First report of Phytophthora niederhauserii causing root rot of carob tree ( Ceratonia siliqua L.) in Spain.

Carob tree (Ceratonia siliqua L.) is a crop native to the Mediterranean Basin mainly being used as livestock feed, in pharmaceutical industry, and also a valuable source of human food due to the high dietary fibre and sugar contents. In January 2023, one-year-old 'Duraio' carob trees grafted on 'Rojal' rootstock on pots showing dark brown to necrotic lesions on the secondary roots, decline symptoms and leading to the death of some plants, were detected in a nursery located at the Valencian Community region in Spain. Disease severity was 40 to 50% of root area and disease incidence was approximately 50% on approximately 1000 plants. Six representative plants were randomly collected. Roots were washed under running tap water to rinse the soil away, and small symptomatic pieces were cut, disinfected with 70% ethanol, and then dried on absorbent paper. Two-mm-long segments were plated on CMA-PARBPH, a Phytophthora-selective medium, and incubated at 25ºC. After 2-3 days, growing colonies were transferred to potato dextrose agar (PDA) medium. A Phytophthora-like organism was consistently isolated (50% of root segments, n= 120). Colonies were whitish with irregular margins, had coenocityc mycelium, irregularly branched hyphal swellings, and chlamydospores were absent. Sporangia were non-papillate, persistent, ellipsoid and measuring 25 to 40 × 50 to 90 μm (average: 35.5 × 74.7 μm, n = 50). They proliferated internally with both nested and extended proliferation. These morphological features were similar to those of Phytophthora niederhauserii (Abad et al., 2014). Internal transcribed spacer (ITS) and cytochrome c oxidase I (COX1) regions of a representative isolate CF3 were sequenced to confirm the identity. Both sequences were deposited in GenBank under accession numbers OR763816 for ITS and OR783697 for COX1. OR763816 showed 99.87% sequence identity to P. niederhauserii strain Ex-type MG865552 and OR783697 showed 99.85% sequence identity to P. niederhauserii strain Ex-type MH136944 (Abad et al., 2023). Pathogenicity test was performed on one-year-old carob tree seedlings (Duraio/Rojal) grown in 13 cm-diameter pots to fulfill Koch's postulates. The inoculum was prepared in 1 L glass flasks with a mixture of 200 ml vermiculite, 20 ml oat grains, and 175 ml of V8 broth (20% V8 juice and 0.2% of CaCO3 in demineralized water) (Jung et al., 1996). Glass flasks with vermiculite/oat/V8 mixture were autoclaved three times for 20 min at 120 ºC. These mixtures were inoculated with the isolate CF3 which was previously grown on V8 agar medium and incubated for six weeks in the dark at room temperature (Pérez-Sierra et al., 2013). For inoculation, twenty-gram inoculum were mixed with 200 g autoclaved potting mix (peat, vermiculite and sand; 1:1:1, v/v/v), and added to the pots to plant the seedlings. Seven plants were inoculated and non-infested vermiculite/oat/V8 mixture was used to prepare seven control plants. Two months after inoculation one of the inoculated plants died. Six months after inoculation, only the inoculated plants showed decline symptoms with dry leaves and root necrosis. Isolates resembling P. niederhauserii were recovered by plating the roots from all inoculated plants on CMA-PARBPH, and the identity of the isolates was confirmed as P. niederhauserii based on ITS and COX1 sequencing. To our knowledge, this is the first report of P. niederhauserii causing root rot on carob tree. The detection of this pathogen in nurseries is relevant because its dissemination to orchards could have a negative impact in carob crop production.