Abstract
Crown rot impacted olive plants (cv. Koroneiki) in an orchard in Chakwal, Punjab, Pakistan (32° N, 72° E), with a prevalence of 60%. Observable symptoms included leaf chlorosis, defoliation, wilting, and twig dieback in 6-8-year-old plants, ultimately resulting in their demise (Fig. 1 A, B). It was initially observed in June 2020. In September 2020, 48 samples (3 root and 3 crown stem part sample per plant) of diseased olive plants were randomly chosen from the orchard for detailed analysis. Bark pieces from the diseases portion were sectioned into smaller pieces and placed on corn meal agar supplemented with PARP. The culture was maintained at 22°C in the dark. Within 4 days, coenocytic white fluffy mycelium emerged. Hyphae were subsequently transferred to carrot agar medium supplemented with beta-sitosterol. A total of 15 isolates were obtained and characterized, yet a particular two isolates consistently stood out. These isolates did not show sexual reproduction in culture (Ann 1988). Instead, they reproduced asexually through sporangia borne on aerial sporangiophores, originating directly from the internal mycelium. Each branch of the sporangiophore featured a sporangium at its tip, with sporangia varying in shape from pyriform to ellipsoid and being non-papillate and non-caducous (Fig. 2B, C, D). After morphological study, the isolates were identified as Phytophthora species and named BPK-01 and BPK-02. For the molecular identification, we amplified and sequenced their internal transcribed spacer and beta-tubulin regions using universal primer pairs ITS1 and ITS-4, as well as TUB-F and TUB-R primer pairs (Safaiefarahani et al. 2013). Sequences of 816 bp and 940 bp were obtained for the ITS and beta-tubulin regions, respectively. These sequences were deposited in NCBI GenBank, resulting in accession numbers OL423596 and OL423597 for the ITS region of BPK-01 and BPK-02, and OL771190 and OL771191 for the beta-tubulin region of BPK-01 and BPK-02, respectively. BLAST analysis revealed 99.88% identity (816bp out of 837bp) for ITS and 100% (940bp out of 940bp) for TUB between our isolate and P. inundata reference isolates (GenBank Accessions MK326518.1 and EF210202). Phylogenetic analysis using MEGA X software further confirmed the identity of the olive crown rot isolate as P. inundata, aligning it with reference isolates from various Phytophthora species (Fig. 3) (Tamura et al. 2013). The fungal isolates have been officially deposited as living cultures at the Barani Agricultural Research Institute's fungal culture collection center under the designations BAPI.3550 and BAPI.3551. The pathogenicity test involved inoculating isolated strains on 2.5-year-old olive trees. Each isolate was used to inoculate five Koroneiki plants, while five plants served as controls. Inoculation, performed on the stem, included surface disinfection, wounding, and placement of a mycelium plug, with all plants maintained in controlled conditions (25°C, 12h light). After 15 days, symptoms such as leaf chlorosis, collar rot, and stem rot began to appear, and data was recorded up to 45 days. Symptoms were assessed at the conclusion of the experiments using a 0-4 scale, with the following criteria: 0: No symptoms observed (0%), 1: Symptoms present on 1-33% of the plant (1-33% affected), 2: Symptoms present on 34-66% of the plant (34-66% affected), 3: Symptoms present on more than 67% of the plant (more than 67% affected), 4: Complete root death observed (González et al. 2017). Notably, the symptoms induced by strain BPK-02 were more severe than those caused by BPK-01, ultimately resulting in plant wilting and death (Fig. 4)(Santilli et al. 2020). Control plants inoculated with sterile CA plugs remained healthy. Reisolation and morphological identification of P. inundata from diseased tissues fulfilled Koch's postulates. To the best of our knowledge, this is the first report of P. inundata infecting olive plants in Chakwal region of Pakistan, and it is a serious threat to the olive plantation in Pakistan, posing a significant threat to the olive plantations throughout the country.