Mycorrhizal fungi transfer nitrogen from tree to maize in subsistence 1 farmers ’ fields 2


 Trees within farmers' fields can enhance systems' longer-term productivity e.g., via nutrient amelioration, which is indispensable to attain sustainable agroecosystems. While arbuscular mycorrhizal fungi (AMF) are known to improve plant access to soil nutrients, the potential of AMF to facilitate nutrient transfer from trees to crops is unclear. We used the 15N (nitrogen) natural abundance technique together with root and AMF exclusion plots to assess if Faidherbia albida (faidherbia) trees deliver N to maize via associated AMF in smallholder fields. We show, here, that within one cropping season, maize obtained approximately 35 kg biologically fixed N ha-1 from faidherbia and AMF significantly contribute to this transfer of N. One third of tree-derived N in maize leaves was attributed to transfer via AMF and two thirds were explained by tree leaf litter input. Thus, the faidherbia-AMF association can enhance agroecosystem functioning and as such, attain greater sustainability of low-input cropping systems.


Introduction 40
Food security is at continuous risk in sub-Saharan Africa where the majority of people live 41 from subsistence farming 1 . Traditional fallow practices have been abandoned due to 42 increasing land pressure and continuous maize cultivation has become the norm 2,3 . This has 43 resulted in soil degradation 4 and a negative feedback on food security. Ecologically sound 44 management practices are essential to attain food security. Agroforestry can provide a 45 framework for sustainable farming: trees distributed throughout farmers' fields can enhance 46 soil fertility via above-and belowground organic matter inputs [5][6][7][8][9] . Nutrient availability of 47 5 respectively) or distance from faidherbia (F4,94 = 0.91 and F4,94 = 1.47, respectively) and were 100 on average 0.16 ± 0.002 % and 5.74 ± 0.04 ‰, respectively ( Table 1). The proportion of tree-101 derived N in maize as a result of litter-, AMF-, and root-mediated processes did not differ 102 with distance from faidherbia trees (F4,27 = 1.24, F4,27 = 0.30, and F4,25 = 0.90, respectively; 103 Fig. 2). The effect of roots on tree-derived N in maize was negligible (Fig. 2). Biomass, yield, 104 N content and total tree-derived N in maize leaves on a per plot basis did not significantly 105 differ between plot types (F2,14 = 0.35, F2,14 = 0.86, F2,14 = 0.32, and F2,14 = 2.09, respectively; 106 Table 2). Total tree-derived N in maize grown within 5 m from faidherbia across all three 107 plot types summed up to approximately 35 ± 7 kg N ha -1 out of an estimated total N content 108 of 120 ± 7 kg N ha -1 thereby making up about 30 % of total N in maize. 109 110

Discussion 111
Capitalizing on agroecological processes such as those provided by the faidherbia-AMF 112 association in maize-based agroforestry systems in Malawi is essential to improve 113 agroecosystem functioning on smallholder farms. Our results confirm the importance of 114 faidherbia trees in improving the N budget in farmers' fields and further highlight that maize 115 obtains tree-derived N not only via faidherbia litter inputs but also through N transfer from 116 faidherbia to maize via mycorrhizal mycelia. Tree-to-maize N transfer via mycorrhizal 117 mycelia accounts for one third of the tree-derived N in maize leaves while two thirds of the 118 tree-derived N in maize leaves come from faidherbia litter inputs (Table 1, Fig. 2). 119 Consequently, AMF substantially contribute to making faidherbia-derived N inputs available 120 to maize. 121

Microdose N fertilization by Faidherbia albida 122
Incorporating N2-fixing trees in agroecosystems can benefit crop yields by providing high-123 quality above-and belowground organic matter inputs to the soil e.g. in the form of tree litter 124 6 input, root exudates, and root turn-over 5-7 . Faidherbia litter input alone can provide 50 to 80 125 kg N ha -1 to the soil under faidherbia trees within a given season 22 . However, how much of 126 this tree litter-derived N is effectively incorporated into the soil and subsequently 127 incorporated into crop biomass is unclear. The distinct isotopic N signature i.e. 15 N: 14 N ratio 128 of N2-fixing faidherbia allows distinguishing between biologically fixed tree-derived N from 129 residual soil N. Thus, the 15 N natural abundance technique allows tracing tree-derived N into 130 maize. Here, we demonstrate that in total, over the course of one season, tree-derived N 131 accounts for 35 kg N ha -1 in maize which makes up about 30 % of total N in maize. 132 Therefore, our results confirm the importance of faidherbia trees in improving the N budget 133 of crops in farmers' fields. The recommended rate of N fertilization in Malawi is 96 kg N ha -1 134 but on average only 18 kg N ha -1 are being applied by farmers 23,24 . Consequently, faidherbia 135 provides more than one third of the recommended fertilization and almost twice the amount 136 that is on the average applied by farmers. 137 Microdose fertilization has been shown to result in significant yield increase e.g. 138 microdose fertilization of 24 kg N ha -1 resulted in a 64 % increase in maize grain yield 139 relative to an unfertilized control 17 . We found yields within 5 m of faidherbia were 52 % 140 greater compared to yield away from faidherbia. Specifically, maize grain yield was 3.7 ± 0.4 141 t ha -1 under faidherbia compared to 2.5 ± 0.6 t ha -1 away (i.e., ~35 m ) from faidherbia. We 142 note that the yield obtained away from faidherbia was based on green cob dry weight while We conclude that faidherbia trees are effective in providing a microdose N fertilization to 150 maize in subsistence farmers' fields and have the potential to increase maize yields. 151

Arbuscular mycorrhizal fungi mediate tree-to-crop N transfer 152
Greater crop yields previously observed around N2-fixing trees within agricultural fields have 153 been mostly ascribed to high quality organic matter inputs to the soil 18-22 . The contribution of 154 AMF in making these inputs available to crops, specifically via interplant nutrient transfer 155 has gained much less attention. There has been some evidence that AMF transfer N from 156 trees to surrounding plants 11 but verification of this mechanism under field conditions on 157 smallholder farms has not been done. The combined use of the 15 N natural abundance 158 technique and root and AMF exclusion plots allowed us to disentangle the effect of AMF-159 facilitated transfer of tree-derived N from other belowground transfer mechanisms between 160 maize and trees (i.e., root-to-root contact and direct uptake of tree root exudates by maize 161 roots), and further quantify their respective magnitudes in farmers' fields. In our study 162 system, AMF-mediated N transfer accounted for 28 % of the total tree-derived N in maize 163 leaves within a 5-m radius around faidherbia (Table 2). Tree litter was responsible for most 164 of the tree-derived N in maize and tree roots had a negligible effect on the tree-derived N in 165 maize leaves (Fig. 2, Table 2). 166 The experimental plots were located under the tree crown (average crown diameter of 167 10 m) and therefore, it was expected that the proportion of tree-derived N obtained by maize 168 as a result of tree litter input was the same within the 5 m around faidherbia (Fig. 2). The 169 contribution of AMF-facilitated N transfer from tree to crops versus uptake via root-to-root 170 contact and direct uptake of tree root exudates by maize roots to the crops' N budget likely 171 depends on the tree root system architecture and distance from tree. We did not examine the 172 tree root system architecture, but observed no fine tree roots within a radius of 5 m from 173 faidherbia (at a depth of 0 to 50 cm). Even if maize roots usually grow deeper than 50 cm in 174 8 the absence of our experimental plots, the lack of fine tree roots within the top 50 cm 175 suggests that root-to-root contact between faidherbia and maize is typically minimal and 176 explains why we found no additional increase in tree-derived N obtained by maize grown in 177 the Litter&AMF&Roots plot relative to the Litter&AMF plot (Table 2). Further, the uniform 178 litter input and lack of fine tree roots within the area of the excavated plots explain why the 179 proportion of tree-derived N obtained by maize as a result of litter and tree roots, and 180 subsequently transfer via mycorrhizal mycelia did not change with distance from faidherbia 181 (Fig. 2). Given the apparent spatial separation of faidherbia and maize roots, our results 182 highlight the importance of mycorrhizal mycelia in bridging the space between the rooting 183 space of faidherbia and maize, for maize to obtain N from faidherbia that would otherwise 184 not be available. 185 Despite the contribution of AMF-facilitated N transfer to the proportion of tree-186 derived N obtained by maize leaves, maize biomass and yield were not significantly 187 increased by AMF (Table 2). This is probably linked to the fact that total foliar N content was 188 not affected by plot type i.e. type of interaction between tree and maize (Table 2). Total tree-189 derived N in maize leaves was also not significantly different between plot types but the data 190 follow the same trend as shown by the proportion of tree-derived N ( Table 2)

Tree selection 215
We focused our study on faidherbia (Faidherbia albida, Fabaceae) because it has been highly 216 promoted as an agroforestry species due to its "reverse phenology". The trees' foliage is shed interaction between tree and maize, and to control for the potential effect of the disturbance 235 caused by excavation during plot construction (Litter&AMF&Roots plot). Which plot type 236 was placed at 0°, 120°, and 240° around the tree varied for each tree to account for potential 237 differences in microclimate. Prior to excavating the plots, leaf litter that had accumulated on 238 the soil surface was removed. The excavated soil was piled up next to the plot and after the 239 lining had been put in place, the soil was placed back into the plot, moving from the top to 240 the bottom of the pile (roughly placing the soil back to the original depth). After completion 241 of the construction of plots, maize was sown along two lines throughout each plot and within 242 a 10-m radius circle around each tree. Farmers weeded, harvested, and eventually prepared 243 the fields for the next growing season, following their common practices (described above). 244 No measurements were taken in this year to let the system recover from the disturbance 245 caused by the plot installation. 246

Sample-collection (year 2) 247
At the beginning of the growing season of 2017/2018, maize was sown into each plot and 248 within a 10-m radius circle around each tree. In each plot, maize was sown along two lines 249 11 (0.6 m apart) at every meter, from 1 to 5 m from the base of the tree. Fields were weeded The proportion of tree-derived N (fracN(tree)) in maize leaves was determined on a per 260 distance basis (i.e. at each distance from the tree) using the following equation: For each tree, total biomass and mature cob fresh weight per plot were recorded in the 274 field and composited, homogenized subsamples per tree were oven-dried to determine dry 275 weights. Dry weight of each fraction i.e., leaves, stalks, grain, and cob per tree were 276 determined based on proportional weights obtained for maize previously 27 . Composited 277 maize stalk, grain, and cob subsamples were analyzed for total N and δ 15 N as described 278 above to determine N content of each fraction (i.e., stalks, grain, and cob) per tree and to 279 estimate total tree-derived N in maize grown within 5 m from faidherbia (see below). The 280 total tree-derived N (totalN(tree); [kg N ha -1 ]) in maize leaves was calculated on a per plot basis 281 as follows: 282 where Nmaize is the total N content in maize leaves [kg N ha -1 ], and fracN(tree) the proportion of 284 tree-derived N in maize leaves as calculated by equation (1). To obtain total tree-derived N in 285 maize on a per tree basis, we estimated total tree-derived N per fraction and subsequently, 286 determined the sum of total tree-derived N per fraction of all fractions (leaves, stalks, grain, 287 and cobs) combined. Hence, we adjusted equation (4) to have Nmaize and fracN(tree) represent 288 the total N content and the proportion of tree-derived N in maize stalk, grain, or cob to 289 determine total tree-derived N (totalN(tree)) in maize stalk, grain, or cob, respectively for each 290 tree. The proportion of tree-derived N (fracN)tree)) in maize stalk, grain, and cob on a per tree 291 basis were calculated using equation (1)  , AMF-, and root-mediated processes was assessed with linear mixed-effects models. The 308 Cook's distance measure was used to detect outliers. We obtained no significant interaction 309 of plot type and distance from faidherbia but in case the linear mixed-effects model revealed 310 significant main effects, we applied post hoc pairwise means comparisons using Tukey's test 311 to calculate least-squares means (function 'lsmeans' in package 'lsmeans'). 312 313