ARTÍCULOS DE REVISIÓN
Received: 01 March 2020
Accepted: 01 September 2020
Published: 01 November 2020
Abstract: Agriculture is passing through a global crisis; productivity and sustainability are two challenges for farmers as they strive to produce more food, preserve the environment and maintain the health of living beings. Developed and developing countries need to adopt new productive systems to improve the quality of food, preserve the environment and ecosystems. Currently, the abuse and misuse of chemical products has caused a series of problems including contamination of soil and water and pest resistance to active ingredients. Climate change and other transformations that agriculture is going through, have generated over recent decades the emergence of an industry of biological inputs, which are environmentally friendly products with little risk to human health. These biological products have characteristics and modes of action that provide pest control with fewer risks than from the use of chemical pesticides. This review covers the importance, use and applications of biological products based on beneficial fungi in Latin America, highlighting the project "Biocontrol for Sustainable Farming Systems" carried out in Ecuador, funded by MFAT New Zealand, which has demonstrated the efficiency of microbial products in agriculture through the research results obtained during the project. In addition, aspects related to quality control of bioproducts and regulations for their registration are also mentioned.
Keywords: Agricultural production, beneficial microorganism, biological control, Ecuador, quality control, regulation, sustainable agriculture.
Resumen: La agricultura atraviesa una crisis mundial, la productividad y la sostenibilidad son dos factores que los agricultores están desafiando para producir más alimentos, preservar el medio ambiente y la salud de los seres vivos. Los países desarrollados y subdesarrollados necesitan adoptar nuevas alternativas productivas, que mejoren la calidad de los alimentos, preservando el medio ambiente y los ecosistemas. Actualmente, el abuso y mal uso de los productos químicos ha causado una serie de problemas como contaminación (suelo y agua) y resistencia de las plagas a los ingredientes activos. El cambio climático y otras transformaciones por las que atraviesa la agricultura, generaron hace décadas la aparición de la industria de insumos biológicos, que son productos amigables con el medio ambiente y la salud humana. Estos productos tienen características y modos de acción que ayudan al control de plagas con menos riesgos que el uso de pesticidas. Esta revisión recopila la importancia, el uso y las aplicaciones de productos biológicos basados en hongos benéficos en América Latina, destacando el proyecto "Biocontrol para Sistemas Agrícolas Sustentables" ejecutado en Ecuador, financiado por MFAT Nueva Zelandia, el cual ha demostrado la eficiencia del uso de microorganismos en la agricultura a través de los resultados de las investigaciones llevadas a cabo durante la ejecución de este Proyecto. Además, se mencionan aspectos relacionados con el control de calidad de productos biológicos y las regulaciones para su registro.
Palabras clave: Agricultura sustentable, control biológico, control de calidad, microorganismos benéficos, Ecuador, normativa, producción agrícola.
The growing global concern about the damage that excessive use of synthetic pesticides is causing human health, environment, biodiversity and food security, has provoked a general rejection of chemical control of pests (CCP) in agricultural production1. In this context, contemporary agriculture faces the challenge of intensifying agricultural production, while ensuring the protection of the environment, human health, with sustainable solutions, in which the safe and rational use of pesticides may be a key factor, to guard against future food shortages2.
The soil, which provides the base for agriculture, is one of the habitats with the greatest biodiversity of microbes in nature with about 104 to 109 microorganisms of different species per gram of soil. To maintain soil health, environmentally friendly technologies such as the use of biological control (BC) within integrated pest management (IPM)3 have been promoted.
The last decades have seen a worldwide trend to restrict the application of agrochemicals to reduce their negative impacts on the environment4 and reduce residues in harvested products5. At the same time, resistance has been generated by pests and pathogens to many pesticides preventing effective control and inducing farmers to overuse them6. This has stimulated the search and implementation of control alternatives friendly to human health and the environment, as is the case of the BC.
Biological control was conceived at the beginning of the 19th century, when naturists from different countries observed the important role of entomophagous organisms in nature and following the example of these “biological controllers” attempted to restore the disturbed ecological balance created by agriculture by using living organisms or their metabolites to eliminate or reduce the damage caused by harmful organisms7.
Biological control agents (BCAs) are being developed worldwide, including organisms such as fungi, bacteria, viruses and insects that reduce the populations of pests that affect crops. Fungi in particular, have aroused the interest of companies and research organizations due to their role in controlling insects and plant diseases, without damaging the environment or human health. Sustainable development must guarantee future generations natural assets equivalent to those that present generations inherited and the advancement towards environmental sustainability will require behavioral changes in people as consumers, which will have consequences on the traditional roles of the family, the community, government agencies and the market, as well as require a complete reorganization of traditional farming techniques8.
Pests are the main limitation of agricultural production, and pest control (PC) can represent up to a third or more of the cost of production, depending on the severity of the attack. Traditionally, PC has been carried out with the use of synthetic chemical products (CP), many of which cause deterioration of the environment, human health, the evolution of more aggressive pest populations, the loss of pollinators and natural predators. The negative effects of pesticides have driven the search for new control strategies that prioritize recovery of natural environmental regulation mechanisms within IPM9,10.
BC is a friendly and natural alternative for the environment, which can involve the use of beneficial microorganisms (BM) such as fungi, bacteria, nematodes and viruses that act to reduce the population of pests to lower, manageable densities, either temporarily or permanently11. The fundamental purpose of biological management of agricultural pests is to miti gate the harmful effects and economic losses of insect pests and plant diseases, reduce or replace the use of chemical pesticides, and integrate compatible and synergistic strategies to optimize the effectiveness of IPM12.
The advantages of BC are clear since it has no negative effects on workers in the field, it acts permanently within the natural biodiversity and the agricultural products marketed are perceived of the highest quality because they do not have chemical residues, concepts that are closely related to IPM13. Additionally, there are advantages with little competition for products in the market and the global trend towards the preservation of the environment and the consumption of foods free of pesticide chemicals11. In general, for effective implementation of a BC strategy, the ecological and social components must be considered from the moment a BCA is discovered and throughout the development process12.
The microorganisms used for BC must present specific characteristics such as having rapid growth, high capacity for reproduction and survival, different levels of dormancy, be free of natural antagonists, have high competitive ability, adaptability to the treated plant and high versatility in the environment. There are a great variety of potentially useful microorganisms which can be used as BCAs, among the most studied and of which a greater number of products have been developed are Trichoderma spp., Bacillus thuringiensis, Bacillus subtilis, Paecilomyces lilacinus and Verticillium lecanii11.
The most common BC strategies are12: i). Conservation, defined as modifying the environment or existing practices to protect and enhance the activity of specific natural enemies (NE) or others that reduce the harmful effect of pests, ii). Classic, which is the international introduction of an exotic BCA, usually coevolved, for an invasive pest leading to its permanent establishment and long-term PC, iii). Augmentative, involving supplemental release of NE, iv). Inoculation, defined as the intentional inoculation of a BCA with the expectation that the agent will multiply and control the pest for a prolonged period of time, but not permanently, and v). Inundative, which refers to the release or application of BCA in large quantities to decimate pests when their population increases in such a way that the crop is put at risk.
The objective of this review is to describe the importance of BC as a vital component of sustainable agriculture that preserves natural resources and the environment through the use of microorganisms selected for their high efficiency and safety. Successful cases in Latin America, mainly in Ecuador, will be reported.
Biocontrol can be defined as the use of living organisms to suppress pest populations14. There are several historical references to the use of BC worldwide. Subsistence agriculture has presented practices that suppressed pest populations, but rather than being based on scientifically proven knowledge, these practices were based on experience and tradition15. The same author mentions that despite the lack of interest in improving the effectiveness of NE, some progress was made in the mid-20th century. In recent years, around 2700 NE have been introduced in 196 different countries or islands for PC, as well as more than 440 species are being produced and sold commercially worldwide16. In the past two decades, BC scientists have studied the risks of the introduction of exotic species. Foreign companies that have justified effectiveness for this type of control and have developed risk assessment to reduce the risks17, consequently the development of products with native species is a recommended alternative. The science behind BC has made remarkable progress in the last 50 years, shifting from trial and error to more predictive approaches based on theories of controller-pest interactions and population dynamics16. The BC has a long history of success in developed and developing countries. In Ecuador BC was studied and applied before 1937, however, there are not many detailed reports or reports on this issue18.
Beauveria used as an entomopathogen. Beauveria sp., belongs to the Division Ascomycota, Order Hipocreales, it is the most widely used entomopathogenic fungus in biological formulations for control of pests in the orders Lepidoptera, Coleoptera, Hemiptera, Diptera and Hymenoptera19-22.
Infection by this fungus begins with the adhesion of the conidia to the host's cuticle, followed by germination, which fixes it in the cuticle producing a hypha that allows it to penetrate the insect with the help of hydrolytic enzymes (proteases, lipases, chitinases), mechanical pressure and other factors23. Within the hemolymph, hyphal bodies produce blastospores that disperse throughout the body, destroying tissues, evading the immune system, and producing toxins that contribute to the death of the host24. After the host dies and with favorable environmental conditions, the hyphae emerge, sporulate on the surface of the corpse, and action of wind, rain or other abiotic and biotic factors will spread the spores to new hosts and a new cycle of infection may start25.
Efficacy of infection is mediated by factors environmental such as humidity, temperature, precipitation, solar radiation, biotic factors such as age of the host and pathogenicity of the biological control agent26-28. Therefore, for BC products propagules must be formulated with additives, such as inert diluents, dispersants, adherents, protectants, among others to increase the useful life time, and ensure its efficiency in the field29,30. The species of the genus Beauveria used as insecticides are B. bassiana and B. brong niartii in the most common presentations, wettable powders followed by suspension concentrates and emulsifiable suspensions22.
These formulations have been used in various crops to control mites (Tetranychus urticae), whiteflies (Bemisia tabaci), beetle (Gonipterus scutellatus), coffee borer (Hypothenemus hampei), banana borer (Cosmopolites sordidus), defoliator worm (Dione juno), thrips, aphids, bed bugs, grasshoppers22,25,31-33.
Trichoderma is the main fungal genus used in biological control. Due to the efficiency and ease of production, Trichoderma is among the most successful BCAs in agriculture. Throughout the world, countless phytosanitary products based on this fungus are marketed, which acts as a biopesticide, biofertilizer, growth promoter and inducer of natural resistance, whose application can be foliar, before sowing in seeds or propagation material, treatments after pruning, incorporation into the soil during sowing or transplanting, irrigation, among others34. The most common formulations available on the market are wettable powders, granules, emulsifiable concentrates and concentrated suspensions35.
Trichoderma is the most studied and used biological fungicide in agriculture. It is a genus of fungi that live freely in soil and root ecosystems. Its antagonistic properties are based on the activation of very diverse mechanisms. Trichoderma can exert the biocontrol of phytopathogenic fungi (PF) indirectly competing for space and/or nutrients, producing antibiotics or stimulating the growth of plants and their defense mechanisms, on the other hand, it can carry out biocontrol directly by means of mycoparasitism36.
Some members of this genus have symbiotic associations with plants such as legumes, grasses, nightshades and others, while other species are used as biocontrol agents against PF such as Rhizoctonia, Pythium, Sclerotium and Phytophthora, among others, which affect many crops of commercial interest such as rice, corn, onion, tomato, beans, wheat, cocoa, etc.37. They are also considered able to degrade highly persistent pesticides in the environment, serving as a soil decontaminating agents38, as well as having antibiotic action and acting as a plant growth promoters (PGP).
Among Trichoderma commercial products the species reported are: T. harzianum, T. asperellum, T. viri de, T. atroviride, T. gamsii, T. hamatum, T. koningii, T. lignorum, T. polysporum and many labels indicate a generic Trichoderma (sp. or spp.)39. Many products consist of a single species of Trichoderma and others with combinations of two or more species, or a mixture with other species of fungi and even bacteria that, when combined with inert materials such as powders, microtalcs, solvents, emulsifiers and other additives, improve the stability of the microorganism, protect it from environmental conditions, giving it a longer viability time during storage, ensuring its effectiveness when applied in the field40.
According to one study41, T. inhamatum has an insecticidal and enzymatic against the fruit fly Drosophila melanogaster, colonizing 12 flies and maintaining chitinolytic enzymatic activity for 24 days after inoculation. Other authors3, argue that species such as T. harzianum are not only used as BCAs, but are also excellent PGPs, increasing the germination of seeds such as sunflower and papaya by 30%, which allows us to deduce that the seeds easily assimilate the products synthesized by the microorganisms, accelerating the germination process.
It has been noted42 that endophytic Trichoderma strains of a plant species such as quinoa produce positive effects on the crop, improving the plant growth and the ability to reduce the severity of Peronospora variabilis, due to the production of substances that directly attack the pathogens or by inducing systemic resistance, which in turn reduces the incidence of the pathogen in the plant.
In crops of high economic importance such as cocoa, the need to change the chemical control strategies against Moniliophthora roreri and M. perniciosa for others more friendly with the environment such as the use of Trichoderma spp. has been recognized because of their ability to inhibit growth of these diseases due to their myco-parasitic activity and the production of various metabolites active against the disease43. Other authors44 have noted that application of T. koningiopsis and T. stromaticum improved cocoa crop health, reducing the incidence of diseases and increasing the production of healthy pods. Furthermore, in this study, T. koningiopsis was the most effective organism for control, adapting to the agroecosystem and surviving with the plants during the dry season.
Studies in roses45 have indicated that Trichoderma spp., being an antagonist fungus, can be combined with other BCAs such as Penicillium sp., to combat diseases such as Sphaeroteca pannosa, and present a positive cost benefit, obtaining a positive return of USD 1.54 with a recovery of USD 0.54 for every dollar invested. Research in Ecuador with the MD-2 pineapple showed that the height, number of leaves and the fresh weight of the fruit increased positively with the application of T. harzianum pre-sowing46.
Trichoderma spp., can be formulated in different formats and applied using different technologies. The field application of T. koningiopsisTh003 formulated as dispersible granules significantly reduced plant mortality by 35% within an integrated management program of the neck and root rot of tomato, an important result given the difficulty in controlling the disease with fungicides47.
The use of different types of biological formulations (powders, granules, liquids) based on microorganisms has consistently increased in response to the need to reduce the use of agrochemicals in PC, but it is necessary to evaluate the quality of these products by determining their biological efficacy, viability and purity which are important characteristics to ensure success in the field48-50. Research on bioformulations based on T. asperellum showed that the microorganism had greater stability during storage in dry solid release systems compared to liquids51.
However, more detailed studies based on conidia of T. asperellum carried out with four prototype bioformulations (wettable powder, emulsifiable concentrate, covered and dispersible granule) have determined the main physicochemical (water activity, percentage humidity, pH, rammed density, suspensibility, particle size, wettability, stability of the emulsifiable concentrate) and microbiological (viability, percentage germination, purity, efficiency of the formulation process) parameters necessary for an effective quality control52.
Research on Trichoderma in Latin America. In Bolivia, one of the methods for BC of insects that has gained importance in recent years is the use of microorganisms such as entomopathogenic fungi which produce secondary metabolites and enzymes: proteases, amylases, chitinases, etc., - key factors in biocontrol41.
The application of Trichoderma as a biocontrol agent of Helminthosporium solani for three years, produced a partial effect, reducing the severity of the disease between 10 to 22%, which although not sufficient for commercial purposes is a biological alternative for the construction of IPM in potato cultivation53.
In Chile, the efficiency of Trichoderma sp. strains on different pathogens has been evaluated, observing different degrees of control depending on the pest, in addition to specificity of action and the safest use of the biocontrol agent. For example, one strain showed excellent behavior in the control of Fusarium solani, F. oxysporum, Phythophtora spp., and Venturia inae qualis in commercial apple and tomato orchards, while the Sherwood strain exhibited efficient control of Botrytis cinerea both in laboratory and in experimental lettuce cultures54.
In Argentina, the antagonistic behavior in vitro of 15 native strains of Trichoderma isolated from soil samples was tested against strains of Sclerotinia sclerotiorum using the dual culture methodology. Four isolates were selected as the most efficient in inhibiting the growth and formation of sclerotia of S. sclerotiorum. One of the isolates expressed a superior behavior, managing to cancel the, in addition to having registering the maximum growth in the dual culture and preventing formation of sclerotia. On the basis of this study, the most promising strains are being produced on cheap substrates (broken corn and wheat grains) and formulated before evaluation of their effects in vivo as a biocontrol agents and growth promoters55.
In Colombia, isolates of Trichoderma sp., have shown effects on the phytopathogenic fungi Fusarium sp. and Rhizoctonia sp. producing different degrees of antagonism. The greatest impact was noted on the growth of Fusarium sp., with 78.30% inhibition compared to 18.67% of Rhizoctonia sp. Additionally, the research showed that banana flour with 5% and 2% yeast without buffer is an efficient medium as a substrate for the multiplication of Trichoderma sp., producing high numbers of conidia, with a final concentration of 1.1x109 conidia/mL, and pH 5.7 seven days after processing56.
In Venezuela, the inhibitory effect of three native isolates of T. harzianum was tested against phytosanitary problems caused by Sclerotium rolfsiiin Aloe vera plantations. The isolates produced a good antagonistic effect, inhibiting the mycelial growth of the pathogen (50-90%) and reducing sclerotia formation (86%)57.
In Peru, Peronospora variabilis affects the quinoa crop and can cause a reduction in the yield of up to 99%. A BC strategy for this pest has been the use of Trichoderma because it has an endophytic capacity of up to 60% and produced an increase in yield (3127.30 kg.ha-1) compared to the control (1141.27 kg.ha-1)42.
Trichoderma has also been used as a phosphate solubilizer, an effect proven in Brazil through studies carried out to determine the increase in rice biomass under greenhouse conditions. The influence of Trichoderma inoculation on rice was determined after 30 and 45 days from sowing, measuring different growth parameters, including height, root length and dry matter. Plant growth increased significantly (24.9 cm) in the presence of natural phosphate stimulated by Trichoderma in comparison with the control (18 cm). In general, the relative efficiency of biomass (RE) was significantly higher, with a high content of available P and higher P utilization efficiency (P-UEF), raised from 10 to 61% and from 12 to 62%, for the treatments respectively58.
Regulations for the control of bio-inputs. The stability and quality of the microbial active ingredient are the main requirements for the registration and commercialization of bio-products. Each country has its own agency that is responsible for developing regulations and procedures that allow for registration and ensure the quality of the products that are marketed in their territory59.
The national market for BC in the Andean countries is growing, however, there is no established regulation in most of these countries for the development, elaboration, production and commercialization of biological products (BP)13.
At the Latin American level, Colombia is the country whose regulations can serve as an example for the registration of bioformulations. The agency in charge of regulating and guaranteeing the quality of agricultural inputs is the Instituto Colombiano Agropecuario (ICA) through Resolution 000698 of February 4, 201135. Until December 2018, 303 bio-products have been registered in this country, of which 38 are based on Trichoderma60.
However, in countries such as Argentina, Chile, Bolivia, Peru, Ecuador and Venezuela, the regulations and procedures for the regularization of bioproducts are in the process of being improved. It is estimated that only 38% of the bio-products marketed are registered39.
In Argentina, the Servicio Nacional de Sanidad y Calidad Agroalimentaria (SENASA), through Resolution 350/99, has so far registered 27 BP, of which 3 are based on Trichoderma61.
In Bolivia, the Servicio Nacional de Sanidad Agropecuaria e Inocuidad Alimentaria Unidad Nacional de Sanidad Vegetal (SENASAG) through Administrative Resolution No. 055/2002 and No. 012/2006 has registered 10 BP of these 3 are based on Trichoderma until June of 201162,63.
In Chile, the Servicio Agrícola y Ganadero (SAG), through Resolution 3670/1999 and 2229/2001, has registered 21 products based on microorganisms, of which 9 have Trichoderma as an active ingredient64.
In Peru, the Ministerio de Agricultura Servicio Nacional de Sanidad Agraria (SENASA), has registered around 120 products based on microorganisms, of which 17 are based on Trichoderma65.
In Ecuador, on August 5, 2019, the Agencia de Regulación y Control Fito y Zoosanitario (AGROCALIDAD) issued the regulations for the registration of biopesticides and related materials, through Resolution 143 and issued the document called "Manual of Procedures for the Registration and Control of Biological Control Agents, Vegetable Extracts, Mineral Preparations, Semiochemicals and Andean Products Related to Agricultural Use”. Previously, commercial BP were registered in the category of fertilizers and of 13 products that appear in the database up to April 2019, 4 are based on Trichoderma66. Through the new resolution, it is expected to formalize the commercialization of biopesticides, guarantee quality inputs in the national market, provide agricultural inputs for all productive contexts, regulate the registration based on requirements according to new technologies and generate tools for the benefit of the agricultural sector.
Experiences in Ecuador: Project "Biocontrol for Sustainable Farming Systems". In Ecuador, the use of BCAs has begun to gain more space with the conduct of research by public and private companies that have tested this type of input in various crops such as flowers, bananas, sugar cane, coffee, potatoes, broccoli, blackberries, among others. Favourable results in pest control and growth promotion have led to the adoption of biopesticides and other beneficial microbes reducing the negative impact of overuse of agricultural chemicals on the environment and obtaining food free of contaminants67-70.
In 2009, the "Biocontrol for Sustainable Farming Systems" Project began in Ecuador, executed by the Instituto Nacional de Investigaciones Agropecuarias (INIAP) and the Instituto Interamericano de Cooperación para la Agricultura (IICA), with financing from MFAT-New Zealand. It had the advice of specialists from AgResearch, Plant and Food Research, Lincoln University, INIFAP (Mexico), CORPOICA (Colombia) and USDA (USA), with the aim of generating beneficial microbial formulations and biocontrol technologies that could be implemented in agricultural crops under the environmental conditions of Ecuador71. The main achievements achieved through this project during the last ten years are presented below, among which are:
i). Creation of a biological control laboratory for research in INIAP. A Biological Control Laboratory was created at the INIAP Santa Catalina Research Site as part of the Department of Plant Protection. This laboratory has developed several research processes, mainly on producing microbial formulations based on beneficial fungi and prospecting, isolation and multiplication of beneficial microbes from soil samples and infected pests. In addition, methods for quality control of bio-products based on fungi have been standardized. The quality control carried out includes the implementation of microbiological tests of the product (concentration, viability, percentage of germination and purity) and physicochemical analysis (water activity, percentage of humidity, pH, rammed density, suspension, particle size, wettability of dry products and stability of emulsifiable concentrates)72. On For the implemented methods, continuous training has been provided for technicians from bio-input production laboratories of the Ministry of Agriculture (MAG), private companies, as well as students and teachers from agricultural schools and universities.
In addition, a Biocontrol Laboratory Technicians Network was formed, which is made up of researchers from INIAP, laboratory technicians from private companies, and government technicians from MAG and AGROCALIDAD. Various workshops have been held on methods for the multiplication, production and analysis of the quality of bio-inputs based on beneficial fungi, in order to generate high-quality bio-inputs that favor sustainable agriculture in Ecuador72. It is expected to formalize this Network of Biocontrol Technicians to give it continuity and greater representation.
ii). Evaluation of the quality of commercial bio-inputs. During the first years of the project, the need to evaluate the quality of bioproducts available in the Ecuadorian market was identified, in order to understand the situation and establish a baseline. In 2011, a survey of commercial bio-inputs was carried out including quality control, through microbiological tests, to determine the concentration, viability and purity of microbes in the product. Eighteen commercial products were evaluated, of these 64% of the products did not have viable microorganisms as declared on the label, and 42% showed a high percentage of contamination. Thirty five percent contained viable conidia but the quantified concentration of the microorganism was lower than that reported on the label. Based on the results obtained in the analyzes, the commercial products evaluated were clearly deficient in their quality.
Once the limitation in quality of the commercial products had been determined and recognized as a fundamental factor in their inefficiency, a training program and dissemination of results began, integrating the different actors (AGROCALIDAD, MAG, bio-input producers, farmers and universities) involved in the production of bio-inputs. Ongoing training was carried out in the course of this project on the methods that were defined in INIAP to unify criteria and standardize processes for evaluating the quality of bio-inputs and improve production methods.
In 2019, a new evaluation was carried out to determine impact of the training component of the project in influencing improvement of production systems and quality of bio-inputs available in the Ecuadorian market. Of 13 products evaluated based on Trichoderma spp., 46% complied with what was established on the product label. Technicians from the companies producing the products reaching standards had attended project workshops but the continued presence of sub-standard, un-registered products on the market shows that it is still necessary to reach more producers with training workshops. In this evaluation, a new parameter called “water activity” was considered, which represents the amount of water available for microorganisms to develop their metabolism, which influences their growth and viability. To maintain the viability of a biological product based on fungi, water activity should be between 0.2 to 0.7 aw72, 61%of the evaluated products showed optimal values of this parameter.
iii). Diagnosis of laboratories for quality control of bio-inputs in Ecuador. An evaluation of public sector laboratories and private companies that could perform analysis of quality of bio-inputs was carried out. These included biological control agents (66.67%), plant extracts (14.29%) and related products for agricultural use (19.04%). BCAs analyzed included fungi and bacteria (43.75%), predators and parasitoids (43.75%) and entomopathogenic nematodes (12.5%). Ninety percent of the laboratories reported that they have experience and facilities to carry out research bioassays and 71.43% of the laboratories claimed to have the capacity to evaluate the quality of bio-inputs. The 80.95% of the surveyed laboratories were located in the Sierra region and 19.05% in the Coast. Ninety percent of the laboratories expressed interest in being part of the Bio-Input Laboratory Network coordinated by AGROCALIDAD to carry out quality control analysis; an activity providing a service that will generate an economic income.
iv). Development of prototypes of bioformulations based on beneficial fungi. The concentration of active ingredient, feasibility of production and purity of the final product are important characteristics to ensure the success of a formulation. Several formulations: dispersible granule, covered granule, wettable powder and emulsifiable concentrate, were evaluated to determine the stability and survival of T. asperellum and Purpureocillium lilacinum spores. The bioformulations were made after mass production of spores on rice substrate and use of formulation additives available nationally. Storage stability tests showed that dry formulations of T. asperellum, covered granules and soluble granules, were the most stable with viabilities of 1.6x108 and 1.7x108 CFU/g, respectively and purities greater than 98%, after storage at room temperature (16 °C) for two months. In the P. lilacinum formulations, the wettable powder showed best stability after two months storage, with a viability of 1.1x108 CFU/g and purity of 99%51. In addition, physicochemical and microbiological parameters of prototype T. asperellum formulations (wettable powder, emulsifiable concentrate, coated granule and dispersible granule) were evaluated under ambient (30 °C) and refrigerated (4 °C) temperature conditions during six months of storage. The wettable powder and covered granules stored at 4 °C stood out with conidial viability exceeding 85% germination and colony concentration maintained above 1x108 spores/g. However, the wettable powder at 30 °C maintained a germination of 81% after 6 months storage52. In general, the results of the evaluation of the physicochemical characteristics (stability, wettability, moisture percentage, water activity and pH), determined the stability of these prototypes. Currently, the scaling-up of these technologies is being developed in Ecuador in collaboration with private companies, which is the scaling model adopted by other countries such as New Zealand.
These studies showed quality could be maintained for powder and granule formulations, which preserved the stability of microorganisms at ambient temperatures without the need for refrigeration during transport and storage.
v). Investigations in agricultural crops. The Andean potato weevil (Premnontrypes vorax) is considered one of the most important pest insects of potatoes due to the damage it causes feeding on the tuber. To control this pest, farmers have applied insecticides of high toxicity, in some cases in excess, which have negative impacts on the environment and the health of producers and consumers73. In a study of alternatives to chemical insecticides, the efficacy of two bioformulations of B. bassiana (clay and rice substrate) and the system of application (traps with plants bait with the bioformulate, foliar applications of the bioformulate at 40, 60 and 80 days; application of granules to the base of the plants at 40, 60 and 80 days) were studied. It was observed that with a population of 17 adults of Andean potato weevil/m2, in a clay-silty soil, with organic matter content of 9.8%, treatment with a clay based granular formulation of B. bassiana in foliar applications and at the base of the plants at 40, 60 and 80 days, resulted in 8.7% weevil damage in tubers, compared with 23.7% tuber damage in the control without application. This entomopathogen, therefore, reduced the percentage of damage in harvested tubers, and could provide a biological alternative for incorporation into management programs for this pest, provided there are good humidity conditions and presence of high levels of organic matter in the soil74.
Broccoli (Brassica oleracea) is Ecuador's second most important non-traditional export product and Trichoderma sp. has been applied for seedling protection and crop management. Trichoderma sp. fungus has been applied to the substrate of broccoli seedlings and monitored to determine its effect from the sowing of the seed until before transplantation. At 32 days, and prior to transplanting, the seedlings treated with Trichoderma sp., had higher fresh weights than treatments without application. By quantifying the concentration of the fungus in the substrate, it was found that the population was quite stable but was reduced (3x103 CFU/g of soil) in treatments that received the application of CP, while the treatments with Trichoderma sp. without application of CP maintained a higher population of the fungus (1x104 CFU/g soil). Additionally, the compatibility of Trichoderma sp., with CP used in the seedling production system was tested. The Trichoderma sp. isolate was compatible with the pesticides: Hymexazol, Propamocarb, Betaciflutrin, Metolachlor, Lambdacyha-lothrin, Lambdacyhalothrin + Thiamethoxam, Radian, Chlorpyrifos, Dimethoate, Emamectin Benzoate and Acetamiprid; while, incompatibility was observed with Thiram, PCNB, Captan, Carboxin + Captan, Carbendazim, Iprodione, Pyraclostrobin, Chlorpyrifos, Chlorothalonil and Ciprodinil + Fludioxonil was observed. The results show that Trichoderma sp. can be included in intensive production systems of broccoli seedlings, favoring their growth75.
Avocado (Persea americana) is a fruit tree of economic importance in Ecuador with export potential. The application of T. harzianum (0.18 g/plant at a concentration of 4x108 CFU/g of product) in the seedling substrate of the Criollo cultivar was evaluated. It was determined by nutrient absorption analysis that the inoculated plants showed higher level concentrations of macronutrients such as nitrogen, calcium and magnesium and micronutrients such as manganese and copper in the root system compared to the control without application, indicating that this beneficial fungus acts as a plant growth promoter76.
Andean blackberry (Rubus glaucus), is a native fruit of the Andes with great productive potential. Two technological systems were tested for the management of the blackberry crop: a) clean (with moderate use of synthetic products and chemical fertilization) and b) organic (with use of organic fertilizers (OF) and BP), both systems were managed with and without application of Trichoderma sp. Clean management, with applications of the fungus produced the best results when evaluating the number of buds per branch, fertilized flowers, fruits per branch and yield, obtaining a yield between 19.04 t.ha-1 and 24.97 t.ha-1, depending on the production area77. The blackberries are badly affected by black foot disease, an important disease in producing areas, which was found to be caused by the fungus Dactylonectria torresensis78. The antagonistic capacity of isolates of Trichoderma spp. against the pathogen was determined. Under in vitro conditions, a 68% inhibition of radial growth of the pathogen was observed, while in the greenhouse there was a reduction in the percentage of root neck necrosis of 35%80. Studies were also carried out to determine alternatives for the control of this disease. Carbendazim and alternative products such as Myrtaceae extract showed the lowest incidence of the disease at the root and neck level of the plant, with 22% and 36% respectively, compared to 74% in the control79.
In a further trial, on the productivity (yield and weight of the fruit) of the blackberry crop the application of T. asperellum (1.53 x 109 CFU/g) produced an increase in yield between 13% and 29% compared to the control. Application of the fungus resulted in larger berries with an increase of 1 g/berry in the treatments where the beneficial fungus was applied78,50. During the trials, is was confirmed that, with periodic (monthly) inoculations of Trichoderma sp. to the soil, the establishment of the fungus was achieved, reaching high population levels in the rhizosphere of the crop81.
A survey was made on the use of microorganisms by small farmers in blackberry production. The information was collected in two of the main producing provinces of this fruit in Ecuador (Tungurahua and Bolívar). It was concluded that farmers who have been beneficiaries of the Project have adopted the practice of using BM as part of the integrated management of their crops. It was established that 53% of the bioproducts used have bacteria of the Bacillus genus as active ingredients, 31% are based on Trichoderma and 16% the remainder correspond to other types of with the addition of combinations of microorganisms. Farmers stated that microorganisms are used for different functions such as soil conditioning (53%), control of downward wilt disease (41%) and to prevent diseases (6%). The cost of microorganisms varies according to the bio-inputs used, finding that 42% of farmers spend less than 30 USD/200-liter tank, 35% spend $50/tank, 10% spend $40/tank, and 13% spend more than $50/tank. Eighty-one percent of blackberry producers apply microorganisms twice a year, 16% three times a year and 3% once a year. Sixty-six percent of the blackberry producers indicated reaching a crop yield of 12 to 18 kg/plant, 25% of 18 to 27 kg/plant and 9% had yields below 12 kg / plant, values that in all the cases are higher than the national average reported by MAG of 4 kg / plant. Of the farmers surveyed, 80% stated that they had received training from INIAP in the use of microorganisms.
In tests carried out in organic banana crops (Musa acuminata), the effect of the inoculation of beneficial microorganisms (Trichoderma asperellum, Purpureocillium lilacinum and Arthrobotrys sp.) on the populations of phytopathogenic nematodes was evaluated. While application of the fungi did not reduce the populations of pathogenic or free-living nematodes, plants treated with T. asperellum showed an increase in the number of healthy roots, which favored the increase in productivity of the crop82.
In addition, beneficial fungi (Beauveria and Paecilomyces) were evaluated for the control of the thrip Chaetanaphothrips signipennis which causes red rust damage that causes severe losses to small and medium-sized organic banana producers and those producing plantains for export83. Unfortunately, these beneficial fungi caused a mottling of the fruit when applied directly to the bunch and could not be recommended84. As a consequence, products accepted in organic agriculture were evaluated, finding a 90% reduction in damage from red rust banana thrips on fruits through the application of pyrethrin85, and up to 100% with the application of Saccharopolyspora spinosa83. In a survey of two associations of small banana producers in Ecuador (Asociación Tierra Fértil and ASOPROLIFLO), 95% of the producers consider that the losses due to red rust banana thrips are important and all are willing to use the technology that INIAP offers for the control of the red rust banana thrips; but they want to continue training through continuous knowledge update workshops.
Biological control is the control of pest or plant diseases by applying biological agents suppress a pest or prevent the development of a disease by a pathogen. It is a sustainable approach to pest management, contributing to the reduction in the use of pesticides as part of an IPM strategy86. This type control is based on the premise that it is important to counteract habitat loss and environmental disturbance associated with intensive crop production by conserving and managing biological control agents87. These organisms provide a valuable ecosystem service through their contribution to BC88. Therefore, the conservation of BM is of fundamental importance for the sustainability of crop production89.
Trichoderma spp., is the main antagonistic fungus that has been formulated and is widely used in agricultural applications due to its well-known BC mechanism. It is also known for its beneficial impact on plant growth, decomposition processes and bioremediation90. These benefits bring enormous advantages to the agricultural industry by applying environmentally friendly agricultural practices.
Recent work has pointed out that common plant diseases such as root rot, seedling disease, wilting, fruit rot, among others, can be prevented and/or controlled with Trichoderma spp.90. Commonly, BCAs do not affect to non-target organisms, however, it has been reported that Trichoderma spp., can also affect other microorganisms91. For this reason it is recommended to apply Trichoderma sp. alone and not in combination with other biocontrol agents.
The widespread use of Trichoderma spp. in formulated products (wettable powder and granules) has attracted the attention of researchers to discover more about other potential benefits of Trichoderma spp., especially as PGP and its impact on plant yield50,76,82,84,92, for which its application is recommended as part of agricultural crop management programs. The use of formulations allows the generation of products in which the microorganism has a substrate that allows it to have stability, maintaining concentration and viability, and prolonging its shelf life51. More research is needed to generate new prototypes that allow the production of quality products that obtain efficient results when applied to agricultural crops.
The regulatory issue is of utmost relevance to regulate the production of bio-inputs in a country66. For this reason, the regulations implemented in Latin America allow the establishment of evaluation parameters for the registration of products, guaranteeing their quality. These control processes must be rigorous in order to avoid the commercialization of deficient products that cause the farmer to lose confidence and credibility in bio-inputs.
In Ecuador, there is a trend towards the adoption of biological controls in the production systems of small farmers in which their benefits have been justified through the results of the project “Biocontrol for Sustainable Farming Systems”93. This approach leads to the economic growth of poor rural communities and the development of access to local and international markets. Furthermore, by adopting and promoting biological production systems, Ecuador can promote its agricultural sector not only for production, but also as an attractive place for ecological and community tourism. To a large extent, this agricultural development has allowed Ecuador to work from within towards success in achieving the ONU Sustainability Goals93.
The use of beneficial fungi, mainly Trichoderma sp. and Beauveria sp., has increased in agriculture due to the positive results that have been confirmed through research generated in different Latin American countries that have justified the efficiency of beneficial fungi in PC as well as their functionality as PGP, affecting plant nutrition. In addition, it has been confirmed that the use of biological control assists the development of a sustainable agriculture that conserves natural resources such as soil and water, as well as reducing pollution of the environment by reducing the use of agrochemicals in agricultural crops. However, regulation of the commercialization of these products is an issue that must continue to be strengthened so that users of bio-inputs obtain high quality, efficient products through quality control procedures that allow formulations and product efficacy to be verified.
In Ecuador, the BC issue has taken on importance and currently there is a favorable attitude among farmers to use bio-inputs and reduce the application of agrochemicals due to the results obtained in the Project “Biocontrol for Sustainable Farming Systems”. These results have generated trust and credibility in farmers on the use of microorganisms in agriculture as a component of integrated crop management that protects the environment and offers clean products benefiting consumer health.
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Finally, thanks to all the farmers and technicians who were part of the activities developed in the Project.
William Fernando Viera-Arroyo E-mail address: email@example.com