Please check the PDF attachment.

Not  exactly  a pulse breeder,  I have been involved in soybean nitrogen fixation ecology.  In principle, I agree with Singh's positions: a selection approach both at high and low N supply should be more informative. Rhyzobium strain diversity might also matter... Of course a pulse breeder cannot multiply trials to accomodate different strain by line combinations. To get information about the ability of N fixing capability in pulse lines, the availability of  a non-nodulating line  would be a good check, and a leaf  chlorophyll reader may be practical to have a quick index of plant N status.

Thats a good point Dr Rabindran . But , i have a slightly  different view on the issue.  Presence of rhizobium responsive gene would make pulses, perhaps  more  responsive to  external N-input, simply because of the transcriptional ( Epigenomics) attitude of m-RNAs associated nitrogen fixing genes. 

No doubt , screening needs to be performed under both N-deficit and N-surplus conditions , both.  

of course, the Rhizobium is not essential for growth, just take care you supply enough nitrogen.

…But why bother? Breed pulses in the absence of rhizobium?

What an important ecological niche pulses occupy, by being able to ‘fix’ the most of the N needed (close to 80%) from the atmosphere.  They get the balance from native soil N mineralized in a cropping season (in Ontario, Canada, no N fertiliser is recommended for soybeans, yet they yield 3-4 Mg grains/ha).

I agree with Prof. Antoun. Best thing is to use natural soil containing those microbes to do pulse breeding.

Dr.Rabindran,longtime back,may  be around three decades,I was told about a high-yielding and nitrogen responsive green  gram variety developed at Agricultural Research Station, Lam,Guntur.I think it is possible to breed a high yielding and Fertilizer responsive  and slightly long duration pulse crop by selecting appropriate gene for N response.No harm even if utilizes biologically fixed N.

The element nitrogen, or “azote,” meaning “without life,” but lifeless, since it is a component of food, poisons, fertilizers, and explosives.Nitrogen from Rhizobium is very less quantity so additional N2 have to be supplied by Organic/chemical or bio fertilizers,the Bio fertilizers containing rhizobium also supplied and this is eco friendly.  The current technology for fertilizer production and the inefficient methods employed for fertilizer application, both the economic and ecological costs of fertilizer usage will eventually become prohibitive. Organisms that can fix nitrogen, i.e., convert the stable nitrogen gas in the atmosphere into a biologically useful form, all belong to a biological group known as prokaryotes. Several environmental conditions are limiting factors to the growth and activity of the N2-fixing plants. A principle of limiting factors states that “the level of crop production can be no higher than that allowed by the maximum limiting factor”.In the Rhizobium-legume symbiosis, which is a N2-fixing system, the process of N2 fixation is strongly related to the physiological state of the host plant. Therefore, a competitive and persistent rhizobial strain is not expected to express its full capacity for nitrogen fixation if limiting factors (e.g., salinity, unfavorable soil pH, nutrient deficiency, mineral toxicity, temperature extremes, insufficient or excessive soil moisture, inadequate photosynthesis, plant diseases, and grazing) impose limitations on the vigor of the host legumes. Inside the nodule, there is a limited oxygen and the bactericides are protected by a peribacteroidal membrane in a symbiosome. However, for the oxidation processes, the oxygen is transported to the different compartments through the leghemoglobin... I think you know all the different details of these processes.

https://www.google.co.in/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&cad=rja&uact=8&ved=0ahUKEwjY5eG-tvrNAhXFOI8KHSpYCEEQFggwMAE&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpmc%2Farticles%2FPMC98982%2F&usg=AFQjCNH0fyMhwSRKRs4XpyoTojHXppjx3A&sig2=n6_bz5vnIwt5q3tZnV14SA

https://www.researchgate.net/post/Can_nitrogen_fixation_occur_in_liquid_media_in_the_presence_of_Rhizobium_but_without_plant_roots?_tpcectx=profile_questions

 The expression of "nodulation" genes in the bacteria is activated by signals from plant roots and as a result the bacteria synthesise signals that induce a nodule meristem and enable the bacteria to enter this meristem via a plant-made infection thread. The chemical signals synhesised by the bacteria are based on a modified amino acid (homoserine lactone) carrying a variable acyl chain substituent, and are called acyl homoserine lactones (AHLs). By detecting and reacting to these chemicals, individual cells can sense how many cells surround them, and whether there are enough bacteria, i.e. a quorum, to initiate the change towards acting in a multicellular fashion. This is known as 'quorum sensing'.

Within the nodule meristem, the bacteria induce specialised genes required for nitrogen fixation. Within the nodule and infection thread, plant products including glycoproteins and glycolipids are synthesised de-novo. Monoclonal antibodies directed against such plant products have been isolated and used to analyse the spatial and temporal expression of plant-made components important in the developing nodule. Specific pea genes induced during the symbiosis have been identified and have given new insights into novel roles of various gene products expressed during the infection process that leads to nodule development.

 For details see link: https://www.jic.ac.uk/science/molmicro/Rhizo.html

 The formation of nitrogen fixing nodules on legumes requires co-ordinated expression of several bacterial and plant genes. Initial stages of nodule formation require expression of specific nodulation (nod) genes by rhizobia. The nodABCFELMN gene products are involved in the synthesis of a group of signal molecules (Nod factors) that induce nodule morphogenesis. These signals are acylated derivatives of an oligo N acetyl-glucosamine polymer (4 or 5 residues long). NodF and NodE determine the type of N-acyl group (C18:4 in Rhizobium leguminosarum biovar viciae) on the first glucosamine on the oligomer, whilst NodL carries out an O-acetylation of the first glucosamine residue. The role of these Nod factors in efficient nodulation are needed to be analysed with regard to effects on competitive nodulation in different varieties of pea. In parallel, R. leguminosarum biovar viciae secretes a Ca2+ binding protein (NodO) that probably interacts directly with root cell membranes to stimulate the infection process. The mechanism of secretion of this protein will be analysed by identifying the genes that encode the protein translocation machinery. Subsequent stages in nodulation involve intimate contact between the Rhizobium and plant cell surfaces, and the molecular interactions that occur between them are needed to be studied. Bacterial surface components include the lipopolysaccharide (LPS); the role of its structure in the development of normal nodules are needed to be studied using monoclonal antibodies and bacterial mutants altered in LPS structure. Also important for symbiotic nitrogen fixation is a specialised set of cytochromes required for respiration in the low oxygen concentration found in legume nodules. The assembly and regulation of these cytochromes will be analysed with a view to improving the efficiency of nitrogen fixation. Plant-made determinants important in the symbiosis include glycoproteins and glycolipids including inositol derivatives. Using monoclonal antibodies or specific antisera, individual components have been localised to the infection thread matrix, peribacteroid space and peribacteroid membrane. Future studies will focus on the role of host defence systems in controlling the process of tissue and cell invasion by Rhizobium. In addition, the differentiation of the peribacteroid membrane will be studied as a model system for membrane biogenesis and vesicle targetting in plants. These studies will exploit the wide range of symbiotically defective mutants that are available for both the bacterial and higher plant partners. Experimental techniques will range from molecular cell biology (immunolocalisation, in situ hybridisation), through to standard molecular genetics (PCR, cDNA cloning, transformation and the use of transgenic plants). Molecular mapping of pea genes required for nodulation is underway. For details see link: https://www.jic.ac.uk/science/molmicro/Rhizo.html

Above given explanation makes it clear that pulses varieties without nodulation can be bred. There is one assumption that due to nodulation in pulses, these crops become more sensitive to excess water and therefore irrigation frequency is reduced. If there is no nodulation, irrigated crop can be vigoursouly taken just like other crops paddy, wheat . Yield may also increase. Moreover this nodulation becomes faded, once the crop reaches to reproductive phase and it is also assumed that crop becomes susceptible to root diseases which is also a limiting factor for low yield. These all are assumptions without any scientific evidence. This may be a new area of future research. Compliments to Dr. Ramalingam for out of the box thinking.

Very good point Dr Malhotra , pulses are for sure ,  nodulation in pulses is very sensitive to excess water , and pulses will perform better in terms of yield without nodulation , but other side of the coin is , pulses are mechanistically made to fix atmospheric nitrogen . Whether or  not , nodulation process in pulses turn them more sensitive to various abiotic stresses and a handicap to higher production..?

Fellow Researchers,  As per my understanding, the mode of N acquisition in any pulse crop could be readily switched from ‘biological fixation’ to ‘soil dependence’ (acquisition of available N from soil).   We only need to apply inorganic N fertiliser to well-functioning pulse crop; the crop will stop ‘biological fixation’ and start taking up available N from the root zone.  That is because biological N fixation is known to be highly sensitive to inorganic N.  Therefore, (in my view) it is not essential to breed pulses responsive to fertiliser N.  What may be more useful is to breed pulses that can sustain a well-functioning nodule system, late into the pod-filling stage.

N acquisition of any crop (whether it is a pulse or not) is sensitive to moisture stress.  No matter what amount of available N present in the root zone, if there is not enough soil water, crops will not respond to applied N, unless we supply irrigation. If irrigation is not a limiting factor, why breed pulses without rhizobium?

Dr.Jayasundara, I appreciate you reply. I believe that we need pulses with high yield potential and are able to utilize efficiently   soil residual N, N fixed  by Rhizobium and applied N externally.Also pulses express their full yield potentil under irrigation or assured rainfall.I think if a very high yield potential variety is bred, it will utilize all the  available resources efficiently and give reasonably high yield.

Susantha , thats a very good point to breed pulses having functional nodule system late in the crop season . Question is whether pulses sense the externally applied nitrogen  more effectively , instead of  relying on their own inbuilt system of nitrogen fixation . For a moment , lets assume , there is no nodulation system , the pulses will respond to externally applied nitrogen with  irrigation . In that case , what kind of consequences , we can anticipate with regard to yield performance ..?

 Bacteria of the genus Rhizobium play a very important role in agriculture by inducing nitrogen-fixing nodules on the roots of legumes such as peas, beans, clover and alfalfa. This symbiosis can relieve the requirements for added nitrogenous fertilizer during the growth of leguminous crops. The Rhizobium group is studying the bacterial and legume genes involved in establishing and maintaining the symbiosis. This will provide background knowledge for use in applied objectives as well as yielding a wealth of fundamental knowledge with wide implications. Underpinning the work is a continuing investigation of the bacterial and plant genes specifically induced during the symbiosis. The communications that occur between the plant and the rhizobia during nodule formation and maintenance constitutes a novel opportunity to study signal transduction in a plant system. The expression of "nodulation" genes in the bacteria is activated by signals from plant roots and as a result the bacteria synthesise signals that induce a nodule meristem and enable the bacteria to enter this meristem via a plant-made infection thread. The chemical signals synhesised by the bacteria are based on a modified amino acid (homoserine lactone) carrying a variable acyl chain substituent, and are called acyl homoserine lactones (AHLs). By detecting and reacting to these chemicals, individual cells can sense how many cells surround them, and whether there are enough bacteria, i.e. a quorum, to initiate the change towards acting in a multicellular fashion. This is known as 'quorum sensing' and this laboratory is coordinating an EU Consortium on 'Rhizosphere Communication' to investigate the extent to which specific plant-growth-promoting bacteria use AHL-based quorum sensing regulation of important physiological traits, and the degree of cross-talk with plant pathogens.

SOURCE:https://www.jic.ac.uk/science/molmicro/Rhizo.html

Some fascinating stuff Dr Malhotra. an eye opener , following this discussion . Legumes have inbuilt mechanism to trigger on  the epigenomics to be able to accelerate the process of biological nitrogen fixation . Do you feel , presence of rhizobium responsive genes will be  an impediment to response to inorganic  nitrogenous fertilizers..?

I feel , it could be an impediment , for the simple reason , plants have tendency to sense their stored nutrients more effectively than exogeneously applied nutrients , legumes no exception in this regard. However , it remains to be seen, at later stages of plant  growth, how effectively plant senses the nitrogen available  within its rhizosphere zone , what quorum sensing , Dr Malhotra highlighted .

Yes it can be bred, but what will be the future of such varieties.

Nodulated legumes are the largest contributors of biologically fixed nitrogen (N) to both natural terrestrial and agricultural environments and are a key component of sustainable agriculture. The root nodulating symbioses that they form with a diverse range of soil bacteria, collectively termed “rhizobia” are capable of fixing more than 200 kg N ha-1 yr-1 in agricultural systems in both tropical and temperate regions. These systems include legumes grown for their high protein grains as animal forage and fodder, and as “green manures”, intercrops and under stories. If appropriately managed, legume crops can achieve high yields without applications of mineral N-containing fertilizer; in addition, they have the potential to contribute substantial amounts of fixed N to the succeeding non-legume crop or directly when used as green manures, intercrops or understory’s.