Sustainable Methods Based on Microbial Biofertilizers and Plant Repellent Extracts in the Cultivation of Aloe Vera

Domenico Prisa^1*, Marco Gobbino²

¹CREA Research Centre for Vegetable and Ornamental Crops, Council for Agricultural Research and Economics, Via dei Fiori 8, 51012 Pescia, PT, Italy.

²Welcare Research srl, Via San Giovanni sul Muro 18, 20121 Milano, MI, Italy.

Abstract

This work aimed to develop a biological cultivation and defense protocol that can be used by companies growing medicinal succulent plants. The protocol was characterized by the use of microbial biostimulants and plant extracts with repellent action (plant growth-promoting rhizobacteria, in particular, Effective microorganisms and extracts of neem, propolis, and horsetail) able to improve the growth and quality of Aloe vera plants especially for the production of gels for cosmetic and medicinal use. The experimental trial at Welcare Industries S.P.A. (Orvieto) showed a significant improvement of agronomic parameters analyzed on Aloe vera plants treated with Effective microorganisms and a significant reduction of the presence of mealybugs following treatments with repellents of plant origin. The application of symbiotic microorganisms and plant extracts for defense in agricultural operations can therefore ensure higher production standards, with a possible improvement in the agronomic quality of plants, also reducing the use of water and fertilizers. This experiment may be of particular interest to farms that want to focus on the production of succulents for medicinal and cosmetic use where plant quality levels must be high.

Keywords: Medicinal plants, Sustainable agriculture, Effective microorganisms, Succulent plants, Microbic biofertilizers

Introduction

The name Aloe vera was used for the first time by Linnaeus, as Aloe perfoliata var. vera (1753). However, Linnaeus' description of Aloe vera as a separate species is after Miller's description of Aloe barbadensis (1768), although they are surely specimens of the same species (Reynolds, 1966). Moreover, the name barbadensis was given by Miller to specimens collected on the island of Barbados, in the Lesser Antilles, surely descendants of some Aloe plant left on the island by Portuguese or Spanish navigators more than two centuries before. For this reason, according to the rules about the attribution of names, it must be used the name Aloe vera Miller, (not: Linnaeus) (Bassetti & Sala, 2003).

Before the binomial classification introduced by Linnaeus, the species was called Aloe vera vulgo or Aloe vera Vulgaris sive sempervivum marinum, and later Aloe foliis spinosis confertis dentatis, vaginantibus planis maculatis. This indicates that the name vera is to explain that the plant was considered by the "vulgo" the true Aloe (Miladi & Damak, 2008).

In the world, Aloe is mainly cultivated in Africa, Australia, North America, Central America, South America, Russia, Japan. There is a large production in the Dominican Republic and Mexico. In Europe and the Middle East, Aloe vera is cultivated in Spain, Italy, Greece, and Israel (Marshall, 1990). The use of Aloe is very ancient, as witnessed by the cuneiform text of some clay tablets found at the end of the nineteenth century by a group of archaeologists in the Mesopotamian city of Nippur, near Baghdad, Iraq, and dated back to about 2000 BC. Aloe was also known and used by the Egyptians for embalming preparations (hence "plant of immortality") or the care and hygiene of the body or as a cicatrizant and mentioned several times in the Bible as an aromatic plant or for the preparation of ointments before burial (Boudreau & Beland, 2006). Plants over the millennia have developed associations and interactions with different groups of microorganisms in the soil (Cakmakci et al., 2007). These associations can be different depending on the areas that are colonized near the roots, in the surface areas, or even within the plant tissues. Interactions between plants and bacteria can be positive or negative and based on these interactions, it is possible to highlight if they are beneficial effects on soil fertility, growth, productivity, and plant defense (Glick, 1995). Many microbial agents can lead to plant death and have determined the development of defense strategies in plants to control pathogens (Elsharkawy et al., 2015). Several are the known microorganisms that can influence the life cycle of plants (rhizosphere bacteria such as Effective microorganisms, endo- and ectomycorrhizae such as Glomus sp., biocontrol agents such as Trichoderma sp.) (Condor et al., 2007).

 The stability of such positive plant-microbe relationships results in the development of eco-sustainable agroecosystems and the improvement of the quality of plant growth and development, allowing a possible reduction of synthetic fertilizers and pesticides.

Aims/objectives of the study

This work aimed to develop a biological cultivation and defense protocol that can be used by companies growing medicinal succulent plants. The protocol was characterized by the use of microbial biostimulants and plant extracts with repellent action (plant growth-promoting rhizobacteria, in particular, Effective microorganisms and extracts of neem, propolis, and horsetail) able to improve the growth and quality of Aloe vera plants especially for the production of gels for cosmetic and medicinal use.

MATERIALS AND METHODS

The experiments, started in March 2021, were conducted in the greenhouses of Welcare Industries S.P.A., Orvieto (TR), Umbria, Italy (42°13′N 12°6′E) on Aloe vera (7-8-year-old plants) (Figure 1). The plants were placed in soil, 21 plants per thesis, divided into 3 replicas of 7 plants each. All plants were fertilized with “Grena olive special”, 150 g per plant in April, June, and October. In the treated thesis the propogem products based on propolis 1% + equisetum 1% (7 treatments every 30 days) and neem extract 1% (7 treatments every 20 days) of the company La Praglia have been used to control mealybug (Figure 2b). A conventional mineral oil-based product was used in the control. While the microbial biofertilizer based on Effective microorganisms (6 treatments) was purchased from the company Emagea.

Preparation/activation effective microorganisms (Ema)

Ingredients:

• 5% EM-1 (mother of microorganisms)
• 3% sugar cane molasses
• 50 L of water

Good quality water and work in aseptic conditions as possible. Use a fermenter (wine-beer) with aquarium heating element/serpentine. Fermenter with water at 30°C where molasses will be added, insert EM-1 and leave for 10 days at 30°C, degassing at least 2-3 times. This will yield 50L of Effective microorganisms. For the Aloe treatment 5L of Ema was taken from the 50L obtained, in 20L of water. From the 20L use 200ml x plant, once every 40 days. The remainder is kept in the fridge at 4°C and can be used every time for new multiplications with the procedure indicated above.

The experimental groups were:

• Group control (CTRL) irrigated with water, fertilized, and treated with mineral oil-based;
• Group with Effective microorganisms (EM) (peat 50% + pumice 50%) irrigated with water, fertilized, and treated with propolis, equisetum, and neem.

On November 8, 2021, leaves length (Figure 2a), leaves width, pH gel, fresh leaves weight, fresh gel weight, optical density on gel nutrient and chemical composition were analyzed. In addition, the content of sugars, aloin, and proline, has been evaluated. 2 leaves per plant, 3 plants per treatment for the evaluation of sugars, proline, and aloin have been selected (Bates et al., 1973). The presence of mealybugs was also evaluated on the leaves (total percentage of leaves per thesis in which the presence of the pathogen was detected).

Statistics

The experiment was done in a randomized complete block design. To analyze the collected data, one-way ANOVA was used through GLM univariate procedure, to assess significant (P ≤ 0.05, 0.01, and 0.001) differences among treatments. Then, mean values were separated by LSD multiple-range test (P = 0.05). Graphics and statistics were supported by the programs Costat (version 6.451) and Excel (Office 2010).

rESULTS AND DISCUSSION

 The experimental trial at Welcare Industries S.P.A. (Orvieto) showed a significant improvement of agronomic parameters analyzed on Aloe vera plants treated with Effective microorganisms and a significant reduction of the presence of mealybugs following treatments with repellents of plant origin. In particular, there was a significant improvement in leaf length and width, fresh weight of leaves, and gel. On the Em-treated gel, there was also a significant reduction in optical density (indicative of greater gel purity) and an increase in sugar content. In particular, there was a significant increase in the content of fructose, glucose, proline, and aloin. On the other hand, there were no significant differences between treatments in pH, soluble solids, and fiber content

In (Table 1), there was a significant increase in leaf length in (EM) 54.86 cm compared to (CTRL) with 50.52 cm and leaf width 9.73 cm (EM) in comparison to 8.84 cm (CTRL) (Figure 3). There was also a significant increase in leaf fresh weight, 382.93 g (EM) concerning 375.11 g (CTRL), and gel fresh weight 138.24 g (EM) in comparison to 132.03 of the untreated control (Figure 4). In contrast, no differences were found in gel pH.

In (Table 2) there was a reduction in the optical density of the gel treated with Effective microorganisms 1.03 (abs) compared to the untreated control 1.09 abs, indicating higher purity. In terms of sugar content, the EM thesis was also better with 1336.59 mg/L compared to 1324.60 mg/L for the control. It was also shown that the treatment with plant repellents based on propolis, neem, and horsetail significantly reduced the presence of mealybug on the leaves, 9.56% (EM), compared to 12.61% (CTRL). There were no significant differences in soluble solids and fiber in the two treatments.

All treatments significantly increased fructose, glucose, proline, and aloin content compared with the untreated control (Table 3). Specifically concerning fructose, the thesis (EM) with 80.75 (mg (g DW)^-1 was better than (CTRL) with 69.89 (mg (g DW)^-1. Also for glucose the thesis (EM) with 34.35 (mg (g DW)^-1 was better than (CTRL) with 32.24 (mg (g DW)^-1. The thesis (EM) also increased proline content with 0.63 (mg (g DW)^-1 compared to the untreated control 0.52 (mg (g DW)^-1 and aloin where the thesis (EM) with 147.01 (mg (g DW)^-1 was superior to (CTRL) with 143.12 (mg (g DW)^-1.

The rhizosphere is the fraction of soil that surrounds plant roots (Kennedy, 1999). This definition was introduced by the German agronomist Lorenz Hiltner in 1904. Today we know that in the rhizosphere lives a wide variety of microorganisms, fungi, bacteria, which can be useful or harmful for plant growth (Ortas et al., 2001). Despite the positive progress in the scientific field, the aspect and the role of 90% of the microbial species present in the soil is still unknown. The rhizosphere is undoubtedly actively influenced by the plant. Plant roots continually excrete a complex mixture of substances that can locally alter the soil (water and pH content, oxygen) and better capture nutrients. Shoots secrete up to 40% and adult plants up to 20% of their fixed carbon into the rhizosphere, increasing the viability of microbial colonies in the rhizosphere. The microbial flora around the plant root differs significantly from that of the surrounding soil in both its composition and activity (Apte & Thomas, 1997).

PGPR (Plant Growth-Promoting Rhizobacteria) represents a group of bacteria of the rhizosphere able to stimulate plant development, both by improving mineral nutrition and by producing biocontrol factors (Prisa, 2018). They include bacteria belonging to different genera: Acinetobacter, Alcaligenes, Arthrobacter, Azospirillum, Azotobacter, Bacillus, Burkholderia, Enterobacter, Erwinia, Flavobacterium, Proteus, Pseudomonas, Rhizobium, Serratia, Xanthomonas. Stimulation of plant growth occurs through direct and indirect mechanisms (Kawalekar, 2013; Olle, 2014).

PGPRs can act directly on the nutrients present in the soil making them available to plants, thus performing a biofertilizer action (Kour et al., 2019). Nitrogen, phosphorus, potassium, and iron are indispensable elements for plant survival and growth. Although they are particularly abundant in the soil, very often they are not directly usable by plants, as they are present in a non-assimilable form. The metabolic activity of numerous bacteria in the rhizosphere converts these molecules into soluble forms (Kumar et al., 2016). Direct mechanisms include nitrogen fixation, phosphorus solubilization, production of phytohormones (auxins, cytokines, and gibberellins), and production of siderophores (Nielsen et al., 2002). In this test, plants treated with (EM) showed significant improvement in agronomic parameters of Aloe vera plants and gel quality characteristics. In particular, there was a significant increase in sugars, fibers, and an improvement in gel purity. This improvement in the quality of Aloe plants caused by the activity of microorganisms has also been observed in previous trials on other vegetable and ornamental species (Prisa, 2019a, 2019b; Prisa & Gobbino, 2021a, 2021b). These aspects are probably related to the microbial influence on the stimulation of root growth, the efficiency of nutrient assimilation by the plant, and the increased solubility of mineral elements in the medium (Rodriguez et al., 2006; Thomas & Singh, 2019). It is also known that microorganisms can improve plant resistance to abiotic stresses, in particular water and nutrient stress (Yadav et al., 2017). Plants have developed during their evolution the most varied defense mechanisms against fungi, bacteria, viruses, insects, and also herbivorous animals. The chemical substances recognized by the plant and that induce its defense reactions are called inducers and promote in the plant defense mechanisms capable of protecting it from the attacks of pathogenic microorganisms, without producing hypersensitivity reactions. Moreover, inducers are also able to act even in absence of the pathogenic agent. Numerous molecules of biological origin are being tested: from proteins to lipids, up to polysaccharides.

Among the plant, metabolites have effect SAR salicylic acid, linoleic acid, active on potato against Phytophthora infestans, galacturonic acid and m-hydroxybenzoic acid on cucumber for anthracnose, jasmonates (including jasmonic acid and methyl jasmonate) active on oat powdery mildew, potato blight, and tomato (El-Seedi et al., 2012). Laminarins selected extracts of algae, seem to have an interesting activity against Botritys cinerea and Plasmopara viticola on the grapevine, P. herpotrichoides, Septoria tritici, Septoria nodorum, Erysiphe graminis, Fusarium roseum and Puccinia recondita on wheat and Phytophthora infestans on potato and Venturia inaequalis on an apple tree (Caballero-Gallardo et al., 2011). Extracts of ivy (Hedera helix) applied to apple and Cotoneaster plants seem to have a certain SAR activity against Erwinia amylovora agent of the bacterial fire blight of pome fruits (Frances & Wirtz, 2005). Oligosaccharins are a small group of oligosaccharides that cause hormonal effects on plants (Elmhalli et al., 2009). They are effective at nanomolar to micromolar concentrations thus giving them the status of phytoregulators (Feuerstein et al., 1986). The effects that some of these carbohydrates produce include stem elongation, stimulation of ethylene production, auxin inhibitory action, and finally stimulation of various defensive actions that the plant can put in place (Chang et al., 2001).

Also in this trial, the use of propolis, neem extracts, and horsetail allowed a significant reduction of the presence of mealybug on Aloe vera leaves. An interesting aspect is that the pathogen can often be lethal for Aloe and other succulents. The repellent effects are certainly to be found in the very strong-smelling volatiles that can repel insects, their antibiotic, antifungal, and antiviral action, and their resistance-inducing action in plants.

Table 1. Characteristics of fresh Aloe vera leaves and gel

One-way ANOVA; n.s. – non significant; *,**,*** – significant at P ≤ 0.05, 0.01 and 0.001, respectively; different letters for the same element indicate significant differences according to Tukey’s (HSD) multiple-range test (P = 0.05). Legend: (CTRL): control; (EM): Effective microorganisms; LL: leaves length; LW: leaves width; FLW: fresh leaves weight; FGW: fresh gel weight

Table 2. Chemical properties and mealybug detection of Aloe vera leaves

One-way ANOVA; n.s. – non significant; *,**,*** – significant at P ≤ 0.05, 0.01 and 0.001, respectively; different letters for the same element indicate significant differences according to Tukey’s (HSD) multiple-range test (P = 0.05). Legend: (CTRL): control; (EM): Effective microorganisms; OD: Optical density; SS: Soluble solids; S: Sugars; F: Fibre; MB: mealybugs

Table 3. Chemical properties of Aloe vera leaves

One-way ANOVA; n.s. – non significant; *,**,*** – significant at P ≤ 0.05, 0.01 and 0.001, respectively; different letters for the same element indicate significant differences according to Tukey’s (HSD) multiple-range test (P = 0.05). Legend: (CTRL): control; (EM): Effective microorganisms; FR: Fructose; GL: Glucose; PR: Proline; AL: Aloin

CONCLUSION

The trial showed that the use of rhizosphere bio stimulating microorganisms can improve growth and leaf production in Aloe vera. In addition, the use of microorganisms can influence the quality of the gel produced by these plants and increase the presence of useful metabolites for cosmetic and pharmaceutical companies. The use of plant extracts in the defense of succulent plants can be a valid alternative to synthetic substances, especially in biological protocols. The repellent substances present in some plants can be used to control the presence of pathogenic insects, fungi, and viruses, strengthening plants in the presence of abiotic stresses such as the absence of water, saline soils, heat, and cold. The application of symbiotic microorganisms and plant extracts for defense in agricultural operations can therefore ensure higher production standards, with a possible improvement in the agronomic quality of plants, also reducing the use of water and fertilizers. This experiment may be of particular interest to farms that want to focus on the production of succulents for medicinal and cosmetic use where plant quality levels must be high.

ACKNOWLEDGMENTS: Thanks to Welcare Research srl for their collaboration, in particular to Dr. Fulvia Lazzarotto (CEO Welcare Research) for her availability and participation in the research. Thanks to Simone Bartoli and Serban Olaru for their help during the field trials.

CONFLICT OF INTEREST: None

FINANCIAL SUPPORT: The research has been funded in the project "BIOALOV: Biostimulants and microorganisms for the qualitative improvement of Aloe production and biological control".

ETHICS STATEMENT: None

REFERENCES               

Apte, S. K., & Thomas, J. (1997). Possible amelioration of coastal soil salinity using halotolerant nitrogen-fixing cyanobacteria. Plant and Soil, 189(2), 205-211.

Bassetti, A., & Sala, S. (2003). Il grande libro dell'aloe. Trento, Zuccari.

Bates, L. S., Waldren, R. P., & Teare, I. D. (1973). Rapid determination of free proline for water-stress studies. Plant and Soil, 39(1), 205-207.

Boudreau, M. D., & Beland, F. A. (2006). An evaluation of the biological and toxicological properties of Aloe barbadensis (miller), Aloe vera. Journal of Environmental Science and Health Part C, 24(1), 103-154. doi:10.1080/10590500600614303

Caballero-Gallardo, K., Olivero-Verbel, J., & Stashenko, E. E. (2011). Repellent activity of essential oils and some of their individual constituents against Tribolium castaneum Herbst. Journal of Agricultural and Food Chemistry, 59(5), 1690-1696.

Cakmakci, R., Dönmez, M. F., & Erdoğan, Ü. (2007). The effect of plant growth promoting rhizobacteria on barley seedling growth, nutrient uptake, some soil properties, and bacterial counts. Turkish Journal of Agriculture and Forestry, 31(3), 189-199.

Chang, S. T., Chen, P. F., Wang, S. Y., & Wu, H. H. (2001). Antimite activity of essential oils and their constituents from Taiwania cryptomerioides. Journal of Medical Entomology, 38(3), 455-457.

Condor A. F., Gonzalez P. & Lakre C. (2007). Effective microorganisms: myth or reality?. Revista Peruana de Biología, 14(2), 315-319.

Elmhalli, F. H., Pålsson, K., Örberg, J., & Jaenson, T. G. (2009). Acaricidal effects of Corymbia citriodora oil containing para-menthane-3, 8-diol against nymphs of Ixodes ricinus (Acari: Ixodidae). Experimental and Applied Acarology, 48(3), 251-262.

El-Seedi, H. R., Khalil, N. S., Azeem, M., Taher, E. A., Göransson, U., Pålsson, K., & Borg-Karlson, A. K. (2014). Chemical composition and repellency of essential oils from four medicinal plants against Ixodes ricinus nymphs (Acari: Ixodidae). Journal of Medical Entomology, 49(5), 1067-1075.

Elsharkawy, M. M., Shivanna, M. B., Meera, M. S., & Hyakumachi, M. (2015). Mechanism of induced systemic resistance against anthracnose disease in cucumber by plant growth-promoting fungi. Acta Agriculturae Scandinavica, Section B—Soil & Plant Science, 65(4), 287-299.

Feuerstein, I., Müller, D., Hubert, K., Danin, A., & Segal, R. (1986). The constitution of essential oils from Artemisia herba alba populations of Israel and Sinai. Phytochemistry, 25(10), 2343-2347.

Frances, S. P., & Wirtz, R. A. (2005). Repellents: past, present, and future. Journal of the American Mosquito Control Association, 21(sp1), 1-3.

Glick, B. R. (1995). The enhancement of plant growth by free-living bacteria. Canadian Journal of Microbiology, 41(2), 109-117.

Kawalekar, J. S. (2013). Role of biofertilizers and biopesticides for sustainable agriculture. Journal of Bio Innovation, 2(3), 73-78.

Kennedy, A. C. (1999). The rhizosphere and spermosphere. In: Principles and Applications of Soil Microbiology, Upper Saddle River, New Jersey, 389-407.

Kour, D., Rana, K. L., Sheikh, I., Kumar, V., Yadav, A. N., Dhaliwal, H. S., & Saxena, A. K. (2019). Alleviation of drought stress and plant growth promotion by Pseudomonas libanensis EU-LWNA-33, a drought-adaptive phosphorus-solubilizing bacterium. Proceedings of the National Academy of Sciences, India Section B: Biological Sciences, 1-11.

Kumar, V., Baweja, M., Singh, P. K., & Shukla, P. (2016). Recent developments in systems biology and metabolic engineering of plant–microbe interactions. Frontiers in Plant Science, 7, 1421.

Marshall, J. M. (1990). Aloe vera gel: What is the evidence. The Pharmaceutical Journal, 24, 360-362.

Miladi, S., & Damak, M. (2008). In vitro antioxidant activities of Aloe vera leaf skin extracts. J Soc Chim Tunisie, 10(10), 101-9.

Nielsen, T. H., Sørensen, D., Tobiasen, C., Andersen, J. B., Christophersen, C., Givskov, M., & Sørensen, J. (2002). Antibiotic and biosurfactant properties of cyclic lipopeptides produced by fluorescent Pseudomonas spp. from the sugar beet rhizosphere. Applied and Environmental Microbiology, 68(7), 3416-3423.

Olle, M. (2014). Effect of Effective microorganisms on yield, quality, and preservation of vegetables. In: Gardening Forum, 10-13.

Ortas, I., Kaya, Z., & Cakmak, I. (2001). Influence of arbuscular mycorrhizae inoculation on growth of maize and green pepper plants in phosphorus-and zinc-deficient soil. In Plant Nutrition (pp. 632-633). Springer, Dordrecht.

Prisa, D. (2019a). Effect of chabazitic-zeolites and effective microorganisms on growth and chemical composition of Aloe barbadensis Miller and Aloe arborescens Miller. International Journal of Agricultural Research, Sustainability, and Food Sufficiency (IJARSFS), 6(01), 315-321.

Prisa, D. (2019b). Effective microorganisms for germination and root growth in Kalanchoe daigremontiana. World Journal of Advanced Research and Reviews, 3(3), 047-053.

Prisa, D. O. M. E. N. I. C. O. (2017). Italian chabazitic-zeolitite and Effective microorganisms for the qualitative improvement of olive trees. Atti del Convegno di Calci (PI), 13-17. doi:10.2424/ASTSN.M.2018.2

Prisa, D., & Gobbino, M. (2021). Biological treatments for quality improvement and production of Aloe vera gel. GSC Advanced Research and Reviews, 9(1), 054-063.

Prisa, D., & Gobbino, M. (2021). Microbic and Algae biofertilizers in Aloe barbadensis Miller. Open Access Research Journal of Biology and Pharmacy, 1(2), 1-9.

Reynolds, G. W. (1966). The Aloes of Tropical Africa and Madagascar. Mbabane Swaziland. The Trustees The Aloes Book Fund.

Rodríguez, A. A., Stella, A. M., Storni, M. M., Zulpa, G., & Zaccaro, M. C. (2006). Effects of cyanobacterial extracellular products and gibberellic acid on salinity tolerance in Oryza sativa L. Saline Systems, 2(1), 1-4.

Thomas, L., & Singh, I. (2019). Microbial biofertilizers: types and applications. In Biofertilizers for sustainable agriculture and environment (pp. 1-19). Springer, Cham.

Yadav, A. N., Verma, P., Singh, B., Chauhan, V. S., Suman, A., & Saxena, A. K. (2017). Plant growth promoting bacteria: biodiversity and multifunctional attributes for sustainable agriculture. Advances in Biotechnology & Microbiology, 5(5), 1-16.