Cassava (Manihot esculenta Crantz) is a Central and South American native plant with tuberous roots rich in starch. It is a staple food for over 800 million people living in developing countries (Burns et al., 2010). In Côte d'Ivoire, it is the second most important food crop with an estimated annual production in 2013 of 2.5 million tons (FAO, 2014). This crop plays an important role in food security and income generation for many smallholder families. Despite its importance, cassava productivity is low in Côte d'Ivoire where yields are around 6 to 8t/ha compared to a global average level of 13t/ha (FAO, 2014). This low yield appears to be due to several factors. Firstly cassava cropping systems in Côte d'Ivoire are intensive and result in a rapid loss of soil fertility usually requiring long fallow periods (up to 7 years) to restore phosphorus and nitrogen levels. Secondly, cassava productivity is affected by pests, with root-knot nematodes being of major importance (Caveness, 1982; McSorley et al., 1983; Jatala and Bridge, 1990). Cassava yield losses due to nematode damage can be up to 87% (Caveness, 1982; IITA, 1990). Root-knot nematode damage can now be considered as a threat to the production of this major crop plant in Côte d'Ivoire where producers are mainly small farmers who cannot afford to buy nematicides. A third problem is the effect of climate change on crop productivity; notably the impact of drought, as unreliability of rainfall is a factor limiting cassava cultivation in tropical and subtropical areas (N'Guettia and Bernard, 1986). Consequently, although known for its ability to withstand drought, the net biomass production of cassava is reduced in times of water stress, irrespective of variety (Connor et al., 1981). Therefore, in order to sustain cassava productivity for farmers in tropical regions, it is important to develop a technology that can confer simultaneously on this plant (i) better growth and yield, (ii) a better tolerance to nematodes and (iii) a better tolerance to water deficit. Compounding the effects of disease and water stress is the increased vulnerability of rural families and smallholder cassava producers who often do not have access to appropriate technologies, services and markets. In developed countries, farmers rely extensively on industrial fertilizers to maximize crop productivity. Unfortunately, because of their financial and environmental costs, industrial fertilizers are not a solution for underdeveloped countries (Sanchez, 2002).

Cassava farmers could benefit from the multiple services offered by soil microorganisms such as arbuscular mycorrhizal fungi (AMF). Indeed, AMF belonging to the phylum Glomeromycota (Schüßler et al., 2001), constitute a multifunctional partner in the mutualistic interaction they develop with most land plants. The major function of AMF is to provide the mycorrhizal plant with water and essential nutrients such as phosphorus and nitrogen (He et al., 2003; Smith and Read, 2008). In addition to this nutritional function they provide, AMF can enhance plant tolerance to both biotic and abiotic stresses (Augé, 2001, 2004; Ortas et al., 2001; Plenchette et al., 2005; Al-karaki, 2006; Pozo and Azcón-Aguilar, 2007; Porcel et al., 2011; Augé et al., 2015). This multifunctional ability of the partner fungi has led to the development of mycorrhizal inoculants as biofertilizers in agriculture. Mycorrhizal inoculation has been applied for decades to promote better plant growth for various crop plants (Osonubi et al., 1995; Carretero et al., 2009). Cassava is highly mycorrhizal (Sieverding, 1989; Oyetunji and Osonubi, 2007) and there is evidence that AMF play an important role in increasing the productivity of cassava (Sieverding, 1989; Cardoso and Kuyper, 2006; Ceballos et al., 2013). Despite this positive impact of AMF inoculation on cassava productivity, and the known positive impact of mycorrhizal inoculation on root-knot nematode infection in crops such as yam and grapevine (St-Arnaud and Vujanovic, 2007; Tchabi, 2008; Hao et al., 2012; Veresoglou and Rillig, 2012), studies of AMF effects on root-knot nematode and water deficit in cassava remain scarce. In other studies, the impact of AMF on water stress has been documented for several crop plants, including cassava (Augé, 2001; Oyetunji et al., 2007). Although these studies point out the importance of AMF, there is no report of an AMF species that can (i) promote cassava growth and yield (ii) alleviate root-knot nematode damage and (iii) alleviate water stress. Therefore, the identification of AMF exhibiting these three traits could be a step forward to sustain cassava productivity in tropical regions.

The objective of this work was to recover native AMF species from smallholder farms and evaluate their potential to promote cassava growth and enhance resistance to root-knot nematode and water stress. Criteria such as ubiquity and relative abundance in field soils and baited soils were used to select native AMF species that were subsequently further evaluated for plant growth promotion in greenhouse and field conditions.

This work aimed to select an abundant native AMF capable of improving cassava crop productivity via several mechanisms, namely improved plant growth, water stress tolerance and nematode resistance. This is an improvement on previous studies, which tended to focus on one aspect affecting cassava yield, without studying the possible interactions with nematodes and drought.

During this study, there was a difference in the way the two native AMF species impacted cassava plant growth in greenhouse conditions. It was shown that only A. colombiana significantly increased the plant growth parameters, such as foliar surface area, plant height and biomass (Table 3). It has been reported that several factors, such as environmental conditions and functional diversity, can affect nutrient exchange between the fungi and plant partners (Walder and van der Heijden, 2015). The experimental conditions used in this work might have been favorable to A. colombiana, which significantly improved P uptake compared to A. appendicula. Variable effects among endogenous single species due to the use of different culture media were also observed in other studies (Williams et al., 2012; Ortas and Ustuner, 2014). Also, the two native AMF species may differ in terms of regulation of genes involved in P uptake. Such observations were made when maize plants were individually inoculated with different AMF species (Tian et al., 2013). In our study, single inoculation with A. appendicula had no effect. Meanwhile dual inoculation with both species positively improved P uptake and cassava plant growth. Similar observations were made when citrus was treated with different AMF species using a dual inoculation approach (Ortas and Ustuner, 2014). It could mean that when used together as dual inoculants, the two native AMF species induce phosphate transporters in cassava plants, as reported for different AMF species used to inoculate maize plants (Tian et al., 2013).

The study on the interaction between the two native AMF and Meloidogyene spp. in greenhouse revealed that negative effects of the AMF against the nematode (reduction of egg and nematode densities) were clearly observed, whether or not AMF and nematodes were co- or post-inoculated (1 month later). Interestingly, the presence of the nematode exerted a negative effect on the AMF, by reducing mycorrhizal intensities and frequencies in the case of simultaneous inoculation. Both types of interactions between nematode and AMF have already been reported. These mutual negative effects occur when fungi and nematodes are competing for space and nutrients (Schouteden et al., 2015). For example, the fungus Scutellospora heterogama exerted a biocontrol effect on the sedentary endoparasitic nematode Meloidogyne incognita (reproduction was reduced) only when it was pre-inoculated whereas co-inoculation had no effect (Dos Anjos et al., 2010). Such observations have also been made for migratory endoparasitic nematodes. For example, it was shown that Radopholus similis and Pratylencus coffeae affected the frequency of Funneliformis mosseae colonization in banana, but not the intensity (Elsen et al., 2003a,b). In contrast, root colonization by R. irregularis in vitro banana plantlets was not affected either by R. similis (Koffi et al., 2013) or by P. coffeae in transformed carrot roots (Elsen et al., 2003c).

Overall, in the presence of these native AMF, cassava plants continued to grow even though nematodes were present. It appears that the mycorrhizal cassava plants were either resistant (e.g., suppression or reduction of the nematode reproduction) or tolerant (low or no suppression in cassava plant growth) to nematodes, as reported in other studies (Hussey and Roncadori, 1982; Affokpon et al., 2011). However, the mechanism of the bioprotection conferred to cassava plants by the native AMF against the root-knot nematode Meloidogyne spp. is not yet understood. It may be due to the production of phytochemical inhibitors of nematodes, as was observed elsewhere. Indeed, in this work it was observed that phenolic compounds were significantly increased in cassava plant roots when nematodes were post-inoculated. Previous work has shown production of phenolic compounds to be a plant defense mechanism against nematode attacks (Zhu and Yao, 2004; Xu et al., 2008). Elsewhere, accumulation of phenolic compounds has been observed in mycorrhizal Impatiens balsamina, an ornamental plant, in presence of M. incognita (Banuelos et al., 2014). Singh et al. (1990) concluded that the pre-inoculation of plants, coupled with biochemical changes are responsible for resistance to nematodes. In contrast, when the cassava plants were co-inoculated with the AMF and the nematodes, there was no significant increase in phenolic compounds. Obviously there may be another mechanism involved in the inhibition of Meloidogyne spp. activity. For example, there was an up regulation of mycorrhiza-induced plant defense genes against the ectoparasitic nematode Xiphinema index in grapevine plants pre-inoculated with R. intraradices (Hao et al., 2012).

Besides its capacity to promote cassava growth and enhance resistance and tolerance to the root-knot nematode, A. colombiana also conferred water stress tolerance to cassava plants under severe drought condition. This AMF species significantly improved cassava plant growth under water stress. It was observed that mycorrhizal frequencies decreased gradually during the period of drought for all treatments, compared to the 100% FC water regime. However, despite severe water stress, the mycorrhizal colonization frequencies of A. colombiana remained higher than the control and stable over time. This water stress tolerance could be the result of A. colombiana promoting specific plant stress resistance response during the drought period, as suggested by others (Augé, 2001). For example the presence of this AMF may enhance photosynthetic activity due to the high levels of chlorophyll a and total sugars in A. colombiana colonized cassava plants compared to non-mycorrhizal plants (Mathur and Vyas, 1995).

Overall, this study clearly showed the multiple functions of the native AMF species A. colombiana. Importantly, A. colombiana was dominant in all three study areas and was persistently found and easily produced in trap culture. Abundance and persistence of AMF species are very important for efficient AMF species selection to ensure potential inocula are not lost during trap culture propagation (Trejo-Aguilar et al., 2013). This is essential, as the most widespread method for inoculum propagation is the use of trap plants (Berruti et al., 2016).

Under field conditions, A. colombiana showed a good potential for improving cassava productivity. The dual inoculation using the two native AMF species also increased cassava yield under field conditions. This opens up the possibility of using single and dual inoculation of these two native AMF species to improve cassava productivity in the field. During this study the native inoculants performed better than the commercial inoculant. Indeed, the origin and the composition of AMF are very important factors to take into account for inoculum development (Berruti et al., 2016). It has been shown that native AMF have higher efficiency in terms of plant protection against nematode (Affokpon et al., 2011) and stress tolerance (Ruiz-Lozano and Azcón, 2000) than commercial inoculants generally used in the field. Commercial inoculants are generally comprised of AMF species that can be considered as exotic species in tropical and subtropical regions (Oliveira et al., 2005; Schreiner, 2007). One main drawback in the use of commercial inoculants is the fact that the species used might not survive the competition with local AMF communities. Rodriguez and Sanders (2015), who discussed this issue, recommended research to understand local communities through metagenomics and genetic studies. The use of native inoculants comprised of native AMF like A. colombiana is highly recommended as an alternative to exotic species (Oliveira et al., 2005). As a persistent and abundant generalist, A. colombiana may have been a good competitor under field conditions, as in the greenhouse. Moreover, since commercial inoculants can be either ineffective (Faye et al., 2013) or badly formulated (Corkidi et al., 2004), the use of A. colombiana is more likely to be affordable and effective for cassava farmers in tropical and subtropical regions.

In conclusion, this study clearly points out the potential of A. colombiana as a native AM fungus suitable for inoculating cassava. The process developed in this study to select the multipurpose (plant growth improvement, water stress tolerance and nematode resistance) AMF species A. colombiana for cassava could be applied to efficiently select effective AMF inocula for other crops.

This work is done is the scope of a project in the Laboratoire de Biotechnologie Végétale et Microbienne under the supervision of ZA. SJ designed and run all the experiments as a Ph.D. student. KC helped in designing the PCR amplification protocols and sequence analyses. VR was a cosupervisor of this work as a collaborator on this project. ZA is the coordinator of the project and SJ supervisor.

We are grateful to the West African Agricultural Productivity Program (WAAPP) for funding the Project IVO-RHIZE (Projects 047/PPAAO/2012 and 028/CS/PPAAO/2015), within which this study was conducted. We are also grateful to this program for funding SJ scholarship and the Agence Universitaire de la Francophonie (AUF) for awarding him a biotechnology mobility program.