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Begomovirus is a major and economically important genus of the Geminiviridae family. It comprises a wide range of viruses that infect a number of dicot plants including the horticulture crops, cereal crops, aromatic plants, vegetable crops, medicinal plants and weeds in various regions of the world. This study aims to investigate and correlate the various symptoms of begomovirus / satellites in different plants grown in the vicinity of Kohat, Pakistan. Furthermore, the characterization of the selected virus-associated satellites at the molecular level is also studied. Samples of suspected plants showing begomoviral infection were collected from the Kohat District. Genomic DNA was extracted from the infected plants and subjected to PCR using DNA-1/DNA-2 and Beta01/Beta02 for alpha satellites and beta satellites, respectively. The amplified PCR products were cloned and sequenced commercially. After sequencing, in silico sequence and phylogenetic analysis was also performed. Our study discovered that many plants in the Kohat District display begomovirus and satellite disease symptoms with mild to extreme disease severity. Disease incidence is especially high in okra. Beta satellites were isolated and sequenced from Spinacia oleracea and Capsicum annum plants and they showed more than 90% sequence similarity with chilli leaf curl and tomato leaf curl beta satellites. The existence of betasatellites in spinach and chilli plants was discovered for the first time in the Kohat region. Moreover, the distribution of these highly pathogenic variants of chilli leaf curl and tomato leaf curl betasatellites in the district Kohat has been reported previously. Copyright (c) The Author

Begomovirus is a major and economically important genus of the Geminiviridae family. It comprises a wide range of viruses that infect a number of dicot plants including the horticulture crops, cereal crops, aromatic plants, vegetable crops, medicinal plants and weeds in various regions of the world. This study aims to investigate and correlate the various symptoms of begomovirus / satellites in different plants grown in the vicinity of Kohat, Pakistan. Furthermore, the characterization of the selected virus-associated satellites at the molecular level is also studied. Samples of suspected plants showing begomoviral infection were collected from the Kohat District. Genomic DNA was extracted from the infected plants and subjected to PCR using DNA-1/DNA-2 and Beta01/Beta02 for alpha satellites and beta satellites, respectively. The amplified PCR products were cloned and sequenced commercially. After sequencing, in silico sequence and phylogenetic analysis was also performed. Our study discovered that many plants in the Kohat District display begomovirus and satellite disease symptoms with mild to extreme disease severity. Disease incidence is especially high in okra. Beta satellites were isolated and sequenced from Spinacia oleracea and Capsicum annum plants and they showed more than 90% sequence similarity with chilli leaf curl and tomato leaf curl beta satellites. The existence of betasatellites in spinach and chilli plants was discovered for the first time in the Kohat region. Moreover, the distribution of these highly pathogenic variants of chilli leaf curl and tomato leaf curl betasatellites in the district Kohat has been reported previously.

Etiological patterns of uropathogens is different in different regions due to continuous evolution, of bacteria, antibiotics sensitivity patterns, misuse and overuse of antibiotics. Therefore, it is important to know the antibiotic susceptibility patterns for prescription of suitable antibiotic. This study was conducted to determine the prevalence of uropathogens and their antimicrobial sensitivity pattern from Kohat region of Pakistan. In this study 100 samples were collected from both males and females of all ages in which 70 samples contained microbes. In 30 samples no microbial growth was recorded. The Percentage of positive culture from both male and female were 57% and 43% respectively Both Gram (+) and Gram (-) bacteria were found in UTI but E.coli (34.21%) was predominant followed by K. pneumoniae (10.52%), P. aeruginosa (9.21%), K. oxytoca (6.57%), C. albicans (5.26%), E. faecium (5.26%), E. faecalis (3.94%), S. aureus (3.94%), E. cloacea (2.63%), C. freundii (2.63%), P. mirabalis (2.63%) and A. baumannii (1.31%). Many of the isolates showed resistance to commonly used antibiotics. The sensitivity percentage of different commonly used antibiotics against both Gram (+) and Gram(-) bacteria were Ampicillin 13%, Ceftriaxone 25%, Amikacin 77%, Gentamicin 41%, Augmentin 44.77%, Fosfomycin 64%, Cotrimoxazole 36%, Nitrofurantoin 68%, Ciprofloxacin 37%, Imipenem 78%, Meropenem 67%, Cefepime 25% and Tetracycline 40%. The most effective antibiotics against both Gram (+) and Gram (-) bacteria were Fosfomycin, Imipenem, Meropenem Amikacin and Nitrofurantoin. In light of the findings of this study, it is strongly recommended to discover new antimicrobial compounds and evaluate the resistant pattern at genomic and proteomics level to discover the genes which are responsible for antibiotics resistant pattern.

Parthenium hysterophorus [commonly known as Carrot grass; native to American tropics; family Asteraceae] is a flowering, short-lived perennial or an annual invasive-weedy plant. In the recent few years, the plant is spread (in epidemic proportions) vigorously, at least, in the two provinces (KP and Punjab) and the twin capital cities (Islamabad and Rawalpindi). The weed came in the lame light soon after the monsoon rains and floods of September 2012 and August 2013 that hit larger areas of KP and Punjab provinces of Pakistan. The massive boom in the weed was witnessed in areas under floods of the river Kabul and the Indus Ocean. It is speculated that after initial entry into the flood zones, the seeds then germinated and dispersed into the near and farther areas in the country, including hilly areas. The plant is categorized as a poisonous weed, and a source of skin allergies and itching. It may be a cause of increased reports of asthma, cough, fever, and allergies related to eyes in these areas [1, 2]. Due to its vigorous growth and allelopathic effects, the plant soon dominated over all other weeds and crop plants. Furthermore, it has a tremendous potential to withstand abiotic and biotic stresses. Its vigorous growth has resulted in the loss of local floral biodiversity [3]. Specifically in Islamabad, it has dominated completely the wild cannabis (weeds). However, this year, the cannabis weed seems to overcome gradually the parthenium dominance. The coming years may witness the revival of other plant species suppressed by parthenium. Although the plant seems to be resistant/tolerant to biotic stresses, very few plants were identified with leaf rolling, vein yellowing, stunting and bunchy-top like diseases, indicative of begomovirus infection. PCR amplification and sequencing confirmed a symptomatic parthenium plant positive for the viral infection (unpublished observation; Figure 2). Previously, only a single report is available of parthenium being infected with geminiviruses [4]. Currently, no weedicide is available to eradicate the weedy plant. The only available alternative is to drag the plants out of soil along with their roots before they bear seeds. The presence of the plant everywhere on barren lands, streets, along the drains, hilly terraces, orchards, in lawn grass, and in crop fields makes complete eradication almost impossible (Figure 1a-c). Increased ailment linked with parthenium like – toxicity in the livestock and insect pollinators, skin allergies in humans and reduced agricultural productivity necessitates that the government and non-government stakeholders should take stringent measures to save human health, livestock and agricultural production. Copyright (c) Muhammad Ali, Riaz Ahmad Afridi, Sadiq Ali, Malik Nawaz Shuja, Hasan Riaz

The increased and non-judicious use of pesticides and fertilizers has associated complications on the human, animal and plant health. They may accumulate in water and soil to reduce diversification of croplands, limit nutrients availability and diminish genetic diversity of the associated microbes, flora and fauna. Chemical pesticides and fertilizers are also known to impart global warming, insect, animal and human genetic disorders and diseases. Plant probiotics, thereby enhancing plant health, growth and production, not only reduce/minimize the use of chemical pesticides and fertilizers but also are helpful reclaiming the soil beneficial for all living organisms. However, there are certain shortcomings and challenges associated with the use of plant probiotics. This chapter circumvents the studies covering, in general, the origin, classification, mechanism of action, and in specific, the perspectives and challenges using plant probiotics.

Bacillary diarrhea caused by Shigella flexneri is mediated by various virulence factors which make it the leading agent of diarrhea in developing countries. Previously, a high prevalence of S. flexneri, associated with diarrhea has been reported in Pakistan but no data is available on their virulence profile. The present study reports for the first time analysis of various virulence factors among S. flexneri serotypes isolated from clinical (diarrheal stool) and non-clinical (retail raw foods and drinking water) sources. A total of 199 S. flexneri (clinical: 155, raw foods: 22, water: 22) belonging to various serotypes were subjected to virulence genes detection and virulence profiling. The most frequent virulence gene was found to be ipaH (100%), followed by sat (98%), ial (71.3%), set1B (65.8%) and set1A (38.7%). A high level of virulence was detected in serotype 2b as compared to other serotypes as 32.3% of all serotype 2b have the entire set of five virulence genes including ipaH (100%), ial (100%), sat (37.7%), set1A (89.3%), and set1B (100%). Seven different virulence gene profiles (V1 - V7) were detected and the most frequently observed to be V1 (ipaH+, ial+, sat+, set1A+, set1B+) followed by V3 (ipaH+, ial+, sat+, set1B+). The predominant virulence gene pattern in serotype 2b isolated from clinical and non-clinical samples were V1 and V3. Furthermore, about 32% strains belonging to serotype 2b contain the complete set of five virulence genes isolated from patients with high disease severity. In conclusion, the current finding revealed for the first times that serotype 2b was the most virulent strains in both clinical and non-clinical samples in Pakistan. In addition, the virulence of serotype 2b was well correlated with high disease severity.

Luteoviruses are economically important plant viruses. Specifically, barley yellow dwarf virus is epiphytotic to almost all small-grain cereal growing areas. The disease cycle is complex. This luteovirus has evolved several intelligent mechanisms to communicate with both plant and phloem-feeding insect-vector aphid. Environmental cues influence disease severity, aphid infestation and viral load. Within an aphid, virus circulates persistently in a non-propagative manner and is transmitted selectively to the host plants. Selection of viruses within aphids has a role in virus isolate prevalence over a specific area. In the host-plant system, the virus has to release its single sense-strand RNA genome (approx. 5.6 to 6 kb), translate and subsequently replicate its genome using its own replicase and host machinery. This review summarizes our current understanding of disease epidemiology and reviews the current literature encompassing viral infectivity, economic impact and control measures.

Ficus virens (family: Moraceae) is a deciduous tree found in the Indian subcontinent, some other Southeast Asian countries, and northern Australia. It is valued for its latex, wood, and medicinal and edible products. In December 2018, during a routine survey to record begomoviruses other than cotton infecting viruses in Pakistan, curling of young leaves, which is a typical symptom of infection by viruses of the genus Begomovirus, was observed in F. virens trees in a park in Lahore, central Punjab, Pakistan. The total DNA of three trees was extracted using the Viral Gene-spin Viral DNA/RNA Extraction Kit (iNtRON Biotechnology, Seongnam, Korea) and used as a template in PCR amplification with begomovirus-specific primers: Beg-F (5′-CCGTGCTGCTGCCCCCATTGTCCGCGTCAC-3′) and Beg-R (5′- CTGCCACAACCATGGATTCACGCACAGGG-3′). The amplified 1-kb DNA was cloned into the pGEM-3Zf (+) vector (Promega, Madison, WI) and sequenced by a commercial sequencing service (Macrogen, Seoul, Korea). NCBI BLAST results revealed a nucleotide (nt) sequence identity of 95, 93, and 92% with chilli leaf curl India virus, rose leaf curl virus, and papaya leaf crumple virus, respectively. Based on the sequence obtained, new primers, DCP-F (5′-AGCACATTGGTAAAGTCATGTGTG-3′) and DCP-R (5′-GGATATCATGTCTGGACTCAAATG-3′), were designed to obtain full-length DNA-A of the begomovirus. An approximately 2.8-kb product was amplified and cloned. The resulting sequence (GenBank accession no. MN537564) showed 97.43% nt identity with duranta leaf curl virus (DLCV; KT948069), a Begomovirus species reported in the ornamental shrub Duranta erecta in Pakistan by Iram et al. (2005) and by Anwar et al. (2017). In the former case, only DNA-A of DLCV was found without any associated molecules such as DNA-B or DNA β, whereas, in the latter case, tomato leaf curl New Delhi virus DNA-B was found in association with the DNA-A of DLCV, like in many other Old World begomoviruses. The attempt to amplify DNA-B through PCR using newly designed primers NDB-F (5′-AAACACAGGAGGGATCGGAG-3′) and NDB-R (5′-CACAGATTTCCTTACGCGTATATT-3′) based on the sequence KT948071 was unsuccessful. Similarly, efforts to detect alphasatellite and betasatellite molecules in infected F. virens using universal primers (Bull et al. 2003; Briddon et al. 2002) were unsuccessful. Furthermore, Southern blot hybridization was carried out using [³²P]-radiolabeled PCR product of 1 kb obtained with Beg-F/R primers as a probe. This produced dsDNA bands of different conformations as well as a ssDNA band, which confirms the presence of DLCV in F. virens. Members of the family Geminiviridae, to which begomoviruses belong, mainly infect herbaceous hosts, but they have also been reported in a few woody hosts, such as citrus chlorotic dwarf associated virus in citrus (Loconsole et al. 2012), grapevine red blotch virus in grape (Al Rwahnih et al. 2013), jatropha mosaic virus in Jatropha multifida (Polston et al. 2014), and apple geminivirus 1 in apple tree (Liang et al. 2015). To our knowledge, this is the first report of DLCV infecting F. virens in Pakistan. The results obtained here indicate that F. virens may serve as an alternative host of DLCV and a possible source of infection to the commonly grown D. erecta ornamental garden hedge.

Cotton leaf curl disease (CLCuD) is the major biotic limitation to cotton production in Pakistan and northwestern India. The disease is caused by various distinct viruses of the genus Begomovirus (family Geminiviridae) in association with a disease-specific betasatellite - cotton leaf curl Multan betasatellite (CLCuMB). Since 2001, when resistance to CLCuD in cotton was broken, only one virus was consistently identified in cotton exhibiting CLCuD symptoms in Punjab province (Pakistan) - cotton leaf curl Burewala virus (CLCuBuV). An analysis of all CLCuBuV isolates available in the databases showed these to represent only a single strain, based on currently applicable criteria. Virus and betasatellite clones were obtained from a leaf sample (isolate C49) collected from a CLCuD symptomatic cotton plant in Layyah district, Punjab province, in 2012. Analysis of the sequence of the betasatellite showed this to be an isolate of CLCuMB containing the recombinant fragment typical of this satellite post-resistance-breaking. The virus was shown it to be an isolate of CLCuBuV but to be distinct from all previously characterised isolates and to represent a distinct strain. In common with previous CLCuBuV isolates, the virus from C49 is a recombinant containing sequences derived from viruses of two species that were prevalent in cotton pre-resistance-breaking but with distinct recombination break sites. As was the case with the earlier CLCuBuV, the newly identified strain of CLCuBuV lacks an intact transcriptional activator protein.