RHIZOSPHERE BACTERIA STRAIN R-12 AGAINST

Transcription

1 PROTEIN PROFILLING ANALYSIS of ANTIANTHRACNOSE-PRODUCING RHIZOSPHERE BACTERIA STRAIN R-12 AGAINST Colletotrichum gloesporioides at DIFFERENT ph CONDITIONS S. N. Aisyah, Fatchiyah, A. Bakhtiar, Jamsari

2 Scientific backgrounds Low productivity of chili pepper due to diseases Anthracnose, one of major disease in chili pepper Need of safe and eco-friendly controlling techniques Development of bacterial biofungicide

3 Research aims Identify dynamic protein profile of R-12 bacteria related to its antianthracnose compounds productions. Determine the best ph condition for R- 12 bacteria s antagonistic ability against Colletotrichum gloesporioides.

4 Research benefits Academical: Development of proteomic-based study and researches for agricultural developments Economical: Low cost antiantrachnose commercial production. Environmental: Production of safe and ecofriendly biofungicides leads to better fruit and enviroment quality Social: Reducing farmer s tendency in using high amounts of synthetic pesticides

5 Materials and methods Culture of fungi and bacteria Antagonist assay in ph mediums Dual culture assay in ph mediums Protein extraction (Zhang et al., 2009) SDS-PAGE

6 a) Culture of fungi and bacteria Fungi Colletotrichum gloesporioides from red chili pepper (Ilahi, unpublished) Culture condition: PDA ph 7 30 o C 7 days Bacteria Rhizosphere bacteria strain R12 (Riwani, 2012 unpublished) Culture condition: NA ph 7 30 o C 18 hours

7 b) Antagonist assay in different ph mediums Medium used was PDA with different ph ranged from 6-8. Method used was spotting method. 5 μl R12 liquid culture (LC) were spotted at 4 different positions 3 cm in length from 2 days aged fungi growth position.

8 Figure 1. Bacterial spot positions in antagonist assay

9 c) Dual culture assay in ph mediums Two days mycelial pellet of fungi and one day R-12 pellet were cultured together in LB with ph ranged 6-8. Control: fungi only and bacteria only. Culture conditions: 160 rpm 30 o C 2 or 3 days.

10 d) Protein extraction and SDS PAGE Method used: Zhang et al., For intracellular protein: bacterial pellet. For extracellular protein: cell-free supernatant Gel concentration: 12% SDS-PAGE gel Running condition: 200 volt 50 minutes. Marker used: Prestained protein ladder (Thermo scientific)

11 RESULTS AND DISCUSSIONS

12 a) Antagonistic assay in modified ph medium Figure 1. Growth of C. gloesporioides (Cg) in antagonist assay at different ph conditions on 5 days after treatment. Cg (control) Cg :: R12 (6) Cg :: R12 (7) Cg :: R12 (8)

13 ph 8 ph 7 ph 6 Table 1. Effect of different ph conditions towards the growth of C. gloesporioides, both the treated and control samples. ph Isolates Diameter of fungi (cm) per days after treatment Control Cg :: R Control Cg :: R Control Cg :: R

14 Table 2. Percentage of R-12 inhibition capacity against growth of C. gloesporioides in different ph conditions. ph Inhibition (%) / Days after treatment

15 b) Protein profilling of fungal-bacteria interaction Figure 2. Proteome profile of extracellular (a) and intracellular (b) of UBCR- 12 in co-culture with C. gloeosporioides under different ph levels.

16 Acknowledgements This work was fully funded by General Directorate of Higher Education through PMDSU Research Grant fiscal year