Citrus is economically an important crop in Pakistan and good source of vitamin C with great antioxidant potential. Citrus production is affected throughout the world by a disease “Huanglongbing (HLB)”, causal organism for which is “Candidatus liberibacter asiaticus” (CLas), Africanus and Americanus. Survey of citrus growing regions of central Punjab were conducted from 2014-2015. Bacterial diversity in the leaves of citrus trees and their associated weeds were explored using a cultured based approach. Based on the analysis of the 16S rRNA gene sequences, all the bacterial isolates were classified into two major phyla of the Domain “Bacteria”. Thirty seven endophytic bacterial isolates from citrus foliage fall in fifteen genera on the basis of 16S rRNA sequence. The culturability of these bacteria revealed that they are Firmicutes and Proteobacteria especially towards the fruiting stage. The species of Bacillus, Enterococcus, Staphylococcus, Brevibacterium fall in the Firmicutes whereas alpha (Rhizobium) and beta (Comamonas, Burkholderia); gamma Proteobacteria (Enterobacter, Yersinia, Klebsiella and Psychrobaer) were the predominant ones. The bacteria which were isolated and sequenced from different weeds belongs to the class Firmicutes (Bacillus, Enterococcus, Staphylococcus, Lysinibacillus, Brevibacterium, Paenibacillus) and Proteobacteria (Pantoea, Pseudoacidovorax, Escherichia, Enterobacter, Pseudomonas), respectively. This study will help understand the bacterial diversity of pathogenic strains on all the alternate weeds that can be a host for pathogens and become source of inoculation and disease spread. Next generation sequencing (NGS) supports quick exploration of the diversity of bacterial populations in numerous environments. NGS based diversity analysis revealed Proteobacteria, Firmicutes, Actinobacteria, Cyanobacteria and Bacteriodes as the predominant genera in all samples. The leave samples of Citrus aurantifolia, Citrus paradisi had almost equal diversity of bacteria but it varied in Citrus reticulata Blanco with higher proportion of Proteobacteria and Cyanobacteria as compared to others. NGS technology provides the evidence of the microbiota present in plant tissues and through this technique understanding about host pathogen interaction can be enhanced. By Far this is the first 16S rRNA based NGS study from Citrus leaves using Illumina (Hi Seq) in Pakistan. ix Plant growth promoting bacterial endophytes (PGBEs) constitutes an alternative to enhance crop yield and resistance of plants against diseases. In vitro screening the isolated strains for (PGBEs) characteristics were carried out. In this study screening of sixteen novel bacterial endophytes isolated from the leaves of citrus were performed for plant growth promoting activities including Indole acetic acid, siderophores, and Phosphate solubilization. Isolated bacterial endophytes were also tested for the screening of cell wall hydrolyzing enzymes production such as cellulase, chitinase, protease, pectinase and lipase as well as antibacterial activity. According to results 75-80 % of the test strains were positive for PGP and enzymatic activities of test strains. However antibacterial activity was found positive in 68.75% of test cultures while five spp. Enterococcus faecalis, Klebsiella pneumonia, Staphylococcus haemolyticus, Bacillus megaterium and Bacillus cereus did not show any antibacterial activity against any of the targeted bacterial strains. Thus the data presented in this manuscript is unique and in the sense that these bacterial species has not been reported before from citrus. The findings are likely to use of these endophytes to enhance the plant growth and yield. Endophytes have a symbiotic relationship with the different parts of plants and could play a very important role in improving the plant growth. Ten morphologically different bacteria belongs to different genera were selected for the single and mixed inoculation of sour orange seedlings by three different (Injection, soil mix and spray) methods. After five weeks of inoculation, different morphometric parameters were recorded i.e., shoot/root length, shoot/root fresh and dry biomass, Bio-physical analysis (Relative water content were also performed to detect the effect of these endophytic bacteria on physiology of host plant and also to check whether these bacterial species reduce or enhance the plant growth. The selected strains were members of firmicutes (Staphylococcus haemolyticus, Enterococcus faecalis, Bacillus safensis, Bacillus megaterium, Bacillus cereus, and Brevibacillus borstelensis) and Gama Proteobacteria (Pseudomonas sp., Pseudomonas aeruginosa, Enterobacter hermachei, and Proteus mirabilis). x Five bacterial strains named as Bacillus cereus, Bacillus safensis, Bacillus megaterium, Pseudomonas aeruginosa, Proteus mirabilis imparted beneficial effects on growth and physiology of sour orange seedlings. The best results in terms of plant growth were shown by Bacillus cereus and Pseudomonas aeruginosa. These strains can be used as bio-fertilizers as alternate to chemical fertilizers after checking their impact on field conditions but in case of mixed inoculation of bacterial strains 5M (Bacillus safensis , Pseudomonas sp., Bacillus megaterium) and 6M (Pseudomonas aeruginosa, Brevibacillus borstelensis, Bacillus cereus, Proteus mirabilis) performed best in terms of increase of plant growth promoting activity. According to results of all three methods of inoculation showed variable trends for different parameters with different strains, but soil mixing, or injection method were found to be good for the inoculation of bacteria in to plants. Real time PCR was performed to determine the relative quantification of bacterial inoculums. The increased amount of bacterial cells in inoculated treatments indicated the successful survival and colonization of these inoculated bacterial strains as well as their competitiveness on the native bacterial community. Isolated endophytes were applied on six seasonal winter vegetables (Cabbage, Chili, Spinach, Salad, Brinjal, Turnip) to know the extent of colonization of these bacterial endophytes on these vegetables as a host or non-host tissues bacterial suspension (108 CFU mL-1) were inoculated on the ventral side of vegetables leaves by injection. Plants were analyzed after five weeks of inoculation, for the physical, bio-physical and physiological parameters. According to Results Bacillus cereus, Bacillus safensis, Bacillus megaterium, Pseudomonas aeruginosa, Proteus mirabilis impose beneficial effects on growth and physiology of vegetable hosts. The results pointed out that bacterial endophytes isolated from citrus leaves own an important role to improve plant growth and could be used as inoculants to establish a sustainable crop production system.
Penelitian ini bertujuan untuk mengetahui dan menganalisis hubungan pengaruh kinerja kepegewaian dalam administrasi perkantoran di kantor camat kecamatann Tuhemberua kabupaten Nias Utara. Populasi dalam penelitian ini adalah pegawai Kantor Camat Kecamatan Tuhemberua yang berjumlah 12 orang. Jenis data yang digunakan dalam penelitian ini adalah data primer. Teknik pengumpulan data dengan angket, wawancara, observasi, ujian (test) dokumentasi. Teknik analisis data dengan melakukan verifikasi data, pengelolaan angket, dan pengolahan data: (a). Uji validitas data, (b). Uji reliabilitas data, dan (c). Pengujian hipotesis. Dari hasil perhitungan koefisien korelasi diperoleh rhitung (rxy) = 0,834 jika dikonsultasikan pada tabel harga kritik r produk moment untuk interval kepercayaan 5% setelah di hitung rhitung = 0,834 > rtabel = 0,576. Berdasarkan regresi linear sederhana maka sumbangan kinerja kepegawaian terhadap administrasi perkantoran di Kantor Camat Gunungsitoli Tuhemberua 2,668. Dari perhitungan koefisien determinasi besarnya pengaruh variabel x terhadap variabel y di Kantor Camat Gunungsitoli Tuhemberua 69, 48%. Berdasarkan kriteria pengujian hipotesis ternyata Ha adanya pengaruh dan H0 tidak adanya pengaruh, sebab thitung = 7, 20 > rtabel = 2,160 sehingga dapat dinyatakan adanya pengaruh kinerja kepegawaian terhadap administrasi perkantoran pada kantor camat Tuhemberuaa kabupaten Nias Utara.
Malaria is a devastating disease for the world and it is also endemic in Pakistan. This study was undertaken to determine the current epidemiology and antimalarial resistance of Plasmodium species, across Pakistan. This malariometric population survey was conducted in 2011 using blood samples collected from 801 febrile patients of all ages in four provinces and the capital city of Islamabad. Microscopically confirmed Plasmodium-positive blood samples were later reconfirmed by PCR. Of the 707 PCR-positive samples, Plasmodium falciparum infection was found in 128, Plasmodium vivax in 536 and mixed infection of P. falciparum plus P. vivax in 43 isolates. Ninety-four microscopy-positive samples were PCR-negative and results were reconfirmed by repeating PCR. None of the isolates was positive for Plasmodium malariae or Plasmodium ovale DNA. Although microscopy is gold standard for malaria diagnosis but in our study PCR showed diagnostic sensitivity and specificity over microscopy in malaria speciation. Limited numbers of studies have been carried out in Pakistan to establish the efficacy for treatment of P. falciparum and P. vivax with chloroquine (CQ) and sulfadoxine/pyrimethamine (SP), as CQ plus primaquine is recommended for P. vivax treatment and SP in combination with artesunate to P. falciparum. So we investigated molecular resistance markers in the pfcrt, pfmdr1, pfdhfr and pfdhps genes and determined the origin of chloroquine-resistant in P. falciparum parasites across several sites in Pakistan. Pyrosequencing, microsatellite typing and real-time PCR techniques were used for molecular characterization. The pfcrt K76T mutation was found in all samples as part of the SVMNT haplotype. Microsatellites flanking pfcrt showed high similarity to the signature found in India and Papua New Guinea. Pfmdr1 N86Y was found in 20% of samples and all samples carried a one copy of the pfmdr1 gene. The pfdhfr double mutation C59R+S108N was present in 87% of samples while the pfdhfr triple mutant (N51I+C59R+S108N) was not detected. Pfdhps A437G was found in 60% of samples. Pure pfdhps K540E was rare, at 4%, but mixed genotype 540K/E was found in 77% of samples. Similarly, pure pfdhps A581G was found in 4% of the isolates while mixed 581A/G was found in 39% of samples. Plasmodium vivax is the most common human malaria species in Pakistan. This study presents pattern of polymorphisms in the pvdhfr, pvdhps and pvmdr1 genes conferring drug resistance in P. vivax among Pakistani isolates. Out of 579 PCR-confirmed (536 P. vivax and 43 P. vivax/P. falciparum mixed infection) samples, 372 isolates were randomly selected for further molecular characterization by sequencing and real-time PCR. Seventy six of the isolates (23%) were double mutant at positions S58R and S117N in pvdhfr. Additionally, two mutations at positions N50I and S93H were observed in 55 (15%) and 24 (7%) of samples, respectively. Three 18 base pair insertion-deletions (indels) were observed in pvdhfr, with two insertions at different nucleotide positions in 36 isolates and deletions in 10. Ninety-two percent of samples contained the pvdhps (S382/A383G/K512/A553/V585) SAKAV wild type haplotype. For pvmdr1, all isolates were wild type at position Y976F and 335 (98%) carried the mutation at codon F1076L. All isolates harboured single copy of the pvmdr1 gene. Malaria epidemiological results of this study indicate that malaria infections in Pakistan are largely attributed by P. vivax then P. falciparum and mixed species (P. vivax + P. falciparum) infections are also prevalent. In addition, regional variation in the prevalence and species composition of malaria is high in this region. The P. falciparum drug resistance results suggest an emerging problem of multi-drug resistant in Pakistan. The chloroquine resistance genotype has reached complete fixation in the population, with a microsatellite pattern indicative of a selective sweep. Moreover, the incidence of mutations in both pfdhfr and pfdhps, albeit without the presence of the pfdhfr triple mutant, indicates that therapeutic efficacy trials are warranted to assess whether SP remains efficacious drug in combination with artesunate for the treatment of P. falciparum. P. vivax molecular studies showed that the prevalence of mutations associated with sulphadoxine-pyrimethamine resistance is low in Pakistan. The high prevalence of P. vivax mutant pvmdr1 codon F1076L indicates that efficacy of chloroquine plus primaquine could be in danger of being compromised, but further studies are required to assess its clinical relevance. These findings will serve as a baseline for further monitoring of drug-resistant P. vivax malaria in Pakistan.