Effects of inoculation with some native arbuscular mycorrhizal fungi on tomato (Solanum lycopersicum L.) growth

In Burkina Faso, tomato (Solanum lycopersicum L.) sector plays a very important socio-economic role. However, its production is confronted by many constraints, among which is the soil poverty in mineral elements such as nitrogen and available phosphorus resulting in an increase in the area of land that is sown for this crop and an increase in the use of chemical inputs. In addition, chemical inputs have shown their limits with several environmental negative impacts. Therefore, this study was initiated to help improve sustainable agricultural production. In this study, tomato was grown in the greenhouse and inoculated with three natives’ mycorrhizal inocula. The growth parameters were measured at 30 and 60 days after sowing. Above-ground, root and total biomass were assessed at 60 days after sowing. The results showed an improvement in height of 164.44%, in the collar diameter of 75.25%, in above ground biomass production of 540%, root biomass of 1061.97% and a total biomass of 638, 1% after inoculation. This study had shown promising results and merits further investigation in situ.


INTRODUCTION
Tomato (Solanum lycopersicum L.), is an herbaceous plant widely cultivated for its fruit and it represent one of the main food crops in the world. This crop is the second most important vegetable produced and consumed in Western countries (Willcox et al., 2003). With its products, tomatoes are one of the main food sources of carotenoids providing around 80% of the daily intake of lycopene, folate, ascorbic acid, flavonoids, a-tocopherol and potassium in the Western diet (Bramley, 2000;Willcox et al., 2003). In Burkina Faso, tomato is one of the most important vegetable crops. However, its production encounters enormous problems such as the poverty of soils, particularly in phosphorus (P) and nitrogen (N) (Diem et al., 1981;Mikola, 1987), water deficit, wind and water erosion, as well as fungal, bacterial and viral diseases. Its production increased from 1,000 t in 2000 to 12,635 t in 2017 (FAO, 2020). The improvement in this production is linked to the area planted (from 1,000 ha in 2000 to 1,254 ha in 2017) since the yield has remained around 10 t/ha (FAO, 2020). In  addition to the increase in the cultivated area, there is also the intensive use of chemical fertilizers to correct soil poverty and fight against tomato diseases. This results in huge losses for farmers. In addition, intensive agriculture has shown its limits: Soil and water pollution, the emergence of pesticide-resistant pathogens, high pesticide costs, risks to farmers' health, and weakening ecosystems (Pierre, 2012). Regulations are now in place to limit the use of these inputs. Therefore, maintaining crop productivity through agriculture that substitute chemical inputs use by the mobilization of biological processes is at the heart of the challenges of current agricultural research. Thus, tomato cultivation remains to this day, a major concern because its important contribution to food security and the increase in income of producers, especially in family farming. The current agriculture turns towards an ecological intensification which leans on the promotion of the ecological mechanisms by practices such as crop rotation, the cultural association or the biological control. The use of beneficial microorganisms such as arbuscular mycorrhizal fungi (AMF) is increasingly considered as one of the best sustainable organic farming practices (Haro et al., 2017). It is well established that the tomato forms a symbiosis with arbuscular mycorrhizal fungi (Dodzia et al., 2012;Khalloufi, 2017) while the beneficial effect of mycorrhizal symbiosis on plant growth and production has been the subject of several studies (Haro et al., 2016a(Haro et al., , 2012(Haro et al., , 2015(Haro et al., , 2017(Haro et al., , 2016b. Exploiting this symbiosis would be a possibility to improve tomato productivity. Thus, we became interested in the tomato mycorrhizal symbiosis with the aim of evaluating the effect of inoculation of this plant with arbuscular mycorrhizal fungi native to Burkina Faso on its growth.

Plant and fungal materials
Tomato variety AMIRAL F1-hybrid [variety of tropical, semi-tropical and Sahelian zones (produced/imported, packed and marketed by SAKATA SEED INDIA PVT., LTD)] was used. Tomato seeds were surface disinfected by soaking in 96% ethanol for 3 min, rinsed thoroughly with sterile distilled water and then disinfected in calcium hypochlorite solution (CaCl2O2 at 3.3%, w/v) for 3 min and finally rinsed thoroughly with sterile distilled water before sowing. These seeds were sown at a rate of 4 seeds per pot.

Culture substrate
The growing substrate was a sterilized soil of Ouagadougou and its physical and chemical characteristics are presented in Table 1. Culture substrate was homogenized, sieved with a 2 mm sieve and sterilized at 121°C for 1 h.

Test implementation
The test consisted of growing tomatoes in 2 L pots containing 2 kg of sterilized culture substrate (Table 1). The inoculation was carried out at the sowing time with 10 g of inocula [each inoculum constituted of spores, mycorrhizal root fragments and soil and was kept at room temperature (about 25°C)] (Haro et al., 2017) for each inoculated treatment. The inoculation consisted to place in the middle of each pot containing the culture substrate, 10 g of inoculum at 2 or 3 cm deep. Control pots were not inoculated. The experiment unit was composed of 3 treatments (two inoculated treatments (M1 and M2) and one control)] with 5 replicates per treatment. The tomato was sown at the rate of 4 seeds per pot and a wedge was carried out two weeks after the plants emergence to allow only one plant per pot. The experimental design used was a simple randomization complete block design. This experiment lasted 60 days (flowering stage).

Grow parameters measurement
To estimate the effect of mycorrhizal inoculation on the tomato, the height, the diameter at the collar, the rate of relative growth in height and the rate of relative growth of the diameter at the collar were calculated at the 30th and at the 60th day after sowing. The relative growth rate in height (RGRH) was calculated according to the following formula:
The collar diameter was measured using a caliper at the separation zone between the root system and the aerial part at the30th and at the 60th day after sowing. The relative growth rate of the collar diameter (TCRdc) was calculated by the following formula: With Dc = Diameter at the collar, I = initial and f = final. For the same parameter, data followed by the same letters are not significantly different according the Newman-Keuls test (p < 0.05). Standard error of the mean (n = 5). For the same parameter, data followed by the same letters are not significantly different according the Newman-Keuls test (p < 0.05). Standard error of the mean (n = 5). Height 1 and 2: Height measured respectively at 30 and 60 days after sowing. Diameter 1 and 2: diameter at the collar measured respectively at 30 and 60 days after sowing. NS: Not significant.

Shoot, root and total biomass measurement
At 60 days after sowing, each plant was carefully removed in order to recover the aerial part and all the roots of the plants. All these parts were dried in an oven at 70°C for 72 h for the measurement of shoot, root and total biomass. After the biomass measurement, the roots were used for the mycorrhizal infection study.

Staining for mycorrhizal colonization
About 10 g of roots from each treatment were thoroughly washed and placed in falcon tubes and then cleared using 10% KOH. They were heated in 90°C water bath for one hour. The roots were washed with tap water. Staining was then done by adding 0.05% trypan blue in lactic acid and heating in 90°C water bath for 30 min (Phillips and Hayman, 1970) and the observation was done under microscope (OLYMPUS BH-2) (magnification = 10x). The mycorrhizal frequency and intensity were estimated by Trouvelot et al. (1986) method.

Statistical analysis
Data were statistically analyzed using a one-way analysis of variance (ANOVA) with XLSTAT 2018 statistical software, and the means were compared using the Newman-Keuls test (p < 5%). Table 2 shows the frequency and intensity of mycorrhization of the tomato. The frequency of mycorrhization was quite high (86% for M1 inoculum and 82% for M2 inoculum) while the intensity of mycorrhization remains low (40.5% for M1 inoculum and 35.3% for M2 inoculum) ( Table 2). Statistical analyzes show significant differences between the different treatments. However, the roots of the control treatments are not mycorrhized ( Table 2). Table 3 presents the results of the measurements of the height, the relative growth rate in height, the collar diameter and the relative growth rate of the collar diameter of the tomato inoculated with native arbuscular mycorrhizal fungi. The results showed that the plant height varied according to the inoculum used and over time. At 30 days after sowing, the statistical analysis does not show any significant difference (P> 0.05) between the different treatments, neither for the height nor for the collar diameter (Table 3 and Figure 1). At 60 days after sowing, the differences appear between the different treatments for both the height and the collar diameter and the highest values were obtained for inoculation with M1 inoculum (35.7 cm for the height and 3.5 mm for the collar diameter). This inoculum improved the tomato height growth by 164.44% and that collar diameter by 75.25% compared to controls (Table 3). However, M2 inoculum did not improved tomato growth compared to the control (Table 3). Moreover, the statistical analysis did not show any significant difference (P> 0.05) between the different treatments either for the relative growth rate in height, or for the relative growth rate in the collar diameter. In general, the M1 inoculum improved the tomato growth (Table 3 and Figure 1).

Assessment of shoot, root and total biomass
The results on shoot, root and total biomass are presented in Table 4. These results varied according to the treatments. Statistical analysis showed significant differences between the different treatments. The highest values were obtained with M1 inoculum (Table 4). This inoculum improved the shoot biomass by 540%, the root biomass by 1061.97% and the total biomass by 638.1% compared to the control (Table 4). However, M2 inoculum did not improved tomato biomass production compared to the control (Table 4).

DISCUSSION
The objective of this study was to assess the effects of mycorrhizal inoculation with native strains from Burkina Faso on tomato growth. The mycorrhizal results showed that the roots of the tomato were highly colonized by the mycorrhizal strains used. The absence of mycorrhizal infection on the controls roots showed that this treatment was free from any mycorrhizal contamination. The growth and biomass stimulation between the different treatments and the control could be attributed to the effect of arbuscular mycorrhizal fungi inoculation.
The results on tomato growth show that inoculation improved the growth of this plant. Statistical analysis of plants height measured at 30 days after sowing did not show any significant difference between inoculated plants and control. This can be explained by the fact that the substrate used contained the necessary nutrients which were directly accessible to the plant's roots. These results are in agreement with those of Haro et al. (2012) who showed that the plant will not find a need to form the mycorrhizal symbiosis if the nutrients are available in the environment. This also explains the absence of significant differences in the rate of relative growth in height and the rate of relative growth in the collar diameter of the tomato. However, the statistical analysis on the height and the collar diameter of the plants measured at 60 days after sowing show significant differences between the different treatments. This can be explained by the fact that the growth of the plants over time has led to the depletion of the mineral elements directly accessible by their roots. Thus, the mycorrhizal symbiosis was established and developed gradually with the exhaustion of the mineral elements in the soil; hence For shoot, root and total biomass, M1 inoculum showed the best efficiency. As with the height and collar diameter measured at the 60th day after sowing, the effectiveness of the M1 inoculum on stimulating the production of biomass could be explained by the improvement of the tomato mineral nutrition by this inoculum. These results are in agreement with those of Ndoye et al. (2016) who showed that inoculation with G. verriculosum, G. manihotis and R. irregularis significantly improved the biomass of fonio plants. Similar results were found by Laminou Manzo et al., (2009) who showed the effectiveness of Glomus intraradices on the production of total biomass of Acacia raddiana, Acacia nilotica, Acacia senegal and Prosopis chilensis.
It appears that M1 was the most effective of all the inocula used in this study. This could be explained by the fact that M1 contains three AMF genera (Scutellospora sp., Gigaspora sp., Glomus sp.) whose effects can be added compared to M2 which has only one genus (Glomus sp.). Similar results were found by Haro et al. (2012) who showed that native strains containing at least 2 genera have effects that can add up compared to monospecific inocula.
The M2 inoculum did not improve either the tomato growth or the biomass production. This can be explained by the ineffectiveness of Glomus sp. in the tomato mineral nutrition improvement, which is justified by a host preference for endomycorrhizal fungi. Similar results were found by Haro et al. (2012) on cowpea.

Conclusion
From these results, it generally appears that mycorrhizal inoculation improves the growth and production of tomato biomass. This study showed that Burkina Faso native mycorrhizal strains can improve both the production of biomass and the growth in height and in the collar diameter. Although, it has shown promising results, this study deserves to be supplemented by in situ tests which would make it possible to assess the effect of this inoculation on the tomatoes production.