“Isolation and Characterization of endophytic microbes from Chrysanthemum plants showing wilting symptoms”
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Out of 21 bacterial isolates, 8 were found pathogenic on the basis of enzyme production and compatibility test. All 6 fungal isolates were pathogenic on the basis hydrolytic enzyme production. In a pathogenicity trail, treatments T8, T9, T12 and T19 gave maximum disease incidence and disease severity was maximum in treatments, T2, T8, T9, T12, T15 and T19. Fungal isolates N1, N2 and N6 and bacterial isolates H10, H19, H20 were more pathogenic as compared to other isolates. On the basis of restriction fragment length polymorphism (RFLP) of 16S rDNA gene with two restriction enzymes AluI, and BsuRI bacterial isolates H3 and H18 were identical. Two bacterial strains H2 and H20 were similar in restriction enzyme BsuRI and different in restriction enzyme AluI. The Chrysanthemum isolates were placed into 6 genotypes on the basis of restriction endonuclease AluI and 5 genotypes on the basis of BsuRI. In all five bacterial isolates (H3, H6, H7, H10, and H20) were selected for 16S rDNA sequence analysis and identification. The results awaited. All six fungal isolates (N1, N2, N3, N4, N5 and N6) would be identified on the basis of ITS region sequence analysis.
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“Isolation and Characterization of endophytic microbes from Chrysanthemum plants showing wilting symptoms”
- 1. “Isolation and Characterization of endophytic microbes from Chrysanthemum plants showing wilting symptoms” Anamika Id No. 49672 M.Sc. Microbiology
- 3. Known as chrysanths or mums It belongs to ◦ Family Asteraceae ◦ Genus Chrysanthemum L. Native of China, first cultivated their as a flowering herb. Introduced in India 1290 AD In India, its local name is “Guldaudi” ◦ White mums ◦ White spider mums ◦ Pink chrysanthemum ◦ Red chrysanthemum • In India, natural blooming season is from July to February.
- 4. Disease Causal Organism Leaf Spot Septoria chrysanthemi, Septoria chrysanthemella, Alternaria sp., Cercospora chrysanthemi, Pseudomonas cichorii Rust Puccinia chrysanthemi Wilt Fusarium oxysporium f. sp. chrysanthemi, Verticillium albo-atrum Powdery Mildew Erysiphe cichoracarum Ray Blight Ascochyta chrysanthemi Ray Speck Stemphylium sp, Alternaria sp. Gray Mold Botrytis cinerea Stem rot Rhizoctonia solani, Fusarium solani Root rot Pythium sp., Phytopthora sp., Phoma chrysanthemicola Bacterial Blight Erwinia chrysanthemi Stem necrosis Pseudomonas cichorii Bipolaris leaf spot Bipolaris setaria Charcoal stem rot Macrophomina phaseolina Cylindrosporium leaf Cylindrosporium chrysanthemi Crown gall Agrobacterium tumefaciens Fascination Corynebacterium fascians Mosaic CMV, Chrysanthemum virus-B Chlorotic mottle Chrysanthemum chlorotic mottle viroid Chrysanthemum stunt Chrysanthemum stunt viroid Table1 List of major disease incidents of Chrysanthemum
- 6. Isolation of fungal and bacterial isolates from stem of diseased Chrysanthemum spp. Biochemical characterization and identification of endophytic fungal and bacterial isolates. Pathogenicity trial on homologous host. To evaluate biocontrol efficacy of Pseudomonas koreenses strain against fungal and bacterial isolates pathogens of chrysanthemum plants
- 7. Isolation of fungal and bacterial isolates from stem of diseased Chrysanthemum spp.
- 9. Isolation of micro-organism Bacterial isolates were isolated and maintained on plate count agar ; fungal isolates were isolated and maintained on potato dextrose agar. 21 bacterial isolates (Cho et al. 2007), 6 fungal isolates (Bao et al., 2004) were recovered from the stem of chrysanthemum plant used in this study.
- 10. 21 bacterial isolates 14 were gram’s negative 16 were rod shaped 7 were gram’s positive 5 were coccus shaped
- 11. Fig. 1 Morphological characteristics of fungal isolates recovered from Chrysanthemum plant A) Growth of fungal mycelium on PDA agar plates. B) AS viewed under compound microscope (40X). FUNGAL ISOLATES N1 N2 N3 N4 N5 N6
- 12. Table 2 Morphological characteristics of fungal isolates recovered from diseased Chrysanthemum plants (Bao et al., 2004). S. No. Fungal isolates Colour of mycelium on PDA Mycelial characteristics under microscope(40X) Type of spore Identification on basis of mycelial characteristics 1. N1 Brown Septate Club shaped Alternaria sp. 2. N2 Green Multinucleate and aseptate Ascospore Penicillium sp. 3. N3 Black and white (grey) Septate No spore Fusarium sp. 4. N4 Black and white (red) Septate No spore Fusarium sp. 5. N5 White and yellow Coenocytic No spore Verticullium sp. 6. N6 Green and white Septate Round Aspergillus sp.
- 13. Biochemical characterization and identification of endophytic fungal and bacterial isolates.
- 14. Screening for production of cell wall degrading enzymes • Tortella et al., 2008Xylanase • Tortella et al., 2008Cellulase • Beily et al., 1985β – 1,3- glucanase • Passari et al., 2016Protease • Reetha et al., 2014 Hydrogen cyanide • Hankin et al., 1975 Pectinase
- 15. Result of cell wall degrading enzymes Out of 21 bacterial isolates 17 were positive Protease 9 were positive Xylanase 6 were positive β – 1,3- glucanase Cellulase 5 were positive Pectinase No isolate positive HCN production
- 16. Xylanase Cellulase β – 1,3- glu canase Protease Pectinase Hydrogen cyanide Fig. 2 Screening of cell wall enzyme production by xylanase (H9, H10, H11 and H12); Cellulase (H18, H19, H20 and H21); β – 1,3- glu canase (H6, H7, H8 and H10); Protease (H3, H18, H19 and H20)Pectinase (H10, H20, H7 and H5); HCN production (H20).
- 17. Xylanase Tortella et al., 2008 Amylase Sunitha et al., 2013 Lipase Sunitha et al., 2013 Pectinase Sunitha et al., 2013 Cellulase Sunitha et al., 2013 Protease Sunitha et al., 2013 Laccase Sunitha et al., 2013 Chitinase Wiwat et al., 1995 Hydrolytic Enzymes produced by fungal isolates
- 18. All 6 fungal isolates were positive for xylanase and chitinase activity. Only 4, 3, and 2 isolates were positive for lipase, protease and cellulase activity. None of the fungal isolates were positive for production of pectin and laccase enzyme. Result hydrolytic enzyme production
- 19. Xylanase Amylase Lipase Pectinase Cellulase Protease Laccase Chitinase Fig. 3 Fungal isolates produced Hydrolytic enzymes (xylanase (N2); Amylase (N6); Lipase (N5); Pectinase (N2); Cellulase (N2); Protease (N3); Laccase (N1) and Chitinase (N5)).
- 20. In vitro compatibility of bacterial isolates (Essarts et al., 2016)
- 21. Bacterial isolates used for lawn preparat ion Growth of bacterial isolates spot inoculated H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15 H16 H17 H18 H19 H20 H21 H1 - - - ++ - + - - + - ++ - - - - - - + - - H2 - - - - - - - - - - + - + - + - - - - - H3 + ++ + ++ + + +++ + + + ++ + ++ ++ + ++ - + - +++ H4 - - - - - - - - - - - - - - - - - - - - H5 - - - - - - - - - - - - - - - - - - - - H6 - - - - - - - - - - - - - - - - - - - - H7 - - - + - - - + - - - - - - - - - - - - H8 - - - - - - - - - - ++ - + - - - - - - - H9 - - - - - - - - - - - - - - - - - - - - H10 - - - - - - - +++ - - - - - - + - - + - - H11 - + - - ++ - + - - - ++ - - - - - - + - - H12 - - - - - - - - - - - - - - - - - - - - H13 - - - - - - - - - - - + - - - - - + - - H14 - - - - - - - - - - - + - - - - - - - - H15 - - - - - - - - - - - - - - - + - - - - H16 - - - - + - + - - - - + - - - - - + - - H17 - + - + + - - - - - - + - + + - - - - - H18 + + - - ++ + + +++ + + + ++ ++ ++ ++ + + + - ++ H19 + - - - - - - - + - + ++ ++ ++ + + - - - ++ H20 - - - - - - ++ - - - - - - - - + - - - - H21 - + - + + + - - + - - + - + + - - - - - + growth observed - no growth Table 3 In vitro compatibility of bacterial isolates
- 22. Fig. 4 In vitro compatibility test of bacterial isolates. All 21 isolates have been spot inoculated on lawn of bacteria A (H3) , B (H5), C (H6) and D (H8) A B C D
- 23. In vitro antagonism of bacterial isolates against fungal isolates Dual culture assay (Abdeljalil et al., 2016)
- 24. Bacterial isolates Zone of inhibition (in cm)* against pathogenic fungi N1 N2 N3 N4 N5 N6 Fusarium oxysporum cd at 5% H2 - - - - - - - - H3 - - - - - - - - H6 - - - - - - - - H7 - - - - - - - - H10 3.96±0.14b 7.16±0.14e 2.96±0.14a 3.16±0.14b 3.56±0.14b 5.66±0.14c 5.86±0.14d 0.66 H18 - - - - - - - - H19 3.27±0.02d 5.17±0.02f 2.27±0.02a 3.07±0.02c 3.07±0.02c 3.97±0.02e 2.87±0.02b 0.27 H20 2.34±0.38b 6.14±0.38f 2.74±0.38c 3.04±0.38c 1.84±0.38a 5.34±0.38d 4.34±0.38e 0.34 *= All values are the means of three replicates±SD; letters in superscript showing overlap of significance Table 4 In vitro antagonism of bacterial isolates against fungal isolates through dual culture plate assay.
- 25. Fig. 5 In vitro antagonism of bacterial isolates recovered from chrysanthemum plant against phytopathogenic fungi A) H19 against N3 B) H10 against Fusarium oxysporum C) H20 against N5. Control A B C
- 26. Molecular Characterization • DNA isolation and quantification (Ausbel et al., 2003) • Amplification of 16S rDNA (Laguerre et al., 1994) • Restriction analysis of 16S rDNA: AluI and BsuRI*
- 27. Fig. 6 Genomic DNA of bacterial isolates from chrysanthemum plant lane 1 to 8. . Lane 1 (H2) Lane 2 (H3) Lane 3 (H6) Lane 4 (H7) Lane 5 (H10) Lane 6 (H18) Lane 7 (H19) Lane 8 (H20) 1 2 3 4 5 6 7 8
- 28. Fig. 7 16S rDNA amplicon of bacterial isolates from chrysanthemum plant lane 1 to 8. Lane 1 (H20) Lane 2 (H2) Lane 3 (H3) Lane 4 (H6) Lane 5 (H18) Lane 6 (H7) Lane 7 (H10) Lane 8 (H19) Lane M, marker (lambda Hind III/ EcoRI double digest) 1 2 3 4 5 6 7 8 M 21,226 bp 5,148 bp 2027 bp 1584 bp
- 29. Fig. 8 ARDRA of bacterial isolates from chrysanthemum isolates lane 1 to 8. . Lane 1 (H20) Lane 2 (H2) Lane 3 (H3) Lane 4 (H6) Lane 5 (H18) Lane 6 (H7) Lane 7 (H10) Lane 8 (H19) Lane M, Marker (100 bp step up ladder) with restriction endonuclease, A(AluI) , B (BsuRI*) 1 2 3 4 5 6 7 8 M A B 1,000 bp 900 bp 800 bp 700 bp 600 bp 500 bp 400 bp 300 bp 200 bp 1,000 bp 900 bp 800 bp 700 bp 600 bp 500 bp 400 bp 300 bp 1 2 3 4 5 6 7 8 M
- 30. Pathogenicity trial on homologous host.
- 31. Pot trail (Gosh et. al., 2009 )
- 32. Table 4 Detail of various treatment given in pot trials Treatments T1 Control T2 N1 T3 N2 T4 N3 T5 N4 T6 N5 T7 N6 T8 Fusarium oxysporum T9 Sporous fungus consortia (N1, N2, and N6) (SP) T10 Non- sporous fungus consortia (N3, N4, and N5) (NS) T11 H2 T12 H3 T13 H6 T14 H7 T15 H10 T16 H18 T17 H19 T18 H20 T19 Consortia of bacterial isolates (H10, H19, and H20)
- 33. Treatment Disease index(percentage) At 30 days At 45 days Disease severity At 30 days At 45 days T1 0 0 0 0 T2 45 65 5 7 T3 20 55 4 6 T4 0 35 2 5 T5 35 60 3 5 T6 15 50 3 6 T7 35 65 4 6 T8 50 75 5 7 T9 50 85 5 7 T10 45 65 3 5 T11 25 60 4 6 T12 35 70 5 7 T13 0 35 2 5 T14 0 55 3 4 T15 35 55 5 7 T16 40 65 4 6 T17 0 50 3 5 T18 0 65 4 6 T19 30 70 5 8 Table 5 Percent disease index and severity in chrysanthemum plant treated with various bacterial and fungal isolates.
- 34. A D E F CB Fig. 10 Pot trial to assess pathogenic potential of fungal and bacterial isolate on Chrysanthemum plant under glass conditions. A) With T1 treatment B) with T8 treatment C) with T2 treatment D) with T7 treatment E) with T11 treatment F) with T12 treatment.
- 35. To evaluate biocontrol efficacy of Pseudomonas koreenses QFR5 strain against fungal and bacterial isolates pathogens of chrysanthemum plants
- 36. In vitro antagonistic activity of Pseudomonas koreensis strain Dual Culture assay
- 37. Biocontrol agent Isolates Pseudomonas koreensis (Zone of inhibition in cm)* N1 4.84±0.12c N2 2.94±0.12a N3 - N4 - N5 4.14±0.12b N6 Fusarium oxysporum 2.94±0.12a H2 - H3 - H6 - H7 - H10 H18 - H19 - H20 0.56±0.5b Table 6 Inhibition of radial growth of fungal and bacterial pathogens in dual culture assay by Pseudomonas koreensis
- 38. N1 N2 N3 N4 N5 N6 F. oxysporum A B H10 H20 Fig. 12 In-vitro inhibition of different fungal and bacterial isolates using Pseudomonas koreensis A)Control of fungal isolates N1, N2, N3, N4, N5, N6 and F. oxysporum B) corresponding fungal isolates + P. koreensis C) P. koreensis show inhibition zone in bacterial isolates. C
- 39. Conclusion Out of 21 bacterial isolates, 8 were found pathogenic on the basis of enzyme production and compatibility test. All 6 fungal isolates were pathogenic on the basis hydrolytic enzyme production. In a pathogenicity trail, treatments T8, T9, T12 and T19 gave maximum disease incidence and disease severity was maximum in treatments, T2, T8, T9, T12, T15 and T19. Fungal isolates N1, N2 and N6 and bacterial isolates H10, H19, H20 were more pathogenic as compared to other isolates. On the basis of restriction fragment length polymorphism (RFLP) of 16S rDNA gene with two restriction enzymes AluI, and BsuRI bacterial isolates H3 and H18 were identical. Two bacterial strains H2 and H20 were similar in restriction enzyme BsuRI and different in restriction enzyme AluI. The Chrysanthemum isolates were placed into 6 genotypes on the basis of restriction endonuclease AluI and 5 genotypes on the basis of BsuRI. In all five bacterial isolates (H3, H6, H7, H10, and H20) were selected for 16S rDNA sequence analysis and identification. The results awaited. All six fungal isolates (N1, N2, N3, N4, N5 and N6) would be identified on the basis of ITS region sequence analysis.
- 40. Future prospects The pathogenic potential of selected fungal and bacterial isolates should be validated in vivo conditions. The identification of fungal and bacterial pathogens of chrysanthemum plant would help in effective management of disease through biocontrol agents. The Pseudomonas koreensis strain could be developed as effective biocontrol agent.
- 41. Reference Singh, P. K., & Kumar, V. (2014). ‘Fusarium Wilt of Chrysanthemum–Problems and Prospects. Plant Pathology & Quarantine, 4(1), 33-42. Sunitha, V. H., Devi, D. N., & Srinivas, C. (2013). Extracellular enzymatic activity of endophytic fungal strains isolated from medicinal plants. World Journal of Agricultural Sciences, 9(1), 01-09. Abdeljalil, N. O. B., Vallance, J., Gerbore, J., Bruez, E., Martins, G., Rey, P., & Daami, R. M. (2016). Biocontrol of Rhizoctonia Root Rot in Tomato and Enhancement of Plant Growth using Rhizobacteria Naturally associated to Tomato. Journal of Plant Pathology & Microbiology, 7, 1-8.