First Report of Fusarium brachygibbosum Causing Cankers in Cold-Stored, Bare-Root Propagated Almond Trees in California

A canker disease of dormant bare-root Prunus trees in cold storage or in the field soon after planting was first observed in California in 1997-98, resulting in the death of numerous trees over the next 4 years (Marek et al. 2013). Fusarium acuminatum and F. avenaceum were identified as the main causal agents and were isolated from both symptomatic and asymptomatic tissue. The disease reemerged in 2011 in one nursery in Sutter County. In fall 2013, samples were taken from various steps of the nursery production system. Surface-sterilized bark and root pieces of asymptomatic almond (Prunus dulcis [Mill.] Webb) tissue were placed on Komada’s selective medium (KOM, Komada 1975) and incubated at 25°C under constant illumination (67.8 μmol/sec m²) for 7 days. Emerging mycelium from wood pieces was transferred to carnation leaf agar (CLA) to induce sporulation (Leslie and Summerell 2006; Padwick 1945). In addition to the aforementioned Fusarium spp., fungal growth and conidia resembling F. brachygibbosum emerged from some samples, from which four single-hyphal-tip isolates (BL32, BL33, BL56, and BL66) were obtained and grown in potato dextrose broth for 7 days to be used for DNA analysis and pathogenicity tests. The Ef-1α and ITS regions were amplified using primers ef1/ef2 and ITS4/ITS5 (Geiser et al. 2004; Marek et al. 2013). Ef-1α sequences were deposited in GenBank (KX421376-79) and were nearly identical to F. brachygibbosum strain NRRL 25093 from GenBank except for 5 SNPs occurring in all four isolates (99% identity with NRRL 25093 for BL33, BL56, and BL66) and an additional 17 SNPs occurring only in BL32 (97% identity). ITS sequences shared 100% identity with NRRL 25093. Culture morphology on PDA was similar to that described for F. brachygibbosum (Padwick 1945), with abundant aerial mycelium and colonies white to slight yellow or pink. On CLA, abundant sporodochia were produced with macroconidia that conformed to the shape and size described by Padwick (1945). These were slightly curved, with 3 to 5 septa, wide central cells, slightly sharp apexes, and distinct basal foot cells. Terminal and intercalary chlamydospores were abundant on CLA after 4 weeks. Microconidial production was absent or sparse under our culture conditions. All four F. brachygibbosum isolates were tested for pathogenicity by inoculating 2-year-old detached almond branches cv. Monterey. For each isolate, three surface-sterilized (0.6% NaHClO₄ for 5 min and rinsed twice in deionized water) branch segments were inoculated with a colonized filter paper (4 mm²) placed over a 1-mm² puncture wound to the cambium (Marek et al. 2013). Branch segments were incubated upright in sterile test tubes fitted with foam plugs and containing 5 ml sterile water at 15°C in the dark. Canker lesions were measured after 2 weeks incubation and ranged from 26.7 to 83.0 mm², comparable to lesion areas resulting from inoculations conducted in parallel with isolates of F. acuminatum and F. avenaceum (Marek et al. 2013). F. brachygibbosum was reisolated from cankers on KOM from all inoculated branches, completing Koch’s postulates. Upon further sampling at the original nursery and two other commercial nurseries, F. brachygibbosum was isolated from asymptomatic stems and peach rootstocks of grafted bare-root almond trees, soil in almond orchards, wheat plants in rotation with nursery tree production fields, and surfaces of nursery equipment. To our knowledge, this is the first report of F. brachygibbosum causing disease in the United States and the first report of this species causing disease on almond trees.