**4. New approaches of application of mutually beneficial plant-microbe systems in sustainable agriculture**

#### **4.1. Development of multicomponent inocula containing BSM**

Very little is still known about the plant regulation of mycorrhization process. In split-root systems on alfalfa (*Medicago sativa*), inoculation of one half of a split-root system with the fungus *Glomus mosseae* significantly reduced later AM colonization on the other half. A similar suppressive effect on mycorrhization was observed after inoculation with *Sinorhizobium meliloti* [215]. Furthermore, prior addition of purifed rhizobial Nod factors on one half signifcantly reduced mycorrhization on the other half of the split-root system, and reciprocally, prior mycorrhization on one side suppressed nodule formation on the other side of the splitroot system. Together these data point to a common autoregulation circuit for both symbioses [210]. It was suggested that Nod factor signaling, as well as mycorrhizal Myc factor signaling, induces expression or post-translation processing of CLE peptides, which likely function as ascending long-distance signals to the shoot [210]. Also, it was demonstrated that mutations in *HAR1* of *Lotus* and corresponding orthologues in other legumes increase both nodulation and mycorrhization suggesting the shared role of these orthologous genes in controlling the rate of root colonization by microsymbionts. Thereby, not only the local signal transduction

(CSP) but the systemic autoregulation is common for the RN and AM symbioses.

interactions with the use of plant and microbe mutants is also in progress.

result of low relative efficiency (electron allocated to N2 versus H+

The next-coming step of development the genetics of symbioses is studying gene networks on intergenomic level, i.e. the coordinated expression of plant and microbe genes. For AM, with use of the new molecular approaches, in particular transcriptomics, a series of AM fungal genes has been identified, having altered expression levels during the AM formation [216-219]. Still it is not well studied at which stages of fungal-plant interaction the complementary partners' genes are induced or repressed, and so the use of plant mutants impaired at different steps of AM development might be a challenging approach to reveal the pattern of plant and fungal genetic cooperation [220]. The same research aimed at identification of plant-rhizobial gene

It has been recently observed that *Medicago truncatula* showed significantly lower efficiency of nitrogen fixation than its close relative *Medicago sativa* L. [221]. The number of nodules formed on the roots of *M. truncatula* was less than that of *M. sativa*, and the nitrogen fixation measured on plants at the beginning of flowering (as well as specific N2 fixation (μgN h-1 mg nodule-1)) was significantly lower. The reasons for the low efficiency in nitrogen fixation were partially a

from nodules in *M. truncatula* when compared to *M. sativa*. This might be connected with a low malate concentration in the nodule tissue of *M. truncatula*, and thus insufficient carbon provision for asparagine formation (fixed nitrogen is to be added to malate to form asparagine) [221]. Therefore, Sulieman and Schulze [221] suggest that improvement the malate formation

According to these data, genes encoding for enzymes of malate synthesis should be good candidates for markers to be used as selection and breeding aimed at improvement of symbiotic properties in *M. truncatula*. But, in different species potential markers of symbiotic effectiveness could be found among genes of different functional groups. Our original data on sequencing alleles of symbiotic genes in pea (*Pisum sativum*) varieties with different symbiotic

in *M. truncatula* nodules could help improving the effectiveness of nitrogen fixation.

), and slow nitrogen export

**3.4. Next stage of development the genetics of symbioses**

182 Plant Breeding from Laboratories to Fields

An existence of plant genes [148, 150, 222] and their molecular products [223] common for both AM and RN symbioses led to a conclusion that system of legume symbiotic genes should be considered as a single whole, controlling the development of a tripartite symbiosis (legume plant + AM fungi + rhizobia). This fact along with the demonstration of synergistic activity in beneficial soil microbes (reviewed in: [103]) and a suggestion that plant genetic systems controling the development of RN and, probably, of some other beneficial plant-microbe associations evolved on the basis of that of AM [35] have great importance for the application of tripartite or even multi-partite symbiotic systems in low-input sustainable environmentallyfriendly agrotechnologies.

The use in sustainable agriculture of inocula based on beneficial soil microbes as described above allows the improvement crop productivity with decreased doses of mineral fertilizers and pesticides (reviewed in: [224, 225]). These days the majority of commercial inocula contain pure cultures of single microorganisms and only occasionally multiple combinations. There are several objections to the use of mono-inoculation. Firstly, endemic microbial communities are stable and the introduced microbe may be allowed to occupy a very small niche in the whole community or even get lost in a first week after introduction. Secondly, genetic material in microbes is very plastic, and consequently strains introduced into natural ecosystems can rapidly lose their beneficial traits. Thirdly, the existence of microbial cooperation in the rhizosphere [103] as well as in natural synergistic associations of different microbes including those between AM fungi and their endocellular or superficial symbionts [103, 132] question the possibility and expediency of applying mono-inoculants and even use of the term 'monoinoculation' itself. Finally, plants possess relatively stable genomes and this fact contributes significantly to the effectiveness of symbiosis [226]. Therefore, for industrial plant production in sustainable systems we should use plants having highly effective interactions with all kinds of beneficial soil microbes, which can encourage the development of multiple niches hosting microbes and regulating their activity. For this it is necessary to develop new multi-component microbial inocula which increase the content and biodiversity of beneficial soil microbes in agricultural land.

There is experimental evidence of the effectiveness of simultaneous inoculation of legumes with AM fungi and nodule bacteria leading to increased productivity and quality of the yield, e.g. groundnut [138], pea [139-141, 227], albaida (*Anthyllis cytisoides*) [95], and soybean [142]. The effect achieved equalled or exceeded that achieved with mineral fertilizers [140, 141, 227]. The effect also exceeded that of mono-inoculation with AM fungi or with rhizobia either in model experiments or under field conditions [139, 140, 142]. In long-term experiments in a desertified Mediterranean ecosystem, it was found that simultaneous inoculation with AM fungi and rhizobia enhanced the establishment of key plant species and increased soil fertility and quality; increased soil nitrogen content, organic matter content, and soil aggregate hydrostability and enhanced nitrogen transfer from nitrogen-fixing to non-fixing species associated with the natural succession of the plants [95].

There is an example of application of triple inoculum (AM fungi, rhizobia and PGPR) to the legume *A. cytisoides* which was successful only when the microorganisms used were isolated from local environment [94]. In collaboration with an innovation company "Bisolbi-Inter" (Russian Federation) the All-Russia Research Institute for Agricultural Microbiology (AR‐ RIAM), Saint-Petersburg, Russian Federation, has developed technology for the production and application of a new multifunctional biopreparation BisolbiMix [228] containing a complex of the most effective isolates of endosymbiotic microbes (AM fungi and rhizobia) and associative bacteria (PGPR) from the collection held at ARRIAM. A non-sterile substratecarrier which is derived from washing-filtration by-products of a sugar-beet factory contains its own microbial community including all the above groups of beneficial microbes. The preparation can be formulated into a seed dressing (not effective for all the crop plants tested) or granules. The efficacy of BisolbiMix was demonstrated in field trials with legumes, e.g. pea [227] or non-legumes such as wheat, pumpkin and potato (Chebotar V.K. et al., unpublished results). The use of microbial formulations containing rhizobia for non-legumes seems to be sensible because it is known that nodule bacteria which do not form nodules on a non-host legume as well as non-legume roots can operate as PGPR [229, 230]. Thus, the selection of rhizobia with both PGPR activity and efficient symbiotic nitrogen fixation should be advan‐ tageous in crop rotations or intercropping systems using legumes and non-legumes.

It is possible, therefore, to develop effective multi-microbial inoculants, but it is necessary to use local communities of beneficial microbes because this exploits the natural biological and genetical adaptations of the partners to their environment [94, 231].

#### **4.2. Breeding for improving legume symbiotic effectiveness**

During development of plant-microbe systems for low-input sustainable ecologically friendly plant cultivation it is necessary to be guided by conclusions of EC experts about global productivity of legumes (http://www.grainlegumes.com/aep/; http://ec.europa.eu/research/ biosociety/food\_quality/projects/002\_en.html) for sustainable agriculture. The use of legumes in agriculture is leading to: improved soil fertility and increased diversity of crops and soil microbial communities; reductions in the use of non-renewable natural resources; decreased negative effects from intensive agrotechnologies on the natural environment due to decreased requirement for mineral fertilizers and pesticides and decreased production of animal protein and associated wastes; local production of pollution-free food and forage; and a more stable income for the agricultural producers. This is why it is necessary to breed legumes which have highly effective interactions with beneficial soil microbes.

e.g. groundnut [138], pea [139-141, 227], albaida (*Anthyllis cytisoides*) [95], and soybean [142]. The effect achieved equalled or exceeded that achieved with mineral fertilizers [140, 141, 227]. The effect also exceeded that of mono-inoculation with AM fungi or with rhizobia either in model experiments or under field conditions [139, 140, 142]. In long-term experiments in a desertified Mediterranean ecosystem, it was found that simultaneous inoculation with AM fungi and rhizobia enhanced the establishment of key plant species and increased soil fertility and quality; increased soil nitrogen content, organic matter content, and soil aggregate hydrostability and enhanced nitrogen transfer from nitrogen-fixing to non-fixing species

There is an example of application of triple inoculum (AM fungi, rhizobia and PGPR) to the legume *A. cytisoides* which was successful only when the microorganisms used were isolated from local environment [94]. In collaboration with an innovation company "Bisolbi-Inter" (Russian Federation) the All-Russia Research Institute for Agricultural Microbiology (AR‐ RIAM), Saint-Petersburg, Russian Federation, has developed technology for the production and application of a new multifunctional biopreparation BisolbiMix [228] containing a complex of the most effective isolates of endosymbiotic microbes (AM fungi and rhizobia) and associative bacteria (PGPR) from the collection held at ARRIAM. A non-sterile substratecarrier which is derived from washing-filtration by-products of a sugar-beet factory contains its own microbial community including all the above groups of beneficial microbes. The preparation can be formulated into a seed dressing (not effective for all the crop plants tested) or granules. The efficacy of BisolbiMix was demonstrated in field trials with legumes, e.g. pea [227] or non-legumes such as wheat, pumpkin and potato (Chebotar V.K. et al., unpublished results). The use of microbial formulations containing rhizobia for non-legumes seems to be sensible because it is known that nodule bacteria which do not form nodules on a non-host legume as well as non-legume roots can operate as PGPR [229, 230]. Thus, the selection of rhizobia with both PGPR activity and efficient symbiotic nitrogen fixation should be advan‐

tageous in crop rotations or intercropping systems using legumes and non-legumes.

genetical adaptations of the partners to their environment [94, 231].

**4.2. Breeding for improving legume symbiotic effectiveness**

It is possible, therefore, to develop effective multi-microbial inoculants, but it is necessary to use local communities of beneficial microbes because this exploits the natural biological and

During development of plant-microbe systems for low-input sustainable ecologically friendly plant cultivation it is necessary to be guided by conclusions of EC experts about global productivity of legumes (http://www.grainlegumes.com/aep/; http://ec.europa.eu/research/ biosociety/food\_quality/projects/002\_en.html) for sustainable agriculture. The use of legumes in agriculture is leading to: improved soil fertility and increased diversity of crops and soil microbial communities; reductions in the use of non-renewable natural resources; decreased negative effects from intensive agrotechnologies on the natural environment due to decreased requirement for mineral fertilizers and pesticides and decreased production of animal protein and associated wastes; local production of pollution-free food and forage; and a more stable

associated with the natural succession of the plants [95].

184 Plant Breeding from Laboratories to Fields

For more than twenty five years the authors' laboratory has specialized in the genetics of plant-microbe interactions using pea (*P. sativum* L.) as a model plant. Our experience for improving the effectiveness of beneficial plant-microbe systems with pea is consequently given as an example. At the same time, the authors' team knows only single record of other activity of this nature: genetic variability of onion (*Allium* spp.) has been shown with respect to its responsiveness to AM fungi inoculation which indicate that onion breeding for improving efficacy of associations with AM fungi is possible [232]. The ne‐ cessity for this sort of plant breeding is also considered, mainly with respect to the effec‐ tiveness of RN symbiosis [233-236].

#### **4.3. Analysis of genetic variability of pea with respect to its effectiveness of interactions with beneficial soil microbes**

A high level of genetic variability was demonstrated in analyses of the symbiotic effectiveness under double inoculation with AM fungi and nodule bacteria of 99 land-races and outclassed heritage cultivars of *P. sativum* from the collection N.I. Vavilov's All-Russia Research Institute of Plant Industry, Saint-Petersburg, Russian Federation, of different geographical origin [139, 141]. In a few genotypes considerable increases in plant dry weight (about 300 %), seed productivity (more than 650%), phosphorus and nitrogen content (more than 900 and more than 300 %, respectively) were observed. The most promising highly symbiotically effective genotypes and those with low symbiotic potential were included in the Pea Genetic Collection (ARRIAM) to be used for experiments studying the functioning of tripartite/multipartite symbiosis. Types identified as highly symbiotically effective genotypes were involved in breeding programmes to create commercial pea cultivars with great potential for interactions with beneficial soil microbes (in collaboration with All-Russia Institute of Leguminous and Groat Crops (ARILGC), Orel, Russian Federation).

The most promising highly symbiotically effective pea genotypes previously selected and dif‐ ferent commercial pea cultivars created without consideration of symbiotic effectiveness were involved in three-year field trials (Orel district) [227]. Seed productivity and plant dry weight were chosen as the main criteria for the evaluation of symbiosis effectiveness in legume crops. The double (actually multiple, see above comments on the nature of AM fungi) inoculation was shown to increase seed productivity and plant dry weight in most of the pea genotypes studied and sometimes this could exceed the effect of mineral fertilizers. The effectiveness of legume breeding to improve the symbiotic potential of legume cultivars was proven therefore under field conditions and the genotypes to be used in such breeding programmes were identified. The genotype K-8274 (non-commercial) was selected as a standard of symbiotic effectiveness. Additionally, it was demonstrated that highly effective genotypes can be also found among commercial pea cultivars created without consideration for effectiveness of interactions with beneficial soil microbes. Taking into account that most commercial legume cultivars have acci‐ dentally lost their abilities for symbiotrophic nutrition without selective pressure during breeding of intensive crops, the latter constitutes a very important finding for plant breeders and gives them the possibility for concurrent generation of cultivars with required pea plant architecture, other agriculturally important traits and high effectiveness of interactions with all types of beneficial soil microbes in a single breeding programme.

#### **4.4. Breeding to improve pea symbiotic effectiveness**

In order to cultivate plants with improved symbiotic potential a special breeding nursery was created in the experimental trials ground of ARILGC on land where for the 5 years before nursery establishment mineral fertilizers had not been applied. To reduce the incidence and severity of root pathogens a 6-field crop rotation was used where cultivation of winter wheat was followed by peas. The multi-component preparation BisolbiMix was used for the inocu‐ lation of test plants.

Using the breeding nursery as well as a breeding protocol developed from long-term collab‐ oration of ARRIAM with ARILGC the first (in the whole history of legume breeding) pea cultivar "Triumph" having increased potential of interactions with beneficial soil microbes was intentionally created [237]. It arose as a result of crossing a commercial cultivar 'Classic' (donor of agriculturally important traits) and the genotype K-8274 (donor of symbiotic effectiveness trait) and subsequent individual selection of genotypes with high productivity and capacity for supporting various beneficial microbes.

The cultivar "Triumph" is of middle stem height, semi-leafless and has stable productivity un‐ der different climate conditions, it is comparatively resistant to root rots and pests. Its produc‐ tivity is not lower than those of the productivity standards for Orel district using the conventional production technologies and 10% greater in comparison with the standard culti‐ vars when inoculated with BisolbiMix. As a result of two-year state trials (2007-2008) the pro‐ ductivity of "Triumph" was shown to be comparable with those of standard regional cultivars enabling recommendation for commercial cultivation in the Central region of Russian Federa‐ tion (unpublished results). Thus, the innovative concept of the authors' research team for plant breeding (applicable not only for legumes, but also for non-legumes) is bearing its first fruits.
