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Introduction
In Mexico, coffee has been considered one of the most economically, socially and culturally important crops (Escamilla et al. 2005). The state of Veracruz is the second largest coffee producer in the country, with coffee being the state’s third most important agricultural product after maize (Zea mays) and sugarcane (Saccharum officinarum L.) in terms of cultivated area and the first in terms of the value of crop exports (SIAP 2015).
The species Coffea canephora var. robusta is a cross-pollinated plant, which implies a high variability in the type and production of plants obtained by seed. To adequately multiply robusta coffee one option is to use the asexual method, using vegetative parts obtained from selected mother plants (Abrego 2012). The advantages of this technique are: 1) production of homogeneous plants from selected genotypes with high-yield robusta coffee materials improved by up to 30%; 2) uniform plantations with good productive potential of smaller size that make the harvest and crop tasks easier, 3) more plants per area unit, and 4) shortening of the plantation’s pre-productive period (INIFAP 2010). The cutting is a separate portion of the plant, endowed with cauline buds and leaves and induced to form roots and shoots through chemical, mechanical and/or environmental manipulations. The acquisition of new plants through cuttings is possible thanks to two characteristics of the plant cell, which are totipotency and dedifferentiation (Hartmann and Kester 1997).
One of the most important factors influencing asexual growth of seedlings is the choice of substrate since it is the rooting medium whose functions are to keep the cuttings fixed in place, provide moisture to their base and act as a buffer in chemical and pH reactions. The substrate’s support function is essential to provide good characteristics such as water-holding capacity, bulk density, electric conductivity, neutral pH, macro and micronutrients (Dumroese et al. 2011); the most notable organic
substrates are peat-moss, compost and vermicompost; the last two are obtained by biological processes that transform organic remains of different materials into a relatively stable organic matter with chemical, physical and microbiological characteristics for the reproduction of plants in a nursery (Velasco-Velasco et al. 2011, Quintero et al. 2003). Similarly, Mendoza-Hernández et al. (2014) mention that vermicompost outperformed compost and peat for rooting cuttings, the presence of hormone-like substances in the vermicompost might be behind this effect; the same authors mention that the vermicompost-based substrates gave acceptable results for growing plants, though none performed as well as the control.
The raw material used to make compost and vermicompost has a direct impact on the final concentration and behavior of the nutrients, so the final quality of these products is expected to be very different from each other, such as their effect on the soil and crops after application (Duran and Henríquez 2007). In relation to Coffee canephora propagation, different results have been published; on the one hand Guisolfi et al. (2020) studied physiological variables by using different proportions of agricultural residues as part of the substrate compared to the commercial substrate in the production of Conilon coffee seedlings, and these authors did not found significant differences between them. On the other hand, Quartezani et al. (2019) observed that proportions of composted urban waste higher than 50% added to the soil substrate promoted the highest plant growth rates; hence the use of stabilized organic matter in substrates for clonal plant propagation is essential to promote favorable conditions for the development of both shoots and roots. Therefore, there is an opportunity area for studying different types of compost and vermicompost made from local raw materials, and evaluate them as potential material in the clonal C. canephora propagation.
The aim of this study was to determine the effect of four types of substrates based on vermicompost and compost made from different types of organic matter, three robusta coffee clones (INIFAP 00 28, INIFAP 00-24 and INIFAP 97-14) produced by the Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP) and their interactions on the development of physiological variables of robusta coffee (Coffea canephora P.) cuttings.
Materials and methods
Description of the study area
The experiment was carried out in a greenhouse at the INIFAP “El Palmar” Experimental Station, located at 18° 32’ LN and 96° 47’ LW, at an elevation of 180 masl in the municipality of Tezonapa, Veracruz. This region is characterized by a warm humid climate, summer rains, annual precipitation of 2,885 mm, and a mean annual temperature of 24.4 °C, with a minimum and maximum of 16.1 °C and 35.4 °C. The municipality of Tezonapa is located in the central region of Veracruz and its physiography is characterized by flat lands and rolling hills with slopes of 5 to 20%. The soils of the region are red laterites, deep with excellent natural drainage, clayey-sandy loam texture and pH 4.8 (INIFAP 2009).
Study factors
This research had two study factors: a) type of substrate and b) type of robusta coffee clone. Three substrates based on vermicompost were evaluated: 1) Rabbit manure vermicompost + sand at a ratio of 70:30 v/v (RaVc), 2) Sheep manure vermicompost + sand at a ratio of 70:30 v/v (ShVc), 3) Cow manure vermicompost + sand at a ratio of 70:30 v/v (CoVc) and 4) Compost made on the basis of sugarcane filter cake + coffee husk + sand at a ratio of 33:33:33 v/v (INIFAP); the last-mentioned substrate is what INIFAP usually uses to grow robusta coffee cuttings. With regard to plant material, three types of robusta coffee clones were used: INIFAP 00-28, INIFAP 00-24 and INIFAP 97-14. Table 1 shows the list of treatments resulting from the combination of both factors.
Table 1: Evaluated treatments
| Treatments | Identification | Description |
| TRT1 | RaVc/00-28 | Rabbit manure vermicompost + sand (70:30 v/v)/clon INIFAP 00-28. |
| TRT2 | RaVc/00-24 | Rabbit manure vermicompost + sand (70:30 v/v)/clon INIFAP 00-24 |
| TRT3 | RaVc/97-14 | Rabbit manure vermicompost + sand + arena (70:30 v/v)/clon INIFAP 97-14. |
| TRT4 | ShVc /00-28 | Sheep manure vermicompost + sand (70:30 v/v)/clon INIFAP 00-28. |
| TRT5 | ShVc /00-24 | Sheep manure vermicompost + sand (70:30 v/v) /clon INIFAP 00-24. |
| TRT6 | ShVc /97-14 | Sheep manure vermicompost + sand (70:30 v/v) / clon INIFAP 97-14. |
| TRT7 | CoVc/00-28 | Cow manure vermicompost + sand (70:30 v/v) / clon INIFAP 00-28 |
| TRT8 | CoVc/00-24 | Cow manure vermicompost + sand (70:30 v/v) / clon INIFAP 00-24. |
| TRT9 | CoVc/97-14 | Cow manure vermicompost + sand (70:30 v/v) /clon INIFAP 97-14. |
| TRT10 | INIFAP/00-28 | Compost made on the basis of sugarcane filter cake + coffee husk + sand (33:33:33 v/v) / clon INIFAP 00-24. |
| TRT11 | INIFAP/00-24 | Compost made on the basis of sugarcane filter cake + coffee husk + sand (33:33:33 v/v) / clon INIFAP 00-28. |
| TRT12 | INIFAP/97-14 | Compost made on the basis of sugarcane filter cake + coffee husk + sand (33:33:33 v/v)/ clon INIFAP 97-14 |
Preparing the rooting chamber
A rooting chamber measuring 6.0 m x 1.3 m equipped with a sprinkler irrigation system was designed to house the coffee clone cuttings. The prepa ration of the seedbed within the rooting chamber is described below in chronological order: 1) 10 cm of sand was placed, 2) 10 cm of gravel were placed on the sand layer, and 3) 15 cm of the substrate to be evaluated were placed over the entire chamber surface. Divisions perpendicular to the length of the chamber were made with blocks lined with transparent polyethylene to prevent the flow of water and nutrients between divisions where a clone (30 cuttings) was placed. The substrates that include vermicompost were mixed with sand at a 70/30 ratio (ver- micompost/sand v/v), except the one corresponding to INIFAP (Table 1). Before sowing the cuttings a prolonged irrigation was applied to field capacity following the method stated in NOM-021-SEMARNAT-2000 (SEMARNAT 2002), and then a fungicide was applied to prevent the growth of any fungus.
Collection, sowing and acclimatization of cuttings
The cuttings were collected in a clonal coffee garden located at the INIFAP “El Palmar” Experimental Station on September 11, 2015 at 6:30 h. The methodology used by INIFAP for selecting and sowing cuttings was the “rooted cuttings” technique proposed by Méndez et al. (2015). Young shoots of good size, preferably primary branches and without disease, were selected. The shoots were separated from the mother plant with a bevel cut made from the base with pruning shears, the lateral axillas were removed and the leaves were cut back to three-quarters. The bevel cut was made to shoots of 3 to 4 internodes at 1 cm above and 5 cm below the internode. Once the cuttings were obtained, they were treated with an antioxidant solution (15 L of water mixed with 150 mL L-1 of ascorbic acid, 100 mL L-1 of citric acid and 30 g L-1 of sugar) and then immersed in a commercial fungicidal solution (Amistar 1.5 g L-1). Initially, irrigation was applied to the substrate up to field capacity, following the method mentioned above, and then the water applied was done as it was required. The cuttings were inserted into the substrate and spaced 5 cm apart. After the sowing work, the rooting chamber was covered with 600-gauge clear plastic, which was secured into place. The irrigation and weeding were conducted three times a week by adding water through a pipe with an ending sprayer and weeding manually. The cuttings underwent acclimatization for three months inside the rooting chamber.
Evaluated variables
Physical and chemical analysis of the substrates was carried out in the soil science department at the central laboratory of the Universidad Autonoma Chapingo. The evaluated variables were: pH: potentiometer in suspension (sample:water ratio 1:5); Electrical conductivity (EC): conductivity bridge in suspension (sample:water ratio 1:5); Organic matter (OM): Walkley and Black. Nitrogen (N): extracted with 2N potassium chloride and determined by steam distillation; Assimilable phosphorus (P): (P): Bray P1, Potassium and Sodium (K, Na): extracted in 1.0 N ammonium acetate, pH 7.0, 1:20 ratio and determined by flame emission spectrophotometry; Calcium, Magnesium (Ca, Mg): extracted in 1.0 N ammonium acetate, pH 7.0, a 1:20 ratio and determined by atomic absorption spectrophotometry; Iron, Copper, Zinc, Magnesium (Fe, Cu, Zn, Mg): extracted with a 1:4 DTPA ratio and determined by atomic spectrophotometry; Boron (B): extracted with 1.0M CaCl2 calcium and determined by photocolorimetry with azomethine-H; Bulk density (Bd) test-tube method; Carbon: Nitrogen (C:N) and CIC: estimated by calculation. The methodology for analyzing these variables was carried out in accordance with Mexican Official Standard NOM-021-SEMARNAT-2000, which was used to interpret the results.
Robusta coffee clones
The physiological variables evaluated in each of the cuttings were: mortality percentage, axillary bud and rooting, axillary bud length (cm) from the base of the cutting to the terminal part of the shoot, number of shoots, number of internodes, number of leaves, number of roots at the end of the trial and taproot length (cm) from ground level to the root tip. All variables were measured after three months of the experimental period.
Rooting chamber
Maximum and minimum temperatures were recorded every other day during the three-month cutting acclimatization period, as well as the relative humidity inside the rooting chamber. These variables were measured using a datalogger Thermohygrometer TFA Dostmann 30.5000.02.
Experimental design and data analysis
We used a strip-plot design with a factorial structure in the treatment (substrate x clone) distributed in a randomized block design. ANOVA and Tukey’s mean comparison (p ≤ 0.05) was performed. Thirty robusta coffee cuttings were used in each treatment. The model that describes this design is as follows:
i = 1,2,3, j = 1,2,3,4, k = 1,2,3,4
where yijk is the ij-th response observed in block k, µ is the overall mean, bk is the random effect of block k assuming bk ~i.i.d.N(0, δ 2 b ), ɑ i is the fixed effect due to clone i, ω ik is the random effect due to clone i within block k assuming ω ik ~i.i.d.N(0,δ 2 ω ), ϓj is the fixed effect of substrate j, φ jk is the random effect due to substrate j within block k assuming φ jk ~i.i.d.N(0,ɑ2 φ), (ɑϓ) ij is the effect of the interaction due to clone i and substrate j, and ɛ ijk is the experimental error assuming ɛijk ~i.i.d.N(0, δ 2 ). To compare the length of shoots (Sl) and roots (Rl) and the number of internodes (Ni), shoots (Ns), leaves (Nl) and roots (Nr), two types of statistical models were used. One was a mixed linear model for a continuous response (Sl and Rl). The linear predictor for Sl and Rl is ή ijk = µ+ ɑ i + ω ik + ϓ j + φ jk + b k ; where ή ijk is the response observed in the ijth clone - substrate in the k block, and µ, ɑi, ω ik , ϓj, φ jk and b k defined above. Sl and Rl have a normal distribution, that is y ijk |ωik, φ jk , b k ~N(0, δ2). The other was a generalized linear mixed model with Poisson response for Ni, Ns, Nl, and Nr whose linear predictor is ή 𝑖𝑗𝑘 = µ+ ɑ 𝑖 + 𝜔 𝑖𝑘 + ϓ 𝑗 + 𝜑 𝑗𝑘 + 𝑏 𝑘 . The response variables Ni, Ns, Nl, and Nr have a Poisson distribution y ijk |ωik, φ jk , b k ~N(λ ijk ) with link function ήijk= log(λ ijk ) and the verse is λ ijk = e(ήijk).
Results
Physical and chemical analysis of substrates
The highest pH values were found in the ver- micompost substrates; according to NOM-021 the substrate with vermicompost based on cow manure (CoVc) showed a moderately alkaline pH, the RaVc and ShVc substrates showed neutral pH, and the substrate based on INIFAP compost had a moderately acidic pH (Table 2). In the case of EC, all substrates were found in a very slightly saline range with values from 1.17 to 1.94 dS m-1. The OM content in the RaVc and CoVc substrates was found in the middle content, while the ShVc and INIFAP substrates were in the low content. The N content in all substrates was in a range from 2.0 to 3.7%, this was probably due to the different types of organic materials used in each of the substrates. The available P content in each of the substrates was high, according to Official Mexican Standard NOM-021-SEMARNAT-2000, with values ranging from 357.14 to 647.24 mg Kg-1 (Table 2). The K and Na content results were high in the substrates analyzed according to the standard used. The available content of micronutrients such as Ca, Mg, Fe, Cu, Zn and Mn were found suitable for use in each of the substrates analyzed. However, the Fe, Cu and Mn micronutrients obtained a higher content in the INIFAP substrate than the other substrates (Table 2).
Table 2 Chemical and physical characteristics of substrates.
| Parameters Chemical and physical | Types of substrates | |||
| RaVc | ShVc | CoVc | INIFAP | |
| pH | 7.15 | 7.29 | 7.70 | 6.07 |
| EC dSm-1 | 1.638 | 1.949 | 1.778 | 1.172 |
| N% | 2.7 | 3.5 | 2.2 | 2.0 |
| P mgKg-1 | 489.00 | 647.24 | 357.14 | 610.19 |
| K mgKg-1 | 3552 | 3792 | 4236 | 2412 |
| Na mgKg-1 | 1632 | 1572 | 1452 | 576 |
| Ca mgKg-1 | 3272 | 4065 | 2516 | 2516 |
| Mg mgKg-1 | 1610 | 2021 | 1154 | 940 |
| CEC Cmol (+)kg-1 | 21.8 | 25.7 | 21.1 | 33.8 |
| Fe mgKg-1 | 21.64 | 35.96 | 21.78 | 124.36 |
| Cu mgKg-1 | 9.18 | 9.62 | 4.87 | 13.23 |
| Zn mgKg-1 | 30.20 | 17.22 | 13.05 | 9.65 |
| Mn mgKg-1 | 5.54 | 9.22 | 5.64 | 21.34 |
| B mgKg-1 | 4.34 | 1.75 | 3.34 | 3.43 |
| Bd g/cm3 | 0.95 | 0.98 | 1.14 | 1.08 |
| OM% | 1.68 | 1.28 | 2.69 | 0.67 |
| C:N | 2.8 | 4.7 | 1.4 | 5.1 |
Physical and chemical variables: pH (hydrogen potential), EC (Electrical conductivity), N (Nitrogen), P (Phosphorus), K (Potassium), Na (sodium), Mg (magnesium), CEC (cation exchange capacity), Fe (Iron), Cu (Copper), Zn (Zinc), Mn (Magnesium), B (Boron), Bd (Bulk density), OM (Organic matter) and C: N (Carbon Ratio, Nitrogen).
As regards CEC, RaVc and CoVc obtained an average value, while the ShVc and INIFAP substrates showed a high one. This variable increases notably with the presence of organic matter and it could be the basis of the substrate fertility. The highest Bd values were found in the CoVc substrate and the lowest in the RaVc one, it worth to mention that no statistical analysis was done on this variable.
Effect of substrate type on the physiological development of coffee cuttings
Substrates based on vermicompost and compost had significantly different effects on the physiological development of some variables, as shown in Table 3. Based on the analysis of variance, the average percentages for plant mortality (M), sprouting (S) and rooting (R) did not show significant differences in the different types of substrates used (p = 0.53,0.91 and 0.22, respectively). However, it should be mentioned that the INIFAP substrate showed the highest average percentages in sprouting and rooting, with 83.02 and 63.1% respectively, while the highest mortality percentage was observed in the CoVc substrate. In terms of the average outbreak length (Ol) in cuttings, the INIFAP substrate showed the greatest average length with 2.01 cm, which was significantly different (p ≤ 0.05) from the other substrates, whereas the RaVc and ShVc substrates had average lengths of 1.88 and 1.84 cm, respectively, which were statistically equal; the shortest average length was observed in the CoVc substrate, with 1.46 cm.
Table 3: Means and standard errors of the physiological variables of coffee cuttings evaluated in the different types of substrates.
| sustrato | M (%) | S (%) | R (%) | Ol (cm) | Ni (cm) | Nl | Nr | Rl (cm) |
| RaVc | 4.54 ± 1.7a* | 82.0 ± 3.5a* | 54.7 ± 4.48a* | 1.88 ± 0.15ab+: | 0.89 ± 0.06a* | 1.71 ± 0.12a* | 1.71 ± 0.09ab* | 11.72 ± 0.32a* |
| ShVc | 6.9 ± 2.6a | 80.2 ± 3.4a | 49.6 ± 4.19a | 1.84 ± 0.15ab | 0.76 ± 0.05ab | 1.43 ± 0.10a | 1.56 ± 0.10b | 11.21 ± 0.35ab |
| CoVc | 8.3 ± 2.9a | 80.0 ± 4.1a | 53.8 ± 5.1a | 1.46 ± 0.15b | 0.63 ± 0.05b | 115 ± 0.09b | 1.95 ± 0.12a | 11.40 ± 0.36a |
| INIFAP | 5.3 ± 2.0a | 83.0 ± 3.4a | 63.1 ± 4.28b | 2.01 ± 0.11a | 0.83 ± 0.05a | 1.52 ± 0.11a | 1.72 ± 0.09ab | 10.96 ± 0.28b |
* Averages and standard deviation in columns with the same letters are statistically equal (p ≤ 0.05). M (Percentage of mortality), S (Axillary bud yield percentage), R (Percentage rooting), Ol (Outbreak length), Ni (Number of internodes), Nl (Number of leaves), Nr (Number of roots) and Rl (root length). VcCo (70% vermicompost rabbit dung + 30% sand); VcBo (70% vermicompost of sheep manure + 30% sand); VcVa (70% vermicompost cow dung + 30% sand) and INIFAP (33% coffee husk + 33% sugarcane compost + 33% sand.
Regarding the number of internodes (Ni), the different types of substrates showed highly significant differences (p = 0.005). In the RaVc and INIFAP substrates the cuttings showed the highest average number of internodes (0.89 and 0.83 respectively), followed by the ShVc substrate (0.76) and lastly the CoVc one (0.63) per cutting. For the number of leaves, analysis of variance showed highly significant differences (p = 0.003) among the substrates used, with the RaVc and INIFAP substrates having the highest number of leaves with 1.71 and 1.52 respectively, while the CoVc substrate had the lowest number of leaves with 1.15 (Table 3). As for the number of roots, the cuttings showed no significant differences among the different types of substrates used (p = 0.09). In the root length of the cuttings, there were also no significant differences (p = 0.08); however, the RaVc and INIFAP substrates had the longest length with means of 11.86 and 11.86 respectively, while the ShVc substrate showed the shortest root length with 11.31 (Table 3).
Effect of the type of robusta coffee clone on the physiological development of cuttings
In general, significant differences were observed in the physiological development of the different cuttings from robusta coffee clones (Table 4). Analysis of variance of the mortality percentage showed statistically significant differences in at least one clone (p = 0.025). The highest mortality (M) percentage was observed in the INIFAP 97-14 clone (8.76%) followed by the INIFAP 00-24 clones with 7.59%, both clones were different statistically to INIFAP 00-28 (3.35%). In the percentage of axillary bud (S), the coffee clones showed significant differences (p = 0.031), with the INIFAP 00-28 clone obtaining the highest average percentage (89.2), followed by the INIFAP 00-24 and INIFAP 97-14 clones with statistically equal means (77.8 y 74.2, respectively). Significant differences (p = 0.008) were observed among the clones in rooting percentage: the INIFAP 00-24 and INIFAP 97-14 clones showed the highest percentage (70.60 and 63.80%, respectively) and were statistically equal to each other, while the INIFAP 00-28 clone had the lowest percentage (30.60). In outbreak (OI), the robusta coffee clones showed statistical differences (p = 0.001), with the INIFAP 00-28 clone with a mean of 2.10 cm obtaining the greatest outbreak length compared to the rest, and the INIFAP 97-14 clone having the shortest average length of 1.49 cm. Regarding the number of internodes (Ni) the clones showed significant differences (p = 0.019). The INIFAP 00-28 clone had the highest number of internodes (0.87) and the INIFAP 97-14 clone the lowest (0.69). In the number of leaves (NI), analysis of variance showed that the clones had significant differences among them (p = 0.02). The INIFAP 00-28 clone had the highest average number of leaves (1.63) compared to the leaf mean observed in the INIFAP 00-24 and INIFAP 97-14 clones (1.47, and 1.25, respectively). For the number of roots (Nr), the clones showed significant differences (p = 0.026); the INIFAP 97-14 clone cuttings showed the highest average number of roots (1.95), followed by the INIFAP 00-24 and INIFAP 00- 28 clones which had a lower average number of roots (1.78 and 1.50, respectively). Finally, the clones showed significant statistical differences in average root length (RI) (p = 0.002), with the INIFAP 00-24 and INIFAP 97-14 clones having an average root length (12.48 and 11.99, respectively) statistically equal to each other but different from that of the INIFAP 00-28 clone (9.57).
Table 4: Means and standard deviations of physiological variables of coffee cuttings in different types of substrates.
| Robusta coffee clones | M (%) | S (%) | R (%) | Ol (cm) | Ni | Nl | Nr | Rl (cm) |
| INIFAP 00-28 | 3.35 ± 1.1b | 89.2± 2.3a | 30.6± 6.57b | 2.1 ± 0.091a | 0.87 ± 0.05a | 1.63 ± 0.11a | 1.5 ± 0.1b | 9.57 ± 0.39b |
| NIFAP 00-24 | 7.59 ± 2.2a | 77.8 ± 3.36b | 70.6± 6.57a | 1.79 ± 0.093b | 0.77 ± 0.05ab | 1.47 ± 0.10ab | 1.78 ± 0.07ab | 12.48 ± 0.24a |
| INIFAP 97-14 | 8.76 ± 2.5a | 74.2± 3.6b | 63.8 ± 6.57a | 1.49 ± 0.095c | 0.69 ± 0.04b | 1.25 ± 0.09b | 1.95 ± 0.08a | 11.99 ± 0.25a |
Columns with the same letters are statistically equal (p ≤ 0.05). M (Percentage of mortality), S (Axillary bud yield percentage), R (Percentage rooting), Ol (Outbreak length), Ni (Number of internodes), Nl (Number of leaves), Nr (Number of roots) and Rl (root length).
Interaction of substrate and coffee clone on the physiological development of cuttings Mortality
Cutting mortality was statistically different (p ≤ 0.05) among the substrate/coffee clone combinations used (Table 5). Higher cutting mortality percentages were observed in the ShVc and CoVa substrates than in the RaVc substrate, while the INIFAP 00-28 clone showed better adaptability in the substrates tested.
Table 5: Comparison of physiological variables between treatments
| Treatment | Mortality | Sprouting | Rooting | Outbreak length (cm) | Number of | Number of roots | Root length (cm) | |
|---|---|---|---|---|---|---|---|---|
| Internodes | leaves | |||||||
| RaVc/00-28 | 2.5d | 91.4ab | 30.9de | 1.9a | 0.9a | 2.2abc | 1.6bcd | 9.8d |
| RaVc/00-24 | 6.2abcd | 74.2c | 63.9abc | 1.8ab | 0.9a | 2.6a | 1.6bcd | 13.2a |
| RaVc/97-14 | 5.9abcd | 75.8bc | 67.5ab | 1.4bc | 0.8a | 2.6a | 1.8abc | 12.5a |
| ShVc/00-28 | 4.1dc | 86.2abc | 20.7e | 1.9a | 0.9a | 2.2abc | 1.4cd | 9.9cd |
| ShVc/00-24 | 7.1abcd | 80.3abc | 72.7ab | 1.7ab | 0.8ab | 2.3ab | 1.3d | 12.4a |
| ShVc/97-14 | 11.3ab | 72.5c | 56.7bc | 1.3c | 0.5c | 2.1bc | 2.1ab | 12.0abc |
| CoVc/00-28 | 5.4bcd | 86.1abc | 31.1de | 1.8ab | 0.8a | 2.2abc | 1.7abcd | 10.0bcd |
| CoVc/00-24 | 7.1abcd | 76.1abc | 64.2abc | 1.3c | 0.6bc | 2.2abc | 2.3a | 12.1ab |
| CoVc/97-14 | 14.4a | 76.7abc | 65.0abc | 1.1d | 0.5c | 2.1bc | 1.9abc | 12.3ab |
| Inifap/00-28 | 2.2d | 91.8a | 42.0dc | 2.1a | 0.8a | 2.0c | 1.3d | 8.7e |
| Inifap/00-24 | 10.6abc | 80.4abc | 77.1a | 1.8ab | 0.1ab | 2.3ab | 2.0ab | 12.2ab |
| Inifap/97-14 | 5.9abcd | 71.7c | 67.1ab | 1.8ab | 0.9a | 2.4ab | 1.9abc | 12.0abc |
Columns with the same letters are statistically equal (p ≤ 0.05). Treatments: Rabbit vermicompost 00-28 clon (RaVc/00-28), Rabbit ver- micompost 00-24 clon (RaVc/00-24), Rabbit vermicompost 97-14 clon (RaVc/97-14), Sheep vermicompost 00-28 clon (ShVc/00-28), sheep vermicompost 00-24 clon (ShVc/00-24), sheep vermicompost 97-14 clon (ShVc/97-14), Cow vermicompost 00-28 clon (CoVc/00-28), Cow vermicompost 00-24 clon (CoVc/00-24), cow vermicompost 97-14 clon (CoVc/97-14), INIFAP Compost 00-28 clon (Inifap/00-28), INIFAP Compost 00-24 clon (Inifap/00-24), INIFAP Compost 97-14 clon (Inifap/97-14).
Sprouting
The axillary bud percentages were significantly different (p ≤ 0.05) among the different treatments (Table 5). Results show that the INIFAP 00-28 clone in combination with the different substrates showed the highest sprouting, followed in descending order by the INIFAP 00-24 and 97-14 clones.
Rooting
The rooting percentages in the substrate-clone interaction were significantly different (p ≤ 0.05); the cuttings from the INIFAP 00-24 and 97-14 robusta coffee clones (Table 5) showed a greater number of roots in all substrates, while fewer roots were observed in the INIFAP 00-28 clone.
Outbreak length
In the substrate-clone interaction the cuttings showed a significantly different (p ≤ 0.01) average outbreak length in the different combination of factors (Table 5). The highest average length in cuttings was observed in those planted in the INIFAP substrate followed by the RaVc and ShVc substrates, while the shortest length observed was in the cuttings planted in the CoVc substrate. It is important to note that the INIFAP 00-28 clone showed the greatest average length in each of the substrates used and the INI- FAP 97-14 clone showed the shortest length in most substrates except the INIFAP one.
Internodes
In the variable number of internodes, highly significant differences (p ≤ 0.01) were found in the different substrate-clone combinations (Table 5). Better interaction of the two factors on the number of internodes was found in the cuttings planted in the RaVc, ShVc and INIFAP substrates, whereas a lower interaction effect was observed in the cuttings planted in the CoVc substrate.
Number of leaves
Similarly, in the number of leaves, significant differences (p < 0.01) were obtained in the different treatments evaluated (Table 5). Cuttings sown in the RaVc substrate showed the highest number of leaves and those planted in the CoVc substrate produced the smallest number. This result correlates with the variable number of internodes since each internode usually has a pair of leaves.
Number of roots
The number of roots produced by the coffee cuttings showed significant differences (p < 0.05) among the different treatments. The INIFAP 00 24 and 97-14 clones produced a higher number of roots in the four substrates, while the INIFAP 00-28 clone obtained a lower number of roots in each of the substrates used (Table 5). This could have been influenced more by the characteristics of the species itself rather than the nutrient content of the substrates.
Root length
The average root length among the different substrate-clone combinations was significantly different (p = 0.05). It can be seen that the INIFAP 00-24 clone cuttings showed the greatest root length in all substrates tested, followed by the INIFAP 97-14 clone and the INIFAP 00-28 one, which produced the shortest root length (Table 5).
Temperature and relative humidity inside the rooting chamber
Figure 1 shows the maximum and mínimum temperatures as well as the relative humidity inside the rooting chamber during the three-month cutting acclimatization period. The minimum temperature ranged from 18 to 34 °C, and the maximum from 26 to 48 °C; these temperature ranges together with the applied irrigations caused the relative humidity to vary between 80 and 98%.
Discussion
In addition to pH and nutrient availability, the most important chemical properties of a substrate are cation exchange capacity (CEC), electrical conductivity (EC), C:N ratio and bulk density (Bd) (Cruz et al. 2014). Gines and Mariscal (2002) state that a slightly acidic pH provides better assimilation of essential elements (N, K, Ca, Mg). Barbaro et al. (2014) state that the higher the electrical conductivity the higher the salts concentration, and that low EC facilitates fertilization and prevents crop phytotoxicity problems, since the presence of some ions (such as those of calcium, magnesium and sodium chlorides and sulfates) in the soil solution at certain concentrations causes toxic effects to the plant. High Bd facilitates substrate compaction and root compression, also affecting irrigation efficiency and fertilization (Abad et al. 2004). Martinez and Roca (2011) mention that a Bd < 0.75 g cm-3 is an optimal substrate level. In this work, high values of Bd ranging from 0.95 to 1.14 g cm-3 could affect the evaluated variables such as percentage of mortality (M); for example the substrate CoVc had an M percentage value of 8.3 (Table 3), at the same time this substrate presented 1.14 of Bd (Table 2), and the RaVc had the lowest Bd value (0.95 g cm-3) and it has the lowest mortality percentage. According to Abad et al. (2004), a C:N ratio < 30 in organic substrates is considered suitable for cultivation, and is an index of a mature and stable material. Therefore, the C:N ratios obtained by the different types of substrates were all found in optimal ranges.
According to the results, the vermicompost substrate made from rabbit manure (RaVc) and the compost substrate made from coffee husk and sugarcane filter cake compost (INIFAP) showed higher physiological development than the sheep ma nure vermicompost (ShVc) and cow manure vermicompost (CoVc) substrates. This effect could be attributed to several characteristics of the study substrates; pH can be one of them, which were INIFAP (6.07) and RaVc (7.15), EC could be another characteristic that could affect positively on the physiological variables, other two are Bd and CEC; however, it is not clear which one had greater influence on the physiological variables. In this regard, Santamaría et al. (2001) mention that most studies on the use and efficacy of vermicompost at nursery level in fruit and ornamental species show better results than the implementation of other composted organic materials, although they may have similar chemical and biological characteristics. Nevertheless, Contreras et al. (2008) evaluated the effect of vermicompost application and mineral fertilization on coffee, observing higher dry weight in plants with mineral fertilization, followed by treatments with 20 and 30% vermicompost. It has been observed in coffee cultivation that vermicompost induces a better response in the vegetative development of the coffee tree as compared to bokashi (Mosquera et al. 2016). Some results may differ, considering that the quality of the substrate is related to the source material and handling during the preparation process, having variation in the content of nutrients and microorganisms. In general, in coffee cultivation, a positive and significant effect has been observed with the application of various organic fertilizers at the nursery stage (Julca et al. 2002).
Overall, in this work, the INIFAP 00-28 clone exhibited the greatest physiological development of the photosynthesis part (axillary bud, outbreak length, number of internodes, number of leaves), the INIFAP 00-24 clone obtained the least development, and the INIFAP 97-14 clone had the greatest root development. In accordance with the above, in many growing plants a balance between the photosynthesizing surface area and the root area is maintained. This confirms what was stated by Raven et al. (1999), who mention that in seedlings the adsorption (root) area greatly exceeds the photosynthesizing area. This behavior is very important, since if the sprouting of the buds occurs before root emission, they would compete and could deplete the water and nutritional re serves of the cutting (Ruiz and Mesen 2010). In the same way, Alfenas et al. (2009) argue that the root formation process may be influenced by the genetic constitution of the matrix plant. In this sense, some species present great differences in rhi- zogenic capacity and it is probable that the rooting capacity of robusta coffee is genotypically conditioned. Nonetheless, Alves et al. (2016) mention that the organic substrate is a favorable alternative that stimulate biometric characteristics compared to the soil and commercial substrate in the formation of clonal coffee canephora seedlings during the formation stage in the nursery. This factor is important for future cloning work, where the genotype/clone to be propagated must be taken into account (Abanto et al. 2014).
Saenz et al. (2010) mention that the root length is the most important quality characteristic that allows predicting the survival of the plant in the field, which is why these two variables are key for deciding which clone should be propagated. Additionally, Rodriguez et al., (2017) state that some physiological variables had significant effects on the initial development of coffee seedlings with substrates using cattle manure with 5.25 kg m-3 of liThoThamnium compared to filter cake with 1.75 kg m-3 of liThoThanmium. On the other hand, plants grow continuously, producing new cell organs from their meristems; consequently, postembryonic development is a continuous process in plants that depends on environmental factors and the type and quality of the substrate, resulting in a high degree of phenotypic plasticity because the plant cells are unable to escape from their environment, and are forced to cope with changing and often unfavorable growth conditions. Thus, mechanisms regulating tissue cells can facilitate stable changes in gene activity and adjust gene expression patterns, allowing plants to survive and reproduce successfully in an unpredictable and changing environment.
Conclusions
Vermicompost made from rabbit manure (RaVc) and compost based on coffee husk and sugarcane filter cake (INIFAP) showed a greater effect on the physiological development of coffee cuttings compared to substrates based on cow manure vermicompost (CoVc) and sheep manure vermicompost (ShVc). pH and EC were the substrates characteristics that affected the physiological variables and reduce the mortality percentage of coffee cuttings, and Bd and CEC had not clear influence on the physiological variables. The INIFAP 00-28 clone showed the greatest physiological development of the studied coffee cuttings. Therefore, according to this research, the physiological development of the cuttings depended on the origin of the organic residue used in making the vermicompost and compost, and the genotypic characteristics of the propagated clone. The 00-28 coffee clone had less mortality percentage, the highest axillary bud yield percentage, high outbreak length, high number of in-ternodes and number of leaves.
