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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. This open access article funding is provided by the Free University of Bozen-Bolzano, Library.

Potato ( Solanum tuberosum L.) cultivation in Pakistan faces challenges, with black scurf disease caused by Rhizoctonia solani Kühn being a significant concern. Conventional methods like chemical fungicides partially control it, but an effective solution is lacking. This study explores the potential of biofertilizers and soil amendments from Asteraceae weed biomass to manage the disease. Two potato varieties, Karoda and Sante, were chosen, and two biofertilizers, Fertibio and Feng Shou, were tested alone or with Xanthium strumarium biomass. Disease pressure was highest in the positive control, with significant reduction by chemical fungicide. X. strumarium biomass also decreased disease incidence significantly. Fertibio showed better efficacy than Feng Shou. Physiological and biochemical attributes of plants improved with biofertilizer and biomass application. Tuber weight, photosynthetic pigments, total protein content, and antioxidant enzymes (CAT, POX, and PPO) were positively correlated. Combined application of Fertibio and S. marianum biomass effectively managed black scurf disease. These eco-friendly alternatives could enhance disease management and yield. Future research should explore their cost-effectiveness, commercialization, and safety., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Rafiq, Shoaib, Javaid, Parveen, Hassan, Nawaz and Cheng.)

Currently, the number of patients with cancer is expanding consistently because of a low quality of life. For this reason, the therapies used to treat cancer have received a lot of consideration from specialists. Numerous anticancer medications have been utilized to treat patients with cancer. However, the immediate utilization of anticancer medicines leads to unpleasant side effects for patients and there are many restrictions to applying these treatments. A number of polymers like cellulose, chitosan, Polyvinyl Alcohol (PVA), Polyacrylonitrile (PAN), peptides and Poly (hydroxy alkanoate) have good properties for the treatment of cancer, but the nanofibers-based target and controlled drug delivery system produced by the co-axial electrospinning technique have extraordinary properties like favorable mechanical characteristics, an excellent release profile, a high surface area, and a high sponginess and are harmless, bio-renewable, biofriendly, highly degradable, and can be produced very conveniently on an industrial scale. Thus, nanofibers produced through coaxial electrospinning can be designed to target specific cancer cells or tissues. By modifying the composition and properties of the nanofibers, researchers can control the release kinetics of the therapeutic agent and enhance its accumulation at the tumor site while minimizing systemic toxicity. The core-shell structure of coaxial electrospun nanofibers allows for a controlled and sustained release of therapeutic agents over time. This controlled release profile can improve the efficacy of cancer treatment by maintaining therapeutic drug concentrations within the tumor microenvironment for an extended period.

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., Competing Interests: The author(s) declare no conflict of interest.

Crown galls were observed on one-year-old olive plants (Olea europaea cv. Manzanilla) in the District Layyah (30.9693° N, 70.9428° E) of Punjab, Pakistan. Large tumors were evident on collars region, causing growth stunting, leaf yellowing, and overall plant dieback (Supplementary fig. 1). Total 900 of olive plant were grown including 300 young plants in five hectare orchards, around 25% of the young plants in orchard had gall formation with varying in size (2-15cm), majority of the infected plants were grown near the water channel, where soil moisture level were high (90-100%). Other olive orchards in the same area have not crown gall problem and the tumorigenic strains of bacteria can cause crown gall on plants (Nemanja Kuzmanović et al. 2015). This study was aimed to determine the pathogen of disease. The randomized collected samples were rinsed with tap water and galls were sterilized with 10% sodium hypochlorite solution for 1.5-3.0 min, washed with sterilized Distilled Water (SDW) then chopped and immersed overnight in SDW at room temperature. Isolations were carried out by plating the internal gall tissues on fresh Luria Bertani agar (LB agar) supplemented with natamycin. After incubating at 28°C for 5 days, 10 single colonies were transferred on new LBA plates for further cultivation at 28°C. After 48 to 72 h, three strains showed white to cream-colored, smooth, convex, glistening, circular with entire edges, and mucoid bacterial colonies resembling Agrobacterium spp. These three strains (BAT01, BAT02, BAT03) also showed biochemical and physiological characteristics similar to A. tumefaciens, including oxidase positive, growth at 35°C and in 2% NaCl, and alkalinity from litmus milk. They were tested negative for utilization of citrate and acid production on potato dextrose agar (PDA) supplemented with CaCO3 (Young et al. 2015). Amplification and sequencing of these three strain's 16S rRNA region and chromosomal recA gene with the universal primers fD1/rP2 and F2898/F2899 verified the identification at species level (Weisburg et al. 1991) . BLAST analysis revealed 100% identity for 16S rRNA and recA gene between the olive crown gall strains. Accession No. of deposited sequences were given in table 1 and the reference sequences GenBank Accessions No. of A. tumefaciens is FM209485.1 and KY913787 respectively. Phylogenetic analysis based on 16S rRNA of the strains from the crown gall and reference strains of various species of Agrobacterium by Maximum-likelihood method with Tamura's three-parameter model using the MEGA X software program confirmed the strain from olive was A. tumefaciens (Supplementary fig. 2). Inoculating the crown part of the plant through wounds of sterile needles plunged into young (2 to 3 day) bacterial culture (107 CFU/ml) and sterile distilled water (SDW) was screened for pathogens on 10 one-year-old olive plants cv. Manzanilla. Plants were grown at 23 ± 3°C, and tumor formation was observed 4 weeks after inoculation. Typical tumours formed and no symptoms found in control plants at inoculation sites and Koch's postulates were fulfilled with re-isolation and amplification of bacteria with recA gene region. This data shows that A. tumefaciens causes crown gall in olive plants. though it is reported before in different olive growing region in the world but This is first time reported in Layyah, Punjab, Pakistan.

Colletotrichum gossypii is a causal agent of anthracnose in upland cotton (Gossypii hirsutum). C. gossypii belongs to the C. gloeosporioides species complex. However, the high level of genetic similarity among the species within the C. gloeosporioides species complex makes the development of a diagnostic assay for C. gossypii challenging. Furthermore, the spore size and cultural characteristics of C. gossypii from different geographical areas can vary. In this study, we examined the morphological variance, growth pattern, and pathogenicity of C. gossypii and developed a molecular diagnostic assay to detect C. gossypii in cotton plants from different regions of China. To overcome any ambiguity in morphological and pathogenic characteristics, a set of primers targeting the β-tubulin (TUB) gene of C. gossypii was designed. Single-nucleotide polymorphisms (SNP) were used to identify C. gossypii at the species level using DNA sequence alignments of the TUB gene. The SPSCG/F and SPSCG/R primer pair only amplified C. gossypii, and was able to amplify C. gossypii in mixtures of other Colletotrichum spp., even when DNA concentrations were up to 10 times lower than that of the other species. This is the first report of the development of SNP-based markers for the specific identification of C. gossypii.

Anthracnose disease in the cotton plant caused by fungal pathogen Colletotrichum gossypii. It is supposed to be most critical diseases in the cotton crop as it causes infection and leads to complete damaging of the cotton crop by infecting the leaves, stems, and bolls in the field. The disease control is challenging due to the absence of an effective fungicide without damaging the farmer health and environment. So the series of experiments were designed to assess the antagonistic activity of biosurfactant released by strain Bacillus licheniformis OE-04 against the anthracnose causing agent in cotton and this strain was screened out from forty eight strain of rhizobacteria. We also estimated the heat stability and pH range and toxicity of biosurfactant produced by strain 0E-04. The results showed that biosurfactant has maximum antifungal activity against C. gossypii. In vitro study concluded that the biosurfactant can reduce fungal activity by inhibiting the spore germination of C. gossypii. Moreover, the biosurfactant also has wide pH and temperature range. We observed Antifungal activity of biosurfactant at 5 to 10 pH range and temperature range was also wide from room temperature to 100 °C. We also observed the toxicity of biosurfactant produced by Bacillus licheniformis against zebra fish (Danio rerio). We were noticed that biosurfactant have least harmful effect with maximum concentration. The study confirmed that biosurfactant of Bacillus licheniformis have high pH and heat stability range with least harmful effects so it can be a good replacement of chemical pesticides for cotton anthracnose control., (Copyright © 2018 Elsevier Inc. All rights reserved.)