A New Methodology to Isolate Endophytic Bacteria from Hard Sugarcane Internode V. Jayakumar1 and I.

Samson Praisy2
1

Crop Protection Division, Sugarcane Breeding Institute, Coimbatore

2

Karunya university, Coimbatore

Endophytic bacteria occupy internal tissues of plants without causing damage to their hosts (Hallmann et al., 1997). The beneficial bacteria are consistently reported to be present in the root, stem, leaf, fruit, and other parts of many agricultural, horticultural and forest species. The crop specific endophytes can fight against pathogen in the interior location of crop where the pathogen infects and also bring about induced systemic resistance against multiple pathogens attacking the same crop. These endophytic bacteria colonize an ecological niche similar to that of phytopathogens, which makes them as suitable biocontrol agents (Berg et al., 2005). Several procedures and techniques have been employed and developed by many researchers to effectively isolate the endophytic communities from various parts of both monocotyledonous and dicotyledonous plants. In general the plant samples are thoroughly surface sterilized by different techniques to achieve successful elimination of the epiphytes. A review by Zinniel et al., (2002) highlights the methods to isolate the endophytic bacterial strains from root, stem, and leaves. Normally the plant leaves, stems and roots are individually surface sterilized with 90% ethanol and sodium hypochlorite containing 0.1% Tween 20 for 60 sec and rinsed with sterile distilled water and each part is dissected into several segments. To remove the disinfectant, sections are rinsed five times in sterile water and dried with sterile paper towels. The above protocol varies for plant to plant and also kind of plant parts to be used for isolation of endophytic bacteria, which needs to be standardized. Extraction of internal sap material of plant and isolation of bacteria from sap is another area of importance to get more no of endophytic bacterial isolates. Normally the plant tissue is macerated with sterile mortar and tissue extracts were then serially diluted in potassium phosphate buffer and plated to recover the endophytic bacteria. Takahashi et al., (2011) reported that they could get maximum no of endophytic bacteria by collecting inter cellular fluid in rice. Trituration of plant materials can be done by mechanical breaking the plant parts with blender, tissue pulverizer, vacuum & pressure extraction, and also extraction of sap material by

centrifugation. But in all these methods maintaining sterile condition throughout the process is tedious. Without mechanical breaking, extraction of sap from hard woody and fibrous crops is very difficult and that hinders the isolation process of endophyte. Sugarcane stem (internode) is such a hardy fibrous plant part that needs specific protocol for surface sterilization and extraction of sap for isolation of endophytic bacteria. In view of that a non-mechanical methodology has been standardized for surface sterilization and extraction of sap from sugarcane stem. The methodology for surface sterilization and non-mechanical extraction of sap for isolation of endophytic bacteria was standardized in various combinations and then compared with mechanical trituration. The standardized protocol that showed best surface sterilization and sap extraction and recovery of endophytic bacteria on par with mechanical method is given below. The shoot (internode) portion of sugarcane was cut and brought to the laboratory. The hard rind (skin) of the cane was removed using sterile knife. The remaining surface sterilization was done in laminar air flow chamber. To surface sterilize the shoot, the rind removed canes were first rinsed with 70 per cent ethanol for 3 min and the treatment was repeated for 3 times. Then shoot material was washed with sterile distilled water for 3 times. The second stage surface sterilization was done with 30 per cent hydrogen peroxide for 3 min and then washed with sterile distilled water for 3 times. Then the shoots were once again surface sterilized with 70 per cent ethanol and shoots covered with ethanol were flame sterilized finally (fired over flame: care should be taken while firing, hold the cut ends of cane with sterile iron holders). The sterilized shoots were cut in to less that 1cm3 blocks (approximately) with a sterile knife and the cut materials were pooled as sample. The cut tissues were then macerated with phosphate buffer in a sterile pestle and mortar (it breaks the hard fibrous cane tissue partially). The macerated samples were collected in a sterile centrifuge tubes (with cap) and centrifuged at 10000rpm for 15 min. After centrifugation the supernatant was transferred to separate sterile collection tube and the centrifugation and collection was repeated 5 times. The sediment fibrous shoot materials were then transferred to sterile pestle and mortar and added 5 ml of sterile water and crushed again. The materials were again transferred to collection tube and again centrifuged and this process was repeated again (totally 3 maceration was done). The supernatants were pooled and used for plating in the culture media as per requirement.

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