First report of Phytophthora taxon walnut on Pistacia vera in California

Pistacia vera L. trees planted in 2002 in a Kern County, California (CA), research block developed bleeding stem cankers 2 years after planting. Over the past 14 years, canker development and subsequent mortality has resulted in the loss of approximately 45 trees. Cankers first appeared in summer months, and affected trees declined and died 2 to 3 years after symptoms first manifested. Tissues from canker margins were sampled and embedded in CMA-PARP (Jeffers and Martin 1986). White colonies with coenocytic hyphae grew from samples and isolates were transferred to 1/3 strength V8 juice agar and sent to the CDFA-PPDC lab for identification. Agar plugs of 5 mm were taken from the margin of a 3-day-old culture and placed into soil water under light at 22°C for 2 days. Ovate, apapillate sporangia ranged from 20 to 75 μm × 17 to 40 μm (34.8 × 24.9 μm average) with l:b ratio of 1.4 (n = 74) and were formed on long pedicels. The internal transcribed spacer region (ITS) of rDNA was amplified from one isolate using ITS1 and ITS4 primers as described by White et al. (1990), and the amplicon was sequenced (GenBank accession no. KY094490). BLAST analysis of the 817-bp amplicon showed 100% identity with the ITS sequence of Phytophthora taxon walnut (KC291550) from Italy (Ginetti et al. 2014). Additionally, a region of the cox1 gene was amplified with primers OomCoxILevup and Fm85mod and sequenced (KY552668). BLAST analysis of the cox1 amplicon also showed 100% identity with the sequence of P. taxon walnut from Italy (KC291584) (Ginetti et al. 2014). Koch’s postulates were completed by inoculation of detached woody (n = 5) and nonwoody stems (n = 5), as well as in situ inoculations on woody (n = 5) and nonwoody stems (n = 5) on mature trees. Inoculations on detached stems were conducted twice; in situ inoculations were conducted once. All inoculations were made by slightly wounding the stem with a sterile scalpel prior to introduction of the pathogen on 1/3 V8 agar plugs. Uncolonized agar plugs were utilized to establish uninoculated control treatments. All inoculation sites on stems were wrapped with Parafilm. Detached stems were incubated in moist chambers. Lesion development was measured 4 and 10 days post inoculation on detached nonwoody and woody stems, respectively. In situ lesion development was assessed 15 and 111 days post inoculation on nonwoody and woody stems, respectively. Average lesion length on inoculated, detached, nonwoody stems averaged 34.6 and 127.0 mm in the first and second experimental runs, respectively. Lesions on inoculated nonwoody stems in situ averaged 37.8 mm. Uninoculated stems and inoculated woody stems remained asymptomatic. The pathogen was routinely reisolated from symptomatic tissues and was never recovered from uninoculated tissues. The current study demonstrates the pathogenicity of P. taxon walnut on nonwoody stems of P. vera and is the first report of the pathogen in CA. Until recently, P. taxon walnut and other Clade 6 taxa have only been associated with disease in natural areas (Brasier et al. 2003). The current study, in conjunction with the report of foliar disease of walnut (Juglans regia) in Italy (Ginetti et al. 2014), demonstrate expansion of the host range of P. taxon walnut to agricultural crops. The current study also extends the range of symptoms associated with the pathogen.