Dear Dr. Anoop Kumar ,

To inoculate several strains as mixtures of strains on plant, before any assay, it is essential to find isolates without any antagonistic effect with each other.

An in vitro antagonistic assay on isolates to find such isolates can be as follows; briefly:

Spread 100 µl of suspension of each strain on the NA plate’s surface and allow it to be dried.

Then, inoculate 10 µl of suspension of another strain on sterile assay disks (5-mm diameter) placed on the agar.

Incubate the plates at 28 ͦ C for 48 h and check for signs of clear zones indicating growth inhibition.

Isolates without signs of clear zones can be inoculated with each other on plant

Best regards,

Hassan Etesami

Dear Dr. Anoop Kumar ,

To inoculate several strains as mixtures of strains on plant, before any assay, it is essential to find isolates without any antagonistic effect with each other.

An in vitro antagonistic assay on isolates to find such isolates can be as follows; briefly:

Spread 100 µl of suspension of each strain on the NA plate’s surface and allow it to be dried.

Then, inoculate 10 µl of suspension of another strain on sterile assay disks (5-mm diameter) placed on the agar.

Incubate the plates at 28 ͦ C for 48 h and check for signs of clear zones indicating growth inhibition.

Isolates without signs of clear zones can be inoculated with each other on plant

Best regards,

Hassan Etesami

Sir,

Microbes while in storage condition, need to be under dormant stage. So, the mixture must contain very little moisture.  On application to soil / seed / plant / organic residue, immediately they need to be supplied with food, say sugar solution.

Then we can get more efficacy.

Thanks

Dr Hassan , We tested these microbes complementarity by allowing them stay  together for six months in a broth , and  followed the population of each of these five microbes  , whether or not , there is any change in population of any of these five microbes . Interestingly , the population of each of the all the five microbes  followed an increasing trend upto six months , but thereafter , the re was some reduction in population of Trichoderma .   Our another point  of intent is , that we intend to inoculate soil not the plant  . 

Dear Annop,

I am not a specialist in this field but I try to present my approach from an ecological perspective: Microbial consortium are a better mimicry of natural support system of roots compared to single microorganism inoculations. However, entering to rhizosphere, their fate depends on the mutual interactions between them and other players there mainly the roots. Therefore adding external nutrients, even if works in short term could not be considered the best option. We have to think about shaping the tiny rhizosphere ecosystem (of course in volume, while possibly the one with the most diversity of organisms). The successfulness of microorganism would depend on the shaping of lasting food chains in this ecosystem. Therefore the role of roots as the only main donors of food becomes prominent. I believe the management of plants nutrition is the key to lasting and diversity of rhizosphere ecosystem. By supplying the plants with ready to use nutrients we reduce their dependence on rhizosphere ecosystem so they cut or reduce their supply of food toit, as a result the ecosystem degrades by time. supplying of nutrients in forms that necessitates the microorganism intervention to make it available to roots powers the symbiotic relations and increases the diversity and makes it a more stable ecosystem. Humic substances can do this and some foliar treatments by organic acids could encourage the plants to invest more on their root and relations with the rhizosphere communities.

Regarding developing the ideal mixtures, I believe that if we use a basic sample from rhizosphere of plants with old cultivation history and try to add our suggested microorganism(s) and see whether they could find a place in the already diverse rhizospher ecosystem. This way we might reach to a ecosystem that is closer to its climax state and therefore more stable.

Excellent Ebrahim .  I am responding to you from bottom of your answer  to top. We followed exactly the same while isolating and characterizing the microbes from those orchards having excellent past history of quality production and the prolonged orchard life , called elite orchards in horticultural terms. As I said previously , we had large number of accessions of these diversity characterized from rhizospherel of such orchards.  Screened them first in-vitro for various nutrients , we are looking at . It was then followed by biometric response in nursery as pre-evaluation . Shortlisted them further to have most efficient microbes , then made them as  microbial  consortium . Thereafter , studied their complementarity in-vitro , whether they click with each other when brought together. finally , we tested them again in consortium mode in nursery plants as well as grown up trees , with excellent response in both the cases ( One of the prime reasons for such response is simply , they are isolated from th esame rhizosphere , they originally belong to , called native isolates ) . Now , my intension is to extend its shelf life , which is nothing but a function of food availability to microbes . And best food is nutrients or any other form of nutrients , for example humic acid . We have  yet not tried foliar spray of humic acid to test an  expansion in microbial diversity of rhizosphere  ?. I will appreciate , if you have any publication , please pass on to me .

Dear Anoop,

We work on foliar application of diverse organic acids. We have noticed that applied foliar organic acids can affect on ion absorption from the root. In attached article we showed this phenomena in a hydroponic culture condition.The positive effect of foliar organic acids on root exudation of organic acids is confirmed by An et al (2014).

  In the second article we observe that the root to shoot ratio could change by more investment in root system in response to foliar organic acids. We have a large amount of still-unpublished works that support this phenomena. 

There are many reports showing the effect of foliar humic substances on increasing of root growth. There are reports on ion uptake as well. However larger roots means more place for soil microorganisms as well. However I am not sure that a direct effect on organic acid exudation is already shown or not but it seems very likely to happen. 

 An, Y., Zhou, P., Xiao, Q., and Shi, D. (2014). Effects of foliar application of organic acids on alleviation of aluminum toxicity in alfalfa. J. Plant Nutr. Soil Sci. 177, 421–430. doi: 10.1002/jpln.20120044

Article Foliar sprays of citric acid and salicylic acid alter the pa...

Article Foliar Sprays of Citric Acid and Malic Acid Modify Growth, F...

Dear Ebrahim , I appreciate your efforts , and the publications enclosed therein. Whats your opinion  about the foliar application of microbial consortium with or without humic acid  , and testing the response on the root and nutrient dynamics, including the root: shoot ratio.  

Sir,

Pl see Plant Soil (2014) 383:1–41

Journal of Food Agriculture and Environment 1313(11):131-138 ·

Scientific Research, Vol.5 No.1, January 2015

In all these article they have used humic acid treatment and said about increased population and activity.

Thanks

Sir,

I have answered with some references. In all those articles there is usage of Humic acid. It favours rhizosphere microbes (all). My addition to the topic of discussion is that the microbes are soil pH specific, hence along with nutrients soil pH should also considered while preparing consortium, It must be for the specific soil.

Thanks

Ms Reyhaneh, there has been some attempts to develop microbial consortium  having AMF as one component , but we do not find much  published literature on such efforts. one of then prime reasons , I anticipate , the lack of synergisms  , especially with Pseudomonas . Probably our colleagues may throw better lights on this issue.

Dr Rajakumar and Ebrahim , thanks for giving the discussion to a new direction , which is so exciting . Do we expect some improvements in shelf life of microbial consortium with addition of humic acid into the broth ? .

Dr Rajakumar , you have raised a very specific, but equally pertinent  point that a microbial consortium developed by isolating them from a specific soil ( Acidic , alkaline or salt affected  soil type) using specific plant type/crop type. Will we have the same magnitude  of plant  response , if a microbial consortium developed from Alfisol applied to Aridisol and vice-versa ?. For example, seed spices prefer salinity prone areas ( Aridisol) , you isolate rhizo-competent microbes, and prepare a consortium , and  inoculate the same into rhizosphere of potato( Entisol) or citrus ( Vertisol), do we expect the same magnitude of  plant response?.

Hi all!

I would also like to add! I have used different Bacillus strains for consortium formation to inoculate rhizosphere.

In my case the shelf life of cultures was good (more than 1 year). I just grown pure cultures of individual strains on L-agar plates and after ~20 hours incubation at 37 C, I just sealed the plates with para-film and shifted the culture at 4C in refrigerator. 

Dr Yasin  very nice information , can you share the names of those Bacillus  strains please. And the plant rhizosphere from which , you isolated those microbes , bit elaborative response , if you can pass on , it will be good for our other friends.   

Hi again

Dear Anoop the type of Bacillus stains I used were heavy metal resistant, especially selenium resistant. these strains were isolated form industrially contaminated soils and from the rhizoshere of grass plants, grown on that soil.I have used more than 30 strains, but not all were able to survive for 1 year on L-agar plates, according to above mentioned conditions of the media. However, if you want me to tell you the exact names of strain then give me please some time, so I may confirm it.

Secondly, to my understanding, if you are able to control the moisture content of the solid culture media, as i did by wrapping with para film sheets, the shelf life of the culture can be improved. Although the broth culture can also be a good options but there are more chances of contamination and it is again difficult to understand either after one year culture is contaminated or not. Because in refrigerator at such low temperature 4 C it is easy to have contamination.

Hope this will work for you, Cheers

Thanks Yasin , you are really adding some distinction to the quality of  discussion . It will be wonderful experience to our colleagues as well,  to know such heavy metal resistant Bacillus species . Developing microbial consortium is the call of the day . And such efforts will be more paying,  if we develop such consortium based on certain soil conditions , contaminated soils and salt affected soils are surely one these, what earlier our another colleague Dr Rajakumar expressed.

As I said earlier Ms Reyhaneh ,  I agree with you without any second thought . But , these exercises require truly very exhaustive work  and consumes lot of time . But , again , there is no short cut to  success.

One strategy probably developing instant consortia, the microbes are made in to consortia just at the nick of application time. During storage, each component organism may be stored in independent compartments of single storage space. The antagonistic effects may probably minimal once they reach rhizosphere which is dynamic environment and store house of various micro-flora and fauna. 

Interesting Dr Kalidasu , but how to exercise this arrangement to store each microbes in an independent  compartment of single storage space .And , moreover  that's the purpose of the consortium  where different microbes must express themselves while interacting with each other. Such consortium when inoculated in field , finds altogether a different dynamism .

Dear Anoop,

Some days earlier you had mentioned "foliar application of microbial consortium with or without humic acid  , and testing the response on the root and nutrient dynamics, including the root: shoot ratio". In this case there is no need for applying them foliar cause at least for bacteria there is no advantage to be applied this way. Applying together to soil is a better approach in my view. Sorry for the delay in response.

Enough evidence is there proving PGPR foliar sprays help in management of diseases, probably residing in phylloclade. However, growth promotion effects were least studied. There are certain works indicating PGPR sprays changed composition of leaves related to certain compounds, when used against diseases. Taking this cue, we can theorize that growth promotion is also possible, to what extent is unknown.

Ebrahim, yes I still remember , there are plenty of literature worked out to expand the  microbial diversity of the  rhizosphere through foliar spray of humic acid blended with different  microbes , may not be necessarily   microbial consortium,  so that phylloplane is activated to  effectively  trap  the   incident sun light , eventually improving the photosynthetic efficiency of the crop . Hope , Dr Kalidasu , you will be in agreement on this issue. Role of PGPRs in offering various changes in nutrient as well as microbial pool including carbon pool of the rhizosphere is well documented . But , there is hardly any simultaneous study to look at both the ends , growth promotion on one hand , and offering some control the soil borne diseases on the other hand.  In this context only , the use of biosurfacetants started claiming their relevance since they are so effective on rhizoplane than either  rhizosphere or phylloplane  .

One of the better ways of improving the functionality of the microbial consortium is to find a suitable competence of AM species , add real efficiency.

One of the better ways of improving the functionality of the microbial consortium is to find a suitable competence of AM species , add real efficiency.

Yes  Dr Singh I agree with you , introduction of AM will surely boost the efficacy of the microbial consortium , but needs another very massive efforts , to screen the diversity within AM , and then study their compatibility in a consortium of already  existing microbes  ... but good suggestion .  

I request our friends to throw some lights about the diversity of AMFs in exploiting them to provide the better dynamism to microbial consortium.

Dr Singh , few days back , in some of  RG questions , I responded by saying do we have some indicator microbes specific to specific soil conditions in order to judge the general health of the soil microbiologically . Likewise we have probably yet not attempted to analyse the AMs diversity of different crop rhizopshere , annual versus perennial  fro example. Rhizosphere of one crop to another crop . Can we sythesize a consortium of rhizocompetent microbes including AMs using a process called rhizosphere  hybridization .

Friends , I am still waiting for some clue (s) , as how to extend the shelf life of microbial consortium , since our current microbial consortium spans for about 6-9 months only . In order to bring broader spectrum  to the effectiveness of the microbial consortium, this is a on-goin process to keep on identifying the more rhizocompetent microbes , and keep on adding them ....so on. 

Dr Srivastava , why don't you try some growth regulators added

> exogeneously , and see how the microbial population in a consortium mode

> undergo reorientation through a in-vitro study ?.