Anthracnose of olive fruit caused by Colletotrichum acutatum was a severe epidemic disease in Pakistan that occurred in September 2020. The estimated disease incident was recorded as 59%. Anthracnose causes a significant reduction in yield and quality traits. Anthracnose has been found in several orchards. Agricultural practices, environmental factors, and disease aggressiveness vary between orchards. Therefore, we looked at spore size, cultural traits, morphological variation, growth pattern, and pathogenicity of different strains of C. acutatum from various orchards. Molecular and phylogenetic analysis confirmed the isolated strains as C. acutatum. In all, 15 C. acutatum isolates from olive orchards were tested for susceptibility to four commercial fungicides (P < 0.001). The examined isolates’ in vitro fungicide sensitivity varied with fungicide concentration. The concentration at which conidial germination was hindered by 50% compared with the control values was observed for difenoconazole, tebuconazole, carbendazim, and cyprodinil, ranging from 0.12 to 2.69 g ml⁻¹. Based on the findings of the fungal growth inhibition studies, carbendazim has been found to be the only fungicide that effectively reduces (P < 0.001) anthracnose caused by C. acutatum strains. Additionally, results revealed that preharvest site treatments of different fungicides greatly decreased anthracnose infections on olive fruit (70 to 90%), and postharvest site applications significantly reduced disease prevalence and severity (75 to 95%). The fungicide carbendazim significantly decreased pre- and postharvest anthracnose infection on olive cultivars. This study suggests that the latter compound might be used to control olive anthracnose in Pakistan while lowering environmental impact and fungicide resistance.

Post-harvest Fruit rot of apple (Malus domestica) was observed on apple cultivar “amri” (local variety grown in swat) during a survey of fruit and vegetable markets in Lahore, Pakistan from June to October 2015. Symptoms appeared as soft watery lesions of yellowish-brown colour and oval in shape on skin of fruit. The lesions become large and size varied from 56-67 mm in diameter. White and velvety growth of fungi was observed on the surface of the fruit, which becomes green with maturity. Infected samples were arbitrarily collected, cut into 5 cm small pieces, surface-sterilized in 1% NaOCl solution. The pieces were placed on Malt Extract Agar (MEA) and incubated at 28 oC for 4-5 days. A single spore culture of the isolated fungi was prepared and maintained on MEA. On MEA, fungal colonies ranged from 12-19 mm diameter after 5 days incubation and were centrally umbonate, velutinous, marginate and sulcate. Colony mycelium was white initially and became dull green to blue green with maturity. Underside of th...

In October-December 2015, dark brown, circular to oblong lesions with chlorotic halos were observed on the leaves of Solanum melongena L. (eggplant) variety Dilnasheen in District Lahore, Pakistan. Early symptoms appeared as dark brown circular spots on the leaves. Over time, spots merged and formed oblong lesions. Entire fields were severely affected by the disease. Pathogen isolations were made on potato dextrose agar (PDA) medium. Symptomatic leaf samples were cut into 3 to 6 mm² pieces, surface sterilized with 2% NaOCl for 2 minutes and rinsed in sterilized water several times, followed by air drying. These samples were plated onto PDA and incubated at 28 ºC for 5 days in the dark. A dark brown colony appeared on the medium. The reverse side of the colony was zonate, black at the center, and lighter outward. Under microscope, dark brown, septate, geniculate, simple or unbranched conidiophores were observed. Conidia were light to dark brown; straight to pyriform; with 3 to 4 cells, with large and curved central cells; boat-shaped; rounded at the tip, smooth walled; with three septa; conidia range in size from 18-26.5 x 8.5-14 μm and are produced apically in a sympodial manner leaving a dark brown scar on conidiophore. Based on morphological characteristics, the pathogen was identified as Curvularia lunata (Manamgoda et al. 2011). The fungus was further identified by amplifying the internal transcribed spacer region (White et al. 1990) by using ITS1 and ITS4 primers. The resulting sequence was submitted to GenBank with Accession No. KU836756. This sequence showed 99% homology with C. lunata (GenBank Accession No. KR870876). To confirm pathogenicity, a C. lunata spore suspension (5 mL) of 4•5x10⁵ conidia/ mL was sprayed onto wounded leaves of variety Dilnasheen in Petri dishes containing furrowed moist cotton. Wounded Dilnasheen leaves were sprayed with 5 mL of sterile distilled water as a negative control. Both treatments were incubated at 26 ºC near-UV light. After 7 days’ incubation, typical necrotic spots were appeared on the inoculated leaves similar to those previously observed in the field. By completing Koch’s postulates, the fungus was consistently re-isolated from infected leaves on PDA, whereas the control leaves did not show any symptoms. To our knowledge, this is the first report of Curvularia lunata leaf spots on Solanum melongena in Lahore, Pakistan. This pathogen is of significant importance as it reduces the production and quality of the eggplant fruit and may become an alarming treat to the production of eggplant in upcoming years if left uncontrolled.

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.