First Report of Phytophthora cinnamomi Causing Trunk Canker of Almond in California

Abstract

Almond (Prunus dulcis) is the most economically important nut crop in California, accounting for $5.9 billion value in 2016. During fall of 2015, declining 2-year-old almond trees on clonal Hansen 536 (peach × almond) rootstock were observed in a commercial orchard in Kern County, California. Affected trees expressed early defoliation with amber-colored gummosis on the trunk. Cankers developed at the bud union and extended into the scion in trees planted with their bud union placed at or below the soil surface. To isolate the causal agent, necrotic pieces of bark and subtending vascular tissue were cut from the canker margin, surface sterilized in 0.5% sodium hypochlorite solution for 3 min, rinsed twice in sterile water, and plated on corn meal agar amended with selective antibiotics (PARP) (Jeffers and Martin 1986). After several days of incubation at 25°C, Phytophthora-like colonies grew from the diseased tissues and were transferred onto acidified potato dextrose agar medium (APDA). The isolates were tentatively identified as Phytophthora cinnamomi Rands based on morphological characteristics. Sporangia were nonpapillate and ovoid to obpyriform or ellipsoidal to elongate-ellipsoid with an apical thickening, avg. 57.2 × 36.6 µm with a length-width ratio of 1.56; mycelium with abundant coralloid hyphal swellings. Globose chlamydospores formed abundantly in culture on the parent hyphae or on new hyphal branches (18 to 42 [avg. 27.65] μm in diameter) (Erwin and Ribeiro 1996). For all isolates, the internal transcribed spacer (ITS) regions of the rRNA gene and a portion of the β-tubulin (TUB) gene were amplified and sequenced with primers ITS1/ITS4 and TUBUF2/TUBUR1 (Lan et al. 2016), respectively. Two representative sequences for each locus were deposited in GenBank (ITS; KY924650 and KY924651, TUB; KY924648 and KY924649). BLASTn analysis against Phytophthora Database (www.phytophthoradb.org) showed that the ITS sequences had 99% identity and the β-tubulin sequences had 97 to 98% identity to reference sequences of P. cinnamomi (ITS: GU259227; TUB: EU079894). Two of the almond isolates of P. cinnamomi were tested for pathogenicity by wound inoculating 2-year-old potted almond trees of cv. Nonpareil on rootstocks of Hansen 536 and Nemaguard (Prunus persica × Prunus davidiana). Five-millimeter-diameter mycelial disks from PDA cultures of each isolate (and sterile PDA as a control) were used for inoculating wounds of the same size (5 mm) made in stems of: i) the almond scion on Hansen 536 rootstock, ii) the almond scion on Nemaguard rootstock, iii) Hansen 536 rootstock below the scion, and iv) Nemaguard rootstock below the scion. Five replicate trees were inoculated per combination of the three inoculants (two of P. cinnamomi, one control) and the four inoculation treatments in a randomized complete block design. After inoculation, the wounds were covered with petroleum jelly and wrapped with Parafilm. Two months after inoculation, all plants inoculated with the control remained healthy, whereas all scions and rootstocks inoculated with either isolate developed stem cankers. None of the trees inoculated on the rootstocks died during the experiment, but 40% of the trees inoculated on the scion were killed. P. cinnamomi was reisolated only from the infected plants. More than 10 other species of Phytophthora were reported to cause root and crown rots as well as aerial cankers on almond (Browne and Viveros 1999; Browne et al. 2015). To our knowledge, this is the first report of P. cinnamomi causing trunk canker disease of almond in California.