Asian Research Thesis Index

Abstract

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.

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