I think single sampling wont be enough to determine the bacterial and archeal community. Better to go for multiple sampling.

If you perform just one sampling you will get the bacterial and archaeal community structure of your rhizosphere sample but not from the rhizosphere of your plants. You need at least 3 distinct sampling to get a representative microbial community structure of your rhizosphere. If you want to compare the rhizosphere between 2 plants species for example, you will need at least 3 samples per plant species (6 samples in total), each sample on different plants. You can do more samples if your results show high variability.

Another advice: when you design your experiment and plan to use DGGE, you need to carefully think about how many samples you will have because you can only process a limited number of samples per DGGE (between 15 to 20 in general). It can be extremely difficult to compare results between different DGGE gels, so if you have a small experiment, sometimes it will make your life easier to fit all your samples in 1 DGGE gel.

Hope that help

Aimeric

Hi dhamodharan sir and Aimeric sir , thanks for your suggestion.

Aimeric sir, I have 3 different rhizosphere samples of same plant collected from a equidistance( like 1-2 km distance) in triplicates. Like wise for the other plant also the same method has been followed for sample collection. My doubt is since the plants will be the same and undisturbed, can one time sampling is enough for describing the total microbial community from that rhizosphere since the plants will remain the same throughout their life time..

The microbial community from the rhizopshere will not be the same over time even if it is from the same plant. The different life stage of your plants, the seasonal changes, changes of the soil physic & chemistry will influence the rhizosphere community. So if you want to compare the microbial communities from the rhizosphere from different plant species for example, you will need to take your samples in the same time. You can only have one sampling time and you will get the structure of your communities at this specific time of the year. If you are interested in the dynamic of your communities, you can do sampling 2 times during the year or after a rain event if you want to have a look on the effect of rain/soil moisture on your rhizospheric microbial communities from different plant species... So, the need of doing different time sampling depends of your research question. You will not be able with just one sampling time to get the total microbial community structure from a specific rhizosphere. For that, you would need potentially many sampling over seasons and at least over 2 years to see how constant the microbial community is. So, I would expect in your case it is enough to have one sampling time to compare the microbial communities from different plant species unless you want to understand the environmental drivers of your communities over time.

You can have a look on this article if you want to see how plant development can affect your microbial community:

Houlden et al (2008), Influence of plant developmental stage on microbial community structure and activity in the rhizosphere of three field crops. FEMS Microbiology Ecology, 65, 193-201

Hope that help,

Aimeric

Thanks for your valuable suggestion Aimeric sir. As per your suggestion i have two different sampling time one is post monsoon and another is during monsoon. I just tried with one plant sample for comparison of two samples in which i got almost a same profile. My doubt is since there is not much variation can we conclude that the rhizosphere of the plant has the same microbial community and there is no shift or change in community like that?

I am not sure I fully understood what you did. Did you do a DGGE with the 3 samples you took of your plants post monsoon and the 3 samples of the same plants during the monsoon, or just 1 sample post monsoon and during the monsoon? To conclude on the effect of monsoon on your microbial community, you need to run the 3 samples you took of the rhizosphere of your plants post monsoon and during monsoon. You need to have your triplicates; you can’t conclude anything based on 1 replicate. Then you need to analyse your DGGE to extract the data of your profiles (using software such as TotalLab, BioRad or Bionumeric etc.) and do a cluster analysis to determine if your profiles/bacterial community structure are actually similar or not. Unless your DGGE profile is extremely simple with less than 10 bands per sample profile (which should not be the case as you work on bacteria from the rhizosphere), you can’t really determine by eyes if 2 profiles (i.e. two microbial communities) are similar. DGGE profiles are considered to differ greatly with only few bands difference (keep in mind that DGGE focus only on the dominant microbial taxa).

If you do not have access to any software to analyse your gel, have a look if any bands appear or disappear between the profile post and during monsoon. If you see few bands appearing of disappearing between the 2 sampling time, that mean your community are different and there is a shift in your microbial community structure due to the monsoon. And inversely, if you do not see bands appearing or disappearing between the 2 sampling time, there is no shift in your community. It is difficult to judge how similar are you profile without seeing the DGGE gel…

OK Aimeric sir, I got clarity in what you said. I just observed the gel directly by running the samples next to each other, collected during monsoon and post monsoon in which i found the same kind of profile. As per you said i will upload in the software and see whether the profile is same or different.