master seminar ppt phage mediated management.pptx

Phage mediated regulation of bacterial diversity of plant pathology

1. PAT 591- MASTER’S SEMINAR (0+1) Topic- Phage Mediated Regulation of Bacterial Diversity on Phyllosphere TAMIL NADU AGRICULTURAL UNIVERSITY, COIMBATORE Chairman: Dr. P. Muthulakshmi , Ph.D Professor (Plant Pathology) Center for students welfare, TNAU, Coimbatore Student: Ankita Ghosh ID: 2022515023 II M.Sc. (Department of plant pathology)

2. CONTENTS

3. Introduction Microbial communities Soil Leaf Ocean Human body (Everything is everywhere, but the environment selects) Bacteria in any given environment face strong selection pressures from other microbes, predators, viruses, and in the case of bacteria living within another organism, the host immune response. Unlike abiotic factors, which are non-living elements like temperature or pH, biotic factors are living organisms or their products that can influence the survival and adaptation of bacteria. Bacteriophages (phages) represent perhaps the most ubiquitous of these biotic drivers (the most abundant biological agent on earth). Koskella. et.al,. 2013

4. Early study with phages Viruses that replicate within the bacteria Discovery in 1915 by Fedrick. W. Twort Term Bacteriophage coined by Felix d’ Herelle (1917) Before the discovery reports that indicate/suggest the presence of phages. Successfully used in control of plant pathogens Fedrick. W. Twort (1915) Felix d’ Herelle (1917) Summers, 2005

5. Understanding Bacteriophage Specificity in Natural Microbial Communities For phages to alter the composition of a microbial community, there must exist a degree of specificity. Like all viruses, bacteriophages are very species-specific with regard to their hosts and usually only infect a single bacterial species or even specific strains within a species.  Koskella. et.al ,.2013 Specificity Governed by Surface receptors on bacterial cells bacterial defense mechanisms against phage infect Not all bacteria are infected by all phages, and indeed that most phages can only infect a subset of bacterial species

6. Phages have important impacts on the competitive dynamics and structure of bacterial communities (Bohannan and Lenski, 2000; Koskella and Brockhurst , 2014; Rodriguez-Valera et al., 2009; Weinbauer and Rassoulzadegan , 2004) Morella.et.al,.2018 Maintain bacterial diversity through variety of mechanisms Evolutionary and coevolutionary dynamics “Kill the winner”

7. Phyllosphere microbiome assembly Over the course of evolution, plants have been equipped with various mechanisms that select specific microorganisms in the phyllosphere . The phyllosphere comprises all the above ground sections of plant. This habitat is colonized by complex microbial communities, including bacteria, fungi & archea . It is in general a nutrient poor environment that undergoes fluctuations in temperature, UV, and moisture .The most abundant members of this community are considered to be phyllosphere -associated bacteria with an estimate of to per square centimeter.   Morella et al , 2018; Mandal and Jeon, 2023 Most abundant: PHYLLOSPHERE ASSOCIATED BACTERIA

8. Host Genotype Environmental Factors Anthropogenic Factors Microbe–Microbe Interactions: Intraspecific Interspecific These interactions are either cooperative (mutualism and commensal), parasitic, or competitive (antibiosis, competition for nutrients or space) and can be formed within or between bacteria–bacteria , fungus–fungus , bacteria–fungus , bacteria–virus , etc. (Summers, 2005) Major factors influencing microbiota assembly in the phyllosphere

9. EPIPHYTES Larger bacterial aggregates on the trichomes, veins and epidermal cell groves (leaf exudates containing nutrient-rich regions) LEAF CUTICLE LAYER Determines the official chemical properties of the leaf surface and renders the permeability and wettability, which facilitate the appearance of microorganisms. (plays a major role in survival of the epiphytes) Structure of phyllosphere microbial assemblage: a.) Stage for microbial community structure development. b.) Regulation for the microbial community structure in phyllosphere . (Sivakumar et al, 2005)

10. In plant rhizosphere they can reduce the prevalence of harmful bacteria Phage Mediated Regulation host-associated microbiomes Bacteriophages, as predator of bacteria are significant players in shaping microbial communities. Morella et al , 2018; Chevallereau et al, 2021; Mandal and Jeon, 2023 Virulent phages can shape the microbial community composition by influencing the colonization success of bacteria. Temperate and filamentous phages are also important determinants of pathogen colonization success. Phages may promote the expression of innate immunity genes Lyse the bacterial cells impacting the success of bacterial colonization. Carry genes such as virulence factor or genes for biofilm formation May promote the expression of innate immunity genes or Prevent the activation and proliferation of immune cells

11. Chevallereau et al, 2021 AMGs (auxiliary metabolic genes) have been best described in marine phages Modulation of bacterial mutation rate. Phage-mediated evolution of bacterial communities Found in phages that are involved in various metabolic processes these genes play roles in processes such as photosynthesis, carbon metabolism, and nitrate reduction. In changing and stressful environments, high mutation rates can be advantageous for bacteria. This is because mutations can lead to genetic variation, allowing bacteria to adapt more quickly to new conditions Phages can influence the evolution of bacterial communities by altering mutation frequencies, promoting the selection of hypermutator bacteria

12. Life cycle: Lytic and lysogenic

13. Impact of lytic phages Direct cell lysis ( Koskella & Brockhurst , 2014). Indirect effects on competition ( Koskella & Brockhurst , 2014). Nutrient release (Weitz & Wilhelm, 2012) Integration into bacterial genomes ( Kraushaar et al., 2017). Facilitating horizontal gene transfer ( Weinbauer & Rassoulzadegan , 2004). Morella.et.al,.2018 Understanding these interactions between phages and bacterial diversity could have implications for various systems, such as agriculture and the environment, and may potentially be harnessed to increase productivity or understand ecosystem functioning (Zhang et al., 2017). However, further research is needed to fully understand these dynamics and their applications.

14. Limitations Technical limitations have also impeded our ability to understand the role of phages in host associated microbiomes. Challenges with Phage Identification Metagenomics Sequencing Culturing Phages Unique Interactions in Host-associated Microbiomes Manrique.et.al,.2016

15. SCIENTIFIC STUDIES

16. Objective: In this study, the aim was to control bacterial leaf blight on Welsh onion using phage biocontrol in Vietnam. Phages isolated from X. axonopodis pv . allii ( Xaa ) infected onion leaves were screened, and three promising ones were selected based on host range and plaque/halo size. These were then characterized by whole-genome sequencing. The selected lytic phages were evaluated for their potential to control X. axonopodis pv . allii both in vitro and in greenhouse and field conditions. SCIENTIFIC STUDY- 1

17. Different Xanthomonas axonopodis pv . allii strains and phages isolated from infected Welsh onion leaves collected from different provinces in the Mekong Delta of Vietnam.

18. Evaluation of the host spectrum of ten Xaa phage isolates against twelve Xaa strains. Broadest Host Range Showed activity against all the tested Xaa strains Appears to be more susceptible to phage infection (selected as host for multiplication of the phages) ɸ16, ɸ31 and ɸ17A showed largest halo diameter in 72 hrs

19. Phage Characterization They have 81 %similarities with Xylella phage Paz and 9% with Xylella Phage prado Also showed that they belong to Autographiviridae family

20. Efficacy of Phages against Bacterial Leaf Blight on Welsh Onion in Greenhouse Conditions The three selected lytic Xaa phages were evaluated for their biocontrol potential in a greenhouse experiment After 9 days after inoculation ( dai ), the percentage of infected leaf area (%) was highest in the control group, followed by ɸ17A and ɸ16, at 67.5%, 37.2%, and 43.3%, respectively. The density of bacteriophage on the leaf surface (pfu/g leaf) after 72 hours after inoculation ( hai ) was highest in ɸ31, followed by ɸ17A, ɸ16, and the Three-phage cocktail, at 9.03, 7.89, 7.35, and 6.97, respectively. Development of leaf blight caused by X. axonopodis pv . allii on Welsh onion in greenhouse conditions. Five different treatments were tested for their efficacy to control leaf blight symptoms nine days after infection: (A) Oxolonic acid ( Starner ), (B) ɸ 31, (C) ɸ 16, (D) ɸ 17A, (E) three-phage cocktail (ɸ 16, ɸ 17A and ɸ 31) and (F) Control (bacteria only) A: Oxolonic acid B: ɸ31 C: ɸ16 D: ɸ17A E: three phage cocktail F: Control Pfu: (Plaque forming unit)

21. Efficacy of Bacteriophage in Controlling Bacterial Leaf Blight in Field Conditions Phage Φ31 and the phage cocktail were tested for their efficacy in a field trial where the plants were artificially inoculated. The plants treated with the bactericide and φ31 still showed higher disease control. This shows that the phage treatment performs equally well as a commercially available bactericide treatment.

22. Highlights In this study, lytic phages Φ16, Φ17A, and Φ31 , specific to X. axonopodis pv . allii and belonging to a new phage species and genus within the Autographiviridae , were isolated and characterized from four provinces in the Mekong Delta of Vietnam. Moreover, their efficacy for the biocontrol of leaf blight in greenhouse and field conditions was evaluated. When the three highly related phages were individually applied or as a three-phage cocktail at 108 PFU/mL in greenhouse conditions , the results showed that treatment with Φ31 alone provided higher disease prevention than the two other phages or the phage cocktail.

23. Furthermore, phage concentrations ranging from 105 to 108 were compared, and optimal disease control was observed at 107 and 108 PFU/ mL. Finally, under field conditions, both phage Φ31 alone and the phage cocktail treatments suppressed disease symptoms, which were comparable to the chemical bactericide oxolinic acid ( Starner ). Phage treatment also significantly improved yield, demonstrating the potential of phage as a biocontrol strategy for managing leaf blight in Welsh onion.

24. SCIENTIFIC STUDY- 2 Objective: To know bacteriophage profiling of phyllosphere of wheat and their link with potential bacterial host

25. Virus operational taxonomic units ( vOTUs ) recovered from the four viral metagenomes from winter wheat leaves. Viral metagenomes from flag and penultimate leaves were sequenced without (FL, PL, respectively) and with MDA amplification ( FLmda , PLmda , respectively). Total Shows the % of reads that mapped back into their respected contigs Overview of total assembled contigs, identified viral contigs, and species-rank viral clusters ( vOTUs ), in terms of the number of contigs, cumulative contig length, and the fraction of mapped reads.

26. Predicted genome completeness of the recovered vOTUs . From the recovered vOTUs i.e., from 876 number of sequences: 12.2% (high quality) 15.9% (medium quality) 62.6% (low quality) 7.4% (complete) 1.9% (not determined)

27. Venn diagram of the presence of vOTUs in the four viral metagenomic samples.

28. The Wheat Phyllosphere Microbial Fraction Is Dominated by Members of the Pseudomonadaceae and Xanthomonadaceae Family The taxonomic profile indicates that: The dominant phyla on the wheat phyllosphere samples are Proteobacteria, Bacteriodetes , Actinobacteria, and Firmicutes . The most abundant members of this community belong to the Xanthomonadaceae and Pseudomonadaceae families as 33.2% and 25.1% of the reads are assigned to those taxa, respectively. (A) Relative abundance of the dominant bacterial families on the bacterial fraction. Taxonomy was assessed at the read level from the microbial fraction samples ( FLmf and PLmf ).

29. Diverse phyllosphere associated bacteriophage communities can be successfully recovered using viral metagenomics. They are predicted to Infect the dominant bacterial families on the microbial fraction. The wheat phyllosphere microbial fraction is dominated by members of the Pseudomonadaceae and Xanthomonadaceae family. Wheat phyllosphere harbours a distinct phage community. Highlights

30. SCIENTIFIC STUDY- 3 Objective: This study aimed to investigate the effect of phage therapy on Xanthomonas oryzae pv . oryzae ( Xoo ) in the rice phyllosphere community and how it affects the phyllosphere microbial community.

31. Alpha diversity Chao1 index

32. Bacterial communities in the epiphytic samples were mainly composed of Proteobacteria, Firmicutes, Cyanobacteria, Bacteroidota , Actinobacteriota , and others . Endophytic samples also had Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes, Deinococcus Thermus, and others. The C223E treatment significantly enriched certain bacterial groups in the epiphyllous community compared to C2, including Sphingomonas , Exiguobacterium , Stenotrophomonas, Phyllobacterium , and others.

33. Highlights Here, the glasshouse pot experiment results showed that phage combination could reduce the disease index by up to 64.3%.  High-throughput sequencing technology was used to analyze the characteristics of phyllosphere microbiome changes and the results showed that phage combinations restored the impact of pathogen invasion on phyllosphere communities to a certain extent, and increased the diversity of bacterial communities. In addition, the  phage  combination reduced the relative abundance of epiphytic and endophytic  Xoo  by 58.9% and 33.9%, respectively.

34. In particular, Sphingomonas and Stenotrophomonas were more abundant. According to structural equation modeling, phage combination directly and indirectly affected the disease index by affecting pathogen  Xoo  biomass and phage resistance. In summary, phage combination could better decrease the disease index. These findings provide new insights into phage biological control of phyllosphere bacterial diseases, theoretical data support, and new ideas for agricultural green prevention and control of phyllosphere diseases.

35. CONCLUSION AND FUTURE ASPECTS

36. Reference: Manrique, P., Bolduc, B., Walk, S. T., van der Oost, J., de Vos, W. M., Young, M. J., & White, B. A. (2016). Healthy human gut phageome . Proceedings of the National Academy of Sciences, 113(37), 10400-10405. Koskella , Britt, and Nicole Parr. "The evolution of bacterial resistance against bacteriophages in the horse chestnut phyllosphere is general across both space and time."  Philosophical Transactions of the Royal Society B: Biological Sciences  370.1675 (2015): 20140297. Summers, William C. "History of phage research and phage therapy."  Phages: Their role in bacterial pathogenesis and biotechnology  (2005): 1-17. Koskella , Britt, and Sean Meaden . "Understanding bacteriophage specificity in natural microbial communities."  Viruses  5.3 (2013): 806-823. Morella, Norma M., et al. "The impact of bacteriophages on phyllosphere bacterial abundance and composition."  Molecular ecology  27.8 (2018): 2025-2038.

37. Chevallereau , Anne, et al. "Interactions between bacterial and phage communities in natural environments."  Nature Reviews Microbiology  20.1 (2022): 49-62. Forero -Junco, Laura Milena, et al. "Bacteriophages roam the wheat phyllosphere ."  Viruses  14.2 (2022): 244. De Mandal, Surajit , and Junhyun Jeon. " Phyllosphere Microbiome in Plant Health and Disease."  Plants  12.19 (2023): 3481. Nga, Nguyen Thi Thu, et al. "phage biocontrol of bacterial leaf blight disease on welsh onion caused by Xanthomonas axonopodis pv . allii ."  Antibiotics  10.5 (2021): 517. Sivakumar, Natesan, et al. " Phyllospheric microbiomes: diversity, ecological significance, and biotechnological applications."  Plant microbiomes for sustainable agriculture  (2020): 113-172.