Ayesha Baig

High-value horticultural crops grown under protected cultivation are highly susceptible to soilborne and aerial diseases. Greenhouse conditions, including high humidity, controlled temperature, and adequate soil moisture, favor the development and interaction of oomycetes, fungi, bacteria, and plant-parasitic nematodes, resulting in multi-pathogen disease complexes that reduce plant vigor, yield, and crop quality. This chapter examines the ecology, epidemiology, and environmental drivers of greenhouse disease complexes, emphasizing nematode-induced root damage that predisposes plants to fungal, bacterial, and aerial diseases. It links soilborne and aerial pathosystems and evaluates next-generation diagnostics and disease surveillance, including omics-based tools, biosensors, remote sensing, artificial intelligence (AI)-assisted image analysis, hyperspectral and thermal imaging, and Internet of Things (IoT)-based smart sensor networks. The chapter also outlines a smart Integrated Disease Management (IDM) framework incorporating cultural practices, resistant cultivars, biological control agents, microbiome-based approaches, and precision agriculture to improve crop resilience, productivity, and sustainable crop protection under protected cultivation.

Long non-coding RNAs (lncRNAs) have emerged as pivotal regulators in plant immune responses, yet their roles in rice resistance against Magnaporthe oryzae (M. oryzae) remain inadequately explored. In this study, we integrated translatome data with conventional genome annotations to construct an optimized protein-coding dataset. Subsequently, we developed a robust pipeline (“RiceLncRNA”) for the accurate identification of rice lncRNAs. Using strand-specific RNA-sequencing (ssRNA-seq) data from the resistant (IR25), susceptible (LTH), and Nipponbare (NPB) varieties under M. oryzae infection, we identified 9003 high-confidence lncRNAs, significantly improving identification accuracy over traditional methods. Among the differentially expressed lncRNAs (DELs), those unique to IR25 were enriched in the biosynthetic pathways of phenylalanine, tyrosine, and tryptophan, which suggests that they are associated with the production of salicylic acid (SA) and auxin (IAA) precursors, which may be involved in defense responses. Conversely, DELs specific to LTH primarily clustered within carbon metabolism pathways, indicating a metabolic reprogramming mechanism. Notably, 21 DELs responded concurrently in both IR25 and LTH at 12 h and 24 h post-inoculation, indicating a synergistic regulation of jasmonic acid (JA) and ethylene (ET) signaling while partially suppressing IAA pathways. Weighted gene co-expression network analysis (WGCNA) and competing endogenous RNA (ceRNA) network analysis revealed that key lncRNAs (e.g., LncRNA.9497.1) may function as miRNA “sponges”, potentially influencing the expression of receptor-like kinases (RLKs), resistance (R) proteins, and hormone signaling pathways. The reliability of these findings was confirmed through qRT-PCR and cloning experiments. In summary, our study provides an optimized rice lncRNA annotation framework and reveals the mechanism by which lncRNAs enhance rice blast resistance through the regulation of hormone signaling pathways. These findings offer an important molecular basis for rice disease-resistant breeding.

WRKY transcription factors play an important role in transcriptional reprogramming associated with plant abiotic stress responses. In this study, the role of Solanum tuberosum (S. tuberosum; St) WRKY transcription factors StWRKY4 and StWRKY56 were explored in response to salt stress by generating transgenic potato lines using RNAi. The results showed that the total chlorophyll content in transgenic StWRKY4 was 6.1 mg/g at 200 mM after 35 days; however, in StWRKY56, an elevated 12.6 mg/g total chlorophyll was observed which indicated different operating mechanisms of these StWRKY transcription factors under salt stress. Proline content increased to 1.0 mg/g in StWRKY4 while it decreased to 0.54 mg/g in StWRKY56 as compared to their respective control plants after 35 days at 200 mM of salt stress. For Na⁺/K⁺ ratios, StWRKY4 and StWRKY56 showed 32.3 and 5.5 values, respectively, in silenced plants under similar conditions. This shows contrasting trends in StWRKY4 and StWRKY56 for Na⁺/K⁺. However, the expression analyses of StSOS1s were found to be upregulated, whereas for StNHX3s these were found to be downregulated in StWRKY4 and StWRKY56 under salt stress. Thus, this study, for the first time, demonstrated the different but critical roles of StWRKY4 and StWRKY56 for fine-regulating salt stress tolerance in complex signaling network of potato plant.

Potato tubers collected from different areas showed the prevalence of dry rot with characteristic white mycelia like symptoms of Fusarium pathogen. Fusarium species from diseased tubers were isolated based on morphological features. 18 S-Internal Transcribed Spacer (ITS) and Translation Elongation factor 1-α (TEF1-α) based analysis followed by phylogenetic tree constructed using ITS, TEF1-α and RNA polymerase II subunit B (RPB2) identified Fusarium isolates as Fusarium verticilloides, Fusarium soloni, Fusarium falciforme and Fusarium oxysporum. According to our knowledge, this study is the first report of the occurrence of Fusarium falciforme causing dry rot on potato tubers in Pakistan. The pathogenicity test confirmed the re-appearance of dry rot symptoms with Fusarium falciforme on potato tuber. The formation of Fusarium falciforme Ag NPs was confirmed using UV-Visible spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and Field-emission scanning electron microscopy (FE-SEM) analysis. The synthesized Ag NPs showed color change with Fusarium falciforme and accordingly, UV peak was observed at 431 nm. The XRD revealed Ag based specific angles at 36.14°, 44.26°, 64.42° and 77.44° corresponding to face cubic structure. Similarly, FTIR band absorption at 3297 cm−1, 1626 cm−1 and 1057 cm−1 ascribed the presence of various biomolecules with O-H, N-H and C-N stretching vibrations based on Ag NPs synthesis. SEM indicated 29 nm synthesized mainly spherical Ag NPs and EDS analysis showed the presence of Ag in NPs. Maximum zone of inhibition with Fusarium falciforme Ag NPs was observed against Pseudomonas aeruginosa, Escherichia coli, Pseudomonas syringae and Staphylococcus aureus at 0.05 LSD significance.

This study aimed to assess the water quality, prevalence of waterborne diseases, and hygiene practices in selected Union Councils (UCs) of Abbottabad District. A small number of studies on water contamination and its effects on health have been carried out by various researchers and government organizations in Abbottabad. Considering how much time has passed since the last study, this one will provide up-to-date information on water quality and disease prevalence, taking into account any developments or patterns over the last ten years. The areas of high and low prevalence of waterborne diseases were identified based on secondary data from district hospitals. The high-prevalence area included UC Nawanshehr, UC Mirpur, and UC Sherwan, while the low-prevalence area comprised UC Bakot, UC Moolia, and UC Tearch. Water samples were collected from these UCs and analyzed for physical parameters (pH, turbidity, TDS, EC, and temperature) as well as biological parameters (E. coli, Salmonella typhi, and Vibrio cholera) to assess the drinking water quality. Questionnaires were also administered to gather information on water supply, storage, treatment, disease prevalence, and hygiene practices. The findings indicated that all physical parameters fell within the permissible limits; however, all water samples were found to be contaminated with biological agents. UC Mirpur and UC Bakot exhibited the highest disease prevalence rates of 55% and 33%, respectively. Correlation analysis revealed strong positive correlations (correlation coefficients of 1, 0.8, and 0.5) between the presence of E. coli, Salmonella typhi, and Vibrio cholera and disease prevalence. The questionnaire data revealed that the main sources of water supply were tube wells, small streams, and water pumps, with minimal water treatment practices. The water treatment level was, however, noted to be far below expectations, even with the required hygiene practices being followed in all Union Councils. Above all, these findings indicate the necessity to promote best practices for water treatment in the studied UCs and inform efforts to improve drinking water quality at the population level. Public health measures need to focus on improving access to safe drinking water sources, appropriate water treatment, and hygiene. The identified findings from this study nurture innovative affordable solutions that can lead to decreased waterborne diseases and enhanced public health protections in the Abbottabad District.

Citrus Huanglongbing (HLB), caused by the phloem-inhibiting bacterium Candidatus Liberibacter asiaticus (CLas), is the most devastating citrus disease, intimidating citrus production worldwide. Although commercially cultivated citrus cultivars are vulnerable to CLas infection, HLB-tolerant attributes have, however, been observed in certain citrus varieties, suggesting a possible pathway for identifying innate defense regulators that mitigate HLB. By adopting transcriptome and small RNAome analysis, the current study compares the responses of HLB-tolerant lemon (Citrus limon L.) with HLB-susceptible Shatangju mandarin (Citrus reticulata Blanco cv. Shatangju) against CLas infection. Transcriptome analysis revealed significant differences in gene expression between lemon and Shatangju. A total of 1751 and 3076 significantly differentially expressed genes were identified in Shatangju and lemon, respectively. Specifically, CLas infected lemon tissues demonstrated higher expressions of genes involved in antioxidant enzyme activity, protein phosphorylation, carbohydrate, cell wall, and lipid metabolism than Shatangju. Wet-lab experiments further validated these findings, demonstrating increased antioxidant enzyme activity in lemon: APX (35%), SOD (30%), and CAT (64%) than Shatangju. Conversely, Shatangju plants exhibited higher levels of oxidative stress markers like H₂O₂ (44.5%) and MDA content (65.2%), alongside pronounced ion leakage (11.85%), than lemon. Moreover, microscopic investigations revealed that CLas infected Shatangju phloem exhibits significantly more starch and callose accumulation than lemon. Furthermore, comparative sRNA profiles revealed the potential defensive regulators for HLB tolerance. In Shatangju, increased expression of csi-miR166 suppresses the expression of disease-resistant proteins, leading to inadequate defense against CLas. Conversely, reduced expression of csi-miR166 in lemon plants enables them to combat HLB by activating disease-resistance proteins. The above findings indicate that when infected with CLas, lemon exhibits stronger antioxidative activity and higher expression of disease-resistant genes, contributing to its enhanced tolerance to HLB. In contrast, Shatangju shows lower antioxidative activity, reduced expression of disease-resistant genes, significant ion leakage, and extensive callose deposition, possibly related to damage to plant cell structure and blockage of phloem sieve tubes, thereby promoting the development of HLB symptoms.

Potato is one of the highly consumed vegetable crop grown in different regions across Pakistan that is affected by fungal diseases. The current research was conducted to identify fungal pathogen causing mold-like disease of potato in Khyber Pakhtunkhwa (KP), Pakistan. For molecular identification and characterization of the fungal disease; potato tuber samples were collected followed by culturing on potato dextrose agar (PDA). Based on morphological features, the pathogen was identified as a Penicillium species. This result was obtained in 45 different isolates from potato tubers. Molecular identification was done using β-tubulin primers and ITS5 sequencing of 13 different isolates that releveled 98% homology with BLAST (GenBank accession no. KX958076) as Penicillium solitum (GenBank accession nos. ON307317; ON307475 and ON310801). Phylogenetic tree was constructed that showed Penicillium solitum prevalence along with Penicillium polonicum and Penicillium citrinum on potato tubers. Based on this, Penicillium solitum based silver nanoparticles (Ag NPs) were synthesized and characterized using UV-visible spectroscopy, Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), energy dispersive X-ray (EDX) and field emission scanning electron microscopy (FE SEM). UV-analysis showed a characteristic peak at 410 nm confirming synthesis of Penicillium solitum based Ag NPs. This was further confirmed by XRD followed by EDX and SEM that showed face cubic crystal structure with Ag as major constituent of 18 nm formed spherical Ag NPs. FTIR showed band stretching of O-H, N-O and C-H of biological origin. Similarly, Penicillium solitum based Ag NPs presented strong anti-bacterial and anti-fungal activity at 0.5 level of significance LSD. According to our knowledge, this is the first report of Penicillium solitum identification in Pakistan, its Ag NPs synthesis and characterization to be used against pathogens of agricultural significance.

This research was carried out to analyze variations in indoor and outdoor ozone concentrations and their health impact on local communities of megacities in Pakistan. For indoor ozone measurements, industrial units of an economic zone, Hattar Industrial Estate, Haripur, KPK, Pakistan, were selected. For outdoor ozone measurements, maximum and minimum peaks from different selected stations of three megacities (Islamabad, Abbottabad, and Haripur Hattar) in Pakistan were analyzed for paired comparisons. The tropospheric ozone levels were measured with the help of a portable SKY 2000-WH-O₃ meter from December 2018 to November 2019. According to the findings of this investigation, the indoor ozone concentrations at Hattar Industrial Estate exceeded the permissible limit devised by the WHO. The highest concentration (0.37 ppm) was recorded in the month of May in the food industry, while the lowest concentration (0.00 ppm) was recorded in the cooling area of the steel industry in the month of December. For outdoor ozone concentrations, the maximum concentration (0.23 ppm) was detected in Islamabad in the month of March 2019, whereas the rest of year showed comparatively lower concentrations. In Haripur, the maximum concentration (0.22 ppm) was detected in the month of February 2019 and a minimum concentration (0.11 ppm) was found in the month of November 2019. In Abbottabad, the maximum concentration (0.21 ppm) was detected in the month of March 2019 and the minimum concentration was 0.082 ppm. Increasing tropospheric ozone levels might be harmful for local communities and industrial laborers in the winter season because of the foggy weather. In the Abbottabad and Hattar regions, since COVID infection is indirectly related to low temperature and high emission of gases may compromise the respiratory systems of humans. The results of the present study were shared with industrialists to set precautions for ambient air quality and support the adoption of low emission techniques in industries for the safety of labour and nearby residents.

Background: Taxus wallichiana is an evergreen tree species found in the Himalayan region of Pakistan. The tree possesses important secondary metabolites such as Taxol that has been implicated in treating breast, ovarian and colon cancer. Therefore keeping in view the importance of this plant species, silver nanoparticles were synthesized using Taxus wallichiana aqueous leaf extract and evaluated for their anti-bacterial and anti-cancer properties. Methods: Silver (Ag) nanoparticles (NPs) were characterized for their optical, morphological and structural features using techniques such as UV-visible spectroscopy, X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) and were evaluated for their antibacterial activity and anti-cancer activity using U251 cell line. Results: The study showed that the UV-absorbance peak of Ag₂O NPs at 450 nm shifted to 410 nm, affirming the formation of leaf extract Ag NPs. Similarly structural studies revealed the crystalline nature of the cubic structure of the Ag crystal with an average crystallite size of 29 nm. FTIR analysis exhibited the existence of different functional elements including O-H and N-H and phenolic groups. Non-spherical glomerular shaped Taxus wallichiana Ag NPs were observed from SEM studies and EDX profile showed Ag as the main element along with constituent of biological origin. The synthesized Ag NPs showed significant antibacterial activity against Salmonella typhi, and Staphylococcus aureus. The cytotoxic activity of Ag NPs on U251 brain cancer cells showed a synergistic effect with 10 ug/mL concentration after 48 and 72 h incubation based on cell viability assay indicating promising glioblastoma drug potential.