Abstract

Arbuscular mycorrhizal fungi (AMF) are beneficial for plant growth and development by facilitating the absorption of water and minerals from the soil. Symbiotic relationship between AMF fungi and plant roots is of paramount importance and could reduce crop production dependence on fertilizers. Nutrient deficiency in the soils of the western highlands of Cameroon especially N and P is a limiting factor to grain-filling in cereals. This study sought to investigate the effect of AMF combined with different levels of mineral fertilizer on the growth, root colonization and yield of wheat (Triticum aestivum). The experiment was conducted in a complete randomized block design arranged in split-plots using two varieties (Irad 1and Irad 2) and five treatments: control, F100%, AMF, AMF+F50% and AMF+F25% repeated three times. Results showed that all traits were significantly affected by treatments, except the plant height and spike length. Sole AMF treatment or in combination with low level fertilization (AMF+F25%) indicated better results for dry aboveground biomass, intensity of root colonization and yield. Contrarily, increased fertilization levels (F100% and AMF+F50%) reduced biomass, intensity of colonization and yield. No significant difference was observed between AMF and AMF+F25% treatments as well as between the two wheat varieties. These results suggest that wheat inoculation with AMF can reduce or simply replace the use of mineral fertilizer in wheat production in the western highlands of Cameroon.

1. Introduction

Wheat (Triticum aestivum L.) is a cereal crop of the family Poaceae, and amongst the most cultivated in the world in terms of area and production after rice ¹. It is a staple food of nearly 70% of the world's population that is highly consumed in a variety of food products (bread, cookies, porridge, couscous…), animal feeding and biofuel production ^{2, 3, 4}. Increasing population in many African countries has resulted in an increase in wheat consumption as a staple crop in the past twenty years and such a move has been associated with changes in food preferences with urbanization ⁵. The world production is estimated at 734 million tonnes ⁶ and production in Africa is relatively low (26 million tonnes) with Egypt being the leading producer of 9.4 million tonnes ⁶.

In Cameroon, wheat production was estimated in 2019 to be 900 tonnes annually, highly inferior to the country’s demand. In the same year the country imported 955 thousand metric tonnes with an estimated import value of 185,914.00 US Dollar to fully satisfy the demand ⁷. This suggests a high need to improve wheat production in the country. Based on the following, recommendations have been made to Government and non-governmental organizations to support and promote local wheat production in Cameroon ⁷.

The Agroecology of Cameroon is highly diversified with five different zones: Sudano-Sahel, Guinea Savanna, Bi-modal Equatorial, Mono-modal Equatorial and Western Highland. This agro-ecological diversity indicates the possibility to produce major crops grown in other African countries including wheat. ⁸ showed that prevailing climatic conditions in Cameroon is favorable zones for wheat cultivation. Generally, one of the limiting factors regulating the productivity of grain crops is availability of plant nutrients in soil. Particularly in acidic soils, phosphorus uptake is limited due to its tendency to complex with cations such as iron and aluminum rendering it unavailable to the plant ⁹. Phosphorus and nitrogen deficiency due to unavailability deficiency is one of the main limiting factors in agricultural production ¹⁰, and in the case of wheat cultivation, this deficiency leads to poor grain filling and consequently yield reduction ¹¹. Grain-filling is the main constraint of wheat production in the western highlands of Cameroon. The application of nitrogen and phosphorus based fertilizers could be considered an efficient approach to improve grain-filling thereby improving the yield of cereals in low-nutrient soils. However, the high cost of these fertilizers makes them not affordable to poor resource farmers ¹². Also, its production requires high amounts of non-renewable energy resulting in greenhouse gases release in the atmosphere ¹³. In addition, P efficiency use is low due to its fixation capacity in the soils ⁹. In these situations, effective agronomic management is important, particularly, the management of soil microbiota including arbuscular mycorrhizal fungi (AMF) is an option to increase the availability and utilization of soil-derived nutrients ¹⁴.

AMF are soil microorganisms that belong to the phylum Glomeromycota and form symbiotic association with the roots of over 80% vascular plant species ¹⁵. They are ubiquitous in soils around the globe and have been associated with improve plant growth for over 100 years through improved mineral absorption ^{16, 17}. Many studies have reported that AMF was able to improve the absorption of minerals such as phosphorus, nitrogen and zinc ¹⁸. In addition, AMF improves plant resistance to biotic and abiotic stresses ¹⁹. Inoculation with AMF can supply up to 90% of plant P and 20% of plant N due to AMF extraradical hyphal network which ensures the intensive exploration of large soil volume ^{19, 20}. The degree of plant benefits from AMF inoculation depends, among others factors, on the nutrient conditions in the soil, especially the availability of nitrogen and phosphorus, which are considered the most restrictive nutrients for plants growth ²¹. These two elements negatively affect AMF root colonization when they are present at high levels in the soil. The high P–fertilizer application decreases the supply of soluble carbohydrate in roots and hence reduces the appresorium formation and fresh infection ²². Also, the arbuscle formation and active P transfer to plants is reduced in high P content in soils ²³. High applications of N fertilizer reduces AMF colonization in plants with mainly hyphal, vesicles and spore colonization only, though colonization intensity is low. However, the highest mycorrhizal efficiency was observed when the soil contained between 7.8 and 25 mg kg^-1 of P ²⁴. In addition, low level of nitrogen increases the AMF colonization, plant growth and root formation ²⁵.

The objective of the present study was to investigate the effect of AMF inoculation combined with different levels of N-P fertilizers on the growth and yield parameters of Wheat. The proposed study envisaged to test the hypothesis that combining AMF with mineral fertilizers would result in reducing fertilizer application and thus improve the profitability of farmers in subsistence cropping systems.

2. Materials and Methods

The experiment was conducted at the Research and Application Farm of the Faculty of Agronomy and Agricultural Sciences of the University of Dschang, West region of Cameroon. The farm is located in the Western Highlands Agro-ecological Zone (Latitude 5.5° N, Longitude 10.05° E and Altitude of 1,410m) characterized by a rainy season that runs from mid-March to mid-November and a dry season that runs from mid-November to mid-March. The evolution of meteorological data recorded during the experiment (March 6th to July 17th 2020) are presented on Figure 1. The soil in the study site is ferralitic with chemical properties (0-20 cm layer) as follows: pH (H₂O) 5.5; carbon (%) 2.61; nitrogen (g.kg^-1) 0.46 and P Bray II (ppm) 2.66.

Seeds of two wheat (T. aestivum) cultivars, Irad 1 and Irad 2, obtained from the Institute of Agricultural Research for Development (IRAD) Nkolbisson, Centre Region, Cameroon, were chosen based on their adaptation at high altitude (western highlands).

The biofertilizer (AMF inoculum) consisted of sandy soil, spores, hyphae and infected root segments of fungal genera Glomus, Gigaspora and Acaulospora at 200 propagules g^-1 of inoculum obtained from GIC AGRIBIOCAM (Agriculture Biologique du Cameroun). Mineral fertilizers used in this study were urea (46% N) and NPK (20-10-10).

A randomized complete block design arranged in split-plot with three repetitions including two cultivars (Irad 1 and Irad 2) as the main plots, and five treatments (Control, F100%, AMF, AMF+F50% and AMF+F25%) as the sub-plots, was adopted. F100% was the recommended fertilization for optimum wheat growth and development. Treatments are defined on Table 1.

Two seeds hole^-1 were manually sown at the depth of 2-3 cm. The planting distance was 25 cm between the lines and 15cm between plants, giving a planting density of 266667 plants ha^-1. At sowing, six grams of the AMF inoculum (1200 propagules) were placed in each planting hole. Split application was done for mineral fertilizers, the first, NPK (20-10-10) two weeks after sowing and the second, urea, at flowering, in the appropriate dose each. Weeds were controlled manually during the experiment.

Growth parameters were collected 8 weeks after planting (WAP) on eight randomly chosen plants. Data were collected on plant height, number of tillers, dry aboveground biomass and AMF root colonization. At harvest (20WAP), the number and the length of spikes were measured. The seeds from each plant were collected, counted and weighed to determine the dry weight per plant and of 1000 seeds. Yield was calculated using the formula:

(1)

AMF root colonization was assessed on roots from five randomly selected plants in the middle of each plot. Root colonization was determined on root subsamples. Roots were cut into 1-2 cm pieces, stored in 10% KOH overnight at room temperature. They were washed several times with de-ionized water and soaked in alkaline 3. 5 % H₂O₂ for 30 minutes ²⁶. The roots were subsequently stained at room temperature for 45 minutes with a solution of blue ink (Parker^P Quink®) diluted in 1 % HCl at 1:50 proportions ²⁷. Thirty root fragments (1 cm long) of each root subsample (from 5 plants) per treatment were considered to estimate treatment means. For each fragment, the abundance and diversity of the mycorrhizal structures (Arbuscules, vesicles, intracellular hyphae, auxiliary cells and spores) were noted. The frequency (F%: the percentage of root fragments that contains either hyphae, arbuscules or vesicles/spore) and the intensity (I%: the abundance of hyphae, arbuscules or vesicles/spores in the root system) of the mycorrhization were evaluated as described by ²⁸.

All data collected were subjected to analysis of variance (ANOVA) using the R software. Differences between treatments were separated using Fisher's least significant difference (LSD) at a probability threshold of 5%. Pearson’s correlation analysis was performed for growth and yield parameters in SPSS version 21.

3. Results and Discussion

Results showed that wheat growth represented by the number of tillers and dry aboveground biomass indicated significant differences between the varieties, treatments and interactions (variety x treatment) (Table 2). The plant height was not significantly affected by treatments and varieties. The assessment of plant height was done probably before the complete establishment of the symbiosis and thus could reduce the possibility of detecting significant differences among treatments. ²⁹ observed that the development of AMF symbiosis on upland rice in the field started 3 weeks after sowing. Moreover, plant height may be a genetically-mediated character. In addition, similar results were obtained on ryegrass ³⁰. However, the interaction between variety and treatment was significant (P˂0.05). The maximum plant height (93.85cm) was observed in Irad 1 for the treatment AMF+F50% and the minimum heights (72.75cm and 77.76cm) for the control and AMF+25% treatments of Irad 2, respectively. The number of tillers per plant was significantly affected by the fertilizer treatments. The maximum number of tillers was observed on AMF (15) and AMF+F25% (15) of Irad 1. The same trend was observed for Irad 2 with 19 and 16 tillers for AMF and AMF+F25% treatments, respectively. Increased mineral fertilizer application in AMF inoculated plants (AMF + F50%) reduced the number of tillers to 13 and 15 for Irad 1 and Irad 2, respectively as compared to AMF and AMF+F25% (Table 2). However no significant difference was observed between the AMF+F50% and F100% treatments. The lowest number of tillers was obtained in the control treatments independent for the variety.

The dry aboveground biomass followed the same trend with the highest biomass obtained in AMF (204.77g and 347g) and AMF+F25% (204 77g and 349g) for Irad 1 and Irad 2, respectively. Similarly, the lowest biomass was obtained in the control treatments for both varieties. Thus, AMF inoculation or inoculation with 25% fertilizer significantly improves wheat (Irad 1 and Irad 2) growth in terms of number of tiller and dry aboveground biomass.

Vegetative growth mostly relies on N availability and many scientists have reported the role of AMF in nutrient uptake from the soil nutrients, especially N, which can effectively promote the growth of the host plant ³¹. In addition, the number of tillers and dry aboveground biomass were strongly correlated to the intensity of root colonization suggesting that the extra radical mycelium formed after the establishment of intraradical structures (especially arbuscules) can effectively improve nutrient uptake, thereby improving plant growth and development ³². The results are in agreement with those of ³³ who showed that the root colonization levels were positively correlated to the growth of tomato. The increase in fertilization level improves the soil nutrient availability and the plant can directly absorb enough nutrients to reduce its dependency on AMF symbiosis, resulting to lower plant growth. Similar results were also observed on maize ³⁴.

Mycorrhizal colonization was observed in both AMF inoculated and non-inoculated plants whatever the fertilization level. The colonization frequency in wheat roots is significantly influenced by applied fertilization for the two cultivars (Figure 2). In the non-inoculated treatments (control and F100%), the values were considered high and varied from 63% to 77% suggesting the occurrence and the acceptance potential of the soil native strains of AMF of the two cultivars. This is due to the ubiquitous character of AMF which can form symbiotic association with the roots of over 80% of terrestrial plants ¹⁵. But the frequency of mycorrhizal colonization was significantly high in the inoculated plants.

The highest frequency was observed in AMF treatment (96% and 86% Irad 1 and Irad 2, respectively) and AMF+F25% (90% and 86% for Irad 1 and Irad2, respectively). High fertilization level (F100% and AMF +F50%) had a significant negative impact on the frequency of root colonization especially for Irad 2 compared to low fertilization level (AMF and AMF+F25%). Unfortunately, the frequency of root colonization is not the efficient variable for mycorrhizal colonization evaluation. The intensity of colonization represents the entire transport network that will provide the potential quantities of nutrients available to the plant. When root cells are penetrated by hyphae, they branch and form arbuscules, which improve the nutrient exchange between partners ³⁵. Root infection intensity was significantly influenced by treatments (Figure 3).

AMF inoculation alone (AMF) or combined with 25% fertilizer (AMF+F25%) increased significantly the intensity of root colonization by 30% over the control in both varieties. AMF recorded 36% and 31% for Irad 1 and Irad 2, respectively while AMF+F25% showed 36% in both varieties. Increasing the level of fertilizer from 25 to 50% (AMF+F50%) reduced the colonization intensity (from 30% to 12%) compared to AMF and AMF +F25%. This is in agreement with the findings of ³⁶ for durum wheat under field condition who found a reduction in mycorrhizal root colonization in plants grown in nutrient-rich soils. Excessive fertilization significantly reduced the AMF colonization due to the higher nutrient availability in the soil after the chemical addition ³⁷.

In this condition the plant is better able to satisfy its nutritional needs without having to transfer photosynthates to the mycorrhizae and thus reduce the AMF colonization ³⁸.

Contrarily, in low fertilizer conditions, the plant is stimulated to develop a symbiotic relationship to increase its chances of intercepting more nutrients. However no significant difference was observed between Control, F100% and AMF+F50% and between the cultivars.

Data on yield components (number of spikes per plant, spike length and number of grains per plant) are shown on Table 3. The number of spikes per plant contributes directly towards yield. Results indicated that the highest number of spikes were obtained in AMF (10 and 12) and AMF+F25% (10 and 11) treatments for Irad 1 and Irad 2, respectively (Table 3). Increasing the fertilizer level from 25% to 50% and 100% result to significantly decrease in the number of spikes per plant for both varieties. Also, the spike length showed a similar trend. Data regarding the number of grains per plant indicated that all the mycorrhizal inoculation treatments had the highest number of grains per plant (630, 586 and 551 for AMF, AMF+F50% and AMF+F 25%, respectively) for Irad 1. There was a slight difference for Irad 2 with 776 grains per plant for the obtained AFM+F25% treatment (Table 3).

Analysis of variance revealed that grain weight per plant, the weight of 1000 grains and yield were significantly enhanced over the control treatment for both cultivars (Table 4). Grain weight per plant and the weight of 1000 grains showed the highest values in AMF and AMF+F25% and the lowest in the control treatments. The yield in F100% (3.11t. ha^-1 and 3.24t. ha^-1 for Irad 1 and Irad 2, respectively) was significantly similar to that of AMF+F50% (3.29t. ha^-1 and 3.47t. ha^-1 for Irad 1 and Irad 2, respectively). The AMF and AMF+F25% treatments significantly produced higher yield over control treatments. The AMF produced grain yield of 5.82t. ha^-1 and 5.17 t. ha^-1 for Irad 1 and Irad 2, respectively while AFM+F25% yielded 5.64t. ha^-1 and 5.33t. ha^-1 grain for Irad 1 and Irad 2, respectively. The lowest yield was obtained in the control treatments for both cultivars.

The percentage increase produced by AMF alone was 150% and 125% over the control for Irad 1 and Irad 2, respectively. AMF associated with 25% fertilizer (AMF+F25%) increased yield by 144% and 132% over the control for Irad 1 and Irad 2, respectively. There was a significant interaction effect (P<0.05) between variety and treatment on grain weight per plant, 1000grains weight and yield (Table 4). Results showed that AMF inoculation alone or combined with 25% mineral fertilizer improve wheat grain yield by at least 100%. Thus, limiting chemical sources of nutrients stimulates plants in the development of a highly permissive root system toward symbionts and the more efficient exploitation of soil resources ³⁹. Moreover, the application of F100% reduced grain yield by 80% over AMF treatment. Interestingly, no significant difference was observed between these treatments suggesting the possible replacement of mineral fertilizer by AMF inoculation. Many authors, demonstrated that increased photosynthetic activities and other leaf functions are directly related to improved growth frequency of AMF inoculation that is directly linked to the uptake of N, P, and carbon, which could promote the development of spikes. ⁴⁰ have demonstrated AMF-mediated increased allocation of shoot biomass to spikes and grains through increased N and P redistribution to spike particularly under low fertilizer levels. N and P are considered nutrients mediating grain filling and yield increase of cereals in low nutrient soils. Plants colonized with AMF are able to produce extensive underground extraradical mycelia ranging from the roots up to the surrounding rhizosphere, thereby helping in improving the uptake of nutrients specifically N and P ⁴¹. Results obtained in this study corroborates that obtained in the inoculation of maize, durum wheat and tomato ^{34, 36, 39}.

Pearson’s correlation analysis showed significant correlations among the variables studied (Table 5). The number of spikes per plant showed significant positive correlation with number of tillers per plant (0.984^**). The aboveground biomass was positively correlated with number of tillers (0.837^**) and number of spikes (0.868^**) at high level of significance. Grain yield had highly significant positive correlation with number of tillers (0.76^*), number of spikes (0.73^*), aboveground biomass (0.65^*) but had highly significant positive correlation with spike length (0.82^**), grain weight per plant (1.00^**) and thousand grain weight (0.97^**). These results were in agreement with ⁴². For arbuscular root colonization, the results showed that the intensity root colonization was significantly correlated with plant growth parameters and highly correlated with yield and yield component (Table 5). These findings are supported by the other scientists of the world. The development of plant growth and the increment of yield are linked to the increased uptake of minerals especially N and P, and other beneficial traits helping extraradical mycelium exchange nutrients toward roots ^{33, 34}.

4. Conclusion

The present study showed that the AMF-inoculation alone or with 75% reduction of the recommended quantity of mineral fertilizer has the potential to improve the growth, AMF root colonization and grain yield of wheat in the western highlands Cameroon. AMF and AMF combined with 25% recommended mineral fertilizer gave significantly the same grain yield, suggesting that AMF can replace mineral fertilizer in wheat production at the level of the field. Consequently, the AMF inoculation could be used in wheat production in the Western Highlands of Cameroon to reduce mineral fertilization applications thereby reducing production cost and limiting environmental issues associated with chemical fertilizers. This will in return reduce the cost of farm inputs while enhancing wheat production through optimization of biological resources. Thus, it could be used as an eco-friendly approach to improve wheat production in the country and for the enhancement of global food production.

Acknowledgements

The authors are grateful for material and scientific contributions of members of the Genetics, Biotechnology, Agriculture and Plant Physiology Research Unit of the University of Dschang. Other support not mentioned are equally appreciated.

References