Tools for innovative organic breeding arise from rhizosphere microbial ecology
Bosco, M., Picard, C.1.
Key words: Plant genotype; below-ground potential; Organic breeding; Plant-probiotic microorganisms.
Abstract 
Research on soil microbial ecology is beginning to elucidate how and how much beneficial soil micro-organisms (i.e. plant-probiotics) contribute to plant integrity and plant environmental fitness. The differences so far highlighted among crop varieties show highly positive interactions with plant-probiotic microflora (PPM), and upgrade the role of soil PPM at the level of other essential factors for sustainable plant breeding. Current research efforts, aimed to rapidly achieve crop varieties fitting for low-input and organic production systems, finally take into account the capacity of each individual variety to efficiently exploit indigenous PPM.
Introduction 
In natural environments, the nutrients uptake of plants, as well as their health, is greatly regulated by the presence and activity of beneficial micro-organisms, i.e. those known to enhance plant growth by fixing atmospheric nitrogen, solubilizing phosphorus, nitrogen, iron and other nutrients, by producing bioactive compounds that stimulate root proliferation and by suppressing root diseases. These beneficial micro-organisms are now called “plant probiotics” (Picard and Bosco, 2007), and include mycorrhizal fungi, antagonistic fungi and the large group of Plant Growth Promoting Rhizobacteria (PGPR). The term “probiotic” has been borrowed from another ecosystem, the gastrointestinal tract, where probiotic bacteria and yeasts can exert health-promoting properties, such as solubilizing nutrients, producing vitamins in situ, reducing the symptoms of diarrhea and of inflammatory bowel diseases. The common idea for plant- and gastrointestinal- probiotics is that in both cases a beneficial microflora minimizes a range of biotic and abiotic stresses. 
Despite the fundamental role of plant-probiotic microflora (PPM), its interactions with plant-root systems have been largely ignored in agro-ecosystems. In fact, modern agricultural soils are almost universally maintained at high fertility, and the selection of most of the current crop varieties has been made under these conditions. Furthermore, resistance toward soilborne pathogens has been in general ignored. Possibly as a consequence, modern breeding programs may have yielded cultivars highly dependent on fertilizer and pesticide supply, and that have diminished capacity to form synergistic microbial associations. For example, modern strawberry cultivars, selected under utilization of methyl bromide soil fumigation, resulted to be non-adapted to the sublethal effects of organisms in non-fumigated soils (Fort et al., 1996). Similarly, Hetrick et al. (1995) found that older cultivated wheats, developed prior to 1950 and thus before the widespread use of inorganic fertilizers in breeding programs, were more reliant on mycorrhizal symbiosis than modern wheat varieties. This is in agreement with Engelhard et al. (2000), who found that wild rice species and older rice varieties were preferred over modern rice cultivars by endophytes of roots such as Azoarcus spp. Confirming the hypothesis of the loss of beneficial root microbial associations during modern breeding programs in highly fertilized systems are the rare cases of varieties selected in low fertilized soils, such as the current Brazilian cereal and sugar cane genotypes. In fact, these cultivars can especially benefit from associative N2 fixation (Baldani et al., 2002), allowing the reduction or complete elimination of N fertilization for these crops.
All these findings highlight the fact that it may be difficult to select cultivars for lower-input agriculture from the elite cultivars currently used in conventional agriculture. In order to increase or maintain the rate of production despite less input of H2O, N, P, Fe or pesticides, it seems thus primordial to breed new crop varieties able to obtain their nutrient supply and their root protection mainly from an efficient association with PPM.
Materials and methods 
Host variation in responsiveness to beneficial micro-organisms generally has been expressed as microbial root-colonization density and diversity, as well as effective plant growth stimulation and protection (Tanksley and McCouch, 1997). Concerning atmospheric nitrogen fixation, root nitrogenase activity and quantification of plant N derived from the atmosphere have also be taken in consideration, both for rhizobial-legume symbiosis and plant associations with free-living microorganisms. Specifically for legumes, number of nodules was also a quantitative trait measured to assess host reaction (Herridge and Rose, 2000). Finally, the mycorrhizal responsiveness was defined in terms of Pi uptake and mycorrhizal dependency (MD) (Tawaraya, 2003).
Results and Discussion
Independent of the beneficial association studied, it has been established that host genotype has a substantial impact in determining the extent of microbial colonization. For example, it has been shown that there is significant genotypic variation in the responsiveness of legume cultivars to Rhizobium. In fact, a range of bean, soybeans as well as of Lucerne (Hungria and Phillips, 1993) genotypes differed in relative nodulation. Furthermore, high variability in nitrogen fixation was observed among crop legume genotypes, varying from 0 to 97% of crop nitrogen derived from nitrogen fixation (Herridge and Rose, 2000).
Concerning the mycorrhizal association, the majority of information on variability in MD was obtained in studies with the cereals Zea mays (maize), Hordeum vulgare (barley) and Triticum aestivum (bread wheat) (summarized by Smith and Read, 1997). For example, diversity in MD level was observed among wheat genotypes by Hetrick and colleagues (Hetrick et al., 1995). Interestingly, it was reported that diversity in capacity of wheat to sustain AM colonization was associated with yield responses, varying from zero to positive or negative values (Xavier and Germida, 1998). Furthermore, MD is often negatively correlated with root morphological traits, such as root length, root dry weight, root hair length and density of root hairs, traits known to improve the ability of the non-mycorrhizal plant to acquire Pi directly from the soil (reviewed in Tawaraya, 2003).
Finally, differential capacity to support associative PGPR has been clearly established among cereal species as well as among genotypes within cereal species. Maximal nitrogenase activity was reported to be dependent upon maize genotype (Ela et al., 1982). By comparing 69 rice lines from diverse backgrounds, Shrestha and Ladha (1996) demonstrated that nitrogen fixation differed significantly amongst the various lines, ranging from 1.3 to 20%. Those with high nitrogen fixation were mostly traditional varieties. More recently, Azevedo et al. (2005) observed that the genetic structure in populations of root-associated diazotroph colonizing rice, maize or sorghum was indeed plant-species dependent. Furthermore, Picard et al. (2008) gave recently clear evidence that maize genotype influences the size of PGPR communities involved in nitrogen fixation and plant protection, as well as the diversity of the AMF colonizing population.
Interestingly, all these variations among cultivars in interactions with PPM seem to have resulted from evolution over generations (Engelhard et al., 2000). More importantly, it was evidenced that root colonization by PPM could be an inherited trait (Smith et al., 1999), probably related to heterosis (Picard et al., 2008). Moreover, at least for the rhizobium-legume symbiosis, the specificity of the beneficial association also resembles a gene-for-gene system. In fact, the strain- and host-genotype-specific interactions are characterized by unique patterns of signal release and response (Pueppke et al., 1998).
Knowledge of the available genetic variability should be utilized for implementing a selection procedure adapted for low input environments. This selection can be carried out with conventional breeding procedures, i.e. by the direct detection of effective root association with beneficial micro-organisms, or can be integrated with the use of molecular marker tool. In that light, more and more studies were conducted to identify quantitative trait loci (QTLs) underlying the plants ability to establish a favourable rhizosphere microbial community. For example, by analyzing 197 recombinant inbred lines (RIL) in a mapping population of maize, Kaeppler et al. (2000) demonstrated that host variation on AM colonization was associated with two QTL. In the same manner, three quantitative trait loci in tomato associated with suppression of root infection by Pythium torulosum in response to introduction of the rhizobacterium Bacillus cereus strain UW85 explained 38% of the phenotypic variation observed (Smith et al., 1999). 
Conclusions
From recent findings we understand that future efforts for breeding sustainable varieties must consider the below-ground genotype potential of each crop, often misunderstood. In particular, the scientific community of soil microbial ecologists supports very strongly the idea that a lot of new knowledge will be produced in the near future. Innovations in sustainable crop varieties will undoubtedly pass through the exploitation of plant-probiotic micro-organisms, by developing and testing, at laboratory, field, and biostatistic levels, a brand new breeding strategy targeted to crop varieties that rely the most part of their productivity, environmental fitness on PPM.
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