Genetically Modified Plants Based on Bacillus Genes and Commercial Bacillus-Based Biopesticides for Sustainable Agriculture

Abstract

Due to the increase in the global population, there is an urgent call to enhance the crop production through sustainable agriculture. Biological control is a possible solution. There are many examples of biological control agents applied to different crops that have improved their yield or quality, including vegetable and fruit crops and ornamental plants. The Bacillus species have been used as powerful tools since they suppress plant pathogens and promote plant growth as well. During the last five decades, B. thuringiensis has been used as biopesticide in several crops. However, it has some disadvantages such as its instability under field conditions due to sunlight; therefore, frequent applications are necessary, making its use more expensive. To solve this problem, genetically modified crops have been employed to allow the plant to express the toxin in the whole plant. Genetic engineering is a method used to make changes in the genetic material of an organism using scientific techniques. Therefore, genetic engineering opens up opportunities for creating genetically modified plants to increase crop yields and avoid pests. Genetically modified (GM) crops have been cultivated during the last three decades. Transgenic plant technology can be used to address global food scarcity, particularly in developing countries. Genetically modified organisms are a controversial topic that needs to be considered more carefully. Many toxicology studies have confirmed that Bt microbial formulations are safe for consumption. In this review, we will revise the application of Bacillus genes and Bacillus formulations to crops and their safety for human health to provide a more comprehensive understanding of this topic.

1. Introduction

The worldwide population is increasing; therefore, to meet growing food demand, sustainable agricultural production is needed. The use of chemical pesticides has been the best way to control pests in recent decades. However, the use of these chemicals has caused several health problems in humans, since these chemicals enter the food chain and eventually the human body, some of them being carcinogens [1]. Concerns about human health and environmental problems prioritize safer and more sustainable crop production.

Biological control represents a possible solution, as a strategy based on the use of microorganisms to control some risk factors, such as pathogens or pests affecting plants and crops, in a safe and natural manner [2]. There are several microorganisms that have been used as biological control agents, Bacillus spp. being one of the most employed in agriculture as a biopesticide in integrated pest management programs. Bacillus use direct and indirect mechanisms that can be functioning simultaneously during plant growth, either through achieving nutrients or modulating plant hormone levels and secreting chemical compounds to act against plant pathogens, or by inducing resistance to pathogens.

The production of biopesticides has improved with the help of molecular biology, genetics, protein engineering, and genome sequencing. Microbial pesticides have been effectively used for pest control due to their cost-effective mass production, specificity, persistence in the environment, and safety. However, synthetic pesticides can be more competitive than bacterial insecticides because the latter have a narrower activity spectrum, rapid environmental degradation, and lower efficacy. Bacillus thuringiensis is one of the most used biopesticides worldwide; however, in recent years more Bacillus species have demonstrated their potential as biopesticides used in agriculture due to the vast array of chemical compounds produced by these bacteria.

This genus has the capacity to secrete several metabolites, which act against a variety of plant pathogens or promote plant growth. The direct and indirect mechanisms employed by Bacillus spp. to improve the plants’ growth include nitrogen fixation, solubilization and mineralization of nutrients, phytohormone production, production of siderophores, antimicrobial compounds and hydrolytic enzymes, induction of systemic resistance (ISR), and tolerance to abiotic stresses [3].

Taking these relevant points, in this review we will revise, as an introductory topic for the readers, the role of this genus in horticultural crops focusing on the genetic modifications to improve its efficacy against crop pests. Additionally, the developments realized regarding the genetic engineering in Bacillus species applied to plants were revised as well as the safety of Bt-based genetically modified organisms.

2. Bacillus in Horticultural Crops

The horticultural sector improves land use for food and promotes crop diversification, employment generation and poverty alleviation. Among the horticultural crops, fruits and vegetables are the most numerous crops; however, there are also flowers, aromatic plants, spices, and plantation crops. There are many examples of different crops that improved their yield or quality after the application of a biological control agent, including vegetable, fruit, and ornamental plants [4].

One of the most important aspects of achieving the good quality and yield of horticultural crop production is the availability of nutrients in the soil. For this reason, over the years, chemical fertilizers have played the main role in increasing the productivity of crops. This practice has led to several problems, such as environmental pollution and therefore impacts on human health. The best green alternative has been the development of biofertilizers, which are supported by microorganisms that are nitrogen fixers, solubilizers of phosphates, and phytohormones and growth promoters. All of them use different strategies to facilitate plant growth. Biofertilizers have been evaluated in a wide variety of crops, including rice, cucumber, wheat, sugarcane, and corn, among others [5]. The application of biofertilizers in horticulture implies the improvement of the yield and quality of crops. Beneficial microorganisms improve the rhizospheric region, making nutrients available for plants or producing phytohormones.

Bacillus spp. use direct and indirect mechanisms that can act simultaneously in plant growth. The direct mechanisms include achievement of nutrient supplies and modulation of plant hormone levels. The indirect mechanisms include the secretion of chemical compounds to act against phytopathogens or the induction of pathogen resistance [6].

There are several examples of the role of Bacillus as a biofertilizer. Among vegetables, some examples are included. The yield of mustard and tomato was increased after applying Bacillus- or Trichoderma-based fertilizers [7,8]. By applying individual inoculants of Bacillus, Brevibacillus, and Rhizobium, the macro- and micronutrient content in broccoli was improved [9]. Bacillus and Pseudomonas improved the biomass of lettuce seedlings [10]. Similarly, other crops such as spinach and flax, also exhibited improvements after treatment with Azotobacter, Bacillus, or AMF [11,12]. B. subtilis was applied as a biofertilizer to increase cotton yield [13]. Treatment with B. subtilis and B. megaterium resulted in growth and yield increasement, and improved seed quality of maize [14].

Bacillus strains have been intensively used against Fusarium [15,16,17,18,19] and Aspergillus [20,21,22] species. Also, Bacillus-based biocontrol has been used to decrease mycotoxin contamination in crops [23,24,25]. The studies devoted to lower mycotoxin content in GM-Bt plants (maize) [26,27,28] should also be mentioned.

Generally, fruit crops have received more attention than vegetables and ornamental crops. The application of Bacillus strains has reduced the crop maturation days of strawberry plants [29]. The inoculation with the commercial product Rhizocell C containing B. amyloliquefaciens improved the photosynthetic capacity of strawberry plants, increasing the fruit yield and biomass compared to other commercialized products [30]. The inoculation with B. amyloliquefaciens has improved the yield of banana, infested with the fungal disease, under field conditions [31]. A liquid B. subtilis commercial microbial fertilizer was applied to citrus groves of the Tarocco blood orange (Citrus sinensis), exerting positive effects on fruit quality [32]. Bacillus spp. was applied as a biofertilizer to treat nutmeg seeds, showing an improvement in the growth of nutmeg seedlings [33].

Bacillus species also use indirect mechanisms to inhibit plant pathogens. The genus Bacillus spp. secretes several secondary metabolites that act against phytopathogens causing plant diseases, promoting plant growth [34,35,36,37]. In addition, these bacteria induce systemic resistance in plants [38]. There are some mechanisms to control pathogens causing plant diseases. Some of these mechanisms include (a) competition; (b) antibiosis; (c) predation or parasitism; and (d) induction of host plant resistance [39].

Biofungicides have several advantages in use against the crop pests compared with chemical pesticides. The first is that they have a strong selectivity, being safe for humans and animals. Second, they are safe for the environment since they are derived from natural ecosystems. Moreover, they are easily decomposed by sunlight, plants, or various soil microorganisms, completing a natural life cycle. This guarantees that these products do not persist long in the environment, which reduces the risk to non-target organisms [37]. Bacillus-based biopesticides control crop pests, improving soil quality and health, and the growth, yield, and quality of crops [40].

Several Bacillus-based biopesticides that have been commercialized are shown in

Table 1. Commercial Bacillus-based biopesticides currently in use.

Microorganism	Action	Brand Name	Producer
B. amyloliquefaciens	Fungicide	Serifel Integral Taegro Companion Maxxx Serenade Amylo-X Aveo	BASF Ag products Syngenta Group pH Douglass Plant Health Bayer CropScience LP Certis Valent BioSciences
B. pumilus	Fungicide	Ballad plus Sonata AS YieldShield	Bayer CropScience LP
B. sphaericus	Insecticide	VectoLex	Valent BioSciences
B. subtilis	Fungicide	Kodiak Cillus Biotilis	Bayer CropScience LP Green Biotech, Korea Agri Life
B. thuringiensis var. aizawai	Insecticide	XenTari Agree Turex Solbit	Valent BioSciences Certis Green Biotech, Korea
B. thuringiensis var. israelensis	Insecticide	Bactimos Teknar VectoBac VectoMax Aquabac Bacticide BTI granules	Valent BioSciences Becker Microbial Biotech Int’l Clarke Mos. Cont.
B. thuringiensis var. kurstaki	Insecticide	Dipel Foray Cordalene Lipel Sp Lipel Biolep BMP 123 Baturad Belthirul Deliver Delfin Condor Crymax Javelin WG Lepinox WG Turex Turicide Safer BTK Rapax Lepinox plus	Valent BioSciences Agrichem Som Phytopharma Agri Life Biotech international Becker microbial Agrindustrial S.A. Probelte S.A. Certis Woodstream Canada Ecogen/Intrachem
B. thuringiensis var. tenebrionis	Insecticide	Novodor	Valent BioSciences

.

B. thuringiensis (Bt) has been the most used and commercialized biopesticide [41]. It has been widely used in agriculture since it is eco-friendly and safe for non-target organisms, but it is effective and highly specific against insect pests affecting crops belonging to the Lepidopteran, Dipteran, and Coleopteran insect orders [41]. Many commercial products of Bt bioinsecticides have been developed over the decades and are available on the market [42]. This biopesticide capacity is due to the production of crystalline proteins called Cry proteins along with spores during the sporulation stage, which are toxic to different insect orders. This capacity of Bt is important to the application of this bacterium as a green biopesticide against crop pests.

3. Genetic Engineering in Bacillus Applied to Plants

Genetic engineering is a method of making changes to the genetic material of an organism using scientific techniques. Genetic engineering has become an intervention in the field of agricultural and industrial biotechnology including different types of plants, animals, and microorganisms [43]. In agriculture, these techniques have been applied to achieve modified crops by integrating sequences of DNA into the germplasm of crop plants to obtain new crops with better characteristics than wild-type crops, such as appearance, yield, size, and resistance to pests. The integrated DNA sequences encode insecticidal proteins in transgenic plants to resist insect pests [44].

To understand how B. thuringiensis was genetically modified for introduction into several crops for pest control, it is necessary to know how this bacterium acts against several pests through its insecticidal proteins derived from toxin genes. The mechanism of action of insecticidal toxins is basically described in Figure 1. Briefly, the insecticidal toxins of B. thuringiensis need to be ingested by the insect larvae to be effective [45]. After ingestion, the toxins are solubilized by the alkaline conditions and then are converted into toxic core fragments, which bind to the receptors of epithelial midgut cells. Then, the toxin adopts a specific conformation allowing its insertion into the cell membrane and forming transmembrane pores, which cause an osmotic imbalance causing cell rupture. This leads to insect death caused by bacteremia [46].

These insecticidal toxins are derived from cry genes [47], which have been classified and organized in a systematic nomenclature. Cry toxins have been effective for the control of several insect pests affecting important crops. Naturally occurring cry genes and several mutations show varied specificity and novel/improved toxicity against a specific insect group. However, some insects can acquire resistance to the cry gene product [46]. Traits of Bt, such as pest resistance and herbicide properties, are most extensively used in plant genetic engineering. Bt toxin genes have been used in genetic engineering for application to many crops to act against specific pests [48]. For several years, the development of new toxin genes in new Bt strains was the main aim of researchers on this topic. After the toxin mechanism of action was studied and elucidated, research centered on altering the amino acids of the main domains of toxin. In this way, new proteins could be created.

Bt biopesticides have some advantages, such as specificity and their environmental safety and they are cheap and easily formulated [49]. However, they can have some disadvantages, such as their instability under field conditions due to sunlight; therefore, frequent applications are necessary, making their use more expensive. As has been seen, short persistence and low residual activity are two factors limiting the wide use of Bt products. Different formulations and strategies have been developed to protect Bt biopesticides from sunlight [50].

Another problem is their restricted field application since they have been applied mainly to the aerial parts of the plant. To solve this problem, genetically modified crops have been employed to allow the plant to express the toxin throughout the plant. These transgenic Bt crops are protected from insect attack by expressing Bt toxins in plant tissues. During decades of improvement of B. thuringiensis strains as biopesticides, the main issue has been their application to crops. For this reason, cry genes were manipulated to achieve genes with a wider target spectrum or higher toxicity than wild-type strains [47,51,52]. Therefore, several genetic tools were developed.

The first genetic exchange system available in B. thuringiensis was generalized transduction [53]. The second important advance in genetic exchange was the discovery of a conjugation-like process involving plasmid transfer. This tool permitted the development of strains with crystal protein gene combinations that are active against some insect species. The transfer of recombinant plasmids was possible from one strain to another. However, this technique has some limitations such as plasmid incompatibility, location of cry genes on non-transmissible plasmids, the presence of undesirable genetic material, and eventual plasmid loss [54]. Using the molecular method of in vivo homologous recombination, this structural instability or loss of plasmids can be avoided. Homologous recombination utilizes integrational vectors and thermosensitive plasmids along with chromosome fragments. These fragments are homologous to the B. thuringiensis chromosome and are introduced into the integrative plasmids to realize recombination with the chromosome. When the transformation does not occur naturally, alternative methods such as electroporation and biolistic bombardment have been effectively used on Bt transgenic crops (Figure 2). Plant expression systems such as cauliflower mosaic virus 35S promoter, maize promoter, or chloroplast promoter, were the key to expressing Bt genes in plants [55,56]. Effective promoters and expression cassettes probably could improve gene expression instead of “transformation” methods.

The best option to avoid plant toxicity is to transform truncated toxin genes. For this reason, truncated cry1A genes have been used to achieve transgenic tomato, tobacco, and cotton. Several companies, such as Monsanto or Mycogen, have made several probes changing the promoters, antibiotic resistance genes, transformation-tissue culture systems, and developing novel insecticidal proteins [57]. In this way, some genetically modified products were sold by several companies, such as Ecogen. New Leaf was the variety of potato expressing the cry3A toxin gene. This was the first commercially available product of its kind, manufactured in 1995 by an affiliate of Monsanto (NatureMark) [57]. After this, other transgenic Bt crops were commercialized, including maize and cotton [58,59]. Some crops like potato, tomato, tobacco, rice, maize, and broccoli have been genetically modified to express cry genes to kill the pests causing damage to the plant. This caused great controversy about their safety for human health [60].

Similarly, genetically modified organisms, generated via transfection of B. thuringiensis subspecies genes, produce biotechnological products that have various applications. B. thuringiensis var. kurstaki (Btk) is a bacterium, which protects crops from insect pathogens and is available as a registered formulation in the marketplace (DiPel and Forey). These two formulations are applied by spraying crops in agriculture, horticulture, and woodland plants. Moreover, the insect and fungal pathogen, B. thuringiensis subsp. israelensis (Bti), is a biologically active strain that acts against mosquito species but is harmless to humans [60]. Therefore, Bti is employed for the effective treatment of stagnant ditches, ponds, lakes, and wastewater settling tanks.

Corn expressing Cry1Ab and cotton expressing Cry1Ac are other genetically modified crops [61]. Transgenic crop cultivation expressing the insecticidal cry gene derived from B. thuringiensis, the most successful bioinsecticide, is now a common practice worldwide [62]. These transgenic plants include cotton, cauliflower, tomato, corn, chilli, and eggplants, products famous for their resistance against insects [63]. Two toxins, Cry1Ac and Cry2Ab, have been commercialized for the cotton crop with the name of Bollgard II, protecting against lepidopteran pests [64]. Another strategy was to produce GM crops with several different genes active against different target insect pests such as Monsanto’s YieldGard Plus maize expressing cry1Ab1, which is active against lepidopteran insects, and the cry3Bb1 gene, active against the coleopteran corn borer pest [65]. Another modification has been the expression of Vip3 proteins, which are vegetative insecticidal proteins active against lepidopteran insects, along with Cry proteins [66,67,68]. Using double stranded RNA (dsRNA), a transgenic cotton has been developed against H. armigera [69]. A generalized strategy that is followed to clone Bt genes into plant genome is schematized in Figure 3.

4. Safety of Bt Based Genetically Modified Organisms (GMO)

Bt is a very useful microorganism for attacking crop pathogens, since it is a natural bioinsecticide and it has been demonstrated to do little damage against non-target organisms due to it being very specifically active against certain pests [70]. Genetically modified (GM) crops have been cultivated during the last three decades. The global population is growing exponentially; therefore, it can be important to develop transgenic plant technology to avoid food scarcity, especially in developing countries. In this way, farmers can achieve greater crop yields in a shorter time [71]. The commercial production and distribution of Bt crops, such as corn, cotton, potato, and tobacco were approved in 1995, by the Environmental Protection Agency (EPA) in the USA [42]. Although several crops have been genetically modified with Bt toxins, currently, the most common Bt crops are corn and cotton [71].

However, genetically modified organisms are a topic of debate worth considering [72,73]. In this sense, the main concern about the potential risks of recombinant Bt strains and GM-Bt crops is the impact on non-target beneficial species [70], the effects of introducing genes to the relevant microorganisms and unpredicted effects among others, especially food safety (Figure 4). To avoid the problem of their impact on non-target beneficial species, the main aim is to increase host specificity and persistence to achieve better efficacy and lower insect resistance [74,75,76,77]. Laboratory and field studies have been conducted on the effects of GM crops on the environment and human health, resulting in little risk for human health [76,77,78]. Risks are assessed by performing laboratory tests, followed by small- and large-scale field trials to ascertain environmental safety before the release a commercial transgenic crop. To achieve this, the procedure has been performed in laboratory and greenhouse conditions, in small- and large-scale trials, and commercial release tests [79]. Risk management needs to include evaluation of identified risks and some measures that are important for determining how harms to human health and the environment can be mitigated. This must be introduced into the decision making on and regulatory approval of transgenic crops. In the first instance, the gene flow from transgenic crops to humans or the environment needs to be assessed; however, cross fertilization rates influenced by several factors such as pollen viability and longevity or wind direction also need to be considered [79].

The food safety of Bt transgenic crops has been profoundly studied. Toxicity, allergenicity, and genetic hazards are the three major health risks potentially associated with GM foods. Besides, introduced genes might alter the nutritional value of foods in unpredictable ways by reducing some nutrients levels while raising the levels of others [80,81,82]. The assessment of potential allergenicity tests should be carried out by comparison between the introduced protein characteristics with the characteristics of known allergens. The Cry proteins of Bt crops have to follow the recommendations of the codex [83]. A study about allergic reactions in workers who manufacture or apply Bt microbials was conducted showing no evidence of any reaction [84]. A comprehensive food safety assessment approach involves several analyses as well as elucidation of the basic mechanisms of food allergy [85,86]. Any eventual negative health impact needs to be ruled out; therefore, toxicological tests should be developed [87]. The major concern is about several lethal effects and adverse influences on fertility [88].

There have been several studies regarding human health and how Bt food can influence health in humans, demonstrating that Bt toxins are easily degraded in the presence of digestive fluids of the human gut within 0–7 min. Therefore, the risk of Bt foods for human health is small [42,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90]. A recent review gives evidence that Bt maize is highly selective against pests with relatively few negative consequences, especially when compared with chemical insecticides [78,80]. In peer-reviewed studies on GM food safety/security there have no significant health risks have been reported [91]. However, more studies are needed on the GM effects on human consumption [91].

5. Conclusions and Future Perspectives

As has been mentioned before, Bacillus sp. especially B. thuringiensis has been used in agriculture as biopesticide during the last decades. Some improvements have been applied over the years. In the first instance, genetic engineering was held hostage by the lack of technological progress; however, advances in molecular biology and methodologies made it possible to address this problem, enabling the manipulation of insecticidal toxins to produce more specific strains of B. thuringiensis. Then, the genes could be cloned inside the plant avoiding some issues associated with spraying techniques and making more specific Bt toxins against crop-associated pests. Biotechnology is related to recombinant DNA techniques or genetic engineering.

However, in practice, biotechnology includes not only genetic engineering but also manipulation of reproductive processes. These tools are used to improve global food availability at reasonable prices for the rural poor worldwide. It is necessary to understand genetics to search for answers to several questions or problems. In this sense, the genetics of Bacillus has been studied for years. Genetically modified crops or transgenic crops have many benefits that initially pushed chemical pesticides into the background. However, the voices against transgenic crops quickly began to be heard, alleging environmental damage and risks to human health. The demonstrations against GMOs carried out by Greenpeace against the Monsanto Company, accusing it of destroying monarch butterflies, were very well known and continue to the present day.

Thousands of studies have been carried out regarding the risks of transgenic crops. In some of them, minor effects on non-target beneficial species were reported. Therefore, increasing host specificity and long-term persistence can be good ways to achieve better efficacy and lower insect resistance. However, in most of the studies no impact or significant damage to human health or the environment was demonstrated. Many toxicology studies conducted with Bt microbial formulations have confirmed their safety for consumption. It is thought that GM crops are quite safe and even less harmful than using crops treated with chemical pesticides and fertilizers that have been shown to be carcinogenic. Many scientists are protected by the reasonable doubt that the study of the consequences of introducing transgenic crops into the food chain will take years to be completely deciphered.

The most recent advances have come with CRISPR technology, which has been developed to accurately modify the genes of living organisms and is now allowing more and more researchers to use gene editing in agriculture. Researchers have used CRISPR to develop fungus-resistant wheat, drought-resistant corn, and tomato plants with a larger fruit size.

This can be very opportune for the companies that control the world production of chemical pesticides, which take advantage of these doubts to continue to monopolize the market. If there is little doubt about the risk of consuming GM foods, the use of GM foods will be put on the back burner until the necessary knowledge is gained to dispel any doubts about their efficacy and safety. Genetic improvement has been a strong tool to develop and characterize the genes and proteins responsible for plant growth promotion, as well as the suppression of plant pathogens, allowing genetic modifications to increase their efficacy. Despite great advances that have been achieved to date, there is still a lot of work to be done.