In case of Bacterial blight disease of rice , there is always a controversial statement about the existence of the pathogen . If i want to check this invitro condition how should i proceed this? Treating the seeds with the pathogen will answer the question or Is their any other standardized procedure to check? i wish to study the path of its entry into the seed when the infected seeds are planted. Can anyone suggest methods to study apart from fluorescent tagging?
Dear Ranju,
The best way to confirm the association of a pathogen with the seed is by agar plating method. You can do by this way: properly sterilize the seed samples and inoculate sterilized seed samples on to the nutrient agar followed by incubation overnight. within 24-48 hours of incubation period, you can check the growth of bacterial pathogens. Identify the bacteria using available techniques.
To confirm whether it is associated with the disease or not, you have to do artificial inoculation.
Further to validate the results, you can have a original isolate from diseased plant and one from seed and compare both.
Remember, to confirm the identity of bacteria, 16SrDNA sequences and biochemical tests are very much essential which help us to differentiate from one another.
Dear Ranju,
The best way to confirm the association of a pathogen with the seed is by agar plating method. You can do by this way: properly sterilize the seed samples and inoculate sterilized seed samples on to the nutrient agar followed by incubation overnight. within 24-48 hours of incubation period, you can check the growth of bacterial pathogens. Identify the bacteria using available techniques.
To confirm whether it is associated with the disease or not, you have to do artificial inoculation.
Further to validate the results, you can have a original isolate from diseased plant and one from seed and compare both.
Remember, to confirm the identity of bacteria, 16SrDNA sequences and biochemical tests are very much essential which help us to differentiate from one another.
I can write similarly to colleague Mahadevakumar Shivannegowda that the best way to confirm the association of a pathogen with the seed is by agar plating method. You can do by this way: properly sterilize the seed samples and inoculate sterilized seed samples on to the nutrient agar followed by incubation overnight. within 24-48 hours of incubation period, you can check the growth of bacterial pathogens. Identify the bacteria using available techniques.
To confirm whether it is associated with the disease or not, you have to do artificial inoculation.
Further to validate the results, you can have a original isolate from diseased plant and one from seed and compare both.
I can write that biochemical tests are very much essential which help us to differentiate from one another.
Dear Ranju,
For sure agar plating mehod could be a valid in vitro assay. However, we have to keep in mind that in vitro tests often do not correspond to the natural infection (in terms of survival). In addition, seedborne pathogen should be distinguished in two groups. Those that contaminate the seeds only on the surface and can survive for a certain period of time (generally for a short period) and those that can also get accessed into seed and survive for years. Often, pathogens that have a systemic movement could get accessed into seed surface, Clavibacter michiganensis subsp michiganensis on tomato for example, through a naturally infected plant. In this latter case, production and trade of healthy seeds is really a big challange.
If you want to follow the entry path of the pathogen, as far as I know, fluorescent tagging is the only method that allows us to do so.
There is a small problem here, but because unsterilized seeds yield several fungi on blotter it does not mean they are all pathogens. We can get several fungi even after surface sterilization (shows real seed borne fungi), some are already designated as pathogens but they may lead endophytic life within seed. Some time it is difficult to understand which is pathogen, endophyte and saprophyte as they switch their lifestyle in a given condition (i mean edaphic factors).
I also agree with the statements of Dr Mahadevakumar and Dr Igor - Styriak that the Agar plating of surface sterilized seed is the best way to confirm weather the pathogen is seed born or not. Further the pathogenicity of the pathogen can be confirmed by inoculating the plant/host, if the symptoms are similar with origin fungus or not. (this is the best way of confirmation). Fluorescent tagging is the best way to follow the entry path of the pathogen.
The answers you have provided are adequate, but you may have to try different agars for a bacterium from a seed. The other possibility is to make an external seed cleaning, soak and then make a DNA extraction, PCR with primers specific if you suspect on a especial bacterium or primers of 16s. so you can get a more precise answer.
The previous answers are all fine and should be considered. My comment is just to reinforce the fact that saprophytes can grow faster and overgrow eventual pathogens present in the sample, leading to false negatives. Be careful also with sample size in relation to expected seed-transmission rate of the main pathogens being considered and the detection precision of the methods already indicated.
Yes, Sp Ranju you are correct to say Xanthomonas campestris causing bacterial blight of rice is not confirmed for its seed transmission. However, its seed contamination (external association ) is confirmed. To see the entry path, I suggest component plating of seeds on agar plate i.e. separate different parts of the seeds (lemma, palea, awn, embryo, endosperm etc) and plate them separately and observe the growth of the bacteria.
Dr. Carlos Lopes
I 100% agree with your point , in such a case how can i carry out this? i treat the seeds with the pathogen and then plant the same , after the seeds are formed, if i want to check the presence of pathogen in the seeds i cant go for plating method , since it will get false result , then how is it possible to prove whether its seed borne? Can you suggest a point
Surface sterilize each component before plating. Please take off the water drop adherent to seed part before plating. You can do by transferring the seed parts to a dry sterile petri-plate.
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence
- Fluorescence