait (Themann and Werres 1998). Necrotic lesions from bait were cut into 5 mm sections and plated as described above. When whitish hyaline colonies developed in selective media from soil and root samples, outgrowing hyphal tips were subcultured onto corn meal agar to obtain pure cultures. Morphological characteristics of the colony and reproductive structures were analyzed: a colony with a rosette aspect developed in APDA medium, hyphal swellings were abundant, and persistent ellipsoid to ovoid nonpapillate sporangia of 58.4 × 38.7 μm were obtained, accordingly to Erwin and Ribeiro (1996) for P. cinnamomi. From 23 orchards positive to Phytophthora isolation, in 20 of them the Phytophthora species was tentatively identified as P. cinnamomi. From these, six isolates were analyzed molecularly. DNeasy Plant Mini Kit (QIAGEN) was used for DNA extraction. PCR amplification of the ITS region of rDNA was conducted using primers ITS4 and 5 and the obtained products were sent to Macrogen, Korea, for purifying and sequencing. The six obtained sequences were compared with GenBank using BLAST searches and returned a 99% nt identity to P. cinnamomi from Taiwan (GU111594). Sequences were deposited in GenBank (KU961897 to 98, KU961902, KU961904, and KU961906 to 07). The other Phytophthora isolates are under identification process. Three of the P. cinnamomi isolates were used for pathogenicity testing using 3-month-old ungrafted walnut plants grown in 3-liter pots with sterile substrate. Sixteen plants were used (four per isolate and control plants). Each plant received 50 ml of SDW with 3 × 10⁵ zoospores ml^-1 and the controls only 50 ml SDW. Plants were saturated for 48 h at 25 ± 5°C. No injuries were done to the plants. Seven days later, all inoculated plants showed decline and leaf chlorotic symptoms, leaf fall, and cankers in the crown and stem with a mean length of 2.5 cm. Control plants were symptomless. P. cinnamomi was reisolated from rotted roots and cankers in all the inoculated plants, fulfilling Koch’s postulates. This species was previously reported in Chile by Rojic and Cancino (1975) causing crown canker in the Valparaíso Region and no later work was done until now. Thus, this survey and work illustrates an important gap in terms of controlling walnut root and crown rot. This disease is a serious problem for the Chilean walnut industry, but the high percent of P. cinnamomi prevalence on commercial orchards permit to focus on the control strategy, specially oriented to introduce Phytophthora-resistant rootstocks.