Development of a Technical Itinerary for the Production of Avocado (Persea américana Mill.) Seedlings with Biofertilizers ()
1. Introduction
Avocado (Persea americana Mill.) is a plant of Lauraceae family. Its fruit is considered to be the world’s most nutritive fruit [1]. It is also considered to be the world’s most energy value fruit [2]. This plant is a tropical species that adapts perfectly to subtropical climates with mild and tropical winters [3]. Mexico, with a production of 2,300,889 tonnes is the world’s leading producer of avocado, followed by the Dominican Republic (661,626 tonnes) and Peru (535,911 tonnes). Kenya is the leading African producer with a production of 364,935 tonnes (FAOSTAT, 2019). Avocado imports in 2020 grew by 6.9% to around 2.3 million tonnes. The main importing countries, the United States and the European Union, absorbed some 48% and 25% of world exports respectively (FAOSTAT, 2020).
Cameroon, which has all the assets for the massive production of fruit trees, is not a competitor on the world market. The development of orchards is limited by the lack of quality seedlings. Supply of agricultural inputs is not appropriate. The regulations, even if they exist on paper, are not observed in most cases. There are no manufactured substrates for agriculture on the market as is the case in developed countries. The production of avocado seedlings is done in most cases according to a production itinerary that includes a germination phase in the germinator followed by transplantation in the nursery. The substrates used in the germinator are soil, sawdust and sand.
The role of the substrate in the germination is documented. Some results are contradictory. For example, wood sawdust is reported to negatively affect the germination of Sunflower (Helianthus annus L.) [4] while Singh et al. [5] state the contrary. Among the soil microbial communities, mycorrhizal fungi are a “key” component in plant-soil relationships. These fungi, present in the soils of most ecosystems, form symbiotic associations with the roots of many terrestrial plant species (about 80%) [6] [7]. They play a major role in the amelioration of soil fertility and organic agriculture [8]. In exchange for the carbon resources received from the host plant, mycorrhizal fungi improve plant nutrient uptake and transport (mainly phosphorus) with very low mobility [9]. Biological fertilizers are commonly used in seedling or crop production processes [1] [10] [11]. The sowing date is known to influence the yield of plant such as onion [12] [13] [14] [15], rice [16] [17], strawberry [18] and fennel [19].
In the tropics, biotic and abiotic pressures are such that successful orchard establishment requires very vigorous seedlings at planting. The technical itineraries used in the production of avocado plants are not scientifically documented. This study aims to optimise the use of these materials to produce vigorous avocado plants from biofertilizers. This objective is realised by identifying the best substrate for the germinator, the most appropriate transplanting date and the biological fertilisation in the nursery that could guarantee the production of the most vigorous avocado seedlings. The three specific objective of the study are to: 1) Evaluate the effect of different substrates currently used by seedling producers of the region on germination; 2) Determine the best stage for transplanting germinated seeds from the germinator to the nursery; and 3) Measure the effect of different biofertilizers on growth in the nursery.
2. Materials
2.1. Study Site
The experiment was conducted in Yaounde in the Centre region, Mfoundi administrative Division, more precisely in the locality of Simbock (Latitude: 3˚49'13.76'' Longitude 11˚28'13.52'', Altitude: 694 m. The average annual rainfall varies between 1500 and 2000 mm/year. The average annual temperature is between 23˚C and 27˚C, and the relative humidity and average humidity are above 80%.
2.2. Biological Material
For plant material, we used: Avocado seeds consisting of an accession of the species Persea Americana obtained from vendors in the town of Mbouda in western Cameroon.
2.3. Organic Fertilisers and Mycorrhizae Strains
Two types of fertiliser were used in the study: 1) organic fertilizer and 2) biological fertilizer. The Aburcular Mycorrhizae Fungi (AMF) species used in this study are Gigaspora margarita and Acaulospora tuberculata.
2.4. Experimental Set-Up
In a germinator: The experimental set-up chosen was the randomised complete bloc design with two repetitions. The studied factor is the substrate with three modalities (soil, white wood sawdust and sand), each consisting of 100 seeds, with a total of 600 seeds (Figure 1).
In the nursery: The experimental design used is the split-plot factorial design with two factors and three repetitions including transplanting date (TransD/ main factor) and biological fertilisation (Fert/secondary factor). Transplanting date included: Trans40 (40 days after sowing), Trans 65 (65 days after sowing) and Trans75 (75 days after sowing) and fertilisation Fert1: control (soil + sand + fertilizer treatment); Fert2 (T1 + Gigaspora margarita); Fert3 (T1 + Acaulospora tuberculate and Fert4 (T1 + Gigaspora margarita and Acaulospora tuberculate, that is Fert2 + Fert3). Three repetitions of 10 plants/repetition, giving (3 × 4 × 10) × 3 plants; that is 360 plants in total (Figure 2).
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Figure 1. Germination of avocado seeds on different substrates. (a) Sawdust, (b) Soil and (c) Sand.
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Figure 2. Seedlings transplantation at transplanting date 40 (TransD40). (a) Sowing; (b) Nursery.
3. Methods
3.1. Preparation of the Substrate
The substrate used for the germplasm experiment in each compartment consisted of three basic elements that were used to prepare the substrates. These were: the S1 substrate made up of fine sand; the second substrate S2 consisted of black humus soil. The third, S3 substrate was white wood sawdust. The soil was taken from the locality of Etoudi in the city of Yaounde to a depth of 15 cm and then sieved. The wood sawdust was taken from a saw mill and the sand from a sand deposit and then sieved to eliminate residues. The different tubs of the germinator were filled with each of the substrates.
3.2. Preparation of the Grains
After removing the seeds from the fruits, they were cleaned to remove the fruit tissue and then washed with tap water before disinfection by soaking in a solution containing a mixture of insecticide (Cigogne 360 EC, pyrethroid insecticide; 50 ml/15L of water); and fungicide (Mancomax bleu 800 WP 80 g/15L of water) for 15 minutes.
3.3. Maintenance of the Germinator
The germinator was established in a sunny place, sheltered from the wind. The maintenance of the germinator consisted in watering every day in the evening or very early in the morning, with a watering can to maintain moisture in the substrate; and removing weeds that compete with the plants. Once the seeds germinated, they were transplanted.
3.4. Setting up the Trial in the Nursery
3.4.1. Preparation of the Substrate
The substrate used for the experiment was a mixture of black soil and fine sand and fowl droppings. The loam soil used was dried, sieved to obtain uniform particle sizes and mixed with sieved fine sand and fowl droppings in proportions of 1/2, 1/4 and 1/4 respectively to obtain good drainage, permeability, as well as better water retention capacity. The characteristics of this soil are indicated in Table 1.
3.4.2. Filling the Bags
The 70 micron bags (30 by 21 cm) were filled completely so that the walls of the bag could not be folded over the free surface of the substrate.
3.4.3. Transplanting
Once the seeds have germinated in the germinator containing only white wood sawdust as substrate, the seedlings were extracted from the germinator and transplanted. The mycorrhizae were applied to the roots of the seedlings (by coating them) before transplanting into the bags, at 2/3 depth in the substrate.
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Table 1. Physical and chemical properties of the soil used for the experiments.
3.5. Measurement of Agronomic Parameters
3.5.1. Evaluation of the Effect of the Substrate on Germination in the Germinator
On each block of the germinator containing the different substrates (black soil; sand and white wood sawdust). Three parameters for this evaluation were recorded including the germination rate (GR, in percentage of seeds), the daily average germination (DAG) and the root volume (RooTV) of avocado seeds sown on the different substrates. Germination Rate (GR) indicates the number of seeds that are likely to germinate in a given period (Germination power). It is given by GR = Number of germinated seeds/Total number of seeds sown*100.
The average daily germination rate (DAG) was calculated according to Osborne and Mercer [20]. DAG = Final germination percentage/Number of days to final germination. Ratings were recorded every four days over a period of 65 days, for a total of 11 ratings.
Root volume (RootV) was measured with a measuring cylinder, by dipping all the roots from each substrate separately in a graduated cylinder containing water. The volume of water displaced gives the root volume. Root volume = Final volume of water displaced − Initial volume of water. A total of 20 × 3 seedlings were used for this evaluation, 20 seedlings from each substrate.
3.5.2. Evaluation of Different Transplanting Dates (TransD) and Fertilisations (Fert) on the Growth of Seedlings in the Nursery
The two factors evaluated here are transplanting dates (TransD40; TransD60; TransD75) and the biological fertilisation applied. Fert1 (10 gr 20-10-10 plus 10 gr chicken droppings), Fert2 (Acaulospora tuberculata), Fert3 (Gigaspora margarita) and Fert4 (Mixed mycorrhizal strains of Gigaspora margarita and Acaulospora tuberculata) were analysed for growth in the nursery. Four variables were used for their evaluation: the gain in plant height (GainPltH) and diameter (GainPltD), the leaf area (LeafA) and the chlorophyll index (ChlorInd). The GainPltH and GainPltD were calculated as follows:
Gain = Final data − Initial data/Initial data. The gain was calculated because the plants were not of the same size at transplanting. The sizes of the seedlings were measured with a tape at one-week intervals.
The leaf area (LeafA) of the seedlings was obtained by measuring length and width with a tape measure and calculating using the formula:
S = 2/3(L + l);
S: leaf area, L: length and l: width.
The chlorophyll index of the different plants was measured with a Chlorophyll meter (SPAD 502plus) at the 11th week.
3.6. Root Colonisation in Relation to the Different Treatments
3.6.1. Root Isolation
In order to verify the effectiveness of mycorhyzae colonisation in each treatments, 10 plants were selected randomly and the soil was crumbled to obtain roots. The roots obtained (the finest) were cut into fragments of 1 to 2 centimetres in length.
3.6.2. Root Staining and Observation for Mycorrhizae Colonisation
Root staining was done according to the modified Grace and Stribley [21] method. Arbuscules and vesicles of mycorrhizae were observed using 10×, 20× and 40× objectives of a light microscope.
3.7. Statistical Analysis
The collected data were subjected to analysis using the SPSS version 20 software and the means were separated with the Least Significant Difference (LSD) at 5% threshold. The graphical representations of the data were made with Microsoft Excel 2016. The model used is a univariate general linear model whose dependent factors are the treatment and the date of transplanting of the seedlings. The relationship between growth parameters and chlorophyll index was established using Pearson correlation coefficient at 5%.
4. Results and Discussion
4.1. Results
4.1.1. Substrates versus Germination Rate
Seedling germination started on the 10th day after sowing and reached its maximum between the 40 - 50th day. This is illustrated on Figure 3 on which the sigmoid shape of the germination can be observed. Results here indicate that there is a difference between germination rates of seedlings on different types of substrates. The germination rate is lower in sand (83.64%) than on soil (98%) and wood sawdust (100%) after 65 days of germination.
4.1.2. Substrates versus Daily Average Germination (DAG) and Root Volume (RootV)
Results indicated on Table 2 and Table 3 reveal a significant effect of substrate (p = 4.80E−02) over daily average germination (DAG) and root volume of
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Figure 3. Evolution of the germination of avocado seeds on soil, sawdust and sand.
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Table 2. ANOVA of impact of substrate on Daily Average Germination (DAG).
seedling (RootV; p = 5.00E−03) (Figure 4). This is illustrated on Figure 4 by a gain of 8 days (43 - 35) when seeds are germinated on wood sawdust or soil in comparison with those germinated on sand. This results to a gain of 49.42% of root volume on wood sawdust over sand, and 19.53% of wood sawdust over soil (Figure 5 and Table 4).
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Figure 4. Root volume of seedlings from different substrates (in cubic millimetres).
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Figure 5. Daily average germination (DAG) of avocado seeds on soil, sawdust and sand. from the 11th to the 65th day after sowing.
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Table 3. ANOVA of impact of substrate on Root Volume (RootV) of seedlings.
R Squared = 0.72 (Adjusted R Squared = 0.65).
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Table 4. Multiple comparison of substrate effect on Root Volume (RootV) based on LSD indicating a differenceof root volume in relation with type of substrate. Sawdust versus sand (p= 0.001) and sand versus soil (p= 0.042).
Based on observed means. The error term is Mean Square (Error) = 7.607*. The mean difference is significant at the 0.05 level.
4.1.3. Transplanting Date and Biological Fertilisation versus Growth Parameters
The combined effects of the transplanting dates and the biological fertilisation used are indicated on Table 5. It can be seen from Table 5 that the transplanting date has a very significant influence on studied parameters. Gain in plant height (GainPltH; p < 0.0001), Gain in plant diameter (GainPltD; p < 0.050), Leaf area (LeafA); p < 0.0001) and Chlorophyll index (ChlorInd; (p = 0.021). Fertilisation had a significant effect on leaf area (SurtF) (p = 0.013). No significant interactions were detected. The separation of the means between the different transplanting dates is indicated in the Table 6. Transplanting dates have a very significant influence on seedling growth parameters. The 40-day duration was the one that resulted to the most vigorous plants appropriate for grafting.
4.1.4. Fertlisation versus Growth Parameters
A significant fertilisation effect was detected on the leaf area variable (Table 5). Biofertilisers reduced the leaf area from 5% - 10% compared to the treatment with manure (Table 7).
4.1.5. Verification of the Effectiveness of Avocado Seedling Symbiosis
The presence of hyphae and vesicles was observed in the plants of treatments Fert2, 3 and 4, in contrast to the roots of the plants of treatment Fert1 where no mycorrhizal symbiosis was observed under the microscope (Figure 6).
4.2. Discussion
The development of research on the establishment of a technical itinerary is of primary interest in agronomy. The results obtained from this work have made it possible to appreciate the effect of different substrates, transplanting dates and biofertilisers on the nursery growth of avocado plants. Different substrates used for the germination of P. americana show different behaviours, as a function of
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Table 5. ANOVA of plant growth parameter Gain in plant high (GainPltH), Gain in plant diameter (GainPltD), Leaves area (LeafA), and Chlorophyll index (ChlorInd).
aR Squared = 0.67 (Adjusted R Squared = 0.29); bR Squared = 0.90 (Adjusted R Squared = 0.79); cR Squared = 0.80 (Adjusted R Squared = 0.56); dR Squared = 0.59 (Adjusted R Squared = 0.12).
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Table 6. Multiple comparisons of transplanting date on chlorophyll index (ChlorIndex), leaf Artea (LeafA), Gain in Plant Height (GainPltH) and Plant Diameter (GainPltD).
**Correlation is significant at the 0.01 level (2-tailed).
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Table 7. Influence of fertilisation on leave surface (Leaf A).
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Figure 6. Verification of the effectiveness of avocado seedling symbiosis with non mycorhyzed (a) and mycorhyzed seedlings (b).
their varying structures and compositions. A higher germination rate (100%) was obtained in the wood sawdust growth substrate, followed by soil (98%) and finally sand substrate (92%).
The 65-day mean germination rate was higher in the wood sawdust (1.79), followed by soil (1.72) and finally sand (1.28). This difference in germinative behaviour of avocado seeds shows that wood sawdust by its ability to retain moisture and its mineral composition, favours the development of avocado seeds. As regards the soil and sand substrates, these results corroborate those of Sounon et al. [22] who worked on the best substrate for nurseries and showed that the ferralitic substrate presented the best results on the growth of Artemisia annua plants compared to the sand substrate. The wood sawdust contains moisture and mineral elements [23] while sand is siliceous in nature, with granules that are hard and heavy (http://umc.edu.dz, 2015); it is therefore assumed that these characteristics can cause dehydration of the seeds by preventing the passage of oxygen and water to the seeds. Similarly, the relatively low root volume (5.20) and higher in wood sawdust (7.77) which is the best substrate, could explain the high germination rate in wood sawdust, as a large root volume would would favour the development of the plants and therefore their germination thanks to the wood sawdust with available water and mineral elements. In an unconstrained environment, a few roots can be sufficient to meet the plant’s water and nutrient requirements [24].
The variation in transplanting dates of avocado seedlings significantly influenced the plant’s gain in height, diameter, leaf area of the seedlings and the chlorophyll index. At the end of our experiment, we observed that the TransD40 transplanting stage had the best results in terms of leaf area and chlorophyll index than TransD65 and TransD75, which had better results in terms of plant height and diameter gain; this could be due to the fact that the late transplanted plants required a longer adaptation phase due to the trauma suffered by the roots. It is noted that the white sawdust substrate used in the germinator is rich in mineral elements with a high humidity and oxygenation rate, which are favourable to root development. These results corroborate those of Satapathy et al. [25], Tahir et al. [26] and Goita et al. [16] who found that paddy yield decreased with delaying the transplanting time.
Plenchette and Morel [27] stated that whether plants are mycorrhized or not, they all feed from the same phosphorus (P) pool since the ions dissolve in the soil solution; in other words, the P released from the NPs can be used by mycorrhised or non-mycorrhised plants. On the other hand, the control treatment effect shows a significant difference in leaf area, with results significantly higher than those of the mycorrhizal treatments in the case of leaf area. This confirms the work of He and Cui [28] who observed no significant difference in avocado biomass when applying AMF on sterile and non-sterile soil. Indeed, the AMF should perform their function through the strongly branched outer hyphae which increase the plant’s absorption capacity [29]; this absorption capacity which is conditioned by the type of crop grown and the nature of the soil can influence the AMF [30]. In our case, the nursery study in bags could reduce the exploration zone of the AMF and therefore their action. This could explain why the mycorrhizal fertilisation effect did not show any significant difference on the growth parameters. Viera et al. [31] working on native mycorrhizae of the rhizosphere, established a correlation between the growth of avocado seedlings and the amount of accumulated phosphorus. The only significant difference for the fertilisation effect was noted only for the leaf area parameter, knowing that nitrogen plays a major role in the multiplication of chloroplasts (green foliage) and also in the increase of the leaf surface and that the Fert1 treatment composed of fowl droppings, very rich in nitrogen and phosphorus, increases the protein synthesis and the phosphorylated compounds in the plants, decreases the content of soluble sugars in the roots, and consequently the rate of mycorrhizal colonisation [32]. Phosphorus input in the soil decreases the mycorrhization rate of the host plant [27]. The organic amendment applied to the Fert1 non-mycorrhizal treatment possibly favoured the development of endogenous soil fungi and their symbiosis with avocado roots. Our results corroborate with those of Okur et al. [33] who stated that soil amendment with organic fertilizers increases the activity of the soil microbial biomass, hence competition when a mycorrhizal amendment is added such as mycorrhizal fertilizer.
5. Conclusion
The general objective of this research work was to define a technical itinerary for the production of avocado seedlings using biofertilizers. With the aim of producing organic avocado seedlings, this work had a two-fold objective: 1) The evaluation of the effect of the substrate on the germination and the vigour of the avocado seedling on the one hand and 2) on the other hand the evaluation of the effect of the organic fertilization (based on mycorrhizae and hen droppings) and of the stage of transplanting on the development of the avocado seedlings. At the end of this work, it appears that: 1) Wood sawdust being the best substrate compared to sand increases the root volume by 49.42%. This gain in root volume is 19.53% compared to soil. Soil increases root volume by 25% compared to sand. Also, the germination rate is lower in sand (83.64%) and higher in wood sawdust (93.64%). 2) Early transplanting dates (TransD40) significantly influence the growth (diameter and height) of the seedling and therefore better for rootstocks; 3) The non-mycorrhizal treatment shows a gain in leaf area, although the beneficial effects of the mycorrhizal treatments do not yet translate into a significant difference in growth parameters. The use of mycorrhizal treatments in the field on young seedlings could allow a better discussion of the results obtained in the nursery. These results are to be used to elaborate a technical sheet for the production of avocado seedlings with bio-fertilisers.