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Cultivos Tropicales

Print version ISSN 0258-5936On-line version ISSN 1819-4087

cultrop vol.40 no.1 La Habana Jan.-Mar. 2019

 

Original Article

Effect of foliar aqueous extract of moringa in the initial acclimatization phase of pineapple

Lianny Pérez-Gómez1  * 

Iris Capote-Betancourt1 

Lelurlys Nápoles-Borrero1 

Danilo Pina-Morgado1 

Claudia Linares-Rivero1 

Maribel Rivas-Paneca1 

Maritza Escalona-Morgado1 

Romelio Rodríguez-Sánchez1 

Aurora Terylene Pérez-Martínez1 

1 Centro de Bioplantas. Universidad de Ciego de Ávila Máximo Gómez Báez, Cuba. CP 69450

ABSTRACT

Moringa oleifera Lam. leaves have several chemical constituents such as: amino acids, mineral ions, ascorbate, phytohormones and secondary metabolites. Consequently, these extracts can be used to improve growth in other plants. In addition, it is awarded antimicrobial activity against pathogens of agricultural interest. The objective of this work was to evaluate the effect of the aqueous extract obtained from moringa leaves on the morphological changes of the Ananas comosus var. comosus ‘MD-2’ plant in the initial stage of acclimatization. It was evaluated the effect of the concentration (dilutions of 1:4 and 1:8) and the immersion time (0, 24, 48 and 72 hours) in the aqueous extract of moringa previous to the acclimatization of pineapple seedlings. The aqueous extract of moringa and Previcur® Energy LS 84 was compared in the acclimatization of pineapple seedlings. It was achieved 100 % survival with the immersion of the pineapple seedlings for 24 and 48 hours in aqueous extract diluted 1:4 and for 72 hours in aqueous extract diluted 1:8. Morphological indicators were higher with the immersion of pineapple seedlings for 72 hours in aqueous extract diluted 1:8, after 42 days in acclimatization. Significant differences weren’t observed in morphological indicators for treated plants only with Previcur® Energy LS 84 and aqueous extract diluted 1:8. In these treatments weren’t observed visible symptoms of diseases.

Key words: Ananas comosus; biochemical compounds; biostimulant; growth

INTRODUCTION

In recent years, the Moringa oleifera Lam., has been widely used in human and animal nutrition, in traditional medicine, in the production of biodiesel and in the pharmaceutical and cosmetic industry 1-3. In addition, their extracts are attributed biostimulant activity because they are rich in amino acids, mineral ions, ascorbate, and phytohormones 4,5. This makes it favor the growth of some plants, which allows its use in different branches of agriculture. However, in most studies, extracts of moringa leaves have been used to stimulate germination of legume and cereal seeds, as well as to evaluate their effect on plant growth and development 6,7. The presence in moringa extracts of phytochemicals such as coumarins, flavonoids, terpenoids, carotenoids, tannins and phenolic compounds confers antimicrobial activity against pathogens of agricultural interest 8-10.

The pineapple (Ananas comosus var. Comosus) is the third fruit with the highest production in the world. It has a pleasant taste and aroma, as well as vitamins C, B1, B6, folic acid and minerals such as K+ (11. In 2016, its global production amounted to 25,809,038 tons and in Cuba to 48,501 tons 12. For pineapple producing countries and in particular for Cuba, the deficiency of propagation material is a problem that arises when it is desired to promote new areas or introduce a new variety.

The hybrid 'MD-2' has characteristics of great economic importance, such as high yields and the quality of the fruit. It is one of the cultivars with the highest import volumes as fresh fruit in the markets of the United States and the European Union 13. However, plants grown in the field average only two propagules per productive cycle. This makes it necessary to establish schemes for the production of seeds through biotechnology and to evaluate their introduction in the conditions of Cuba and specifically in Ciego de Ávila territory.

The development of micropropagation techniques has had highly advantageous results in the rapid and quality propagation of several economically important plant species 14. In this environment, the Bioplant Center developed a micropropagation protocol based on the use of liquid medium and temporary immersion technology together with the implementation of a semi-automated system 15, which makes it possible to reduce the time needed to generate sufficient quantities of vitroplants destined to the creation of basic seeds banks that allow the promotion of pineapple plantations with high quality seed.

Acclimatization is due to the fact that the plants undergo physiological changes that can influence the following stages. The pineapple has a slow growth, which delays the duration of this phase. In addition, pineapple plants during acclimatization can be affected by oomycetes and phytopathogenic fungi, mainly of the genus Phytophtora, Fusarium and Rhizoctonia, producing important losses 16.

The objective of this work was to evaluate the effect of aqueous extract obtained from leaves of Moringa oleifera Lam. in the morphological changes of the pineapple plants 'MD-2' in the initial stage of acclimatization.

MATERIALS AND METHIDS

Plant material

The plant material of pineapple (Ananas comosus var. Comosus) 'MD-2' was supplied by the Technological Scaling and Transfer Laboratory of the Bioplant Center. Plants that came from the in vitro rooting stage (30 days) and were in a culture medium containing: MS salts 17, 100 mg L-1 of myo-inositol, sucrose at 30 g L-1, were used. , 1 mg L-1 of thiamin-HCl and 2.69 μmol L-1 of naphthalene acetic acid (ANA).

Obtaining and characterization of the aqueous extract of moringa leaves

To obtain the aqueous extract, the leaves of Moringa oleifera Lam were used. cv. Supergenius from adult plants of three years grown in the “Juan Tomás Roig” Experimental Station, Center of Bioplants that possessed the phytochemical components shown in Table 1.

Table 1 Phytochemical components detected in the leaves of Moringa oleifera Lam. cv Supergenius 

Compound Test or reagent used Leaves
Volatile coumarins NaOH +
Saponins Foaming, -
Triterpenes and steroids Liebermann-Buchard reaction +
Salkowski reaction +
Flavonoids Reaction of Shinoda -
Anthracene derivatives Reaction of Borntrager -
Phenolic compound FeCl3 +
Tannins Gelatine-NaCl -

- Absence of the compound, + Presence of the compound

To obtain the aqueous extract, 400 g of fresh mass of moringa leaves were milled with liquid nitrogen in a commercial blender. The proportion of plant material and water was 1:2.5 (m:v). The extraction was carried out for one hour with agitation and then filtered with gauze to eliminate the plant material. Next, the sample was centrifuged for 20 minutes at 15,000 x g and the supernatant was collected. The concentration of proteins 18, the concentration of carbohydrates 19 and the concentration of soluble phenolic compounds 20 were determined to the aqueous extract and are shown in Table 2.

Table 2 Characterization of the aqueous extract obtained from leaves of Moringa oleifera Lam. cv Supergenius 

Aqueous extract
Proteins (mg g-1 MF) 8,27
Carbohydrates (mg g-1 MF) 53,8
Phenolic compund (mg g-1 MF) 6,50

(mg g-1 MF= Fresh mass)

Effect of concentration and time immersion in aqueous extract of moringa in the acclimatization of pineapple plants

Homogeneous plants rooted in vitro were selected for acclimatization, which had between 0.8-1.62 g of fresh mass, 8-10 cm in length, 4-6 leaves and 3-4 roots, as established in the technical instructions for the propagation of the pineapple of the Bioplants Center 21. Two solutions of the aqueous extract of moringa were prepared: extract diluted four times (1: 4) and extract diluted eight times (1: 8). The roots of the plants were submerged during: 0, 24, 48 and 72 hours in the solutions. 7.5 mL of the dilutions of the aqueous extract/plant were used.

They were placed for the duration of the immersion in culture chambers (KOXKA, mod EC-1200F) with controlled environmental conditions. Temperature (25±1 °C), photosynthetic photon flux (PPF = 80 mol m-2s-1) and relative humidity (RH = 70 %) were fixed throughout the experiment to promote crop growth, photoperiod it was of 16 light hours and eight hours of darkness.

The plants were planted in plastic containers with a volume of 222.59 cm3 with a mixture of the sifted substrate of Ferralitic Red soil and filter cake (derived from sugarcane) at a ratio of 1: 1 (v/v). They were acclimatized in a house of culture under conditions of 80 ± 3 % relative humidity, 30 ± 2 °C temperature, natural light with photosynthetic photon flux of 400 ± 25 umol m-2s-1 and atmospheric conditions of concentration of CO2 and natural photoperiod. These were sprayed every 10 days with a foliar fertilizer mixture containing 16.0 g of crystalline N-P-K and 1.0 g of Multimicro Combi (Haifa Chemicals Ltd., Haifa Bay 26120).

At 42 days the survival (%) of the plants was determined as the quotient of the number of live plants at the end of this stage between the initial numbers of plants (30 plants). In addition, the morphological indicators were evaluated: fresh mass (g), dry mass (g), length of the plant (cm), number of leaves, length of the longest root (cm) and number of roots per plant.

Comparison of the aqueous abstract of moringa leaves and the Previcur® Energy LS 84 in the aclimation of pineapple plants

Four treatments were designed: roots submerged in distilled water for 72 hours (control), roots submerged in moringa extract diluted eight times (1: 8) for 72 hours, roots submerged in Previcur® Energy LS 84 (Bayer CropScience, 1.0 ml) L-1) for three minutes and a combination of immersion of the roots in moringa extract diluted eight times (1:8) for 72 hours and then in Previcur Energy® for three minutes 22.

The plants were sown and cultivated under the same conditions as in the previous experiment. At 42 days, the same indicators described in the previous experiment were evaluated.

Statistical analysis

In the statistical processing of the data, the utility SPSS Version 21 for Windows, SPSS Inc. was used. The analysis of variance (ANOVA) simple and bifactorial (immersion time and dilution of the aqueous extract) was used. The means of the treatments were compared using Tukey's multiple range test (p <0.05) after checking the assumptions of normality and homogeneity of variances. In the indicators survival, number of leaves and number of roots it was necessary to transform the data to achieve the assumptions of the parametric tests carried out. In both experiments, 30 plants were used per treatment with a completely randomized experimental design. The experiments were repeated three times. The percentage of survival represents the average of the three repetitions (n=3). For the rest of the indicators evaluated, each plant was considered as an experimental unit and represents the average of all the individual measurements (n=90).

RESULTS AND DISCUSSION

Effect of the concentration and immersion time in the aqueous extract of moringa in the aclimatization of pinapple plants

The Figure 1 shows the survival of pineapple plants after 42 days in acclimatization, after immersion of the plants in extract of Moringa oleifera Lam diluted four (1:4) and eight times (1:8) during 24, 48 and 72 hours. The best results were obtained with the immersion of the roots of the pineapple plants for 24 and 48 hours in the aqueous extract diluted 1: 4, without significant differences with the dilution 1: 8 for 72 hours. In these treatments 100 % survival of the plants was achieved. On the other hand, the plants whose roots were submerged in aqueous extract of moringa (1: 4) for 72 hours, significantly decreased their percentage of survival. However, the dilution (1:8) required 72 hours of immersion to provide 100 % survival.

Figure 1 Effect of the time of immersion of the roots of the plants of Ananas comosus var. Comosus 'MD-2' in dilutions of the aqueous extract of leaves of Moringa oleifera Lam. cv Supergenius in survival at 42 days under controlled environmental conditions 

Plants that died during the experiment showed flaccidity in the leaves with symptoms of rot and brownish-black coloration towards the base of the leaves. This could be associated with the presence of Phytophthora parasitica Dastur, which is the main pathogen that affects the cultivation of the 'MD-2' pineapple during acclimatization 16. However, it is necessary to perform laboratory analyzes to identify if the damage is due to the presence of this pathogen.

Table 3 shows the values of the morphological indicators of the pineapple plants in response to different immersion times in dilutions of the aqueous extract of moringa leaves. The number of leaves and the fresh mass were significantly higher when the plants were submerged for 72 hours in a diluted 1: 8 extract, without statistical differences with the rest of the treatments where the aqueous extract of moringa leaves was used, regardless of the dilution and the time of immersion. The length of the plant reached the highest values when the diluted extract was used 1: 4 for 48 hours and the extract diluted 1:8 for 72 hours, without statistical differences with the treatments where the aqueous extract of moringa leaves was used regardless of Immersion time. The length of the longest root was greater when the immersion in the aqueous extract of moringa leaves was carried out for 48 and 72 hours, independently of the dilution. The dry mass did not show significant differences between the dilutions used, except when the immersion was performed in the 1: 4 dilution for 72 hours. The number of roots showed no difference between the treatments.

Table 3 Effect of the immersion time and dilution of the aqueous extract of Moringa oleifera Lam. leaves cv Supergenius on morphological indicators of plants of Ananas comosus var. Comosus 'MD-2' after 42 days in acclimatization 

Dilution extract Time (hours) Number of leaves Plant length (cm) Number of roots Longest root length (cm) Fresh mass (g) Dry mass (g)
1:4 0 3,61 (13) b* 9,36 b 2,65 (7) 3,09 b 3,78 b 0,27 b
24 3,74 (14) ab 11,51 ab 2,65 (7) 3,19 b 4,02 ab 0,34 ab
48 3,74 (14) ab 13,88 a 2,65 (7) 3,83 ab 4,77 ab 0,33 ab
72 3,74 (14) ab 12,23 ab 2,65 (7) 4,07 ab 4,22 ab 0,30 b
1:8 24 3,74 (14) ab 11,76 ab 2,65 (7) 3,18 b 3,97 ab 0,34 ab
48 3,87 (15) ab 11,47 ab 2,65 (7) 3,87 ab 4,13 ab 0,31 ab
72 4,13 (17) a 14,48 a 2,83 (8) 4,58 a 4,97 a 0,39 a
ESx 0,24 1,7 NS 0,58 0,51 0,04

* Averages with unequal letters differ statistically for each indicator (n=90, bifactorial ANOVA, Tukey, p≤0.05). For the statistical treatment, the leaf number and root number data were transformed according to x'= x0.5. The table shows the transformed data and the original data appear in parentheses

The fact that the immersion in the highest dilution of the aqueous moringa extract (1:8) at 72 hours showed the best results in terms of survival and the morphological indicators could be related to the lower viscosity of this solution, which favors the entry of water and the penetration of mineral ions and other nutrients that benefit growth. For this reason, this treatment was selected to continue the experimentation because the survival and the morphological indicators evaluated in the plants showed a superior quality.

Comparison of the aqueous abstract of moringa leaves and the Previcur® Energy LS 84 in the aclimation of pineapple plants

After 42 days of culture, the survival of the plants treated with Previcur® Energy LS 84, aqueous extract diluted eight times (1:8) and the combination of both was 100 %; while that of the control treatment of 81 % (data not shown).

Table 4 shows the values of the morphological indicators of pineapple plants as a response to immersion prior to acclimatization in the aqueous extract of Moringa oleifera Lam. leaves and in the Previcur® Energy LS 84. The length of the plant and the length of the longest root were higher in the plants treated with Previcur® Energy LS 84, without significant differences with the aqueous extract diluted 1: 8. The fresh mass showed the best results in the treatments with Previcur® Energy LS 84 and the aqueous extract diluted 1:8, without significant differences with the control. The indicators number of leaves, number of roots and dry mass did not show differences for the treatments evaluated.

Table 4 Morphological indicators of the plants of Ananas comosus var. comosus 'MD-2' in response to immersion in the aqueous extract of leaves of Moringa oleifera Lam. cv Supergenius and in Previcur® Energy LS 84, after 42 days in acclimatization 

Treatments Number of leaves Length of plants (cm) Number of roots Longest root length (cm) Fresh mass (g) Dry mass (g)
Control 3,74 (14) 9,38 c 2,64 (7) 3,30 b 3,39 a 0,29
Aqueous extract (1:8) 4,00 (16) 14,60 a 2,82 (8) 4,53 a 3,55 a 0,37
Previcur®Energy LS 84 4,00 (16) 15,35 a 2,82 (8) 4,95 a 3,66 a 0,35
Aqueous extract (1:8) + Previcur®Energy LS 84 4,00 (16) 12,30 b 2,64 (7) 2,60 b 2,72 b 0,25
ESx NS 1,33 NS 0,52 0,24 NS

* Averages with unequal letters differ statistically for each indicator (n = 90, one-way ANOVA, Tukey, p≤0.05). For the statistical treatment, the leaf number and root number data were transformed according to x'= x0.5. The table shows the transformed data and the original data appear in parentheses

The morphological indicators of the plants that were treated with immersion for 72 hours in aqueous extract diluted 1: 8 followed by three minutes in Previcur® Energy LS 84 were affected. It is possible that the phenolic compounds and the aromatic amino acids of the proteins present in the raw extract of moringa leaves interacted with the active ingredient of Previcur® Energy LS 84 (propamocarb fosetilate) generating chemical compounds of the phenylcarbamate type. These compounds are known to have herbicidal activity and can act as inhibitors of mitosis or electron transport in chloroplasts affecting the growth of plants 23.

The results of this investigation showed the beneficial effect of the aqueous extract of moringa leaves on the morphological indicators in the acclimatization of the 'MD-2' pineapple. The direct contact of the aqueous extract of moringa leaves for 72 hours with the absorbent structures of the roots of the pineapple plants 'MD-2' could have favored the entry of minerals and organic compounds present in the extract. Some authors recognize the stimulatory effect of the growth of aqueous extracts of leaves of Moringa oleifera Lam., associated with the presence of amino acids, mineral ions such as K+ and Ca2 +, ascorbic acid and phenolic compounds 24.

In addition, when the aqueous extract diluted 1: 8 was applied, the Previcur® Energy LS 84 and the combination of both treatments showed no visible symptoms of fungal diseases that appear at this crucial stage of acclimatization, where it is recognized that the highest mortality of plants is associated with Phytophthora parasitica Dastur 16. This behavior observed with the aqueous extract could be associated with the presence of phenolic compounds (Table 1), which have recognized antimicrobial activity 25.

CONCLUSIONS

The aqueous extract of moringa leaves favored the initial stage of the acclimatization of the 'MD-2' pineapple. The immersion of the roots of the pineapple plants for 72 hours in an aqueous extract diluted eight times (1: 8) benefited the growth of the plants and no differences were seen with the Previcur® Energy LS 84.

ACKNOWLEDGEMENT

The investigations were carried out in the framework of the national project P131LH003028 “Obtaining bioactive plant extracts, rich in secondary metabolites for the control of pests and diseases of crops of agricultural importance”, coordinated by Dr. Martha Hernández de la Torre. The authors are grateful for the contribution to the work of Degree. Arturo Matos Ruíz.

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Received: September 10, 2018; Accepted: January 29, 2019

*Author for correspondence. lianny@bioplantas.cu

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